Biosafety in Industrial Biotechnology

Biosafety in Industrial Biotechnology
Biosafety in Industrial
Biotechnology
Edited by
P. HAMBLETON, J. MELLING
Centre for Applied Microbiology and Research
Porton Down
Wiltshire
UK
and
T.T. SALUSBURY
Science and Technology Section
British Embassy
Japan
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

First edition 1994
© 1994 Springer Science+Business Media Dordrecht
Originally published by Chapman & Hall in 1994
Softcover reprint ofthe hardcover 1st edition 1994
Typeset in 1O/12pt Times by Cambrian Typesetters, Frimley, Surrey

ISBN 978-94-010-4590-2 ISBN 978-94-011-1352-6 (eBook)
DOI 10.1007/978-94-011-1352-6
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Preface
As an industry, biotechnology may be likened to the Hymn Book, being

both ancient and modern. Whereas activities such as baking, brewing, the
fermenting of foods date from our earliest attempts to control and utilise
the environment, the application of recombinant DNA technology is
recognised as being at the forefront of novel industrial development.
Perhaps because of its association with processing foodstuffs together
with the benefits derived from applications in the early organic chemistry
and pharmaceutical industries, biotechnology has been regarded as being
inherently safe. Yet unlike other modern industries, such as chemical and
nuclear, where regulation has followed from incidents or accidents,
modern biotechnology has been subject to close scrutiny and regulation
almost from its inception.
The process of regulation itself is somewhat unusual in that it was
initially self-imposed by the very scientists who developed the fundamental
techniques of recombinant DNA technology. They recognised the signific-
ance of their development but were concerned of the effects on humans
and the environment of uncontrolled application of the new, powerful
technology. Concern about the possible consequences of genetic manipula-
tion has undoubtedly been the driving force behind the regulations that are
now in place in many parts of the world and which are the subject of this
book. Safety issues in the biotechnology industry can be categorised under
three headings: worker, environmental and consumer (product) safety.
Product safety, in the pharmaceutical industry at least, is adequately
covered by existing requirements for quality, safety and efficacy. The new
regulations are largely concerned with affording protection to the worker
and the environment, despite the fact that there is no direct evidence that
the new industry represents a threat to either. The failure of the perceived
risks to become apparent is now stimulating moves towards a degree of
deregulation. Nevertheless, there are now in place around the world many
regulations governing the biotechnology industry. This book aims initially
to provide a practical overview of the present regulatory scene. Knowing
the regulations is one thing, being able to meet their requirements is
another and the other aim of this book is to provide the reader with
practical information to achieve this.
In considering engineering solutions to the problems of meeting
regulatory requirements emphasis has been placed on dealing with
microbiologically-based biotechnology processes. Release of bio-hazardous
VI PREFACE
materials from the process probably represents the most likely risk of
contaminating the workplace/environment and chapters in this book
provide information on means of controlling and monitoring release and
consider possible consequences of release.
In the event, the initial fears about the undesirable impact of
biotechnology upon health and the environment have not been realised.
Some may see this as justifying the introduction and retention of
regulations. Others may feel that this justifies releasing the industry from
their control. In either event the industry will need to maintain its good
safety record if it is to succeed in selling its products to a world that is
becoming increasingly sceptical of expanding industrial activities.
Many thanks to Dr. Peter Hammond for assistance in creating the index
and Ann Bennett for administration help.
P.H.
1.M.
T.S.
Contributors
G.D.J. Adams Freeze Drying Research and Development Section,

Centre for Applied Microbiology and Research, Porton
Down, Salisbury, Wiltshire SP4 OJG, UK.
J.E. Benbough Biosafety Investigation Unit, Centre for Applied
Microbiology and Research, Porton Down, Salisbury,
Wiltshire SP4 OJG, UK.
A.M. Bennett Biosafety Investigation Unit, Centre for Applied
Microbiology and Research, Porton Down, Near
Salisbury, Wiltshire SP4 OJG, UK.
A.N. Cottam Health and Safety Executive, Safety Policy Division
(Branch E), Magdalen House, Stanley Precinct,
BootIe, Merseyside L20 3QZ, UK.
J.R. Court Facilities Management Group, Centre for Applied
J.S. Deans Health and Safety Executive, HPDI, Baynards House,
Rose Court, 2 Southwark Bridge, London, SEl 9HF,
UK.
P. Hambleton Director of Production, Centre for Applied Micro-
biology and Research, Porton Down, Salisbury, Wilt-
shire SP4 OJG, UK.
D.T. Kingsbury Johns Hopkins School of Medicine, Genoma Data
Base, 2024 East Monument Street, Baltimore, MD
21205, USA.
G. Leaver AEA Technology, Biotechnology Services, Building
353, Harewell, Didcot, Oxfordshire OXII ORA, UK.
J. Melling Chief Executive and Director, Centre for Applied
K.P. Norris 53 Bouverie Avenue, Salisbury, Wiltshire SP2 8DU,
UK.
V11l CONTRIBUTORS
A. Rimmington Centre for Russian and East European Studies, The

University of Birmingham, PO Box 363, Birmingham
B15 2TT, UK.
T. T. Salusbury First Secretary, Science and Technology Section,
British Embassy, 1 Ichiban-cho, Chiyoda-ku, Tokyo
102, Japan.
I.W. Stewart AEA Technology, Biotechnology Services, Building
353, Harewell, Didcot, Oxfordshire OXll ORA, UK.
A.J. Taylor Department of Health, Wellington House, 133-155
Waterloo Road, London SE1, UK.
Contents
1 The development of European legislation on genetically

modified organisms 1
A.J. TAYLOR
1.1 Introduction I
1.2 The development ofthe EC directives I
1.3 The EC Directives on genetic modification 2
1.3.1 Directive 90/219/EEC - Contained use of genetically-modified
micro-organisms 3
1.3.2 Directive 90/220/EEC - Deliberate release of genetically-modified
organisms 5
1.4 The situation in the twelve Member States (as of February 1993) 8
1.4.1 United Kingdom 8
1.4.2 Federal Republic of Germany 8
1.4.3 Netherlands 9
1.4.4 France 10
1.4.5 Denmark 10
1.4.6 Belgium II
1.4.7 Remaining EC Member States II
1.5 Situation in the EFTA nations (as of February 1993) II
1.6 Europe vs. the USA 12
Disclaimer 13
References 13
2 Occupational and environmental safety: the UK legislative

framework 14
A.N. COTTAM
2.1 Introduction 14
2.2 International influences 14
2.2.1 The UK approach IS
2.3 Occupational health and safety legislation 16
2.4 The Health and Safety at Work Act 18
2.4.1 Duties of employers 18
2.4.2 Duties of employees 19
2.4.3 Duty not to misuse 19
2.4.4 Duties of manufacturers and suppliers 20
2.4.5 Management systems 20
2.5 The Environmental Protection Act 21
2.6 Specific regulations 21
2.6.1 Control of Substances Hazardous to Health Regulations
1988 (COSHH) 21
2.6.2 Genetically Modified Organisms (Contained Use) Regulations
1992 and Genetically Modified Organisms (Contained Use)
Regulations 1993 22
2.6.3 Pressure Systems and Transportable Gas Containers Regulations
1989 23
2.6.4 Electricity at Work Regulations 1989 24
X CONTENTS
2.7 Advisory committees 24

2.7.1 Guidelines 25
2.7.2 Local involvement 26
2.8 Inspection and enforcement 26
2.8.1 Aims of inspection 27
2.8.2 Powers of inspectors 27
2.8.3 Enforcement 28
2.9 Thewayforward 29
References 31
3 Regulation of biotechnology in the United States, Canada,

and Latin America 32
D.T. KINGSBURY
3.1 Introduction 32
3.2 The United States 32
3.2.1 Federal regulatory structure 33
3.2.2 Food and Drug Administration 36
3.2.3 Environmental Protection Agency 43
3.2.4 Department of Agriculture 49
3.2.5 Coda 51
3.2.6 State regulatory bodies 51
3.3 Canada 52
3.3.1 Veterinary biologics 53
3.3.2 Genertically modified plants and micro-organisms 54
3.4 Latin America and the Caribbean 54
4 The legal and regulatory framework for biotechnology in

Japan 57
T. SALUSBURY
4.1 An overview ofthe Japanese biotechnology industry 57

4.2 Government attitudes to biotechnology 57
4.3 The government bodies involved in biotechnology 58
4.3.1 Laboratory-scale experiments 58
4.3.2 Monhusho and STA Guidelines 58
4.4 Industrial-scale applications of r-DNA technology 61
4.4.1 Industrial uses of r-DNA technology 61
4.4.2 Guidance for the use of r-DNA technology in the pharmaceutical
sector 62
4.5 Agricultural applications of r-DNA technology 63
4.6 Foods and food additives 64
4.7 Deliberate release of genetically-modified micro-organisms 65
Acknowledgements 66
References 66
5 Biotechnology and industrial microbiology regulations

in Russia and the former Soviet republics 67
A. RIMMINGTON
5.1 Introduction 67
5.2 Regulations governing work with micro-organisms containing
recombinant DNA 68
CONTENTS Xl
5.2.1 History 68
5.2.2 Thecurrentguidelines 70
5.2.3 Regulatory authorities 74
5.3 Regulations governing labour safety in biotechnology research
institutes and the microbiological industry 77
5.3.1 Labour safety standards 77
5.3.2 Rules governing the release of micro-organisms into the workplace 78
5.3.3 Regulations governing the release of micro-organisms into the
environment 79
5.4 Adherence to regulations governing the containment and safe use of
micro-organisms 80
5.4.1 The environment 81
5.4.2 Waste water 83
5.4.3 Industrial personnel 84
5.5 Conclusions 86
References 87
6 Physical aspects of the uncontrolled release of material in

biotechnology operations 90
K.P. NORRIS
6.1 Introduction 90
6.2 The generation of aerosols 92
6.3 Persistence of aerosols in a closed space 95
6.4 Persistence of aerosols in the atmosphere 98
6.5 Retention, clearance and absorption in the respiratory tract 99
6.6 The biological behaviour of airborne particles 100
6.6.1 The stability of the organism 100
6.6.2 Particle size 101
6.6.3 Relative humidity and temperature 101
6.6.4 Oxygen 102
6.6.5 Sunlight 102
6.6.6 Protecting agents 103
6.7 Airborne allergens 103
6.8 Conclusions 104
References 105
7 Health hazards in biotechnology 109

A. M. BENNETT
7.1 Introduction 109

7.2 Health hazards 110
7.2.1 Laboratory-associated infection III
7.2.2 Allergic reactions 112
7.2.3 Endotoxin reactions 116
7.2.4 Toxic reactions to products or by-products 118
7.2.5 Hazards posed by genetic modification 119
7.2.6 Hazards posed by animal cell culture 120
7.2.7 Hazards posed by plant cell culture 121
7.3 Hazards ofbioprocessing equipment 122
7.3.1 Fermentation 122
7.3.2 Centrifugation 122
7.3.3 Cell disruption 122
7.3.4 Filtration 123
7.3.5 Product handling 123
7.3.6 Risk assessment 123
7.3.7 Prevention 124
XII CONTENTS
Acknowledgements 124
References 125
8 Containment of unit processes 129

P. HAMBLETON and J. MELLING

8.2 Unit processes in biotechnology 130
8.3 Categories of containment 131
8.4 Safety cabinets 132
8.4.1 Classification 132
8.4.2 Air filtration 134
8.4.3 Class I cabinets 135
8.4.4 Class II cabinets 136
8.4.5 Class III cabinets 136
8.4.6 Laminar flow work stations 137
8.4.7 Application of Class III cabinets to process containment 137
8.4.9 Other processes 139
8.4.10 Flexible film isolators 143
8.5 Design engineering for secondary containment 144
8.5.2 Other processes 145
8.6 The cost of containment 146
8.7 Conclusions 146
References 147
9 Containment in downstream processing 149

J.S. DEANS and I.W. STEWART

9.2 Regulations and guidelines 151
9.3 Cell separation 152
9.3.2 Centrifugation 154
9.4 Cell disruption 166
9.4.1 Physical methods 166
9.4.2 Non-physical techniques 172
9.5 Fluid handling 173
9.6 Discussion and conclusions 174
9.7 Recommendations 175
References 176
10 Freeze-drying of biohazardous products 178

G.D.J. ADAMS

10.2 Principles ofthe freeze-drying process 178
10.2.1 Product preparation 178
10.2.2 Prefreezing 179
10.2.3 Primary drying 179
10.2.4 Secondary drying 179
10.2.5 Stoppering and removal 180
10.2.6 Storage and reconstitution 180
10.2.7 Technical features of the freeze-drier 180
CONTENTS XIll
10.3 Risk assessment 181

10.3.1 Potential hazards 181
10.3.2 Processing biohazardous materials 181
10.3.3 Liquid and particulate aerosols 182
10.4 Hazards associated with product dispensing and handling finished
m~~~ 1~
10.4.1 Dispensing pumps 183
10.4.2 Dispensingneedles 183
10.4.3 Filling reservoirs 184
10.4.4 Tray dispensing 184
10.4.5 Ampoules 184
10.4.6 Vials 185
10.4.7 Container breakage 185
10.4.8 Spillages 185
10.4.9 Stoppering 186
10.4.10 Product removal 186
10.4.11 Container sealing 186
10.4.12 Product storage 186
10.4.13 Container leakage during storage 187
10.4.14 Leak testing of sealed vials and ampoules 187
10.4.15 Reconstitution 188
10.5 Formulation 188
10.5.1 General concepts 188
10.5.2 Prefreezing 189
10.5.3 Freeze-drying 189
10.5.4 Freeze-drying excipients 189
10.5.5 Container breakage and miscellaneous consequences of product
freezing 190
10.5.6 Storage 190
10.5.7 Comparison of protection during aerosolation or freeze-drying 191
10.6 Ablation 191
10.6.1 Loss of contents 191
10.6.2 Influence of product formulation on ablation 191
10.6.3 Ablation and spillage 192
10.6.4 Ablation and back-migration of vacuum pump oil 192
10.7 Practical aspects of the design and operation of freeze-driers and
associated equipment 192
10.7.1 Freeze-drier design 193
10.7.2 Freeze-drier fabrication 194
10.7.3 Chamber/condenser geometry 194
10.7.4 Protective devices 195
10.7.5 Electrostatic precipitation and ultraviolet irradiators 195
10.7.6 Incineration 195
10.7.8 Selection and position offilters 196
10.7.9 Decontamination of the interior of the freeze-drier 199
10.7.10 Biocides and sanitising agents 199
10.7.11 Sterilisation by gaseous biocides 200
10.7.12 Dryheat 204
10.7.13 Atmospheric pressure steam (live steam) 204
10.7.14 Press uri sed steam 204
10.7.15 Integrated approach to safe freeze-drying ofbiohazardous materials 206
10.7.16 Factors affecting operational safety 206
10.7.17 Dispensing product 207
10.8 Conclusions 207
Acknowledgement 209
References 209
XIV CONTENTS
11 Interpretation of regulatory requirements to large scale

biosafety - the role of the Industrial Biosafety Project 213
G. LEAVER

11.2 Regulatory issues 213
11.3 Risk assessment 214
11.4 Human health and safety 215
11.4.1 Estimation of GMO hazard 215
11.4.2 Elaboration of containment principles 217
11.4.3 Equipment containment design principles 218
11.4.4 Measuring and monitoring containment 228
11.4.5 Maintenance and training 232
11.5 Environmental safety 232
11.6 Conclusions 235
Acknowledgements 236
References 236
12 Managing the emuent from bio-industrial processes 240

J.R. COURT

12.2 Regulatory background 241
12.3 Assessment of risk and appropriate action 241
12.4 Categories of waste 242
12.5 Liquid effluent 242
12.6 Choice oftreatment method 244
12.7 Containment considerations 244
12.7.1 Multiplicity of containment devices 245
12.7.2 'Dead legs' and crevice avoidance 246
12.7.3 Leaktesting 246
12.7.4 Standard operating procedure (SOPs) and process records 247
12.7.5 Planned preventative maintenance (PPM) schedule 248
12.7.6 Commissioning and validation 248
12.8 Practical treatment methods 248
12.8.2 Disinfection using chemical agents 249
12.8.3 Heat treatment using steam 250
12.9 Testing effluent for sterility 251
12.10 Design and qualification of a heat treatment effluent plant 252
12.10.1 Design considerations 252
12.10.2 Qualification of effluent treatment plant 256
12.11 The approach to effluent at CAMR 257
References 266
13 Sampling methods for testing and monitoring biosafety of

biotechnology equipment and activities 268
J.E. BENBOUGH

13.2 Sampling microbial aerosols 269
13.3 Sampling methods 270
13.4 Air sampling devices 272
13.4.1 Inertial collectors 273
13.4.2 Air centrifuges 280
CONTENTS xv

13.5 Sample assessment 286
13.5.1 Background 286
13.5.2 Detection methods 286
13.6 Practical applications of sampling techniques 288
References 291
Index 293
1 The development of European legislation
on genetically modified organisms
A.l. TAYLOR
1.1 Introduction
The call in the 'Berg letter' I for a moratorium on the further development
of certain classes of work involving recombinant DNA (r-DNA) had
effects across Europe. The UK, the Netherlands and the former West
Germany, produced voluntary guidelines to cover such work during the
1970s. Only the UK established a statutory system of notification when the
Health and Safety (Genetic Manipulation) Regulations, 1978 came into
force, the first specific law dealing with the 'new biotechnology'. 2
Most of Europe controlled the technology, if at all, by voluntary systems
of oversight well into the 1980s. Interest in harmonised control can be seen
to have been catalysed by the Organisation for Economic Co-operation
and Development (OECD). OECD had issued a report entitled
Biotechnology: International Trends and Perspectives 3 and from this
stemmed a programme of work controlled by OECD's Committee for
Scientific and Technological Policy to study safety issues. An ad hoc Group
of National Experts worked from 1983 to 1986 to produce the now familiar
guidelines entitled 'Recombinant DNA Safety Considerations,.4 This
established principles for safe operation when using genetically modified
organisms (GMOs) and these became accepted globally. The OECD
report recognised "that there is no scientific basis for specific legislation to
regulate the use of r-DNA organisms". If that recommendation had been
followed this review would cease at this point. However, a harmonised set
of principles was attractive to many who saw it as but a short step to
regulation. The OECD report had hardly been read and fully digested
before Europe took that first step.
1.2 The development of the EC directives
In November 1986, at a time when only the UK and Denmark had any
national legislation specifically to exert control on genetic modification,
the Comission of the European Communities (EC) issued a communication
to the Council entitled 'a Community Framework for the Regulation of
2 BIOSAFETY IN INDUSTRIAL BIOTECHNOLOGY
Biotechnology' in which it set out proposals for Community regulations.

This would have the aim of providing "a high and common level of human
and environmental protection throughout the Community". Preliminary
work began early in 1987, and by June of that year a set of four proposed
Directives were circulating for comment within the Communities. Two
came from the Environment Directorate (DG XI). These dealt with (a) the
deliberate release of genetically-modified organisms into the environment;
and (b) the control of accidents and waste management from the
contained use of dangerous and genetically-modified micro-organisms.
A third proposed Directive was developed by the Social Affairs
Directorate (DG V) dealing with the protection of workers from the risks
related to exposure to biological agents at work. The fourth proposal came
from the Industry Directorate (DG III) and dealt with "the contained use
of genetically modified micro-organisms which do not cause human
disease". This Directive was designed to take the OECD's principles for
Good Industrial Large Scale Practice (GILSP) and translate them into a
regulatory scheme.
The early DG XI contained-use proposed Directives set out five
groups of micro-organisms on a scale of ascending order of hazard which
would deal with both natural and modified microbes in a single piece of
legislation. Waste streams were to be monitored and final disposal
notified. As the Commission's internal discussions developed, the proposals
DG XI from DG III were combined into a single Directive which was to
deal with the contained use of genetically-modified organisms alone.
The new package was led by DG XI (Environment Directorate), which
inevitably changed the nature of the developing regulatory structure.
Instead of industry-driven legislation, dealing with laboratory and
commercial-scale operations, the Directive was to be influenced in its
development by those concerned with the harm to the environment that
might occur as a result of "escape from containment".
The DG V Directive dealing with issues of worker protection was put to
one side and did not re-emerge until 1990 as a separately-negotiated piece
of European legislation. 5 It was adopted by the Council in November 1990
with implementation due in November 1993 (1994 for Portugal). Its
provisions include the development of a Communty-wide classification
scheme for all biological agents following the familiar four hazard groups
used internationally.
1.3 The EC Directives on genetic modification
On 23 April 1990, the Council of Ministers adopted legislation in the form

of two Directives which set out procedures and conditions for work
involving genetic modification. These Directives apply to all Member
DEVELOPMENT OF EUROPEAN LEGISLATION 3
States of the EC who were obliged to implement this Community

legislation by 23 October 1991.
1.3.1 Directive 9012191 EEC - Contained use of genetically-modified

micro-organisms 6
The legal basis of this Directive is Article 130 of the Treaty of Rome, which
established the European Economic Community. Article 130 deals with
Community action relating to the environment; protecting and improving
the quality of the environment; protecting human health and promoting
prudent use of natural resources. A key aspect of this Article of the Treaty
is that it permits (subject to certain restrictions) individual Member states
to implement further measures, i.e. to go beyond the Directive. When
examined alongside the second Directive, concerning Deliberate Release
(see below), it will be seen that the Contained Use Directive (CUD) is
somewhat curiously the more environmentally-directed of the two pieces
of legislation, at least in terms of its Treaty basis.
The objective of the CUD is summed up in its first Article. It provides a
harmonised framework for all types and scales of contained uses of
genetically-modified micro-organisms (GMMOs). Here we see another
apparent anomaly between the two Directives: the CUD is restricted to
micro-organisms. This does not leave as large a legislative gap as first sight
might suggest, for Article 2 defines micro-organisms as including plant and
animal cells. Thus, many contained uses of higher organisms will be
covered by the CUD as the work will involve construction of modified
plants and animals at the level of cell culture.
The CUD defines a minimum list of techniques that are considered to be
within the meaning of the term 'genetic modification' - r-DNA techniques
using vector systems; direct introduction of nucleic acids by methods of
micro-injection; and cell fusion/hybridisation. Member States can include
additional techniques under national controls, if they see fit.
In addition, Article 2 sets out a list of techniques which are to be
considered as genetic modification only if they involve the use of r-DNA or
existing GMMOs - in vitro fertilisation, polyploidy induction, conjugation,
transduction, transformation or "any other natural process". This last
inclusion leaves a great deal to national interpretation.
Article 3 specifically excludes from the scope of the Directive the
techniques of mutagenesis, work with somatic animal hybridomas, cell
fusion of certain plant cells, and self-cloning of low risk micro-organisms.
However, Member States are again free to go further than the Directive in
these areas, if they so wish.
The concept of containment requires that there be physical barriers that
limit contact of the modified organism with the environment. Biological
barriers alone appear not to be sufficient under the CUD.
The CUD separates operations with GMMOs into two types, A and B.
Type A operations must fulfil two criteria, its purpose should fall into one
of five categories, teaching, research, development, non-industrial or non-
commercial operations; and it must be small-scale. All operations falling
outside these five categories are classified as Type B.
The CUD gives 10 Iitres as indicative of a limit for "small-scale" but this
is not binding on Member States. The European Commission's Group of
National Experts (an ad-hoc group of Member States representatives
which has met since 1990 to progress implementation of the Directives)
agreed to a set of guidelines to clarify further the concept of 'small-scale'.
These recognise that, in many sectors and applications, volumes greater
than 10 Iitres will still qualify as Type A operations.
Just as scale is separated so are GMMOs themselves. The CUD classifies
all GMMOs as either Group I or Group II.
Group I organisms are intrinsically 'low-risk' and the CUD has used the
criteria for GILSP status developed for the 1986 OECD report. For
industrial-scale use, the Directive has simply equated Group I classification
with GILSP status. During negotiations on the CUD, it was recognised
that the GILSP criteria did not directly translate to small-scale work. In
1991 the European Commission, together with the Member State
authorities, established guidelines to define the criteria for classifying
GMMOs into Group I which are particularly relevant to type A (small-
scale) operations. 7
The CUD sets out obligations for Member States to establish the legal,
administrative and practical measures to implement the Directive and to
lay down measures to be taken by users of GMMOs in containment. All
persons undertaking such work must carry out a risk assessment (Article
6), the principles of which are set out in Annex III of the CUD, a summary
of this assessment being submitted in any notification of work. The
Directive recognises that not all of the factors in its Annex III will be
relevant to all GMMOs.
The Member States are to designate a competent authority/authorities to
implement the Directive and to receive notifications from users. The
system of notifications (set out in Articles 8, 9, 10 and 11) takes account of
whether the work is in a new or established centre, whether it is type A or
B and whether Group I or II GMMOs are involved.
A table of the notification requirements of the CUD appears in Table
1.1. It should be noted that individual Member States may choose to apply
variations according to their national requirements.
The CUD gives 60 or 90 days as the maximum time periods for the
competent authority of a Member State to respond. However, it allows the
'clock to stop' when additional information is requested or for time taken
for public consultation.
The safety principles for work with GMMOs in Group I are set out in
Table 1.1 Notification requirements of the Contained Use Directive
Use Notify
First use of installation

(a) with group I GMMOs 90 days in advance, then proceed
(b) with Group II GMMOs 90 days in advance: Consent required
Subsequent uses of GMMOs

(a) Group I GMMOs in Type A Keep records
(b) Group I GMMOs in Type B 60 days in advance, then proceed
(c) Group II GMMOs in Type A 60 days in advance, then proceed
(d) Group II GMMOs in Type B 90 days in advance: Consent required
Article 7 and were taken from the OECD 1986 Report. Containment
categories for work with Group II GMMOs are detailed in Annex IV of the
CUD and again these were been taken from OECD.
The remainder of the CUD sets out a series of obligations on both users
and Member States. Users must notify their authorities of any accident - "a
significant and unintended release of GMMOs ... which could present an
immediate or delayed hazard to health or to the environment".
National Competent Authorities may set up schemes of public consulta-
tion or inquiry on proposed contained uses (Article 13); must ensure that
emergency plans are drawn up, where necessary, before work with
GMMOs commences (Article 14); must alert other Member States of
accidents that might affect them (Articles 15/16); must set up a scheme of
inspectors (Article 17); and must send a summary report to the
Commission every three years (Article 18).
The provisions regarding confidentiality are of extreme importance.
Whilst it recognised that commercial confidentiality is essential, Article
19.4 sets out what information cannot be kept confidential - the
description of the GMMOs, the name and address of notifier, purpose and
location of use, monitoring and emergency plans and evaluation of healthl
environmental effects.
The Directive also sets up a committee (the Article 21 committee) of
Member States' representatives which is to assist the Commission and
which may, by qualified majority voting, amend Annexes II to V of the
Directive - 'adaption to technical progress'.
1.3.2 Directive 9012201 EEC - Deliberate release of genetically-modified

organisms. 8
The legal basis of the Deliberate Release Directive (DRD) is Article 100a
of the Treaty of Rome, which is concerned with the adoption of measures
for approximating law, regulation or administrative actions of Member
States with the object of the establishment and functioning of the internal
market. Thus, the DRD is legally concerned with preventing barriers to
trade between Member States and not primarily about environmental
protection. However, Article 100a is drafted in terms of ensuring a high
level as a base for health, safety, environmental protection and consumer
protection. Thus the DRD is drafted in terms of a harmonised regulatory
framework for both experimental and commercial releases of GMOs and
to provide for protection of human health and the environment.
As detailed above, the Directive covers a somewhat wider definition
than the CUD, a GMO here being any modified biological entity capable
of replication or genetic transfer. At the time of adoption of the DRD,
some confusion remained over what constituted a 'release' to the
environment. The committee of Competent Authorities has further
defined "intentional introduction" and a potential grey area between the
CUD and the DRD in respect of waste streams containing live GMMOs
from industrial facilities has been avoided by sensibly including wastes in
the notification information provided for a Type B activity with a Group I
GMM under the CUD.
A second area of uncertainty stemmed from the structure of the DRD.
Split into four parts, Parts Band C are the important sections. Part B deals
with releases "for research and development purposes or for any other
purpose than for placing the market", whilst Part C covers "placing upon
the market of products containing GMOs". The Part C mechanisms for
product clearance are described below. "Placing upon the market"
however is not defined in the way that commerce might expect but as
simply "supplying or making available (GMOs) to third parties". Strictly
interpreted, this could prevent transfer of a GMO from one research
laboratory to another unless the entire Part C system was adhered to with
a five-month notification period! Similarly, products intended for contained
use, such as diagnostic test kits, are in a somewhat grey area. Clearly, they
are "placed upon the market", but no release as such is intended. These,
and other similar issues, were the subject of discussion by the Commission
and Member States during 1992.
The range of GMOs exempt from the provisions of the release Directive
is more restricted than the equivalent exemptions under the CUD. In
particular, the release/placing on the market of somatic animal hybridoma
cells and of self-cloned Group I GMMOs do fall within the scope of the
DRD.
Research or trial releases to the environment fall within the restrictions
of the 'Part B' clearance system. Proposers must submit a technical dossier
to the competent authority of the country in which the trial is to take place.
The dossier concerns the risk assessment and notification detailed in
Annexe II of the Directive. It is possible for the notification to deal with
more than one GMO for a site of release or the same GMO on different
sites. Subsequent releases of a GMO are still to be subjected to separate

notification (Article 5).
The Member State has 90 days to respond to the proposer, and again the
clock can 'stop' for additional information to be sought or for public
consultation to take place. In addition, within 30 days of receiving a
proposal, a Member State must send a summary to the European
Commission for forwarding to the other 11 States. Each then has 30 days to
present observations or to ask for more information. It should be noted
that the Directive does not give the other Member States any power to
veto, or to demand any change in a trial release in the first State. The
consent to release such trials remains a national decision. The format of the
summary dossier for information exchange was agreed at the Council of
Ministers and has now been published. 9 For the future it is envisaged that if
sufficient experience has been obtained for a particular type of GMO a
'fast track' procedure may be agreed (Article 6.5) as recently introduced in
the UK.
Part C of the DRD sets out a community-wide system for product
clearance. This concerns products consisting of, or containing, GMOs and
deals with the potential risks to humans, plants, animals and the general
environment. It is envisaged that in order to reach this stage, a GMO
product would have already received consent for testing (under Part B of
the DRD) or will have undergone a risk assessment based upon that
required in Part B. The latter will be required of products developed
beyond the European Communities.
There is a provision in the DRD for Part C to be replaced by Community
Product legislation as this is developed. The first such Directive deals with
pesticides lO and was adopted in 1991 with a two-year period for
introduction of a product risk assessment scheme similar to that foreseen in
the DRD.
In order to market a product, an importer or manufacturer will have to
deal with only one competent authority - that is, the first Member State in
which marketing is intended - the 'gateway' authority. Notification must
contain the information indicated in Annexes II and III of the DRD with
appropriate supporting data.
The committee of Member States representatives has established a
common notification format which will be used by all countries. After the
notification has been made and after approval, the notifier is still obliged to
provide any relevant new information with regard to risks.
Upon receipt the 'gateway' authority has 90 days either to forward the
notification summary dossier to the European Commission with its
"favourable opinion" or to reject the proposal as not fulfilling the
requirements of the DRD. For successful proposals, the next step is for the
Commission to forward the summary to the other Member States which
have 60 days in which to raise any objection.
Should any disagreement take place, which cannot be resolved within

the 60-day period, the Commission shall use the Committee of Member
States representatives which will deliver an opinion by qualified majority
voting (Articles 12, 13 and 21).
One area of possible difficulty with this scheme is found in Article 16,
which empowers any Member State to restrict or prohibit the use or sale of
a product on its territory if it has "justifiable reason to consider that a
product ... constitutes a risk to human health or to the environment". In a
paper published in November 1991, the Danish Government is reported to
be prepared to take cases to the European Court if necessary in order to
protect any additional environmental safeguards. II
As with the CUD, there are provisions for reports from Member States
(Article 18), and for protection of confidentiality (Article 19) and it is
envisaged that the committee of Member States will carry forward the
adaption of the Annexes to technical progress (Articles 20 and 21).
In addition, a list of approved products will be published in the official
Journal of the European Communities every three to six months.
For those Member States that had existing comprehensive legislation
specific to genetic modification work (UK, Germany, Denmark and the
Netherlands), the major impact of the Directives is the new pan-
European clearance mechanisms for products. For the remainder of the
States, the Directives will establish a new statutory framework.
1.4 The situation in the twelve Member States (as of February 1993)
1.4.1 United Kingdom

A major change comes with the introduction of the Department of the
Environment as Joint Competent Authority alongside the Health and
Safety Executive for both Directives. The post-Directives national legisla-
tion reinstate a requirement for notification on GILSP operations which
had been dropped from UK legislation in 1989. It also introduced a
positive consent scheme for higher-risk, large-scale operations and
releases. These categories were previously subject only to notification and
review by advisory committees. Draft Regulations were prepared follow-
ing two rounds of public consultation in October 1991 and August 1992.
Made under Part VI of the Environmental Protection Act (1990), and the
Health and Safety at Work etc Act (1974), these came into force in
February 1993. 12 . 13
1.4.2 Federal Republic of Germany

In 1990, what was then West Germany enacted its Gene Law. Until then,
research had been conducted under the German 'Guidelines for the Safe
Handling of r-DNA'. These were binding on publicly-funded work and

applied voluntarily to the private sector. Industrial production facilities
using GMOs were licensed under the Federal Nuisance Act.
The 1990 law applies to all installations using GMOs and is relatively
complex. Contained uses are classified into four levels of safety and further
differentiated between commercial activities and research. Even the lowest
risk level of research is subject to prior notification and in levels 2-4 a
permit is usually required. The commercial sector is further subject to
controls that require public hearings by the state authority. Experience
with the Federal Nuisance Act (on which much of the Gene Law is based)
has led to fears that public hearings will continue to delay commercial
projects (a delay permitted in the EC Directives).14 However, provided
proposals for industrial-scale use remain in the GILSP categories the
public hearing process will not be required and such fears may be without
foundation.
A problem appears to have developed in Germany, arising both from
the Act and from the structure used to implement the two EC Directives.
Licences for contained applications are controlled by the State legislatures
rather than the Federal Government. The Federal Republic now comprises
16 State governments (,Lander') and for each at least two agencies
comprise the competent authority for the Contained Use Directive.
Reports during 1992 suggest that there is considerable variation in the
approach to the CUD between the state authorities. 15
Until 1992, political pressures had restricted trial releases of GMOs in
Germany. For the Deliberate Release Directive, the control is at a central
level with the Federal Health Ministry acting as the main competent
authority for handling release notifications. The Federal Environment
Office and the Federal Biological Office also cooperate.
1992 saw considerable lobbying from both academia and industry in
Germany aimed at revision of the 1990 Law. This has been successful, at
least in part, with a Parliamentary decision to amend the Law. 16
1.4.3 Netherlands
The Netherlands had adapted existing national legislation to control the
contained use of GMOs and some field tests under the Nuisance Act. This
was supported by guidelines on safe practice, and by amendment of the
1985 Chemical Substances Act to control all activities with GMOs. For
both CUD and ORO, the Competent Authority is the Dutch Ministry of
Housing, Planning and Environment Protection. Advice is obtained from
the 'Provisional Committee on Genetic Modification' which was reconsti-
tuted during 1992. The administrative provisions of the ORO were in place
by October 1991, the implementation date. However, full implementation
of the CUD did not occur until 1992.
1.4.4 France
France has based its control of r-DNA technology on a framework of
product controls under the Commission de Genie Biomoleculaire (CGB)
created by order of the Ministry of Agriculture in 1986. A second inter-
ministerial decree in 1989 established the Genetic Engineering Commission
(CGG) under the Ministry for Research and Technology with responsibility
for classification of work with GMOs. The promotion of safe working
practices has been the responsibility of AFNOR (the French Standards
Institution) .
Implementation of the EC directives had been advanced by the proposal
from the Ministries of Research and for the Environment, that gene
technology be regulated under an existing law dealing with 'installations
classes'. Contained use would be under the control of Departmental
authorities who will license production facilities whilst research scale
operations would be dealt with by the Ministry of Research.
France has been the most active of the 12 Member States of the
Communities in respect to releases to the environment with 80 applications
in 1990 alone. Releases will be dealt with under national 'vertical'
legislation being the responsibility of the appropriate sector ministry. The
two existing committees, CGG and CGB, continue to advise. The French
Parliament passed the legislation to formally implement the two Directives
during 1992.
1.4.5 Denmark
Denmark became the second country to have specific legislation for
biotechnology when in 1986 its Environment and Gene Technology Act
became law. The Act is administered through the Ministry of Environment's
National Agency of Environmental Protection. It covered all aspects of
gene technology in research, production and release work. The Act deals
with environmental protection, human health and safety. Research is
permitted only in classified laboratories and specific approval is required
for production-scale operations.
The 1986 Act effectively banned the deliberate release of GMOs, but in
1989 the Minister of the Environment, with the specific authority of the
Danish Parliament, agree to permit two small-scale trials with modified
plants. In the same year, an amendment to the 1986 Act was passed to
permit large-scale research to be regulated by notification only.
In many ways, Denmark was in the best position to implement the two
EC Directives. What is seen as a tightening of regulation in the UK is
conversely viewed by some as a relaxation of the pre-existing Danish
system.
Denmark will divide regulatory responsibility between its Labour
Inspectorate for health and safety and the Environment Ministry.

However, the latter has been formally nominated as the competent
authority for both the CUD and the DRD.
Denmark does not operate an advisory committee of experts such as
those found in the UK, France, Germany and the Netherlands, but obtains
all its advice from Ministry of Environment officials. Denmark's system of
law allowed it to be the only Member State to have reasonably claimed to
have fully implemented both the CUD and DRD on 23 October 1991.
1.4.6 Belgium
The last of the six Member States of the European Community who have
significant industrial and environmental experience with GMOs, Belgium
was in the least developed position in regards to implementation. Well into
1992, there was still no regulatory framework in place, nor had agreement
been reached on the subject of competent authority status.
Belgium's experience in GMO applications has been based entirely upon
voluntary arrangements. This appears to have worked well; all release
trials have been notified in advance to the Department of Agriculture
(crop plants) or the Department of Public Health (animal vaccines). The
draft EC Directives were used as the basis of review, as have the OECD
guidelines.
Belgium's regional structure means that there are multiple ministries, all
with legal competence. There was some reluctance to agree either a
national authority or a national advisory committee in respect to the CUD
or DRD. The latter part of 1992 had seen some progress towards
implementation.
1.4.7 Remaining EC Member States

Provisional nominations of competent authorities have been made from
Italy, Greece, Luxembourg, Spain, Portugal and Ireland but no advanced
regulatory system appeared to be in place by the end of 1992.
Ireland operates a r-DNA committee which produced a "Guide on r-
DNA Regulation in Ireland" in 1987. Italy and Spain responded to a 1989/
1990 OECD survey that neither had national guidelines nor specific
regulation in existence. Portugal and Luxembourg were the only EC
Member States not responding to that survey.
1.5 Situation in the EFT A nations (as of February 1993)
1991 saw agreement in principle between the EC and EFTA (the European
Free Trade Association) on the formation of the European Economic Area
(EEA), the principal benefit being an extension of the single trading

market to 19 countries. Under these arrangements, the EFTA nations
(Iceland, Norway, Sweden, Switzerland, Austria, Liechtenstein and Finland)
were due to implement EC legislation induding environmental and
internal market measures during the mid 1990s.
Advisory Committee review exists in Finland (r-DNA Advisory group-
National Board) and Switzerland (Swiss Interdisciplinary Commission for
Biological Safety in Research and Technology). Norway controls the
release of GMMOs and GMOs under a range of existing sectorial
legislation including the Pollution Control Act, Wildlife Act, Inland
Fisheries Act etc. Austria has established a Parliamentary Commission to
determine its regulatory policy for work with GMOs. This Commission was
due to report in late 1992.
1.6 Europe vs. the USA
1993 was a watershed in the development of regulatory controls for

European research and industry. The two EC Directives should provide a
harmonised approach for research and product development whilst
maintaining the high safety record of biotechnology.
However, there will still remain regulatory and political differences in
approach between Member States. There are fears that the entire EC
package will do more than drive innovation beyond its geographical area of
control. Only time and experience will tell if such fears are justified. The
opportunity for a 'level European playing field' for all involved in the
technology is attractive, provided that the disadvantages in terms of
comparison with opportunity beyond the Ec/EFT A are not too great.
The system of regulation described above is essentially 'process driven',
since it is comprised of horizontal legislation across all sectors using
techniques of genetic modification. The picture in the USA is somewhat
different. Here the three Federal agencies, Environmental Protection
Agency (EPA), Food and Drug Administration (FDA) and the US
Department of Agriculture (USDA), control various aspects of products
through existing legislation which has been adapted to cover the products
of gene technology. (See also chapter 3.)
In 1992, the US Office of Science and Technology issued the long-
awaited policy white paper on Federal oversight of biotechnology - the
"Scope paper". 17 This endorsed the approach of a risk-based review of
products, rather than being control-based upon the process of production.
Under the then Bush Administration, the approach of the US Government
was to press the line that GMOs did not bring any novel hazards either to
humans or the environment. At the time of writing, the Clinton
Administration's approach to this area of technology is still to be seen.
The regulatory schemes of the US and Europe are somewhat different,
but it is not at all clear whether such differences of approach will make one
rather than the other easier for industry to utilise.
Disclaimer
The views expressed in this paper are those of the author and do not
necessarily reflect the policies of the Health and Safety Executive or any
UK Government department.
References
1. Berg, P., Baltimore, D., Boyer, H. W. et al. (1973). Potential biohazards of recombinant
DNA molecules, Science, 185, 303.
2. Genetic Manipulation Regulations, 1989. Statutory Instruments No. 1810, HMSO.
3. Bull, A.T., Holt, G. and Lilly, M.D. (1982). Biotechnology -International Trends and
Perspectives. OECD, Paris.
4. Organisation for Economic Co-operation and Development. (1986). Recombinant DNA
Safety Considerations. OECD, Paris.
5. Council Directive on the protection of workers for risks related to exposure to biological
agents at work. (90/679/EEC). (1990). Offic. 1. Europ. Commun., L374, 1-12.
6. Council Directive on the contained use of genetically modified micro-organisms. (90/2191
EEC). (1990) Offic. 1. Europ. Commun., L1l7, 1-14.
7. Commission Decision of 29 July 1991 concerning the guidelines for classification referred
to Article 4 of Directive 90/219/EEC. (911448IEEC). (1991). Offic. J. Europ. Commun.,
L239, 23-26.
8. Council Directive on the deliberate release into the environment of genetically modified
organisms. (90/220/EEC). (1990). Offic. 1. Europ. Commun., L1l7, 15-27.
9. Council Decision of 4 November 1991 concerning the Summary Notification Information
Format referred to in Article 9 of Directive 90/220/EEC on the deliberate release into the
environment of genetically modified organisms. (911596/EEC). (1991). Offic. 1. Europ.
Commun., L322, 1-16.
10. Council Directive concerning the placing of plant protection products on the market. (911
414/EEC). (1991). Offic. J. Europ. Commun., L230, 1-32.
II. Shackley, S. and Hodgson, J. (1991). Biotechnology Regulation in Europe, Bioi
Technology, 9, 1056-1061.
12. The Genetically Modified Organisms (Contained Use) Regulations 1992. Statutory
Instruments No. 3217, HMSO.
13. The Genetically Modified Organisms (Deliberate Release) Regulations 1992. Statutory
Instruments No. 3280, HMSO.
14. Schubert, G. (1990). Much more discussion needed. The current state of the debate on
the West German genetic engineering bill. Proc. Eur. Workshop on Law and Genetic
Engineering, pp 28-31, BBU Verlag: Bonn.
15. Fritsch, F. and Haverkamp, K. (1991). The German Genetic Engineering Act. Bioi
Technology, 9, 435-437.
16. Abbott, A. (1992). Germany will ease requirements of gene law in bow to researchers,
Nature, 360, 286.
17. Office of Science and Technology Policy. (1992). Exercise of Federal oversight within
scope of statutory authority: Planned introductions of biotechnology products into the
environment. US Office of Science and Technology Policy, Washington DC.
2 Occupational and environmental safety: the UK
legislative framework
A.N. COTTAM
2.1 Introduction
Biotechnology is an age-old process with its origins in processes such as

brewing and breadmaking. Historically the technology has generated no
unique considerations over its hazards to human health and safety or the
environment. Indeed, in contrast to many industrial operations a bio-
technology process usually operates under moderate conditions. Organisms
are typically grown in dilute solutions under moderate pH, at close to
ambient temperature and pressure and have by-products that are usually
biodegradable. Consequently the hazards to humans or the environment
that may be associated with large inventories of flammable or toxic
materials, exothermic reactions, high process temperatures, pressures, and
toxic by-products are usually avoided. Workers are, however, potentially
exposed to biological and chemical hazards and may also be exposed to
physical hazards. Most of the organisms used are aerobic, utilizing oxygen
and evolving carbon dioxide, consequently the vessels used for growth may
accumulate excessive levels of carbon dioxide. Some parts of the plant may
be heated or sterilised by steam, with the potential for burns or scalding. In
addition, some stages of the process may be accompanied by high noise
levels.
The introduction of the modern biotechnology of genetic modification
(genetic manipulation or recombinant DNA techniques) in the last two
decades has generated some concern. This highlights the need to ensure
that what are sometimes seen as the special hazards from the organisms
used in biotechnology are adequately assessed and that the risks are
controlled.
2.2 International influences
The approach taken to the assessment and control of industrial hazards is

enshrined within legislation in many countries (see chapters 1, 3, 4 and 5).
The traditional applications of biotechnology have usually been within the
scope of general health and safety law with specific legislation for
UK LEGISLATIVE FRAMEWORK 15
biotechnology not being generally developed. The introduction of genetic

modification techniques, however, has focused regulatory attention leading
to a number of international and national developments.
The Organisation for Economic Cooperation and Development
(OECD) has provided the main focus for developing scientific principles
underlying the safety of biotechnology. The OECD has produced
international guidance 1 on the safe use of genetically modified organisms
in the laboratory, industry and the environment and has an on-going
programme of work in safety in biotechnology. Although the guidance
produced by OECD is non statutory many of its concepts have been
implemented globally in specific legislation or published guidance.
Many of the recommendations of the OECD have formed the basis of
the European Directives on Genetic Modification. The European Commis-
sion has adopted two directives concerning genetic modification work. 2 ,3
• The Contained Use of Genetically Modified Micro-organisms
• The Deliberate Release into the Environment of Genetically Modified
Organisms
A third Directive on 'Biological Agents' covers work with all organisms,

including traditional organisms and again includes requirements for risk
assessment and control of hazards but covers all biotechnology processes, 4
These Directives are implemented within the European Community
through national legislation. Much of the content of the directives is
already included in the present UK regulatory scene and this in part
reflects the success of the UK approach in influencing international
initiatives.
2.2.1 The UK approach

Despite the rapid development of the technology, in particular the
techniques of genetic modification, the fundamental role of the legislation
has been and will remain unchanged, that is to:
1. Promote high standards of safety.

2. Reassure the public that the appropriate controls are in place.
3. Enable the technology to develop.
In the last decade the Health and Safety Executive (HSE), its advisory
committees and indeed the UK as a whole has gained a reputation for the
balance which has been achieved in dealing with the potential hazards from
laboratory and industrial applications of biotechnology. We are now at a
particularly important and interesting stage and are on the brink of a new
regulatory structure which will introduce harmonised systems of control
throughout the European Community for such work.
",""" ""d
Safety at
Work Act
~Environmental
.
ProtectIOn Act
(1974) (1990)
I
Regulations
I
Guidance
I
Inspection
Figure 2.1 UK framework of legislation and guidance.
Within the UK the main emphasis historically has been on human health
and safety. The environmental impact of the technology now has to be
considered. The safety aspects of biotechnology are the subject of a
framework of legislation and guidance (Figure 2.1) that has had an
important influence on the prudent and successful development of the
industry. This framework of control includes site inspection by the Health
and Safety Executive.
2.3 Occupational health and safety legislation
Legislation in pursuance of health and safety dates from the Health and
Morals Apprentices Act (1802) which was designed to protect young
children working in cotton woollen mills, and other factories where more
than 20 persons were employed. Various minor legislation was passed in
ensuing years and in 1833 the Factory Act was passed. In view of the
multiplicity of legislation and as a result of recommendations in 1876 by a
Factory Commission in 1901, the Factory and Workshop Act was passed
and provided a comprehensive piece of legislation where all the previous

law was consolidated into one statute. The Act gave the power to the
Secretary of State to make Regulations for particular industries. In 1916
this power was extended to permit Welfare Orders to be made dealing with
washing facilities, first aid etc. In 1937 the Factories Act was passed which
eliminated the distinctions between the different types of premises and
made detailed provisions for health, safety and welfare. Minor amendments
were made in 1948 and 1959 and the various statutes were consolidated by
the Factories Act (1961) which is still in force in Great Britain today. In
1970 a Committee on Health and Safety at Work was appointed under the
chairmanship of Lord Robens. This committee recommended that a
system be devised whereby all employers and all employees became aware
that health and safety was the concern of everyone. It was also
recommended that there was a need for a single comprehensive framework
of legislation which would cover all work activity, supported and
supplemented by a series of controls to deal with specific problems, and
assisted by voluntary standards and more flexible Codes of Practice. Its
final recommendation was the establishment of a unified enforcement
authority having overall responsibility for initiating legal proposals, giving
assistance and advice, possessing stronger enforcement powers and with
the ability to delegate its enforcement functions when necessary.
The result was the passing of the Health and Safety at Work Act (1974).
The Act applies to all persons who are employed and has brought into the
protective umbrella some 8 million new entrants who were not hereto
covered by previous legislation. As well as laying down duties for
employers and employees the Act imposes certain legal requirements on
those who manufacture, import, design or supply articles or substances
which are to be used at work. Some of the provisions are designed to bring
about a greater personal involvement of those concerned. The Act has
acted as a catalyst for considerable management activity and a greater
awareness of responsibility has brought about increased concern for the
health and safety of employees.
This Act also established the Health and Safety Commission (HSC) and
the HSE. The Act introduced a regulatory framework which imposed
general duties to protect the health and safety of workers and also
meml:lers of the public who may be affected by work activities. These
duties apply to employers, the self-employed and to workers themselves.
They are qualified by the concept of reasonable practicability, which
implies that a judgement is made of the risks involved in a particular
process and, dependent on these risks, the appropriate systems of control
are required to be implemented (see Figure 2.2). The greater the risk the
greater the amount of time, trouble, money and effort required to control
the risk. All industry, including biotechnology, is subject to these controls.
Risk Controls
• within workplace • cost
• outside workplace • effectiveness
• public perception
'Reasonably practicable'
Figure 2.2 The balance between risk and cost of controls.
2.4 The Health and Safety at Work Act
The aim of the Act is to:

• secure the health, safety and welfare of persons at work;
• protect persons other than persons at work against risks to health or
safety arising out of or in connection with the activities of persons at
work;
• control the keeping and use of explosive or highly flammable or
otherwise dangerous substances, and generally preventing the unlawful
acquisition, possession and use of such substances; and
• control the emission into the atmosphere of noxious or offensive
substances from premises of any class prescribed for the purpose of this
paragraph.
2.4.1 Duties of employers

The general duties of employers to their employees are set down in Section
2 of the Act.
Section 2(1) "It shall be the duty of every employer to ensure, so far as
reasonably practicable, the health, safety and welfare at work of all his
employees" .
Section 2(2) (a) "the provision and maintenance of plant and systems of
work that are, so far as is reasonably practicable, safe and without risks to
health" .
This is a general requirement covering all plant, which the Act defines as
including machinery, equipment and appliances used at work. It does not
supersede the more detailed and specific provisions covering certain
equipment contained in other legislation, but it applies to all plant used in
any work activity, whether or not subject to existing safety legislation.
Section 2(2)(b) "arrangements for ensuring, so far as is reasonably

practicable, safety and absence of risks to health in connection with the
use, handling, storage and transport of articles and substances".
This subsection is concerned with the materials and substances, whether
in solid or liquid form or in the form of a gas or vapour, so that the
subsection covers everything used at work and all work activities.
Section 2(2)(c) "The provision of such information, instruction, training

and supervision as is necessary to ensure, so far as is reasonably
practicable, the health and safety at work of his employees".
Section 2(2)(d) "So far as is reasonably practicable as regards any place

of work under the employer's control, the maintenance of it in a condition
that is safe and without risks to health and the provision and maintenance
of means of access to and egress from it that are safe and without such
risks" .
2.4.2 Duties of employees

The duties placed on "employed persons" are in Sections 7 and 8 of the
Act. These read as follows: "It shall be the duty of every employee while
at work:
• to take reasonable care for the health and safety of himself and of other
persons who may be affected by his acts or omissions at work; and
• as regards any duty or requirement imposed on his employer or any
other person by or under any of the relevant statutory provisions, to co-
operate with him so far as is necessary to enable that duty or
requirement to be performed or complied with".
2.4.3 Duty not to misuse

Section 8 of the Act places a duty on all persons, whether they be
employers, employees or self-employed, and states:
"No person shall intentionally or recklessly interfere with or misuse anything
provided in the interests of health, safety or welfare in pursuance of any of the
relevant statutory provisions".
2.4.4 Duties of manufacturers and suppliers

Section 6 of the Act places duties on persons who design, manufacture,
import or supply articles for use at work. They are required to ensure as far
as is reasonably practicable that any plant, machinery, equipment or
appliance is so designed and constructed as to be safe and without risk to
health when used. They must also carry out any testing or examination
necessary to achieve this and they must ensure that adequate information
will be available about the use for which it was designed and about any
conditions necessary for its safe use.
A person who erects or installs such plants etc. must ensure so far as is
reasonably practicable, that it is installed so as not to be unsafe or a risk to
health when used.
A person who manufacturers, imports or supplies any substance for use
at work must ensure that, as far as is reasonably practicable, it is safe and
without risk to health when used. There are requirements for testing,
examination and research. They must also ensure that there is adequate
information available about this and about any conditions necessary to
ensure that it will be safe and without risks to health when used.
2.4.5 Management systems

Much guidance has been published on the importance of effective
management systems in assessing and controlling risks. 5 •6
The Management of Health and Safety at Work Regulations (1992f now
make explicit in law the importance of effective planning, organisation,
control, monitoring and review of the preventative measures needed to
minimise risks. The regulations also make clear that the starting point for
effective health and safety management is a suitable and sufficient
assessment of the risks to both employees and non-employees. The main
requirements are for employers to:
• assess the risks to the health and safety of their employees and others who
may be affected in order to identify the measures needed to comply with
relevant health and safety law. Employers with five or more employees
will need to record the significant findings of the risk assessment;
• make arrangements for implementing the health and safety measures
identified as being required by the risk assessment. Arrangements for
planning, organisation, control, monitoring and review will need to be
covered. Again, employers with five or more employees will have to
record their arrangements;
• appoint competent people (either from inside the organisation or from
outside) to help with the implementation of the health and safety
arrangements;
• set up emergency procedures;

• provide information to employees which can be understood, as well as
adequate training and instruction; and
• to work together with other employers sharing the same workplace.
Some of these duties, such as the duty to assess risks, also apply to the self-
employed. There are also specific duties on employees to use equipment
only in accordance with the training they have received and to report
dangerous situations and any shortcoming in their employers' health and
safety arrangements.
2.5 The Environmental Protection Act
The HSE's regulatory responsibilities are concerned with human health

and safety and it was recognised that a gap existed in relation to
environmental legislation. New powers were required to protect the
environment in order to implement the provisions of the EC Directives on
GMOs. To close this gap the Secretary of State for the Environment
introduced primary legislation in Part VI of the Environmental Protection
Act (1990). Part VI is aimed at preventing or minimising damage to the
environment which may arise through activities involving GMOs and
contains a general duty on persons undertaking activities involving GMOs
to employ the best available techniques not entailing excessive cost
(BA TNEEC) for preventing damage to the environment. Other parts of
the Act may also apply to the wastes from biotechnology processes.
2.6 Specific regulations
2.6.1 Control of Substances Hazardous to Health Regulations 1988

(COSHH)
These regulations 8 apply not only to chemicals but also to biological agents
as they include micro-organisms in the definition of 'substances hazardous
to health'. The requirements of COSHH include:
• assessment of health risks (including pathogenic, toxic and allergenic
risks), from organisms, their components and products;
• prevention or control of exposure;
• maintenance, examination and testing of control measures;
• environmental monitoring;
• health surveillance;
• provision of information, instruction and training.
2.6.2 Genetically Modified Organisms (Contained Use) Regulations 1992

and Genetically Modified Organisms (Contained Use) Regulations
1993
The Genetically Modified Organisms (Contained Use) Regulations (1992)9

repeal and replace the earlier legislation in this field, the Genetic
Manipulation Regulations (1989). They implement within Great Britain
EC Directive 901219/EEC on the contained use of genetically modified
micro-organisms, which was adopted on 23 April, 1990. The Regulations
came into force on 1 February, 1993.
The Regulations have been made under the powers of the Health and
Safety at Work Act (1974)5 (the HSW Act) and the European Communities
Act (1972) and are concerned with protecting both human health and the
environment. They require, with certain exceptions, that anyone carrying
out any activity involving genetic modification must do so in conditions of
contained use which satisfy the Regulations.
For genetically modified micro-organisms the Regulations cover both
human health and environmental risks. For larger genetically modified
organisms, such as plants and animals, they cover human health risks only.
The environmental risks associated with work with larger organisms are
covered separately by section 108(1)(a) of the Environmental Protection
Act (1990) (the EP Act) which came into force for this purpose on 1
February, 1993 by commencement order No 12 and with the Genetically
Modified Organisms (Contained Use) Regulations (1993).10 Section 108(1)
requires anyone creating a genetically modified organism which is not an
approved product under the Deliberate Release Regulations or obtairing
one from elsewhere, to carry out an assessment of the environmental ris\-' s
and make it available for inspection.
The Contained Use Regulations are administered jointly by HSE and the
Department of the Environment. Notification of intention to carry out
GMO work is to be made only to HSE however, and enforcement also falls
only to HSE including in those premises where local authorities enforce
other HSW Act provisions.
Genetic modification involves altering the genetic structure of organisms
to change some of their characteristics. It opens the way for advances in
science, and in the production of food, pharmaceuticals and other
products, and in pollution control. Often it is little more than an extension
of the traditional drive to develop better strains of plants and animals and
to use the properties of micro-organisms in useful processes, like the
production of bread, wine or cheese.
The Genetically Modified Organisms (Contained Use) Regulations (1992)
interpret genetic modification as "the altering of the genetic material in
that organism by a way that does not occur naturally by mating or natural
recombination or both" and list examples of techniques that are regarded
as genetic modification. Contained Use means any operation involving

genetically modified organisms (GMOs) under conditions of containment.
The containment must be provided by physical barriers, whether or not
they are supplemented by chemical or biological ones. The containment
must limit the contact of the GMO with the general population and the
environment.
The activities covered by the Contained Use Regulations include
laboratory operations, housing and/or breeding of modified animals in
animal houses or farm animals restrained by appropriate fencing, the use
of growth rooms and glasshouses of appropriate specification and the use
of fermenters. Waste streams from contained facilities also fall under the
Contained Use Regulations ..
The main requirements of the Contained Use Regulations provide for:
• human health and environmental risk assessment;
• the need to keep records of risk assessments;
• the need to establish a local genetic modification safety committee to
advise on risk assessments;
• categorisation of work on the basis of risks to human health and safety
and of damage to the environment, taking into account the nature of the
organism and the type of activity;
• advance notification to the HSE of an intention to use premises for
activities involving genetic modification for the first time and, for some
activities, consent from the Executive before work can start;
• notification to the Health and Safety Executive of individual activities
involving genetic modification and, for some activities, consent from the
Executive before they can proceed;
• standards of occupational and environmental safety and levels of
containment;
• notification of accidents and, where appropriate, the drawing up of
emergency plans;
• disclosure of information and public registers, with provision for
confidentiality;
• fees for notifications.
The Regulations modify the meaning of the term self employed so that
these regulations apply to any person who is not an employer or an
employee. Thus they apply to research students, for example, who are
engaged in any GM activity.
2.6.3 Pressure Systems and Transportable Gas Containers Regulations

1989
These regulations deal with the risks arising from the energy stored in
pressurised systems. They require that pressure systems should be properly
designed, constructed, repaired and modified. Systems should be marked

in accordance with the regulations, and only operated within the safe
operating limits of the system. Detailed guidance is given in the Approved
Code of Practice. 11
2.6.4 Electricity at Work Regulations 1989

Guidance on these regulations is given in the Memorandum of guidance on
the electricity at work regulations (1989).12
2.7 Advisory committees
Developments in the legislative framework concerning the safety of

humans and the protection of the environment have been influenced by the
advice from independent advisory committees. These committees are an
important feature of the present system, providing expert advice not only
to the HSC and HSE but also to Ministers including those in the
Departments of Environment, Trade and Industry and Agriculture. Their
importance cannot be underestimated.
Until the 1970s there was little regulation in the specific area of
microbiology. An outbreak of smallpox in London in 1973 13 which
originated from a laboratory-acquired infection, led to a succession of
committees which were convened to investigate the cause and, further, to
provide guidance on the risks of handling micro-organisms and to produce
codes of practice. One of the best known of these was the Code of Practice
for the Prevention of Infection in Clinical Laboratories and Post-mortem
Rooms,14 commonly known as the 'Howie Code' after its chairman Sir
James Howie. In addition, the Dangerous Pathogens Advisory Committee
(DPAG) was set up to oversee the use of pathogens which are not normally
handled in clinical laboratories. Unfortunately there was another fatal
incident with smallpox, in Birmingham, in 1978. 15 .16 As a result of
investigations into a review of this incident a new body called the Advisory
Committee on Dangerous Pathogens (ACDP) was constituted in May 1981
to replace the DPAG.
The UK was one of the first countries to establish a national advisory
committee, the Genetic Manipulation Advisory Group (GMAG) in 1976.
The GMAG was superseded in 1984 by the Advisory Committee on
Genetic Modification (ACGM) established by the HSC. Its terms of
reference include the provision of advice to the HSC, the HSE and the
Health, Agriculture, Environment, Industry and Northern Ireland
Ministers on particular aspects of GM work.
In April 1990 the HSC together with the Secretary of State for the
Environment announced the establishment of a new advisory committee,
the Advisory Committee on Releases into the Environment (ACRE). This

committee is responsible for providing advice on the release of GMOs to
the environment. All three advisory committees are based on a tripartite
structure with equal representation of nominees from employer and
employee organisations, together with representatives from the scientific!
medical community: The ACGM and ACDP are serviced by the HSE. The
ACRE is serviced by the DoE. The tripartite structure on which these
committees are based has proved to be most successful in the provision of
information, the development of standards, the encouragement of open-
ness, the identification of priorities and in the provision of advice to
Government ministers about methods for risk assessment and appropriate
controls. Much of the work of the three committees, in particular the
production of guidance material, is delegated to small working groups
which often contain, in addition to members of the advisory committee,
members co-opted from the scientific community. This approach ensures
that the guidance produced has proven scientific credibility and is of real
practical assistance to those involved with the work.
2.7.1 Guidelines
The HSW Act allows for the development of approved codes of practice
and of published guidance, for example dealing with specific processes or
substances. This sort of guidance can be changed in step with developments
in the industry itself. As in any activity where a risk to health may exist the
fundamental principles of occupational hygiene (risk assessment, substi-
tution and control- as enshrined in the COSHH Regulations) apply. These
general principles have also influenced the approach taken to the
development of guidance on the evaluation and control of risks from work
with biological agents.
A particularly important role in the development of guidance has been
played by the Advisory Committee on Genetic Modification (ACGM) and
Advisory Committee on Dangerous Pathogens (ACDP). These independ-
ent, 'watchdog' committees with employer, employee and specialist
representatives have been set up to advise HSE and other government
departments, including health, environment and industry. Guidance
produced by ACGM includes detailed guidelines on the 'approved'
methods of risk assessment, laboratory containment facilities, large-scale
use of GM organisms, as well as the handling of oncogenes, eukaryotic
viral vectors and transgenic animals. ACDP has produced guidance on
laboratory containment of dangerous pathogens, HIV and flexible film
isolators. The British Standards Institution (BSI) and its equivalents
internationally have also produced standards on topics such as micro-
biological safety cabinets and disinfection.
2.7.2 Local involvement

Whilst the HSE relies heavily on the provision of advice from its advisory
committees, the HSE, ACGM and ACRE have recognised the importance
of local review of GM work by appropriately constituted local safety
committees. From the early days the establishment of such committees
played an important role in the regulatory framework. A statutory duty
was introduced in the 1992 GMO Regulations 9 which required the
establishment of a local Genetic Modification Safety Committee at all
centres undertaking GM work. Although the membership of such
committees will be influenced by the types of proposal being reviewed,
guidance is available on the constitution of such committees. 17 Of
particular importance is the role of the biological safety officer who carries
responsibility for the provision of advice concerning the safety of GM work
as well as providing a focus for contact with regulatory authorities. This
local involvement provides a forum to discuss GM activities locally and to
communicate legislative controls on such activities.
2.8 Inspection and enforcement
HSE has several roles including site inspection and where necessary
enforcement to ensure that in-house risk assessment is being done
consciously, correctly and conscientiously and making certain that the
actual arrangements for control are satisfactory and consistent with the
risk. To do this effectively HSE must, particularly in a relatively new area
such as biosafety learn with those developing that technology. HSE
welcomes the opportunity to discuss with researchers, manufacturers and
users in the industry their plans and proposals at an early stage and to enter
into technical dialogue. HSE believes that over the years a good
relationship has been established with those in academia and industry and
is anxious that this should continue. Prevention is better than cure.
In nearly all circumstances the first contact with HSE is through one of
the 20 area offices. Inspectors in these area offices have a general
responsibility for inspection of all work premises and processes. The
Education National Interest Group (NIG) based in London has an overall
co-ordination responsibility for inspection throughout the education
section and there are equivalent NIGs for the food, drink and chemical
(including pharmaceutical) industries. Every Factory Inspector in the areas
has access to specialist biosafety and chemical engineering inspectors to
provide specific expertise and knowledge as required.
The exception to these general arrangements is that responsibility for the
inspection of genetic modification facilities, as well as facilities propagating
HIVor handling ACDP group 4 organisms throughout the country, rests
with the biosafety unit of HSE's Technology and Health Sciences Division
based at the headquarters of the HSE in Merseyside. These inspectors
enforce and advise on standards of health and safety in facilities, under the
general duties of HSWA. Working under this legislation the remit of this
group of inspectors has primarily been the protection of workers and the
public. For GM work, however, under an agency agreement with the
Department of Environment (DoE), which will give HSE responsibility for
enforcing those sections of the Environmental Protection Act dealing with
GM, their duties will be extended to include protection of the environment.
2.8.1 Aims of inspection

The duty to comply with the law and control risks rests on the employer not
the enforcing authority. The primary goal of the Health and Safety
Executive (HSE), as the enforcing authority is to influence the behaviour
of employers and achieve a high degree of compliance. The aims of
inspection can be summarised as:
• to assess whether the organisation has taken the necessary measures and
provided a structured means to identify, rectify and prevent deficiencies
that might present risks;
• to stimulate the organisation to ensure that health and safety at work and
protection of the environment receive a high priority;
• to encourage workers and their representatives to play their part in
achieving a safe and healthy working environment;
• to provide guidance and appropriate technical information;
• to ensure that the relevant law is adhered to, where necessary using
enforcement procedures;
• to make sure that the enforcing authority is kept informed of any defects
in the legal and administrative requirements.
2.8.2 Powers of inspectors

The powers of an Inspector to enable him to undertake his duties under
this Act are given in Section 20 of the Act. They include power to enter, at
any reasonable time, any premises which he has reason to believe it is
necessary for him to enter for the purpose of carrying into effect any of the
legal provisions within the field of responsibility of his enforcing authority.
He may take with him any duly authorised person and any equipment
that he needs and he may take measurements, photographs and recordings
that are necessary for any examination or investigation. He may take
samples and can require any person to give him information relevant to his
examination or investigation, to answer questions and to sign a declaration
of the truth of his answers. He can require any person to afford him such
facilities and assistance, within that person's control or responsibilities, as

are necessary to enable the Inspector to exercise any of the powers
conferred on him. An Inspector is appointed in writing by his enforcing
authority and must when required to do so produce a copy of his
instrument of appointment.
Duties are placed on Inspectors by Section 29(8) of the Act to disclose
certain information to persons employed at premises to which the Act
applies, to assist in keeping work people adequately informed about
matters likely to affect their health, safety and welfare. This duty is in
addition to that placed on the employer by Section 2(2)(c) (mentioned
above) and may include factual information obtained by the Inspector
which relates to the premises or anything being done there, and
information with respect to any action which he has taken or proposes to
take in, or in connection with, the premises. The Inspector is required to
give the employer the same information as he gives to the employed
persons.
During a site inspection inspectors would review both the local
organisation for work with biological agents as well as the physical
containment facilities with reference to the relevant guidance. Inspectors
are able to provide advice on the interpretation and implementation of
guidance in particular circumstances and frequently made recommenda-
tions for improvements in facilities or procedures. A range of more formal
enforcement actions can be taken where this is necessary.
2.B.3 Enforcement
If an Inspector discovers a contravention of one of the provisions of health
and safety law he can:
Issue a prohibition notice if there is a risk of serious personal injury, to

stop the activity giving rise to this risk, until the remedial action specified in
the notice has been taken. The notice can be issued whether or nor there is
a legal contravention, and it can take effect immediately or at a later time.
It can be served on the person undertaking the activity, or on a person in
control of it at the time the notice was served.
Issue an improvement notice if there is a legal contravention of any of the

relevant statutory provisions, to remedy the fault within a specified time.
This notice may be served on any person who, in the opinion of an
inspector is contravening or has contravened a relevant statutory provision.
This could include not only the employer but (by virtue of S36(1) ) any
other person such as a manager or foreman whose act or default has
apparently caused the contravention by the employer. Notices may also be
served on employed persons where appropriate. A person on whom a
notice is served may appeal against the notice, or any terms of it, to an
industrial tribunal.
Prosecute any person contravening a relevant statutory provision

instead of, or in addition to, serving a notice. Certain offences may be
prosecuted only summarily in a magistrates court in England and Wales or
a Sheriff court in Scotland. The majority of offences may be prosecuted
either summarily, or on indictment in the Crown Court in England and
Wales or the Sheriff court in solemn procedure in Scotland. In most cases,
where offences are triable either way, they are nevertheless prosecuted
summarily if the court and the defendant consent to this.
The maximum fine, on summary conviction, for most offences is
£20 000. There is no limit to the fine on conviction on indictment.
Imprisonment for up to two years can be imposed for certain offences. In
addition to any other penalty, the Court can make an order requiring the
cause of the offence to be remedied. If a person on whom an improvement
or prohibition notice is served fails to comply with it, he is liable to
prosecution and failure to comply with the prohibition notice could lead to
imprisonment.
Seize, render harmless or destroy any substance or article that he

considers to be the cause of imminent danger or serious personal injury.
Inspection is the means by which Government assures itself that the
legislative system is working effectively to ensure the protection of people
and the environment from risks arising from GM work. It provides a
springboard for enforcement and enables checks to be made of compliance
with the law at the level of the workplace, and decisions to be taken about
actions necessary to remedy deficiencies or to punish offenders. Through
the information gathered by inspectors, it provides a feedback loop to the
law makers about the effectiveness of the law, and allows a two-way flow of
information about technical developments between the regulator and the
regulated, thus ensuring common, high standards throughout the industry.
The credibility of the industry's health, safety and environmental record
depends, at least in the eyes of the public, the media and politicians, on this
external Inspection process. Its role in the system is therefore essential:
prevention is better than cure.
2.9 The way forward
The tasks facing legislation in dealing with these complex technologies are
difficult and frustrating. It has to be accepted that most new technologies
have had their safety problems and there is concern that although both
traditional and new biotechnologies have excellent safety records, uncritical
acceptance may increase the chances of mistakes. Any accident of a serious

nature could seriously hinder further development, causing the imposition
of unrealistic legislative restrictions. Those who devise regulations find that
the path they have to tread between protection of the public and the stifling
of innovation is rather narrow. There is a need for industrial and
environmental pressure groups to talk to one another and to try to
understand each other's views. In many ways these groups have the same
targets: biotechnology may be less capital intensive and more environ-
mentally benign than the chemically-based technology it may replace. If
biotechnology is to survive, however, and to grow in today's climate, the
companies which employ it will have to take a long-term approach,
patiently developing relationships with all concerned parties. Questions
will be asked about the necessity, not only of certain types of experiment,
but also of the value to society of the products of biotechnology.
Assurances will be sought from scientists that innovative biotechnology
and its products are safe. Industry needs to develop guidance on safe
working practices but these should be built on objective assessments of the
hazards posed and the risks involved (see chapters 7 and 11). Biotechnology
is the one industry where the development of appropriate practices and
control measures seems to have established a safe commercial activity
without having had the compulsion of a major accident. Industry has to
recognise that while the level of risk of a technology may remain the same
the acceptability of that risk to the public and governments may be subject to
change. Although there is a false expectation that scientists can measure in
absolute terms whether something is safe or not they will be expected to
measure risk; the acceptability of that risk will then be judged by many
interested parties.
The approach taken in the UK has been pre-emptive, an approach not
driven by cases of human ill health or harm to the environment. Indeed
after more than a decade of laboratory work and industrial application
under controlled conditions, any risk that might exist has not been realised.
There is no substantive evidence of excess mortality, morbidity or adverse
environmental effects. This proactive approach contrasts with certain other
areas where it has been said that Government has usually legislated as a
direct response to accidents or incidents. Quite apart from the possible
costs in human and financial terms of such an approach there are also
plainly other undesirable effects of a predominantly reactive approach. On
the one hand public confidence is destroyed and even subsequent controls
are seen as 'too little too late'. On the other hand sudden changes and
hurried regulatory reaction can lead to considerable upheaval and the
danger of over-reaction or misplaced resource.
The challenge for the future for the industry will be to ensure further
exploitation of the benefits of biotechnology whilst ensuring that any
hazards to human health or the environment are controlled to the
satisfaction of both regulatory authorities and the general public. The

balance between development of the technology and the controls placed in
the industry appears to have been reached in the 'traditional' biotechnology
sector. The magnitude of this challenge will, however, increase as the
environmental and agricultural applications of biotechnology multiply. In
particular as the issues of developing realistic methods for risk assessment
and the maintenance of objectivity come under increasing public interest.
These issues are likely to be of global importance and future progress will
rely heavily on the openness of the industry as well as the work of bodies
such as the Organisation for Economic Co-operation ad Development and
European Community in the establishment of internationally agreed safety
principles and procedures for all in the industry to follow.
References
1. Organisation for Economic Co-operation and Development. (1986) Recombinant DNA-

Safety Considerations, HMSO Publication Centre, London.
2. European Community (1990) Council Directive on contained use of genetically modified
micro-organisms (90/219/EEC). Off. J. Eur. Commun. U17.
3. European Community (1990) Council Directive on Deliberate Release Into the Environ-
ment of Genetically Modified Organisms. (90/220/EEC) Off. J. Eur. Commun. U17.
4. European Community (1992) Council Directive on the Protection of Workers from Risks
Related to Exposure to Biological Agents at Work. (90/679IEEC). Off. J. Eur. Commun.
L374.
5. Successful Health and Safety Management. Health and Safety Executive (1991). HMSO
Publications Centre, London.
6. Health and Safety Management in Further and Higher Education: Guideline on Inspection,
Monitoring and Auditing. (1992) HMSO Publications Centre, London.
7. Management of Health and Safety at Work Regulations. (1992) HMSO Publication
Centre, London.
8. Control of Substances Hazardous to Health (Health and Safety Executive) (1988). HMSO
9. Genetically Modified Organisms (Contained Use) Regulations (1992). HMSO Publica-
tions Centre, London.
10. Genetically Modified Organisms (Contained Use) Regulations (1993). HMSO Publica-
tions Centre, London.
11. A Guide to the Pressure Systems and Portable Gas Containers Regulations (1989). HMSO
12. Memorandum of Guidance on the Electricity at Work Regulations (1989). HMSO
13. Report of the Committee of Inquiry into the Smallpox Outbreak, London in March and
April 1973. (Command 5626 1974) Publications Centre, London.
14. Code of Practice for the Prevention of Infection in Clinical Laboratories and Post Mortem
Rooms (Howie Code) (1978). HMSO Publications Centre, London.
15. Report of the Investigation Into The Cause of the 1978 Birmingham Smallpox Occurrence
(1980). HMSO Publications Centre, London.
16. Report of the Working Party of the Laboratory Use of Dangerous Pathogens. HMSO,
17. The Safety Representatives and Safety Committee Regulations (1977). HMSO Publications
Centre, London.
3 Regulation of biotechnology in the United States,
Canada, and Latin America
D.T. KINGSBURY
3.1 Introduction
Over the past several years there has been a concerted attempt to
coordinate the regulatory policies of the countries of the Americas. This
attempt has not been fully realized. However, there are in place a wide
range of generally compatible systems. These range from the relatively
structured regulatory environment in the United States and Canada to the
recommendations for guidelines made by the collective bodies of the Pan-
American Health Organization (PAHO), The Organization of American
States (OAS) and The Inter-American Institute for Cooperation in
Agriculture (IlCA). These various policies will be reviewed in this chapter.
The United States, followed closely by Canada, has been the leader in the
development of biotechnology in the Americas, and in the development of
regulatory policy and practice, reacting to the changing conditions as new
biotechnology products came out of research laboratories and into the
marketplace.
3.2 The United States
The regulation of biotechnology products in the United States falls

principally to three government agencies: the Food and Drug Administra-
tion (FDA), the Department of Agriculture (USDA), and the Environ-
mental Protection Agency (EPA). The present underlying regulatory
philosophy and the mechanism of the inter-agency coordination was first
outlined in the "Coordinated Framework for the Regulation of Bio-
technology" published in the US Federal Register in 1986 (51 FR, 23,302,
June 26, 1986), and further clarified in the 1992 publication "Exercise of
Federal Oversight Within Scope of Statutory Authority: Planned Introduc-
tion of Biotechnology Products into the Environment" (57 FR, 6753,
February 27, 1992). The Coordinated Framework provided a road map for
the movement of products within the regulatory system and incorporated
the principle that products were to be regulated on their characteristics and
risk, and not the process used in their production. These principles applied
REGULATION IN THE AMERICAS 33
to both living and non-living products. In the Coordinated Framework the

FDA announced that it did not need specific or special procedures for
products of biotechnology, but that its existing authorizations were
adequate. The EPA and the USDA both announced that they felt that
additional rules and guidelines were necessary in order to clarify how
products would be reviewed by the agency. The first USDA rules were
published in final form in 1987 (52 FR, 22892, June 16, 1987) and since that
time there has been additional clarification and fine tuning, the most recent
published in March, 1993 (58 FR, 17044, March 31, 1993). The final rule
making by EPA has not yet appeared; however, a proposed rule was
published in January, 1993 (58 FR, 5878, January 22, 1993).
Guidance for continued refinement of the regulatory process was
provided by The President's Council on Competitiveness in a publication
entitled Report on National Biotechnology Policy issued in February 1991.
In that report the President's Council decried the still present inconsist-
encies in regulatory oversight, especially in cases of living organisms to be
used in the open environment, and urged agencies to reaffirm a "risk-
based" and not "process-based" approval process. The Report outlined the
following four principles of regulatory review:
"1. Federal government regulatory oversight should focus on the charac-
teristics and risks of the biotechnology product - not the process by
which it is created.
2. For biotechnology products that require review, regulatory review
should be designed to minimize regulatory burden while assuring
protection of public health and welfare.
3. Regulatory programs should be designed to accommodate the rapid
advances in biotechnology. Performance-based standards are, there-
fore, generally preferred over design standards.
4. In order to create opportunities for the application of innovative new
biotechnology products, all regulation in environmental and health
areas - whether or not they address biotechnology - should use
performance standards rather than specifying rigid controls or specific
designs for compliance."
3.2.1 Federal regulatory structure
3.2.1.1 An historical perspective

Any examination of the topic of regulation of biotechnology products must
begin with a focus on one of the most controversial, and still unresolved,
issues of regulatory policy, the regulatory focus on the 'process' of
biotechnology and not the products. To understand this issue it is necessary
to step back and take a brief look at the origins of biotechnology
regulation.
In 1973 Herbert Boyer and Stanley Cohen first described their

development of recombinant DNA (r-DNA) technology. Even during that
first year, concerns were arising regarding the potential hazards of this new
technology, regardless of the fact that recombinant-like processes were
already recognized in nature. The discussion expanded throughout 1973
and continued to grow over the next few years. In the well-known Asilomar
meeting in 1975 a group of scientists called for the National Institutes of
Health (NIH) to develop 'guidelines' for the use of this technology,
including a set of safety procedures to be followed. In response,
"guidelines" which included a series of containment levels, matched to
experimental procedures, were developed, and experiments were
categorized by their predicted level of hazard. Some experiments were
banned based on their perceived danger, since no real dangers had been
identified. The debate over the safety of r-DNA technology became
extremely emotional and politically charged, and several cities banned all
r-DNA work within their jurisdictions. A large number of environmental
and social activist organizations joined in the debate.
The release of the NIH Guidelines in 1975 had two lasting effects. First,
they helped to stabilize the scientific environment, but unfortunately failed
to silence the ongoing debate regarding the suitability of the technology.
The other effect of the Guidelines was the establishment of the concept,
which remains in the public policy debates even today, that the technology
needed to be regulated, rather than the specific results of the technology.
This issue continues to dominate the discussions related to the regulation
of products to be introduced into the environment as living organisms,
even though it was very clear that the hypothesized hazards were not real,
and no problems have arisen as a result of r-DNA research.
In the mid-1980s the US Government moved to re-examine the
regulatory environment, especially that related to commercial develop-
ment, and to replace the NIH Guidelines with a regulatory policy that was
more in keeping with the statutory authorities given to the regulatory
agencies, and to extract the NIH from acting as a de facto regulatory body.
The result of that effort was the "Coordinated framework for the
Regulation of Biotechnology" which appeared in June, 1986. Although it
has evolved over time, that policy statement remains the basis for the
current US regulatory environment.
As the traditional agencies took a more active position in the
biotechnology arena, the research agencies, lead by the NIH, continued to
re-examine their position. In recognition of the safety record of the basic
research community, and the increased recognition that many of the
perceived hazards of r-DNA technology were ill founded, the NIH
continued to relax the guidelines, and to place more of the oversight at the
local level. The NIH Recombinant Advisory Committee (RAC), which at
one time reviewed many proposed experiments in detail, now focuses its
attention on topics related to human gene therapy, and does not deal with
laboratory-based research problems, with the only exception being those
few experiments involving the introduction of extremely dangerous
bacterial toxins into common bacterial hosts. Even at the local level, the
guidelines have been relaxed to such an extent that most r-DNA activities
are considered routine, and no particular oversight is imposed. The
Human Genome Project is a particularly good example of the revision in
perceptions. Initially under the NIH guidelines cloning human DNA was
considered a hazard requiring special containment, now laboratories all
over the world are mapping and sequencing human genes. The development
of physical maps is based on a variety of cloned human DNA constructs
ranging from megabase sized Yeast Artificial Chromosomes (Y ACs), to
large collections of 50-70 kilobase sized cosmids. The goal is to develop
'sequence ready' clones covering the entire human genome. These
reagents are widely available and exchanged freely.
3.2.1.2 Large-scale production

The demand for large-scale production of recombinant organisms continues
to grow as the commercial side of biotechnology matures. Under the
original NIH guidelines, a quantity greater than 10 liters was considered
large-scale. More experience with recombinant organisms, combined with
a better understanding of the large-scale production and down stream
processing has led to greater relaxation of these controls. At the moment,
in the United States, the approval to move each large-scale processing of a
recombinant micro-organism is left at the local level. Each organization
proposing such activity is required to appoint an Institutional Biosafety
Committee (IBC) which has the responsibility to review the proposal and
make a final determination regarding its suitability. The IBC is composed
of institutional members together with suitable representatives of the
surrounding communities. Community representation generally includes
clergy, lawyers, business leaders, and non-technical community members.
This mechanism has been very effective, and production facilities are
functioning efficiently and safely in many parts of the country. There are a
few examples of communities which did not wish to have such facilities in
their midst and this advisory mechanism has proven to be an effective
means of safeguarding everyone's interests. The current shortage of
manufacturing facilities in the United States has not resulted from
regulatory barriers, but rather from the explosive demand for production
capacity as the industry rapidly develops. The two most serious barriers
have been a shortage of capital, and the time delays associated with the
design and construction of a facility which safeguards the environment on
one hand, and the product on the other.
3.2.2 Food and Drug Administration

The Food and Drug Administration (FDA) has consistently taken the
position that no new procedures or requirements are necessary for the
oversight of products resulting from the new biotechnology. The FDA has
steadfastly regarded the techniques of the new biotechnology as simply
extensions or refinements of older forms of genetic manipulation, and the
products have been subject to the same regulatory requirements as other
products.
Therefore, there are no statutory provisions or regulations that address
biotechnology specifically. The laws and regulations under which the
agency approves products place the burden of proof of safety as well as
effectiveness of products on the manufacturer. The agency possesses
extensive experience with these regulatory mechanisms and applies them
to the products of biotechnological processes. An expanding fraction of the
products the FDA regulates represents the results of new technological
achievements, and the products of biotechnology have led to the marketing
approval of more than 750 products (mostly diagnostics, but including 19
therapeutics). In addition more than 1,200 clinical trials of drugs and
biologics are under way.
The marketing of new drugs and biologics for human use, and new
animal drugs, requires prior approval of an appropriate new drug
application (NDA), biological product license, or new animal drug
application (NADA). For new medical devices, including diagnostic
devices for human use, either a Premarket approval application (PMA) or
reclassification petition is required. If the device is determined to be
substantially equivalent to an already marketed device, a premarket
notification under section 51O(k) of the Federal Food, Drug, and Cosmetic
Act (the FD&C Act) is required. For food products, section 409 of the
FD&C Act requires FDA pre-clearance of food additives including those
prepared using biotechnology. Section 706 of the FD&C Act requires pre-
clearance of color additives. The implementing regulations for food and
color additive petitions and for affirming generally recognized as safe
(GRAS) food substances are sufficiently comprehensive to apply to those
involving biotechnology. Genetic manipulations of plants or animals may
enter the FDA's jurisdiction in other ways; for example, the introduction
into a plant of a gene coding for a pesticide or growth factor may constitute
adulteration of foodstuff derived from the plant, or the use of a new micro-
organism found in a food such as yogurt could be considered a food
additive. Such situations are evaluated case-by-case and in cooperation
with the USDA, where appropriate.
In May 1992 the FDA published an updated statement of policy
regarding the oversight of new varieties of food plants, again stating the
principle that the techniques of their construction was not the trigger for
special review. The FDA has identified scientific and regulatory issues
which may require a consultation between the developer of a new variety
and the FDA. These issues are related to characteristics of foods that raise
safety questions and would trigger a higher level FDA review. The issues
identified in the FDA statement included the presence in the new variety
of a substance that is completely new to the food supply, the presence of an
allergen in an unusual or unexpected setting, changes in levels of a major
nutrient, or the increased level of a toxin normally found in food. New
varieties without these characteristics are subject to lower level scrutiny.
The technique employed in the development of the new variety does not in
itself determine the need for, or the level of, review.
3.2.2.1 The regulatory process

Congress has provided FDA authority under the FD&C Act and the
Public Health Service (PHS) Act to regulate products regardless of how
they are manufactured. Each request for product approval is considered
using the appropriate statutory and regulatory criteria. The following
sections summarize general requirements for various kinds of products.
General requirements for new drugs and biologics for human use. A new
drug is a drug not generally already recognized by qualified scientific
experts as safe and effective for the proposed use. New drugs may not be
marketed unless they have been approved as safe and effective for their
intended uses. Clinical investigations on human subjects by qualified
experts are a prerequisite for the determination of safety and effectiveness.
Sponsors of investigations of new drugs or new uses of approved drugs file
a Notice of Claimed Investigational Exemption for a New Drug (IND) to
conduct clinical investigations on human subjects. The IND must contain
information to demonstrate the safety of proceeding to test the drug in
humans, including, for example, drug composition, manufacturing and
controls data, results of animal testing, training and experience of
investigators, and a plan for clinical investigation. In addition, assurance of
informed consent and protection of the rights and safety of human subjects
is required. FDA evaluates IND submissions and reviews ongoing clinical
investigations. Significant changes in the conditions of the study, including
changes in study design, drug manufacture or formulation, or proposals for
additional studies, must be submitted to FDA as amendments to the IND.
FDA approval of an NDA or an abbreviated New Drug Application
(ANDA) is required before the new drug can be marketed. The NDA
must contain, among other information, the following:
1. A list of components of the drug and a statement of the composition of
the drug product.
2. A description of the manufacturing and packaging procedures and

controls for the drug product.
3. A description of the non-clinical studies concerning the drug's pharma-
cological actions - and toxicological effects.
4. A description and analysis of each clinical study.
5. A description and analysis of any other data or information relevant to
an evaluation of the safety and effectiveness of the drug product,
including commercial marketing experience.
NDA holders who wish to market an approved drug under conditions

other than those approved in the NDA must submit a supplemental NDA
containing clinical evidence of the drug's safety and effectiveness for the
added indications. Extensive changes such as a change in formula, manu-
facturing process, or method of testing differing from the conditions of
approval outlined in the NDA may also require additional clinical testing.
Biological products must also be approved by the FDA prior to
marketing, as required by section 351 of the PHS Act. A biological product
is 'any virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood
component or derivative, allergenic product, or analogous product
applicable to the prevention, treatment, or cure of diseases or injuries of
man.' Unapproved biological products are regulated under the same
regulations as new drugs during the IND phase. Prior to marketing,
separate licences are issued for the manufacturing establishment and the
biological product. The manufacturing establishment and the biological
product must meet standards (including any FDA standards specific for the
product) designed to ensure the safety, purity, potency, and efficacy of the
product. To obtain a licence, the facility must also pass a prelicensing
inspection. Licensed products are subject to specific requirements for lot
release by FDA.
Manufacturers of new drugs and biologics must operate in conformance
with current good manufacturing practice (CGMP) regulations. These
regulations require adequately equipped manufacturing facilities,
adequately trained personnel, stringent control over the manufacturing
process, and appropriate finished product examination. CGMPs are
designed to protect the integrity and purity of the product. The sponsor's
process techniques are also considered in FDA's reviews and communica-
tions for the development of appropriate information on which the
submission of an NDA, ANDA, or biological product licence application
would be based. For example, the use of r-DNA technology to manufacture
new drugs or biological products may result in products that differ from
similar products manufactured with conventional methods. Determination
of the extent of testing required will depend upon the nature of the particular
product. In some instances the molecular structure ofthe product may differ
from the structure of the active molecule in nature. For example, the first
human growth hormone manufactured using recombinant micro-organisms
had an extra amino acid, an amino-terminal methionine; hence it is an

analogue of the native hormone. Such differences could affect the drug's
activity or immunogenicity and, consequently, could affect the extent of
testing required.
Another consideration in the review of new drugs or biological products
produced by recombinant techniques is whether the manufacturing process
includes adequate quality controls. For example, the occurrence of
mutations in the coding sequence of the cloned gene during fermentation
could give rise to a subpopulation of molecules with an anomalous primary
structure and altered activity. This is a potential problem inherent in the
production of polypeptides in any fermentation process. As with conven-
tionally produced products, assurance of adequate processing techniques
and controls is important in the manufacturing of any biotechnology-
produced new drug or biological product. Review of the production of
human viral vaccines routinely involves a number of considerations
including the purity of the media and the serum used to grow the cell
substrate, the nature of the cell substrate, and the characterization of the
virus. In the case of a live viral vaccine, the final product is biologically
active and is intended to replicate in the recipient. Therefore, the
composition, concentration, subtype, immunogenicity, reactivity, and
non pathogenicity of the vaccine preparation are all considerations in the
final review, whatever the techniques employed in 'engineering' the virus.
However, special considerations may arise based upon the specific
technology employed. For example, a hepatitis B vaccine produced in
yeast (via r-DNA techniques) would be monitored for yeast cell contamin-
ants, while distinctly different contaminants would be of concern in a
similar vaccine produced from the plasma of infected patients.
Nucleic acids or viruses used for human gene therapy are subject to the
same requirements as other biological drugs. At present scientific reviews
of these products are also performed by the National Institutes of Health.
General requirements for animal food additives and drugs. Animal food
additives and drugs are subject to similar mandatory requirements of the
FD&C Act as the like products for use in humans. Animal biologics,
however, are licensed by the USDA under the authority of the Virus-
Serum-Toxin Act of 1913. Questions as to whether a product is an animal
biological subject to USDA licensure, or a new animal drug to be regulated
by FDA are referred to a standing committee of representatives from
USDA and FDA.
New animal drugs must go through the Investigational New Animal
Drug (INAD) and New Animal Drug Application (NADA) process, a
procedure similar to that required for human drugs, as discussed earlier.
However, IN AD regulations do not require advance Agency approval for
clinical investigations for the drug, although authorization is required for
use of edible products derived from food-producing animals in which the
drug has been used. The data must be specific for each animal species for
which the drug is intended. For NADA approval, it must be shown that the
product is safe and effective when used in accordance with approved label
directions. Also, it must be shown that those drugs which are intended for
use in food-producing animals and used in accordance with approved label
directions, do not accumulate as unsafe residues in the edible tissues of the
animal at the time of slaughter. Moreover, the manufacturer must submit
acceptable methods for measurement of any drug residue in edible tissues.
Further, animal drugs, including premixes for use in medicated feeds, must
be manufactured in conformance with CGMPs. Substances that are used in
animal feeds, other than drugs, and that are produced by r-DNA
technology, are considered to be food additives and require approval of a
separate food additive petition (FAP), even though a similar substance is
currently approved as a food additive.
A number of the early biotechnology products have been identical to a
previously approved animal drug held by the same applicant. The FDA's
Center for Veterinary Medicine (CVM) has stated that, when the new
substance produced by biotechnology is identical or virtually identical to an
approved substance produced by conventional technology, only a supple-
mental application is necessary. This policy only applies when the sponsor
of the biotechnology product is also the sponsor of the conventionally
produced product. If, on the other hand, the new substance produced by
the biotechnology is significantly different from that produced by conven-
tional means, an original application will be needed.
Two examples, each involving the adoption of r-DNA technology as an
alternative means of producing a substance that is currently the subject of
an approved NADA, will illustrate. In the first example, the drug is (or
appears to be) unchanged by the new production method. Under the
current regulations, such a departure in manufacturing procedure requires
a supplemental application which requires approval before implementation.
The supplement would be a Category II supplement under CVM's
supplemental policy in that it involves a revised method of synthesis or
fermentation for the new drug substance. However, in accordance with the
CVM's supplemental policy the underlying safety and effectiveness data
supporting the original NADA usually would not be reviewed (for
compliance with contemporary standards) since there is likely no increased
risk of human exposure to the drug. Data may be required to demonstrate
the new animal drug product is essentially biologically equivalent to the
drug product for which approval has already been granted. Approval of
such a supplemental NADA is not required to be published in the Federal
Register.
In the second case, a new method of manufacture changes the molecular
structure or chemical composition of the active ingredient. Such a change
in the identity of the new animal drug normally will require an original new
animal drug application and subsequent publication of a notice of approval

in the Federal Register. Ordinarily, an original NADA requires complete
safety and effectiveness studies, meeting contemporary standards. How-
ever, reference to data in another NADA sometimes suffices to support a
separate NADA approval, where the existing NADA is owned by the
applicant of the new NADA, or where the new applicant obtains
authorization to refer to another NADA. In this case reference might be
made to data contained in the NADA supporting approval of the drug as
produced by conventional means.
It may be possible to regard the new application as if it were a category II
supplement. This finding would be dependent upon data showing the new
substance to be sufficiently similar to the original in terms of its
pharmacology, toxicology, bioequivalence, and metabolism.
Thus, regardless of the type of application required, there is no legal
requirement for the generation of new safety and effectiveness data if the
applicant has access to previously submitted data, and there is no scientific
need.
General requirements for medical devices. Medical devices for human

use are regulated by requirements of the FD&C Act as modified by the
Medical Device Amendments of 1976. In general, a device is a health care
product that does not achieve any of its principal intended purposes by
chemical action in or on the body or by being metabolized. Devices include
diagnostic aids such as reagents, antibiotic sensitivity discs, and test kits for
in vitro diagnosis of disease. The FD&C Act establishes three classes of
devices: class I (general controls), class II (performed standards), and class
III (premarket approval). Classification of a device is determined by the
level of regulatory control needed to provide reasonable assurance of the
safety and effectiveness of the device. A class I device is a device for which
the 'general controls' authorized by or under various sections of the FD&C
Act are sufficient to provide reasonable assurance of the safety and
effectiveness of a device. A class II device is a device for which general
controls by themselves are insufficient to provide reasonable assurance of
the safety and effectiveness of the device, for which there is sufficient
information to establish a performance standard to provide such assurance,
and for which it is therefore necessary to establish a performance standard
to provide reasonable assurance of its safety and effectiveness. A class III
device is a device that cannot be classified into class I or class II and that is
purported or represented to be for use in supporting or sustaining human
life or for a use which is of substantial importance in preventing
impairment of human health, or that presents a potential unreasonable risk
of illness or injury. Premarket approval obtained in accordance with
section 515 of the FD&C Act is required to provide reasonable assurance
of the safety and effectiveness of a class III device.
Before a manufacturer may introduce into commerce any medical device

it has not previously marketed, the manufacturer must submit to the FDA
a premarket notification. This notification requirement is designed to
assure that manufacturers do not intentionally or unintentionally circum-
vent the automatic classification into class III of devices not on the market
prior to enactment of the Medical Device Amendments and not sub-
stantially equivalent to pre-amendment devices.
A new device, that is, one not substantially equivalent to a pre-
amendments device, remains a class III device requiring FDA approval of
a premarket approval application (PMA) unless FDA reclassifies it into
class I or class II, usually in response to a manfuacturer's petition. In the
premarket approval process the manufacturer must establish by valid
scientific evidence that the device is safe and effective for its intended use.
This evidence is usually data from clinical investigations.
For a significant risk device, as defined in the FDA's regulations, the
sponsor must submit an application to the FDA for approval to conduct a
clinical investigation. This application seeks an Investigational Device
Exemption. When the manufacturer believes that there are sufficient data
to establish the safety and effectiveness of its device, the manufacturer files
a PMA.
General requirements for foods. Several sections of the Food, Drug and
Cosmetic Act apply to the Agency's regulation of food. No particular
statutory provision or regulation deals expressly with food produced by
biotechnology. Accordingly, when confronted by an issue concerning the
regulation of food produced by new biotechnology, the Agency will apply
the relevant statutory or regulatory provisions. Most issues concerning the
safety of a food will involve the application of either section 402(a)(1) or
section 409 of the FD&C Act.
Section 402(a)(1) of the FD&C Act provides, in part, that a food is
adulterated if it bears or contains any poisonous or deleterious "added
substance which may render it injurious to health." Courts have agreed
with the agency's interpretation of this section that any substance that is
not an inherent constituent of food may be regulated as an added
substance. Furthermore, if the quantity of the constituent exceeds the
amount that would normally be present because of some technological
adjustment to the product, that excess quantity may also be viewed as an
'added substance' within the meaning of the section. Thus, section
402(a)(1) applies to most of the harmful substances that may occur in
human food. For example, if a food produced with a new technology
contains a higher level of a substance than it might ordinarily have, then
that level "may be injurious to health" and the agency could regulate the
product under section 402(a)(1). Similarly, if a food produced by
biotechnology contains, as a result of the production process, a harmful or
deleterious substance not contained ordinarily in the food, the food could
be in violation of the section.
The other primary statutory provisions that the FDA relies upon in
determining the safety of food and food constituents are sections 201(s)
and 409, the food additive provisions of the FD&C Act. The definition of
food additive appears in section 201(s) of the FD&C Act and includes
both artificial and natural substances. The definition provides that:
the term food additive means any substance the intended use of which results or
may reasonably be expected to result, directly or indirectly, in its becoming a
component or otherwise affecting the characteristics of any food (including any
substance intended for use in producing, manufacturing, packing, processing,
preparing, treating, packaging, transporting, or holding food; and including any
source of radiation intended for any such use), if such substance is not generally
recognized as safe by qualified experts.
If the substance is generally recognized as safe (GRAS) for a given food
use the product is not a food additive. Specific factors which trigger
additional review were mentioned earlier.
The included material here can only be a general guide to the
complexities of FDA product regulation. Additional information specific
to a particular product may be obtained by contacting the Agency directly
at:
The Office of the Senior Advisor for Science
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857
USA
Telephone + 1 (301) 443-5839
Fax + 1 (301) 594-6777
3.2.3 Environmental Protection Agency

The Environmental Protection Agency's statutory authority for regulation
of microbial products falls under two Federal statutes, the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA), and the Toxic
Substances Control Act (TSCA). These statutes have differing require-
ments for regulatory oversight, and differing procedures regarding
notification and permitting. Neither statute was specifically written to
address the problems of biotechnology, and without additional rule making
the EPA feels that the procedures are not explicit. In the 1986
"Coordinated Framework" EPA outlined a policy and announced their
intention to release "a significant new rule" to cover this new group of
organisms. Because of the difficulties associated with making the appro-
priate definitions based on process rather than biological characteristics,
the EPA has only recently been able to come forward with proposed new
rules. They are continuing to work on these rules, and publication of the
final rules should occur some time in 1994.
At present two EPA requirements are in place as outlined in the 1986
notice. First is a notification and reporting requirement for small-scale field
tests, and the experimental use permit and registration requirements under
FIFRA, and second, the premanufacture notice requirements under TSCA
for "new" micro-organisms, as defined in the EPA section of the
"Coordinated Framework." While it is prudent for anyone with a product
subject to Federal regulation to make a preliminary contact with the
appropriate agency, that is even more essential for products subject to
EP A regulation due to vagaries of the FIFRA and TSCA language and
their interpretation for enforcement purposes.
3.2.3.1 The regulation of microbial products under FIFRA

A variety of biological agents, including viruses and micro-organisms, may
be used as pesticides and in this context they are subject to regulation
under FIFRA, unless they have been specifically exempted by regulation.
Before EPA can register a pesticide, it must have sufficient data to be
confident that when used in accordance with widespread and recognized
practice, it will not cause unreasonable effects on humans or the
environment. The specific kinds of data and information that are required
to support registration of various microbial pesticides under FIFRA are
too detailed to include here but may be found in 40 CFR 158.65, and
162.163. EPA has also published guidance for developing these data in
"Pesticide Assessment Guidelines: Subdivision M - Biorational Pesticides,"
which may be obtained from the Agency.
EPA holds a thorough review of all submitted data prior to pesticide
registration. However, prior to registration, producers may test their
products under an experimental use permit (EUP). The regulations
governing EUPs for most microbial pesticides assume that certain small-
scale experimental uses of new pesticides and new uses of previously
registered pesticides will not require an EUP. However, EPA has
imposed restrictions on small-scale testing of microbial pesticides defined
as "nonindigenous and genetically altered" (49 FR, 40659, October 17,
1984). Under EPA policy the small-scale provision of FIFRA would not
automatically apply to genetically altered microbes and that the Agency
should be notified before the initiation of any field testing of genetically
altered organisms. This original policy was expanded in the "Coordinated
Framework" and the most recent proposal (58 FR 5878-5902, January 22,
1993) provides guidance into the current thinking.
The current policy remains that outlined in the "Coordinated Frame-
work" published in 1986. In that document the EPA explicitly outlined the
policies for review of microbial pesticides, and the data requirements. Unit
II.D of that publication (51 FR, page 23320ff, June 26, 1986) provides
detailed instructions.
Small-scale field testing. The EPA requires notification prior to the use
of microbial pesticides for small-scale testing for those organisms which are
defined as nonindigenous or genetically altered. The purpose is to screen
for possible risk to human health or the environment, and to determine if
an EUP is required. Small-scale tests are defined as terrestrial field studies
that involve 10 acres or less of land, or 1 surface acre or less of water.
In order to correlate the level of review with the potential level of risk
the EPA adopted a two-level review system based on its evaluation of the
potential risks posed by various types of micro-organisms. This system
provides the Agency with information about all types of micro-organisms,
both those posing low or negligible risk (Level I), and those of greater risk
requiring greater attention (Level II). This overall system would be slightly
modified under the proposed rule published in January, 1993. However,
three options were presented in that proposal and it is uncertain which of
the three will be adopted. Until those changes are made the 1986
procedures remain in effect.
Level I reporting. Level I reporting for small-scale field testing applies

to all genetically engineered or nonindigenous microbial pesticides not
covered in the definition for Level II oversight (described below). In many
cases experience has led the Agency to modify its original views of
potential risk; under those circumstances a case-by-case review is necessary
for final determination. Level I reporting requires the following informa-
tion, or an explanation of why such information is unnecessary.
1. Identity of the micro-organism, including its characteristics and the
means and limits of detection.
2. Description of the natural habitat of the organism or its parental strains,
including information about natural control mechanisms in the eco-
system.
3. Information on the host range of the parental strain(s) or non-
indigenous organism.
4. Information on the relative environmental competitiveness of the
organism, where available.
5. If the micro-organism is genetically engineered, information about the
methods of the genetic modification(s); the identity and location of the
rearranged or inserted/deleted gene segment(s) in question; a descrip-
tion of the new trait(s) or characteristic(s) that are expressed;
information on the potential for genetic exchange or transfer to other
organisms; and on genetic stability of any inserted sequence.
6. A description of the proposed testing program, including site location,
crop to be treated, target pest, amount of test material to be applied and

method of application.
Once these data are submitted EPA has 30 days to review the
information to make a preliminary determination of the need for an EUP.
If the applicant is not notified within the 30-day period that an EUP is
required, then the applicant is free to conduct the proposed trial. If, on
initial examination, questions arise regarding the safety of the test then an
EUP is required. The applicant has two options at this stage: to apply for a
permit, providing the information necessary to support such an application;
or to provide all of the additional data outlined below for a Level II
application. If the applicant selects the second option, the Agency has an
additional 60 days to review the package and determine if a full EUP is
necessary.
Level II reporting. Level II reporting is required for microbial pesticides
formed by deliberately combining genetic material from organisms of
different genera, genetically engineered microbial pesticides derived from
source organisms that are pathogens, and nonindigenous pathogenic
microbial pesticides. The definition of the terms pathogen and non-
indigenous are complex and can be found in the "Coordinated Frame-
work" publication. These definitions are being refined as experience
accumulates and a producer is best advised to contact the Agency if there is
any question between Level I and Level II reporting. Like Level I
documentation, reporting requires the following information, or an
explanation of why such information is unnecessary.
1. Background information on the micro-organism, including:
(a) identity of the micro-organism with a table of characteristics;
(b) means and limits of detection of the micro-organism using the best
available techniques;
(c) a description of the natural habitat of the organism, including
information on naturally occurring control factors;
(d) information on host range;
(e) information on survival and the ability of the organism to increase
in number in the environment; and
(f) if the organism is genetically engineered, the following additional
information is required:
(i) information on the methods used to genetically alter the
micro-organism;
(ii) the identity and location of the inserted/deleted gene
segment(s) in question (host source, nucleotide sequence or
restriction map), the expressed phenotype of the new informa-
tion and the background of the host.
(iii) information of the control region(s) of the gene(s) and the
new traits that are expressed;
(iv) information on the potential for genetic transfer to other

organisms, and the stabilty of the new information;
(v) information on relative environmental competitiveness.
2. Description of the proposed field test:
(a) the purpose or objective of the testing;
(b) detailed description of the proposed test, including test para-
meters;
(c) a designation of the pest organism(s) involved, both common and
scientific name;
(d) a detailed statement of the composition of the formulation to be
tested;
(e) the amount of pesticide proposed for use and the method of
application;
(f) the State(s) in which the test will be conducted, and the exact
location within those States;
(g) the crops, fauna, flora, geographical description of sites, modes,
dosage rates, frequency and situation of application for the
pesticide application;
(h) a comparison of the natural habitat of the micro-organism with the
test site;
(i) details of the application site including size, structures present,
and means of controlling movement into and out of the site(s);
(j) the proposed date and duration of the testing, and means of
supervision of the site( s);
(k) procedures for monitoring the micro-organisms within the site,
and in surrounding areas;
(I) the method of disposal of plants, animals, soils, etc. that were
exposed during or after the trial; and
(m) means of evaluating potential adverse effects and methods for
controlling the micro-organism if spread is detected beyond the
test site.
Once the supporting data have been submitted, EPA has 90 days to
review each submission and approve the trial or rule that an EUP is
required. EPA encourages applicants to contact the Agency and discuss
proposed trials prior to beginning the application process in order to
determine the exact requirements for the particular trial under considera-
tion. Since all applications are examined on a case-by-case basis, the
experience of the Agency will impact on the details of the information
necessary for a thorough review.
Pesticide registration. Before a pesticide can be commercially marketed

it must be registered as outlined in FIFRA. Generally, large-scale field
testing is necessary prior to registration in order to obtain the data
necessary to support the registration application. Such testing generally

requires an Experimental Use permit (EUP) issued for limited use of an
unregistered product. The data needed for EUP applications generally are
similar to those for registration as outlined in section 3 of FIFRA. The data
requirements generally resemble those outlined above for level II
reporting, although they may be more extensive in some cases depending
on the exact nature of the product. Direct consultation with the Office of
Pesticide Programs is strongly recommended. The office can be reached at:
Environmental Fate and Effects Division (H7560C)
Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460
USA
Telephone + 1 (703) 305-6307
3.2.3.2 Regulation of microbial products under TSCA

The EPA interprets micro-organisms to be "chemical substances" for the
purpose of regulation under section 3 of TSCA. Under that provision the
scope of their regulation excludes micro-organisms used as pesticides, or
organisms used to produce foods, food additives, drugs cosmetics, and
medical devices. Likewise, plants and animals are not subject to regula-
tion under TSCA. With these exceptions, all other micro-organisms
produced for environmental, industrial, or consumer uses are potentially
subject to regulation. TSCA is a complex statute and there are many
organisms which have been in common industrial use for many years and,
therefore, are not subject to TSCA regulation because they appear on an
approved list. However, these organisms are approved only for those uses
which are on the list, and any 'significant new use' of the organism is
subject to regulation. Furthermore, 'new' organisms are subject to
regulation and the definition of 'new' is subject to the judgment of the
agency.
TSCA requires a Premanufacturing Notification (PMN) before the use
of a regulated organism for manufacture or in the environment. For the
purposes of TSCA the Agency has defined "new" as any organism that,
through deliberate human intervention, contains genetic material from
dissimilar source organisms. Organisms are considered dissimilar if they
are from different genera using standard and accepted sources of
taxonomy.
In the "Coordinated Framework" the EPA spells out in great detail the
requirements for PMN application, and the conditions for approval. EPA
expects manufacturers and importers to contact EPA well in advance of
PMN submission, to allow for pre notice consultation. The Agency feels
that this consultation will provide ample opportunity for the discussion and
solution of any potential problems and will expedite the review. Section
REGULA nON IN THE AMERICAS 49
5( d)( 1)( A) of TSCA specifies the information required for PMN submission
and includes information on the production, workplace exposure, and
release of the organism. In addition, extensive risk assessment data are
also necessary, but the extent of data submission will vary according to the
specifics of each case and close consultation with the Agency is required to
expedite the review. One major consideration is the level of containment
in the manufacturing process, and the potential for worker exposure.
All PMN reviews follow a strict pattern of administrative steps,
regardless of the substance being reported. Within five days of receipt
EPA will announce the filing in the Federal Register. Submitters may list
some of the details of the submission as "confidential business information"
which will not be listed, but in the case of micro-organisms the Agency will
list a generic description of the organism if the producer feels that the
detailed identity and use of the microbe is confidential. EPA encourages
producers to be as open as possible in sharing product information with the
public. Following the Federal Register notice, the Agency has 90 days to
review the PMN, and has the option of extending the time by an additional
90 days if necessary. If no action is taken during the review period the
producer is free to use the micro-organism and once it is approved for use it
enters the registry and no additional approvals are needed unless the
organism is used for a significantly different purpose.
The "Coordinated Framework" outlined the Agency's interpretation of
the Significant New Use Rule (SNUR) and called for voluntary reporting
of significant new uses of previously approved organisms. The focus of the
SNUR guidelines was environmental introductions of micro-organisms,
especially those that have been genetically manipulated. The EPA
encourages users to be as comprehensive as possible in the interpretation
of new non-agricultural uses of micro-organisms, especially for pathogens
and genetically-modified organisms. In cases of questions about the
applicability of the SNUR it is best to contact the TSCA branch of the
Agency. For all contacts related to regulation under TSCA contact:
The Office of Toxic Substances
(TS-794)
Environmental Protection Agency
401 M Street, S.W.
Washington, DC 20460
USA
Telephone + 1 (202) 382-3852
3.2.4 Department of Agriculture

The Department of Agriculture outlined its approach in the 1986
"Coordinated Framework" and has subsequently published several state-
ments of clarification and rule making. The central office within the
Department with jurisdiction over biotechnology products is the Animal

and Plant Health Protection Service (APHIS). In 1987 rule making,
APHIS established procedures for the issuance of permits for the field
testing of new plant varieties derived from r-DNA technology. Subse-
quently hundreds of trials have been safely performed on a number of
plant varieties. In light of this extensive experience with field trials in
November 1992 APHIS published a proposal that would require only
notification to APHIS, rather than prior approval, for field trials of
transgenic plants that meet certain criteria designed to insure that the
plants are not plant pests. These proposals have now been modified and
published in the form of final rules. Extensive information is available in
the Federal Register notice of March 31, 1993 (58 FR, 17044, March 31,
1993). These new rules are too extensive to duplicate here, so only
summary information is given.
3.2.4.1 Definition of a 'regulated article'

Under the Federal Plant Pest Act the definition of a 'regulated article' is
the definition of the scope of the regulatory process. In the March 31,1993
notice a 'regulated article' was defined as:
Any organism which has not been altered or produced through genetic
engineering, if the donor organism, recipient organism, vector or vector agent
belongs to any genera or taxa designated in Sect. 340.2 of the Code of Federal
Regulations (7 CFR Part 340) and meets the definition of plant pest, or is an
unclassified organism and/or an organism whose classification is unknown, or
any product which contains such an organism, or any other organism or product
altered or produced through genetic engineering which the Director, Bio-
technology, Biologics, and Environmental Protection (BBEP) division of
APHIS, determines is a plant pest or has reason to believe is a plant pest.
Excluded are recipient microorganisms which are not plant pests and which have
resulted from the addition of genetic material from a donor organism where the
material is well characterized and contains only non-coding regulatory regions.
The revised procedures include a list of regulated articles which may be
introduced without a permit, but simply following notification of the BBEP
Director. Principally six plant species have been placed in the notification
category, corn, cotton, potato, soybean, tobacco, and tomato. It is
anticipated that additional plant species will be placed in this list as the
USDA gains additional experience. In addition to being on the above list,
a number of other criteria are necessary for a product to fall under the
notification umbrella. The criteria are spelled out in great detail in the
Federal Register notice.
The Research and Education division of the Department of Agriculture
had intended to develop its own set of 'guidelines' for field research.
However, in light of the effectiveness of the procedures then in effect, it
announced that it had abandoned these earlier intentions. This then, leaves
in place a mechanism of oversight for research with field trials subject to

the jurisdiction of various agencies, depending in the characteristics of the
organism in question or its intended use, and not its source of funding. For
example, veterinary vaccines and plant pests would be subject to USDA/
APHIS oversight, human vaccines by FDA, and microbial pesticides by
the Environmental Protection Agency.
For detailed information about specific product, or for more information
contact:
Office of the Director
Biotechnology, Biologics, and Environmental Protection
Animal and Plant Health Inspection Service
US Department of Agriculture
Federal Building, room 850
6505 Belcrest Road
Hyattsville, MD 20782
USA
Telephone + 1 (301) 436-7602
3.2.5 Coda
The 1992 US election changed the political climate for regulatory policy.
The Council on Competitiveness has been eliminated, and no clear picture
has emerged regarding the coordinated formation of regulatory policy.
Vice President Gore has a well-established track record of encouraging
excessive regulation in this area, and his domestic affairs advisor was the
author of the congressional bill for the comprehensive regulation of field
research with recombinant DNA-manipulated organisms, which was
defeated in 1990. We must wait and see if these old habits will persist now
that Mr. Gore has a new venue. Biotechnology is rapidly developing as a
tool in so many areas of potential benefit to the environment that the
Administration cannot afford to interfere with one of the strongest
elements of US technology driven industry.
3.2.6 State regulatory bodies

Generally speaking, state laws are superseded by Federal statute and
regulation. However, strictly interpreted this is limited to activities
involving interstate movement and commerce. Therefore, the only time
that an investigator or company will have to be especially cognizant of state
laws is in the case of field trials which do not involve interstate movement.
This clearly leaves the FDA regulated areas out of this dilemma and limits
investigators to field trials involving the USDA and the EPA. Both
agencies are very sensitive to a particular State's interest and responsibility
and State authorities are brought into the review process at an early stage.
However, it is essential that investigators become familiar with the

situation in the State in which a field trial is under consideration in order to
avoid unforeseen problems. One example of such a problem occurred
when a group wished to test a recombinant rabies vaccine on an isolated
island on the coast of North Carolina. The trial did not involve any
interstate jurisdiction and the State imposed a regulatory burden in excess
of the Federal regulations and delayed the trial for many months. Most
States accept that Federal oversight preempts the need for state regulation
and work closely with the Federal authorities. However, before planning a
field trial of an experimental product, an investigator should contact the
State Environmental Agency where the trial is being considered, and
determine if specific regulatory burdens have been imposed.
In a survey done by the Pharmaceutical Manufacturers' Association in
1992, they found that the number of States considering legislation to
regulate biotechnology had dropped from a peak of 10 in 1988 to only two
at the time of the survey. The two states considering legislation at the time
were Connecticut and Vermont, and at this time those bills have not passed.
The Vermont bill was only to appoint an advisory board to recommend
future actions to the State.
Beyond State regulations, there are still a few local governments that
have specific restrictions on biotechnology products. Most jurisdictions
which passed restrictive legislation in the 1970s have repealed those laws;
however, there are still a few local restrictions. Even when all of the
regulatory barriers have been passed, unusual products continue to attract
public attention. If a field trial is being contemplated of a truly unique
product it is well worth the investigators' investment of time to make
contact with the local community and its leaders, and to open an active
dialogue with the community so that questions and concerns may be dealt
with before they become magnified by misunderstanding or misinforma-
tion.
3.3 Canada
The dominant Agency in the regulation of environmentally released

biotechnology products is Agriculture Canada. This Agency, especially
through its offices of Pesticides and of Veterinary Biologics has developed
a comprehensive set of regulations dealing with the introduction of
biotechnology products into the environment. Agriculture Canada has
worked closely with the Organization for Economic Cooperation and
Development (OECD) in the development of a coordinated regulatory
policy. The Canadian philosophy, while a little more process oriented than
that in the US, does not differ markedly. Non-living products of
biotechnology are regulated in much the same way as their traditional
counterparts. Living products that are derived from the biotechnology

receive a more stringent review, triggered in part on the method used for
their development. Therefore, the use of r-DNA technology, by itself, is a
regulatory trigger.
3.3.1 Veterinary biologics

Veterinary biologics are placed into two Classes, based on the properties of
the preparation. Class I products include: inactivated r-DNA-derived viral
vaccines; inactivated r-DNA derived bacterial vaccines; viral, bacterial,
cytokines or other subunits, monoclonal antibody products; and vaccines
containing live organisms modified by gene insertion or deletion, without
introduction of 'foreign' DNA.
Class II products include: vaccines using a live vector to carry
recombinant-derived foreign genes; and vaccines containing live organisms
modified by gene insertion or deletion, but including the introduction of
'foreign'DNA.
Class I products are deemed to have no new or unusual safety concerns,
and are reviewed without special precautions. Class II products, however,
involve the introduction of r-DNA constructions into live organisms and
additional information is needed for full review. Issues include the
competitiveness of the organism in the environment, its survivability and
persistence, and whether it will transplant the wild-type organisms from
the environment. The added genetic information must be well charac-
terized, and the full nucleotide sequence may be requested. The fully
documented identification of the entire insert from the "foreign" source is
essential. Concerns for safety to humans and animals, and impact on the
environment, must be addressed in an environmental assessment or
environmental impact study to be evaluated by an ad hoc committee before
live products can be considered for a limited field trial or licensing.
Agriculture Canada has broken the development process into four
stages, and oversight at each stage is reflective of the potential hazard to
the environment. Stages I and II involve contained laboratory and other
contained facility testing. The level of Stage II containment may be
negotiated with the regulatory body based on the review of the Stage I
data. The movement of the live r-DNA product from Stage II to Stage III,
for a limited trial outside of a fully contained facility, requires a complete
submission of all available data on the product, including safety data from
Stages I and II. Based on the submitted material, the regulators will
determine the need for an ad hoc committee review, and any additional
data. Stage IV is the product licensing stage and the decision will be based
on the complete submission of the data developed in Stages I-III. The
submissions are examined by an ad hoc committee, which may request
additional information from the producer.
3.3.2 Genetically modified plants and micro-organisms

The Seed Division of Agriculture Canada has produced a publication,
"Information to be Requested in Applications for Field Testing of
Genetically Modified Plants" (Kalous, M.J. and Duke, L.H., 1989. The
Regulation of Plant Biotechnology in Canada, Part 2, The Environmental
Release of Genetically Altered Plant Material. Seed division, Agriculture
Canada, Ottawa, Ontario. pp. 20-26). This document describes the
philosophy of the current policy development and some of the specific
information to be submitted. It is clear that as the experience with
modified plants increases that the specifics of regulatory decisions will be
changing. Rather than describing the background material here, it is much
wiser to potential producers to contact Agriculture Canada to obtain up-to-
date information about the particular product in question. Like the United
States, progress in the genetic engineering of plants has been rapid and the
regulators have gained much empirical knowledge of the properties of
these modified plants.
Like genetically modified plants, modified micro-organisms are regulated
by Agriculture Canada, generally through the Pesticides Division.
Guidance was published in 1988 regarding field trials of micro-organisms,
but considerable experience in dealing with microbes has made a few
changes in the details of data submission. Direct interaction with the
regulatory bodies is strongly recommended at an early stage of planning for
field tests.
Agriculture Canada may be contacted at:
Pesticides Directorate
Agriculture Canada
PO Box
Ottawa, Canada KIA OC6
Telephone + 1 (613) 993--4544
Fax + 1 (613) 998-1312
Veterinary Biologics and Biotechnology

Agriculture Canada
810 Fallowfield Road
PO Box 11300 - Station H
Nepean, Canada
Telephone + 1 (613) 998-9320
3.4 Latin America and the Caribbean
While no strict international regulatory agreement has been ratified, a

common set of guidelines has been proposed to the governments of the
Latin American and Caribbean countries. Issued as "Guidelines for the

Release Into the Environment of Genetically Modified Organisms," by the
International Office of Epizootics, of the Organization of American States
(OAS) and the Inter-American Institute for Cooperation on Agriculture/
Canadian International Development Agency Project (I1CA/CIDA),
(I1CA Miscellaneous Publications Series, ISSN-0534-5391, PO Box 55-
2200 Coronado, Costa Rica), the goal was to strengthen the overall
development plans for agriculturally related biotechnology in the Americas.
These guidelines, published later in 1991, added to the earlier (1988)
endorsement of the NIH Guidelines for laboratory based research.
The context for this action was described in the prologue as follows:
No biotechnology regulations exist in Latin America and the Caribbean, with
the exception of a few research institutes that have established internal biosafety
assessment procedures for work with biotechnological techniques. This is not
only because of the small research effort currently being undertaken in the
region, but also because of the lack of political or public pressure to establish
these regulations. But with the rapid advent of commercial live products
obtained through biotechnology, it is urgent to establish in each country of the
region adequate mechanisms and norms to safeguard public health and the
environment from any foreseeable and significant risks. The guarantee of
standards equal to the ones applied in developed countries is an important
objective, so as to maintain the confidence of the scientists and the general
public in the new technologies. This is in the best interest also of companies and
research institutes which need clear guidelines for their work. The rapid access
to the latest technology by Latin American and Caribbean countries, essential
for maintaining and increasing the productivity and competitiveness of their
agriculture industry, will depend heavily on the existence of this regulatory
framework.
The policy and approaches towards biosafety in developed countries are not
necessarily the most adequate for developing countries. There is therefore a
need for adapting them to the local circumstances, on the basis of the experience
of the most advanced countries. This can be achieved best on a regional basis,
due to the lack of national expertise and resources, as the cooperative action of
the Inter American Institute for Cooperation on Agriculture, the Pan American
Sanitary Bureau, the Organization of American States, the International Office
of Epizootics and the US Department of Agriculture in this matter shows.
This description of the condition in the majority of the countries of the
Americas indicates the desire of each to develop biotechnology as a
commercially important source of new development, but the lack of full
understanding by the political leaders of what this technological revolution
really means. At the time of this writing, the 'guidelines' have not been
placed into statute in any of the affected countries, although serious
consideration of the issues has been approached. Commercial producers
with an interest in entering the market place in this region are best advised
to contact the I1CA office referenced above to obtain the guidelines, and
then to contact the specific regulatory or environment bureaus of the

potential target countries for detailed guidance. The regulatory environ-
ment in most Latin American and Caribbean countries is not well enough
established to be able to provide detailed guidance. This issue, like many
others, is captive to other issues of North-South tension in the Americas.
Product introductions, and advanced field trials are often welcomed, but
the decisions for approval are very much case-by-case.
4 The legal and regulatory framework for
biotechnology in Japan
T. SALUSBURY
4.1 An overview of the Japanese biotechnology industry
Biotechnology is now one of the three main pillars of Japan's advanced

technologies (the others being the electrotechnologies and new materials).
Since the introduction of biotechnology in 1973, Japan has made
remarkable progress in this field. New technologies such as gene
recombination, cell fusion and immobilized enzymes have broadened
Japan's fermentation-based industries. Pharmaceutical, chemical, food
and seedling companies launched aggressive research programmes to
develop new production technologies and to enter new business areas
(such as plant breeding, livestock and fish farming). Indeed, a biofever led
companies from many, very different sectors to enter the field with high
expectations (many of Japan's steel companies, for example).
Technologies such as genetic recombination and cell culture are now
basic tools in pharmaceutical research. These technologies are responsible
for the marketing of products such as erythropoietin, tissue plasminogen
activator, colony stimulating factor, urokinase, interferons, human growth
hormone, insulin and hepatitis B vaccine. In 1992, the Japanese market for
these drugs was estimated to be worth about Y100 billion per year. 1
The Japanese chemical industry was quick to adopt biotechnology,
although the actual impact in this sector has been much smaller than
expected. Biotechnology is now used in the production of fine chemicals
such as acrylamide and adipic acid, as well as anthocyanin and shikonin
pigments. The cosmetics industry is a major user of the technology, using it
to produce hyaluronic acid, cyclopentadecane and fragrances. Detergent
manufactures were quick to use proteases, lipases and cellulases in their
products. Japan's huge amino acid industry has established mass production
technologies for making most of the world's supplies of L-tryptophan, D-
alanine and L-Iysine. I
4.2 Government attitudes to biotechnology
The overall climate for biotechnology is a favourable one. Over 30 000

r-DNA experiments have been carried out without incident. By September
Table 4.1 Guidance on the regulation of r-DNA technology in Japan
Ministry/Agency Date Title
Monbusho 1979 Guidelines for r-DNA Experiments in Research Organisa-

tions such as Universities (revised 1991)
STA 1979 Guidelines for r-DNA Experiments (revised 1991)
MITI 1986 Guidelines for Industrial Application of r-DNA
Technology
MHW 1986 Guidelines for the manufacture of Drugs Using r-DNA
Technology
1992 Guidelines for Foods and Food Additives Produced by r-
DNA Techniques
MAFF 1989 Guidelines for the Application of r-DNA Organisms in
Agriculture, Forestry, Fisheries, the Food Industry and
Other Related Industries
MAFF 1992 Guidelines for Foods and Food Additives Produced by r-
DNA Techniques
Source: Japan Bioindustry Association, March 1992.
1992, 313 industrial-scale processes based on r-DNA technology had been

licensed.
Government and the biotechnology industry are of the firm opinion that,
based on the large volume of experimental data collected since 1974, r-
ONA technology presents no particular risks. In general, public opinions
of biotechnology are favourable. Although the regulatory situation may
seem complex, guidance is favoured over regulation. This guidance is
available from five government bodies for the experimental or industrial
work that falls into the appropriate area (Table 4.1).
The Japanese government has been successful in promoting bio-
technology in Japan. However, it has done this without massive subsidies
or strong legislation. The 1993 government budget for all forms of
biotechnology research was YllO billion, compared to private sector
spending of Y235 billion. In most cases, government money is available
only to groups of companies involved in very high-risk projects. More
important, government helps put together research associations of
companies and academics for the cooperative development of specific
technologies.
4.3 The government bodies involved in biotechnology
Table 4.2 shows the responsibilities for safety in biotechnology assumed by

Ministries and Agencies. Monbusho (the Ministry of Education, Science
and Culture), the Science and Technology Agency (ST A), the Ministry of
Health and Welfare (MHW), the Ministry of International Trade and
Industry (MITI) and the Ministry of Agriculture, Forestry and Fisheries
LEGAL AND REGULATORY FRAMEWORK IN JAPAN 59
Table 4.2 Who does what: the framework for the areas of jurisdiction in the regulation of r-
ONA technology in lapan
Research
Monbusho STA
Research work in national and private Research work in national institutes and
universities (with culture volumes of less private sector laboratories (with culture
than 20 litres) volumes of less than 20 litres)
Industrial applications
MAFF MAFF MAFF MHW MITI

Plants Microbes Microbes Microbes Microbes
Release Release Industrial Industrial Industrial
process process process
(Stimulated model environment Foods Diagnostics Enzymes
and open release) Fertilisers Drugs Amino acids
Feed additives Vaccines Fine chemicals
Animal drugs
Agrochemicals
Source: lapan Bioindustry Association, March 1992.
(MAFF) are the major ministries involved in implementing government

biotechnology policies. The Prime Minister's Council for Science and
Technology coordinates the respective administrative objects of each
body.
Monbusho and ST A are concerned with experiments at the laboratory
scale, while MHW, MIT!, and MAFF oversee industrial applications. A
number of support organisations, such as the Japan Human Science
Foundation (JHSF) and the Japan Bioindustry Association (JBA), help to
represent the views of industry to government. They also play an important
role in feeding back information from government to industry. Keidanren
(the Japan Federation of Economic Organizations) has called for the
elimination of overlapping research programmes and the unification of the
guidelines issued by the various government bodies. However, the existing
structure is unlikely to change in the near future.
4.3.1 Laboratory-scale experiments

For laboratory-scale experiments (which can include development work
with cultures of up to 20 I) a comprehensive set of guidelines are available
from Monbusho 2 and the STA? These guidelines follow the US National
Institutes of Health (NIH) Guidelines closely.
4.3.2 Monbusho and STA Guidelines

Both Monbusho 2 and STA 3 give detailed guidance in defining the basic
conditions required in establishing safeguards for r-DNA experiments and

set out management responsibilities and procedures. Guidance is also
given on storage, transport, training and health management issues.
Physical methods of containment are defined for small-scale experiments
(below 20 litres of culture volume) in four levels: PI (lowest), P2, P3 and
P4 (highest). There are about 100 universities with PI and P2 facilities and
a further 50 which can carry out P3 experiments. Guidance is also given on
the physical containment methods, facility design and experimental
protocols required for large-scale culture experiments with culture volumes
of over 20 litres. In this case, three levels of containment are used: LS-C
(lowest), LS-l and LS-2 (highest). Standard for Class I (lowest), Class II
and Class III (highest) safety cabinets and HEP A (high-efficiency,
particulate air) filters and test methods are also defined in detail.
In addition to physical containment methods, biological containment
systems are defined. These are host-vector systems which use hosts which
are only viable under special incubation conditions and vectors which can
only be transferred to specially prepared cells. The level of biological
containment for experiments with viruses is classified according to two
levels based on the host-vector system.
A Bl host-vector system consists of a host with low viability in natural
conditions. The vector must be dependent on the host and incapable of
transfer to other cells. Examples of Bl host-vector systems are EKI (based
on the bacterium Escherichia coli K12 and a plasmid which cannot
conjugate or transfer to other bacteria), SCI (a laboratory-maintained
strain of the yeast Saccharomyces cerevisiae as host and a plasmid or
minichromosome as vector) and BSI (the bacterium Bacillus subtilis
Marburg 168 as host and a plasmid). B2 host-vector systems consist of a
host with especially low viability in natural conditions and a vector which
depends on the host completely. The most common example is EK2 (a
defective strain of E. coli K12 and the well-characterised plasmid
pBR322).
Non-standard experiments which use microbes or cultured cells as hosts
require a separate application in advance and approval from either
Monbusho or the STA. This would include experiments on unidentified
species, species whose non-pathogenicity has not been confirmed or
cloning experiments on genes which code for proteins toxic to vertebrates.
However, the majority of laboratory-scale experiments use EKl, BSl, SCI
or EK2 as a host-vector system with DNA from plants and organisms on an
approved list. Thus no individual approvals are required. In these cases,
the laboratory need only inform the STA or Monbusho of the experiments
that are taking place. As Table 4.3 shows, only a small number of
laboratory-scale r-DNA experiments require an examination.
The guidelines for laboratory-scale experiments were relaxed in 1991,
first by Monbusho (January) then the STA (September). These relaxations
Table 4.3 Number of experiments using r-DNA technology in Japan,

broken down by ministry granting approval
Ministry/Agency Type Cases
STA Examination 1432

Notification only 10526
Monbusho Examination about 2000
Notification only 21061
Note: Data from March 1990.

Examination: A separate application has to be submitted to the STA or
Monbusho in advance for approval.
Notification only: Since the experiment is on an approved list, individual
examinations are not required. However, the organisation must still inform
the ST A or Monbusho.
Source: Japan Bioindustry Association, November 1992.
broadened the scope for experiments which do not require the approval of
either body.
4.4 Industrial-scale applications of r-DNA Technology
Three powerful ministries all playa part in the regulation of the industrial
uses of biotechnology (Table 4.2). MAFF issues guidance for processes
manufacturing agricultural chemicals, fertilisers, feed additives, veterinary
drugs and foods. It also sets guidelines on deliberate release and
recombinant plants. MHW is responsible for r-DNA applications in the
production of pharmaceuticals. Since it has responsibility for food safety, it
also has an interest in foods produced by recombinant technology. MITI
issues guidance for processes which use r-DNA technology to produce
enzymes and fine chemicals.
4.4.1 Industrial uses of r-DNA technology

Table 4.4 shows that 208 industrial processes use r-DNA technology.
These products include amino acids such as L-glutamate, L-Iysine, L-
tryptophan, L-threonine, L-phenylalanine and L-aspartate. Other products
are nucleic acids, vitamins, perfume bases, pigments, hyaluronic acid,
collagen and cyclodextrin. Enzymes such as heat-resistant proteases,
lipase, amylases, cellulase and protease are also produced.
MITI has revised guidance for industrial applications of r-DNA
technology 4 several times. These are based closely on guidance produced
by the Organisation of Economic Cooperation and Development (OECD). 5
This sets the safety evaluation and classification of GILSP (good industrial
large-scale practice), then Categories 1, 2 and 3.
Table 4.4 Number of industrial processes based on r-DNA technology

in Japan, broken down by ministry granting approval
Ministry Product Processes
MHW Medical diagnostics: 31

Drugs: 53
Vaccines: 8
92
MITT Fine chemicals: 124

Enzymes: 66
Amino acids: 15
Others: 3
208
MAFF Amino acids: 3

Veterinary drug: I
Application in a simulated
model environment:
Application in the
environment: 1
Fisheries industry: 1
Recombinant model mice: 6
18
Total: 313
Note: All but four of these industrial processes operate at GTLSP

(good industrial large-scale practice).
Source: Japan Bioindustry Association, September 1992.
4.4.2 Guidance for the use of r-DNA technology in the pharmaceutical

sector
Table 4.4 shows that 92 medical diagnostics, drugs and vaccines have been
licensed by the MHW which use r-DNA technology. These include human
insulin, interferons, hepatitis-B vaccine, tissue plasminogen activator and
erythropoietin.
MHW's approach to r-DNA-derived drugs has been to evaluate them
with the greatest caution. 6 Full sets of preclinical and clinical data are
required, even if the same ingredient of natural origin already has approval
elsewhere. However, as the technology matures, it is allowing some
flexibility into the system. Specific notification requirements are that
processes such as cell cloning and cell fusion should be validated during
manufacture. The structure, uniformity and purity of any new drug should
be determined and special attention must be paid to the possibility of
contaminants from the host system (such as polypeptides or endotoxins
from host systems). The properties of the host-vector system should be
described and all cell cultures be free of infectious viruses. Of course, all
cell cultures must be retrovirus-free. MHW has issued guidance on the
manufacture of drugs using r-DNA technolog/· 8 which follow the OEeD
guidelines closely.
4.5 Agricultural applications of r-DNA technology
As Table 4.4 shows, there have only been 18 agricultural applications of

r-DNA technology which have used MAFF's guidelines. Of these, three
are amino acid manufacturing processes using E. coli and one is for
production of interferon from silkworms for use as a veterinary drug.
No food product based on r-DNA technology has been commercialised
in Japan. However, cell fusion techniques have been used to produce
improved strains of yeast for the production of sake and wine. Soluble fibre
drinks are becoming popular, with manufacturers claiming that they
stimulate the growth of Bitidus bacteria in the lower intestine. These
drinks contain fructo-oligosaccharides produced in bioreactors. Processes
for the continuous production of high-fructose corn syrups using the
immobilised enzyme glucose isomerase are well established. A number of
research teams are attempting to develop technologies to produce wine,
beer, cyclo-dextrin, vinegar, soy sauce and malto-oligosaccharides in
bioreactors.
MAFF's guidelines'ol.lO aim to promote the safe development of agro-
industries by defining general principles for the appropriate use of r-DNA
organisms. The principle of these guidelines is that the safety of a
recombinant plant must be evaluated on the basis of experiments in a
simulated model environment. Experiments start in greenhouses and
progress to limited, small-scale field trials before any use in the outside
environment can be considered.
Table 4.5 shows the systems of tests needed before MAFF can grant
approval for a recombinant plant. The first two stages of tests in laboratory
and enclosed greenhouse take place under the supervision of the STA.
MAFF then supervises simulated environment testing in isolated small
fields. Only then can the final stage begin (testing in open fields). Once
Stage 4 has been completed successfully, commercial plant breeding and
commercial exploitation can begin.
By March 1922, about 100 research groups were at Stage I, working with
plant varieties such as rice, tomato and melon. Five groups were at Stage
II, working with virus-resistant petunia, melon and three varieties of rice.
Two of the groups working on recombinant rice were about to move to
Stage III. MAFF's National Institute of Agro-Environmental Science
(NIAES) had just moved its recombinant tomato project from Stage III to
Stage IV. This tomato contains a gene which expresses tobacco mosaic
Table 4.5 Flow chart of the tests needed for a recombinant plant
Stage Experiments Government

body
I. Closed test system Introduction of new characteristics STA

(laboratory or isolated into plant using r-DNA
green house) technology
II. Open test system Cultivation of recombinant plant STA

(greenhouse) to confirm genetic characteristics
III. Simulated environment Cultivation of recombinant plant MAFF

(isolated, small fields) to evaluate environmental effects
IV. Open Systems Unrestricted cultivation MAFF

(common fields) recombinant plant
Note: By March 1992, about 100 research groups were at Stage I, working with varieties of
rice, tomato and melon. Five groups were at Stage II, with virus-resistant petunia, melon and
three varieties of rice. MAFF's National Institute of Agro-Environmental Science (NIAES)
had just moved a recombinant tomato project from Stage III to Stage IV. This tomato
contains a gene which expresses tobacco mosaic virus (TMV) coat protein.
Source: MAFF, March 1992.
virus (TMV) coat protein. It is expected to be approved by MAFF and be

commercialised in 1995. However, MHW approval will be needed before
they can be used as foods.
4.6 Foods and food additives
MHW's Food Sanitation Investigation Council drew up guidance on foods

and food additives produced by r-DNA techniques in 1992. 8 These
guidelines stipulate that although there is "no scientific basis for saying that
biotechnology itself is dangerous", it is an advanced technology with a
short track record. It is thus necessary to draw up guidelines to ensure the
safety of foods and additives manufactured by this technology, in order to
prevent hazardous impurities from mixing with them.
The guidelines cover r-DNA technologies, cell fusion, tissue culture and
the use of bioreactors. Safety assessment guidelines require compliance
with manufacturing guidelines. Manufacturers and importers must obtain
safety assessment from MHW's Food Sanitation Investigation Council,
who will indicate whether individual products agree with the manufacturing
guideline by examining the necessary information. Safety assessment
guidelines cover the definitions and range of coverage of food and
additives which do not contain recombinant material and which are
identical (or can be considered identical) to the existing foods or additives.
The manufacturing process assessment is based on OECD guidelines for
GILSP. This includes the facilities and materials whose safety assessment
has been established by past experience. Where no safety assessment has
been, experimental tests for toxicity must be made. Products must be
assessment to see whether recombinant material is present and whether the
product could be considered to be identical to existing foods or additives.
For those products having no past safety assessment, tests for toxicity must
be carried out. All these steps await the new plant varieties which will be
approved by MAFF.
In September 1992, MHW announced a study to establish food safety
standards for marine organisms produced by biotechnology. About 20
types of fish and shellfish have been developed (but not yet marketed).
These include oysters, flatfish and salmon which are almost double their
normal size due to the use of techniques such as polyploidy.
4.7 Deliberate release of genetically-modified micro-organisms
Genetically-modified micro-organisms (GMMO) can only be used in

industrial processes under the appropriate containment conditions. These
are the OECD-based guidelines issued by MAFF, MHW and MITI. If
micro-organisms are to be released into the environment, an appropriate
safety evaluation must be carried out in advance under the supervision of
MAFF.9 Although various groups are working on genetically-modified
plants (see above), there have been no attempts to release or even to test
genetically-modified micro-organisms for agricultural or bioremediation
purposes. These developments will only be possible once public acceptance
has been obtained.
In 1991, the Environment Agency's Central Council for Environmental
Pollution Control produced a White Paper on protecting the environment
from the deliberate release of genetically-modified organisms.1O This
White Paper admitted that while there are no inherent risks in the
technology, administrative methods were needed to protect the environ-
ment from the deliberate release of genetically-modified organisms
(GMOs).
While deliberate release in the testing, research and development stages
should be controlled by flexible guidelines, the White Paper argued that
when genetically-modified organisms are marketed, they should be subject
to the appropriate product laws. The White Paper proposed a move from
guidance to regulation and implied that r-DNA technology could be
dangerous or harm the environment. Because of this, it thus attracted
intense opposition from MITI, MHW, MAFF and industrial bodies such as
the lBA. It is thus unlikely to be taken further and the present situation of
guidance from five separate government bodies will continue.
Acknowledgements
I would like to acknowledge the considerable contribution of my colleague,

Ms Yasuko Otsuka, the Embassy's expert on Japanese biotechnology. I am
also grateful to Dr Akihiko Mine of the Japan Bioindustry Association and
representatives of MITI, MHW, MAFF, STA and Monbusho for their
assistance.
The views expressed in this chapter are those of the author. They do not
necessarily reflect the policies of any UK government department.
References
1. Japan Chemical Week (1992). Japan Chemical Annual 1992, The Chemical Daily Co Ltd,
Tokyo (in English).
2. Monbusho. Guidelines for Recombinant DNA Experiments in Universities and Monbusho
Research Institutions, Monbusho, Tokyo, 1979 and 1991 (with minor revisions).
(Monbusho's own English translation.)
3. STA. Guidelines for Recombinant DNA Experiments, STA, Tokyo, 1979 and 1991 (with
minor revisions). (STA's own English translation.)
4. MITT. (1986). Guidelines for Industrial Application of Recombinant DNA Technology,
MITI, Tokyo. (MITT's own English translation.)
5. OECD. (1986). Recombinant DNA Safety Considerations. OECD, Paris.
6. Kawahara, A. (1990) Regulatory aspects of biotechnology in Japan, Drug Information
Journal, 24, 141-152 (in English).
7. MHW. (1986). Guidelines for the Manufacture of Drugs Using Recombinant DNA
Technology, MHW, Tokyo. (MHW's own English translation.)
8. MHW. (1992). Guidelines for Foods and Food Additives Produced by Recombinant DNA
Techniques, Chuo Hohki Shupan KK, Tokyo. (Alternate chapters in Japanese and
English.)
9. MAFF. (1989). Guidelines for the Application of r-DNA Organisms in Agriculture,
Forestry, Fisheries, the Food Industry and Other Related Industries (Japan), MAFF,
Tokyo. (MAFF's own English translation.)
10. Central Council for Environmental Pollution Control. (1991). Report of the Expert
Committee on Biotechnology: Basic Views on Environmental Protection for the Deliberate
Release of Genetically Modified Organisms into the Environment, Environment Agency,
Tokyo. (Environment Agency's own English translation.)
Note: Copies of the English language references cited can be obtained from the Department
of Trade and Industry Overseas Technical Information Service (OTIS). OTIS is administered
by the Production Engineering Research Association (PERA) at Melton Mowbray LE 13 OLX
(telephone 0664--501501).
5 Biotechnology and industrial microbiology
regulations in Russia and the former Soviet
republics
A. RIMMINGTON
5.1 Introduction
This chapter attempts to describe and analyse the regulations governing

industrial biosafety on the territory of the former USSR. Given the recent
dramatic political changes which have resulted in the dismantling of the old
monolithic Soviet state one might question the relevance of such a study to
the situation as it exists in this part of the world today. However, it should
be remembered that despite the ever-increasing momentum towards full
independence, the republics which constituted the USSR are likely, in the
absence of any alternatives, to continue to enforce compliance to Soviet
regulations governing industrial safety and other matters for some
considerable time to come.
Furthermore, the study of industrial biosafety in the former USSR
makes a fascinating case-study in its own right. For here was a country with
one of the most grandiose programmes for the development of industrial
biotechnology in the world, combined with a political system, which
allowed flagrant breaches of regulations governing labour safety and
environmental protection. It was a recipe for disaster. This was exactly
what happened.
The controversy surrounding the production of single cell protein (SCP)
in the former USSR is a very good example of the problems Soviet industry
confronted when it flouted the regulations governing industrial biosafety.
Initially, the Soviet SCP programme had appeared to be a great success.
Writing in New Scientist in June, 1985, I described how huge n-paraffin-
based SCP factories with capacities in excess of 100 000 tonnes per year
were operating at Angarsk, Kirishi, Kremenchug, Novopolotsk, Mozyr,
Svetlyi Yar, Syzran and Ufa. 1 Microbial protein production increased
dramatically from 16700 tonnes in 1960 to 1 799000 tonnes in 1988.
Biotechnology was poised to make a major contribution towards solving
the shortages in the nation's food supply.
By 1987 it had become apparent that SCP production and the
biotechnology industry in general were facing a major crisis. Reports of
environmental pollution at the giant SCP factory at Kirishi (near
St Petersburg) first appeared in the Soviet press in June 1987. Then in

March 1988 Martin Walker, writing in the British newspaper The
Guardian, described how the situation at Kirishi had developed into "the
worst ecological disaster since Chernobyl". 2
Subsequently the biotechnology industry came to assume the kind of
pariah status in the public consciousness in the USSR which had been
assigned to the nuclear industry for so many years in the West. Mass
demonstrations were reported all over the country at sites where
microbiological factories were under construction. Other plants were
forced to close or shut-down production temporarily due to massive public
pressure. It is virtually impossible to build a biotechnology facility
anywhere in Russia or the former Soviet republics today.3
It is against this background that in 1989 the USSR Ministry of Health
published a new set of guidelines governing safety of work with
recombinant DNA molecules. As section 5.2.1 shows, until this time most
laboratories appear to have been working in accordance with a set of
provisional rules issued in 1978. The controversy surrounding industrial
biotechnology in the USSR throughout the late 1980s will undoubtedly
have influenced the scientists who drafted the new set of regulations in
1989. The main innovation in these rules appears to be the establishment of
specific State regulatory agencies to enforce adherence at R&D facilities
and factories (the new regulations are discussed in sections 5.2.2 and
5.2.3).
Section 5.3 discusses the regulations governing safety in industrial
biotechnology as a whole with specific reference to labour safety and the
protection of the environment. Section 5.4 then attempts to examine the
past record of Soviet microbiological facilities vis-a-vis adherence to the
regulations set out above. It is demonstrated that, both with regard to
physical containment, and the safety of personnel working with micro-
organisms, State regulations, in industry at least, have been largely
ignored. An attempt is then made to draw conclusions as to what this may
tell us about the situation regarding safety in microbiological facilities
operating in Russia and the former Soviet republics today.
5.2 Regulations governing work with micro-organisms containing

recombinant DNA
5.2.1 History
The great controversy over recombinant DNA (r-DNA) research which
raged in the USA during the period 1973-1977 also spread to the Soviet
Union. Following the international conference which met at Asilomar to
draw up guidelines on such research, the head of the Soviet delegation,
REGULATIONS IN FORMER SOVIET REPUBLICS 69
Academician Baev, told Soviet scientists on his return to the USSR: "We
in the Soviet Union have no fear of the future, no worries that powerful
and blind forces will direct scientific research in genetic engineering along
an evil path contrary to the intentions and wishes of the broad population.
We are convinced that reason and goodwill will triumph - at least in our
socialist country.,,4 However, secretly the Soviet delegates had confided to
their Western colleagues that the Soviet reaction to the Asilomar
conference was one of anger to the bringing up of the issue of controls over
research. No doubt they feared a repetition of the excessive governmental
interference experienced during the Lysenko period.
Following Asilomar, Baev launched a public relations campaign aimed
at calming any fears concerning r-DNA research which might have been
aroused by the controversy in the USA. He argued that only in the
capitalist countries was the possibility of abuse real. 5 However, one of the
leading party idealogues, I.T. Frolov, rejected this view and talked of "a
new stage in the development of science" and held that the issues raised
would be "inevitably included in a sharp philosophical ideological
struggle".6 This must have sounded very ominous indeed to those
biotechnologists who had lived through the Lysenko era.
Recognising the need for self-regulation, scientists working in the
Russian 'bio-city' of Pushchino issued a set of provisional guidelines,
"Interim safety rules for work with recombinant DNA" (Vremennye
pravila bezopasnosti rabot s rekombinantnymi DNA) governing work with
r-DNA in 1978. 7 The level of containment applicable to work on specific
host-vector systems was determined by the Commission for Recombinant
DNA which came under the control of the Interdepartmental Scientific-
Technical Council on Problems of Molecular Biology and Molecular
Genetics. 8
The guidelines issued in 1978 were applicable only to work carried out in
laboratories and presupposed that the volume of culture fluid would not
exceed 10 litres. 9
According to Zilinskas, writing in 1984, no administrative agency was set
up to enforce the Soviet guidelines. 10 He also reported that, in practice,
rather than follow the domestic guidelines, researchers in several prominent
institutes followed the guidelines formulated by the US National Institutes
of Health. Moreover, it appeared that each laboratory had its own set of
regulations governing the conditions of research and the professional
behaviour of researchers. 11
Zilinskas also reports that Soviet scientists who studied or worked in the
West claimed that the rules they followed at home were similar to those
found in Western laboratories. In support of this claim is the fact that there
would appear to have been subsequent modifications to the initial Soviet r-
DNA guidelines that reflect the general downgrading of regulations in
other nations. 12 It would appear, then, that in the late 1970s and early
1980s Soviet researchers, at least at the laboratory level, were following

much the same safety procedures as their colleagues in the West. However,
the lack of a State administrative agency to enforce the guidelines in
laboratories nationwide would appear to suggest that Soviet researchers at
this time were better able to circumvent these rules than their Western
counterparts if and when the need arose.
5.2.2 The current guidelines

In recent times a growing number of research institutes have been
equipped with containment units for work with potentially pathogenic
micro-organisms containing r-DNA. Thus, in 1990 it was reported that
eight institutes in the USSR possessed facilities with maximum containment
units (i.e. those designated as P4 or Biosafety Level 4 - BL4) and at least
another 12 possessed P3 containment units (see Table 5.1).13
The rapid growth in the numbers of such laboratories may explain why a
new, official set of rules governing work in research centres which possess
these containment units were drawn up by an Interdepartmental Commission
of the USSR Academy of Sciences chaired by Academician Baev. These
were published by the USSR Ministry of Health ("Anti-epidemic rules
governing the safety of work with recombinant DNA molecules" -
Sanitarno-protivoepidemicheskie pravila "bezopasnost' raboty s rekombin-
antnymi molekulami DNK") and came into force on 1 June 1989. 14
Under these guidelines the classification of laboratories in Russia and
the former Soviet republics appears to follow the US pattern and
designates four levels of physical containment: Fl or First level - minimal
containment (Biosafety Level 1 or PI); F2 or Second level - low
containment (Biosafety Level 2 or P2); F3 or Third level - medium
containment (Biosafety Level 3 or P3); F4 or Fourth level - high
containment (Biosafety Level 4 or P4). 15
Table 5.1 Facilities with containment units in Russia and the former Soviet republics
Name of facility Location Subordination Protection

level
All-Union Scientific Obolensk, Formerly to USSR P4

Research Institute of Scrpukhov raion, Ministry of the Medical
Applied Microbiology Moscow oblast', Industry
Russia
All-Union Scientific Vladimir, Vladimir State Agro-Industrial P3
Research Institute of oblast', Russia Committee
Foot and Mouth
Disease
All-Union Scientific Kol'tsovo, Formerly to USSR P4
Research Institute of Novosibirsk oblast', Ministry of the Medical
Molecular Biology Russia Industry
Name of facility Location Subordination Protection

level
Belorussian Scientific Ulitsa Nogina 3, Belorussian Ministry of P4

Research Institute of Minsk, Belorussia Health
Epidemiology and
Microbiology
D.1. Ivanovskii Institute Ulitsa Gamalei 16, Academy of Medical P4
of Virology Moscow 123098, Sciences
Russia
Georgian Antiplague Tbilisi, Georgia USSR Ministry of P3
Station Health
Institute of Poliomyelitis Kievskoe shosse, 27 Academy of Medical P4
and Viral Encephalitis kilometr, Moscow, Sciences
Russia
M.M. Shemyakin Ulitsa Miklukho- Academy of Sciences P3
Institute of Bio-organic Maklaya, 16/10,
Chemistry Moscow 117871,
Russia
"Mikrob" All-Union Saratov, Saratov Ministry of Health P3
Scientific Research ab/ast', Russia
Antiplague Institute
N.F. Gamaleya Institute Ulitsa Gamalei 18, Academy of Medical P4
of Epidemiology and Moscow 123098, Sciences
Microbiology Russia
Rostov-on-Don Rostov-on-Don, Ministry of Health P3
Scientific Research Rostov ablast',
Antiplague Institute Russia
Scientific Research Vologograd, Ministry of Health P3
Antiplague Institute Volgograd oblast',
Russia
Scientific Research Irkutsk, Irkutsk Ministry of Health P4
Antiplague Institute of ablast', Russia
Siberia and the Far
East
Scientific Research Kirov, Kirov ob/ast', USSR Ministry of P3
Institute of Micro- Russia Defence
biology
Scientific Research Zagorsk, Moscow USSR Ministry of P3
Institute of Micro- ab/ast', Russia Defence
biology'S Division of
Virology
Scientific Research Aral'sk, Kzyl'-Orda USSR Ministry of P3
Institute of Micro- oblast', Kazakhstan Defence
biology'S Scientific
Field-Testing
Laboratory
Scientific Research Lesoparkovdya 4, St USSR Ministry of P3
Institute of Military Petersburg 195043, Defence
Medicine Russia
Scientific Research Prospekt Formerly to USSR P3
Institute of Vaccines Timiryazeva 7, Ministry of Medical
and Sera Tomsk, Russia Industry
Scientific Research Ulitsa Dubrovskaya Ministry of Health P4
I nstitute of Viral 15, Moscow,
Preparations Russia
Veterinarian Scientific Kazan State Agro-Industrial P3
Research Institute Committee
For experiments which require medium containment, the regulations

require that only purpose-built laboratories be used and that, in addition,
these should possess protective equipment. The laboratory must also be
isolated from the rest of the building by air-tight interlocking doors. An
autoclave for decontamination of laboratory wastes should be situated
inside the laboratory. The surfaces of walIs, floors, work-tops and ceilings
have to be purified and decontaminated. There should be a ventilation
system that creates a negative pressure in the laboratory. All exhaust air
should pass through independent air flues after preliminary purification
through filters. Both air flues and filters are subject to annual inspection. 16
Entrance to F3 (P3) laboratories is restricted to those listed in
the research programme sent for approval to the Local Regulatory
Commission. 17
Workers in F3 laboratories must work in accordance with the regulations
set out in GOST 12.1.008-76 (Sector Labour Safety Standards. Biological
safety. General requirements) published in Moscow in 1976 (see section
5.3).18 But, as will be discussed in section 5.4, there appears to have been
scant regard for rules governing the safety of personnel working with
micro-organisms in the past.
F4 (P4) laboratories are used for experiments with novel and genetically-
altered micro-organisms which are hazardous to humans, animals and
plants. Such laboratories must be sited away from the main research centre
or be completely isolated within the building in which they are located. 19
F4 laboratories are required to have solid walls, floors and ceilings in
which all openings (air ducts, electrical sockets, etc.) are hermetically
sealed in order to ensure physical isolation and to prevent the escape of
biomaterials from the laboratory. They are also required to have a separate
ventilation system which maintains a negative air pressure. The laboratory
must also have equipment for decontaminating the air before it is expelled
into the atmosphere. 20
Entrance to F4 laboratories is subject to the same restrictions applied to
F3 laboratories (see above).21
Biohazard warning signs must be placed on all external and internal
doors leading into laboratory rooms where experiments are being carried
out. 22
Experiments which require F4 physical containment are only conducted
in cabinets with high-level protection, i.e. a totally enclosed, ventilated,
air-tight cabinet where operations can be carried out through attached
gloves when necessary.23
The assignment of physical containment levels are also set out in the new
rules. Experiments and procedures carried out with micro-organisms and
their DNA (bacteria, rickettsia, fungi and viruses) which are human or
animal Class 1, 2, and 3 pathogens must employ physical and biological
containment levels in accordance with Tables 5.2, 5.3, and 5.4.
Table 5.2 Level of containment required where both vectors and host cells
are Class 1, 2 or 3 pathogens
Class of Physical containment Biological containment

pathogen level (F) level (BI)
1 4 2
2 3 2
3 2 1
Table 5.3 Level of containment required where vector is a Class 1, 2 or 3

pathogen and the host cell is non-pathogenic

1 3 2
2 2 2
3 2 1
Table 5.4 Level of containment required where vector and host cell are non-
pathogenic, and DNA is from organisms which are Class 1,2 or 3 pathogens

1 4 2
2 3 2
3 2 1
Safety measures for recombinant systems, containing toxin genes from

vertebrates, are dependent upon the degree of toxicity and other
properties of these agents. In the case of genes cloned in Escherichia coli
K-12, the following safety measures would be employed:
(a) Where the LDso of a toxin is between 100 nanograms and 1000
nanograms, a combination of F2 plus BI2 (Biological containment
Level 2) or F3 plus BIl (Biological containment Levell) is required
(e.g. abrin, epsilon toxin of Clostridium perfringens).
(b) Where the LDso of a toxin is between 1 microgram and 100
micrograms, a combination of F1 plus BIl is required (e.g. alpha-toxin
and beta-toxin of Staphylococcus aureus, ricin, exotoxin A of
Pseudomonas aeruginosa, Bordetella pertussis toxin, Yersinia pestis
mouse toxin, neurotoxins from snake venom).
(c) Cholera toxin, thermo-labile toxins from E. coli and Klebsiella, and
thermo-stable toxins from E. coli and Yersinia enterocolitica require F1
plus BI1.
In the case of other host organisms containing toxin genes, safety measures
are reportedly being established by the Central Regulatory Commissions
based on the recommendations of the USSR Academy of Sciences'
Commission on Recombinant DNA.
Experiments (processes) using pathogenic Class 1 animal or human
viruses in tissue culture require physical containment level F4. Those using
viruses belonging to Class 2 require F3. Class 3 viruses require F2. For any
other viruses, containment level F1 is sufficient.
For the following recombinant systems, F1 containment is sufficient:
(a) recombinant systems with plant cells in culture;
(b) recombinant systems with animal cells and vectors originating from
sources which are non-pathogenic for humans and animals;
(c) laboratory experiments with transgenic animals and plants.
The 1989 guidelines set the maximum volume of culture fluid for a
laboratory at 50 litres.
The recent increase in the number of scientific and commercial contacts
with Western institutions and companies will probably result in the
elimination of any differences that exist between Western and Soviet
regulations and procedures governing microbiological containment. A
good example of just such an instance is the recent purchase by the M.M.
Shemyakin Institute of Bio-organic Chemistry in Moscow of a P3
containment laboratory from the UK company Port on International. As
part of the contract with Porton, Russian scientists visited the UK to
receive on-site training in the procedures governing the use of their new
laboratory.24
5.2.3 Regulatory authorities

The main innovation in the new guidelines appears to have been the
establishment of regulatory agencies to enforce adherence at research
facilities and factories across the USSR (see Figure 5.1). Since the break-
up of the Soviet Union, virtually all Soviet organizations have simply been
abolished or taken under control by the Russian government. Therefore it
seems likely that the regulatory agencies established by the 1989 guidelines
will have their jurisdiction limited to the Russian Federation. However,
institutes in other former Soviet republics are likely to continue to have
their work approved by these agencies until their governments establish
their own regulatory bodies.
Under the new guidelines, the Central Regulatory Commission
(Tsentral'naya rezhimnaya komissiya) of the USSR Ministry of Health is
the highest organ of control with respect to all research and industrial
production carried out in the medical and microbiological industries
involving the use of r-DNA. Similarly, the Central Regulatory Commission
§~
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of Gosagroprom is the highest organ of control with respect to work in the

livestock, veterinary and horticultural fields. 2s
The USSR Academy of Sciences' Interdepartmental Commission on r-
DNA works as a consultative body and passes on its findings to the Central
Regulatory Commissions. It is appointed by the Presidium of the USSR
Academy of Sciences in agreement with the departments. 26
Control at the institute and factory level is to be enforced by the
Local Commissions on Recombinant DNA (Mestnaya komissiya po
rekombinantnym DNK). These are appointed by the director of an
institute or factory and their function is to register work being carried out
which involves the use of r-DNA and to enforce adherence to the new
guidelines. The directors of institutes or factories (or their representatives)
are responsible for the observance of these rules in establishments which
come under their jurisdiction?7
The deliberate release into the environment of organisms containing r-
DNA requires the authorization of the Central Regulatory Commission of
the USSR Ministry of Health and/or USSR Gosagroprom and a resolution
of the USSR Academy of Sciences' Interdepartmental Commission on
Recombinant DNA?S
Recombinant systems which are considered high-risk are only permitted
by a special decision of the Central Regulatory Commission based on the
findings of the USSR Academy of Sciences' Interdepartmental Commission
on Recombinant DNA. Among the systems assigned to this category are:
(a) Recombinant DNA, containing functionally active toxin genes from
vertebrates with an LDso of less than 100 nanograms per kg of body
weight (for example, botulinin, tetanus and diphtheria toxins).
(b) Host-vector cloning systems relating to group 1 pathogenicity.
(c) The transfer of a gene or genes to group 1 pathogen host cells of group
1 pathogenicity.
(d) The transfer of a gene or genes to human cells.
(e) The deliberate introduction into the environment of any organisms
containing r-DNA (see above).
The current guidelines also lay down procedures governing the use of
micro-organisms containing r-DNA in industry. Authorisation for the use
of such organisms in pilot-scale production has to be sought from the
relevant Local Regulatory Commission. The use of micro-organisms
containing r-DNA for industrial production requires the authorisation of
the USSR Ministry of Health's Central Regulatory Commission. 29
5.3 Regulations governing labour safety in biotechnology research institutes

and the microbiological industry
5.3.1 Labour safety standards

A whole series of labour safety standards and other regulations for use
within biotechnology research institutes and factories belonging to the
microbiological industry (some of these are outlined in Table 5.5) have
been introduced since the late 1970s.
The safety standards include sections on microbiological analyses of the
air and the gathering of dust samples to "ascertain the air's content of
proteins or other components of microbiological synthesis products".
Procedures for measuring the degree of contamination of equipment and
walI surfaces inside a factory and details of special clothing to be worn by
workers were also outlined. 30
The aim of these standards was to improve analytical methods placing
the emphasis on the use of standardised procedures to determine
concentrations of micro-organisms within production facilities. It was
believed that such standardisation would "raise the level of health
inspection and make it possible to compare and analyse working conditions
in the (biotechnology) sector as a whole and at individual enterprises that
use either different or identical technologies and process instrumenta-
tion".31
The application of these standards used to be mandatory for alI
production facilities within the Soviet microbiological industry. Recent
legislation appears to have abolished the system of State standards.
However, it is probably still the case that most factories operating in Russia
and the former Soviet republics are working in accordance with the
regulations set out in Table 5.5.
Table 5.5 Regulations governing work in the microbiological industry. 1976--1989
Regulation Applications
GOST 12.1.008-76 Sector Labour Safety Standards. Biological safety. General

requirements (Moscow, 1976)
OST 59.01.003.01-80 Sector Labour Safety Standards. Health and hygiene evaluation
of enterprises of the microbiological industry. Air-quality
analysis
OST 59.01.003-47-85 Sector Labour Safety Standards. Air quality of the workplace.
Acceptable levels of yeast cells. Methods of ascertaining
concentrations
OST 59.01.003.48-85 Sector Labour Safety Standards. Air Quality of the Workplace.
Acceptable levels of microorganisms used for controlling
agricultural pests. Methods of monitoring
5.3.2 Rules governing the release of micro-organisms into the workplace

In order to prevent the exposure of workers to dangerous levels of
substances in the air of industrial enterprises, lists have been drawn up
which define maximum permissible concentrations (Predel'no Dopustimye
Kontsentratsii - abbreviated to PDK) for potentially harmful substances.
PDKs have been defined for a whole range of products produced in
microbiological factories. Table 5.6 for example shows the maximum
permissible concentrations of microbially synthesised amino acids in the
workplace, which were approved by the USSR Ministry of Health in 1985.
These range from 2 to 10 mg/m 3 of air. 32 Clearly such guidelines have
immense practical importance in Russia and the former Soviet republics
which together, for example, produce around 34000 tonnes, or 25% of
total world output, of lysine. 33
The USSR was also the world's leading producer of single cell protein
(output stood at 1 715 000 tonnes in 198934) and here again, as shown in
Table 5.6 Maximum permissible concentrations

(PDKs) of microbially synthesised amino acids in
the air of the workplace
Amino acid Maximum permissible

concentration, mg/m 3
Alanine 5
Arginine 10
Asparagine 10
Cysteine 2
Cystine 2
Glycine 5
Glutamic acid 10
Histidine 2
Hydroxyproline 5
Isoleucine 5
Leucine 5
Lysine 5
Methionine 5
Phenylalanine 5
Proline 5
Serine 5
Threonine 2
Tryptophan 2
Tyrosine 5
Valine 5
These PDKs were approved by the FSU Ministry of

Health in 1985 (Sidorov, K.K. (1987). Predel'no
dopustimye kontsentratsii vrednykh veshchestv v
vozdukhe rabochei zony, utverzhdennye
minzdravom SSSR v 1985 g., pp. 51-53 Gigiena
truda i professional'nye zabolevaniya, Number 2,
February.)
Table 5.7 Maximum permissible concentrations of single cell protein (SCP) in air of work-
place and the atmosphere
Type of single cell protein PDK, mglm 3
In the air of In atmospheric

the workplace air
Paprin (derived 0.1 0.001

from petroleum
n-paraffins)
Eprin (derived 0.3 0.004
from ethanol)
Gaprin (derived 0.3
from natural gas)
Source: Dalin, M.V., Gukasyan, I.A., Spivak, S.M., Fish, N.G., Kravtsov, E.G. and
Ermolaev, A.V. (1991). Podkhody k razrabotke diagnosticheskikh allergenov dlya
obsledovaniya rabochykh, zanyatykh v proizvodstve mikrobnykh biomass kormovogo
naznacheniya, i naseleniya selitebnykh zan v regionakh raspolozheniya mikrobiologicheskikh
proizvodstv (Obzor), Gigiena truda i professional'nye zabolevaniya (5), 31-33.
Table 5.8 Maximum permissible concentrations (PDKs) of Candida yeast

strains approved by the FSU Ministry of Health for use in the production of
single cell protein
Yeast Strain Maximum permissible

concentration in air of
workplace, cells/m 3
C. rnaltosa BSB 569 1000

C. rugosa BSB 925 300
C. tropicalis BSB 928 300
C. scotti KS-2 1000
Source: see Table 5.7.
Tables 5.7 and 5.8, PDKs have been assigned to a variety of micro-
organisms and products associated with the manufacture of SCPo A
number of other products produced via microbial synthesis have also been
assigned such PDKs.
5.3.3 Regulations governing the release of micro-organisms into the

environment
In order to prevent the release of excessive quantities of dangerous
substances into the environment, lists of PDKs which define maximum
permissible concentrations for potentially harmful substances in the
atmosphere are also drawn up.
Table 5.7 shows the PDKs for various types of single cell protein in the
atmosphere. Thus, the atmospheric PDK for SCP derived from n-paraffins
(paprin) is 1 fAg per m 3 of air, while the atmospheric PO K for SCP derived
from ethanol is 4 fAg per m 3 of air. 35
A potential hazard resulting from the operation of n-paraffin-based SCP
factories (and indeed other biotechnology plants) is the discharge of live
micro-organisms into local water supplies. Sargeant and Evans postulated
that the release of such carefully selected and very specialised industrial
micro-organisms into the natural environment "would be tantamount to
abandoning an over-bred and pampered lap-dog to its fate in Siberia". 36
To lend weight to their argument, they cite a case some years ago where
"the contents of a very large antibiotic fermenter (presumably somewhere
in the West) were inadvertently discharged into the sea, [and] no trace of
the organism could be detected next day, despite a very thorough,
widespread and anxious search". 37
Nevertheless, there is a possibility that micro-organisms released from
factories in effluent could remain viable and find their way into drinking
water, thus threatening the health of the local population. In response to
such concerns, provisional safety levels have been established for micro-
biological pollutants in Russian industrial effluent. Thus, PDKs have
recently been determined for single cell protein, dendrobatsillin (a Bacillus
thuringiensis-based pesticide) and turingin (another microbial pesticide) in
reservoir water. 38
5.4 Adherence to regulations governing the containment and safe use of

micro-organisms
No information is available on the adherence of Soviet research facilities to

the regulations on work with potentially hazardous micro-organisms.
However, reports do suggest that until recently rules governing the
disposal of radioactive material from Soviet research institutes were often
ignored. A similar situation may have existed with regard to work with r-
ONA in microbiological containment facilities. Certainly, the prevailing
"work culture" in the USSR - poor labour discipline and very high rates of
alcoholism and absenteeism - did not encourage strict adherence to state
regulations.
Much more evidence is available concerning adherence to regulations
governing the use of micro-organisms in industry in the USSR. For such
had been the failure of Soviet factories to follow guidelines concerning the
release of microbiological material that a major environmental crisis was
created. 39 Indeed, so strong has become the public opposition aroused by
these releases, that it is today virtually impossible to build a biotechnology
plant anywhere within Russia or the former Soviet republics (see

introduction).40
5.4.1 The environment

Sargeant and Evans, writing in 1979 on the hazards involved in the
industrial use of micro-organisms, point out that the risk to local residents
outside a typical biotechnology plant is unlikely to be any greater than that
posed to those living close to a mechanically aerated sewerage works with
its potential population of human pathogens. They do go on, however, to
say that "the failure of a fermenter's effluent air filter could allow the
escape of micro-organisms into the atmosphere and produce allergic
response in some individuals".41 In the West such failures are reported to
be very rare; this would contaminate the bioreactor. Sargeant and Evans
estimate that during SCP production, the bioreactor must be productive
for about 350 days in the year or the process will prove uneconomical.
Thus, much effort is put into making the system reliable. However, in
Soviet microbiological factories, such failures were comparatively common
in the late 1970s and throughout the 1980s. This put local populations at
serious risk of infection or sensitisation.
Table 5.9 lists some of the factories which have been shut down
following discharges of material into the environment. Looking at just two
of these plants illustrates the apparent general failure of Soviet industry to
follow guidelines concerning release of microbiological material. In the
case of the single cell protein (SCP) factory at Angarsk (in the Irkutsk
oblast', of the Russian Federation), it was reported in the press that the
amount of microbial protein released daily into the atmosphere exceeded
the maximum permitted concentration (determined by the State regulatory
authorities) by a factor of 1.3. 42 In the period from 18 to 26 October 1988 it
Table 5.9 Facilities closed temporarily or permanently following release of microbiological

material
Location Product Capacity
Angarsk, Irkutsk oblast', Single cell protein Unknown

Russia
Kirishi, Leningrad oblast', Single cell protein 100 000 tonnes per annum
Russia
Kremenchug, Poltava oblast", Single cell protein 120000 tonnes per annum
Ukraine
Kstovo, Gorky oblast', Russia Single cell protein 70 000 tonnes per annum
Syzran, Kuibyshev oblast", Single cell protein 120000 tonnes per annum
Russia
Tomsk, Tomsk oblast', Russia Single cell protein 80 000 tonnes per annum
Ufa, Bashkir ASSR, Russia Single cell protein 100 000 tonnes per annum
Zaporozh'e, Zaporozh'e Single cell protein 8000 tonnes per annum
oblast', Ukraine
is probable that the concentration of this material in the city's atmosphere

was even higher, for it provoked an 'epidemic' of uncontrollable coughing
in local inhabitants. During this period, 1008 people were treated in the
Angarsk Department of Medicine and of these 111 were hospitalised. Two
of the patients were considered to be seriously ill. Eighty-two per cent of
those treated said they were suffering from chronic bronchitis, asthma and
other lung complaints. 43
A similar state of affairs appears to have existed at the SCP factory based
at Kremenchug in the Poltava ablast' of the Ukrainian SSR. Here it was
reported that the level of microbiological material in the environment
around the plant exceeded the maximum permissible concentration by a
factor of 30. 44 Once again, there was a sharp increase in the number of
cases of bronchial asthma, allergic dermatitis and bronchitis among the
local population. According to one specialist, child morbidity in the region
of Kremenchug increased by a factor of 1.5-2 and the morbidity of the
adult population doubled as a result of these discharges. 45
One way in which managers at SCP factories are dealing with discharges
of viable micro-organisms and SCP dust is by planting 'green-belts' around
their enterprises. One study of the effectiveness of these screens showed
that they trap a significant quantity of yeast cells and retain protein dust in
their leaves during the spring and summer months. Specialists are quick to
point out that these green-belts are not the solution to the pollution
problem and that both for reasons of health and safety of local populations
and economics, discharges must be prevented in the SCP factories
themselves. 46
It was not just SCP factories which flouted regulations concerning the
release of micro-organisms into the environment in the USSR. Reports
indicate that large quantities of micro-organisms were also released from
Soviet enzyme factories. A study of plants employing surface cultivation
methods recorded levels as high as 100 000 viable fungal spores per m 3 in
the air outside one unnamed facility. 47
A detailed study was also carried out of an enzyme factory in Tukums
(Latvia) in order to determine the level of atmospheric discharges at the
facility and the effect these had on the local population. In this case a high
concentration of Aspergillus awamari spores (used in the production of the
preparation avamorin) was found immediately outside the factory and
concentrations decreased rapidly at greater distances. No spores were
found more than 300-500 m from the factory. Researchers found that
changes in the immunological reactivity and sensitivity of the population
living adjacent to the factory reflected the distance of their homes from the
facility. Some 66% of those unfortunate enough to be living immediately
adjacent to the Tukums plant exhibited allergies when examined by
researchers. At greater distances this figure decreased until at 500 m from
the factory only some 29.6% of the population exhibited allergies. 48
Similar problems have been reported at factories which use submerged

fermentation methods to produce enzyme preparations. At one factory
using Aspergillus niger and Aspergillus batatae, 20-200 fungal spores per
m3 were found 20-30 m from the plant. No spores were found at distances
in excess of 150-180 m. Despite this fact, Soviet experts pointed out that
the micro-organisms used in the submerged fermentation process were
powerful allergens and therefore discharges released in close proximity to
population centres should be purified. 49
This suggests that until very recently, regulations governing the release
of microbiological material from factories in the USSR have been ignored.
The local Party and ministry authorities have then combined to prevent
any considerations of local health hazards caused by such discharges from
interfering with production. The State regulatory authorities (in this case
the State Inspectorate for Protecting the Atmospheric Air, set up in
19835°), which had the power to close factories and prosecute factory
managers for failing to control pollution, proved to be singularly
ineffective in preventing these discharges. It has only been with the advent
of glasnost (free speech) that local protestors have been able to publicise
the situation and bring it to the attention of the top policy-makers.
5.4.2. Waste water

Breaches of regulations concerning the release of micro-organisms into
industrial effluent may also have occurred in Soviet microbiological
factories. However, studies carried out by Soviet scientists appear to
indicate that this presented no serious hazard to local populations.
For example, workers from the USSR Academy of Medical Sciences'
A.N. Sysina General and Communal Hygiene Institute and the Ufa
Institute of Hygiene and Professional Illness made a detailed study of
discharges of waste water from the Ufa n-paraffin-based SCP factory. They
found between 107 and 108 viable Candida guilliermondii cells per litre of
waste water from this plant. A study was then made of the waste water
after it had passed through the local purification works. The first part of
the purification process included passage through sand traps, preliminary
settling tanks, aeration tanks and secondary settling tanks. This halved the
levels of C. guilliermondii. This was followed by repeated biological
purifications and subsequent chlorination of the water. At this stage, after
passage through the complete purification process only a few yeast cells
were detected and none of these were related to C. guilliermondiiY Thus,
provided adequate provision is made for purification, the release of viable
micro-organisms in the waste water of Soviet n-paraffin-based SCP plants
would appear to pose no threat to the water supplies of adjacent towns and
cities.
5.4.3 Industrial personnel

Soviet industry appears to have displayed a complete disregard to
regulations concerning the safety of personnel employed in microbiological
production facilities. A report in March 1988, for example, described how
workers engaged in SCP production at factories based in Angarsk, Ufa,
Nizhnii Novgorod and Kirishi were suffering from a whole range of serious
work-related diseases including fungal infections, diseases of the lungs and
skin, immune system deficiencies, and complications during pregnancy. 52
This state of affairs is perhaps not surprising when one considers that in
one Soviet n-paraffin-based SCP factory studies showed that some of the
workforce were exposed to dense aerosols of up to 436 000 cells per m3 in
their work place. 53 This would appear to exceed the limits set by the USSR
Ministry of Health by a factor of 400 (see section 5.3.2). The escape of such
large quantities of micro-organisms into the work environment at this
(unnamed) plant was ascribed to lack of containment in equipment, the
flow of microbiological material through open chutes and the intake of
contaminated air through the ventilation systems. 54
A similar situation was found to exist in factories producing SCP from
timber and agricultural wastes with levels as high as 364 000 cells per m3
being recorded in the work place. K.1. Kal'chenko (in 1972) and R.M.
Kollo (in 1975) found that the release of such large quantities of micro-
organisms were leading to work-related sensitisation and immunological
shifts among workers in these factories. 55
The dangers for pregnant women working in Soviet single cell protein
factories were revealed in a study carried out by A. V. Litovskaya and N. V.
Mokeeva. They found an increased rate of genital infection (similar to
thrush) caused by micro-organisms (Candida) used in the production
process. A depressed immunological response was also observed in
pregnant workers. They recommended that women should have no contact
with industrial micro-organisms during the period of their pregnancy. 56 It
is, however, difficult to foresee how these recommendations could be
enforced, given the fact that existing Soviet legislation obliging enterprises
to relocate pregnant women away from hazardous environments has had
very limited success.
A major occupational hazard for workers in the microbiological industry
appears to be exposure to dust during the final stages of the manufacturing
process (drying, packing and loading). Thus, for example, during the
loading of waggons with SCP in one particular factory, the concentration of
protein dust in the air reached average levels of 38 mglm 3 .57 This is nearly
100 times greater than the recommended PDK for the level of microbial
dust in factories producing SCP (0.4 mglm 3 ).58 One of the most effective
ways of reducing this source of pollution would be to produce SCP in a
granulated rather than powdered form. 59 However, dust is likely to remain
a major problem for workers in this branch of the microbiological industry,

since the production of granulated SCP was only expected to form 60% of
total output by 1990. 60
Workers from other branches of the Soviet bio-industry have also been
exposed to excessive concentrations of microbiological material. For
example, in the 1970s a detailed study of a facility at Tukums, producing
the enzyme preparations A vamorin and Orizin (based correspondingly on
A. awamori and A. oryzae) , was carried out. Even after improvements had
been implemented to reduce pollution, a level of 5000-27 000 fungal
spores per m 3 were found in some parts of the work place. Little wonder
that a significant number of the workers examined were found to display
changes in their immunological response. They also developed complaints
such as itching, allergic rashes, dermatitis, asthma and vasomotor
rhinitis. 61
As is the case with SCP factories, Soviet enzyme plants are also reported
to have released large quantities of dust. Studies carried out in the 1970s
revealed levels to be as high as 18-175 mg/m 3 in some factories. 62 By the
late 1980s, this situation was reported to have improved but dust released
during the final stages of manufacture remained a major occupational
hazard. The concentration of dust during the manufacture of a pectinase
enzyme preparation in one factory under study was found to be between
2.1 and 5.9 mg/m 3 compared with a PDK of 4 mg/m 3 . PDKs for other
enzymes being produced at this factory were also exceeded and this
resulted in a number of skin, laryngeal, gastrointestinal and broncho-
pulmonary complaints amongst workers. 63
A number of reports suggest that personnel employed in factories
producing microbial pesticides in the USSR were also exposed to large
quantities of microbiological material. In 1985 the Riga Medical Institute's
Occupational Diseases Department made an in-depth study of the health
of workers in two pilot plants belonging to the All-Union Scientific-
Research Institute of Microbial Pesticides which were producing pesticides
based on B. thuringiensis. 64 During medical examinations it was revealed
that 50% of the workers suffered from allergic reactions: itchy skin, rashes,
swollen face etc. These allergic reactions were only found in 15% of the
administrative and supervisory staff. Approximately 40% of workers
examined were also found to suffer from acute and chronic conjunctivitis,
as well as frequent illnesses of the upper respiratory tract. Allergic
dermatitis was found in 27% of workers. Clinical manifestations of allergy
were found to increase in proportion to the duration of service at the
factories. 65
Exposure to microbiological material was reported to result from
discontinuities in the production process, the large number of manual
operations (especially in the latter stages of production) and the lack of
efficient ventilation in production areas. These technical deficiencies
reportedly resulted in a situation where the bacteria count in the work

place of the pilot plants exceeded 40 000 per m 3 of air. This level of
contamination would presumably have been in clear violation of the State-
defined safety norms.
A similar situation has been reported at an unnamed pilot plant (belong-
ing to a Soviet biochemical factory) producing viral insecticides (Virin-
ENSh and Virin-EKS). High concentrations of baculoviral polyhedrons
were also found in this installation, ranging from 50 to 50000 per m 3 . This
led to a situation where 56.2% of those workers tested had antibodies to
baculoviruses and a high degree of sensitisation to viral protein. A lack of
safety precautions in the viral production unit at this facility resulted in
more than 70% of workers employed there contracting diseases such as
rhinitis, pharyngitis, bronchitis, conjunctivitis, and dermatitis, as well as
chronic gastritis, colitis, and liver disorders. 66
All the available evidence points to the fact that Soviet industry had
been largely flouting regulations relating to the release of microbiological
material and the safety of workers handling this material. The new
emphasis laid upon strict adherence to legal norms which was an integral
component of the Gorbachev reforms may help eliminate such abuses by
factory managements in the future. However, it is worth pointing out that
the dire state of the economy in Russia and the former Soviet republics,
together with the transfer of enterprises to self-financing, places additional
pressure on the authorities to flout environmental regulations in the
interests of maintaining industrial output.
5.5 Conclusions
The combination of the obsolete equipment of the vast majority of Soviet

microbiological factories and political pressure to maintain high levels of
output resulted in the creation of serious hazards both for industrial
personnel and the environment. The dramatic changes in the political
climate which we have seen over the past few years mean that we can
expect to see a heightened concern on the part of the authorities, both with
regard to occupational hazards and environmental protection. However, a
vast injection of capital investment is required to modernise microbiological
factories - indeed the industry of Russia and the former Soviet republics as
a whole - and thus reduce the risk of major environmental disasters which
can have a global impact. Unlike the situation prevailing in the West, there
is also a poor tradition of strict adherence to rigorous safety procedures
upon which workers and scientists can draw. All this may point to the need
for support from Western countries both in terms of financial investment
and training of industrial personnel.
Addendum in Proof
New information on biotechnology legislation in the Russian Federation has

been made available to the author since completion of this chapter. As early as
1991, the Academy of Science's Institute of State and Law in conjunction with
the State Committee of Science and Technology drew up a draft law on 'The
Organisation of Work and Provision of Safety in the Field of Genetic
Engineering'. However, recent political turmoil meant that this legislation was
never enacted by the Russian Parliament.
In another attempt to kick-start the legislative process, the Ministry of
Science and Technology Policy has formed a National Committee for the
Elaboration of Legislation for Work with Genetically Modified Organisms.
Headed by one of Russia's leading biotechnology researchers, Prof.
Konstantin Skryabin (based at the Centre of Bioengineering in Moscow), the
committee includes representatives from various organisations, including the
Ministry of Agriculture and Ministry of Ecology and Natural Resources.
They have consulted widely both with international experts and domestic
interest groups such as the Russian Orthodox Church. The Committee hoped
to have a draft of a law ready for consideration by the new Russian Parliament
in March 1994.
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50. Tsaturov, Y. (1987). Keeping the air clean in the cities, Advances of Science and
Technology, (20), 127, 15, July.
51. Nemyrya, V. I. and Vlodavets, V. V. ibid.
52. Razin, S. (1988). "Bomba" pochtal'ona Vasil'eva, Komsomol'skaya pravda, Number 61
(19161), 15 March.
56. Litovskaya, A.V., Mokeeva, N.V., Ispol'zovanie immunomikologicheskikh pokazatelei
pri reshenii voprosov okhrany zdorov'ya beremennykh rabotnits proizvodstv mikro-
biologichcskogo sinteza, Gigiena truda i professional'nye zabolevaniya, Number 7.
57. Nemyrya, V.l. and Vlodavets, V.V. ibid.
58. Sosedova, L.M., Kal'chenko, K.I., Khomutova, V.A. and Muratova, N.M. (1991).
Obosnovanie predel'no dopustimoi kontsentratsii kormovykh drozhzhei, poluchcnnykh
pri utilizatsii otkhodov tsellyulozno-bumazhnogo proizvodstva, Gigiena truda i profes-
siona/'nye zabolevaniya (5), 29-31.
59. Simaev, Yu. (1987). Sud'ba tsennogo produkta, Sotsialisticheskaya industriya, Number 6
(4097),8 January.
60. Rychkov, R. (1984). Mikrobiologicheskaya promyshlennost' v sisteme APK, Ekonomika
sel'skogo khozyaistva, Number 4.
61. Nemyrya, V.l. and Vlodavets, V.V. ibid.
63. Gigienicheskaya otsenka faktorov proizvodstvennoi sredy na zavodakh mikrobio-
logicheskogo sinteza fermentnykh preparatov, Gigiena truda i professional'nye
zabolevaniya, Number 4, 1989.
64. Ivanova, LA. (1985). Characteristics of the effects of microbiological means of plant
protection on an organism, in lzvestiya akademii nauk latviiskoi SSR, Number 6, June,
pp. 76-81, translated in JPRS Report, Science and Technology, USSR: Life Sciences.
65. Ivanova, LA. ibid.
66. Vasil'eva, V.L. et al. (1984). Effect of baculoviruses on health of workers involved in
production of viral insecticides, Vrachebnoe delo (5), 116-119, May. Translanted in JPRS
Report, Science and Technology, USSR: Life Sciences.
6 Physical aspects of the uncontrolled release of
material in biotechnology operations
K.P. NORRIS
6.1 Introduction
The main hazards arising from the uncontrolled release of micro-organisms
and their products in biotechnology operations are from inhalation,
ingestion and by skin contact. Ingestion and skin contact can be avoided by
suitable operating procedures, but inhalation is more difficult to control
since the release of even a small quantity of material which becomes
airborne inside a plant or laboratory enables personnel in and near the
plant to come into contact with a significant amount of material simply by
their continuing to breath the contaminated air. In an eight-hour shift the
average worker and those in the immediate vicinity of the operation inhale
a minimum of 10 m 3 of air and if the average concentration of airborne
material is only 1 part in 100 million a worker can easily inhale a total dose
of 0.1 mg during a single shift. This is the sort of dose which is used
routinely for the delivery of therapeutic medicines by aerosol dispersal and
doses of this order of magnitude or lower are used in aerosol vaccination.
The aerosol route is potentially more dangerous than ingestion because the
effective aerosol dose of most organisms is about 1 million times less than
that by ingestion. I
To prevent this exposure, either the process has to be contained or strict
control has to be exercised over all processes involving the handling of
liquids and powders in bulk. This control must extend also to the process of
waste disposal since this can generate a hazard, possibly at a site remote
from the plant where the active material is produced.
The significance of the airborne route of entry has long been recognised
by those working with pathogenic organisms and the incidence of infection
in laboratory workers handling pathogenic organisms provides some
evidence of the dangers involved in biotechnology operations. Chatigny
and Clinger2 state "that every species of pathogenic micro-organism
studied in the laboratory has at some time or another caused infection of
operators". Wedum and Kruse 3 suggest as a working rule that for the most
infective organisms between one and ten bacteria or viral units will
produce infection. They also say "that in some cases the dose to produce
symptoms in man is less than that in laboratory animals". They state that
PHYSICAL ASPECTS OF UNCONTROLLED RELEASE 91
for many diseases of humans sub-infective doses inhaled daily are

neutralised by the body defences but no evidence is presented to
substantiate this proposition.
The importance of the aerosol route was emphasised by Sargeant and
Evans,4 who stated that if an organism is introduced into a host by a
portal, such as the lung, which is different from that in nature then the
form of the disease which develops may be different from the normal
disease. They described how Francisella tularensis, if ingested, produces a
typhoidal disease, by infection through the skin it produces a bubonic form
and a pulmonary form when produced by an aerosol capable of being
retained in the lung. The form of the disease therefore depends upon the
portal of entry and thus upon the particle size of the challenge. If the
organism is infectious the initiation of disease depends upon the dose being
large enough to overcome the natural immunity of the individual and upon
the state of health of the individual. Large doses will overcome immunity
acquired by vaccination.
Wedum, in a review of the handling of infectious agents,S outlined the
procedures to reduce the hazard but concluded that the safe handling of
infectious agents depended more on the attitude of the laboratory director
and his or her senior assistants than on equipment. He stressed the
importance of education, engineering and enforcement to protect the
experiment, the scientist and the administrator.
The size of an aerosol particle is of paramount importance in
determining the hazard which arises from its release since the size of the
particle determines not only the length of time it remains airborne, but also
the site of deposition in the respiratory tract if it is inhaled, the survival and
infectivity of micro-organisms contained in it and the ease with which it can
be removed from the air by filtration and other means.
Particles of dried biological material in the form of a dust behave
differently from their microscopic size and, along with fibrous materials,
constitute a special hazard because of the ease with which they can
penetrate into the lung.
In considering the risk from exposure to aerosols, the major problem is
the difficulty of identifying the causal relationship between a size fraction
of the variable airborne load to which workers and those living close to a
plant releasing an aerosol are exposed over a long period of time and the
severity of the symptoms in those individuals who are affected. The
problem is compounded because of the absence of aerosol sampling
equipment capable of monitoring the environment over a period long
enough to establish any causal relationship,6 by the considerable delay (6
to 18 months or longer) which can occur before a person exposed to a
highly variable load of material which is not infective either becomes
sensitive to the organism or produces clinical symptoms, and by the
evidence that bacterial samplers are not so efficient as animals in collecting
fine bacterial aerosols. It is not surprising therefore that Muir, 7 in Airborne

Allergens in Clinical Aspects of Inhaled Particles, says "the industrial
causes of asthma are numerous; there is however no information concern-
ing the particle size of the allergens or their concentration in most cases".
When measures are taken to control the dust or aerosol content of the
environment it is not uncommon for other hazards to assume importance,
as Craft8 discovered when investigating workers involved in the production
of quinabarbitone. After efforts to reduce the dust content of the
atmosphere had been successful the level of the drug in their blood
continued at a high level due to skin intake.
6.2 The generation of aerosols
Almost every operation involving the manipulation of microbial suspen-

sions, solutions or suspensions of material or of finely divided powders
generates an aerosol. Operations which are known to generate an aerosol
have been extensively reviewed by Chatigny,9 Darlow lO and Dimmick and
Akers. 10
Pipetting can release up to 10 000 droplets of a diameter of 1-10 !lm. The
expulsion of liquid from nozzles is used routinely in devices such as the
Collison spray12 and in two fluid atomisers as a means of generating fine
aerosol clouds.
The vibration or twanging of a platinum loop used for the inoculation of
a culture of organisms can also generate an aerosol and this technique is
used to advantage in the vibrating reed aerosol generator which is capable
of generating a monodisperse aerosol.
Harper13 examined the leakage from centrifuges used in clinical
laboratories and he found that 27% of the sealed containers (buckets or
rotors) failed to contain aerosols when a test suspension containing
1 X 1010 viable spores of Bacillus subtilis per ml was centrifuged. The
principle of using centrifugal force to produce a monodisperse aerosol is
applied to advantage in the spinning top aerosol generator. 14
Any process such as stirring or bubbling results in the formation of a
thread or film of liquid which subsequently breaks down into small droplets
which then evaporate to form an aerosol. The physics of aerosol generation
was reviewed by Green and Lane 15 and by Hidy and Brock. 16 According to
the former authors the energy required to dissipate 1 ml of water per
second ranges from 2 X 106 calories for 1 !lm drops to 13 calories for 20 !lm
drops. Thus whilst a large energy input is required to generate an aerosol
composed of small droplets, only a moderate energy input will produce a
coarser aerosol. Other principles employed to generate aerosols are
ultrasonics 15 and electrical atomisation. 17
Biological material is normally encountered in an aqueous medium and
once a droplet of biological material is released into the atmosphere the

water in the droplet rapidly comes into equilibrium with the water vapour
in the atmosphere. The greater the radius of curvature on the surface of the
droplet (i.e. the smaller the droplet) the more rapidly it comes into
equilibrium. Hinds l8 produced a graph which shows that a 15 flm droplet of
water will evaporate completely in 300 ms at 20°C and 50% relative
humidity; a 10 flm droplet takes 150 ms and one at 4 flm takes less than
40 ms to evaporate. A 15 flm diameter droplet of a bacterial suspension
containing 2 X 10 10 bacteria per ml (approx 5% by weight of dissolved and
particulate solids) would therefore evaporate in less than half a second to a
volume of 1I20th of its original volume or to a diameter of 15 ..;- (100/5t 3 or
5.5 flm diameter. Aerosols produced by shearing such a suspension tend to
be heterogenous in size so droplets of all sizes lose water until their
equilibrium diameter is (112.7) that of their original size.
A characteristic of a heterodisperse cloud which is often used to define
the particle size of the cloud is the Mass Medium Diameter (MMD). This is
the size for which half the weight of the cloud is contained in particles
greater than that size and half the weight is contained in particles less than
that size. Whilst the MMD conveys some information about the particle
size distribution it fails to give any information about the number of
particles of a given size in the aerosol. Heterodisperse aerosols tend to
consist of a large number of small particles, constituting only a small part of
the mass, and a very small number of larger particles, which constitute a
large part of the mass. Optical and microscopical methods of determining
particle size distribution produce a number distribution. Estimations of the
mass of material in an aerosol based upon such methods can be grossly
incorrect unless great care is taken to sample a representative volume of air
which includes the largest particles present in the aerosol.
Dimmick l9 introduced the valuable concept of the 'spray factor' to
quantify the magnitude of the aerosol generated in one minute by a
particular operation and the concept was developed in a further paper. 20
The spray factor is defined as the number of viable organisms released into
the air per minute divided by the number of viable microbes per millilitre
of the material being handled. The factor has the units mllmin and derived
units are given in Table 6.1. A major deficiency of the spray factor is that it
gives no indication of the particle size of the aerosol generated.
COX 21 gives an example of the use of the Spray Factor. Suppose a
bacterial suspension at 4 X 10 10 bacteria per ml is to be sonicated for
5 minutes in a room of volume 6 X 104 litres, what concentration would be
established in the room? From Table 6.1 the spray factor is 1 X 10-4. The
source strength is 4 X 1010 so that the number of bacteria generated per
minute is 4 X 1010 X 1 X 10-4 = 4 X 106 bacteria per minute. During
5 minutes' sonication the total number of bacteria dispersed would be 5 X 4
X 106 = 2 X 10 7 bacteria. At equilibrium the aerial concentration would be
Table 6.1 Examples of spray factor during aerosol generation
Operation Spray factor
Blender, lid off 6 X 10'

Sonic homogeniser, maximum aeration 1 X 10 4
Sonic homogeniser, minimum aeration 5 X 10 7
Pipetting vigorously 1 X 10-4
Vortex mixer overflow 8 X 10-"
Drop spilled on zonal rotor 2 X 10- 6
Single drop of liquid dropped 1 metre 2 X 10-"
2 X 107 -;- 6 X 104 = 3.3 X 102 bacteria per litre of air, so an unprotected
operator breathing at a rate of 20 litres of air per minute would inhale
approximately 66 000 bacteria per minute.
The ability of a specific component of a bio-reactor to generate an
aerosol was evaluated by Cameron et al. 22 They operated a reactor inside a
cabinet and then carried out operations such as taking a sample of the
contents of the reactor and measured the aerosol which was created by the
operation. Provided the operator technique was correct, no aerosol was
generated but with poor technique it was possible to generate an aerosol
with a peak concentration of 29 organisms per litre. Unfortunately the
spray factor was not calculated.
Accidents involving fermenters were studied by Ashcroft and Pomeroy. 23
They simulated the breakdown of the bacteriological filter, failure in the
anti-foam system, failure in the culture transfer pipework and explosive
breakage of the fermenter. The most dangerous accident in terms of
generating a fine aerosol was failure of the anti-foam system since this
allowed the suspension to bubble through the filter, generating small
droplets of suspension which dried-down rapidly to form particles, many of
which were less than 5 Ilm in diameter. The next most dangerous accident
was the complete disruption of the fermenter. The spray factor measured
by Ashcroft was 0.005. For a reactor containing 10 15 cells an accident in
which the reactor explodes may release as many as 5 X 1010 bacteria. The
other accidents simulated by Ashcroft produced much less aerosol but they
led to the gross contamination of surfaces which required disinfection and
cleansing to ensure that any material dried on the surface was not
subsequently dispersed as an aerosol.
Kenny and Sabel24 provided valuable particle size data on aerosols
created during laboratory procedures and simulated accidents. In their
experience a large part of the material was dispersed as an aerosol of
particle size less than 5 Ilm in diameter.
Processes in which cells are disrupted and specific components are
removed are also prone to produce aerosols since they involve the
application of energy to liquids. In the history of laboratory infections
these devices and centrifuges have caused a large percentage of the
fatalities and illnesses which have resulted. Rotary vacuum filters are
another commonly used cell separation method which can produce
microbial aerosols.
The final processing and packaging of biotechnological products is
perhaps the most dangerous phase of the manufacturing process due to the
presence of the active product in high concentration. Active products are
usually protected against environmental contamination, including the
operator, by placing the filling station under a particulate-free stream of
air. This airflow, whilst maintaining the filling station under aseptic
conditions, can cause the operator to be subject to a constant supply of
aerosol generated during the filling operation. Benbough has pointed out
that operations ensuring the sterility of the product may not be adequate to
protect the worker operating the planes and this particularly applies to
spray-dried materials which are difficult to handle without generating an
aerosol.
The problem of aerosol generation is not confined to the immediate
operations involved in producing the organism or product since waste
water or slurry left after the extraction of the active component can
continue to constitute a hazard. In the USSR biotechnology plants were
closed because they failed to solve the problem of waste disposal. 26 (See
also chapter 5.) Parker et al.27 in the USA studied a case in which waste
water from processing vegetables was allowed to settle in a tank.
Adventitious organisms multiplied in the water before it was sprayed onto
cultivated grass or forests as a fertiliser. An extensive airborne load of
organisms was found at 1 km and even at a distance of 10 km downwind as
many as 127 viable particles/m3 were recovered. Wastewater treatment was
further studied by Fannin et al. 28 who proposed the use of coliphages and
coliform organisms as indicators of the hazard from animal viral contamina-
tion. Teltsch et al. 29 isolated salmonella species and Brenner et al. 30 animal
viruses from waste water sites.
Adams et al. 31 demonstrated the presence of a considerable number of
coliform organisms up to 0.8 miles downwind of a sewage plant. Data given
in this paper enables the spray factor for a sewage works to be calculated as
being 1 X 10- 1°. As some plants handle 25 million gallons a day and the
slurry contains up to 107 organisms per ml the opportunity for the release
of a large numbers of organisms is clear. Spendlove extended the study to
consider the release of bacteria from industrial, agricultural and municipal
operations. 32
6.3 Persistence of aerosols in a closed space
An aerosol particle suspended in a parcel of still air is acted upon by gravity

and when the rate of fall is such that the gravitational forces acting on the
Table 6.2 Unit density particles
Diameter (flm) Terminal velocity (cm/s)
0.01 6.7 X 10- 0

0.3 4.2 X )0-4
1.0 3.5 X 10-3
3.0 2.9 X 10-2
6.0 1.1 X 10- 1
10 3.1 X 10 1
30 2.7 X 10°
100 24.8 x 10°
particle are equal to the viscous frictional forces of the surrounding air the
particle falls at its terminal velocity. Hinds 18 gives a table of terminal
velocities (Table 6.2).
In still air, particles with a diameter greater than 6 /lm will settle out
rapidly but particles below this size are readily kept in suspension by the
smallest air currents.
Particles which sediment onto a surface no longer constitute a direct
inhalation hazard but they can easily be re-aerosolised and so constitute a
secondary hazard. The force necessary to remove a small particle from a
surface was studied by Hidy and Brock.16 Adhesion depends upon many
factors but in general the smaller the particle the greater the adhesion
force. Particles adhering to a surface can be removed by mechanical
disturbance or by aerodynamic displacement. Particles leave a horizontal
surface first by beginning to slide or roll along it until their speed is
sufficient for small deformities on the surface or on the particle to cause
them to jump. This enables the particle to enter the turbulent air near the
surface and to become an aerosol. On a wetted surface a capillary film can
build up between the particle and the surface and this can increase the
adhesive force holding the particle to the surface. For this reason Darlow33
recommended that all surfaces in microbiological areas should be swabbed
regularly with cloths soaked in an appropriate disinfectant to reduce
biological contamination of the surfaces and to prevent re-aerosolisation of
dried material.
Problems associated with surface contamination were reviewed in
Surface Contamination. 34 Keruluk et ai., who were contributors to the
book, provided estimates of the airborne concentration arising from
known contamination densities on the floor. They concluded that the air
filtration systems in clear rooms considerably reduce the microbial
contamination in the air over that present in non-environmental areas but
the amount of microbial surface contamination, particularly at the bench
level and on the floors, was in many instances as high as in non-
environmental areas.
Ventilation theory provides a simple means of estimating the conse-

quences of the release of material inside a building. If a building of volume
V m 3 is ventilated with a volume of v m 3 of fresh air per second, the
ventilation rate or the number of air changes which occur in one hour in the
space (N) = 3600vlV. The ventilation rate for typical structures is given
below.
N h- I
Houses and churches 0.2-3.0
Hospitals and offices 0.5-3.0
Schools 2.0---6.0
Factories 3.0---60
The ventilation rate depends on several factors:

(a) The rate at which fresh air is supplied to the space.
(b) The size of openings.
(c) The external wind speed and direction.
(d) The temperature gradient between the air inside the space and the
outside.
For a given supply of air the ventilation rate depends mainly on the size of
openings, but a change of external wind speed from 5 to 15 mph can
increase the ventilation rate four-fold. A strong temperature gradient has a
much smaller effect.
Dimmick indicated how the calculation on the spray factor could be
extended to room ventilation and the proximity of the operator to the
release. To allow for ventilation a rough rule of thumb calculation is to
reduce the estimated dose by one third (i.e. multiply the value by 0.67) for
every 10 changes of air per hour ventilation, provided that the exposure
time is greater than 3 minutes. To allow for proximity to the source,
whether in a ventilated room or not, then during the first 3 minutes'
exposure a person within 1 metre of the source would effectively be in a
volume of 103 litre rather than the 6 X 104 in the example given above. The
aerial concentration would then be 2 X 107 -:- 103 = 2 X 104 bacteria per
litre, and the dose would be 2 X 104 X 10 X 3 X 0.3 = 1.8 X 105 bacteria.
The final factor of 0.3 is the efficiency of retention of the aerosol in the
lung (see section 6.5). COX 21 states that to attempt to refine these
calculations further is unrealistic but they are a useful way of quantifying
the hazard. Because there are so many factors which influence the survival
and infectivity of bacteria Cox recommends that procedures for handling
bacteria are based upon the assumption that the 1Dso is one bacterium and
measures are taken to reduce the exposure to that level.
Air purification processes can either remove the aerosol or inactivate the
material composing it. An aerosol particle may be removed from the air by
settling, inertial separation, filtering, scrubbing or by electrostatic precipita-
tion, and it may be inactivated by heat, radiation or by chemical treatment.
It is important to realise that whilst inactivation renders the material

incapable of further growth or division it does not necessarily prevent it
from acting as an allergen. The aim in cleaning the air must therefore be
very clear when a particular method is selected for a particular operation.
6.4 Persistence of aerosols in the atmosphere
Once the biological particles of an aerosol enter the atmosphere they are
carried along by the wind and dispersed by the action of turbulent
diffusion. In the vertical direction turbulence mixes the cloud in an ever-
increasing thickness until the turbulent boundary layer near the earth is
uniformly filled. The boundary layer is of variable depth, being typically
some tens to hundreds of metres deep at night and from hundreds to one or
two thousand metres during the day. The depth depends upon the amount
of radiation (sunlight and cloud cover) available to maintain the turbulence
and the dynamic energy (wind speed) of the air flow. The turbulence is
related to what is called the stability of the atmosphere and the
meteorologist Pasquill defined six categories of atmospheric stability.
These range from A, extremely unstable such as on a hot sunny day with
low wind speed through B, moderately unstable; C, slightly unstable; D,
neutral such as on overcast conditions day or night; E, slighty stable to F,
moderately stable such as on a cold clear night when there is no wind. 35 For
these categories of stability Pasquill provided graphs of the cloud
dimensions as a function of distance downwind and these enable the
concentration to be calculated for any release of material. Models based
upon this work have been evaluated and refined so that the effect of a
release at any height, over any terrain, or at any temperature can be
calculated with considerable confidence. 36--42
An accident which gives rise to an instantaneous release generates a
cloud known as a 'puff'. As a puff is carried downwind it expands in width,
length and height and the concentration of the cloud decreases with
increasing distance downwind. The length of time to which a person in the
path of the cloud is exposed increases with distance downwind and is
inversely proportional to the wind speed. As a result the concentration of
particles in a puff is inversely proportional to the cube of the distance
downwind but the dosage, defined as the integral of the concentration X
time of exposure is inversely proportional to the square of the distance
downwind and inversely proportional to the wind speed. A continuous
release such as from an exhaust stack gives rise to a plume of material in
which the concentration is proportional to the square of the distance
downwind.
A simple example indicates that if a reactor with a spray factor of 0.005 ;
and containing 1015 cells suffered an accident on a night when the wind was
2 mls then persons living 1 km downwind from the reactor would inhale
about 800 cells. If the cells were pathogens and only 10% of them were
viable after airborne travel the consequences would still be serious at that
distance. At distances closer to the plant doses will be large enough to raise
antibodies against the bacteria and possibly cause other side effects.
The way in which natural micro-organisms which might contaminate
industrial processes are released into the atmosphere, transported and lost
from the atmosphere has been extensively reviewed by Gregory.43 He
shows that micro-organisms can readily be transported for considerable
distances and that the airborne spread of organisms has played an
important part in the distribution of species and of plant and animal
pathogens in nature.
6.5 Retention, clearance and absorption in the respiratory tract
The respiratory tract of man consists of many structures but they can be
divided into three main parts:
1. The nasophaharynx and mouth.
2. The air passages of the larynx, trachea and large bronchii, by which air
is conducted to and from the lung.
3. The gaseous exchange area of the lung consisting of the bronchi and
alveoli.
The nasopharynx and trachea provides a good filtration system against
particles of diameter greater than 20 ~m since particles of this size tend to
be impacted in the nasal passages. The area is lined with ciliated epithilium
which transfers deposited material upward, and according to Druett 44 is
cleared with a half-time of minutes.
Particles smaller than 20 ~m penetrate into the bronchus and lungs and
the larger particles are retained in the bronchii, whereas particles 0.5 to 3.0
~m in diameter are deposited by impaction in the alveolar region of the
lung. 18 Dimmick and Akers 11 suggest that an average lung retention factor
of 0.3 can be assumed for particles in the size range 1-5 ~m and this was the
factor used earlier in the calculation involving the spray factor. The lung is
cleared of foreign matters by phagocytic and ciliary actions with a half-time
of 24 hours. 44 Elimination via the lymph tract and bloodstream accounts
for one-third of the material deposited, while the remaining two-thirds is
voided via the gastrointestinal tract. Some micro-organisms can resist these
clearance mechanisms and initiate disease through lesions of the ciliated
epithelium.
Particles of unit density and a diameter of 0.5 ~m are deposited relatively
inefficiently in the lung but particles below this size become trapped by
diffusion processes.
Particles composed of extremely hygroscopic material may take up water

as they travel through the respiratory tract and increase in size and thus be
deposited higher in the tract than their size in dry air may suggest.
Most dusts and freeze-dried material are of irregular shape and may be
aggregated into loose clumps. The behaviour of such dusts when airborne
depends entirely upon the mobility or settling velocity of the particles and
not on their apparent microscopic size. A clump of dust may appear to be
15 ~m in diameter, but if its structure is loose it may only sediment with the
same speed as a 7 ~m diameter sphere. When it is inhaled into the lung it
will be deposited in the same manner as if it were a 7 ~m sphere.
Fibres also behave in an anomalous way since the terminal velocity of a
fibre 15 is virtually independent of its length but is approximately equal to
that of a unit density sphere whose diameter is three times the diameter of
the fibre. Such fibres, because of their tendency to align themselves with
the long axis parallel to the airflow, are able to penetrate the alveoli of the
lung and many are trapped at the bifurcations of the small airways. Fibrous
material therefore constitutes a special hazard.
6.6 The biological behaviour of airborne particles
The factors which influence the survival of an organism in an aerosol are

numerous and they include:
• Stability of the organism.
• Particle size.
• Temperature.
• Relative humidity.
• Oxygen.
• Sunlight.
• Protective agents.
6.6.1 The stability of the organism

The stability of the material in suspension before it becomes airborne has a
profound effect upon its subsequent behaviour in the airborne state, yet in
much of the literature concerned with the aerobiology of micro-organisms
little attention is paid to this fact despite the recognition in the early 1950s
that organisms recovered from the stationary phase of growth survived in
the airborne state very much better than cells harvested in the early
logarithmic phase. This work was consolidated by Dark and Callow,45
who, with the advantage of a chemostat, were able to show that changes in
the method of culture and medium can greatly affect the aerosol stability of
cells. They concluded that 'mature' cells of low metabolic activity produced
in batch culture survive best in the aerosol.
Other examples of the importance of the physiological state of the

organism were provided by Hambleton et al. 46 who compared cells grown
in batch culture with those grown in continuous culture using the same
growth medium. They were able to demonstrate that the toxicity of the
cells was quite different. Similarly, Pearson and Elwood,47 who grew cells
in continuous culture, were able to change the culture from being non-toxic
to highly toxic by changing either the growth rate of the culture or the
growth limiting factor in the growth medium.
6.6.2 Particle size

The first indication that the particle size of an aerosol had a significant
effect upon the biological behaviour of the material contained in the
aerosol came from the work of May and Druett 48 who constructed a
vertical wind tunnel into which particles of controlled size could be sprayed
into a dynamic airstream. By its use they were able to show that a 1 !-tm
diameter droplet of Bacillus anthracis was more effective in creating
infection in guinea pigs than in a 12 !-tm particle. Later they reported that a
1 !-tm diameter droplet of Brucella suis was 800 times more effective than a
12 !-tm diameter droplet. Similar results were obtained with F. tularensis.
Later, May and Druett developed the technique whereby bacteria
contained in particles of known size and attached to microthreads could be
exposed to the open air. This lead to the discovery that survival of micro-
organisms in free air is particle size dependent. Bacteria contained in large
particles survive better than those contained in small particles. The reason
being that in the open air the bacteria are subject to an important
environmental factor called the open air factor (OAF).49 OAF probably
occurs in concentrations of parts per hundred millions and no analytical
method is available for estimating it, but it inactivates bacteria, viruses and
phages particularly those contained in small particles (1 !-tm in diameter).
Those contained in larger particles are less affected. Survival in the open
air is therefore particle size dependent but in closed buildings OAF appears
to exercise little effect on the survival of, airborne micro-organisms. 50
Despite the presence of OAF there are many records of bacteria
surviving airborne travel and creating infection at a distance from the
source. Outbreaks of Legionnaires' disease have shown that airborne micro-
organisms can be carried long distances into the ventilation systems of
other buildings and cause infection. There is also evidence that foot and
mouth virus caused infection after many miles of airborne travel. 5 !
6.6.3 Relative humidity and temperature

On being exposed to the environment, micro-organisms lose water
molecules that are replaced during host infection or transfer to some other
aqueous medium such as occurs in most air sampling procedures. These

movements of solvent water are dependent on the relative humidity and
temperature of the air surrounding the aerosol particle and they influence
the way in which the organisms contained in the particle survive.
With bacteria Cox suggests that low survival at high relative humidity
(RH) is due to surface damage arising from denaturation of proteins or
lipoproteins and this results in leakage of ions, and causes reduced RNA,
DNA, and protein synthesis, impaired active transport and greatly
decreased oxygen consumption. Of these factors the loss of ability to utilize
oxygen appears to be the most important since cells which cannot produce
energy are unable to divide and form colonies.
The T series of coliphages survive well when stored in the dark in
purified air at RH greater than 75%. Except for Tl, which survives well
over a range of RH values from 20% to 95%, the survival of coliphages at
RH values below 75% falls progressively with lower humidity.
Harper 52 was one of the first investigators to study viral aerosols and he
showed that survival depended very much on the composition of the
disseminating fluid. Poliovirus and Foot and Mouth virus survive best at
high RH whereas Semliki Forest virus, vaccinia, Venezuelan Equine
Encephalitis and influenza virus are most stable at low RH. The DNA-
containing viruses, pigeon pox virus, and Simian virus 40 maintain their
infectivity over a wide range of RH.
The little work which has been done on the aerosol survival of dry
powders indicates that the survival of organisms disseminated from the wet
and dry state is quite different 53 •54 and Cox concludes that the problem of
aerosol survival is not solely a question of removing and putting back
water.
6.6.4 Oxygen
Micro-organisms such as spores, phages and viruses are not affected by
oxygen in the airborne state but oxygen has an adverse effect upon the
survival of vegetative bacteria and algae particularly when the cells are in
the log phase of growth. Cox suggests that this is due to inactivation of the
cell division process.
6.6.5 Sunlight
Light in the visible and ultra-violet region of the spectrum can be lethal to
bacteria although spores and some viruses such as foot and mouth disease
virus and polio virus are quite resistant to sunlight. The sensitivity of
micro-organisms, contained in an aerosol, to radiation is also dependent
upon the degree of desiccation, the oxygen tension, the particle size and
the nature of the spray fluid. Dry disseminated bacteria are also affected by
sunlight although to a lesser degree than when the aerosol is produced by

dissemination from a fluid.
6.6.6 Protecting agents

Many compounds have been added to the disseminating fluid or powder to
influence the subsequent infectivity of micro-organisms disseminated as an
aerosol. Those found to afford protection include spent culture media, di-
and tri-saccharides, raffinose, dextran, glucose and glycerol, polyhydric
alcohols, sorbitol and inositol and sodium glutamate.
Whilst certain generalisations can be made concerning the survival of
airborne micro-organisms they are of little value in assessing the hazard
arising from the uncontrolled release of a particular micro-organism under
the unique conditions existing in a biotechnology plant.
6.7 Airborne allergens
Even when a process deals with killed cells so that the viability of cells in
the airborne state can be ignored, aerosols of bacterial antigen may still
pose a significant hazard.
According to most authors the mass concentration of the airborne
material is the important measure in allergic response and not the number
concentration irrespective of the particle size, but Muir 7 suggests that the
intensity of the antigen stimulus to the mucosa is closely related to particle
size because this determines the site of action in the respiratory tract.
Spores of about 1 flm in diameter are probably distributed fairly
uniformly throughout the lung so that the concentration of spores per unit
area of lung surface is almost constant in all regions. Larger particles
trapped in the nose result in much more local response. This is well
illustrated by comparing grass pollen grains (32 flm in diameter) with the
spores of the fungus Cladosporium (10 flm X 5 flm in size). Patients
sensitive to grass pollen develop symptoms when the airborne concentration
reaches 50 grains per cubic metre, whereas those sensitive to Cladosporium
react when the concentration reaches 3,000 spores per cubic metre. The
mass of airborne pollen is only twice that of the fungal spores but those
which are trapped in the nose are concentrated over an area of a few
square centimetres of mucosa. The fungal spores on the other hand are
distributed over the wide surface of the bronchial tree.
The allergic manifestations depend upon the concentration of antibody
and antigen at a particular site and on the sensitivity of the tissues at that
point to substances such as histamine. The differential deposition of
inhaled antigens in various parts of the respiratory tract as a function of the
particle size of the inhaled particles results in localised concentrations of
antigen. The water-soluble fractions in the antigen may well be leached off
the particles and obtain access to other regions of the lung or body through
the blood stream so that inhaled allergens may cause eczema or renal
damage. Despite these reservations there is a general relationship in that
large pollen grains which are trapped in the nose tend to cause rhinitis and
conjunctivitis while smaller fungal spores, which can penetrate the nose
more typically cause asthma. Dusts with aerodynamic diameters below 5 or
6 [lm are associated with the allergic alveoli tis group of disorders. Many of
these finer particles are deposited in the upper respiratory tract, however,
and it is not surprising that nasal symptoms, asthma and alveolitus may be
caused by one and the same spore.
The rate at which an insoluble particle is removed from the lung is also a
function of the site of its deposition and hence of its size. Those penetrating
to the alveoli are removed very slowly and thus exert an antigenic stimulus
out of all proportion to their total mass. Those allergens deposited on the
ciliated epithelium nearer to the terminal bronchioles take longer to be
expelled than those which are trapped in the trachea, whereas those
deposited in the nasal areas may be removed in minutes.
It is not known why some airborne pollens and fungi cause symptoms
and others do not. Nor is there any way of predicting this other than by
clinical assessment of each in turn. In general, however, those pollens and
moulds which are present in greatest airborne concentrations for the
longest period of time have been found to be the most common cause of
symptoms.
In addition to the effects of particle size there is some evidence that the
pattern of allergic response is an inherited characteristic. What is clear is
that it is not possible to predict the development of symptoms in an
individual exposed to an aerosol of antigenic material, nor is it possible to
forecast the effect of repeated exposure to that or similar agents.
6.8 Conclusions
There can be no doubt that most biotechnological operations are capable

of generating an aerosol of the material being handled and the inadvertent
introduction of this into a worker in the plant or a person living downwind
of the plant could have entirely unpredictable effects.
In the case of living organisms there is no scientific basis for predicting
the survival of the cells in the airborne state unlessa detailed aerobiological
study has been conducted under exactly the conditions operating in the
plant. There is even the possibility that in ideal conditions cells will
multiply in the airborne state, and when infection can be produced by only
a few organisms the nature of the problem facing those who wish to
establish the hazard associated with biotechnological operations are
brought into sharp focus.
The only generalisation which can be made is that the virulence of a

micro-organism is markedly dependent upon aerosol particle size. This
dependence is not only because survival is affected but also because the
part of the respiratory tract where an aerosol particle is deposited depends
upon the size of the particle.
Modified strains are used in many operations because of the alleged
safety of the avirulent or disabled organism. Sergeant and Evans 4 sounded
a note of caution concerning the use of avirulent strains when they said,
"avirulence is largely a matter of degree related to the dose, portal of entry
and the resistance of the host". In support of this statement they said that if
the dose is large enough organisms not normally regarded as pathogenic
can infect humans; they can also cause disease if they have access to usually
inaccessible parts of the body, i.e. brain or spinal cord following an
accident. In this connection it should be remembered that the aerosol route
of entry is an unusual one for most micro-organisms.
The potential hazard to humans of transformed but disabled bacteria
was considered by a WHO Working Group.55 They concluded that
experimental risk assessment studies specifically designed to test the
hypothesis that host organisms can acquire novel hazardous properties
from DNA donor cells have failed to demonstrate the existence of some
conjectured hazards and they went on to say that these arguments do not
indicate that micro-organisms containing r-DNA molecules are not free
from hazard. Surprisingly, none of the experiments involved a rigorous
aerobiological investigation of the disabled bacteria.
The allergic response of individuals exposed to aerosols of biological
material is difficult to assess since it may take months of repeated low level
exposure for the symptoms to emerge. The experience of those involved in
aerosol vaccination show that untoward side effects might be experienced
in those inadvertently exposed. A consequence of this is that any
modification to plant or working procedures which could change the load
of airborne particles must be documented so that its impact on subsequent
consequences can be evaluated. The importance of the attitude of the
manager and his or her assistants towards safety was recognised and
expressed by Wedum 5 as long ago as 1972 and in 1994 it is still of
paramount importance.
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7 Health hazards in biotechnology
A.M. BENNETT
7.1 Introduction
Although the public perception of micro-organisms may be one of disease-

causing germs, biotechnology is generally regarded as being a very safe
industry with no reported cases of worker mortality caused by exposure to
micro-organisms or their products. However, there have been many cases
of workers being removed from processes or even from a company because
of illness due to exposure to a micro-organism or a microbial product.
Instances of occupational respiratory allergies have been reported in many
countries including Britain, the former Soviet Union, the United States,
the former Czechoslovakia, Italy and China. 1 There are European
biotechnology companies who have incidences of occupational asthma in
the workforce of 12% with the resulting problem of losing highly qualified
staff. In the UK occupational asthma caused by some biological products,
including antibiotics and proteases, is recognised as a compensatable
illness. Expenditure on preventing these symptoms is economic since it
reduces sick leave, compensation and re-training costS.2
In the former Soviet Union a massive programme was undertaken in the
1970s to build single cell protein and lysine plants to provide an alternative
source of animal feed in order to reduce dependency on grain imports. This
programme eventually ground to a halt as a result of public demonstrations
and local political opposition to biotechnology, even though both the
building and operation of the plants provide vitally needed jobs for the
local economy. Due to a series of incidents involving ill health and
environmental pollution in the vicinity of biotechnology plants the Russian
people have become suspicious of biotechnology. These problems seem to
have been exacerbated by pollution from heavy industry in these areas.
The public perception of biotechnology and microbiology was so negative
in the Soviet Union that the Ministry of the Medical and Microbiological
Industry was renamed the Ministry of the Medical Industry.3,4
To prevent such a public reaction to biotechnology it is necessary to
ensure that the industry is subject to appropriate regulation and that the
public is kept informed of both the benefits and possible dangers of
biotechnology. In the European Community (EC) the Directives on
contained use and environmental release of genetically-manipulated
micro-organisms (GMMOs) and on the prevention of worker exposure to

biological agents are being applied. 5 - 7 These apply both in countries with a
history of protest against the use of GMMOs, such as Denmark and
Germany, and those with no history of protests and little regulation. In
Denmark, the government is attempting to allay public fears about genetic
manipulation by encouraging 40% of school children to carry out some
form of genetic manipulation in the c1assroom. 8
The major hazards of large-scale biotechnology are those associated with
exposure to large concentrations of aerosolised micro-organisms or their
products. This is usually caused by lack of good housekeeping, the use of
inappropriate equipment or due to accidents. 1 It is difficult to impose
stringent legislation to prevent incidences of ill health as there are no
threshold limit exposure values for any micro-organism and few for their
products.
This chapter discusses the range of health hazards associated with micro-
organisms and their products, the possible risk of genetic modification and
animal cell culture and identifies areas of bioprocessing that may give rise
to health hazards.
7.2 Health hazards
Although biotechnology is generally regarded as a clean, safe industry,

many hazards are associated with individual processes, even when
biological hazards are disregarded. During sterilisation processes, there
are scalding risks associated with heated vessels. Bioreactors are high-
pressure vessels that need to be carefully constructed and certified to
defined standards to avoid the possibility of explosions. Chemical hazards
include the use of large quantities of highly flammable solvents during
extraction processes, the use of potentially allergenic filter materials and
media components and the use of highly acidic and alkaline solutions in
downstream processing and fermentation. In laboratories there may be the
risk of exposure to highly toxic chemicals and potent carcinogens.
In modern bioprocessing establishments many working procedures are
covered by standard operating procedures that precisely describe each
process. Strict codes of practice are enforced in working areas: these
include the wearing of protective clothing and restrictions on eating,
drinking etc. This good working practice should be effective at preventing
diseases caused by ingestion of micro-organisms or their products or by
skin contact with potential allergens. The one potential mode of
transmission left is therefore the inhalation of aerosols.
It is extremely difficult to prevent aerosol transmission of micro-
or~anisms or their products if one is unaware of the importance of this
hazard. Aerosols are unseen and can persist in the indoor atmosphere for
HEALTH HAZARDS 111
many hours. Section 7.2.1 on laboratory-acquired infection shows the

prevalence of this route of infection and how insidious it can be. It is
therefore essential to have some knowledge of aerosol behaviour and of
the response of the human respiratory tract to aerosolised material to
understand and to prevent occupational disease in the biotechnology
industry.
Health hazards associated with exposure to micro-organisms or their
products in biotechnology can be split into five categories:
1. infection by a pathogen;
2. allergic reaction to viable or non-viable micro-organisms;
3. allergic reaction to a product;
4. reaction to endotoxin;
5. toxic reactions.
Each of these hazards is discussed below in terms of symptoms and

association with parts of bioprocessing. Other potential but unknown
hazards such as dealing with animal cell culture and genetically-manipulated
micro-organisms are also discussed.
7.2.1 Laboratory-associated infection

The history of laboratory infection has been covered by various authors
over the years 9- 11 and has formed the topic of an excellent book by
Collins. 12 In the early days of medical microbiology, the risk of infection
was perhaps regarded as part of the job, with ill health normally arising as a
consequence of careless or thoughtless laboratory practice. Bad practice
possibly arose partly from a belief in the glamour of medical martyrdom
and partly from the under-recognised importance of aerosol transmission.
Pike 10 reported 4079 instances of laboratory infection with 168 fatalities
in his survey of 1979. The actual number of laboratory infections may have
been much greater, as many of the incidents described only came to light as
a result of the surveyor were of obviously laboratory acquired source such
as exotic viruses and other non-endemic pathogens, while illnesses caused
by endemic pathogens may not have been recognised. It has been reported
in the UK that medical laboratory technicians have seven times the
incidence of tuberculosis compared to a control population. Similar
findings have been reported in Austria, the former West Germany and
Switzerand. 13 · 14 Although general sickness rates are low amongst laborat-
ory workers they have three times the amount of sick leave due to
diarrhoea of unspecified origin and also have high rates of hepatitis. 14
Infections may be transmitted by laboratory workers to others either
accidentally or even intentionally .15, 16
Few cases of laboratory-acquired infection have been reported in the
biotechnology industry. Most of the processes use non-pathogenic organ-

isms (although it has to be remembered that some of these organisms,
especially Aspergillus and Pseudomonas species, have been shown to be
the cause of fatal infections in immunocompromised people and serious
lung infections in people with cystic fibrosis). There have also been recent
reports of fatal infections with Aspergillus fumigatus in previously healthy
adults. 17 In fact Seaton regards aspergillosis as being so commonly
occurring that physicians should always bear it in mind in any patient ill
with pneumonia. 18
When pathogenic micro-organisms are processed using conventional
uncontained bioprocessing equipment the results can be disastrous. In 1939
a tubular bowl centrifuge was used to separate Brucella abortus in a
stairwell in the veterinary college of Michigan State University. It
generated an infectious aerosol that caused an outbreak of brucellosis in
which 93 people were affected and one died. 19 A technician in a
pharmaceutical factory involved in the sonication of novel strains of
Pseudomonas contracted melidiosis having been exposed to an incorrectly
identified strain of Pseudomonas pseudomaUei.2u However, this type of
incident should not recur if codes of practice for dealing with potential
pathogens are in place and staff comply with them.
7.2.2 Allergic reactions

Type I allergic reactions result from contact of the inhaled allergen with
cell-bound IgE. This causes the release of histamine and other vasodilators.
These reactions cause localised inflammation leading to irritation of the
nose, skin or respiratory tract depending on where the challenge is
deposited. This is in turn dependent on the particle size of the challenge.
The effect of this irritation on the respiratory system is to constrict the
airways which leads to breathing difficulties and so produces the symptoms
of asthma.
The second type of reaction commonly brought about by exposure to
biological material is Type III hypersensitivity. The symptoms of this type
of response occur six to eight hours after exposure to allergen. Complexes
are formed between the antigen and IgG which can activate the
complement pathway leading to the release of histamine and so the
production of similar symptoms to those mentioned in type I reactions.
This type of reaction makes it difficult for the sufferer to recognise the
cause of their illness due to the delay in the occurrence of symptoms. If this
type of reaction occurs due to occupational exposure, the medical
condition of the sufferers often deteriorates before the cause is eventually
recognised.
Normally, in the case of inhaled allergens, type I reactions are described
as asthma while type III reactions are described as hypersensitivity
HEALTH HAZARDS 113
pneumonitis or extrinsic allergic alveolitis. This picture is complicated by

the fact that both reactions can often occur as a response to the same
allergen. The loss of lung capacity caused by asthma can be measured
easily with forced expiratory volume meters which are commonly used in
the management of asthma. Their use as part of the health monitoring of
the biotechnology workforce could give information on the prevalence and
epidemiology of asthmatic symptoms in the biotechnology industry.
7.2.2.1 Allergy to biological products. Many common allergies have been

linked to biological materials such as pollen, wood dust, tea dust, animal
danders and soya bean flour.z It is well known that many products of the
biotechnology industry, including the antibiotics and enzymes shown in
Table 7.1, are capable of producing strong allergic responses. These
reactions can be extremely serious and occasionally fatal. 21 In the
literature there are many reports of antibiotics causing occupational
asthma and indeed this illness is a compensatable disease. 2 However, there
have been few, if any, reports of outbreaks of asthma related to these
products in the pharmaceutical industry that tell how they started and how
further outbreaks were prevented. Many people in the pharmaceutical
industry can recount cases in which a worker suffering from occupational
asthma had to be shifted to another part of the plant to reduce their
exposure to the causative agent. Others had to leave the company.
The only well-documented outbreak of asthmatic symptoms caused by
exposure to the biotechnological product was the widespread problem of
asthma caused by exposure to Bacillus subtilis proteases in the washing
powder industry. During the later 1960s and early 1970s these outbreaks
were reported by occupational hygienists, company medical officers and
respiratory disease specialists. They were the subject of many papers. 22-26
Table 7.1 Microbial products implicated in occupational asthma
Product Situation Reference
6. APA Antibiotic production 28

Ampicillin Antibiotic production 28
Amylase Baking 29
Amylase Bulk enzyme handling 21
Benzyl Penicillin Antiobitic production 28
Cellulase Enzyme production 30
Cephalosporin Pharmaceutical production 31
Esperase Enzyme production 32
Lysozyme Pharmaceutical production 33
Penicillin Antibiotic production 34,35
Penicillamine Antibiotic production 36
Protein Dust Single cell protein production 37
Subtilisin Washing powder preparation 22
Tetracycline Antibiotic production 2
Tuberculin Pharmaceutical production 38
Soon after the introduction of bacterial proteases into the formulation of

washing powders it was noticed that many workers were suffering from
respiratory disorders. Flindt22 reported a widespread occurrence of
breathlessness accompanied by wheezing with rapid onset that lasted for
periods of hours, days or even months. Associated symptoms included dry
coughs, chest pains, fever and general malaise. Some patients were unable
to leave their beds during attacks in which they thought they were dying.
Symptoms usually occurred approximately eight hours after contact with
the enzyme and so the cause was not immediately recognised as being
associated with the patients' work. Therefore, because of this delay, the
symptoms were recognised as being extrinsic allergic alveoli tis which can
result in fibrosis and chronic lung disease.
Pepys et al. 23 carried out a series of lung function, skin sensitivity and
bronchial challenge tests on three of the worst affected workers. By
performing skin prick tests to a wide range of commonly occurring
antigens, they found that the workers were not atopic, i.e. they were not
especially prone to sensitisation to environmental allergens. All of these
workers produced skin reactions to the lowest concentration of B. subtilis
protease tested. The lung function tests showed that all three workers were
suffering from lung airway obstruction and they all reacted to a bronchial
challenge test showing both immediate and delayed reactions. One patient
had such a severe reaction to the aerosolised enzyme solution that his lung
capacity was reduced to a third of normal and he had to be given an
adrenalin injection to aid recovery.
Juniper et al. 26 carried out a seven-year survey of workers' health in a
washing powder plant from the start of an outbreak of allergic symptoms
until these symptoms were all but eradicated by a ten-fold reduction in dust
levels. In studies on the original workers who were exposed to the highest
dust levels, skin tests showed that 80% of atopic and 40% of non-atopic
workers produced a positive reaction to the enzyme. Sensitisation occurred
between six and 24 months after first exposure. (This same delay has been
reported privately to the author by representatives of biotechnology
companies whose workforce were suffering from similar symptoms.)
Conditions were improved in the light of recommendations of a working
party set up by the soap and detergent industry. 27 Only non-atopics were
hired and conversion levels fell to 29% and finally to 10.5% as enzyme dust
levels were reduced. Respiratory symptoms caused by inhalation of the
enzyme occurred in 3.2% of the workforce employed over the seven-year
period. These symptoms consisted of breathlessness, sweating and wheezing
within minutes of exposure and/or five to six hours after work. There was
no evidence of permanent lung damage.
The problem with these outbreaks of asthmatic symptoms was that by
the time they had been identified many of the workforce were suffering the
symptoms. The use of simple portable spirometers to measure workers'
HEALTH HAZARDS 115
lung capacity could have been a useful method of medical surveillance as

the loss of lung capacity is the first major symptoms of occupational
asthma.
7.2.2.2 Allergy to micro-organisms. Many types of micro-organisms may

cause allergic reactions when inhaled in large quantities. A list of those
organisms responsible for asthmatic outbreaks that have been used in the
biotechnology industry are listed in Table 7.2. However, it must be
stressed that only a few of these micro-organisms have been reported to
have caused allergic symptoms in the biotechnology industry.
The micro-organisms that have been reported as causing the most
outbreaks of allergic respiratory symptoms in the biotechnology industry
belong to the species Aspergillus. Topping et al. 39 surveyed a biotechnology
plant, which used Aspergillus niger to produce citric acid, after one worker
was diagnosed as suffering from occupational asthma. After extensive
medical screening, they found that 4.9% of the workforce were suffering
from asthmatic symptoms. Positive skin test reactions to A. niger culture
fluid extract and the presence of IgE specific to the organism were far more
prevalent amongst the workers suffering asthmatic symptoms than amongst
their healthy colleagues. All the affected workers either worked in the
surface fermentation or the product recovery parts of the plant where they
were exposed to aerosols of spores or extraction waste water.
In the former Czechoslovakia, Horejsi et al. 40 reported a serious
outbreak of bronchial symptoms amongst workers on an open pan citric
acid fermentation process using Aspergillus species. At the end of the
fermentation, a thick layer of biomass was removed manually by the
workers. This resulted in the generation of large concentrations of aerosol
of both Aspergillus spores and also spores of contaminating Penicillium
species. A medical questionnaire revealed that 94% of these workers
suffered symptoms of coughing, breathlessness and fever on finishing
Table 7.2 Micro-organisms implicated in occupational asthma
Organisms Situation Reference
Actinomycetes Mushroom composting 43

Actinomycetes Farming 44
Aspergillus Fermentation 40
Aspergillus niger Citric acid production 39
Aureobasidium pullulans Office. woodworks 45
Bacillus subtilis Ventilation 46
Baculoviruses Pesticide production 47
Candida tropicalis Protein production 48
Paecilomyces varioti Woodworks 49
Penicillium sp. Citric acid production 41
Penicillium citrinum Cheese making 49
Penicillium citrinum A TP production 50
work. Bronchitis was diagnosed in 39% of these workers and in 71 % of

workers who had left due to ill health compared to 12.6% of the national
population. Both Aspergillus and Penicillium spores were retrieved from
workers' lungs but there was no sign of colonisation. Horejsi et al. 40
blamed the Penicillium for the outbreak because it was more commonly
present in the lungs of the invalids. Considering the type of cross
contamination that must have occurred during spore mat removal, it is
likely that the workers could have become sensitised to both organisms.
Lacey et al. 41 quote levels of microbial aerosols in the farming
environment in the region of 106 and 108 per cubic metre as being the dose
needed to cause symptoms of occupational asthma in exposed workers.
Fisher and Rosner 42 report human inhalation challenges with powders of
Bacillus thuringiensis, a commonly used microbial insecticide. Five
volunteers inhaled 100 mg per day of a spore powder containing 3 X 109
spores per gram for five consecutive days with no evidence of any ill
effects.42 The level of airborne micro-organisms needed to cause sensitisa-
tion will depend on length of exposure, particle size of the challenge and
also on the concentration of other air pollutants. Because of the lack of air
monitoring information currently available for airborne micro-organisms
in working environments the creation of threshold limit values for
potentially allergenic micro-organism seems unlikely in the near future.
7.2.3 Endotoxin reactions

Endotoxin is a term often used synonymously with lipopolysaccharide
(LPS), a major component of the Gram-negative bacterial cell wall.
Endotoxin has been estimated to make up between 3 and 4% of the dry
weight of Escherichia coli K12.51 The LPS molecule consists of a Lipid A
component anchored in the outer membrane. This is attached to a series of
saccharide units specific for different strains of Gram-negative bacteria.
LPS is extremely heat stable and needs to be heated to 180 DC for three
hours to ensure inactivation. When injected into the bloodstream, it
activates the alternative complement pathway and produces a pyrogenic
reaction. Studies on laboratory animals have shown that it can produce
severe reactions when inhaled in microgram quantities. 52
Many diseases in many industries have been linked to inhalation of
endotoxin. Some are shown in Table 7.3. 53 .54 A chronic respiratory disease
of textile workers called byssinosis has recently been linked to exposure to
endotoxin in dirty cotton. Castallen et al. 54 and Kennedy et al. s5 have
shown linear relationships between airborne endotoxin concentration and
decrease in lung function. However, there was no relationship between
decrease in lung function and the dust levels in the cotton works. The
organism most commonly found in cotton and linked for these symptoms is
Enterobacter agglomerans.
HEALTH HAZARDS 117
Table 7.3 Micro·organisms implicated in adverse reactions to endotoxin
Micro-organism Situation Reference
Enterobacter agglomerans Cotton milling 61

Flavobacterium sp. Humidifier 62
Methylophilus methylotrophus Single cell protein production 58
Methylomonas methanolica Single cell protein production 57
Pseudomonas aeruginosa Downstream processing 59
Serratia marcescens Military research 56
Paine 56 exposed four healthy naval officers to a challenge of Serratia

marcescens of aerosol concentrations of 3.4 X 106 organisms per cubic
metre and above for a period of two hours. Each of the volunteers suffered
loss of respiratory function and fever with no signs of infection. Two of the
subjects who had been previously been exposed to clouds of S. marcescens
suffered less severe symptoms than their previously unexposed colleagues.
Although the endotoxins of various species tend to vary in their toxicity to
human beings this experiment shows that airborne concentrations of
Gram-negative bacteria that occur in biotechnology can cause adverse
symptoms.
7.2.3.1 Reaction to endotoxin in biotechnology. Inhalation of Gram-

negative micro-organisms occurring in the biotechnology industry gives
rise to a different spectrum of clinical symptoms from that resulting from
inhalation of Gram-positive micro-organisms, fungi or biotechnology
products. The symptoms caused by the latter organisms tend to be of an
allergic nature with symptoms increasing with length of exposure and only
occurring after previous exposure. Symptoms tend to be localised within
the respiratory tract, normally consisting of rhinitis or asthma, with
possibly dermatitis. With Gram-negative organisms inhalation symptoms
can and do occur after the first exposure. Symptoms can include kidney
and stomach pains, conjunctivitis and aching limbs. 57-59
Gram-negative organisms have often been used in single cell protein
(SCP) production. Organisms capable of using methane or methanol as an
energy source were used extensively in the 1970s to produce SCP before
the increase in the price of oil made these processes uneconomic. The ICI
plant at Billingham, UK used the organism Methylophilus methylotrophus
for this process. 58 Extensive toxicity testing of this organism by ingestion
and injection in animals showed it to be non-toxic. The plant was designed
to operate at total dust levels of below 1 mg/m 3 . Where this limit was
breached, protective clothing was worn. However, in some instances,
workers exposed either to the organism, as a result of a breach of the
bioreactor, or exposed to the product in the aerosol form, suffered an
influenza-like illness. The symptoms included headaches, aching limbs,
chest tightness and shivering. Later incidents involving exposure to high
protein dust concentrations led to the development of additional symptoms,

such as sore eyes with discharge.
Ekenvall et al. 57 also reported the development of influenza-like
symptoms in seven out of eight workers after spray drying was undertaken
in a pilot scale bioprocessing plant. The symptoms began between six and
12 hours after exposure. Again, they consisted of fever, shivering, chest
tightness and in some cases coughing and muscle pain. Five of the eight
affected had symptoms of conjunctivitis, rhinitis and coughing immediately
after exposure to the dust. Two of the sufferers were on the first visit to the
plant so the attacks seem not to be only allergic in nature although all those
exposed to dust had antibodies specific to SCP extracts. Dust levels found
in the spray drying room were 4 mg/m 3 and those in the packaging room
were 20 mg/m 3 . Further attacks were prevented by altering the spray
drying technique to prevent the formation of particles of respirable size.
The workers in the packaging room were also provided with protective
clothing and eye protection.
Dunni1l 59 describes an outbreak of illness associated with downstream
processing of Pseudomonas aeruginosa. He states that "the greatest
demands in terms of biosafety occur from the time the broth leaves the
fermenter through to the post-precipitation stage". Gram-negative organ-
isms tend to produce intracellular, not extracellular, enzymes. High energy
homogenisation processes are thus required in order to rupture the cells
and release the enzyme. This can place the workforce in contact with high
concentrations of airborne endotoxin. In the case reported by Dunnill 59
five workers who were exposed to aerosolised Pseudomonas aeruginosa
during downstream processing suffered influenza-like attacks with both
kidney and stomach pains lasting approximately 24 hours.
Although endotoxin seems to be the main cause of these symptoms, it
has to be recognised that other material apart from the endotoxin,
including intracellular products, is being aerosolised by the downstream
processing. These other by-products may cause allergic symptoms over and
above those caused by endotoxin.
Pa1chak et al., 60 after conducting both a literature review on the clinical
effects of inhaled endotoxin and an airborne endotoxin monitoring
campaign in a biotechnology plant, established an action threshold value
for airborne endotoxin of 30 ng/m 3. Although all routine production
processes gave values under this limit, they found that an experimental
batch harvest process gave levels of up to 1.8 f.tg/m3, 60 times their
threshold value.
7.2.4 Toxic reactions to products or by-products

Previous sections have discussed immunological reactions to both micro-
organisms and their products. However, some of the products and by-
HEALTH HAZARDS 119
products of these organisms may be capable of affecting the health of the

exposed worker in other ways. Since the techniques of genetic modification
have been developed, it has been possible to manipulate micro-organisms
to express large quantities of extremely biologically-active agents such as
interferons, hormones etc. These substances can have a deleterious effect
on workers if inhaled even at very low concentrations. The experience of
oral contraceptive manufacturers shows the problems inherent with
handling large quantities of hormones. There have been many reports of
increased incidences of menstrual problems, loss of libido, post -menopausal
vaginal bleeding and gynaeocomastia (growth of large mammary glands in
males) in workers in this industry.:l3·63.64 Indeed, many manufacturers now
have the policy of only employing post-menopausal women in their
facilities.
Baxter et ai. 65 have reported the concentration of a barbiturate in the
blood of workers involved in barbiturate production as being the
equivalent of half the adult therapeutic dose. The use of airline hoods and
gloves seemed to result in lower barbiturate concentrations in workers'
blood samples. However, the drug was still detectable.
Exposure to airborne antibiotics in the pharmaceutical industry can give
rise to a wide range of symptoms. Farina et al. 66 report symptoms of
vitamin deficiency in people working in streptomycin, penicillin and
tetracycline production. An increase in vaginal candidiasis and general
gynaecological problems was found in a study of female workers in the
antibiotic industry. 33
Other organisms used in the biotechnology industry, such as Aspergillus
flavus and Aspergillus oryzae are known to produce highly lethal toxins
under different environmental conditions (temperature and pH values).
Inhalation of these toxins has also been linked with cancers. It is thus
important to ensure that any fermentations involving these organisms are
well controlled and monitored to prevent any toxin being produced. 67 .68
7.2.5 Hazards posed by genetic modification

In the past decade studies of risk and hazard in biotechnology have
concentrated mostly on the use of genetically modified micro-organisms
(GMMOs). The public perception of these organisms as being "unnatural"
and suggestions by pressure groups that their use will possibly lead to some
form of environmental Armageddon have encouraged many governments
to introduce strict codes of practice for their use. A recent popular science
book 69 suggests that "as a result of the application of genetic engineering,
world-wide pandemics caused by newly created pathogens, the triggering
of catastrophic ecological imbalances by the release of novel micro-
organisms into the environment, the creation of new agents of biological
warfare ... may become realities in the near future". Jukes 70 gives further
examples of hysterical thinking about genetic manipulation when he tells of

a pregnant woman fleeing from her house four miles from the release site
of the ice minus deletion strain of Pseudomonas syringae. He also reports
the case of a biological scientist who claimed that biotechnology would lead
to "a giant slaughterhouse, a molecular Auschwitz, in which valuable
enzymes, hormones and so on will be extracted instead of gold teeth".
Partly as a result of this type of attitude by vocal opponents of genetic
manipulation, especially in the United States, Denmark and Germany,
application of regulations covering the use of GMMOs is very stringent.
This is not necessarily because of any risk to health or the environment, but
rather in response to public perceptions of biotechnology. It seems strange
that many of the protesters do not seem concerned that where no
organisms should be able to leave the bioprocessing facility, workers can
be exposed to high airborne concentrations of GMMOs. They could thus
release them into the environment through coughing or defaecation. As
Wheale and McNally69 point out, "the persistent focus of the conversion of
E. coli K12 into an epidemic pathogen [has] allowed considerations such as
hazards to workers inside the laboratory to be peripheralised in the
debate". In the UK a genetically-modified yeast has been approved for use
in the baking industry without raising much of an outcry amongst
consumers or even environmental activists. 7 )
The hazard posed to operators of bioprocessing facilities by these
organisms would seem to be posed by inhalation of micro-organisms
manipulated to produce high levels of potentially hazardous bioactive
product. If these micro-organisms could carryon producing this product at
high enough levels before they are removed from the lung, this could put
the operator at risk. It would seem to be unlikely that the small number of
organisms likely to be inhaled would have enough nutrients or time in the
lung to produce the product in enough quantity to cause damage, unless
they possessed the ability to colonise the lung. It can be argued that the
fuss and furore over the use of GMMOs has served to hide the actual
health problems of occupational asthma and endotoxin inhalation from
public attention and governmental regulation.
7.2.6 Hazards posed by animal cell culture

Large-scale utilisation of animal cell culture has the potential to produce a
wide range of therapeutic products for commercial use. Proteins produced
from mammalian cell culture are normally secreted in their bioactive
glycosylated form unlike those secreted from GMMOs. The development
of genetic modification techniques for use in mammalian cells allows the
production of highly active therapeutic products in large quantities. It is
therefore probable that animal cell culture will be a growth area in the
future and great care must be taken to ensure the safety of workers in this
HEALTH HAZARDS 121
field while also ensuring that any regulations imposed do not affect the
competitiveness of the technology.
Although there have been no reports of ill health due to contact with
animal cell culture, concern has been expressed about potential health
hazards involved with this technology. Animal cells and their products
have been used for many years in vaccine production without causing any
reported cases of ill health. The major hazard of the large-scale use of
animal cell culture is the potential presence of oncogenic or infectious
viruses in the cells, especially those of primate origin. Another potential
hazard is the introduction of adventitious agents such as mycoplasmas and
viruses during handling by laboratory workers. There is also the possibility
of infectious agents of bovine origin being present in the growth media
used, such as the agent causing BSE. Although all cells used in
bioprocessing will be screened for the presence of a wide range of
oncogenic and infectious agents a negative result does not necessarily mean
that these agents are absent. Hence great care must be exercised in
dealing with large-scale animal cell biotechnology in case of some hidden
risk.72
An additional problem with animal cell technology is that many
processes are designed to protect the process fluid from contamination by
the operator. There is normally an airflow away from the cell line that
could possibly channel infectious material or oncogenes into the operators'
breathing zones. It is important therefore to have a mechanical barrier
between process and operator or at the very least some form of air curtain
such as a Class II cabinet. Many of the products derived from animal cell
culture such as interleukins, interferon and hormones are highly bioactive.
Therefore, the downstream processing steps associated with these products
need to be contained to prevent any chance of operators becoming
exposed to bioactive substances. Frommer et al.72 have proposed a method
of linking the type of cell line used to the containment level required for
cell culture and downstream processing which could be a starting point for
any safety regulation.
7.2.7 Hazards posed by plant cell culture

Plant cell culture is becoming a widely used technology for the production
of plant biochemicals and also for the biotransformation of pharmaceuticals.
The dangers posed to exposed workers by this technology will be entirely
dependent on the products of these processes and possibly the by-products
of the plant cells' metabolism. Therefore the hazards of plant cell
culture will probably be similar to those of animal cell culture discussed
above.
7.3 Hazards of bioprocessing equipment
As discussed earlier, the main danger in biotechnology comes from the

accidental creation of aerosols during bioprocessing. Aerosols are produced
when kinetic energy is exerted on packages of fluid causing an increase in
their total surface area. Which areas of bioprocessing are likely to cause
such problems?
7.3.1 Fermentation
The main hazard of fermentation is the large volume of fluid containing
high concentrations of potentially allergenic micro-organisms and bio-
chemicals. However, bioreactors are certified pressure vessels operating at
low pressure. The only energy inputs are from the impeller(s) and the air
input. If the fermenter is well fabricated and the exhaust gas is filtered,
these aerosols should not be released into the environment. The only other
area of concern is the sampling valve. This should be the only place where
the cell growth medium comes in contact with the external environment.
Many bioreactor sampling valves have been designed to prevent release of
aerosols. Even un contained fermentation sampling valves should not
generate significant aerosols, if used with care. 73 However, catastrophic
accidents leading to gross spillage of the fermenter contents could give rise
to skin contact and possible ingestion hazards, as well as generating
microbial aerosols. 74
The preparation, mixing and dissolution of the media components can
generate potentially allergenic dusts. This should be carried out with
effective exhaust ventilation or contained equipment.
7.3.2 Centrifugation
Centrifugation is a process that applies energy to high concentrations of
micro-organisms in order to separate them from solutions of different
densities. Laboratory centrifuges should not generate aerosols as long as
they are used with sealed buckets and rotors that have been micro-
biologically integrity tested. 75
The continuous centrifuges used in large-scale biotechnology can cause
hazards especially if cell paste removal is manual. 73 Even centrifuges with
enclosed desludging and clean-in-place design can generate aerosols if seals
are badly designed or poorly maintained.
7.3.3 Cell disruption

The mode of action of many cell disrupters is to subject cells to very high
pressures (up to 2,700 bar). If this high-pressure fluid is in contact with an
HEALTH HAZARDS 123
ill-fitting seal, this will allow the generation of an aerosol into the external
environment. The aerosol produced may not contain viable cells. However,
it may contain allergenic material or endotoxin. Often this aerosol will
be of low particle size due to the high energy of the process. This type of
equipment is often associated with illness caused by endotoxin exposure.
Most products of Gram-negative bacteria are intracellular and so homo-
genisation is a common recovery method.
7.3.4 Filtration
Most filtration processes used in large-scale biotechnology should not have
the potential to generate microbial aerosols. Filtration columns normally
use gravitational forces to separate products from impurities and so are low
energy processes. The only potential problem can come in either rotary
vacuum filtration or with filter presses if a violent method of biomass
removal from the filter is used. Wickramanayake 76 has reported that it is
often the practice with filter presses to knock the cell mat of the filter with
hammers. This practice has been shown to generate microbial aerosols.
7.3.5 Product handling

Product handling can be the most dangerous part of bioprocessing. The
product is in its most concentrated form and is bioactive. The finished
product in biotechnology is often a dusty solid which is prone to
aerosolisation. The handling and processing of these powders need to be
carried out with well-designed local exhaust ventilation. If necessary, well-
maintained and validated personal protective equipment should be used to
protect exposed workers. However, this should only be a temporary
measure. If possible, it is preferable to manufacture and market the
product as a liquid or at least as a granulated solid. This will decrease the
likelihood of product aerosolisation. If the product has to be manufactured
in solid form, then it should be produced in granules of 20 [lm or larger to
reduce the risk of aerosolisation and hence of inhalation. Special attention
should be given to ensure that both spray driers and freeze dryers are well
contained. Both types of equipment are very efficient aerosol and dust
generators.
7.3.6 Risk assessment

It is important to carry out risk assessments on bioprocesses in order to
estimate the potential health risk to the exposed workers. The most
effective method of assessing the aerosol risk created by a piece of
bioprocessing equipment is the 'spray factor', This is a concept developed
by Dimmick 77 to relate the aerosol-producing capacity of a piece of
biological equipment to the hazard posed to its operator. The spray factor
can be used to calculate the potential inhaled dose for an exposed worker.
The volume of the working environment and the ventilation rate must be
known. A worker's breathing rate of one cubic metre an hour and a lung
retention value of 0.3 are assumed.
Chatigny and Pruisner78 used a hazard rating for particular biological
processes derived from the spray factor and a hazard rating for a biological
agent to define a particular containment level to be used when working
with transmissible spongiform encephalopathy agents. This method of risk
assessment could be adapted for use in the bioprocessing of other micro-
organisms.
7.3.7 Prevention
The symptoms of respiratory allergy referred to in section 7.2 can be easily
prevented in most situations by the adoption of good housekeeping
techniques. The use of the principles of occupational hygiene, taken from
Ager and Nourish 79 and shown in Table 7.4, should ensure the prevention
of occurrences of any symptoms of ill health associated with the use of most
industrial micro-organisms. Health surveillance of workers exposed to
potentially allergenic material should give early warning of potential
problems. This surveillance may involve regular skin tests with potentially
allergenic material. Lung function tests can be used to ensure that workers
have no pronounced reduction in lung capacity. The Health and Safety
Executive have published a useful guide for employers on occupational
lung diseases including those caused by biological agents.80
Acknowledgements
I would like to express my gratitude to Drs P. Hambleton, J.E. Benbough

and K.P. Norris for helpful comments in the writing of the report.
Table 7.4. Principles of occupational hygiene
1. To keep workplace and environmental exposure to any physical, chemical or biological

agent to the lowest practicable level.
2. To exercise engineering control measures at source and to supplement these with
appropriate personal protective clothing and equipment when necessary.
3. To test adequately and maintain control measures and equipment.
4. To test when necessary for the presence of viable process organisms outside the primary
physical containment.
5. To provide training of personnel.
6. To establish biological safety committees or subcommittees as required.
7. To formulate and implement local codes of practice for the safety of personnel.
After Ager and Nourish (79).

HEALTH HAZARDS 125
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8 Containment of unit processes
P. HAMBLETON and 1. MELLING
8.1 Introduction
Industrial biotechnology processes have been considered to pose little or

no hazard to workers on the environment unlike, say, the chemical and
nuclear industries. I This view would seem to be justified in that the
pharmaceutical and biotechnology industries do have good safety records
based on experience and procedures developed over many years. The
increased industrial application of recombinant DNA (r-DNA) technology
has served to focus attention on the safety of the biotechnology process
industry; attention manifest in the burgeoning regulations and guidelines
appearing nationally and internationally (see chapters 1--4). Although
there is really little to suggest that the new biotechnology processes are
inherently any more or less safe than the established ones 2 the current
regulations stress the need to minimise or prevent releases that might
contaminate personnel or the environment. This requires that risk
assessments be carried out on all stages of biotechnology processes and
that the principles of biosafety be applied to reduce or eliminate the
potential for generating biological hazards.
Biohazards may arise because of the release of micro-organisms or their
products from biotechnology processes (see chapter 7). Micro-organisms,
whether they are inherently pathogenic or genetically modified micro-
organisms (GMMOs), pose a threat because they may be capable of
replicating outside the process environment and, as such, it may be
necessary not only to limit but even to prevent their release. The products
of microbiologically-based processes may also be hazardous (see chapter
7), but for these it may be possible to define acceptable levels of
contamination, in which case, it may be sufficient to minimise release.
Prevention of the release of biohazardous materials from biotechnology
processes can be achieved by applying biosafety containment principles.
Consideration must be given to the containment requirements of all
process steps from the initial generation of seed cultures and inocula to the
handling of process effluent. The problems of treating off gases and liquid
effluent are discussed elsewhere (chapter 12). This chapter will consider
the application of containment principles to prevent release of hazardous
material from unit processes at laboratory and pilot plant scales.
l30 BIOSAFETY IN INDUSTRIAL BIOTECHNOLOGY
Containment of larger scale processes are dealt with elsewhere (chapters 9

and 11).
8.2 Unit processes in biotechnology
Irrespective of the operational scale, biotechnology processes typically

comprise a series of linked process areas, each of which may encompass
one or more unit process operations (Figure 8.1). The nature of the unit
operation may vary from the relatively simple (medium preparation) to the
more complex (fermentation, centrifugation, freeze drying), but most have
the potential to generate health hazards by releasing materials as small
particle aerosols or through gross environmental contamination 3 . As
discussed by Bennett (chapter 7), such releases may involve raw materials,
micro-organisms, crude product, by-products, waste materials or pure
product and can give rise to incidents of serious illness. The biosafety risks
Raw Inoculum
materials generation
~/
Upsteam
processing
Bioreactor
1
Biomass
separation
Downstream
processing
Product
purification
1
Product
finishing
Figure 8.1 Linked unit process operations for biotechnology.

UNIT CONTAINMENT 131
associated with biotechnology process operations can be reduced or

effectively eliminated by the application of containment principles.
8.3 Categories of containment
Various categories of containment can be applied, three of which represent

differing levels of physical containment of the processes themselves.
Primary containment consists of the provision of immediate physical

barriers to release and is represented by the basic design and structure of
the equipment item. A glass bottle filled with a screw cap and elastomeric
seal can be seen to represent an example of primary containment. In the
same sense, a more complex container, such as a bioreactor fitted with
appropriate seals and gas filters, serves as a primary container of the
fermentation process. Whilst some primary containers may be intrinsically
robust, the main feature of primary containment is that in the event of
failure of the containment system, there would be release of contents.
Secondary containment serves to provide a back-up in the event of

failure of the primary barrier and most commonly takes the form of a
physical enclosure, although secondary containment can also be achieved
by engineering improvements to the primary containment unit itself.4
Some do not make this distinction between primary and secondary
containment, describing both as primary containment. 5 However, in our
opinion it is important to distinguish between the inherent containment
properties of process equipment and those features that need to be added
in order to cope with the failure of primary containment barriers.
Tertiary containment describes the use of a defined operational facility

to prevent contamination of the environment external to the laboratory or
production area. This is achieved by facility design (e.g. directional
airflows and air filtration), effluent treatment and operational procedures.
Biological containment whereby micro-organisms are genetically modi-

fied so as to restrict their ability to survive or transmit genetic information,
except in defined growth environments, can provide an important adjunct
to physical containment systems by reducing the inherent risks from
release of the organism.
Other. If the possibility of the release of biohazardous material from a

biotechnology process operation has to be accepted, then it may be
appropriate to consider the use of personal physical protection of the
worker in the form of positive (or negative) pressure respiratory protection
(a)
(b)
)
----"'-- _ _ _---'r--- -
(c)
Figure 8.2 Microbiological safety cabinets. (a) Class 1. (b) Class IT. (c) Class III.
devices. This approach should not be accepted as an optional alternative to

containment of the process and should only be considered as a last resort.
There may also be a temporal element to containment: 6 for example,
following sterilisation or fumigation of a containment environment it may
not be necessary to retain the containment barrier once the danger has
been eliminated. This applies particularly to systems relying on secondary
physical containment. In addition to reducing the risk of generating health
hazards, process containment has one other important safety feature,
namely the prevention of product cross-contamination. In situations where
two or more different processes might be taking place simultaneously the
absolute separation of the product is desirable for both product safety and
commercial reasons.
8.4 Safety cabinets
Physical enclosure is perhaps the most obvious means of contammg

emissions resulting from inadequate or failed primary containment. Where
the unit operation is compact, the enclosure can be a metal or plastic box
designed to prevent escape of emissions whilst allowing a worker to
operate the process more or less normally. The most common means of
achieving this type of containment is by the use of enclosures operating as
biological safety cabinets, of which there are three types, Class I, Class II
and Class III (Figure 8.2), each having different operating characteristics.
Safety cabinets are intended to separate the worker and the biological
agent being manipulated and various designs have been produced with this
end in mind. Confusion has arisen because safety cabinets were designed to
meet a variety of needs ranging from the handling of highly infectious
micro-organisms to the prevention of contamination of tissue cell cultures.
Many types of enclosure apparatus have been described in terms such as
exhaust protective cabinets, hoods, glass boxes and laminar flow cabinets;
these all now fall within the accepted definition of microbiological safety
cabinets. It should be remembered, however, that this is a convenient
generic description and the equipment so described may not necessarily
offer adequate worker protection.
8.4.1 Classification
A classification of microbiological safety cabinets has now become
accepted by many authorities including5 ,7 the British Standards Institution,
UK Health and Safety Executive, WHO and USA organisations. According
to the British Standard (BS 5726, 1979) biological safety cabinets are Class
I (open fronted, exhaust), Class II (open fronted, under directional
downward airflow) or Class III (enclosed, exhaust). It should be
remembered that the classification is arbitrary and does not reflect any
order of safety; cabinet classifications do not relate, for example, to
categories of pathogenic micro-organisms 8 or Biosafety Levels. 5 ,9
8.4.2 Air filtration

The effective operation of safety cabinets relies on the use of High
Efficiency Particulate Air (HEP A) filters to remove micro-organisms from
exhaust air and, for Class III cabinets, inlet air also. It is essential that
HEP A filters be mounted such that air cannot bypass the filter. This means
that the filter must be mounted directly against the cabinet wall and not
remotely along an air duct, unless that duct forms an integral and unbroken
component of the cabinet itself.
For compliance with BS 5726 the HEP A filter must have a sodium
chloride or dispersed oil particle (DOP) penetration of not more than
0.003% when tested in accordance with BS3928.
Although HEPA filters with a penetration of less than 0.003% are
considered to remove airborne bacteria, the Code of Practice for the
Prevention ofInfection in Clinical Laboratories and Post-mortem Rooms lO
considered that effluent air from Class I cabinets should be exhausted to
the open air or via a thimble device into a total loss ventilation system; the
cabinet will not recirculate air within any part of the building. Similar
recommendations have been made for Class III cabinets. 5.7 There are good
arguments for not venting filtered air from safety cabinets directly to
outside air. 11 It is assumed that where air is directly vented to the outside,
in the event of a filter passing hazardous material dilution in the open air
could provide an additional safety factor. This may be far less effective
than might be anticipated since building geometry and air turbulence and
eddies might restrict dispersion of the hazard. A safe alternative would be
to exhaust air from cabinets through double HEP A filters arranged in
series and to vent into a work area filled with a plenum air supply and
HEP A filtered exhaust system. In terms of preventing release to the open
air, venting into the work place could be safer than venting direct to the
outside. The latter system offers advantages where several cabinets
operate simultaneously by avoiding the problems involved with total loss
systems where the amount of air exhausted from the cabinets exceeds the
amount of air entering the work area. Further, the arrangement allows for
easy relocation of cabinets within the work area without having to
disconnect exhaust ducts.
8.4.3 Class I cabinets

These are open-fronted cabinets that operate with negative pressure
ventilation (Figure 8.2a) and have a minimum inward air velocity at the
front opening of 0.75 m/s. Exhaust air passes through a high efficiency
particulate air (HEPA) filter before being exhausted to the outside. Class I
cabinets are intended to protect workers carrying out simple routine
microbiological operations and to prevent dissemination of possibly
hazardous materials from the immediate work area.
Although it may be considered appropriate to carry out biotechnology
process operations within Class I cabinets, it should be remembered that
this type of cabinet does not protect materials within it from possible
external airborne contamination. Also Class I cabinets do not, nor are they
intended to, provide total containment. A protection factor, defined as the
ratio of exposure to airborne contamination generated in the open to the
exposure from the same dispersal generated within the cabinet, is required
to set a minimum standard for containment. For BS 5726, this factor
should not be less than 1.5 X 105 . Where high energy processes, such as
centrifugation, are involved, particles of hazardous materials might be
projected out of the cabinet against the airflow. Hazardous materials might
also escape on removal of the gloved hand of an operator or as a result of
spillage. Care must also be taken to prevent undue perturbation of the air
flow into the cabinet by inappropriate positioning of equipment within the
cabinet or by external air movement caused by personnel movement,
l36 BIOSAFETY IN INDUSTRIAL BIOTECHNOLOGY
opening doors etc. Release of airborne material through the work opening
is almost inevitable should the exhaust air system fail. For these various
reasons, Class I cabinets are not suited to contain many biotechnology
process operations.
8.4.4 Class II cabinets

These are open fronted cabinets that not only have inward air movement
(75 ft/min) at the work opening but also provide a HEP A filtered laminar
airflow within the cabinet work space (Figure S.2b). The cabinets are
intended to provide operator protection, as for Class I cabinets, whilst
protecting materials within the cabinet from external airborne contamina-
tion. In these cabinets there is a downward airflow of HEPA-filtered air
over the work surface to which is added air from the work place that enters
across the working opening. This added air does not enter the work area of
the cabinet but is diverted by the descending air stream through the front
of the cabinet floor. Subsequently all air is HEPA-filtered; a portion of the
filtered air is discharged and the remainder recirculated downward over
the work area.
It is not always appreciated that Class II cabinets may not act as effective
safety cabinets 5 in so far as worker safety is concerned, therefore, in the
UK their use for category 3 and 4 pathogens is strictly forbidden. s .11 As
with Class I cabinets, the internal air flows are subject to perturbation by
cross draughts,12 the formation of air bulges at the working opening and
movement within or adjacent to the cabinet. 13 Because of the ease with
which cabinet air flows are perturbated, they should be considered as
inappropriate for containing biotechnology process operations involving
hazardous materials.
8.4.5 Class III cabinets

These are totally enclosed, ventilated cabinets of gas tight construction and
are designed to separate the worker from the cabinet interior at all times
(Figure S.2c). The cabinets have flexible gauntlets attached mechanically
to the cabinet by means of which the operators may carry out work within
the cabinet. In use the cabinets operate at a negative pressure with air
drawn into the cabinet through a single HEPA filter. Exhaust air is drawn
through a HEPA filter (or filters) before being exhausted. The use of an air
inlet filter offers the advantage of preventing release of hazardous
materials in the event of a fan failure. In addition, it prevents external
airborne contamination of materials being handled within the cabinet. This
latter feature suits Class III cabinets for process containment of
pharmaceutical manufacturing processes involving hazardous micro-
organisms or products.
BS 5726 requires there to be an airflow of 0.75 m/s into the cabinet when
gauntlets are detached and at least 3 m 3 /min through the inlet filter when
the gauntlets are attached. In addition the dimensions of the inlet and
exhaust filters should be such as to achieve a minimum negative pressure of
200 Pa within the cabinet under operating conditions.
8.4.6 Laminar flow work stations

It should be remembered that HEP A-filtered laminar flow work stations
designed to provide horizontal or vertical clean air flows are suitable for
aseptic purposes only and offer no protection to the worker. Hazardous
biotechnology processes must not be carried out in such areas.
8.4.7 Application of Class III cabinets to process containment

Whereas Class I and III cabinets designed to meet BS 5726 are effective for
carrying out, safely, a range of microbiological and biochemical techniques
on relatively small scales, for larger scale work, involving the containment
of bulky equipment, special designs are required that meet the particular
process requirements. At CAMR Porton Down, Class III type cabinets
have been designed and constructed that have proved very effective in
providing secondary containment of biohazardous processes both at
laboratory and pilot plant scales and for pharmaceutical manufacture
involving biohazardous organisms or products.
Where microorganisms are grown in substantial quantities (e.g. > 1 litre
at microbial cell densities of ca. lOlO/ml) there is inevitably a requirement
to process the culture further and to isolate component(s) for further
investigation or for use in diagnostic, therapeutic or prophylactic products.
Hence, when hazadous microorganisms or bio-hazardous products are
involved it may be necessary to achieve containment of many, if not all, the
process steps.
8.4.8 Fermentation
The Porton Mobile Enclosed Chemostat (POMEC) described by Evans
and Harris-Smith 14 was the first fermentation system designed and built to
enable stirred batch (20 I) and continuous (2.5 I vessel) culture of
pathogenic bacteria to be carried out without risk of escape of any aerosols
released from the fermenter vessel. A specially designed and constructed
culture apparatus was contained within a purpose-built Class III type
cabinet constituted of glass reinforced polyester resin. The various controls
and measurement indicators were panel-mounted and accessible on the
exterior of the cabinet. An inclined airlock with two UV lights and a liquid
disinfectant lock (dunk tank) were set into the cabinet wall to allow safe
ingress and egress of material. The safe transfer of culture into a transfer
was achieved by passing a tube from the culture vessel to a receiver bottle
through the dunk tank. The cabinet had additional safety features,
including an accident well, to contain gross spillage in the event of culture
vessel rupture and means to decontaminate either by formaldehyde vapour
and/or drenching in formaldehyde.
A system to separate and recover pathogenic bacteria grown in POMEC
using a continuous flow centrifuge was described by Evans et al. 15
Continuous flow centrifuges, particularly those of the vertical rotating
cylinder type, are notorious generators of aerosols and their containment
for harvesting of pathogens is essential. Evans et al. 15 described a novel
arrangement whereby a continous flow centrifuge contained within a Class
III type cabinet was connected to the POMEC. For this, the two cabinets
were connected via two ports fitted to the outside of the disinfectant lock of
the two cabinets. Connecting tubes were passed between the fermenter,
centrifuge and effluent receiving vessel. This system was similarly used to
link cabinets containing other process equipment such as a glass bead
disintegrator. 11
Despite being designed a quarter of a century ago, the principles
established with the POMEC are still used today although cabinet design
and fabrication have been modified to meet current operating standards. A
recent example of a cabinet designed to enclose a modern fermenter is
shown in Figure 8.3. Typically fermenters of this size (42 I) are steam
sterilisable in situ and require steam and water for temperature control. In
Figure 8.3 Cabinet designed to enclose fermenter.

addition they are equipped with sophisticated electronic control and

monitoring systems. A feature of modern cabinets is the need to provide
interfaces at the cabinet wall that allow physical and electronic services to
pass into and out of the cabinet without jeopardising the biosafety integrity
of the cabinet. These interfaces should be capable of being disconnected
whilst maintaining cabinet integrity.
Although the use of disinfectant fluid locks is still an effective means for
safe entry and exit of materials from cabinets, alternative methods are
being increasingly used because of safety regulations for the use of large
volumes of hazardous chemicals such as formaldehyde and the unsuitability
of such chemicals for use in pharmaceutical manufacturing areas. Items can
be safety passed into and out of cabinets through double-ended pass boxes
after being sanitised by spraying with disinfectants (e.g. 70% v/v
isopropanol) according to validated standard operating procedures.
Transfer port systems (such as those from La Calhene (GB) Ltd.
2 Stephenson Road, St. Ives, Huntingdon, Cambs. PE17 4WJ) were
developed for the safe transfer of radioactive materials between contained
handling areas. The lid of the transfer container is designed to fit to a
special port fixed into the cabinet wall such that once locked to the port the
container can be opened directly to the cabinet interior without exposing
the exterior of the container or its lid to the cabinet interior environment.
Whilst such systems offer high safety they have disadvantages, being
expensive and not convenient to use, they impose limitations on the size
and shape of items to be transferred and, most importantly, all cabinets are
required to be equipped with compatible ports.
Fluids can be passed into and out of cabinets using sterilisable male/
female connectors of the type illustrated in Figure 8.4. With such devices,
it is possible to make and break fluid lines aseptically and to allow
sterilisation of all surfaces exposed to fluids both before and after use.
8.4.9 Other processes

Class III cabinets incorporating such features have been designed and
constructed to contain a variety of modern downsteam processing
equipment including continuous flow centrifuges (Figure 8.5), cross-flow
filtration units for concentration steps, bead mill homogenisers (Figure
8.6) for disrupting cells and fast protein liquid chromatography (FPLC)
systems for protein purification (Figure 8.7). These have been used for the
production of hazardous biological substances such as neurotoxins of
Clostridium botulinum. 16 Because of the relatively compact size and large
operating capacity of most modern downstream processing equipment
these cabinets can readily be used for large-scale (up to and possibly
beyond 500 I initial fermentation volume) processes.
140 B10SAFETY IN INDUSTRIAL BIOTECHNOLOGY
(a)
------tt-
(b)
Figure 8.4 (a) Safety cabinet bulkhead with inner (large) screw cap and outer (small) sealing
screw cap. Note elastomer seal between bulkhead fitting and cabinet wall. (b) Inner
connector (left), showing retainer collar and sealing screw cap. Outer connector (right),
showing retaining collar and sealing screw cap. Note '0' -ring seal in process line to seal
connector to bulkhead fitting. (c) Operations sequence for connections to cabinet.
(l)(i) External connector fitted through cabinet wall and cabinet fumigated. Both process
lines close, using blanking caps. (2)(i) Following fumigation, blanking caps removed and
process line connections made. (ii) Cabinet fumigated. (3)(i) On completion of transfer,
connection is broken and both lines capped. (ii) Cabinet fumigated. (4)(i) Inner cap fitted
into bulkhead fitting. (ii) External connector can be removed to complete transfer.
(c)
IN OUT
u-b---ml
6
Figure 8.5 Continuous flow centrifuge.
Figure 8.6 Bead mill homogeniser.

Figure 8.7 Fast protein liquid chromatography (FPLC) system.
S.4.10 Flexible film isolators

The various cabinet systems described above were all of rigid construction.
Experience shows that rigid cabinets offer many advantages; they are
robust, can support heavy items of equipment and peripherals such as pass
boxes and interface panels. However, it is practical to consider the use of
plastic film isolator technology as an alternative approach to providing
physical encapsulation. 17
The use of flexible film isolators is a well established means of providing
barriers between patients or animals 18 whilst allowing essential support
duties, such as nursing or animal husbandry respectively, to be carried out
safely. The technology also offers an effective means of creating aseptic
environments within relatively 'dirty' environments. The isolators can be
operated to the same containment standards as rigid construction Class III
type cabinets and may provide the most effective solution to containment
problems. 19 They offer advantages including good visibility, improved
worker comfort and cost over rigid cabinets but are perhaps best suited to
operations that do not involve heavy, bulky, complex equipment.
8.5 Design engineering for secondary containment
8.5.1 Fermentation
The provision of secondary containment in the form of Class III type safety
cabinets has proved satisfactory for laboratory-scale fermenters of up to
50 I working volume. Complete physical enclosure is, however, not a
practical solution to the problems of containing pilot-plant scale or larger
systems. Features that represent difficulties in achieving effective contain-
ment of a typical stirred bioreactor are shown in Figure 8.8. Design
approaches for containment of large-scale processing of biohazardous
materials have been discussed by several groupS.4, 17,20-24 A 225 I pilot plant
reactor system designed and constructed so as to be suitable for
biohazardous fermentation was described by Hambleton et al. 4 The design
enables the fermenter to be operated at containment levels above the
requirements of good industrial large-scale practice (GILSP) without
secondary (physical encapsulation) containment of the whole plant. Indeed
the system is routinely used at CAMR for operations at ACDP category 3
(Biosafety level B3 large-scale; BL3-LS 5 ) in conjunction with downstream
process steps contained within Class III cabinets (see above).
The main biosafety features of the fermentation system include the use
of steam barriers on double O-ring seals, supply lines and mechanical seals
on stirrer shafts, multiple O-ring seals, piping of condensate lines and
pressure relief systems to a 'kill-tank', double (in series) filtration of inlet
Inlet filters pressure relief valve

Addition lines Off gas filters
Harvest valve CIP
Sight glass
- - - Top plate seal
Sensors _________ Sampling device
Impeller drive seals
Figure 8.8 Stirred bioreactor.

Figure 8.9 Fermentation system allowing localised containment of sample value and probe
entry ports.
and off-gases, elimination of unnecessary piping joins and use of welded

piping and hermetically sealed steam condensate traps. A mobile flexible
isolator unit can also be used to allow localised containment of sample
valve and probe entry ports (Figure 8.9). The safe operation of such a
complex plant requires effective validation and integrity testing4 and the
reader is referred to chapters 11, 12 and 13 for consideration of safety
validation and testing. Inevitably, it is not practical to engineer secondary
containment features on all primary containment barriers without incurring
considerable expense and so planned preventive maintenance (PPM) is an
important aspect of the safe operation of such a system. 4
8.5.2 Other processes

It is significant that manufacturers increasingly are seeking to incorporate
containment features into the design of process equipment such that
physical enclosure may not be necessary, except at the highest containment
levels. Particular examples of containment design for centrifuges and cell
disruptors are described elsewhere in this book (see chapter 9) but the
146 BJOSAFETY IN INDUSTRIAL BIOTECHNOLOGY
Constant Systems (Constant Systems Ltd., Warwick, UK) homogeniser

represents a good example of a design specifically intended to afford high
intrinsic biosafety for a unit process having high potential to generate
aerosols. 25 Undoubtedly, good biosafety design is now seen as imparting
market advantages by manufacturers and users alike. This is likely to result
in the appearance of more inherently safe bioprocessing equipment. It
must be remembered, though, that such designs do need to be tested
effectively to validate their biosafety performance (see chapters 11 and 13).
Additionally, users must consider their process as a whole. It is of no value
to have a series of high contained unit processes if the means whereby such
processes are interlinked do not have the same level of containment.
8.6 The cost of containment
The cost of rigid Class III type cabinets varies depending on the size of the
cabinet and the complexity of the various service interfaces and peripheral
systems (pass boxes etc.) built in. Typically a simple large cabinet having
no specialist modification would currently cost some £10,000-15,000 whilst
more complex cabinets to contain, say, a 42 I fermenter would cost in the
region of £25,000. The cost of flexible isolators will likewise be dependent
on size and complexity but one such as that in Figure 8.8 would currently
cost in the region of £2,500.
The inclusion of any high containment design features into large-scale
fermenters clearly must have cost implications. The extent of the premium
will vary depending on the containment level required and the exact nature
of the design solutions employed. Nevertheless, it has been estimated 23
that basic fermenter costs will increase by at least 30% for each increase in
containment level. A high containment fermenter forms only part of an
integrated containment system that includes upstream and downstream
processing together with effluent handling systems. Furthermore, high
containment plant would need to be sited within an effective tertiary
containment environment. The combined financial consequences of all
these factors could result in costs of two to five times those of conventional
pilot plants. 4 . 23 Careful consideration, therefore, needs to be given to
ways of achieving appropriate process containment without undue and
unnecessary cost consequences.
8.7 Conclusions
The application of containment principles developed for the safe culture

and process of pathogenic micro-organisms can be seen to be appropriate
to modern process biotechnology. Physical encapsulation of fermenters is a
practical solution with fermenters of up to 50 I working volume. Indeed,

some modern plant is of such compact design that containment of 100 I
working volume bioreactors within Class III type cabinets is now feasible.
For larger volume fermenters enclosure may not offer a practical option
and it is more appropriate to consider means whereby secondary
containment may be 'engineered in' to the equipment. For large-scale
operations containment needs to be seen in the context of the total system,
including off-gases, effluent etc. not merely of one element such as the
bioreactor.
Many modern downstream processes involve the use of equipment items
that are relatively compact yet possess high process capacity and are
capable of remote, even pre-programmed control. Enclosure within Class
III type cabinets offers a practical and reliable means of ensuring that such
process steps can be carried out without risk of release of potentially
hazardous products into the workplace environment.
References
1. Ratledge. C. (1985). Is biotechnology safe? 1. Chern. Tech. Biotechnol., 358, 1-2.

2. Leaver, G., Norton, M.G. and Hambleton, P. (1988). Enabling technology for safe
biological processing - a collaborative approach, Proc. Interphex Con., 28. Cahners
Exhibn. Ltd., Richmond, pp. 48-58.
3. Hambleton, P., Bennett, A.M., Leaver, G. and Benbough, J.E. (1992). Biosafety
monitoring devices for biotechnology processes, Trends. Biotechnol., 10,192-199.
4. Hambleton, P., Griffiths, J.B., Cameron, D.R. and Melling, J. (1991). A high
containment polymodal pilot-plant fermenter - design concepts, 1. Chern. Tech.
Biotechnol., SO, 167-180.
5. Meslar, H.W. and Geoghegan, R.F.Jr. (1991). Bioconta'inment facilities theory and
practice, Pharrn. Eng., 11,27-33.
6. Turner, M.K. (1989). Categories of large-scale containment for manufacturing processes
with recombinant organisms. In Biotech. Gen. Eng. Rev. (Russell, G.E. and Tombs,
M.P. ed), 7, 1-43.
7. Collins, C.H. (1988). Laboratory Acquired Infections: incidence, causes and prevention
(2nd edn). Butterworths: London.
8. Advisory Committee on Dangerous Pathogens (1990). Categorisation of pathogens
according to hazard and categories of containment (2nd edn). HMSO, London.
9. Leaver, G. and Hambleton, P. (1992). Designing bioreactors to minimise or prevent
inadvertent release into the workplace and natural environment, Pharrn. Technol., 4,18-
26.
10. Department of Health and Social Security/Scottish Home and Health Department
(1978). Code of practice for the prevention of infection in clinical laboratories and post-
mortem rooms. (Howie Report). HMSO, London.
11. Melling, J. and Allner, K. (1981). The containment of micro-organisms. In Essays in
Applied Microbiology. (Norris, l.R. and Richmond, M.H. eds). l. Wiley & Sons: New
York, Chp. 11.
12. Rake, B.W. (1978). Influence of crossdrafts on the performance of a biological safety
cabinet, Appl. Env. Microbial., 36, 278-283.
13. Clarke, R.P. and Mullan, B.J. (1978). Airflows in and around linear downflow safety
cabinets,l. Appl. Bacteriol., 45,131-135.
14. Evans, C.G.T. and Harris-Smith, R. (1970). The POMEC: An apparatus for growing
dense cultures of pathogenic micro-organisms. In Tech. Ser. 4 Soc. Appl. Bacteriol.
(Baillie, A. and Gilbert, R.l. eds). Academic Press: London, pp. 137-149.
15. Evans, e.G.T., Harris-Smith, R., Stratton, J.E.D. and Melling, J. (1974). Design and
construction of a ventilated cabinet for a continuous flow centrifuge, Biotech Bioeng., 16,
1681-1687.
16. Wadsworth, J.D.F., Desai, M., Tranter, H.S., King, H.J., Hambleton, P., Melling, J.,
Dolly, J.O. and Shone, e.e. (1990). Botulinum type F neurotoxin: Large scale
purification and characterisation of its binding to rat cerebrocortical synaptosomes,
Biochem. 1.,268,123-128.
17. Allner, K. (1985). Laboratory and equipment design for containment of biohazards. In.
Comprehensive Biotechnology. The Principles, Application and Regulations of Bio-
technology in Industry, Agriculture and Medicine. (Cooney, e.L. and Humphry, A.E.
eds). Pergamon Press: New York, pp. 468-485.
18. Trexler, P.e. (1976). The development of isolators, Post-Grad. Med. 1., 52, 545-549.
19. Dunnil, P. (1982). Biosafety in the large-scale isolation of intracellular microbial
enzymes, Chem. Ind., 22, 270-273.
20. Walker, P.D., Narendranathan, T.J., Brown, O.e., Woodhouse, F. and Vranch, S.P.
(1987). Containment of micro-organisms during fermentation and downstream process-
ing. In Separations for Biotechnology (Verrall, M.S. and Hudson, M.J., eds). Ellis
Horwood: Chichester, pp. 469-482.
21. Leaver, G. (1991) Measuring and monitoring containment in bioprocessing equipment.
In Hazards XI - New Directions in Process Safety. I. Chern. E. Symp. SeT. 124.
Hemisphere, New York, pp. 349-361.
22. Chapman, e. (\989). Client requirements for supply of contained bioreactors and
associated equipment. In Proceedings of the DTlIHSEISCI Symposium on Large Scale
Bioprocessing Safety. (Salusbury, T.T. ed). Warren Spring Laboratory report
LR748(BT). Stevenage, pp. 58-62.
23. Pennman, I. (1989) Bioreactors: Technical considerations in containment. In Proceedings
of the DTlIHSEISCI Symposium on Large-scale Bioprocessing Safety. (Salusbury, T.T.
ed). Warren Spring Laboratory Report LR746(BT), Stevenage, pp 63-70.
24. Flickinger, M.e. and Sansone, E.B. (\984). Pilot- and production-scale containment of
cytotoxic and oncogenic fermentation processes, Biotech. Bioeng., XXXVI, 860-870.
25. Foster, R. (1992). Cell disruption: breaking up is hard to do, BiolTechnology, 19, 1539-
1541.
9 Containment in downstream processing
1.S. DEANS and I.W. STEWART
9.1 Introduction
Recent advances in molecular biology and recombinant DNA (r-DNA)

technology have enabled products of animal, plant or microbial origin to
be produced in large quantities by culturing bacteria, yeast, plant or
mammalian cells. A typical bioprocess will consist of growing cells in a
suitable nutrient medium, followed by the recovery and purification of the
product: downstream processing. If the desired product is extra-cellular
then the first stage in processing will be the removal of large solids and cells
by centrifugation or filtration. The broth is then fractionated or extracted
into major fractions; this can be done using processes such as chromato-
graphy, liquid-liquid extraction or precipitation. The fraction containing
the product may then be purified further, often with more specialised
chromatographic techniques. However, the majority of products remain
intracellular, enclosed in a soluble or insoluble form within the cell. Some
of these products are cytoplasmic, others are associated with cell
membranes, cell wall components or the periplasm (where present). In this
case, the cells must first be harvested to form a concentrated slurry or
paste, then disrupted to release their products into solution for subsequent
extraction and purification. 1 Flow sheets illustrating typical fermentation
and downstream processes are shown in Figure 9.1.
Of all process equipment, fermenters and centrifuges are most likely to
release micro-organisms. 2 According to Kearns, it is possible to kill process
micro-organisms after the fermentation is complete so there may be no
need for containment in further processing steps to eliminate the infectious
risk. However, even dead micro-organisms could present an allergenic
risk. Most reported health problems have been associated with downstream
processing. 3 Dunnill 4 states that the greatest demands on biosafety
occurred from the time the broth leaves the bioreactor to the final
processing steps, as this involves dealing with large amounts of cell debris.
Downstream processing frequently involves the use of machinery that
rotates at high speeds (centrifuges) or exerts increased pressure (liquid
extrusion homogenisers, cross-flow microfiltration and ultrafiltration
units). Such energetic processes may generate aerosols of potentially
Intracellular Extracellular
product product
Fermentation
1
Filtration
Fermentation
or
centrifugation
1
Cell
residue
Filtration
or
centrifugation
disruption
supernatant
1
Filtration
or
Product
extraction
and
recovery
centrifugation
L supernatant
Product
extraction
and
recovery
Figure 9.1 Flowsheet of typical fermentation process.
hazardous material and process equipment design should seek to prevent

or minimise such release.
This chapter reviews current legislation and examines the containment
of large and pilot scale equipment used in downstream processing.
Darbyshire 5 defined large-scale operations as those carried out in pilot or
production scale facilities, referring to more than 1 kg of cell paste or 20
litres of supernatant fluid. Processes that employ high pressures or use
high rotational speeds are particularly considered, these being more likely
to generate aerosols should a breach of containment occur. Details of
commercially available equipment, together with any containment features,
are presented.
DOWNSTREAM CONTAINMENT 151
9.2 Regulations and guidelines
Current containment guidelines fall into two areas:

1. Conventional pathogenic organisms; guidelines for small-scale or
laboratory use have been issued by the Advisory Committee on
Dangerous Pathogens (ACDP) in the UK and the Centers for Disease
Control (CDC) in the USA, together with the National Institutes of
Health (NIH).
2. Regulations covering the use of genetically-manipulated organisms in
the UK and USA and elsewhere are described in Chapters 1-5.
Although the guidelines may appear to address primarily the culturing of
hazardous micro-organisms it should be remembered that subsequent
process steps may pose equivalent, or higher, risk as the organism becomes
more concentrated, for example. Hence more stringent control measures
may be needed for the final purification step than for the initial
fermentation. The ACGM 6 introduced the idea of flexible containment.
That is, it may be appropriate to select and combine containment on the
basis of unit operation assessment rather than have a fixed category of
containment for the whole process. The assessment procedure is complic-
ated where intermediate products or by-products are involved in the
process. Whilst the hazard presented by the micro-organism and its final
product may be known or predictable, it is unlikely that the same
information will be available for intermediates.
Before describing equipment in detail it is worth examining the general
containment features that might be necessary to meet the requirements for
a particular category of operation.
A good example of the different levels of primary containment necessary
to meet the different containment categories is in the use of seals. 7 A single
seal system is the basic arrangement to provide a barrier between micro-
organisms and the workplace environment. If the equipment is operating
above ambient pressure, any failure of the seal would result in flow into the
workplace environment. A double seal arrangement offers extra security
although it could be argued that both seals are likely to wear at the same
rate. Failure of the primary seal would be checked by the secondary seal
against emissions into the workplace. Failure of the secondary seal would
not result in any obvious problem unless the primary seal failed. In both
cases, it would be difficult to know if one of the seals failed. It could also be
argued whether the gap between the two seals could be adequately
sterilised. If not, migration by microbial growth could give rise to
contamination problems.
Regular maintenance and/or seal replacement would be the obvious
recommendation for the double and single seal arrangement before seal
failure occurred. A higher security system can be designed by employing a

barrier fluid between the two seals. Steam is often used as the barrier fluid
(steam tracing), so that any micro-organisms breaching the primary seal
are killed and removed from the system. Commonly, the condensate will
be directed to a kill tank rather than to the steam boiler for heat recovery.
Other barrier fluids used are sterile water, biocides and glycerol, usually at
higher pressure than that in the contained device.
Since the commercialisation of genetic modification techniques, contain-
ment of bioprocessing equipment has taken a higher profile. To date,
however, national and international biosafety activity has focused on
defining hazard groups of micro-organisms and the corresponding contain-
ment levels which need to be used, rather than looking at bioprocessing
equipment itself. The formulation of standards for biotechnology is
starting to attract national and international attention. The European
Committee for Standardisation (CEN) has established a technical com-
mittee (TC 233) to investigate standards in biotechnology. There are four
working groups, of which WG2 is related to large-scale biotechnology
activities and WG4, which is addressing bioprocess equipment. The CEN
activities have commenced following a mandate from the Commission of
the European Communities (CEC). The activities are in line with the
general aims of the Green paper on the development of European
standardisation (COM (90) 456) and complement the Commission's
legislative actions in biotechnology. Testing of bioprocessing equipment
would be an integral part of standards work for WG4. Areas relevant to
biosafety include cleanability, sterilisation and leak tightness of a range of
commonly used bioprocessing equipment. The target date set for these
standards is March 1996. These standards could be incorporated into CEC
directives (those already in existence and future directives) in the form of
technical annexes. In the UK, the British Standards Institution (BSI) has
set up four shadow committees to coordinate UK input to the CEN
working groups.
For the purposes of this account downstream processing has been split
into two main areas: cell separation and cell disruption.
9.3 Cell separation
9.3.1 Filtration
Filtration is one of the commonest processes used, at all scales of
operation, to separate suspended particles from a liquid, using a porous
medium which retains the particles but allows the liquid to pass through.
There is potentially a wide variety of filtration devices available for initial
cell separation. However, the choice is restricted in biotechnology due to
limitations imposed by the nature of fermentation broths. The filters used

for initial solids recovery (e.g. recovery of biomass from fermenter broths)
are of two main types: the rotary vacuum drum, a continuous filter, and the
filter press, a batch filter. Generally, filter presses are slow and labour
intensive and are usually only used at small scales. They are often found in
the older style biotechnology processes such as brewing and distilling.
Rotary vacuum drum filters can be used for larger scale continuous
operations and they are more often found in the pharmaceutical and food
industries. It is easier to contain a rotary vacuum drum filter, e.g. using
local exhaust ventilation, than a filter press but it is not possible to operate
a rotary vacuum drum in an aseptic manner. Filter presses usually operate
at pressures between 5 and 7 bar. Rotary vacuum drum filters operate such
that the vacuum pressure is applied internally so the filtrate is drawn
through the filter, into the drum and finally into a collecting vessel.
. Considering the low pressures and low rotational speeds used in such
devices, their operation should not present a problem in terms of
containment and aerosol formation. However, when the cake is removed
from the filter there is potential for considerable release of biological
material.
9.3.1.1 Membrane filtration

Further downstream, in order to concentrate and purify the product more,
it is possible to use membrane filtration. Here, some form of semi-
permeable membrane is used to separate the components of a liquid
stream. In most of the commercially important processes the driving force
is pressure, the solvent (usually water) is driven through the membrane
while the solute(s) are retained. This type of process includes reverse
osmosis, ultrafiltration and microfiltration.
Cross flow membrane filtration has attracted attention in recent years as
an alternative to high g force centrifugation. Scaling up from laboratory or
pilot scale is relatively easy, as additional modules/units can be added to
increase the surface area for filtration; this can, however, be costly. The
major disadvantage of this techniques is the detrimental effect of
membrane fouling on filtration rates and subsequent product recovery.
Generally, membranes are considered to have less potential for the
emission of aerosols or breach of containment, compared with centrifuges.
Difficulties may be encountered when cleaning membranes in situ. It may
only be achieved adequately through the dismantling of the filter units.
This process could be hazardous in terms of aerosol production, so
adequate precautions should be taken, i.e. the use of secondary contain-
ment.
Traditionally, most membranes have been fabricated from plastics such
as polysulp!lOnes and cellulose acetate. In recent years, inorganic
membranes, made from materials such as ceramics and metals, have been
introduced and these have found application in cell recovery. The

robustness of inorganic membranes are generally higher than plastic
membranes, offering higher temperatures (suitable for sterilisation) and
higher operating pressures. Ceramic membranes, however, are vulnerable
to heat shock and mechanical shock, i.e. they are brittle and can be
broken.
A wide range of membrane equipment designs are available for cell
recovery and other applications at both pilot and production scale. The
inherent containment features vary widely. Some systems only make use of
flexible plastic pipe connected by 'Jubilee' clip type fittings. IDF and
Triclover sanitary connections are found on devices with a higher degree of
engineering.
Plate and frame membrane filters rely on seals on each plate and the
clamps on the assembly for containment. Hollow fibre systems are pressure
limited and are often fitted with a pressure switch in order to prevent the
recirculation pump reaching the bursting pressure of the fibres.
Tubular membrane systems appear to offer the best containment
features because they are usually constructed with hard piping and require
fewer seals to the outside environment. The collection shrouds on the low
pressure side would provide a convenient shield should the membranes or
the filter seals fail. Metal membranes can be constructed using welding and
this negates the need for seals.
In some cases of pilot scale filtration, entire units have been enclosed as
a secondary containment precaution (see Chapter 8). A recent commercial
development is the MBR-Sultzer dynamic filter which is available in three
sizes. Dynamic filtration is the same as cross flow filtration with little or no
recirculation. The cross flow effect is derived from the spinning of the inner
surface filter. This type of filter is more efficient, has a lower pump rate
and a much higher linear velocity across the filter surface, than
conventional cross flow filtration units. There is also little or no damaging
effect on sensitive cells. The medium size has the same capacity as the
Westfalia SA-7 separator. Van Hemert and Tiesjema8 concluded that the
dynamic filter is suitable for work requiring strict aseptic and primary
containment conditions. The use of a double mechanical seal on the
rotating shaft could offer a higher degree of containment if required.
9.3.2 Centrifugation
The separation of biomass from growth media is a difficult operation, as
cells have almost the same density as their surrounding medium, are small,
are able to form stable colloids and are cohesive. Sedimentation of cell
debris presents an even more difficult problem for biotechnologists and the
choice of separation technique is limited. Solid bowl and tubular bowl
centrifuges are relatively inexpensive and have in the past been chosen for
use in the biotechnology industry. They are useful for small batches, but
are labour intensive because the solids have to be dug out by hand. The
scroll decanter centrifuge has limited use in the biotechnology industry
because of the low g forces generated. It should, however, be better
contained than the traditional solid or tubular bowl type of centrifuge. In
reality, only the higher g force devices such as disc stack and tubular bowl
centrifuges are used at large scale. Decanter and solid bowl centrifuges are
however used for separating bigger particles, such as yeast or flocculated
bacteria.
9.3.2.1 The Centritech Cell Separator

A recent design for laboratory scale separation is the Alfa Laval Centritech
Cell Separator which is designed for aseptic separation of mammalian cells
in a completely closed system without any rotating seals. 9 It contains a
spinning disposable bladder which lies within the rotor that spins at speeds
up to 1200 rpm. The centrifugal force created within the bladder separates
the culture into cell concentrate and fluid. A system of tubing and pumps
enables the cell culture to enter the bladder directly from the fermentation
vessel. The tubing is connected to the rotating bowl in a way that allows
one end of the tube to rotate while the other end is standing still. Thus the
separation system is totally enclosed. Further primary containment is
provided by a sealed lid on the rotor chamber and an external hood which
acts as built on secondary containment. The separation insert is delivered
as a pre-sterilised disposable plastic bladder. The novel design of the
Centritech Cell Separator, with no openings to atmosphere and no rotating
seal, means that it is unlikely to produce biological"aerosols during normal
operation. The containment of this device can be tested by simulating
rupture of the bladder. Micro-organisms are detected outside the primary
containment of the sealed lid; however, none are detected outside the
secondary containment. If the interior becomes heavily contaminated,
decontamination may be difficult.
The Centritech Cell Separator has a very low separating capacity (100 II
hour) and therefore cannot compete with disc-stack separators (see section
9.3.2.5).
9.3.2.2 Tubular bowl centrifuges

Tubular bowl centrifuges are operated in a semi-batch mode; the
suspension to be separated is fed continuously to the bowl. The solids
collect in the bowl which has to be then dismounted from the centrifuge for
the solids to be recovered and for cleaning. This inevitably involves
exposing operators to biologically active material, unless the cleaning is
carried out in a biological safety cabinet. Inert plastic liners are available
which reduce exposure by discretely packaging the material. Tubular bowl
centrifuges are generally driven through the upper spindle, which also
provides the supporting mechanism. The drag bearing at the lower end is
merely a guide bush which allows the spinning bowl to establish its axis of
rotation through its centre of gravity. Depending on the bowl size, liquid
throughputs of 60 to 4500 IIhour are possible, but solids capacity is limited
to 0.05 to 2.5 kg/hour, due to the need for intermittent discharge.
Alfa Laval-Sharples supply most of the tubular bowl centrifuges used in
bioprocessing at present, particularly for the separation of cell debris. The
traditional Super Centrifuge has been widely used but it is not inherently
contained and is usually used with some form of secondary containment.
There are two principal sources of aerosol generation. The first is in the
drag bearing at the bottom of the bowl. This is not designed to be a tight fit
and liquid can leak from the inlet feed tube and the bowl so that the outside
of the bowl is wetted. The bowl rotates at speeds of up to 25 000 rpm and
therefore any fluid on the outside of the bowl is easily atomised into the
centrifuge casing. The centrifuge casing is open to the outside through the
gap between the top of the bowl and the discharge pans. The second source
of aerosol is from the centrate discharge at the top of the bowl. Centrate
impacts onto the inside of the discharge pans at high speeds. Again, the
aerosols generated escape between the bowl and the discharge pans. The
latest versions of the Alfa Laval-Sharples tubular bowl centrifuges have
addressed both of these problems.
The Alfa Laval-Sharples AS-16VB is shown in Figure 9.2. In this version
the bowl bottom is self sealing through redesign of the drag bearing
assembly. Comparing the new drag bearing assembly with that of the Super
Centrifuge, the new version contains a seal to prevent leakage from the
inlet feed onto the outside of the bowl. Also the new version of the drag
bearing housing is vented via the outlet tube shown. The centrifuge casing
can also be vented via an outlet tube which can be directed to a HEPA
filter. All covers on the centrifuge casing are sealed with single O-rings.
The major feature which minimises aerosol generation is that, in the AS-
16VB, the centrate is removed by a centripetal pump shown at the top of
the bowl. There is a single simple seal above the centripetal pump on the
drive shaft. The manufacturers claim that these features make the AS-
16VB suitable for BL-I-LS operation which is equivalent to OECD
Category 1, where the requirement is to minimise release. This model can
be fitted with a steam inlet and bursting disc assembly so that the device
can be steam sterilised to 15 psig. However, this requires the centrifuge to
be opened to the atmosphere for a short period and the manufacturers
therefore do not recommend this model for aseptic operation.
Alfa Laval-Sharples also manufacture the AS-26SP tubular bowl
centrifuge. The AS-26SP has all the features of the AS-16VB but in
addition the drive shaft is sealed with a triple mechanical seal. This
centrifuge can be steam sterilised before and after operation without
opening it to the atmosphere. The manufacturers claim that these
Single Seal
Centlpetal Pump
1 _____- - - - Bowl
Drag Bearing
Figure 9.2 Sharples AS-16VB, 0, single O-ring seal; X extract via hepa filters if required.
additional features mean that the AS-26SP can operate at a containment

level of BL-2-LS. There is some question whether the single O-rings of the
centrifuge casing would permit BL-2-LS operation. This containment
category is equivalent to OECD Category 2 or ACGM Category LS-2 and
the requirement would be to prevent release. Chapman \0 suggests that to
prevent release a double O-ring, possibly with steam tracing, would be
necessary. It could be argued that the casing O-rings are effectively a back-
up to the primary seals on the bowl. Also, the ventilated housing acts as a
built-in secondary containment during operation. However, the entire
casing is pressurised for sterilisation and in this case only the single O-rings
are preventing a breach of containment.
Models AS-26 and AS-26VB are also available from Alfa Laval-
Sharples. The AS-26 is not contained and the AS-26VB has the same
containment features as the AS-16VB. The manufacturers claim that the
AS-26VB can also operate to containment category BL-I-LS.
9.3.2.3 Solid bowl centrifuges

Solid bowl centrifuges have the feed stream entering from the bottom of
the bowl and moving upwards. Solids are sedimented in the bowl and
centrate flows out over a weir. Single chamber, triple bowl and multi-
chamber devices are available, each with a larger surface area and hence
greater efficiency. Sedimented solids can be removed intermittently
manually or automatically using a plough with the bowl rotating slowly.
Normal operational speeds lie between 450 and 3500 rpm, developing
centrifugal forces in the range of 500 to 1200 g force.
A novel design, the Alfa Laval-Sharples SP-725 Superhelix, is shown in
Figure 9.3. This is a vertical solid bowl centrifuge. The product stream is
fed through a stationary feed nozzle at the bottom of the bowl and gently
accelerated to bowl speed in the conical feed zone. Under the action of
centrifugal force, the solid phase moves to the bowl wall where the helical
conveyor forces it downwards to the beach. Here, the solid phase is further
concentrated. Solids are finally discharged into the solids chute at the
bottom of the centrifuge, and to prevent escape of material the method of
solid collection must be contained. The manufacturers state that they can
supply suitable equipment for solids handling. The centrate is discharged
by a centripetal pump at the top of the bowl. The automatic solids
discharge of the Alfa Laval-Sharples SP-725 represents an improvement in
solid bowl centrifuge design.
The Superhelix has many similar containment features to the Alfa Laval-
Sharples AS-26SP discussed earlier. The manufacturers claim that the SP-
725 is suitable for operation at containment level BL-2-LS but the question
again arises whether the single O-rings of the centrifuge casing seals are all
that are required to prevent release.
The Alfa Laval-Sharples SP-725 Superhelix was planned to be available
in the UK in 1991, but the authors do not know of any such machines in
use. Alfa Laval-Sharples plan to build and supply two larger models of the
SP-725 but no details are available to date.
9.3.2.4 Scroll-discharge (decanter) centrifuges

Scroll-discharge or decanter centrifuges operate with continuous feed and
continuous discharge of both solids and liquid. The solid bowl of these
machines is conical. Settling solids are moved along the bowl wall and
ultimately discharged by a screw conveyer which rotates at a slightly faster
speed than the bowl shell. They are generally used for the clarification of
Gas Purged
Triple Mechanical
Seal
.L Sedimented
, Solids
Figure 9.3 SP-725 Superhelix.
particles larger than 5 flm in feed slurries of up to 60%, solids

concentration.
To date, scroll discharge centrifuges have found limited use in the field
of direct bacterial separation. Recently, Sharples upgraded one of their
models to operate at higher g forces (around 5000 g) for applications in
yeast and flocculated bacteria recovery: the P-3400 HHS continuous
decanter centrifuge. Unlike solids-ejecting disc-stack centrifuges, there is
no shock wave on discharge, since the solids are continuously removed and
therefore aerosol generation is less likely. Also, the downstream end of the
drive shaft is fitted with a triple mechanical seal with sterile gas purge and
centrate discharge is via a centripetal pump. The machine is claimed to
comply with the NIH guidelines on biohazard as well as FDA (Food and
Drug Administration) good manufacturing practice. However, it remains
to be seen whether these machines provide a viable alternative to disc stack
centrifuges.
Westfalia Separator also manufacture a range of decanter centrifuges.
Of these, only the CA 226-290 and the CA 226-290 are supplied with
sealed casings or have optional additions to seal the housings. Only the CA
226-290 has a CIP facility and none of the models are steam sterilisable.
9.3.2.5 Disc-stack centrifuges

Disc-stack centrifuges predominate at production scale in biotechnology.
They consist of a solid bowl containing a series of hollow truncated cones
('discs') stacked one upon another. Feed suspension enters the centrifuge
through a central feed pipe, passes out of the edge of the bowl then
upwards and inwards through the stack of discs. Solids settle onto the
lower surface of each cone and clarified liquid moves inward and upwards
to reach an annular overflow channel, emerging at the neck of the bowl
around the feed pipe. The sedimented solids slide off the disc and collect in
the space between the stack of discs and the bowl wall.
The different types of disc-stack centrifuge are distinguished by the
method in which they discharge solids from the space between the discs
and the wall. II In solid-bowl or solids-retaining disc-stack centrifuges, the
machine has to be stopped for solids to be removed manually. In nozzle-
discharge disc-stack centrifuges solids are discharged continuously. Opening-
bowl, solids-ejecting or intermittent discharge disc-stack centrifuges
discharge solids either at preset time intervals or discharge is automatically
triggered by the load on the bowl.
Nozzle-discharge disc-stack centrifuges. Solids discharge from nozzle-

discharge disk-stack centrifuges is normally continuous. Two different
types exist. In the Alfa Laval BTUX 510, the solids are collected in conical
storage spaces, with concentrate tubes located around the largest diameter
of the bowl in the apex of the cones (Figure 9.4). Solids pass through the
concentrate tubes and the vortex nozzles into the paring tube chamber.
The concentrate is skimmed off by the paring tube and discharged under
pressure. The clarified liquid phase is displaced towards the centre through
the disc-stack. The centrate is then discharged under pressure via a paring
disc pump at the top of the frame hood. In the BTUX 510, the unique
vortex nozzles automatically compensate for variations in feed flow rate or
feed solids concentration to ensure a constant concentration of the
discharged solids phase. 12
In the second type of nozzle-discharge disc-stack centrifuge the solids are
collected in a triangular storage space with nozzles located around the
largest diameter of the bowl. The size and number of nozzles can be
Pairing
Disc Pump
Pairing
Tube
Concentrate
Tube
Vortex
Nozzles
Figure 9.4 BTU X SIO Nozzle discharge DSC; 1st type.
optimised for each application, so that too dilute a slurry is not discharged,
but it is sufficiently fluid to flow through the nozzles.
A further development of the nozzle-discharge disc-stack centrifuge
incorporates an additional annular valve at the periphery of the bowl. 14
This centrifuge therefore has the same type of solids discharge as a solids-
ejecting disc-stack centrifuge. This hybrid is also equipped with extra
nozzles around the bottom of the bowl. As well as giving the centrifuge a
CIP facility, the additional feature means that blocked discs can be cleared
by initiating a full desludging. A typical example of this type of centrifuge

is the Westfalia HDA 300.
Krook 9 described the containment features of the Alfa Laval BTUX
510. Solids discharge is by compressed air, hence the bowl of the centrifuge
is above ambient pressure. Two double mechanical seals are fitted to the
centrifuge spindle (Figure 9.5). Sterile water at a pressure of 3 to 7 bar is
used as a sealing liquid to keep the seals closed. A small amount of sterile
water may leak through the seal faces into the seal housing at 2. Product
(3) will also be present in the seal housing. The contaminated sealing liquid
(4) is discharged to a waste tank. The pressure in the bowl casing is equal to
that in the waste tank which is vented to atmosphere through an off-gas
filter. Solids are discharged through a cyclone with air from the cyclone
outlet recirculated to the bowl casing. The BTUX 510 has a CIP facility
and can be steam sterilised for decontamination.
Opening-bowl (solids-ejecting) disc-stack centrifuges. Opening-bowl, or

solids-ejecting disc-stack centrifuges are very common in large and pilot-
scale biotechnology plants. They have been the most widely researched in
terms of sterile or contained operation and are similar in design to solid-
bowl and nozzle-discharge disc-stack centrifuges, but here peripheral ports
in the solids collection area are held closed by water or air pressure to
retain sedimented cells during separation. At a predetermined time
interval, the feed-stream ceases and the ports open to allow the solids to
Sealing
Sealing - -..
~~ Liquid
Outlet
Liquid -------J~l
Inlet
4~
Figure 9.5 BTU X double mechanical seal. 2, Sealing chamber; 3, product side; 4. leakage
outlet.
eject (termed 'desludging'). They are often the only type of centrifuge
capable of continuous separation of cells and cell debris because the
frequency of solids discharge can be set to maximise the sedimented solids
concentration.
Hemfort and Kohlstette 13 discuss sealing arrangements for Westfalia
centrifuges. The main aim appears to be to prevent contamination or
oxidation of the product streams by ambient air rather than containment of
the centrifuges. However, sealing of the bowl space from ambient air
should provide a degree of containment. Hemfort and Kohlstette describe
three approaches. The first is the liquid seal shown in Figure 9.6. Here,
sealing liquid is pumped into an extra chamber at the top of the bowl above
the centrate discharge pump. As the bowl rotates, centrifugal force holds
the sealing liquid in the chamber. Sealing liquid is fed continuously at (3)
on Figure 9.6 and the overflow discharges at (2). Any contaminating
material from inside the bowl is washed away in the sealing liquid
discharge. This type of liquid seal is also used between the bowl chamber
and gear casing of the Westfalia type SA 160 and SB 80 disc-stack
centrifuges.
The second approach described by Hemfort and Kohlstette is used when
the bowl needs to be pressurised and is shown in Figure 9.7. Here, simple
Figure 9.6 Liquid seal of solids ejecting DSC - Westfalia separator. 1. Feed; 2. scaling-liquid
discharge; 3, scaling-liquid feed; 4, centrate discharge.
Mechanical
Seal
Figure 9.7 Double mechanical seal of 11 Westfalia separator.
mechanical seals such as sleeve rings made of flexible material or slide rings
are used. Inherently, these simple seals will not be as well contained as a
double mechanical seal because material can form a thin film between the
slide ring and the shaft and thus escape. Hemfort and Kohlstette's third
approach is shown in Figure 9.8. They point out that the liquid seal
described above only functions when the bowl is rotating and centrifugal
force holds liquid in the sealing chamber. This problem can be avoided by
using the design shown in Figure 9.8 for the seal between the bowl chamber
and gear housing. Here, sterile inert gas is drawn through the sealing
chamber under vacuum. Any contamination can be filtered out to waste or
contained disposal. Hemfort and Kohlstette do not make any claims for the
containment category of operation of the three types of seal described.
Most devices have cyclone receivers to contain the discharge of sludge.
However, a considerable shock wave is generated by the centrifuge and the
air which is then displaced from the cyclone may contain aerosols of cells or
debris unless suitable vent filters are fitted. Lawrence and Barry l4 report
shock waves during discharge from an Alfa-Laval AX 213 Separator,
thought to be sufficient to allow aerosol to escape from cartridge housing
air vents. Walker et al. 15 describe modifications to a Westfalia CSA 19--47-
476 centrifuge. The vent filter was blocking due to massive aerosol
Figure 9.8 Gas-sealed seal of Westfalia separator. 1, Inert gas inlet; 2, sealing chamber;
3, restrictor to gear casing; 4, restrictor to bowl chamber.
formation during desludging, so it was removed and attached to the main

frame drain, thus increasing the distance between the solids receiver and
the filter. This alleviated the vent filter blockage problem. Another
solution, offered by van Hemert, 16 used a purpose built receiving vessel for
a Westfalia SA 47-476 model. Damping was effected by a rubber
membrane ring mounted concentrically around the connecting pipe from
the centrifuge solids line.
Neither Alfa Laval nor Westfalia claim in their literature that any of
their centrifuges operate to a particular containment category. However,
from the engineering drawings and specifications for the devices it is
possible to make some suggestions. All the steam sterilisable models
supplied by the two manufacturers are fitted with a double mechanical seal
on the drive shaft between the bowl housing and the gear casing. These are
flushed with sterile water or other liquid which could be diverted to a kill
tank. Thus, using Chapman's criteria these devices may be suitable for
operation at OECD Category 2 where the requirement is to prevent
release. The authors know of a Westfalia CSA 8-47-476 which has been
installed at a bioprocessing facility with the manufacturers claim that it will
prevent release.
Five Westfalia models, CSA 160, HSA 200, SA 45, SB 60 and SC 35,
have a liquid seal at the top of the bowl housing. Two models, SA 160 and
SB 80, have a liquid seal between the bowl chamber and the gear casing.
The liquid seal is only formed while the centrifuge is running and
centrifugal force holds the liquid in the chamber. When the centrifuge
stops, the liquid seal breaks down and any aerosol in the bowl chamber
could escape. Therefore, whilst these types of seal may minimise release
during operation, and perhaps be suitable for operation at OECD
Category 1, they will not necessarily prevent release and consequently
cannot be recommended for higher risk operations.
9.4 Cell disruption
Disruption may involve physical, mechanical or chemical steps to allow

intracellular products (usually proteins) to be extracted from cells.
Alternatively, it may consist of merely removing certain components from
the cell wall or membrane, to permit product leakage. There are many
methods of disrupting cells. The suitability of each method depends on the
scale of production, the protein to be isolated, the individual cell
suspension, and the disruption techniques available. The performance of
each technique is dependent on cell type, culture conditions, pretreatment,
and the device used.
Physical or mechanical methods of cell disruption are the most widely
researched in terms of containment. The underlying principle is either by
breakage of the cell wall by mechanical contact, the application of liquid or
hydrodynamic shear forces, or the application of solid shear forces. Cell
disruption by non-physical methods generally involve simple operations
which may be carried out in large tanks or vessels, which mayor may not
require agitation.
9.4.1 Physical methods
9.4.1.1 Agitation with abrasives

Micro-organisms in dry or frozen solid form can be disrupted by
conventional ball or vibratory mills used in the chemical process industries.
Whilst the method of dry milling may be efficient, it raises a number of
problems, including caking of fine powders (at around 1 ~m most bacteria
are smaller than powders that are generally milled in the chemical process
industries), attrition of the mill surfaces including liners and balls (which
leads to contamination of the disruptate), and the generation of heat
energy which can denature the desired product. In the biotechnology
industry, it is much more common to employ wet milling where disruption
is caused by a mixture of hydrodynamic shear forces and mechanical
crushing.
Bead mills are generally operated at near ambient pressure. When
disrupting very thick cell pastes, there may be a slight build up of pressure
in the vessel, but it is unlikely to exceed 0.2 bar, so bead mills are unlikely
to cause aerosols to be released during operation. In the event of seal
failure or a leak, however, even this low pressure is likely to lead to aerosol
formation.
Dyno-Mills (W.A. Bachofen, Basle, Switzerland) are available in glass
for continuous or batch laboratory-scale work and steel for continuous
pilot- or production-scale operation. In Figure 9.9, the grinding container
of the Dyno-Mill is sealed with a single mechanical seal on the drive shaft
and single O-rings on the flanges. Dyno-Mill claim that the devices are
fully contained and that no secondary containment is needed, except when
loading or unloading the charge in batch mode. Large-scale devices such as
the KDL-PILOT, KD5, KD15, KD50CN, KD200C, and KD250C can be
hard-piped to allow feed and disruptate to pass in and out without contact
with the atmosphere, cleaning in place and steam sterilisation (via the
jacket). Using Chapman'slO criteria, the single mechanical seal on the
drive shaft would not make the Dyno-Mill suitable for OECD Category 1
operations. Certainly, for use with hazardous organisms the use of
contained cabinets is advised (see Chapter 8).
9.4.1.2 Liquid extrusion

This method has been widely studied and relies on the principle that
forcing a cell suspension at high pressure through a narrow orifice will
provide a rapid pressure drop. This is a very powerful means of disrupting
cells. It is a relatively simple matter to design equipment to subject the cell
suspension to shear forces before releasing the pressure. By varying the
pressure applied, cells may be completely or only partly disrupted (the
latter usually being sufficient for the release of periplasmic enzymes).
Figure 9.9 Continuous bead mill.

The earliest devices to employ this principle were the French Press and
the Chaikoff Press. 17 Both these devices are relatively crude and simple
which can only disrupt small batches of cell suspensions. The next stage in
the development was the introduction of dairy industry homogenisers of
which the Manton-Gaulin 15M is a typical example.
The 15M consists of a ram pump which forces product through a one-
way valve into a homogenising valve. The feed enters this valve at high
pressure, typically 533 bar. As the feed passes between the homogenising
valve and its valve seat, there is a rapid increase in velocity with a
corresponding decrease in pressure. This results in cavitation, which,
coupled with impaction of the cells against an impact ring, causes cell
disruption. The gap between the valve and the valve seat is adjusted by a
spring-loaded handwheel. The important design features are the suction
valve, discharge valve, pump plunger and plunger packing. On the suction
stroke of the plunger, the suction valve opens, allowing product to enter
the pump chamber. On the compression stroke of the plunger, the
discharge valve opens, the suction valve closes and product is forced along
a bore to the homogenising valve.
Pandolfe IS described three problems with this type of design. First, if the
plunger packing fails, product can pass from the pump chamber along the
plunger. Because of the high pressures involved, any leaks are likely to
form aerosols. Secondly, flat gaskets are used on the cylinder caps. Any
leakage past these gaskets will again form aerosols due to the high
pressures. The third problem concerns the intersection of the bores. The
intersection of two bores in a cylinder block represents a high stress
location. Optimally, cell disruption applications require pressures of 1000
bar and the intersecting bores of the 15M cannot withstand the stresses of
continuous operation at these higher pressures. The APV-Gaulin 30CD
Cell Disrupter was designed with these problems in mind. The principle of
operation of the 30CD is similar to that of the 15M, except that the 30CD
has a triple-action ram pump which allows operating pressures of 1000 bar
to be achieved. The homogenising valve of the 30CD has also been
redesigned to promote more efficient disruption. The gap between the
valve and valve seat was adjusted by a spring-loaded handwheel in this
particular model.
The ram pump stuffing box for the 30CD is shown in Figure 9.10. In the
30CD, secondary seals have been added to the ram pump plungers to
capture any leakage from the primary packing. Water is circulated through
the stuffing boxes of the ram pumps to act as a coolant or lubricant for the
secondary seal. If necessary, a disinfectant can be circulated in cases where
the primary packing is breached.
APV-Gaulin International (Hilversum, NL) manufacture and supply a
wide range of high pressure cell disrupters. Capacities range from 40 to
6000 litres per hour with operating pressures up to 1100 bar. The
Plunger Ring
Plunger
Outlet
Stuffing Box Seat Inlet

Plunger Secondary Seal
Plunger Primary Packing
Figure 9.10 Ram pump stuffing box of 30 CD.
manufacturers state that the MC series has a double packing set on the ram
pump plungers instead of the single set used by the APV-Gaulin 30CD.
The plungers are lubricated by pressurised sterile fluid between the double
packing to maintain asepsis of the product. The double packing and sterile
lubrication should also prevent hazardous aerosol release. The manu-
facturers also claim that the MC series machines are designed to eliminate
or reduce dead areas to improve cleanability and are all steam sterilisable.
To date, their inherent containment has not been tested.
APV-Rannie (Albertslund, DK) manufacture a range of homogenisers
for cell disruption. Capacities range from the Mini-lab, Type 8.30H at 10
litres per hour at a maximum working pressure of 1000 bar up to the Type
50.80H with a capacity of 6600 litres per hour at a maximum working
pressure of 600 bar. The design of the APV-Rannie devices is similar to
that of the APV-Gaulin 30CD with a triple-action piston pump forcing
disruptate through the homogenising valve. In their advertising literature,
APV-Rannie claim that the pistons and packings have a long lifetime even
with very abrasive products. To date, no published information on the
intrinsic containment of the APV-Rannie devices has been found although
it is believed that DECHEMA in Germany has carried out some work (P.
Kramer, personal communication).
Bran and Luebbe (GB) Ltd, Brixworth (part of the Alfa Laval group)
supply high pressure homogenisers which are also similar in design to the
APV-Gaulin 30CD. Again, a triple action ram pump forces cell suspension
through the homogenising valve. Special packings are available for the ram
pump plungers to allow operation at temperatures up to 120°C and
pressures up to 1000 bar. Capacities range from 20 to 34 000 litres per
hour. No details of the sealing arrangements are available and the authors
are not aware of any containment tests that have been carried out on the
Bran and Luebbe machines.
The Ultra High Pressure Cell Disrupter (Constant Systems, Warwick,
GB) has a novel disruption head (no high pressure valve) which allows
microbial biomass disruption in batch or continuous mode. The former is
achieved through a single 10 ml aliquot and the latter through the use of a
500 ml reservoir. The process of disruption does not require high energy
levels so the temperature of the product is raised only slightly. In the
unlikely circumstances that the high pressure seal or cylinder fails (Figure
9.11), the maximum pressure which can be generated is 1000 psi. This is
limited by seal 'a' (shown on Figure 9.11). This seal normally seals against
low pressure in chamber 'b' and has been designed to fail if pressures are
higher in chamber 'c'. When seal 'a' fails any fluid pumped by the high
pressure piston is returned to the inlet side of the system via passage 'd'.
The system has been tested for containment by the Biosafety Unit at
CAMR, Porton Down. 19 It was found that it is possible to operate the
machine in a continuous mode without generating microbial aerosols.
Although some runs did generate aerosols, this was traced to a subsidiary
piece of equipment being used to store the processed fluid. This
emphasises the need for a contained system to be used with contained
ancillary equipment.
9.4.1.3 Ultrasonic techniques

Ultrasonics are sound waves of greater than 16 kHz frequency; when these
are applied to solutions, they cause 'gaseous cavitation', areas of
rarefaction and compression which rapidly interchange. As the gas bubbles
collapse, shock waves are formed. Sonication in batch or continuous
processing has been employed successfully for the disruption of many types
of microbial cells. 17
A number of commercial laboratory-scale and pilot-scale ultrasonic
disruption systems are available. Devices such as the Soniprep 150 (MSE,
Crawley) use glass tubes with rubber sealing caps. This should provide an
efficient seal between probe and tube, preventing the escape of aerosol.
The filling and emptying the tubes will lead to a breach of containment so
this operation should take place within secondary containment, exhausted
through a HEP A filter if high-risk micro-organisms are being disrupted.
Life Science Laboratories Ltd, Luton, UK supply the FLOCELL, a
continuous flow cell manufactured by Heat System Inc, Farmingdale, New
York, USA. The device consists of a chamber which screws onto an
2 SETS DOUBLE '0' RINGS
- = SEAL
~----i HIGH PRESSURE

PASSAGE 'd'7-1~bJ.-J SEAL
DOUBLE '0' RING
CHAMBER 'c'
HIGH PRESSURE
F'==------i CY LI N D ER
CHAMBER 'b'
Figure 9.11 Constant Systems Ultra High Pressure Cell Disrupter.
ultrasonic probe. Cell suspension is fed into the chamber under pressure at
the bottom and flows out of a port after disruption above the orifice. An
overflow port is provided for recycling cell suspension if necessary. The
flow cell is sealed with single O-rings. Operating pressure is up to
approximately 7 bar. The manufacturers do not claim any particular
containment category of operation for the FLOCELL. With the single 0-
rings it should certainly minimise release if they are kept in good condition.
The device should therefore be capable of handling OECD Category 1
micro-organisms. It may be possible to fit double O-rings with flushing in

the gap if required to increase security. FLOCELLS are available in a
range of sizes for operation at flow rates between 40 and 2400 litres per
hour.
9.4.2 Non-physical techniques

There are a number of non-physical methods of cell disruption available
for use in biotechnology. Osmotic shock, freeze/thaw, treatment with
detergents, lytic enzymes or chemicals are all suitable for use at large scale
although they have not been applied.s.2o-22 Lysozyme is an exception in
that it has been used for a number of years at large-scale. However, it is
only effective for Gram-positive bacteria, Gram-negative are resistant
unless treated. Cell permeabilisation using lytic enzymes and chemicals has
not been widely applied to large-scale intracellular product release,
although Asenj 0 23 reports that a number of investigations are under way.
These techniques may be particularly powerful because they offer the
potential for differential protein release from different cell compartments
as well as selective membrane permeabilisation and the release of
recombinant proteins. Molecular biologists are now able to insert genes
into micro-organisms which code for the secretion of the product molecule
in the periplasm. Enzymic treatment allows the desired product molecule
to be released into solution, leaving the rest of the cell contents still
enclosed in the plasma membrane. This dramatically reduces downstream
processing costs, as well as meaning that no physical disruption step is
needed.
Non-physical methods of cell disruption generally involve simple
operations which may be carried out in large tanks or vessels. These may
have an open top and will require some form of secondary containment or
local exhaust ventilation to minimise operator exposure. With high-risk
micro-organisms, vessels will need static seals and possibly dynamic seals if
the vessels are agitated via a drive shaft. Appropriate methods of sealing to
meet the various categories of containment have been described by
Chapman 10 and have been discussed earlier. An alternative would be to
carry out the disruption treatment in the bioreactor which should already
have the containment features necessary for the particular category of
micro-organisms being disrupted.
If non-physical methods of cell disruption can be developed to the stage
where they are cost-effective at large-scale, the requirement for physical
methods will decrease. There is no doubt that high pressures and high
speed rotating shafts increase the risk of a breach of containment and
subsequent production of hazardous aerosols. It is predictable that the
trend of research will be towards non-physical methods of cell disruption
and their application at large-scale.
9.5 Fluid handling
One essential part of downstream processing is the movement of fermenter

broths, supernatant fluids and whole cell slurries around the production
plant. In the pharmaceutical industry, this has been traditionally achieved
by pressure or gravity feed. In general, these methods are slow and
unsuitable for large scale and continuous bioprocesses. The use of pumps is
now the preferred method for most fluid transfer operations.
Pumps for use in the biotechnology industry have been reviewed by
Matthew Hal1. 24 The types thought to be suitable are summarised as
follows.
Sliding vane. Positive displacement pumps in which a multi-bladed

impeller rotates inside an eccentric pump cavity. They are self-priming and
deliver a non-pulsating flow.
Rotary positive displacement pumps. These have two or more impellers

in the form of toothed gear wheels or lobed cams which rotate with an
extremely small clearance between each other and the pump casing. They
have to be primed and they produce a pulsed output.
Peristaltic. These have a flexible tube set in a curved track in the pump
head. A rotor carried three rollers which compress the tube against the
outside of the track as they rotate. Fluid in the tube is pushed forward by
positive displacement.
Progressing cavity pumps. These are self-priming positive displacement

pumps which have a helical metal rotor lying inside a flexible tubular
stator. The rotor maintains a continuous seal along the length of the stator
and as it turns it forces the fluid through the stator to discharge.
Centrifugal pumps. In their simplest form, they consist of an impeller

with a number of blades rotating within a casing. As the blades rotate at
high speed, a low pressure zone forms at the axis as fluid is forced outwards
along the blades at increasing tangential velocity.
In the review the different types of pumps are assessed against a number
of criteria, two of which are the suitability of seals for hygienic and aseptic
operation and the biological isolation of process fluid from the external
environment. Pumps that score well in these categories will prevent ingress
of material so it could be assumed that they will be equally good at
preventing egress, i.e. they will be contained. The types of pumps which do
well in these categories are the centrifugal and peristaltic pumps. The
suitability of rotary positive displacement pumps varies from manufacturer

to manufacturer. Progressing cavity pumps fare the worst out of the types
surveyed.
When dynamic sealing is required, most types of pump are available with
single or double mechanical seals, some with steam tracing. This should
make them suitable for categories of operation where the requirement is to
prevent release. However, as far as is known, none of the types of pump
described have been tested for containment.
9.6 Discussion and conclusions
Since the advent of large scale biotechnology, coupled with the increased
use of genetic manipulation, one of the major concerns with this new
technology has been safety. To date, much of the attention has focused on
the problems associated with the micro-organism, not the process.
Guidelines exist for the use of GMMOs and containment categories are
arrived at by considering the hazard posed by the organism to humans.
Fermentation is usually the only stage in processing that is considered
when deciding on a containment category. However, most of the reported
health problems have been associated with downstream processing. 3
ACGM Note 66 adopts a flexible approach to containment. Large scale
processes are considered in terms of their unit operations and their
characteristics will dictate the physical containment to be used at that
stage. However, the only clue as to how containment should be achieved is
to 'exercise engineering control measures at source', yet very little work
has been carried out to look specifically at the equipment needed to
manufacture these new products. Often the machinery used in large scale
bioprocessing was designed for other industries, e.g. the Manton-Gaulin
15M Homogeniser was developed for use in the dairy industry. It is not
surprising then that much of this type of equipment has been shown to be
uncontained. Containment features may be added as an after-thought by
the user in order to comply with current or imminent legislation; there has
been a lack of guidance in the evolution of large scale bioprocessing.
However, the formulation of standards for bioprocessing equipment has
started to attract a wider audience with the result that the European
Committee for Standardisation (CEN) have now set up a technical
committee to investigate standards in biotechnology. One of the working
groups will be specifically addressing bioprocess equipment.
In spite of the guidelines which are available, only one author lO has
attempted to translate them into suitable designs for containment. He
proposed a combination of primary and secondary containment for static
and dynamic seals in order to meet OECD categories 1,2 and 3 ('minimize'
and 'prevent' release). It could be argued that a double seal without a
barrier fluid would not prevent a breach of containment if both the seals
failed. It would, however, be simple to incorporate a leak detection system
into the sealing arrangement, such as that on the APV-Gaulin 30CD cell
disrupter. It should also be remembered that seals will be included in
maintenance schedules and as such will be replaced on a regular basis. If
the product is of a high value it may be practice to replace the seals each
time the equipment is used to ensure that the product is not contaminated.
Chapman's concept could prove useful when developing equipment
containment criteria together with associated equipment design.
From the range of equipment discussed in this chapter it is clear that
manufacturers are recognising the needs of the biotechnology industry and
developing bioprocessing equipment with containment in mind. Contain-
ment is increasingly likely to be a requirement for manufacturers who wish
to compete. Facilities such as those which exist in the Biosafety Group at
Warren Spring Laboratory (see chapter 11) and in the Biosafety Unit at
CAMR, Porton Down, are ideally suited to assess the performance of
bioprocessing unit operations and equipment components.
9.7 Recommendations
1. Sealing has been identified, throughout this chapter, as crucial to the

containment of centrifugation and cell disruption unit operations.
Chapman has suggested criteria to "minimise" and "prevent release",
but these have not been experimentally investigated. It is recommended
that this is carried out. Investigations should be directed initially
towards mechanical seals and O-rings.
2. There are a number of commercially available centrifuges which are
claimed to operate at high containment categories. Where manufacturers
make these statements it should be possible to validate them using
'standard' methodologies.
3. There are no claims made for the containment levels of commercially
available cell disrupters. Again it is recommended that such devices
should be investigated. Until such time as data are available, the
authors recommend that when disrupting pathogenic or genetically
manipulated organisms, this should take place in an area of secondary
containment or with local exhaust ventilation.
4. If non-physical methods of cell disruption are developed to a stage
where they are cost effective then the requirement for physical methods
of disruption will decrease. Disruption which can be carried out in a
bioreactor or simple stirred tanks that can be adequately sealed to
comply with legislation will reduce the need for high pressures and high-
speed rotating shafts.
References
1. Salusbury, T.T. (1989). Disruption. In Harris, E.L.V. and Angal, S. (Eds), Protein
Purification Method: A Practical Approach. IRL Press Ltd, Oxford.
2. Kearns, M.J. (1989). Containment of biological hazards; effect of guidelines on the
design of pharmaceutical facilities and process equipment; Pharmaceutical Engineering, 9
(4), 1721.
3. Bennett, A.M., Benbough, J.E. and Hambleton, P. (1990). Biosafety in downstream
processing. In Pyle, D. (Ed) Separations for Biotechnology 2. Elsevier, pp. 592-{i00.
4. Dunhill, P. (1982). Biosafety in the large-scale isolation of intracellular microbial
enzymes, Chemistry and Industry, 22, 877-879.
5. Darbyshire, J. (1981). Large scale enzyme extraction and recovery. In Wiseman, A.
(Ed), Topics in Enzyme Fermentation Biotechnology, Ellis Horwood, Chichester,
Chapter 3, pp. 147-186.
6. ACGM (1987). Guidelines for the Large Scale Use of Genetically Manipulated
Organisms. ACGM/HSEfNote 6.
7. Leaver, G. and Hambleton, P. (1992). Bioreactor design considerations to minimise or
prevent inadvertent release, Pharm. Tech., 4, 18-26.
8. van Hemert, S.P. and Tiesjema, R.H. (1987). Safety aspects of closed system filtration
and ultrafiltration in vaccine production, Swiss Biotech, S, 13-18.
9. Krook, G. (1989). Centrifugal separators: technical considerations regarding containment
and sterilisation. In Salusbury, T.T. (Ed), Proceedings of the DTlIHSEISCI Symposium
on Large Scale Bioprocessing Safety, 30 November and 1 December, 1988. Warren Spring
Laboratory Report Number LR 746 (BT), Stevenage.
10. Chapman, C. (1989). Client Requirements for Supply of Contained Bioreactors and
Associated Equipment. In Proc. Symp. Large Scale Bioprocessing Safety, Ed. T.
Salusbury. Warren Spring Laboratory Report LR746 (BT), Stevenage, UK, pp. 58-{j2.
11. Matthew Hall (1987). Centrifugal contactors and separators. Matthew Hall Engineering
(Southampton) Ltd, PPFB1I011987. Available from DTI MT Division, Ashdown House,
123 Victoria Street, London.
12. Alfa Laval (undated) BTUX 510 Contained Separation System for Commercial Biotech
Production Reference Number PB 41116E2, 9005, Alfa Laval Separation AB, Tumba,
Sweden.
13. Hemfort, H. and Kohlstette, W. (1984). Centrifugal clarifiers and decanters for
biotechnology, Technical Scientific Documentation Number 5, Westfalia Separator AG,
Oelde, Germany.
14. Lawrence, A. and Barry, A. (1982). Potential hazards associated with the large-scale
manufacture of bacterial vaccines, Chemistry and Industry, 22, 880-884.
15. Walker, P.O., Narendranathan, T.J., Brown, D.C., Woolhouse, F. and Vranch, S.P.
(1987). Containment of micro-organisms during fermentation and downstream processing.
In Verrall, M.S. and Hudson, M.J. (Eds), Separations for Biotechnology. Ellis Horwood:
Chichester.
16. van Hemert, P. (1982). Biosafety aspects of a closed-system Westfalia-type continuous
centrifuge, Chemistry and Industry, 22, 889-891.
17. Hughes, D.E., Wimpenny, l.W.T. and Lloyd, D. (1971). The disintegration of micro-
organisms. In Norris, 1.R. and Ribbons, D.W. (Eds), Methods in Microbiology, VoI5B.
Academic Press.
18. Pandolfe, W.O. (1989). The cell disruption homogeniser, In Salusbury, T.T. (Ed),
Proceedings of the DTlIHSEISCI Symposium on Large Scale Bioprocessing Safety, 30
November and 1 December, 1988. Warren Spring Laboratory Report Number LR 746
(BT), Stevenage.
19. Bennett, A.M. (1991). The integrity testing of the constant systems ultra high pressure
cell disrupter. Agenda Item 3.2 Industrial Biosafety Project Meeting Notes November
1991. Warren Spring Laboratory.
20. Andrews, B.A., Huang, R.B. and Asenjo, J.A. (1990). Differential product release from
yeast cells by selective lysis. In Pyle, D. (Ed), Separations For Biotechnology 2. Elsevier.
21. Harrison, S.T., Dennis, 1.S. and Chase, H.A. (1990). The Effect of culture history on the
disruption of Alcaligenes eutrophus by high pressure homogenisation. In Pyle, D. (Ed),

Separations for Biotechnology 2. Elsevier.
22. Heppel, L.A. (1967). Selective release of enzymes from bacteria, Science, 156, 1451-
1455.
23. Asenjo, l.A. (1990). Cell disruption and removal of insolubles. In Pyle, D. (Ed),
Separations for Biotechnology 2, Elsevier.
24. Matthew Hall (1984). Hygienic and aseptic pumps for the biotechnology industry.
Matthew Hall Engineering (Southampton) Ltd, PPFB/4/1984. Available from DTI MT
Division, Ashdown House, 123 Victoria Street, London.
10 Freeze-drying of biohazardous products
G.D.1. ADAMS
10.1 Introduction
Freeze-drying (Iyophilisation) is a well-established technique used to

dehydrate labile materials often, but not exclusively of biological origin.
The process is economically expensive compared with other drying
techniques but where the emphasis is on product stabilisation rather than
dehydration, freeze-drying remains a preferred method for preserving
heat-sensitive bioproducts. I The technique was used as early as 1903 by
Vansteenberge 2 for dehydrating rabies virus and the potential hazards
inherent in the process as well as the benefits of freeze-drying have been
recognised since that time. Little has been published, however, that
specifically addresses the problems associated with processing bio-
hazardous materials. 3
While the emphasis in this chapter will be to consider hazards when
freeze-drying pathogenic micro-organisms or their products, it is important
to appreciate that the freeze-drier is an industrial machine and therefore
subject to non-biological hazards which will influence safe operation as
outlined in Table 10.1.
Chatigny and Clinker 4 compared common laboratory accidents and
showed that exposure to a broken vial of Iyophilised bacteria was
particularly hazardous. While the precise magnitude of their assessment
may be a matter for discussions there is no doubt that handling freeze-dried
cultures of pathogens or toxins may constitute a significant hazard. 6
10.2 Principles of the freeze-drying process
The stages in the freeze-drying cycle may be conveniently divided into the
following.
1O.2.J Product preparation

Product preparation includes sample preparation and dispensing of the
formulated product into suitable containers prior to freeze-drying.
Dispensing is a particularly hazardous operation and will be discussed in
greater detail in section 10.4.
FREEZE-DRYING 179
Table 10.1 Potential non-biological hazards associated with freeze-drying equipment
+ Risk from component implosion during evacuation or explosion during steam sterilisation.
+ Risk of frost-bite. burn or scald from exposed pipework or components over typical
operating range of -80°C to 138 dc.
+ Flammability hazard when plant used to remove organic solvents; explosion risk when
sterilising plant with ethylene oxide or when processing samples containing azides (20).
+ Slippage hazard from water condensate or oil leakage from plant.
+ Environmental pollution by vacuum pump exhaust. voiding of sterilising gases or from
hazardous products vented through the vacuum pump.
10.2.2 PreJreezing
Prefreezing immobilises the solutes within the solution, reduces thermal
inactivation of the bioproduct and prevents the product foaming during
subsequent chamber evacuation. Prefreezing is usually completed by
cooling filled containers on the shelves of the freeze-drier, by cooling
containers in a separate freezer cabinet or immersion in a cold bath.
Freezing the sample by evaporating a proportion of the water from the
solution can be used on small-scale equipment. However, such machines
are complicated in design because of the need to dispel product foaming
induced as the chamber is evacuated. To eliminate foaming, it is necessary
to centrifuge the sample containers at approximately 800 rpm 7 and freeze-
driers employing this principle are called spin or centrifugal freeze-driers.
Tube breakage during centrifugation will increase the risk of contamination
when this method of prefreezing is used.
JO.2.3 Primary drying

Throughout primary drying water is encouraged to sublime from the
sample by evacuating the chamber to reduce air impedance to escaping
water vapour molecules, at the same time gently heating the sample to
compensate for evaporative cooling. It is essential to ensure that the
product has been completely frozen prior to chamber evacuation and to
control heat input throughout primary drying to prevent product melting
and contamination of the freeze-drier by solution ejected from the vial.
JO.2.4 Secondary drying

The water content at the end of primary drying is invariably too high for
optimal product stability and the drying cycle is extended by secondary
drying during which additional moisture is removed by desorption,
resulting in a product with a final moisture content normally below 3%.
10.2.5 Stoppering and removal

After the product has been dried to a desired moisture content, the vials
are stoppered and removed from the freeze-drier before oversealing. Vials
may be back-filled with an inert gas such as nitrogen or helium just prior to
stoppering. When open ampoules or trays (which cannot be stoppered
within the freeze-drier) are used instead of vials, it is necessary to use
alternative sealing techniques in order to prevent the ingress of moist air
into the dried product.
10.2.6 Storage and reconstitution

Although the shelf-life of a labile bioproduct will be appreciably extended
by freeze-drying, stored material will not be immune to thermal decay and
it may be necessary to store the freeze-dried product within a refrigerator
to ensure maximum stability. For use, the dried product is reconstituted
with water or an alternative medium and the hazards associated with both
product storage and reconstitution will be addressed in section 10.4.
10.2.7 Technical features of the freeze-drier

Pilot-scale or industrial freeze-driers are constructed from metal sufficiently
robust to withstand evacuation. When smaller laboratory driers, fabricated
from glass, are used, there is an increased risk of breakage or implosion
and for this reason evacuated glass vessels should be enclosed in a jacket,
net or ensheathed with plastic film to prevent operator injury in the event
of breakage. Because freeze-drying is invariably completed at a reduced
pressure, a vacuum pump is included in the system. Pumps do not 'suck
away' water vapour but serve only to remove air initially within the
chamber or leaking into the drier throughout operation. To provide the
pumping force for sublimation a water trap is interposed between the
drying chamber and vacuum pump. On commercial plant, refrigerated
condensers are used as vapour traps and these must be maintained at a
lower temperature than the product to encourage the migration of water
vapour from the frozen mass and prevent sample melting. Condensers can
be either located close to the product within the drying chamber (internal
condenser) or housed in a separate sub-chamber (external condenser) and
the consequences of these alternative designs will be discussed in section
10.7 (see also Figure 10.2).
FREEZE-DRYING 181
10.3 Risk assessment
10.3.1 Potential hazards

The potential hazards to personnel and contamination of the environment
can be summarised as:
• Exposure to liquid aerosols generated during filling or when handling
dispensed solutions.
• Inhalation of or pollution by powder aerosols produced during freeze-
drying.
• Laceration by contaminated glass fragments from broken containers.
• Ingestion of contaminants via soiled clothing or gloves.
La Salle 8 has described contamination as a two-way problem where
bioproducts could contaminate or become contaminated from the freeze-
drier, environment or personnel engaged in filling or freeze-drying
operations. The author then suggests that adequate containment would
eliminate both the problems of product contamination and operator
exposure. While in principle this concept is feasible, it must be appreciated
that on an industrial scale, filling and freeze-drying of biohazardous
pharmaceuticals will invariably necessitate a compromise between the
need to prevent contamination of personnel and environment while
conforming to the requirements of good manufacturing practice (GMP)
where the emphasis is on product protection.
10.3.2 Processing biohazardous materials

When processing biohazardous materials, whether for laboratory or
industrial use, it is necessary to comply with mandatory regulations
relevant to individual substancesY-ll While these regulations include a
great deal of useful information to assist with the development of
processing protocols, it is useful to divide hazardous freeze-drying
operations into product-risk categories as outlined in Table 10.2, ranging
from low risk, where the hazard becomes significant only when a
substantial proportion of the filling bulk needs to be inhaled or ingested, to
high risk (for example, freeze-drying of live pathogens) where personnel
may be endangered when exposed to only trace quantities of product.
Allergens or potentially allergenic agents deserve special mention since
individual responses to allergens often vary widely. Allergic reactions are
characteristically related to the frequency of exposure and may be insidious
in onset. There is often considerable delay between exposure to the
allergen and the development of sensitivity which can present difficulties
during diagnosis, particularly when an operator has handled a number of
Table 10.2 Classification of hazardous agents processed by freeze-drying
Group I: Low risk

Agents which are hazardous at a high dose and toxic only when a substantial proportion of the
filling bulk is inhaled or ingested.
Group II: Medium risk

Agents which may be hazardous if the contents of a single vial are ingested or inhaled.
Group III: High risk

Agents which are potentially hazardous when personnel are contaminated by a fraction of the
vial contents. This category includes radiochemicals. self-replicating pathogens and untreated
or untested blood or tissue samples which may be contaminated with hepatitis virus, human
immune deficiency virus, etc.
Group IV: Unknown risk

Allergenic or potentially allergenic agents not included in groups I to III.
potential allergens or changed employment between exposure to the

allergen and the appearance of symptoms.
GMP regulations require the monitoring of the health of processing
operatives and may disqualify individuals from specific operations which
involve the handling of specified hazardous materials. It is sometimes
argued that the exposure risk can be considered as reduced when operators
are filling and drying a drug of relatively low toxicity which will ultimately
be used therapeutically. This attitude should not be condoned since an
operator could be exposed to a higher total dose than that administered to
a patient and care should always be exercised when exposing personnel
even to low-hazard material.
While it is necessary to provide adequate precautions to personnel when
freeze-drying hazardous products, overcautious risk assessment can result
in unnecessary or even impractical working regimes. An example where
risk assessment may require modification is where live attenuated vaccines
are freeze-dried on an industrial scale, when it may be operationally
impossible to handle such strains with the precautions essential when
processing virulent strains of that organism.
1033 Liquid and particulate aerosols

Liquid aerosols produced during dispensing or powder particles resulting
from freeze-drying have a polydisperse distribution when initially generated
but rapidly change shape, size and density as they lose or absorb water in
response to environmental conditions. 5 Such changes will have implications
on aerosol distribution patterns, particle settling rates and the biological
consequences of inhalation. 5
Particle distribution will be influenced by air flow configurations and
velocities so that under clean room operating conditions particles may be
dispersed further than distances predicted from data derived using
FREEZE-DRYING 183
experimental conditions such as those described by Chatigny and Clinker. 4

Indeed the principle of positive-pressure air hoods and curtains used
during aseptic filling is to deliberately disseminate contaminants away with
the filling station in order to minimise the risk of product contamination
during dispensing. In these cases it may be more appropriate to dispense
hazardous products under negative-pressure containment rather than
under positive air-flow work stations used for filling pharmaceuticals (see
section 10.7).
It is important to appreciate that products intended for freeze-drying will
have been formulated with additives designed to stabilise the active
component to prevent dehydration damage (see section 10.5). Conse-
quently such formulations are likely to be more resistant to decay when
disseminated as an aerosol during filling, freeze-drying or oversealing.
10.4 Hazards associated with product dispensing and handling finished

materials
Product dispensing represents a particularly hazardous stage in the freeze-

drying operation. When only a small number of vials have to be filled and
freeze-dried and the operation can be completed in a containment cabinet
using patent filling equipment, dispensing is a relatively low-risk operation.
Risks increase markedly when large-volume batches of product have to be
dispensed using commercial, high-speed filling equipment particularly
when the product has to be processed for clinical use under aseptic
conditions.
10.4.1 Dispensing pumps

Either piston or peristaltic filling pumps can be used for liquid dispensing
during a filling operation. The piston pump consists of an internal piston
moving within an outer barrel, a series of valves controlling the direction of
fluid flow. To reduce the risk of breakage and leakage, stainless steel
rather than glass barrels should be used when dispensing hazardous
products. The peristaltic pump is much simpler in design and consists
essentially of a length of flexible tubing which can be compressed to
extrude a selected volume of liquid. This type of pump is less susceptible to
leakage than the piston pump because it has no internal moving parts and
for this reason the peristaltic pump is often preferred when dispensing
hazardous materials.
10.4.2 Dispensing needles

The shape and bore of the dispensing needle fitted to the pump requires
careful design to reduce aerosol generation during filling while enabling
precise fill volumes to be delivered at high speed. The distance between the
needle tip and the base of the vial should be adjusted to ensure that the
solution is ejected from the needle close to the vial base. The needle then
travels upward so that the distance between the dispensed liquid surface
and the needle tip remains constant. Operated in this way, splashing and
aerosol generation are minimised while fill accuracy is maintained. A suck-
back mechanism on the pump will further reduce droplet contamination.
Ultra-clean vials may carry a substantial internal static charge which can
attract droplets from the needle tip causing product splashing into the vial
neck and inaccurate filling. Charge and associated splashing characteristics
are influenced by the formulation of the dispensed solution, the static
properties of the dispensing area and relative humidity (V. Endacott,
personal communication). The problem may be reduced by attaching
discharge strips close to the filling head.
10.4.3 Filling reservoirs

These are glass, metal or plastic vessels fitted with a pressure equalising
vent filter and connected to the dispensing pump. To reduce the risk of
breakage, metal or non-fracturing plastic rather than glass receptacles
should be used. For pharmaceutical dispensing, reservoirs may be
operated under positive pressure to prevent the ingress of particulate
contaminants. When dispensing hazardous materials it may be more
prudent to maintain the reservoir at atmospheric pressure. Filling
equipment and vent filters should be integrity tested prior to each filling
exercise to ensure safe operation.
10.4.4 Tray dispensing

Liquid can be dispensed into plastic or metal trays for bulk freeze-drying.
For single-dose applications, freeze-drying in ampoules or vials is more
appropriate.
10.4.5 Ampoules
These are tall, narrow, all-glass containers which are heat-sealed after
removal from the freeze-drier. The risks from product exposure is greater
when ampoules are used rather than vials since ampoules are open when
loaded into or removed from the freeze-drier. Because ampoules are
geometrically unstable, the possibility of toppling further increases the
contamination risk. Constrictions in the ampoule neck make dispensing
more difficult and increases the possibility of droplets remaining in the
neck. During heat-sealing, dried residue resulting from these droplets may
FREEZE-DRYING 185
sputter from the ampoule before reaching a temperature sufficient to

inactivate infectious material.
Ampoules are often packed into metal trays before loading into the
freeze-drier. Trays containing open ampoules or bulk product should be
protected with a filter lid to reduce contamination of the dispensing area.
These filters should be freely permeable to water vapour. Cotton wool,
Gamgee I2 •13 or glass-fibre mat has been used for this purpose but these
materials may be unsuitable for processing pharmaceuticals since they may
shed fibres during use. Trays and boxes used for freeze-drying should be of
a convenient size and weight to enable ease of handling to reduce the risk
of accidental dropping.
10.4.6 Vials
These are small glass bottles which are fitted with a ventilated stopper
which can be partially seated into the vial neck to permit the escape of
water vapour during drying or fully inserted at the end of the process to
seal the vial prior to its removal from the freeze-drier. Vials are perhaps
the most convenient container used for freeze-drying parenteral products.
10.4.6.1 Special stoppering ampoules

These have been developed to combine the convenience of the vial with
the sealing qualities of an ampoule and can be temporarily stoppered
within the freeze-drier prior to final heat sealing.14 These special ampoules
are particularly unstable, presenting problems during filling or sealing
which have limited their application.
10.4.6.2 Plastic ampoules

These provide a safer alterative to glass containers although limited
availability, the need to use specialist filling and sealing equipment and the
requirement to conform to regulatory pharmaceutical requirements has
restricted their use.
10.4.6.3 Double-chambered vials or syringes 1S

These contain both freeze-dried product and rehydrating fluid and reduce
the risk of sample/diluent mismatch and contamination during sample
reconstitution.
10.4.7 Container breakage

Glass containers are obviously subject to breakage which can be
particularly high when automatic filling or sealing equipment is used.
Machine jamming is increased when glass fragments mix with spilled liquid
or hygroscopic powders to form a viscous, abrasive paste which will affect
the operation of the filling unit. Glass containers should be manufactured

to a high degree of dimensional tolerance to ensure a good fit into filling
machines, freeze-drier trays, etc. Precise dimensional tolerances will also
ensure efficient stoppering and uniform heat transfer throughout the
drying cycle. Tolerances are usually better controlled when tube-drawn
rather than moulded containers are used. In addition tube-drawn vials are
also less prone to stress-fracture during sample prefreezing.
10.4.8 Spillages
Spillages during dispensing are often overlooked since liquid product is
invariably transparent. However any spilled liquid on the freeze-drier shelf
will dry to form a thin, friable layer of powder which will be readily
disseminated from the chamber when the vacuum is released and the
product batch removed.
10.4.9 Stoppering
When good quality vials are used, breakages during stoppering are usually
infrequent unless the stoppering plate pressure is excessive, the shelves
loaded unevenly or with only a small number of vials. Occasional
breakages may occur during stoppering when a vial or stopper has become
dislodged from the product batch during loading and consequently rests on
the product load.
10.4.10 Product removal

Potential problems associated with product removal are:
• Vials loaded into the freeze-drier in trays with removable base plates,
which enable direct vial to shelf contact, and which may fracture when
the base plates are relocated into the trays after drying .
• Rubber stoppered vials may adhere to the underside of the stoppering
plate and will be subsequently lifted from the tray as the plate returns to
its rest position. These adhered vials may then fracture as they
subsequently fall from the stoppering plate.
10.4.11 Container sealing
10.4.]].1 Heat sealing ampoules

Glass ampoules are heat sealed outside of the freeze-drier using either
draw-sealing or tip-sealing techniques. 16 It has been suggested that while
draw-sealing will ensure an acceptable and safe seal, tip-sealing may be less
FREEZE- DRYING 187
satisfactory, resulting in the persistence of micropores in the apparently

sealed tip. J7
10.4.11.2 Oversealing of vials

The risk of vial breakage may be significant during oversealing particularly
when automatic equipment is used for this operation. Two types of
mechanical oversealers are commonly used:
• oversealers which function by clamping the security ring in place and
• 'spinning' oversealers which use revolving wheels to crimp the seal onto
the vial neck.
Both types of oversealer require careful adjustment to ensure an
acceptable seal with minimum vial breakage. Alternatively, vials may be
sealed with a screw cap rather than an aluminium overseal and these
alternatives may be safer to use when hazardous products are processed.
10.4.12 Product storage

High standards of stock monitoring during processing, labelling and
packaging are essential to avoid errors occurring when the finished product
is distributed. Extra packaging precautions may be required when storing
and distributing pathogens or hazardous materials to avoid breakage
during transport. Design features may make the container susceptible to
breakage and when this is observed a more robust alternative design
should be substituted. One example of a particularly fragile container is the
ampoule manufactured with a prestressed ring in the neck to facilitate
breakage for reconstitution. These ampoules may exhibit a higher
accidental fracture rate during transit compared with conventional
ampoules.
10.4.13 Container leakage during storage

Leakage of moist air into the container will invariably reduce the stability
of the stored product and will also increase the possibility of environmental
contamination. The stoppered vial is generally regarded as more prone to
leakage compared to the ampoule, particularly when stored at sub-zero
temperatures when the stopper may lose elasticity, 18 while poor over-
sealing can further increase the risk of vial leakage. 19 For these reasons
Cammack and Adams 20 have recommended the use of all-glass ampoules
for containing products requiring long term stability, while vials may be
more appropriate for products which have shorter anticipated shelf-life.
The possibility that ampoule leakage may occur as a consequence of
inadequate heat-sealing has been demonstrated by Greiff et al. 21
10.4.14 Leak testing of sealed vials and ampoules

When containers are sealed under vacuum (ca. 10-20 Pa), the integrity of
the seal can be confirmed by glow discharge using a high-frequency spark
tester. 22 Seal testing becomes more difficult when vials or ampoules are
back-filled with an inert gas such as nitrogen, prior to sealing, since glow
discharge is inhibited by gas pressures above 25 Pa. Gas-filled ampoules
may be tested by immersing the sealed containers in a dye bath (usually
containing methylene blue dye) and alternatively applying a vacuum/
pressure pulse to the bath to encourage the dye to penetrate an imperfectly
sealed ampoule.23 Collapse of the dried cake and the presence of dye
within the ampoule will indicate leakage. Vials could be tested in a similar
manner although the vacuum/pressure pulse may induce satisfactorily
sealed vials to leak, resulting in a false, high rejection rate. Visual
examination, microscopic optical techniques and the use of ultrasound have
been used for estimating leakage but are less discriminatory than the glow-
discharge test. Leakage could be estimated by filling vials with helium,
argon or a radioactive search gas which can then be analysed using
appropriate detectors 24 although such techniques are of limited value for
routine testing of vials filled with pharmaceutical product.
10.4.15 Reconstitution
There is always the possibility of laceration when hypodermic needles are
inserted into rubber stoppered vials or when ampoules are broken prior to
reconstitution. Greiff25 has described a method for enclosing the ampoule
neck in a rubber sheath prior to reconstitution. The ampoule is then
broken within this sheath and diluent injected through the sheath into the
ampoule. This technique reduces both aerosol dissemination and the risk
of laceration. It may be necessary to reconstitute a hazardous product
within an enclosed safety cabinet to ensure personnel protection.
10.5 Formulation
10.5.1 General concepts

Media formulated for freeze-drying hazardous products have to satisfy two
quite different criteria which are:
• media should ensure safe processing and
• the product should be formulated to retain activity after drying and
maintain stability during storage. 26
Often regarded as a bland, dewatering process, the steps in freeze-drying,
FREEZE-DRYING 189
as outlined in section 10.2, should be considered as a series of discrete

though interrelated stresses which are imposed on a micro-organism or
bioproduct throughout the cycle.
10.5.2 PreJreezing
With some exceptions,27 micro-organisms and biopolymers are resistant to
the effects of cold-shock (chilling in the absence of ice formation).
Biomaterials do, however, sustain significant damage when the medium is
frozen.28--30 Freezing will result in a microseparation of the solution or
suspension into ice and solute-rich phase and death or damage results from
an exposure of the bioproduct to the increasing solute concentration as ice
propagation proceeds,3! while other associated solution changes, such as
alterations in the pH value,32 will increase the damaging osmotic effects.
Living cells can be further damaged when cooling rates above an optimum
are used to prefreeze the suspension when ice nucleation within the cell is
encouraged. 33 Micro-organisms are particularly sensitive to freezing
because damage to the sensitive semi-permeable outer membrane will
exacerbate any denaturation sustained by individual cellular components. 34
10.5.3 Freeze-drying
Further damage occurs during subsequent sublimation and desorption
drying/ 5 product storage 36 or when the micro-organism or biopolymer is
reconstituted. 37
10.5.4 Freeze-drying excipients

Micro-organism viability and biopolymer activity may be particularly low
when such samples are freeze-dried in water or a simple salt solution and,
to reduce damage, 'protective' excipients are invariably added to the
solution or suspension before freeze-drying. 38--40 Additives should ideally
protect during all the stages in the freeze-drying cycle but individual
protectants are characteristically effective only during part of the cycle.
Crowe et al., for example, have demonstrated that a wide range of
cryoprotectants are effective during prefreezing while only sugars are able
to protect during drying.4! Protectants must be compatible with the freeze-
drying process and, in this respect, particularly effective cryoprotectants
such as methanol or dimethyl sulphoxide (DMSO) - both of which are
permeating protectants42 - are of limited value in freeze-drying since they
will evaporate from the frozen mass during sublimation.
Sugars which persist in an amorphous state after prefreezing (such as
lactose, trehalose, etc.) are superior as stabilising excipients compared to
additives such as mannitol which crystallise during freezing and are thereby
effectively removed from the system. 43 One problem with sugars which
form amorphous concentrates during prefreezing is that these mixtures
may subsequently dry with collapse of the developing structure 44 .45
resulting in extreme cases in a sticky residue within the vial. A collapsed
structure often exhibits a high moisture content (which may lead to poor
shelf stability), may be difficult to reconstitute,46 and will increase the risk
of ablative contamination 47 (see section 10.6). Solutions containing
excipients which exhibit low collapse or glass transition temperatures (Tc
or Tg ') are particularly suspectible to collapse during drying.48
Micro-organisms stored in sugar-rich media may exhibit poor shelf
stability as a consequence of accelerated, non-Arrhenius decay49 or as a
consequence of Maillard interactions between reducing sugars and free
amino residues resulting in protein inactivation. 50
10.5.5 Container breakage and miscellaneous consequences

of product freezing
Rowe and Snowman 51 have reported that materials which form surface
skins during prefreezing and which adhere strongly to the container wall
may trap unfrozen liquid beneath the skin. Subsequent freezing of this
trapped liquid may then rupture the container wall. Similar vial breakage
can occur when concentrated solutions containing mannitol are freeze-
dried because of the sudden crystallisation of this excipient within the vial.
In this context it should be noted that some additives (including mannitol)
will fail to crystallise when cooled but may do so as the apparently frozen
mass is warmed so that vial breakage is observed during the early stages of
sublimation rather than prefreezing. 52
Rowe (personal communication) has described the formation of a pip of
detached product originating as solution freezes from the vial base and
walls to form a central column of unfrozen liquid which is extruded
upwards to form a bleb on the surface. This bleb will subsequently dry to
form a pip of dried sample which can be dislodged from the cake during
storage or transport. If the pip should remain within the container neck or
stopper it may be disseminated when the ampoule or vial is opened.
10.5.6 Storage
Freeze-dried materials are not immune to decay but remain sensitive to
heat, light, reactive gases,53 Maillard interactions (see above), free-radical
damage,54 etc. Greiff and Rightsel have established a relationship between
water content and the stability of freeze-dried influenza virus and have
demonstrated that stability could be reduced by drying above or below a
critical moisture content. 55
The mutagenic affect of freeze-drying on bacteria has been demonstrated
FREEZE-DRYING 191
by Ashwood-Smith and Grant,56 and while the exact cause of such

mutation remains uncertain, its significance may require consideration
when attenuated vaccines are freeze-dried.
10.5.7 Comparison of protection during aerosolation or freeze-drying

Because aerosolation and freeze-drying are both processes which desiccate
micro-organisms, it is not surprising that data comparing freeze-drying
protectants from Redway and Lapage 40 with aerosol protect ants by Cox 5
illustrates a close similarity between the two groups of protectants.
Practically this similarity is important because suspensions of micro-
organisms, solutions of toxins, etc., formulated for maximal freeze-drying
stability will consequently be more resistant to inactivation when dissemin-
ated as a powder or liquid aerosol.
10.6 Ablation
10.6.1 Loss of contents

Vial contents are lost during freeze-drying not only as a consequence of
water removal but also by the evaporation or sublimation of solvents or
solutes other than water (for example, ethanol, urea, etc.) from the
sample. Friable solids may also be carried out of the vial by ablation in the
vapour escaping from the drying sample. 47 While particle losses may be of
little consequence during routine freeze-drying, they can become a hazard
when pathogens are processed. Ablation losses can be appreciable, leading
to contamination of all internal parts of the freeze-drier.
10.6.2 Influence of product formulation on ablation

Ablation is particularly noticeable during sublimation although losses
can be detected during prefreezing if the product is frozen by evapora-
tion. 57
Ablation can be reduced by including excipients in the formulation
which form a cohesive cake structure, although data from this laboratorl7
using Escherichia coli as a trace organism has demonstrated that ablation
losses are detectable even from an apparently well formulated product.
The results of this study can be summarised as follows:
(a) Ablation occurs during the sublimation stage of freeze-drying.
(b) The escape of organisms is particularly marked in a poorly formulated
medium (for example, saline).
(c) Loss is detectable even from a well-formulated product exhibiting no
visible evidence of cake fracture or escape of debris from the vial.
(d) Ablation is more noticeable from a collapsed product plug.

(e) Ablation can be significantly reduced by half-stoppering the vials
before drying or by freeze-drying the bacterial suspension beneath an
inert, cohesive, overlayer.
10.6.3 Ablation and spillage

Although spillages during product dispensing may contribute to ablation,
the two hazards are quite different. Ablation can occur in the absence of
spillage and will contaminate all internal surfaces of the freeze-drier. As
discussed above, ablation is not associated solely with a poorly formulated
product but should always be anticipated whenever pathogens or toxins are
freeze-dried unless validated physical barriers are interposed between the
product and the interior of the freeze-drier.
10.6.4 Ablation and back-migration of vacuum pump oil

Contamination by ablation may be exacerbated by migration of mineral oil
from the vacuum pump towards the chamber. 58 Under these conditions
infectious particles could be encapsulated by the oil and protected from
inactivation when gaseous biocides such as formaldehyde are used to
sterilise the freeze-drier.
10.7 Practical aspects of the design and operation of freeze-driers

and associated equipment
This concluding section will address some of the practical aspects of freeze-
drying hazardous products based on the theoretical considerations outlined
in the preceding sections.
Overall the aim of safe processing should be to:
(a) Prevent contamination of the freeze-drier by:
(i) incorporating design features into the equipment and
(ii) adopting operational protocols which are intended to eliminate or
reduce contamination followed by:
(b) effective Decontamination of the freeze drier using post-operative
cleaning and sanitising procedures.
Figure 10.1 illustrates the constituent parts of a generalised freeze-drier.

Ancillary components, such as refrigeratory plant, controllers etc., which
do not come into direct contact with the open product, are obviously less
susceptible to contamination than the interior of the machine.
Laboratory freeze-driers can be decontaminated in the filling area using
FREEZE-DRYING 193
+ Steam Inlet
Pressure
gauge ,-+'--__. .
Compensating
shelf and
Pressure
stoppering
mechanism
+gauge
Product
Door with Vacuum

pump
viewing port ---- Main
Valve
Condenser
drain
Shelf
refrigerator - +- Shelf Refrigerator
heater .-
Figure 10.1 Simplified diagrams of steam sterilisable freeze-drier.
a sterilising vapour or topically applied samtIser. To comply with GMP

requirements, large pharmaceutical plant is usually installed so that
ancillary components are excluded from the dispensing area. In practical
terms we are therefore concerned principally with contamination of the
freeze-drier interior although environmental pollution from the product
through the vacuum pump exhaust, or from the condenser drain etc.,
should also be considered.
10.7.1 Freeze-drier design

It would be possible to construct a freeze-drier incorporating features
designed to minimise the risks of environmental contamination and in 1972
Parker and Smith described the construction of a laboratory drier designed
to freeze-dry pathogens. 59 However, economic constraints inevitably result
in adapting commercially available freeze-driers to reduce the risks when
processing hazardous agents.
Modifications and adaptations in design are obviously influenced by the
scale of operation. For example, in this laboratory, safe processing of small
batches of pathogens can be achieved using a modified laboratory freeze-
drier which can be sited, operated and decontaminated in a contained area.
Clearly when processing several thousand vials it is not possible to move
larger industrial machines.
10.7.2 Freeze-drier fabrication

A freeze-drier intended for processing hazardous products should ideally
be fabricated from stainless-steel since this metal displays excellent
corrosion resistance to a wide range of sterilants. The freeze-drier chamber
should be smooth and crevice-free to facilitate topical cleaning.
10.7.3 Chamber/condenser geometry

The two basic chamber/condenser layouts (that is, internal or external
condenser) are illustrated in Figure 10.2. While there has been controversy
over the most suitable layout, Rowe 13 has suggested that neither design has
an overall advantage in terms of safe operation.
Although internal condensers cannot be isolated from the chamber for
separate cleaning/sterilising purposes and result in a more complex
chamber design, they have the advantage of being accessible from the
dispensing area for topical cleaning or gaseous disinfection. External
condensers are less accessible for topical cleaning. They can be fitted with
internal spraying or flooding devices for in-place sanitising of the
condenser coils but direct access to the condenser can only be achieved by
fitting special entry ports onto the plant. Incorporating design features to
enable effective in-place cleaning of the external condenser will invariably
increase the cost of the freeze-drier. Because of the complexities in the
(a)
Chamber
Vacuum
Shelves - 1 ' - - - - - - - ' Pump
Condenser
("'-----t---~=~Coils in
External
Chamber
(b)
Chamber
Shelves
Condenser
Coils t
Vacuum
Pump
Figure 10.2 Freeze-drier illustrating the relative positions of external or internal water vapour
condensers. (a) Freeze-drier with external water vapour condenser; (b) freeze-drier with
internal water vapour condenser.
FREEZE- DRYING 195
freeze-drier design, it is debatable whether effective in-place cleaning

(CIP) can presently be achieved with industrial freeze-drying plant.
10.7.4 Protective devices

As discussed previously, all the internal surfaces of the freeze-drier must
be regarded as contaminated after a pathogen or toxin has been processed,
and an important feature of design and operation should include the
reduction of ablation. Two physical methods have been adopted to reduce
this risk:
(a) to pass the subliming vapour through an incinerator or
(b) to interpose filters between the product and the vacuum system.
Snowman suggested a combination of these protective devices by installing
an incinerator between the product and the pump while fitting an absolute
filter to the pump exhaust to prevent environmental pollution. flo
Alternative physical methods for decontamination including electrostatic
precipitation, ultraviolet irradiation, heat, etc., are reviewed below.
10.7.5 Electrostatic precipitation 6 ) and ultraviolet irradiators 62

Neither of these types of steriliser is suitable for decontaminating the
vapour escaping from the vial because vapour flow rates during sublimation
are so high that the residence time of an infectious particle within the
steriliser would be too brief for decontamination. Ultraviolet irradiation in
the range 2500--2650 A is used in combination with topical sanitising for
decontaminating dispensing cabinets etc. However, the low energy and the
poor penetrating power of ultraviolet light limits the application of this
method of sterilising.
10.7.6 Incineration
In their description of a laboratory-scale freeze-drier, Parker and SmithS'}
described the drier as an array of manifolds each holding sample ampoules,
connected to a liquid nitrogen/phosphorous pentoxide vapour trap. An
incinerator was interposed between the vapour trap and vacuum pump.
When validated at 110°C, using foot and mouth disease virus, the
incinerator described an inactivation efficiency of 99.997% although, as
the authors state, incorrect operation of the heated trap markedly reduced
its efficiency. It would be essential to thoroughly validate any commercial
air incinerator incorporated into larger, industrial freeze-driers to verify
that infectious particles were not carried through cold spots within the
incinerator. In this context it is sometimes argued that pathogens will be
destroyed by residence within the hot vacuum pump oil. This assumption
would require careful confirmation particularly since pump oil is invariably

inhibitory to assay procedures used to test for bacteria and viruses.
10.7.7 Filtration
This appears to be an attractive alternative to incineration partly because
filtration is a more simple and less expensive technology and partly because
filters may be integrity tested with precision.
Depth or screen filters find wide application for sterilising gases but their
installation in freeze-driers is often completed without adequate validation,
resulting in the use of inappropriate or incorrectly positioned filters.
The efficiency of either filter can be assessed by measuring the
percentage removal of test organism or sized particles from the incoming
air. The efficiency of a depth filter is influenced by a number of factors
including gas velocity; filter thickness; the chemical nature of the filter and
the concentration, size and geometry of challenge particles used. 63 Screen
filters can be integrity tested more precisely using bubble-point, forward-
flow, diffusion tests etc. 63
10.7.8 Selection and position of filters

Filter types and their position within the freeze-drier should satisfy two
broad criteria:
(a) the filter should remove contaminants under all operating conditions
and
(b) the filter should not be adversely influence the freeze-drying process.
Ideally filters should be chemically inert and must be resistant to steam
when installed in freeze-driers designed to be steam sterilised. Filters
should remain effective and freely permeable to the flow of air during
preliminary chamber evacuation, to water vapour evolved during sublima-
tion and to steam introduced during sterilisation. Hydrophobic membrane
filters are particularly well suited for filtering air or gases at high humidity.
All filters will offer some impedence to air air or vapour flow but ideally
filters exhibiting a minimal pressure drop, which are unaffected by
moisture and which have high particulate load capacity, should be selected
for installation within the freeze-drier. While it is possible to design a filter
with an increased surface area, the effective filtration area is usually
dictated by design constraints of commercially available filter units.
10.7.8.1 Positioning of filters in a freeze-drier (see Figure 10.3)

The advantages and disadvantages of the possible installation positions of
filters may be summarised as follows.
FREEZE-DRYING 197
A
Chamber
Figure 10.3 Sites for positioning hepa filters with a freeze-drier.
(a) Filter between drying chamber and air/gas inlet. This is the position for
installing filters used to sterilise air or inert gases admitted into the
chamber to regulate the chamber pressure throughout the cycle and for gas
back-filling at the end of the process. Similar filters may also be installed to
filter the steam admitted for sterilisation. Since the function of these filters
is for GMP pharmaceutical purposes rather than safe operation they are
not relevant to this discussion and the reader is referred to the publication
by Wickert64 which describes the installation and in situ integrity testing of
gas-inlet filters.
(b) Filter between vial/ampoule and drying chamber. Cotton wool plugs or
Gamgee caps placed into ampoule necks or onto vials have been used when
freeze-drying cultures of micro-organisms. 51 However, their use is often
based on bacteriological rather than freeze-drying experience. Operation-
ally it is difficult to consistently and effectively plug each ampoule and
impossible to validate the efficiency of plugging for each container. When
considering the large vapour flow rates which are evolved during freeze-
drying (calculated as equivalent to wind velocities in excess of 600 mph),
one should suspect the efficiency of cotton wool or Gamgee plugging of
ampoules. Stainless-steel containers, sealed with FM 004 fibre-glass filters,
have proved efficient bacterial barriers when freeze-drying large volumes
of pathogenic cultures in this laboratory.
Processing a number of vials within a contained box fitted with absolute
filters offers an alternative when uncontaminated freeze-dried cultures are
required. Taylor et al. described a box design in which vials could be dried
and stoppered to maintain sterility. 65 Although the box was designed to
enable sterile processing in a dirty environment rather than for containing
pathogens, validating the box under freeze-dying conditions has indicated
that the escape of test bacteria could be substantially reduced. Contamina-
tion from the box was observed only from a poorly formulated bulk
product which was prefrozen by evaporation. Bacterial escape was
restricted to leakages around gaskets and seals rather than through the
filter matrix and no loss could be detected at Escherichia coli concentra-
tions below 1011 organisms cm-3 , or when the suspension was dried in vials
or frozen before sublimation.
(c) Filter between drying chamber and condenser. Interposing the filter
between the chamber and the condenser appears to offer several
advantages since the filter potentially protects both condenser and pump
system. However, this position does have a number of serious dis-
advantages:
• adherence to good vacuum technology practice specifies that inter-

connections between the chamber and condenser should be designed to
offer minimal resistance to vapour fIoW. 66 Pipework and valves will
always result in some impedance to vapour flow and this may become
significant if a filter is interposed in the connecting pipe;
• subliming vapour flow may carry a particulate load sufficient to block the
filter;
• most importantly, the filter in this position will be exposed to the
maximal quantity of water evolved during sublimation (in addition to
wetting by steam used to sterilise the chamber) and water condensation
on the filter may impede vapour flow. In extreme cases filter blockage
may be sufficient to increase the chamber pressure above a safe level
which causes the product to melt, bubble from the container and
contaminate the freeze-drier. Fitting filters at this position will also
present problems during installation, maintenance or validation.
(d) Filter between condenser and vacuum pump. Installation of the filter at
this position does have the advantage that most of the water vapour will
have been retained by the condenser before passing through the filter.
However, impendance to vapour flow may still be significant and Harris
has suggested that dust filters placed between the condenser and pump can
reduce pump efficiency by as much as 25%? While there are difficulties
with installing and validating filters at this position, their installation has
become increasingly popular both to prevent pump contamination and
reduce the risk to maintenance personnel responsible for servicing
potentially contaminated vacuum pumps.
Installing a filter after the condenser also has the advantage of isolating
the pump from the product, and by including the filter in the steam
sterilisation cycle the slight risk of contaminating the product from the
vacuum pump (which cannot be steam sterilised) is eliminated.
Liquid nitrogen traps or traps containing chemical absorbents such as
activated charcoal or alumina may be interposed between the condenser
and vacuum pump either to remove non-aqueous solvents or to reduce
FREEZE-DRYING 199
mineral oil back-streaming from pump to product. The efficiency of these

traps should be assessed throughout operation to ensure that they do not
block, impede vapour flow and cause product melt.
Oil-free vacuum pumps are now available which will eliminate the risks
of oil back-migration and associated hazards. 67
(e) Filter on pump exhaust. Absolute filters suitable for installing onto the
pump exhaust are commercially available and should be fitted as a routine
to reduce environmental pollution both from containments passing
through the pump and by the pump oil itself. It is necessary to precede
these filters with an oil-mist trap to prevent oil contamination which will
compromise the efficiency of the absolute filter.
10.7.9 Decontamination of the interior of the freeze-drier

Irrespective of whether the freeze-drier has been fitted with filters,
incinerators or other features designed to reduce internal contamination,
freeze-driers used to process biohazardous materials must be capable of
decontamination at the end of the cycle. As well as protecting personnel
and environment from the processed agent, decontamination will prevent
cross-contamination from materials previously processed within the freeze-
drier.
Decontamination may be accomplished by:
(a) Topical cleaning using a detergent/biocide and
(b) Sterilisation using pressurised steam or a biocide vapour.
In one respect, decontamination following freeze-drying of a known
pathogen is a more simple procedure than general decontamination of a
freeze-drier for pharmaceutical processing since the nature of the pathogen
or toxin will be known and its sensitivity to a particular biocide can be
experimentally validated.
10.7.10 Biocides and sanitising agents

The number of proprietary biocides, sanitisers and detergents available for
topical cleaning and decontamination in the pharmaceutical, food or
allied industries is considerable and biocides designed to meet a wide
application range are available. Compliance with Good Laboratory or
Good Manufacturing Practice 6R recommends that no single biocide be used
routinely for decontamination and that biocides should be alternated on a
campaign basis to prevent the establishment of a biocide resistant strain.
When selecting an appropriate biocide, consideration must be given to
the type of organism, its concentration and the nature of the suspending
medium particularly where appreciable amounts of cell debris or protein
are present which could reduce the efficacy of the biocide. 6'1 During
validation, it is important that the sensitivity of the specific pathogen or
toxin should be assessed wherever possible. In some instances it may be
judged as inappropriate to use the infectious agent itself and a less
hazardous simulant examined under laboratory conditions may be substi-
tuted. When a simulant is used, it is important to ensure that the biocidal
and freeze-drying sensitivities of simulant and agent are comparable.
Micro-organisms differ widely in their sensitivities to freezing or dehydra-
tion and Rightsel and Greiff have characterised viruses as more or less
cryosensitive depending on their physico-chemical characteristics. 70 The
morphology of the simulant should also be considered when validating a
sterilising technique. For example, the complex morphology of T2 phage
may make it less typical as a simulant for assessing filter efficiency than a
structurally more simple virus.
The validation exercise should include the incorporation of standard
organisms of known biocide sensitivity into the test. Crowshaw 7l suggests
that organisms used as standards for disinfectant testing should be
preserved by freeze-drying, since these organisms are phenotypically much
more stable than organisms maintained by repeated culture which can
exhibit altered sensitivity to the disinfectant (although see section 10.5.6
and reference 56 which notes the possible mutagenic nature of the freeze-
drying process). The nature of the method and medium used for
resuspending organisms after freeze-drying can influence the survival of
the test organism and the result of decontamination exercise. 37 Contact
times and the temperature/concentration relationship of the biocide/
organism mixture should be carefully monitored and may be more
accurately validated under controlled laboratory conditions.
It is important that instructions on the use, concentration and application
of the biocide as specified by the supplier should be carefully followed.
Residual traces of biocides or sanitising agent must be removed after
decontamination to prevent their entry into cultures or sensitive bio-
materials subsequently processed within the freeze-drier. Finally prudence
should be exercised in the application of topical biocides used during filling
and loading operations. In this context, alcohol sprays used for cleaning
spillages, surfaces etc. should be used sparingly to prevent sufficient
biocide remaining within the chamber which could contaminate the pump
oil during chamber evacuation.
10.7.11 Sterilisation by gaseous biocides

Several biocides which were formerly used to sterilise freeze-driers for
pharmaceutical processing have been prohibited for use because of their
toxicity. Such prohibitions may be reconsidered when these biocides are
used for decontaminating a freeze-drier for processing pathogens or toxins
FREEZE-DRYING 201
where no suitable alternative is available. Clearly any concession to use a

biocide will only be permitted when it can be demonstrated that the
chemical can be used in a safe manner.
10.7.11.1 Advantages and disadvantages of gaseous biocides

The outstanding advantage of gaseous biocides is their ability to permeate
all parts of the freeze-drier which are inaccessible for topical cleaning.
The interior of the freeze-drier can be decontaminated with gaseous
biocides by two methods:
(1) By generating an aerosol of biocide within the closed freeze-drier and
leaving the biocide in contact for a suitable period prior to purging.
(2) By opening the freeze-drier door and decontaminating the chamber
interior as an extension of the dispensing area. This method can also be
used for decontaminating the exterior of laboratory freeze-driers
operated within a contained area.
Stoppered vials can be left in the chamber and exposed to a sterilising
vapour in order to decontaminate the outside of the vials before their
removal from the freeze-drier. In this laboratory we have been able to
demonstrate that formaldehyde vapour will not permeate stoppered vials
which contain freeze-dried samples.
Disadvantages of gaseous biocides are that they may be corrosive,
flammable or explosive, are invariably toxic and require a critical balance
of temperature/relative humidity/biocidal concentration for effectiveness.
Validating the efficiency of a gaseous biocide is often imprecise because
it is difficult to measure the parameters associated with the decontamination
procedure with precision (compare this with steam sterilisation below) and
validation exercises consequently rely heavily on the use of biological
indicators.
A further disadvantage when using gaseous biocides is that the gas or
vapour must be vented from the freeze-drier or dispensing area following
sterilisation. Frequency of sterilisation should be therefore carefully
controlled to avoid unnecessary decontamination while the gas should be
vented into the atmosphere during periods when personnel access is
restricted. During purging it is necessary to use separate venting fans to
remove the biocide from the freeze-drying chamber to avoid contaminating
the vacuum pump oil.
10.7.11.2 Beta-propiolactone72- 74
Beta propiolactone has been used for both room and equipment
decontamination. The biocide is a colourless liquid with an acrid odour
which is usually applied topically. Residue must be removed by water
rinsing after the sterilisation exercise.
The vapour has poor penetrating ability and should be used at high
humidity and elevated temperature (concentration: 2-4 mg per litre 50°C

and 40% RH: dry spores remain viable even after a 4-hour contact period).
Beta-propiolactone is corrosive to brass and copper and will attack
neoprene gaskets when used as liquid or vapour at 40% RH. The biocide is
toxic, causing respiratory irritation and skin blistering.
Method of use. (1) For interior of freeze-drier - the chamber

temperature is raised to 40°C using hot air or steam and beta-
propiolactone admitted to give a final concentration of 2-4 mg per litre.
The biocide is left for a contact time of 1-2 hours (longer for spores), prior
to removal by outgassing for 2 hours.
(2) For room disinfection (including freeze-drier) - the room temper-
ature is raised and RH elevated to 40%, prior to atomising 500-600 ml
beta-propiolactone per 28 mt 3 into the room. After exposure for 2 hours,
the vapour is extracted from the room.
1O.7.ll.3 Ethylene oxide: 72 ,73.74

This is a reactive gas, supplied in pressurised cylinders diluted with an inert
carrier such as carbon dioxide (10% ethylene oxide: 90% carbon dioxide)
of freon. It displays good penetrating power and will, for example,
permeate unbroken egg shells. The gas is used at a concentration of
400-1000 mg per litre and RH of 25-50%. The sterilisation time may be
reduced at elevated temperatures.
Generally, ethylene oxide is non-corrosive although as a precaution
special gaskets are usually fitted to freeze-driers which are intended to be
sterilised with ethylene oxide. The gas is acutely toxic at concentrations
above 50 ppm, causing skin burns and blistering while cytogenetic changes
in exposed workers have recently been reported. 75 The greatest hazard
associated with the use of ethylene oxide is that the gas becomes explosive
under alkaline conditions or when exposed to certain chemicals. Ethylene
oxide should never be exposed to phosphorous pentoxide (occasionally
used as a freeze-drying desiccant) with which it will react violently.
Because of its explosive potential, ethylene oxide cannot be used for
decontaminating dispensing areas but is restricted to sterilising closed
systems such as the interior of freeze-driers.
Method of use. The chamber is evacuated and then warmed to 40 DC with

hot air or steam to give an RH of 25-50%. Ethylene oxide is then injected
into the chamber to a concentration of 400-1000 mg per litre and sterilising
conditions maintained for 4-8 hours. The freeze-drier is then returned to
atmospheric pressure and the ethylene oxide vented from the drier. An
explosion-proof mixture of 60% ethylene oxide and 40% methyl bromide
has been used for decontamination. 76 Ethylene oxide has been more
widely used in the USA than in the UK for sterilising freeze-driers.
FREEZE- DRYING 203
The alternative use of less explosive propylene oxide has not been widely
adopted for decontamination because of its limited effectiveness compared
with ethylene oxide.
10.7.11.4 Formaldehyde 72 ,73,74

This is supplied as a commercial concentrate of 38--40% w/v aqueous
solution and is a colourless, pungent liquid. Alternatively, formaldehyde
can be generated from paraformaldehyde, a white solid (91 % purity
commercially available), which depolymerises upon heating to produce
formaldehyde vapour. Formaldehyde displays a wide biocidal activity
range, at concentrations between 3 and 10 mg per litre but the vapour is
effective only at RH values above 70%. Biocide concentration and contact
time should both be increased when the organic content of the medium is
high. Formaldehyde will inactivate a range of microbial toxins and is
particularly effective for detoxifying botulinum type A toxin. (Note:
ethylene oxide is not effective.)
Formaldehyde is a respiratory and skin irritant,77 may be allergenic and
may be carcinogenic under certain conditions,7R
Disadvantages of using formaldehyde include polymerisation of the
agent to form persistent residues which require removal by rinsing after the
decontamination exercise. Formaldehyde is corrosive to brass at all
temperatures and corrosive to mild steel at high temperatures by the action
of formic acid formed by formaldehyde decomposition. Neoprene, nitrile
and soft rubbers are attacked at temperatures above 40 DC.
Formaldehyde can also be generated by boiling a mixture of 250-500 ml
of commercial formaldehyde (38% w/v) solution in 1 litre of water per
28 mt 3 (quantities of formaldehyde/water vary depending on room
cladding porosity and organism species). Formaldehyde can also be
generated by adding 170 gm potassium permanganate to 250-500 ml
commercial formaldehyde per 28 ft 3 or by heating paraformaldehyde solid
using 5 gm solid per mt 3 .
Method of use. Freeze-driers may be contaminated by: (i) In closed

freeze-drier: procedure is to evacuate the chamber and admit formaldehyde
vapour at an RH of at least 70%. The vapour is left in contact for a
minimum of 4 hours prior to venting the chamber for 2-4 hours.
(ii) The method most commonly used is to open the chamber door and
decontaminate the chamber as an extension of the dispensing room.
Formaldehyde concentrations can be estimated chemically, although for
validation purposes a spore strip assay is commonly used.
10.7.11.5
Vapour phase hydrogen peroxide has recently been introduced as a
method for sterilising pharmaceutical equipment including freeze-driers. 79
10.7.12 Dry heat

Dry heat is an inefficient method of destroying micro-organisms or
inactivating toxins and sterility can only be ensured at temperatures above
IS0°C. These high temperatures prohibit the use of dry heat for sterilising
freeze-driers although components such as glass containers, trays, overseals
etc, are often sterilised by this medium. The destructive potential of heat
becomes more efficient when the relative humidity is increased and
sterilising temperatures and exposure times can be significantly reduced if
steam rather than dry heat is used for sterilisation.
10.7.13 Atmospheric pressure steam (live steam)

Temperatures above 100°C cannot be attained unless the steam is
pressurised. Because bacterial spores are only destroyed at temperatures
above 110°C, live steam is therefore not an effective sterilant and
consequently is only used to decontaminate freeze-driers when combined
with a gaseous biocide such as formaldehyde.
10.7.14 Pressurised steam

Of the methods discussed for decontaminating a freeze-drier, in the
absolute sense only pressurised steam can be regarded as a sterilant. Since
1970, the use of pressurised steam for sterilising pharmaceutical freeze-
driers has become standard and freeze-drying manufacturers are con-
versant with the problems associated with the fabrication of steam-
sterilisable machines. Exhaustive literature exists detailing the temper-
ature/pressure relationship required to destroy individual micro-organism
species and these conditions can be readily achieved within the freeze-
drier.
In addition to its sterilising action, steam has a number of other
properties which make it virtually an ideal cleaning agent/biocide and these
may be summarised as:
• pure steam leaves no contaminating residue in the chamber;
• steam will permeate all inaccessible parts of the freeze-drier;
• steam exhibits excellent cleaning properties;
• steam can be used to defrost the condenser as a prelude to sterilisation
(although when used to decontaminate a freeze-drier the reader is
referred to section 10.7.14.2 below);
• the sterilising efficiency of steam can be validated by physical measure-
ments of temperature, pressure, exposure time and steam penetration.
FREEZE-DRYING 205
10.7.14.1 Disadvantages of using pressurised steam include the following

(a) The capital cost of the freeze-drier will be increased because the plant
has to be fabricated from heat resistant materials such as stainless steel
(epoxy coated mild steel was used as an alternative but can suffer
deterioration after repeated sterilisation). Costs are further increased
since it is a legal requirement to ensure that the freeze-drier conforms
to safety standards applicable to those for a pressure vessel. To satisfy
these legal requirements it is obligatory for the chamber/condenser to
be certificated using a hydrodynamic pressure test. Since the weight of
a water-filled freeze-drier is high when completing these tests, it is
important to consider hydrodynamic revalidation when deciding on
the siting of the freeze-drier.
(b) To the increased equipment costs must be added the expense of
providing and maintaining the steam supply.
(c) Some heat sensitive components (for example the vacuum pump) in
the freeze-drier cannot be steam-sterilised. Vacuum gauges fitted into
the chamber or condenser may be unsuitable for steaming and will
require blanking-off prior to sterilisation. This is poor practice since a
non-sterilised, potentially contaminated reservoir will remain in the
freeze-drier. Steam sterilisable vacuum gauges are commercially
available which overcome these problems.
(d) Only filtered, pharmaceutical quality steam should be provided to the
freeze-drier since iron oxide and other contaminants in the steam will
rapidly corrode the stainless steel used in the manufacture of the plant.
(e) The additional time required to steam sterilise the freeze-drier and
cool the chamber and shelves prior to loading of a product batch will
increase both cycle time and processing costs.
(f) Because the vacuum pump cannot be used to remove steam and
condensate at the end of sterilisation, additional water-compatible
pumps have to be included which will further increase the capital cost
of the drier.
(g) Non-steam sterilisable freeze-driers cannot be economically modified
for pressure-steaming.
(h) It is necessary to perform planned preventive maintenance schedules
more frequently when driers are routinely steam-sterilised since valve
seatings, gaskets, seals etc., may be subject to more rapid deterioration.
10.7.14.2 The use of pressurised steam for decontaminating a freeze-drier

The advantages of using pressurised steam as outlined above strictly apply
only when sterilising freeze-driers prior to processing parenteral products.
When it is intended to use pressurised steam to decontaminate a freeze-
drier which has been used to dry hazardous materials, a potentially serious
disadvantage should be considered related to the need to raise the
temperature of the metal mass in the freeze-drier to sterilising temperature.

Because the persistence of air within the steam will reduce the sterilising
temperature which can be achieved (for example a 50:50 mixture of air plus
steam at 15 psi will exhibit a temperature decrease from 121°C (pure
steam) to 113 °C) it is essential to remove this air from the freeze-drier
either by evacuation or by steam displacement. Associated with the need
to remove air, steam will condense during the early stages of sterilisation as
the metal mass of the freeze-drier is heated. Consequently at the beginning
of sterilisation a large quantity of water condensate and air mixture will
drain from the freeze-drier before sterilising conditions are attained and
precautions must be taken to decontaminate this condensate prior to
discharge. In the situation outlined above the use of only pressurised steam
may not be safe for decontaminating a freeze-drier after processing a
pathogen. Under these conditions it would be necessary to decontaminate
using a topical or gaseous biocide followed by pressurised steam to clean
the interior of the drier, remove any biocide residues and sterilise the
chamber as a prelude to processing a subsequent batch of product.
10.7.15 Integrated approach to safe freeze-drying of biohazardous

materials
It should be apparent from the preceding discussions that no single design
feature or operating procedure can be relied upon to provide complete
protection when processing hazardous products. Equipment design,
operating protocols, etc. must take into account not only the nature of the
product but also the scale of operation, product formulation, ultimate use
of the agent and operator convenience. In this last context, the author has
experience where operating procedures have so reduced the manipulative
competence of an operator that pharmaceutical quality has been comprom-
ised and low risk operations have become unsafe as a consequence of
procedural constraints imposed on the operator.
10.7.16 Factors affecting operational safety
10.7.16.1 Clothing and personal protection

The standard pharmaceutical suit comprising a fibre coverall, hood,
overboots, rubber gloves and eye-shield provides a fairly effective barrier
against glass fragments and occasional spills when processing low risk
hazardous products. Traditional hospital-type face masks provide a poor
barrier to prevent respiratory contamination and helmets which blow a
curtain of positive-pressure air over the face are both convenient to wear
while providing a barrier between operator and product. 80 Positive-
pressure respirators do afford a greater degree of personal protection8 ! but
may reduce the manipulative competence of the operator.
FREEZE-DRYING 207
Clothing the operator completely in an impervious plastic suit represents

the ultimate in personal protection and has the additional merit of isolating
the product completely from operator and such suits have been used to
maintain sterility during pharmaceutical manufacture. Isolator suits should
not be used as the primary means of protecting the operator but must be
used only in combination with effective techniques for safely dispensing,
handling and freeze-drying hazardous products.
10.7.17 Dispensing product

Small-scale filling operations may be completed in safety cabinets
appropriately designed and designated for a specific biohazard classifica-
tion. 82 Even when spillages have not been observed, the exterior of each vial
should be regarded as contaminated and transport of these half-stoppered
vials to the freeze-drier therefore represents a risk. Packing vials into
contained boxes as discussed in section 10.4 will reduce this risk although
the outside of the box should be decontaminated before removal from the
filling cabinet. Safety cabinets83 which can be moved from the dispensing
area to the freeze-drier may reduce the contamination risk although the
possibility of leakage around the docking port gasket on the freeze-drier
will require testing after attachment to ensure safe, leak-free sealing.
Permanently installed tunnels extending from the filling machine to the
freeze-drier avoid the problems associated with making and breaking the
seal when a portable tunnel is used.
When commercial quantities of product are to be filled, dispensing
should be completed in areas with air-flow configurations as illustrated in
Figure 10.4. When toxic material will require dispensing in a negative
pressure room where product cleanliness is compromised in favour of
containment, the hybrid room (type 3) operated at positive pressure with
the hazardous operation completed in a negative pressure cabinet or
tunnel, may be an alternative when processing low-risk products.
Operations which exclude personnel from the dispensing area have been
developed for sterile, pharmaceutical freeze-drying and manufacture.
While the costs of building and operating such a facility is high, there is no
doubt that increasing use of such facilities will influence the technologies
used both in production freeze-drying and when processing toxic materials.
10.8 Conclusions
By way of conclusion, some of the principles discussed are recapitulated by

describing a hypothetical processing chain. It is recommended that a steam
sterilisable freeze-drier should be used incorporating absolute filters
208 BIOSAFElY IN INDUSTRIAL BIOTECHNOLOGY
(a)
(c)
Figure 10.4 Airflow patterns for dispensing areas used for filling pharmaceuticals or toxic
materials. (a) Conventional clean room with air circulation. Operated at positive pressure
relative to atmoshere. (b) Room used to dispense toxic material with total air discharge.
Operated at negative pressure relative to atmosphere. (c) Hybrid room. H, HEPA filter;
A, air circulation fans; B, containment cabinet; F, filling unit; C, laminar flow air hood;
arrow, ---> direction of air flow.
between the condenser and vacuum pump for maintenance purposes and
on the pump exhaust to prevent environmental pollution.
Product filled into vials and possibly contained within filter boxes
designed to reduce ablation and spillage can then be loaded into the freeze-
drier. After freeze-drying and stoppering the vials could be held within the
drier during formaldehyde decontamination to sterilise the outside of the
vials before removal for oversealing. Condensate from the vapour trap
should be drained and sterilised before disposal prior to condenser
decontamination by flooding or internal spraying with a biocide. After
decontamination, the freeze-drier should be steam-sterilised to clean and
sterilise the plant before subsequent batches of product are processed.
As part of a planned, preventative maintenance (PPM) schedule, pump
oil should be drained and sterilised before discarding together with decon-
taminated filters, used gaskets, seals and components which have been
changed during maintenance. After the PPM schedule, the freeze-drier
should be validated to confirm the correct functioning of the drier and
components including specialist items added to ensure safe operation.
The success of freeze-drying in the 1950s resulted in an assumption that
the process was a somewhat innocuous process applicable to dehydrating
any bioproduct. Recently there has been the need to reappraise these
FREEZE-DRYING 209
assumptions in the light of advances in the understanding of the technology

particularly when freeze-drying is used to process biohazardous materials
including pathogens, genetically-manipulated organisms 84 and toxins
under contained conditions.
Acknowledgement
The author wishes to thank Cormac Stanton for production of the

illustrations in this report.
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decontaminant and sterilant, Appl. Environ. Microbiol., 56 (2), 503-506.
80. Kemira Safety, Unit 14B, Harris Business Park, Hambury Road, Stokes Prior,
Bromsgrove, UK.
81. Martindale Protection and Electric Co. Ltd., Neasden Lane, London, NWlO IRN, UK.
82. Melling, J. and Allner, K. (1981). The containment of microorganisms. In Essays in
Applied Microbiology. J.R. Norris and M.H. Richmond (Eds), 1112-11132. John Wiley.
83. La Calhene (GB) Ltd., 22 Hills Road, Cambridge, C82 UP, UK.
84. Adams, G.D.J. and Warnes, A. (1993). The sensitivity of genetically modified organisms
to freeze-drying: influence of recombinant protein "A" on the survival of Escherichia coli
strain JM83 pPAI6 (in press).
11 Interpretation of regulatory requirements to
large scale biosafety - the role of the Industrial
Biosafety Project
G. LEAVER
11.1 Introduction
Safety issues associated with biotechnology, have gathered momentum in

recent years because of the development of powerful new techniques such
as the application of recombinant DNA technology (r-DNA) to micro-
organisms. National and international regulatory developments have
ensued, most notably the studies by the Organisation for Economic
Cooperation and Development. 1 Many of the principles have been
incorporated into EC directives on contained use 2 and deliberate release to
the environment3 and recently been implemented through the Genetically
Modified Organisms (Contained Use) Regulations, 19924 and the Genetic-
ally Modified Organisms (Deliberate Release) Regulations, 1992.5
In 1986, the UK research laboratories Warren Spring (WSL)* and the
Centre for Applied Microbiology and Research (CAMR) jointly established
the Industrial Biosafety Project (IBP). The IBP offered companies a
research and technology transfer programme and aimed to address the
important biosafety subjects relating to large scale bioprocessing. A
number of reports describing the state-of-the-art on various biosafety
subjects have been written. These include containment and monitoring
aspects of bioreactors, centrifugation and cell disruption operations.
Research papers and presentations to members have also contributed to
the knowledge base. The project has provided an opportunity for members
of industry, the regulators, academe, and government research laboratories
to discuss in detail many of the practical issues surrounding large-scale
work. Symposia have also been organised to widen the scope and influence
of the work.
This chapter highlights many of the activities and issues addressed by the
IBP and the future challenges.
11.2 Regulatory issues
Over recent years, the implementation of the EC Directives on genetically-

modified organisms (GMOs) into national laws has been a significant
'see end of chapter
activity for the regulatory authorities. Within the UK, the Health and
Safety Commission (HSC) and the Department of the Environment (DoE)
produced a first consultation document of the regulations. This document
drew a significant number of responses including a detailed reply from the
IBP which commented constructively on the initial structure, complexity
and definitions. A second and significantly improved consultation docu-
ment was produced which incorporated, amongst others, many of the
IBP's recommendations. Detailed accounts of the European and UK
national regulations are given in chapters 1 and 2.
The Contained Use (1992) Regulations 4 came into force in January
1993. The risk assessment requirements of these regulations are the main
focus of this chapter.
A third EC directive (1990)6 was concerned with biological agents and
encompasses all micro-organisms 'at work'. Within the UK, this directive is
being implemented by a revision of the appropriate sections of the Control
of Substances Hazardous to Health (COSHH) Regulations. 7 Similarly, the
IBP has commented on these regulations presenting the view of the
biotechnology industrial membership.
11.3 Risk assessment
Risk assessment is a key requirement of the GMO regulations. For

contained use, the emphasis historically has been on human health and
safety. Guidance notes in support of GMO work have been produced by
the Advisory Committee on Genetic Manipulation (ACGM). Guidance
note 6 particularly relates to large-scale work. 8 More recently, the
environmental aspects of releases from contained use operations have
received greater prominence. The Contained Use (1992) regulations
require an environmental risk assessment to be undertaken for processing
involving recombinant organisms. The biotechnology industry within the
UK has generally perceived the environmental risk assessment to be an
additional feature not previously addressed.
One purpose of risk assessment is to assign appropriate control measures
or containment measures commensurate with the estimated hazard of
the GMO. Revised guidance on risk assessment was published by
ACGM 9 in their note 7. Figure 11.1 summarises a recommended
framework by ACGM for undertaking the risk assessment and assignment
of control measures. The philosophy is that the human health and safety
and assigned control measures are considered initially. The environmental
risk assessment follows based on the assigned control measures for human
health and safety. Additional control measures are implemented if the
environmental risk assessment considers the human health and safety
control measures to be inadequate.
INDUSTRIAL BIOSAFETY PROJECT 215
Human health risk

assessment
Determine containment
level and additional
control measures
Environmental risk
assessment
Implement
additional
control measures
No Yes
Figure 11.1 Framework for risk assessment and assignmeilt of control measures.
11.4 Human health and safety
11.4.1 Estimation of G M 0 hazard

For laboratory scale operations, the system adopted in the UK is
commonly known as the Brenner method which classifies a GMO on the
basis of three factors:
1. ACCESS, the probability that the GMO, or the DNA contained within
it, will be able to enter the human body and survive there
2. EXPRESSION, a measure of the anticipated or known level of
expression of the inserted DNA
3. DAMAGE, a measure of the likelihood of harm being caused to a
person by exposure to a GMO.
Each factor is assigned a probability value and an overall value of the
GMO hazard to human health and safety is calculated by multiplying the
probabilities. The scoring system allows permutations from the highest

hazard of 1, reducing in increments of 10-3 to the lowest hazard score of
10-36 . ACGM in note 8 assigned laboratory containment levels to GMO
hazard levels. 10
For large scale operations, ACGM 8 did not recommend a linkage
between GMO hazard and containment levels or control measures:
Relative to the construction of genetically-manipulated organisms, there is
nothing intrinsically more hazardous about their large-scale use; it is the scale of
operation and hence the potential for a greater degree of exposure to an
organism and its biologically active product that is increased.
At laboratory scale, containment is therefore assigned according to the

hazard of the substance, i.e. the GMO. At large scale (LS), containment
can be considered to be assigned on the basis of risk which incorporates the
hazard of the GMO and the potential for GMO exposure from the process
equipment.
ACGM s advocated a flexible approach to large-scale containment in
terms of the unit operations of the process to take account of the diverse
range of equipment, processes, and types of hazard from the GMO (e.g.
allergenicity, infectiousness, etc.) The concept of selecting appropriate
containment levels commensurate with the level of risk at a particular stage
of processing is another feature of large-scale contained use of GMOs.
The current approach for assigning the GMO hazard category is to
classify initially into Group I or Group II by applying a list of criteria
detailed in the guide to the Contained Use Regulations. 4 Many of the
industrially important GMOs are likely to be of intrinsically low hazard
and fall within the Group I category because the host micro-organism is
well characterised and with an established history of safe use. The vector
will be well characterised and free from sequences coding for hazardous
products. In addition, the GMO may be genetically crippled such that it
has minimal chance of surviving outside the particular conditions of
fermentation in the bioreactor. Because of this inherant low hazard, the
application of Good Occupational Safety and Hygiene (GOSH) principles
apply for group I GMOs and for sound economic reasons, industry prefers
to use Group I GMOs for production purposes.
If the GMO falls within Group II, then three levels of containment are
considered for the operation in addition to GOSH. These range from B2 to
B4 for large-scale applied flexibly as described above. According to
ACGM, Brenner scores obtained from the laboratory scale construction of
the GMO influence the containment levels assigned to commercial
operations.
In the past, the distinction between small- (laboratory) scale and large-
scale operations was arbitrarily set at 10 I. The EC Directives seem to have
confused this issue of scale by introducing type A and type B operations
which seem to coincide with small- and large-scale operations. The UK

contained use regulations interpret type A operations as used for research,
teaching, non-commercial, and non-industrial uses and generally of small
scale. Type B operations are everything else. The distinction between type
A and B operations is a factor which is causing debate between the GMO
users and the regulators responsible for enforcing the contained use
regulations.
For the purposes of this chapter, type B operations are taken to mean
large scale where the containment levels B2 to B4 are applied where
appropriate for Group II GMOs.
11.4.2 Elaboration of containment principles

Process biosafety at the large scale is a mix of good operating practice,
reliable and well maintained equipment, and good equipment design. The
principles of Good Occupational Safety and Hygiene (GOSH) encapsulate
this statement. The principles stated in the contained use regulations 4 are:
(a) to keep workplace and environmental exposure to any physical,
chemical or biological agent adequately controlled;
(b) to exercise engineering control measures at source and to supplement
these with appropriate personal protective equipment when necessary;
(c) to test and maintain control measures and equipment;
(d) to test where necessary for the presence of viable process organisms
outside the primary physical containment;
(e) to provide training of personnel;
(f) to formulate and implement local rules for the safety of personnel.
These principles are recognisable from the OEeD document' which
underpin Good Industrial Large Scale Practice (GILSP). GILSP has
widely been considered as minimal containment and often considered to
involve no containment measures beyond those required for process needs.
Despite initiatives by the OEeD and the Ee to elaborate on the
international and European interpretation of GILSP process plant design
and operation, there is still considerable variation and unresolved debate.
This perhaps is not surprising given the generality and flexibility of the
guidance. In many cases, process plant operating under GILSP often
seems very similar to plant using the 'minimise release' concepts of
containment level B2. This 'blurring' is particularly apparent for bio-
pharmaceutical processes. However, it is often emphasised that standards
of GILSP will vary from industry to industry so that food and brewing, for
example, may deploy fewer measures to protect the product and hence
have less intrinsic containment.
For Group II GMOs, the regulations require releases to be minimised at
B2 containment levels, and release to be prevented at B3 and B4
containment levels. Here, also, precise definition of terminologies have

not been derived since their introduction by OECD. l Perhaps because of
this lack of definition, the biotechnology industry has taken a responsible
and cautious approach to process plant containment often using higher
containment than arguably is necessary.
In addition to the potential GMO hazard risk of the micro-organism,
higher containment could be justified for other reasons:
• The process may produce biochemicals which may cause occupational
diseases or give rise to environmental difficulties.
• There may be insufficient evidence to support an environmental risk
assessment and satisfy regulatory authorities that releases or discharges
will not harm the environment.
• Public and process operator perceptions and concerns exist about
possible effects by release of modified micro-organisms.
• Companies may not wish competitors to obtain their GMOs by sampling
outside the factory.
The 'down-side' of paying too much attention to containment is that a
'harmless' GMO begins to take on the role of a hazardous chemical or
radioactive substance. Containment therefore becomes counter-productive
when process operators and the public question why special measures are
being taken for a substance claimed to be harmless.
The challenge therefore is to strike a sensible balance betwen the GMO
hazard and the control measures. The problem historically has been a lack
of good information to enable the general and qualitative requirements of
the regulations to be translated into acceptable engineering design. The
Industrial Biosafety Project has encouraged the development and collation
of more data and information so that the problem can be solved on a more
rational basis.
11.4.3 Equipment containment design principles

This section summarises some of the important factors which have been
considered and debated within the IBP and the industry and provides
examples of equipment design and operation. The potential for releasing
significant quantity of biological material either inadvertently as a
consequence of an accident, or routinely during its operation, are both
important considerations.
The principle of developing various levels of containment design is
illustrated schematically by Figure 11.2 for a sealing arrangement. The
basic sealing requirement is a single seal providing a barrier between the
micro-organisms and the workplace environment. Failure of the seal would
normally provide an outward t10w to the environment, due to the pressure
differential, protecting the equipment contents but presenting a biohazard.
(a) SEAL
MICRO-ORGANI
PRIMARY SEAL
(b)
M ICRO-OI3GAN ISM
RETURN
(c)
WORKPLACE
MICRO-ORGANISM ENVIRONMENT
BARRIER
FLUID
Figure 11.2 Concepts of containment for seals. (a) Single seal; (b) double seal; (c) double
seal with barrier fluid.
The use of a back-up secondary seal may offer additional security against
release. A steam flush, or another barrier fluid, offers a higher level of
security.
Chapman 11 suggested a simple framework for relating mechanical design
to the level of operational containment. Table 11.1 illustrates this
framework for static and dynamic seals (using the contained use regulations
terminology) .
Table 11.1 Interpretation of containment design and containment level adapted from
Chapman"
Containment level Static seals Dynamic seals
B2 Minimise release Single '0' ring Double mechanical seal

B3 Prevent release Double '0' ring Double mechanical seal with
steam flush
or
Double mechanical seal in
ventilated housing
B4 Prevent release Double '0' ring with Double mechanical seal with
steam tracing steam flush in ventilated
housing
Without being too literal in the use of these interpretations, the scheme
could provide a starting point for assessing the preliminary intrinsic
containment of equipment at the drawing board stage. A more detailed
assessment would then for example consider the seal geometry or the
reliability of the single seal. 12
The literal translation of this framework however has initiated wide
debate and controversy within the IBP and elsewhere. This is particularly
evident when considering static sealed systems (see section 11.4.3.2). It is
an excellent example of the need of the industry to have something
"concrete" to use yet still retain the flexibility built into the current
guidance. Some of these issues are explored in the next sections using
examples of bioprocess equipment components.
]J .4.3.1 Rotating seals

Rotating seals are commonly used on bioreactors and pumps. For stirred
tank bioreactors, initial considerations include whether the drive should be
top or bottom mounted. The use of a magnetic coupling between the drive
and the agitator is also an option on some fermentation equipment.
In general, there seems to be no consensus on the siting of a drive. The
main objections for bottom-driven bioreactors is the possibility of a
catastrophic failure of the shaft seals providing a major release. Many
authors favour top-driven bioreactors from a containment viewpoint. 13- 15
An additional consideration is the reduced lifetime of seals if the
bioreactor contained abrasive suspended solids.
The advantages of bottom-mounted drives are that they provide easier
access to the bioreactor top plate with for example no disconnection of the
drive and lifting of the motor. It also allows more space for installation of
probes, inlet lines and mechanical foam breakers.
Figure 11.3 illustrates a double mechanical seal arrangement for a
bottom-driven bioreactor. The double mechanical seal is a good example
where the dual requirements of sterile operation and containment can be
. FERMENTER'~
-FLUID - -
UPPER SEAL
ASSEMBLY
ROTATING AND ST
SEAL INTERFACE'
,'-
t·,' '-,
STATIONERY SEAL
'~ "- LOWER SEAL
ASSEMBLY
Figure 11.3 Double mechanical seal, tandem configuration (Courtesy of New Brunswick
Scientific, Hatfield, UK).
achieved. The lower seal assembly provides a back-up to the upper seal
assembly. The chamber between the two seal assemblies can then be filled
with a sterile lubricant such as food grade condensate, sterile water or
pressurised steam.
The principle of the sealing can be seen by considering the upper seal
assembly. A rotating seal connected to the shaft via a bellows assembly
interfaces with a stationary seal connected to the body of the bioreactor. In
this arrangement the upper seal is immersed in the process fluid.
Monitoring of the lubricant flows is a useful means of checking for failure
of seals and is practised on many bioprocess plants especially when
culturing GMOs. For high containment processes such as B3, the lubricant
flow is commonly piped to a kill tank.
For most purposes mechanical seals are perceived to be satisfactory for
bioreactors provided they are routinely maintained and replaced at
recommended intervals. Magnetic drives eliminate the need for penetration

of the vessel wall and offer complete containment at this point. The main
objections are related to the maximum torque which can be achieved
before decoupling and whether suspended solids can get trapped between
the stirrer and support in the vessel if the drive is at the base. Top-mounted
magnetic drives are available. Walker et al. 16 describe a magnetic coupling
design used in bioprocessing and designed to reduce the problems of
decoupling. Currently magnetic drives are not widely used compared with
mechanical seal drives. Some manufacturers routinely supply magnetic
drives mainly for cell culture applications, although magnetic drives can be
obtained from many manufacturers on request including magnetic drives
for bacterial cell bioreactors.
11.4.3.2 Static seals

Static seals are generic to many items of bioprocess equipment and a very
high number of seals may be found on a complete process plant. A single
seal arrangement is suggested as being satisfactory for GILSP and B2
containment. However, the need for mechanical containment of increasing
complexity for B3 and B4 operations is not felt to be necessary and on the
contrary could make a plant very complicated and difficult to operate
effectively.
The double '0' ring concept, in particular, is not perceived by many to
offer any advantages over the single seal system since it is argued that both
seals would be subject to the same conditions of failure and hence the same
failure rate. Even if one seal failed, this could probably not be detected in
many instances until the planned maintenance and inspection. The
prospect of a non-cleanable and possibly non-sterilisable gap between both
seals is another disadvantage.
The barrier fluid concept for a two-seal arrangement in theory might
overcome the problems of detection failure and non-cleanability. The
lifetime and the reliability of the seal could be reduced due to the barrier
fluid. For example, a constant steam trace might reduce the seal lifetime
compared with the normal sterilisation cycles of the single seal arrangement.
The concept of every seal being steam traced is a frightening prospect both
economically and operationally.
Despite these comments, examples are found of equipment where
double seals and sometimes steam tracing are used in certain cases. These
include fermenter top plates 16 • 17 and probes and entry ports. 18
Sealing arrangements are commonly required to connect pipework
together. The ideal means to both prevent release and stop contamination
is to butt weld with an orbital welder. This limits the number of potential
failure points and has been used on a high containment facility. 17 Servicing
of some components however requires cutting through the pipework and
rewelding in the new component. For lower containment systems of
INDUSTRIAL BroSAFETY PROJECT 223
GILSP plants, IDF (International Dairy Federation), ILC (In-Line

Cleaning) and Triclover fitting are satisfactorily used. 19 All these designs
provide a seal ring which butts up flush to the internal bores of the
connecting pipework.
If welding is either impractical or not considered necessary, a single seal
arrangement arguably could cover all containment levels. For higher
containment, increased emphasis should be paid to the reliability testing
and maintenance of the seals.
A general problem is the lack of reliability data for bioprocess
equipment. For common pipework connectors used in bioprocessing plant,
the IBP undertook preliminary tests subjecting them to repeated cycles of
sterilisation and cooling. In nearly all cases, we could not detect
containment failure for sterilisation/cooling up to 160 cycles. It was noted
that the internal part of the seal deformed in many cases deviating from the
flush internal assembly. Thus clean ability would become a problem while
containment was still offered. This phenomenon was also observed when a
coupling was subjected to over 1000 sterilisation/cooling cycles. Thus the
results suggest that such seals were likely to be changed from the hygiene
viewpoint before they got to this state. Containment would be maintained
provided the seal was correctly replaced and tested before use.
11.4.3.3 Pressure relief systems

A pressure relief system is normally required on all bioreactors and
pressurised tanks as a safety feature to comply with pressure vessel design
regulations. In a few cases, some companies appear to have overcome the
need to use a pressure relief system on the vessel either by ensuring
pressure relief is provided on relevant pipework to the vessel and/or
ensuring there are no pumps transferring material which could lead to a
build up of pressure if the outlet pipework were closed. The absence of
pressure relief on the vessels can considerably simplify the process plant.
The safety and regulatory requirements, as well as insurance inspection
requirements, for pressure relief is currently a confused area.
Given that a pressure relief system is required, bursting/rupture discs or
spring loaded safety relief valves can be used. The merits of both systems
have been popular discussion topics. Bursting discs are generally favoured
since they are of more hygienic design, operators cannot interfere with the
release pressure setting whilst relief values may stick open by foreign
material lodging on the valve seat.
The need for a pressure relief to operate should be very rare. Van
Houten 20 outlined a plan of events before the bursting disc would activate.
One obvious factor is automatic shutdown of the air supply if the pressure
exceeded a certain value. For some operations, the supply air pressure
need not be greater than the emergency vent pressure.
For GILSP operations, venting the exit line away from the workplace
but into the environment by venting to a chimney (for example) should

satisfy the health and safety requirements. The environmental risk
assessment could then be separately considered. For contained operations,
venting to a kill tank has been described. This however can sometimes
present a conflict between physical safety and microbiological safety where
many local design codes disallow the installation of devices in the discharge
lines which restrict flOW. 21 A kill tank with HEPA filtered venting could be
interpreted as restricting flow. At present this conflict is not satisfactorily
resolved. Relatively large kill tanks providing damping to shock loading
from the bursting disc is one possibility (see chapter 12).
Companies could also look to the chemical industry for solutions. A
number of venting solutions are available in the chemical industry for
disposal of emergency relief system effluents as reviewed by Grossel. 22
11.4.3.4 Bioreactor exhaust gas

Considerable quantities of exhaust gases emerge from fermentation
processes. Most traditional fermentation processes do not treat the exhaust
gases prior to discharge into the environment. This however is not
necessarily considered good practice by many and the introduction of
tighter environmental regulations requiring information such as environ-
mental impact statements could raise the question whether such traditional
practice is justified. For new processes based on low hazard genetica\ly-
modified micro-organisms, Devine 23 provided a UK regulatory inspector's
viewpoint.
If an environmental impact statement provides evidence that an operation is
acceptable to the satisfaction of the various parties (including the local
authorities responsible for pollution and the environment), then this would be
accepted as good practice. Where there is any significant doubt, it would be
prudent to apply additional controls.
Devine also suggested that a plant which proposed using simple water
sprays to reduce the bioburden of exhaust gases would not necessarily
conform with good practice.
Winkler and Parke 24 reported typical bacterial concentrations of lOb/m 3
air in the bioreactor head space which could be reduced to 100/m3 by use of
a simple syphon device in the exhaust gas line. Winkler also provided some
views on the environmental risk assessment of recombinant micro-
organisms released from the exhaust line at the rate of 1000 microbes per
second. For example, using a simple plume dispersion model, it was
predicted that at 100 m distance from the source, the airborne microbial
concentration was less than 11m3 with the dry deposition being of the order
of 0.4 microbes/m 2 /hour (see also Chapter 6).
In general, the industry to date appears to have deployed some form of
exhaust gas treatment for GMO fermentations. Fogglesong25 reported
successful and safe production of human insulin of scales of 40 m 3 or more

using adapted antibiotic fermenters including the provision for filtration or
incineration of all exhaust gases. Filtration is normally the more practical
option for exhaust gas filtration. Pre-treatment of the exhaust streams by
condensers, gas liquid separators, re-heaters is considered necessary for
optimum performance of the filter. 26 Current practice has used one filter to
minimise release but two filters in series have often been used to prevent
release. It is necessary that filters are capable of being tested in situ.
The need for off-gas treatment of Group I GMO fermentations is a
debatable point. From the health and safety viewpoint, exhaust gas could
be vented away from the workplace environment. If this were planned,
then an environmental risk assessment would need to demonstrate the
practice to be acceptable. Winkler and Parke 24 discuss some of the factors
concerned with environmental risk assessment for GMO aerosols.
11.4.3.5 Sampling systems

Regular sampling of the bioreactor contents is a necessary operational
procedure. Sampling requires good mechanical design and good operator
practice to ensure sterility and containment. Nearly all the sampling valve
designs were conceived originally to protect the bioreactor contents against
contamination. Where high containment is required, some authors
considered that safe operation can only be guaranteed by the use of
secondary containment. 17 Many devices which could be considered to offer
a reasonable level of containment use hollow needles and septa. Many how-
ever are disallowed in many operating companies because of the intrinsic
physical hazard. A contained sample collection system was described by
van Houten 20 consisting of eight separate valves shown on Figure 11.4.
Although intrinsically contained, the complexity could lead to operator
error and automation of the valve sequencing could be beneficial. Jefferis
and Schlager27 applied a quantitative risk assessment technique based on a
fault tree analysis to a similar bioreactor sampling valve assembly. Their
analysis illustrated the benefits of automation.
Ideally if prevent release is required, there should be no chance of
residual live micro-organism droplets being present in the workplace
environment. This could occur when connections are broken such as
sample bottle removal. The Bioengineering sample valve is a simple to use
device designed to prevent release and is shown on Figure 11.5. The
German BG Chemie pUblication 28 illustrates P3 (B3) containment level
with this device. The principle is that the sterile hollow needle penetrates a
rubber septum of the partially evacuated sample bottle. After filling, the
needle is withdrawn and the sampling parts are sterilised with steam before
the bottle is removed. The device has the advantage of simple operation
but the operator must ensure that septa are used only once. When the
sample bottle is removed, there is also a risk of injury from the needle
FILTER
FERMENTER
TO WASTE
TO WASTE
BOTTLE
Figure 11.4 Contained sample collection. After van Houten 20 •
NEEDLE, 6mm DIAMETER

BOTTLE
HOLDER
SAMPLING BOTTLE
FERMENTEA INTERIOR
CONDENSATE '0' RING
Figure 11.5 Bioengineering sample valve.

being pushed into the hand and/or a breach of containment. Proper

training is therefore imperative. Some companies have chosen to improve
the device by fitting simple push-on guards both on the hollow shaft to stop
accidental operation and a shroud over the sampling bottle to protect
against possible shattering of the glass.
Some problems can occur when taking samples from fungal or mycelial
fermentations due to the semi-solid nature of the material. Most
commercial sampling devices would not be suitable since they might
quickly block and/or would not be taking a representative sample of
material due to its complex flow behaviour. Representative sampling in
this case requires flow through a pipe of significantly larger diameter.
Sampling using a sequence of diaphragm valves comparable to the pipe
diameter would appear most suitable for these types of material.
11.4.3.6 Addition and harvest systems

As well as sampling, sterile transfer of material to and from the bioreactor
is required and the possibility of containment breach during these
operations should be assessed. Additions include chemicals for pH
adjustment, antifoam, nutrients and inoculum. Some authors have
considered the possible risks of spillage associated with inoculum addition.
Van Houten,20 Vranch 29 and Werner 30 illustrate contained methods of
adding inoculum by gravity and sterile air overpressure methods. Werner
also illustrates less complicated methods considered satisfactory for
GILSP. Elliot 15 et al. recommend that an unbreakable inoculating device
should be designed and used for large bioreactors which are capable of
being sterilised after inoculation prior to removal. We consider it would
also be useful to design special holders for smaller inoculum flasks to
prevent breakage should the flask be inadvertently dropped. Alternatively,
unbreakable small inoculum flasks could be designed.
Harvesting is not normally a problem for most bioreactors but an
internal dip tube can be used 17 to eliminate leakage risks due to wear or
failure of the harvest valve. In this case, overpressure or alternatively
'suction' is required to harvest the cells.
11.4.3.7 Valves
The most popular types for bioprocessing plants are the diaphragm and
ball valves types. The latter are more robust but contain crevices making
sterilisation more difficult. Barnsley 19 stated a preference for diaphragm
valves since they are reasonably crevice free; the material EPDM (ethylene
propylene diene modified) was the preferred diaphragm material.
Provided diaphragms are replaced on a regular basis and not abused or
badly maintained, then they should not cause problems for most
operations.
Sterilisable butterfly valves with provision for an outer chamber filled
with a pressurised barrier medium has also been described by BG

Chemie 28 (1989) for preventing release.
11.4.3.8 Bund walls

The provision of bund walls or dykes around equipment could be
considered to be good practice. They need to be sized to retain the entire
contents of the equipment should catastrophic failure occur. 21,31 Van
Houten 20 recommends that provision should be made for the material to
be pumped to a spare bioreactor or a continuous steriliser. Decontamination
and cleaning up of the residual puddles are then carried out using the
appropriate safety precautions.
11.4.4 Measuring and monitoring containment

The principles of good safety and occupational hygiene include ensuring
that engineering control measures and equipment are tested adequately
and maintained. Tests can be useful for prechecking equipment prior to
operation, or monitoring during operation, or sometimes both. Both
physical and biological methods can be used many of which have been
evaluated and developed by the Industrial Biosafety Project.
11.4.4.1 Pressure hold test

Pressure testing is widely practised in the biotechnology industry particu-
larly for bioreactors, but the criteria for passing or failing a test is
somewhat arbitrary and often depends upon the operator's experience and
the practicalities of the test. The test is relatively simple and involves
pressurising the equipment to a given pressure, for example with air, then
noting any change in pressure in a specified time period. Relatively few
bio-equipment manufacturers provide details of a pressure test for their
equipment. The operating companies generally have evolved their own
operating procedures including pressure testing. The impetus has been to
ensure the process is not contaminated.
The pressure test is of most use if the test volume is small. It becomes
meaningless for large vessels since changes in pressure will not be noticed
even if there is leakage of material.
We estimate that 4 m 3 volume vessels would need to indicate a change of
2 millibar pressure over one hour for a pressure test to have some use. Such
pressure monitors are available 32 and could be considered for large volume
equipment.
11.4.4.2 Leak location

The pressure hold test can be envisaged as a general test to highlight if
there is significant leakage. If this is so, the leaks can be located by several
methods.
Leaver and Stewart 33 demonstrated the use of helium both for a pressure
test and as a highly sensitive method of leak location. By reducing the data
to an equivalent critical orifice leak, a pressure test on a 50 I fermenter
resulted in a leak tightness value of 178 !-tm. The helium detector readily
located misshapen and worn fermenter couplings which were repaired
resulting in a new overall leak tightness value of 74 !-tm. In a survey of
fermenters users by the Industrial Biosafety Project, bubble emission by
the use of soapy water around joints appeared to a popular method of leak
location. One fermenter manufacturer recommends checking for leaks
using 0.5% Savlon solution to check for bubble emission. Sulphur
hexafluoride (SF 6 ) leak detectors are used routinely on high contain-
ment fermenters at CAMR Porton Down. 17 Another potentially useful
technique, which should be considered, is the ultrasonic detector and can
be used with an air pressure hold test. Leakage is readily detected by the
ultrasonic sounds set up by gas discharge. Advantages of the detector
include the relatively low cost (£500-£1000; US$800-1600) and the
ability to check for leaks during equipment operation.
11.4.4.3 Air monitoring

The most likely route of occupational exposure in biotechnology is via
aerosols generated by breaches of containment during cell growth in
bioreactors and subsequent processing to separate, concentrate and purify
the desired product. 34 Hence aerosol measurement is particularly relevant
to both assess and monitor containment.
Testing for the presence of viable process organisms outside the primary
physical containment is a desirable but not a necessary principle of good
occupational safety and health. Companies have become more interested
in collecting routine data particularly on general airborne micro-organism
levels in the processing plant. Data are still relatively scarce and companies
generally are unwilling to disclose levels of process micro-organisms (if
detected). This situation is partly because companies perceive such figures
might be mis-used, resulting in the setting of unreasonably stringent upper
limits of exposure to low hazard micro-organisms.
The collection of data on a regular basis can be useful in several ways.
Should unusually high levels of micro-organism be detected, the cause can
often be traced to a particular item of equipment or work practice not
operating as planned. In some cases, it can be used to demonstrate a better
method of operation to reduce workplace emissions. Monitoring also
provides a back-up to the equipment validation (such as pressure testing)
although the time lag from the event to micro-organism detection does not
allow immediate corrective action. With more data collected, companies
can begin to develop a strategy to decide when a particular level of
exposure is unacceptable.
It is encouraging that more companies are undertaking monitoring for
process micro-organisms. From the company viewpoint, the practicality of
the technique is often more of a consideration that the quality of the data
likely to be produced. The main considerations on the selection of devices
for sampling airborne micro-organisms have been described by DeCosemo
et al. 35 and Hambleton et al. 36 Environmental monitoring of the air for
process micro-organisms using various types of sampling devices have been
reported15.25.37-41 and also used to assess operational containment of
bioprocess unit operations and components. 42-44 Because there is a range
of samplers which will provide varying degrees of quantification, it is
important that the sampler model and type are stated as well as the
recorded measurements.
Within the IBP, many types of sampling devices both from the collection
viewpoint and the subsequent assay viewpoint have been examined to
improve the specificity and detection response time.
Besides microbial methods of monitoring, useful data can be obtained in
some cases using other techniques. Electronic airborne particle counters
provide near instantaneous readings. They are commonly used for
monitoring clean room environments and provide total particle counts
often with size fractionation but cannot discriminate particle types. Stewart
and Deans 44 used a TSI laser light scattering particle counter to measure
the containment of a cell disruptor by surrounding the equipment with a
cabinet supplying HEPA filtered air.
Many bioprocess streams consist of significant quantities of dissolved
salts which are highly electrically conductive. Measurement of electrical
conductivity is a simple yet effective means of measuring release. A range
of air samplers are available which collect the aerosol into liquid media.
When coupled to a conductivity probe, an on-line device can be operated.
Stewart and Deans 44 also used this technique for measuring cell disruptor
containment and were then able to report spray factors as a means of
characterising aerosol release. Conductivity measurement for monitoring
(bio )aerosol release has an added advantage of being able to detect
containment breach without interference from the ambient air since the
workplace generally has a relatively low concentration of salt aerosols.
Environmental monitoring for biochemicals has also received attention.
Behizad et al. 45 developed a prototype on-line sensing technique for
protease and other biochemicals. The monitor was tested in a detergent
factory environment and demonstrated to be sensitive and rapid. It could
well be a useful development enabling enzyme airborne concentrations,
well below the 0.4 ~g/m3 exposure standard (see 11.4.4.4), to be detected.
detected.
11.4.4.4 Exposure limits and use of monitoring data

In contrast to many other industries, there is a paucity of published
information on either occupational exposure or environmental exposure
effects of micro-organisms and their products. This situation presents
difficulties since the process designer wishes to comply with health and
safety legislation yet has no numbers on which to produce an optimum
design solution when total containment is unnecessary.
A risk assessment to comply with the UK COSHH (control of substances
hazardous to health) regulations is more easily undertaken when handling
chemicals for example. It can often be readily estimated whether a given
emission from an operation will exceed the accepted occupational
exposure limit (OEL). If it is well below the figure, then no further action
is necessary.
In the absence of OELs, some companies are moving towards in-house
limits by building up base line data using a selected monitoring or sampling
device. However, few have currently been published in the field of
biotechnology partly because OELs may be set too stringently for harmless
micro-organisms (as outlined in 11.4.4.3).
Rylander et al. 46 made some tentative recommendations based on fever
and influenza-like symptoms observed to be experienced among workers at
wastewater treatment plants. They suggested that until more precise data
on the dose-response relationships became available, values up to 1000
Gram-negative bacteria/m 3, derived from an endotoxin limit of 0.1 ~g/m3,
was acceptable.
Perhaps more relevant to biotechnology was a study by Palchak et al. 47
concerned with recombinant E. coli K-12 (Gram-negative) endotoxins
exposure during production of therapeutic proteins. The authors suggested
a limit of 30 ng/m 3 using a safety factor of 10. They compared published
values of human exposure studies and designated a decrease in the forced
expiratory volume at 1 second (FEV1) of 5% as a clinically significant
endotoxin effect. They concluded that a mean value of 300 ng/m 3 mean
level gave a 5% decrease in FEV1. This is approximately equal to 3000
cells/m 3 and a working limit of 300 cells/m 3 . Measurements of endotoxin on
their own process plant was below the 30 ng/m 3 value except in isolated
instances where engineering controls were not present resulting in a
maximum endotoxin level of 1812 ng/m 3 . In this case, engineering controls
reduced this level below the 30 ng/m 3 level.
For proteases (detergent enzymes) voluntary exposure limits of 0.4 ~g/
m 3 have been set. 45 By comparison, the limits on an 8-hour personal
exposure to total inhalable dusts is 10 mg/m 3 , i.e. 25 000 times higher than
protease and 100000 higher than the endotoxin concentration. 46
The need for more data and consensus within the industry is an
important aspect of process monitoring. The European Committee for
Standardisation (CEN) Technical Committee 233 has attempted to get a
consensus on this problem so that equipment in particular can be designed
and tested to a performance standard. However, there is still debate on
whether there is a need to set limits bearing in mind the diversity and range
of micro-organisms and industries.
ll.4.5 Maintenance and training

Planned maintenance of equipment is an essential part of ensuring that a
minor fault is prevented from becoming a major and costly fault to correct.
The implementation of proper maintenance is an integral part of good
practice. Kleppinger48 recommended a bioreactor preventive maintenance
programme based on checks before each bioreactor operation, as well as
monthly and quarterly preventive maintenance. Training and systems of
work are equally important in ensuring good practice is maintained.
Solderberg49 and Taylor50 provide useful advice on these aspects.
The provision of standard operating procedures (SOPs) is an important
factor underpinning GILSP and ensuring that Good Occupational Safety
and Health is achieved. In many cases, a high number of SOPs may be
required to cover many of the aspects of the bioprocess operation.
Operability studies can also be useful, particularly when new or modified
processes are being implemented. The Hazard and Operability Study51
(HAZOP) is a system widely used in the chemical industry to systematically
explore hazards which may arise from operating process plant and to
implement actions to deal with the hazardous consequences.
Some companies use HAZOP for biotechnological processes. It requires
convening an expert team to meet on a regular basis, to explore ways by
which the design and operating intentions can go wrong. The committee-
based assessment approach is valuable in many respects since SOPs,
maintenance and emergency procedures for example can be designed or
modified involving key persons responsible for safe process operation.
The IBP have tested a modified form of HAZOP for contained use
biotechnology operations. We found that we could systematically address
the interrelated issues of equipment mechanical design, operation,
monitoring, maintenance, and training. Modifications to SOPs and/or
equipment were identified.
11.5 Environmental safety
The second part of the risk assessment and assignment of appropriate

containment levels is concerned with environmental safety. The need for
environmental risk assessment has taken on a higher emphasis than
previous practice having being written into the EC directives and
implemented in the UK by the Contained Use Regulations.
As discussed in section 11.3, a recommended approach is to undertake
the environmental risk assessment once measures to cover human health
and safety have been addressed.
The need to implement additional control measures is based on
considering hazards from a number of factors, incIuding:9
• potential for survival;

• potential for establishment and dissemination;
• transfer of genetic information between GMO and other organisms
• pathogenicity;
• any other identifiable negative effects (target and non-target).
For each hazard, estimates are required on two features
• the likelihood of the hazards being realised;
• the magnitude of the hazardous consequence.
The two features are assessed on the basis of the containment already in
place or assigned using a four-point scale as shown on Table 11.2. From
this table, the estimates are combined to give the risk of causing
environmental harm.
There is no additional need for containment or extra control measures if
the risk is assessed to be low or effectively zero.
ACGM 9 have suggested that the capacity to survive, establish and
disseminate will be the key. They also suggest that, for organisms not
capable of surviving in the environment, none of the other hazard areas
usually considered will come into play and the organism can probably be
considered safe. Examples of such GMOs are many of the multiply
crippled organisms used in containment such as some auxotrophic strains
of asporogenic bacilli or E. coli K12. However, there is also a need to
decide on the potential for the gene product, or nucleic acid construct, to
cause harm by transfer to another organism where it could replicate and be
expressed. This is irrespective of the organism's capacity to survive in the
environment.
Most companies have found the environmental risk assessment require-
ments to be the major new challenge of the 1992 Contained Use
Regulations. Up to this point, companies had undertaken very little
environmental risk assessment. They also indicated that more information
was required to enable them to undertake any meaningful environmental
Table 11.2 Determination of risk of causing environmental harm
Magnitude of Likelihood of the hazards being realised

hazardous
consequence High Medium Low Negligible
Severe High High Medium Effectively

zero
Medium High Medium Medium/low Effectively
zero
Low Medium/low Low Low Effectively
zero
Negligible Effectively Effectively Effectively Effectively
zero zero zero zero
assessment. Some were prepared to undertake some research on the

environmental survival and interactions of their process genetically-
modified micro-organisms.
For large-scale use, an initial consideration is to estimate the likely levels
and quantities of material which could be released to the environment
either routinely or following an emergency response should a spill have
occurred.
From the human health and safety viewpoint, piping liquid and gaseous
wastes away from the workplace could be considered adequate. If the
overall containment requirement is to prevent release to the workplace and
general environment by inactivating the wastes, then the environmental
risk assessment should be straightforward. No 'hazardous' material would
routinely be interacting with the environment and therefore the likelihood
of the hazards being realised could be judged to be negligible.
The difficulty lies with processes where only minimal containment is
necessary. An important question being raised is whether Group I GMOs
could be discharged in liquid streams after harvesting without further
treatment. The argument here is that the GMO is categorised as 'harmless'
with no more hazard than the natural host organism. Why should it require
special treatment? Current practice generally has been to inactivate waste
streams even for Group I GMOs. This contrasts with traditional process
micro-organisms (which have not been genetically modified) where no
special treatment is routinely deployed.
More data on environmental interactions of industrial GMOs are
required to assist in the environmental risk assessment so that it can be
demonstrated that the risk of causing environmental harm is low or
effectively zero. The production of environmental risk assessments for
process GMOs is currently an evolving subject.
Release of a GMO in the waste stream could be construed to be a
deliberate release although it falls within the remit of the contained use
regulations. Nevertheless, information from studies on deliberate releases
of GMOs could be usefully deployed for contained use operations. The
IBP is seeking to collate relevant information for risk assessment purposes.
The Royal Commission on Environmental Protection adapted the HAZOP
technique as a practical and systematic technique to consider, assess and
control possible risks from GMO release to the environment. The
technique, termed GENHAZ,52 has also been tested by the IBP for
contained use assessments. We developed an integrated approach to assess
the operational aspects of the plant and equipment (as described in section
11.4.5) and the environmental aspects of possible GMO releases. We
concluded that a more rigorous environmental risk assessment could be
developed than had hitherto been provided by companies.
Some further notes on assessing risks to the environment from
production strain GMOs are provided by Winkler and Parke. 24
INDUSTRIAL BroSAFETY PROJECT 235
11.6 Conclusions
The safety issues associated with GMO process-based biotechnology have

resulted in a formalised set of regulations. In the UK, the Contained Use
(1992) Regulations have implemented the EC Contained Use Directives.
Risk assessment is a key requirement of the GMO regulations. For
contained use, the emphasis historically has been on human health and
safety but environmental considerations have gained increasing signific-
ance.
A suggested risk assessment framework is to undertake human health
and safety considerations and then to assess the environmental aspects
taking into account containment measures already assigned.
For sound economic reasons, industry prefers to use Group I GMOs for
production purposes. For such GMOs the application of Good Occupa-
tional Safety and Hygiene (GOSH) principles apply.
Process biosafety at the large scale is a mix of good operating practice,
reliable and well maintained equipment, and good equipment design. The
GOSH principles encapsulate this concept.
It is important to strike a sensible balance between the GMO hazard and
the control measures. The problem historically has been a lack of good
information to enable the general and qualitative requirements of the
regulations to be translated into acceptable engineering design. The
Industrial Biosafety Project has encouraged the development and collation
of more data and information so that the problem can be solved on a more
rational basis.
It is not sensible to rigidly adhere to a framework of equipment
containment design. Nevertheless when higher containment is required, a
containment framework can be helpful to assess preliminary designs.
Testing equipment for containment and/or its reliability is also important.
As an example, a very high number of seals may be found on a complete
process plant. A single seal arrangement is suggested as being satisfactory
for GILSP and B2 containment. With sufficient attention to maintenance
and testing, IBP test data suggest that single seals could be considered
satisfactory for higher containment in many cases. Welding is another
option, such as connecting pipework, for high containment.
Companies are beginning to use workplace monitoring as part of the
GOSH principles. As more data are collected, it is possible that companies
will be able to devise in-house strategies to correct equipment or operator
practices not operating as planned.
The environmental risk assessment is perceived to be a new feature of
contained use GMO risk assessment and is an evolving subject. There is a
need for more information to demonstrate environmental risks to be low or
effectively zero. Examples of the potential for environmental releases are
by the fermenter off gas, bursting/pressure release, or discharge of wastes.

The environmental risk assessment is required to show that no adverse
effects will occur should release occur. To date, companies have
implemented GMO processes at relatively high containment even for
Group I.
Biosafety for large scale operation is an important subject. Standards for
equipment and large scale processes are being formulated by the European
Committee for Standardisation (TC233). Much of this work is driven by
biosafety considerations. The Industrial Biosafety Project has a significant
knowledge-base on large scale biosafety issues and is making a significant
input to the formulation of standards ensuring they are workable and
useful to the industry.
On a wider perspective, collaboration and fruitful discussion between all
sectors of the industry is important to ensure that biotechnological
processes can be implemented based on a balanced view of the risks and
the benefits to mankind. A common approach to biological safety and risk
assessment, to embrace GMOs and non-GMOs, is widely perceived by
industry as a logical and desirable strategy. Treating GMO processes as a
'special case' can be counter-productive leading to unjustified speculation
that the GMO is always inherently more hazardous. The Industrial
Biosafety Project offers an important role by providing the forum and the
relevant scientific 'technology transfer' for a consensus approach to be
implemented.
Acknowledgements
This paper is based upon work undertaken within the Industrial Biosafety
Project based at Warren Spring Laboratory (WSL) and CAMR,
Porton Down. Funding from the following sources is gratefully acknow-
ledged: the UK Department of Trade and Industry, Public Health
Laboratory Service, Health and Safety Executive, and Project Members.
Further details on the Industrial Biosafety Project are available from the
author. WSL has since merged with AEA Technology, Harwell from
where IBP now operates.
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49. Solderberg, A.C. (1983). Fermentation design. Ch. 3 in Fermentation and Biochemical
Engineering Handbook, Vogel, H.C. (Ed.), Noyes, pp. 77-118.
50. Taylor, I. (1989). Software and hardware considerations in biotechnology. In Proceedings
of the DTlIHSEISCl Symposium on Large Scale Bioprocessing Safety, Salusbury, T.T.
(Ed.), 30 Nov.-l Dec. 1988, Warren Spring Laboratory Report LR 746 (BT), August,
Gunnels Wood Road, Stevenage, UK, pp. 88-91.
51. Kletz, T.A. (1992). Hazop and Hazan: Identifying and assessing process industry hazards
(3rd Edn). Institution of Chemical Engineers, ISBN 1 56032 276 4, Hemisphere
Publishing Corporation.
52. RCEP Royal Commission on Environmental Pollution (1991). GENHAZ: A System for
the Critical Appraisal of Proposals to Release Genetically Modified Organisms into the
Environment, Royal Commission on Environmental Pollution 14th report, June, cm 1557,
ISBN 0-10-115572-7 HMSO, London.
12 Managing the effluent from bio-industrial
processes
J.R. COURT
12.1 Introduction
In the bioindustries, the disposal of waste materials is of at least as great

importance as in other fields of industry. While biological materials are in
many ways 'natural' and subject to ready biodegradation by natural
processes, nevertheless the environmental impact of large quantities of
biodegradable materials released over a short period of time and/or at high
concentrations must be considered. Moreover, those bioprocesses which
. handle potentially pathogenic or otherwise hazardous material must be
subject to further constraints. Because of the immediate short-term
hazards presented by the uncontrolled release of such organisms, from the
health of the industrial workers, to the local population both human and
animal, a system of management is necessary to ensure the safe disposal of
bioindustrial wastes. Nor must the potential hazard to plant life be ignored;
plant pathogens are also used in industrial processes.
There is considerable concern world-wide on the hazards, real or
imagined, of release into the environment of genetically-modified organ-
isms (GMOs). The designers of industrial processes must be aware of
environmental issues and take account of them in assessing risks and
strategies. Nevertheless, the bioindustries (for example, antibiotic manu-
facture) have for many years discharged large quantities of biologically
active materials into the environment, with little regard for the environ-
mental risks or health hazards such action could involve. That this situation
has been accepted for so long is due mainly to two factors; first, the types
of organisms discharged were widely regarded as of low pathogenicity, and
second, the commonly held view that if the dilution factor is big enough the
consequences are zero. Recognition that the latter assumption is wrong
and that the disease-causing potential of an organism is not the sole
determinant of its risk, have led to the current emphasis on proven safety.
In this paper, 'hazard' means a substance or circumstance which can or
may lead to ill-health in humans, animals or plant life, or significantly
reduces their quality of life. Any process or material which does so may be
described as hazardous or harmful; if it does not, it is regarded as
innocuous or 'safe'.
EFFLUENT MANAGEMENT 241
Hazardous material which has been rendered safe can be treated for
final discharge by the usual methods of sewage disposal, and no further
comments on this aspect are made here. Rather, the handling and make-
safe of potentially pathogenic materials are covered, with some examples
of our experiences at Porton Down. Grady and Lim! present a compre-
hensive account of biological waste processing.
12.2 Regulatory background
Any consideration of plant and process requirements for the disposal of

waste from a biotechnological process must start with the statutory and
regulatory requirements of the host country or district in which the process
will be operated. In the United Kingdom, the relevant legislation includes
the Environmental Protection Act 1990 and the Health and Safety at Work
Act 1974 (see chapter 2). Under these statutes, the operator of a
biotechnology process is obliged to ensure adequate isolation of the
process materials from workers, the public and the environment until the
risks associated with them have been reduced below a stated level. For
processes involving micro-organisms, the risks are generally classified as
those associated with exposure to the micro-organism itself (which may be
pathogenic, or induce hypersensitive reactions) or products of its growth
and metabolism. Additional risks are associated with non-biological
materials used in the process; for example, organic solvents, mutagenic or
carcinogenic chemicals, disinfectants and chemical sterilants which may be
used indirectly to clean up the plant after process completion.
The requirements of the Health and Safety at Work Act 1974 carry
statutory weight via the Control of Substances Hazardous to Health
(COSHH) Regulations 19882 (and chapter 2). For purposes of the
containment of biotechnological processes, of more practical relevance are
the various Guidelines and Notes issued over the last two decades or so by
the Advisory Committee on Dangerous Pathogens (ACDP) and the
Advisory Committee on Genetic Manipulation (ACGM). The initial
assessment of the risks associated with any proposed process starts with
determination of the hazard category according to these Guidelines. 3-5
12.3 Assessment of risk and appropriate action
Processing biological materials into a form suitable for treatment as sewage

is usually thought of in terms of a sterilisation process. This approach uses
proven principles which are relatively easy to apply. A risk assessment
should be carried out to ascertain the level of treatment necessary to
render safe any material likely to be discharged. This assessment will take
into account both the susceptibility of the organism (or 'agent') to the
proposed treatment, and the relative hazard presented by the escape of a
known amount of agent. Formal risk assessment in the biological industries
is a fairly young science, but has received additional impetus from the now-
statutory requirement in the UK for assessments required by the COSHH
regulations. 2 Recent increased interest in risk assessment is reflected in the
appearance of specialised publications. 6 A so-called fault tree analysis
approach has been applied to risk assessment in bioreactor processes;7
other formal procedures for which attempts have been made to apply to
biological processes include the HAZOP (Hazard and Operability Studies)
and FMEA (Failure Modes and Effects Analysis) procedures. s These
approaches involve a systematic search for all possible deviations of the
process from the predicted course, coupled with an examination of the
consequences of these, and the corrective actions required; and contri-
butory component failure to whole systems. Techniques such as these were
developed and are widely used in the nuclear and aerospace industries.
Until formal techniques can be routinely employed, assessments tend to be
somewhat subjective, and the resulting treatments are often overcautious
and in excess of requirements.
12.4 Categories of waste
An industrial biotechnology process will in general discharge materials in

solid, liquid and gaseous states. Some solids - in the form of contaminated
processing materials such as cellulose or membrane materials - may be
recycled, and must be suitably decontaminated perhaps by autoclaving and
cleaning prior to re-use. Other solid waste, such as centrifuge or
ultrafiltration sludge, is usually handled in the form of an aqueous
suspension. Such liquids and those containing soluble materials form the
bulk of what is regarded as 'effluent'. These are dealt with in the next
section.
Gaseous emissions are largely restricted to the off gases emitted during
fermentation. Containment and the elimination of the infective or toxic
hazards of gaseous discharges are dealt with elsewhere in this volume.
12.5 Liquid emuent
Effluent may arise from controlled or uncontrolled sources. The objective

of an effluent management system is to ensure that all sources are brought
under control. The types of liquid effluent generated by a biotechnological
process are:
• Spent culture medium, containing a variety of dissolved organic and

inorganic chemicals. It usually has a pH of between 4 and 9, and the
temperature is usually less than 40°C, although it may be higher if the
effluent has been subjected to heat treatment in the process equipment.
• Process fluids such as buffers, which may contain contaminating particles
or components. The chemical and physical properties may not differ
widely from those of spent culture medium, but the pH may be more
extreme. The temperature is often low, due to process requirements. It
may contain inhibitory substances used in the manufacturing process, for
example DMSO,fi-mercaptoethanol or PMSF. These may affect sterility
tests on treated waste.
• High solids content sludge; for example, waste material from the
centrifuge or ultrafiltration steps. This material may require separate
handling depending on its viscosity. Often, it is sufficiently liquid to be
pumped and combined with the lower viscosity material for make-safe
treatment. If not, separate treatment may be necessary, and for small
quantities autoclaving or incineration may be adequate.
• Steam condensate. Although derived from a 'sterile' source, condensate
becomes contaminated when in contact with the process materials. Even
when the steam is used as a sterilising agent, there is inevitably an initial
or sustained discharge of condensate which can carry a substantial
bioburden. For this reason, such condensate should be regarded as
hazardous effluent, and be contained and treated as process waste.
• Cooling water. Depending on the Hazard Category of the organism,3
and the degree of containment to which the plant has been designed,
there may be a requirement to contain and treat cooling water. If there is
a measurable or detectable risk of, for example, leaks between vessel
and jacket or other heat exchanger, then the possibility of cooling water
becoming contaminated cannot be discounted.
• Discharge of pressure relief systems and 'leaks'. Although strictly not a
category of waste distinct from those above, the design of a containment
and treatment system for effluent must take into account the likelihood
of the unexpected. Further consideration of these aspects is given later in
this chapter.
Whatever the type of liquid waste, the contaminating micro-organisms may
or may not have been first inactivated, inhibited or removed from the
effluent by some part of the manufacturing process. Biotechnological
processes may render organisms non-viable, e.g. by mechanical disruption,
frequently used to extract proteins and other intracellular components.
Treatment of cell suspensions by extremes of pH can reduce the viable
count in a cell suspension by many orders of magnitude. Table 12.1 shows
the effect of treatment of a genetically modified E. coli strain at pH 10-12
(l.R. Court, 1981, unpublished information). However, effluent is
frequently of necessity stored under ambient conditions in the presence of
Table 12.1 Effect of pH on the survival of E. coli during the addition of

sodium hydroxide to a final pH value of 12. Samples were withdrawn
into excess buffer to restore the pH to neutrality prior to viable counting.
du, colony-forming units
pH value Exposure to indicated Viable count

pH (min) cfu cm-3
7.0 1.28 X 1010

10.1 8.8 X 109
12.0 o 15
12.0 5 30
12.0 10 o
12.0 15 o
nutrients near the optimum for microbial outgrowth, so unless such

material can be shown to be hazard-free at the time of disposal, it must be
regarded as contaminated and therefore subject to the same degree of
stringency in containment as the process itself. Inactivation in the
fermenter is required by the United States N.I.H. Guidelines for large
scale fermentations of genetically-modified organisms at containment
categories BL 1 and higherY
12.6 Choice of treatment method
The method chosen to make safe the effluent will depend on the degree of
containment required; this will be dictated to a large extent by local,
national or international regulatory requirements.3-5 It may be necessary
to satisfy the requirements of the destination country for a product, not
merely those of the place in which the waste is generated in manufacture of
the product. The susceptibility of the agent to a given treatment process is
critical to the success of any given inactivation method. The degree of
homogeneity of the effluent - e.g. the solids content - will affect the ability
of a lethal or other agent to reach its target. Finally, the quantity of effluent
to be treated must be considered.
12.7 Containment considerations
Containment of bioprocesses presents a significant dilemma to the process

engineer who, on the one hand, has to provide the environmental and
health lobby with a safe process (implying negative pressure to contain
processes in the plant) and, on the other, the production manager who
requires an uncontaminated product (implying positive pressure to exclude
contaminants) .
Consideration must be given to the likelihood of escape of the agent and

the consequences of such an escape. Whether the effluent disposal system
is a simple autoclave or large multi-vessel plant, certain principles must be
kept in mind when designing or using the installation. Walker et al.lO give
details of containment devices used in industrial fermentation systems and
downstream processing. Georgio and Wu9 list 13 containment require-
ments for large-scale recombinant DNA (r-DNA) fermentations at various
N.I.H. containment levels.
12.7.1 Multiplicity of containment devices

The main consideration here is that components and sub-systems fail even
in the most carefully designed equipment. For high containment, double or
even triple redundancy is often specified. Some containment systems use
so-called 'steam tracing', in which live steam is fed between static double-
seals. This arrangement ensures that, if either seal fails, only clean, sterile
fluid escapes, either into the environment or into the process, satisfying the
most stringent regulatory requirement to 'prevent' rather than simply
'minimise' release.
Rotating seals, such as those on stirrers and pumps, are best avoided
altogether if this is possible, but agitation may be necessary if significant
amounts of solids are present in the effluent, or if the effluent is otherwise
of high viscosity. A popular choice is seals of the double mechanical seals
type; over many years of industrial experience, this type has been shown to
be reliable and offer high levels of containment. Most designs do in fact
specify a fluid to run between the seals, for lubrication purposes. If this
fluid is steam, the design ensures that all leaks are self-cleaning and non-
hazardous. The seal faces are set remote from the vessel and shaft, and are
spring-loaded to ensure that leaks do not arise from normal wear, thus
prolonging the life of the seals as well as other vessel components, and
reducing the risk of escape.
Where possible, components such as valves or joints should be
permanently fixed in place, e.g. by fusion- or solvent-welding. When this is
not possible - e.g. when removability is required for maintenance - the
type of joint must be chosen to be hygienic, that is free draining and easily
cleaned, and resistant to leaks. Temperature cycling stresses pipework and
can cause leaks from plant previously shown to be leak-free.
Filters used to vent high containment plant or laboratories are not dealt
with here. Suffice to say that the necessity to provide series replicate filters
must be assessed in the light of regulatory requirements, the consequences
of agent release and the statistical nature of the filtration mechanism, that
is, the significance of the biological challenge to the filter. The effluent
plant will contain high levels of agent, but the aerosolisation processes
occurring inside the plant may be of minimal effectiveness, especially as
there is no aeration and/or stirring, resulting in low numbers of organisms

reaching the filter medium. Therefore, where a double filter system is
advised - e.g. for ACDP category 4 or ACGM category 3 (BL 3) - the
agent challenge at the filter may be much less than, for example, in the
fermenter.
Valves are, in general, prone to leakage. They are also very good at
trapping dirt and liquid. Contained valves have a secondary seal which
prevents release if the primary seal fails. The choice of valves must be
made on the basis of the degree of containment offered and the
cleanability. If a valve fails, there must be some indication that it has done
so and it must be possible to decontaminate between the seals without
stripping or dismantling. The valve design and installation should be
chosen such that cleaning and decontamination are facilitated. Contain-
ment of process fluids at valves can be provided by steam tracing; two
series valves (normally closed) are separated by a short length of pipework
containing live steam. This arrangement is found in the drains of high
containment autoclaves and in the 'steam barrier' of industrial plants,
where process lines, carrying process fluids to be kept separated, enter a
common manifold. Steam tracing within the body of a single valve is also
offered by some manufacturers to maximise containment capability.
12.7.2 'Dead legs' and crevice avoidance

Pipework, valves and other systems must be designed, where possible, to
be 'self draining' and free from cracks, crevices and other inaccessible
regions. Nothing impedes the access of the treatment process, be it
chemical or physical, as static residual effluent in dead legs, or trapped air.
All pipework, valve and component design should be directed towards
ensuring that liquid free drains to a single, bulk volume where the
treatment process can be controlled. Similarly, a dead leg which may
become superficially contaminated must be designed to allow access to the
treatment agent, even in the absence of liquid effluent. An example of how
this may be tackled is given in section 12.10.
12.7.3 Leak testing

No containment can be regarded as adequate unless a realistic attempt is
made to measure its effectiveness. There are two basic approaches to leak
testing; first, the identification of leak points on the plant and, second,
biological monitoring for aerosol generation in the vicinity of the operating
plant.
Few, if any, standards have been laid down for acceptable leak rates in
bioprocessing plant,11 and it is normally up to the designer to decide on a
maximum permissible value. This is often an arbitrary decision, since there
is no established correlation between leak rate and hazardous aerosol

generation. The Biosafety Unit at CAMR and DTI Warren Spring
Laboratory in the UK are actively engaged in research in this area.
Leak identification is normally carried out by one or more of the
following methods:
• Pressure decay. The plant is pressurised with air to the normal maximum
working pressure or higher, and the rate of pressure decay after isolation
is measured using a precision pressure gauge. This gives a measure of the
overall system integrity.
• Tracer gas analysis. At relatively low system pressures, a small amount
of an easily detected tracer gas (usually sulphur hexafluoride, SF6 , or
helium) is injected into the system to be tested, and leak points
identified with a suitable detector. This method can be extremely
sensitive and is the method of choice when the highest level of
containment is specified. From the rate of tracer gas emission, the
nominal dimensions of the leak can be measured (G. Leaver, 1990,
personal communication), which is a first step in assessing the risk in
biological terms.
• Leak detector spray. After pressurisation with air, point leaks can be
visualised by the application of a low surface tension liquid which forms
bubbles where the gas is emitted.
• Ultrasonics. Various commercial devices claim to be able to detect leaks
by variations in the pitch and intensity of transmitted ultrasonic sound.
Experience with such devices (J. Benbough, 1991, personal communica-
tion) suggests that the technique is of little use for detecting leaks below
a size which would be considered 'massive' in terms of biological
containment.
Monitoring for aerosolisation of organisms from within the plant measures
not primarily the integrity of the system but the consequences of any leak
which may be present. By using an innocuous organism, the likelihood of
an uncontrolled release of agent is assessed. Air in the vicinity of the
operating plant is sampled and the concentration of the tracer organism
measured. Techniques for this type of monitoring are covered elsewhere in
this volume.
12.7.4 Standard operating procedures (SOPs) and process records

A formal written system of operation and a reliable software control
system where appropriate is essential for the maintenance of plant
integrity. SOPs written with the design limitations of the plant in mind are
an essential element in maintaining a secure containment system. Fully
automatic operation with fail-safe interlocks (to prevent, for example,
release of effluent before treatment is complete) can greatly assist in
maintammg integrity, but the software must be robust and reliable.

Evidence that the software is validated should be provided by the
manufacturer. In the absence of full automation, procedural, mechanical
or semi-automatic interlocks must be designed into the operating procedure
where necessary. A full record of each treatment cycle, in the form of a
temperature trace, analytical evidence of disinfectant concentrations or
other evidence, must be generated. This is used to ensure each cycle
complies with stated objectives and that a valid process has been
performed.
12.7.5 Planned preventative maintenance (PPM) schedule

This must be instituted from the beginning of the life of a plant, and backed
up by adequate record keeping. Feedback of details of plant modifications
to those responsible for the maintenance of SOPs is essential. The
elements of the plant requiring PPM will be identified by the manufacturer
or during commissioning. An operating log of some description must be
maintained to enable predictions of failure, or necessary non-routine
maintenance to be carried out.
12.7.6 Commissioning and validation

A formal commissioning and validation procedure with definite objectives
must be laid down prior to operating the plant. This may require both
physical and biological tests on the plant to ensure that it does work
according to the design brief and is effective in operation. Coupled to this
is the necessity for periodic re-testing to a standard procedure. These tests
need not be as stringent as the initial validation, perhaps only requiring the
measurement of one or a few parameters previously identified during
commissioning as critical, but nonetheless, a written procedure and set of
pass/fail criteria are essential. Extensive modifications to the plant, for
example to heat exchangers in a thermal system or mixers in a chemical
disinfection plant, could justify a full re-commission and/or re-validation.
12.8 Practical treatment methods
Microbially contaminated liquid effluent can be made safe in one of three

ways: sterile filtration, chemical disinfection or heat treatment.
12.8.1 Filtration
An adequately validated filtration system can physically remove all
contaminating particles from a liquid or gas. For relatively small quantities
of liquid, this can be a quick and simple procedure, but it has the
disadvantages of relatively high cost and a by-product (the contaminated
filter matrix) which remains hazardous and must be made safe in one of the
other ways. In the laboratory, this method can be used quickly to prepare
safe material, e.g. for analysis, and the filter can be quickly disposed of by
autoclaving. But 'real effluent' usually accumulates in sufficiently large
volumes to make this approach impractical.
12.8.2 Disinfection using chemicaL agents

This has often been the method of choice in the past for small to medium
quantities of liquid waste, as the capital and running costs of the plant can
be kept quite low. The disinfectant usually chosen for this duty is
formaldehyde, due to its low cost, non-corrosive nature and sporicidal
activity, but an 8% w/v concentration of formaldehyde in water is generally
regarded as a minimum for general use. Formaldehyde presents a
significant hazard to the environment and operator, and has been
implemented as a carcinogen. 12 The effectiveness of formaldehyde greatly
increases above 40 o e\3 and a combined treatment of elevated temper-
atures with formaldehyde, either gas or liquid phase, is particularly
effective as a lethal agent. Russell l4 includes useful information about the
use of formaldehyde as a disinfecting agent.
However, the disadvantages of chemical methods are well-known,15
including the following.
1. The necessity for re-validation for each biological/disinfectant agent
combination to be used.
2. The susceptibility to inhibition of the disinfecting action by the presence
of dissolved and suspended solids, pH, low temperatures etc.
3. The neutralisation of the disinfecting agent by unspecified materials in
the effluent.
4. The necessity to retain treated effluent prior to discharge, pending
viability or sterility tests. This inevitably leads to delay, requiring a
higher capacity plant than would otherwise be the case. In addition,
such viability tests are themselves subject to criticism; for example, the
almost impossible task of taking a representative sample from, say, 2000
litres of material which is small enough to be handled in the laboratory.
This criticism is mitigated to some extent by the necessity to show only
presence or absence of growth in a sterility test.
S. The necessity to release into the environment often substantial
quantities of toxic chemicals. This is a consequence of the need to
overdose in an attempt to overcome the neutralisation or inhibition of
the disinfectant.
12.B.3 Heat treatment using steam

Heat as a sterilising agent is the method of choice for most effluent
treatment plants, for a number of reasons:
• it is readily transferred into and removed from the process without the
necessity to compromise physical containment;
• if properly validated, a heat treatment process does not require routine
biological monitoring;
• temperature is readily measured and controlled;
• in terms of overall cost, heat can be the most efficient process;
• the environmental effects can be minimised by heat recovery.
The lethal effect of heat has been extensively and intensively studied for
many years, and its kinetic characteristics well documented. No attempt is
made here to discuss in detail principles of sterilisation or disinfection;
there is a large literature of standard text books 14-17 to which the reader
should refer.
The heat treatment required will depend on the degree of heat resistance
of the agent (usually measured as the decimal reduction time, D. This is
the time required at a specified temperature to reduce the viable count by
90%). D is specific to the organism under consideration, and for a mixture
of organisms the most heat resistant (largest D value) should be used in
designing the process. Smelt and Mossel 18 give examples of D values for a
variety of bacteria, yeasts and moulds.
The initial concentration of organisms will also have a marked effect on
the required extent of processing. Since the thermal death of micro-
organisms follows first order kinetics, the greater the starting bioburden
the longer the treatment time must be at a specified temperature to achieve
a safe level of contamination. By the very nature of such logarithmic death,
in theory an infinite amount of time is required to reduce the viable count
to zero. This difficulty is usually overcome by designing a process which,
starting with a known concentration of organisms of known heat
resistance, results in a reduction in viable count to a level at which there is
a small but finite probability of any given volume containing a specified
number of viable organisms. The probability confidence limit is chosen in
accordance with the acceptable level of risk of release for the agent
concerned. A value of 1 in 106 to 108 is usual.
An empirical rule-of-thumb is often used in the design of sterilisation
processes which uses the principle of substantial overkill to design a readily
validated make-safe process. The MRC Working Party reports on
pressure-steam sterilizers 19 are often quoted as the intellectual source of
the much-used 'sterilisation standard' of saturated steam at 121.1 °C for 15
minutes. This treatment is widely regarded as giving a sufficient margin of
safety in yielding a sterile product irrespective of the type and initial
concentration of the organisms to be killed. For this reason, this standard is

often applied to a make-safe process more through expedience and
ignorance than strict necessity. This said, it is a standard which is readily
achieved and may be applied without the necessity to test for sterility
before discharge of the effluent provided adequate validation of the
process has been carried out before use, and the process is revalidated
periodically to a fixed schedule.
However, a heat sterilisation method which avoids the overkill approach
has for some years been in use, having been developed in the food
industry. This is the so-called 'Fo concept', which assumes a definite
bioburden and microbial contaminants of known heat sensitivity (D value).
An integrating technique, which may be automated, is used to sum the
killing effect of the heating and cooling periods of the thermal process, as
well as any holding period, with reference to a temperature of known lethal
effect for the organism. The resulting process thus delivers the minimum
lethal dose required to achieve a target number of residual contaminants.
Usually the MRC standard temperature is chosen as the reference. Smelt
and Mosselll 9 and McBride 20 give accounts of this technique, particularly
as applied in the pharmaceutical industry.
12.9 Testing emuent for sterility
Where a heat treatment or other make-safe process cannot be validated to

provide a sterile or otherwise safe product under all conditions, a sterility
or contaminant identification test may be necessary before the effluent can
be safely discharged. The usual technique, if the presence or absence of
microbial growth under the conditions of the test is adequate, is to take
samples of treated effluent into an equal volume of suitable double-
strength growth medium such as MacConkey Broth or thioglycollate
medium. The samples are incubated under a variety of conditions (e.g.
temperature, presence or absence of air) designed to demonstrate the
growth of the organisms of interest. A positive control, containing
organisms known to be viable to demonstrate the ability of the medium to
support growth, is included. An estimate of the likelihood of a test giving a
false negative result can be made. For an uncontaminated sample drawn
from an infinitely large volume, the number of contaminating particles in
the separate units of volume follow the Poisson distribution, and the risk of
discharging hazardous material can be directly measured using the
equation:
n = -logeP = 2.303(-logIOP)
where n is the maximum density of contaminating particles and P the
selected confidence limit. 21
12.10 Design and qualification of a heat treatment emuent plant
Assuming that heat is the chosen method of processing the effluent

generated by the bioindustrial process, what design considerations are
there for the effluent plant itself?
12.10.1 Design considerations
The choice of autoclave or dedicated plant. It is easy both to under- or

over-estimate the required processing capacity when designing an effluent
disposal facility. Over-estimation may lead to excess capital and perhaps
running costs; under-estimation may lead to process delays while effluent is
treated, and increased risk of escape of agent. To err on the side of over-
estimation is the safe course, if capital costs allow.
Small unit volumes of effluent (up to 50 litres) can be treated in a steam
autoclave, provided that adequate measures are taken to transfer the
material in a safe and contained manner to the autoclave chamber. A Class
III cabinet line or contained suite in which the work is performed, and to
which the autoclave chamber has direct access provides excellent contain-
ment security, but is not conducive to the processing of more than a few
litres at a time. Transport of liquid in a leak-proof secondary container
permits larger volumes to be handled, but consideration must be given to
the logistical problems associated with the transport, especially if the
autoclave is situated in a relatively remote location, or is inside another
suite. If the autoclave has one door only, procedures must be adopted to
ensure that contaminated and safe materials are not mixed. Double door
autoclaves permit the uni-directional flow of waste out of the facility, and
in the UK are recommended or mandatory for the higher categories of
containment. 3-5
Autoclaves vary in their capacity to contain contamination and,
depending on the categorisation of the agent of interest, the design of the
any disposal plant and its installation must answer the following questions:
1. What liquids or gases are released or evacuated from the chamber
during the sterilisation process, and before the minimum conditions for
a safe load have been fulfilled? If this happens, is there a risk of release
of agent?
2. What provision is made for the containment of the (unsafe) effluent in
the event of autoclave control failure?, i.e. can the material be securely
returned to the source and can the autoclave be made safe for
maintenance/repairs to be undertaken?
3. Is there a significant likelihood of release of agent via contaminated
leaks which may not be detected either by the control system or the
operator?
4. Since the autoclave chamber is a pressure vessel, what provisions can be

made to control the release of agent from the pressure relief devices
which may be required?
In considering these questions, the designer must always keep in mind the
degree of quantitative and qualitative risk represented by the escape of the
particular agent under consideration. No containment system is totally
secure, and the additional expense of a state-of-the-art containment
solution, both in terms of financial outlay and back-up service and testing,
may not in the end be justified for an agent of relatively low hazard
potential. Each case has to be decided on its own merits.
Most of these questions are equally relevant when considering the design
of a large-scale effluent treatment plan, but here, the design must cater for
a number of fixed installations as sources of material, which have to be
engineered into the design of the overall facility. To estimate the required
capacity, start by counting the total number of sources. Depending on the
level of containment to be designed, this may include sinks and showers as
well as the more obvious sources such as autoclave drains, fermenters or
downstream processing tanks. Estimate the total flow from each source per
unit time. The appropriate time unit to be used will depend on the turn-
round time for the process (batch treatment) or the required mean effluent
residence time (continuous process). For a batch process, this turn-round
time will depend on the following engineering parameters:
• The differential between the temperature of inflowing or stored effluent

and the treatment target temperature.
• Heat exchanger efficiency; the use of heat recovery can speed up the
process.
• Allowable discharge temperature.
• Natural or assisted cooling. Using the latter speeds up the process but
requires large quantities of cooling water and/or refrigeration plant
which increases capital and running costs.
• Insulation of the processing tank. This will speed heating rates but slow
down natural cooling. It has the undoubted benefit of reducing heat gain
in the plant room and reduces heating costs while reducing operator risk
(e.g. from scalds).
• Size of the process tank relative to rate of inflow of effluent and whether
interim storage of material is available. A 'sump tank' (see Figure 12.4)
to receive material at times of high production rate can reduce the
minimum necessary capacity of the processing tanks, which can continue
to process material during periods when the production rate may be low
or zero.
• The physical space available for the plant, and the availability of utility
services such as power and steam.
The calculation of the size of the treatment plant required is a compromise

based on an integrated consideration of all these factors, together with
others such as capital, overhead and running costs.
Criteria of an 'effective process'. As explained above, the plant could be

designed either to render harmless the particular agent around which the
overall industrial process is based using a minimal, structured approach,
for example the Fo technique; or to sterilise every known type of industrial
organism that could possibly be used now or in the future - the overkill
approach (e.g. the MRC standards I9 ). If the use of only a single type of
agent of known and constant heat-sensitivity is anticipated throughout the
life of the facility being designed, then the restrictions imposed by the
structured approach can be tolerated and substantial savings may be made
in terms of capital costs and plant complexity. However, if a change to an
unspecified process organism is contemplated for the future, it is often
expedient, and certainly worth considering at the outset, to install a fully
flexible and automated plant, requiring little or no additional validation or
revision of operating procedures, that is, one which uses the overkill
principle. To design a no more than adequate process, one needs to know
the types and sensitivity of all types of organism present in the material to
be treated. This is possible only if the waste is from a defined process,
uncontaminated by any additional undefined effluent; and the sensitivity of
the contaminants for the chosen lethal agent is known for the particular
conditions under which the waste is to be treated. Often these factors are
unknown. In design terms, one has to look hard at the physical nature of
the effluent, and consider the limitations to heat transfer if the material is
viscous or contains large amounts of solids which could impede heat
transfer, and whether these characteristics are constant and predictable.
Further, the effluent may be bi-phasic, perhaps containing variable
amounts of hydrophobic components which exclude moisture and therefore
protect micro-organisms from the lethal effects of heat or other agents.
Heat exchanger design. The use of carefully designed jacketed vessels to

avoid the use of stirrers and other mechanical devices has already been
mentioned. The type of heat exchanger used will have a marked effect on
the efficiency of the process, and the advice of an experienced engineer is
invaluable in choosing a suitable design. The use of a high surface area
shell-and-tube design should be considered for high viscosity effluent if
high rates of heat transfer are necessary, but this type of plant is inevitably
bulky (see section 12.10). Continuous type processes may have consider-
able advantages over batch treatment in terms of running costs, energy
efficiency and simplicity of control systems.
Tank venting system. Vessels for the treatment of liquid effluent must be
vented to atmosphere to enable filling and emptying. Inevitably, therefore,

there must be a way of ensuring that contaminated material does not
escape during venting. The usual method is by filtration. Hydrophobic
filters are usually chosen; the useful life of this type of filter is reduced by
accumulation of dirt in the filter matrix, and by steam sterilisation.
Adequate pre-filtration can reduce blinding with solids, and filter life can
be extended considerably by the use of a collection tank separate from the
treatment vessels. The choice of filter medium must be made taking into
account the following:
• the need for repeated steam sterilisation;
• high humidity;
• little if any entrained liquid or solids content, e.g. foam, in the gas
stream, i.e. low dirt content;
• usually a low microbial challenge.
Two examples are discussed in section 12.10, In which approaches to
minimising these problems are illustrated.
Pressure relief system. In the UK pressure systems such as autoclaves

and process tanks operating above ambient pressure are subject to
statutory requirement under the pressure vessel regulations 22 that a
pressure relief device must be incorporated. This device must be capable of
ensuring that the internal pressure cannot rise above a defined value if, for
example, the normal vent system becomes blocked. In practical terms, this
means that the contents must have free access to discharge to atmosphere if
the system pressure rises above this value. This relief system is usually
some type of valve which either continually relieves the pressure by
'cracking' open, or 'pops' open to relieve the pressure suddenly.
Increasingly, so called bursting discs are incorporated which are designed
to rupture completely at a defined differential pressure, giving unimpeded
flow characteristics. One particular advantage of the bursting disc is that
there is no likelihood of low level discharge via a 'weeping' valve.
Particular care must be taken in choosing a bursting disc as the burst
pressure decreases with rising temperature, as much as 50% over a range
from 20-120 °e, depending on the material of construction. Protection at
all temperatures, within the design pressure of the vessel, is nonetheless
required, and the vessel may have to be overrated in order to comply with
the applicable regulations.
In the context of a contained biological or chemical agent (as opposed
merely to pressure per se) the operation of such a pressure relief system
may be insufficient to ensure safe operation of the plant. Release of the
agent, the primary objective in using the plant, must be prevented. On the
other hand, in the UK, the regulations 23 do not permit any restriction of
the discharge line which must be free to vent to atmosphere. These two
conflicting requirements cannot rigorously be reconciled. A compromise

solution may be to use a high flow rate capability filter to retain
particulates while allowing gases to discharge with relatively low resistance
to flow in the event of relief valve operation. The choice of filter capacity is
therefore determined by:
• anticipated peak flow rate in the event of relief system discharge;

• acceptable design pressure drop across filter for this flow rate;
• acceptable maximum pressure drop (i.e. !J.P, the differential pressure
across the filter membrane, will rise as the filter becomes contaminated
with solids).
The relief system must also take into account the inevitable discharge of
bulk liquid as well as gas. This too must be contained. The discharge from
the relief valves may be contained in a receiving tank which is not routinely
sterilised. Where no such tank is used, the relief valves can discharge via a
filter sufficiently remote that no liquid can reach the filter and compromise
integrity.
There is clearly no single answer to this problem, and as an example of
the various approaches adopted at CAMR, the reader is referred to section
12.11. Grossel 24 gives details of relief system containment and disposal
methods as used in the chemical industry.
12.10.2 Qualification of effluent treatment plant

Validation has been defined as the attainment 'and documentation of
sufficient evidence to give reasonable assurance . . . that the process under
consideration does ... what it purports to do. ,25 It is often used somewhat
loosely confused with and used as a substitute for 'Qualification', which is
concerned with the process of verification that the equipment behaves
according to the design criteria. Validation usually takes place in two
stages; the first in which the physical parameters such as temperature,
pressure, flow rate, volume and other instrumentation are calibrated and
tested, and, second, the whole process is carried through to completion. In
the case of an effluent plant, this would be a biological challenge to
determine that the plant and process will render material safe for
discharge. Aspects of this have been discussed above.
Qualification of the plant design involves:
• Formal commissioning of plant and control system.
• Vessel and pipework leak test, according to a suitable method.
• Control system measurement and accuracy including correct sequencing
of valve operations.
• Alarm systems - verification that alarms are notified correctly and that,
if automatic action results from an alarm, that the plant fails safe.
• Capability of manual operation in the event of automatic system failure,

with identification of a critical path to 'safe plant' .
• Determination of procedures for normal operation and for emergencies.
Temperature measurements are readily made using commercially available

instrumentation. Thermocouples of the copper-constantan (T) type are
usually chosen as the measurement probes. These can be made sufficiently
small to ensure point measurements can be taken. Thermocouple hot
junctions less than 1 mm wide can be used to measure temperatures in
small containers and crevices, regions likely to retain air in sterilisation
processes or into which heat conduction or convection is inefficient.
Kemper26 gives full details of the theory and applications of thermocouple
devices.
All instruments must be calibrated26 before use and be certified by some
independent body. A calibration check on the complete measurement
system, including probes, must be undertaken immediately prior to the
qualification test. A standard temperature source or measurement device,
traceable to an approved national standard such as the National Physical
Laboratory (NPL: UK) or National Bureau of Standards (NBS: USA), is
used.
During qualification of the process, care must be taken to ensure that the
likelihood that part of the plant may not reach or maintain the required
conditions is minimised to an acceptable level. Non-homogeneous condi-
tions may exist if the tank is not mechanically stirred. Even if it is, regions
of imperfect mixing are likely to occur. Despite careful design, there will
be 'dead legs' where heated fluid or steam cannot find access, and where
conduction is inadequate to reach the temperatures required. Careful
consideration must therefore be given to placement of thermocouples
during qualification. Repeated test cycles will be necessary until the largest
practical number of measurements have been made. An assessment of
likelihood of process failure through non-homogeneous conditions can
then be made.
12.11 The approach to emuent treatment at CAMR
The long history of handling dangerous pathogens at this site (CAMR was
formerly the Ministry of Defence Microbiological Research Establish-
ment) has resulted in the installation of a number of heat treatment and
other effluent plants over a period of about 40 years. This chapter
concludes with a brief overview of some of these, to illustrate some of the
principles mentioned above and to show in which ways the approach to
design has changed in this period. The sources of effluent in CAMR are
various, ranging from used agar plates and a few ml of spent culture
Figure 12.1 Large scale semi-continuous effluent heat sterilization plant. Simplified schematic
plan view (1950s design). Mild steel construction, with manual control. Treated material pre-
heats incoming effluent in a heat-exchanger (HE) before passing to three holding tanks,
where it is accumulated pending the results of sterility tests prior to release to drain. SC,
steam chest; S, sample point and throttling valve; P, pump; M, collection manifold; RT,
receiving tank; F, vent filters; ST, storage tank; DP, discharge point.
medium from innocuous bacterial fermentations, to the washings from the

animal room floor, or hundreds or thousands of litres of bioprocess liquor
derived from the extraction of microbial proteins or toxins from low- or
high-containment category agents.
Figure 12.1 is a line diagram illustrating a semi-continuous effluent
disposal plant installed in the animal wing in the early 1950s, and handling
pathogens up to ACDP category 4. Of mild steel construction, the design is
over-engineered to achieve the make-safe of effluent containing contamin-
ated materials from laboratories, animal and post mortem rooms, washing
areas, floor drains etc. Ten such plants provide excess storage and
processing capacity in the event of process failure in any part. The steam
chest (SC), is a large (ca. 3000 I) pressure vessel containing live steam at 2
bar g pressure. Within is a narrow bore heat exchanger coil carrying the
effluent for treatment. The pressure generated by the pump is regulated by
a downstream throttling valve to give a flow rate which ensures the correct
residence time (2 hours) in the coil. The tubular construction and intensive
treatment specification (125°-134°C for 2 hours) was chosen to ensure
adequate treatment for non-homogeneous effluent with significant solids
content. This arrangement has two particular benefits:
1. Maximum surface area for heat transfer into the effluent, eliminating
the need for mechanical stirrers, despite significant solids content in the
effluent.
2. In the event of heat exchanger rupture (most likely in the region
exposed to the highest temperature), leaks of toxic material take place
into steam at 130 C, providing adequate containment.
D
Treated material is passed to holding tanks pending the results of sterility

tests. If these tests fail, the material can be transferred back to the
receiving tank, RT (Figure 12.1), for re-processing. Sterilising grade filters
permit venting of the receiving and holding tanks.
Various disinfection plants are in use at CAMR for the treatment of
pathogenic and non-pathogenic waste. Two of these are illustrated in
Figures 12.2 and 12.3. The former is a heat disinfection plant used for the
treatment of shower water from an ACDP category 4 suite. Installed about
1975, it is designed to heat the effluent containing low solids and an
exceedingly low titre (if any) of heat sensitive agent, to 80 DC for 60
EFF.
V1
~ /-j
L~
H.L.
M.L.
V2
Figure 12.2 Schematic of single tank effluent heat-disinfection plant. Mild steel, ca. 1975
design, manual control. Used for the make-safe of shower water from a ACDP category 4
laboratory. EFF, effluent entry from shower tray; DB, dosing bottle; F, sterilising grade
filter; HB, heater battery; S, steam input; HL, high level alarm level; ML indicates the
half-full sensor.
J
DB
H.L.
c V
D.P.
Figure 12.3 Medium scale chemical disinfection plant. 1981 design. Semi· automatic control.
Polycarbonate/glass fibrc construction. F, vent filter; SP, sample point; SP, discharge to
drain. For symbols, see Figure 12.4.
minutes. Waste water enters the plant directly from the shower tray via an
anti-syphon trap. The tank is vented while filling via a sterilising grade
filter above the shower tray height. The drain valve is locked shut during
filling to prevent unauthorised release. When the effluent content reaches
the high level (approximately 90% full), the fill valve is closed and steam
admitted to the heater battery. After treatment is complete, the supervisor
examines the temperature record and, if the specified treatment has been
applied, authorises the issue of a key to open the drain valve to discharge
the waste to drain. At CAMR, safety policy requires that infected materials
are disposed of by exposure to saturated steam at 121°C for 15 minutes,
the well established MRC (1959) standard. 9 Further, the Health and
Safety Executive requires that all effluent from high category laboratories
be treated to make safe; but even in a ACDP category 4 laboratory it is
acceptable to heat-treat the shower water at only 80°C, i.e. in a
disinfection process, because there is a much reduced risk associated with
this material.
A chemical disinfection plant is shown in Figure 12.3. Dating from the
early 1980s, this plant was designed to handle labile micro-organisms
derived from laboratories working with ACDP category 3 pathogens, in a
low solids effluent, and using formaldehyde. Two glass fibre vessels receive
effluent from laboratory sinks, showers, fermentation equipment etc.

When the automatic level sensor indicates 90% full, formaldehyde is added
manually via a dosing bottle and non-return trap. A pumped re-circulation
system mixes disinfectant with effluent, and treatment continues at
ambient temperature for 6 hours. A sample is taken for sterility checks and
if no evidence of contamination is detected, release to drain is authorised.
The second tank (not illustrated in Figure 12.3) is essentially an
independent duplicate which ensures continuity of laboratory operations.
Transfer between the two tanks is possible in the event of process failure.
The use of a chemical disinfectant requires the presentation of a uniform
quality of effluent, batch operation and pre-release sterility checks. The
efficiency of the procedure depends on solids content remaining low, the
maintenance of a minimum temperature and the nature of the contamin-
ating agent. The use of excess disinfectant inevitably results in the release
of formaldehyde to the environment.
CAMR has two fully automated effluent sterilisation plants to handle
additional ACDP category 3 agents, including spore-formers. These are
illustrated in Figures 12.4 and 12.5. The large-scale plant (Figure 12.4) is
intended to receive effluent from a biological product manufacturing area.
The original design envisaged high solids effluent, and macerators (no
I V.F. #1 V.F.#2 I
F. PRV.D F.D .• S.
FIoor/eve/
~ SteamlTsp
~ Pump
SZ Non-relUm vaM> Symbols
V.L.
I
I #2. I
SUMP TANK ~ __________ ~ 'TR:I:M~NT
VESSELS
Figure 12.4 Simplified schematic of a large (10 000 I) scale batch effluent heat sterilisation
plant. 1981 design. All stainless steel construction pressure vessels,' with full automatic
control. F, PRY, D, FD, S-sources of effluent (fermenter, pressure relief, drains, floor
drains, sinks); VF, vent filter; FL, fill line; VB, vacuum break; VL, vent line.
M.
V.F.
I
V.F. s
tostack~
D.P.
o o
#1 #2
DT.
Figure 12.5 Schematic diagram of a small (10001) scale batch effluent heat sterilisation plant.
1991 design. All stainless steel construction pressure vessels with full automatic control. VF,
vent filters; VL, two-way vent line; DP, discharge point to drain; EI, effluent input; DT, dip
tube for effluent removal; P, thin-walled pocket for control probes and test thermocouples;
S, vent stack.
longer used) were installed to reduce the particle size. The all-welded all-
stainless steel plant, including pumps, stirrers and other moving parts, has
a total of 18 pairs of double mechanical seals, each purged with live steam
at 4 bar g pressure. Steam condensate is returned to the sump tank (Figure
12.4) which serves to receive and store, pending treatment, all waste
material from the laboratory areas. The sump tank is sufficiently large to
be regarded as at atmospheric pressure at all times, so pressure relief
devices discharge to this tank, which is vented by oversize filters (VF #1
and VF #2, Figure 12.4) situated at high level to avoid blinding with liquid.
This design maximises containment by ensuring the minimum microbial
challenge and dirt loading onto the filters, and removes the need to sterilise
the filters routinely,thus prolonging their useful life, as the sump tank
remains contaminated at all times during normal operation. Each process
tank is vented back to the sump tank during filling, liquid transfer being
effected by pump. Effluent, containing water, solids and dissolved
materials, is received in the sump tank from fermenters, pressure relief
systems, downstream processing equipment, floor drains and sinks. It is
pumped to the bottom stirred treatment vessels (#1, #2, Figure 12.4)
which are vented back to the sump tank; only upon final discharge do the
treatment vessels draw in ambient air via a filter (VF #3, Figure 12.4). The
effluent is pre-heated in a heat exchanger (not illustrated in Figure 12.4 for
clarity) using heat recovered from the treated batch in the second vessel. A
steam heated additional heat exchanger is used to pre-heat the first batch
on start-up. Such an arrangement adds additional stages, joints and
pipework (potential leak-points), not strictly necessary to the primary
purpose of the plant, but heat recovery reduces running costs and shortens
the plant cycle time. The effluent is further heated using a fully immersed
steam coil in each tank. After treatment, discharge of treated material
takes place automatically without delay. No samples are taken as the
process has been extensively validated physically (temperature measure-
ments) and biologically using heat resistant spores in a high solids matrix.
Safety devices built into the control system ensure that, if the effluent fails
to pass through the prescribed process, no discharge takes place, an alarm
is given and the plant can be manually operated to a safe status. Normal
operation is fully automatic. All valves are of the air-operated diaphragm
type and installed to be free-draining. For maintenance, the entire plant
including the vent lines and filters (4 in all) can be independently sterilised
and the filters tested using an aerosol challenge. This means that - in
theory - the plant does not need to shut down for routine filter testing or
replacement. Note that the use of a sump tank means that it and all the
vent filters need not be routinely sterilised as part of the normal process. In
designing the sterilisation system for these filters, the following problem
was confronted.
The vent filter installation must provide facilities for filter integrity
testing, in this example by aerosol challenge. The aerosol injection and
sample points are for most of the time closed off and not in use. Since the
installation is on an effluent plant, does one install another such plant to
handle the contaminated condensate generated during steam sterilisation?
The filter housing itself will inevitably generate considerable quantities of
contaminated condensate, so what does one do with it? Since the filters
must be sterilised prior to testing, should the installation provide these
points with separate facilities for steaming and condensate retention?
The resolution of this conundrum was in fact to provide the filter housing
with separate sterilisation facilities and to design and orientate the aerosol
injection points (closed with double sealed screw caps, and of short, wide
aspect ratio) to be self draining into the pipework. Similarly, by providing
two parallel filter installations, it proved possible to sterilise separately
each filter allowing contaminated condensate to drain back into the
(continuously operational) plant. This simplified the whole installation,
and reduced the risk of uncontrolled release (see Figure 12.4, detail).
The most recently designed effluent plant at CAMR is shown in Figure
12.5. In this case, the design emphasis is on containment and operational
versatility in the type of agents which can be accommodated.
A design philosophy, different from the plant of Figure 12.4, was
adopted in which the number of moving parts was kept to a minimum, and
by using gravity for feed and over pressure for discharge, the need for
pumps was eliminated. Inevitably, the compromise was to eliminate
advantages of the sump tank, and each of the two process tanks is a self-
contained steriliser. The whole plant is fully automated with a compre-
hensive array of microprocessor-based control and alarm systems. Each of
the processing vessels (duty and stand-by) is of all welded AISI316 stainless
steel construction with no penetrations below the effluent level. No
mechanical stirrers are fitted; mixing is promoted by the jacket design and
vessel aspect ratio, which are intended to set up a single convection cell in
the contained effluent. Such a cell is characterised by a cool point at a
definite location in the vessel which can be precisely determined by spot
thermocouple measurements. From this single point control of the
sterilisation process can be effected. Effluent with a low solids content
from fermenters, centrifuges, sinks and showers flows into the plant under
gravity via an independently steam sterilisable manifold. A transfer line
allows material to be diverted between the two treatment vessels (#1, #2)
if necessary, but both vessels are intended to operate independently of
each other and are isolated by double series valves. Because in this case no
sump tank is used (cf. Figure 12.4), each vessel must be vented
independently via its own filter which must be sterilised along with the
batch. This filter is used to displace air during filling and feed air under
pressure for final discharge.
The requirement of the make-safe process must therefore be that the
entire plant, including the filter, must be subject to the minimum process,
otherwise viable organisms could be reintroduced during passage of air
through the filter during discharge. The filters, containing elements of inert
hydrophobic materials (PTFE), are installed to be free-draining, and can
be sterilised by exposure to steam for a relatively short period. They are
isolated from the process vessel early in the treatment cycle and an
independent minimum steam sterilisation, separate from the bulk fluid in
the process tanks is automatically carried out during the effluent heating
stage. After a short period (approximately 20 minutes, to include heating
and holding times), the filter is flash de-pressurised and flushed with air.
This approach has the dual advantages of prolonging the useful life of the
filter and ensuring that a dry filter is available for providing ballast air at
the end of the treatment cycle. Tank venting during effluent heating and
holding (while the filter is isolated) is via upward displacement using a
steam trap and by-pass arrangement (see Figure 12.5). All heating of the
effluent and vessel interior is indirect via the vessel jacket. In this way, all
contaminated condensate generated during effluent heating is passed to
the stand-by tank during sterilisation of the duty vessel. Meanwhile, the
stand-by vessel is also filling with effluent, allowing uninterrupted work in
the laboratory. The plant is simplified and containment enhanced by the
following additional design features:
• absence of heat recovery system and associated pipework; while this
inevitably results in total heat loss, the relatively small capacity of the
plant (d. Figure 12.4) would make the cost savings of heat recovery
marginal.
• no mechanical stirrers or pumps.
• minimal static seals; all valves are double 'O'-ring sealed or diaphragm
type. One small (150 mm diameter) inspection hatch has double 'O'-ring
seals, but it was felt unnecessary to use steam tracing. The remainder of
each tank is all welded.
• All penetrations are welded to the fixed vessel lid. Although not
illustrated in Figure 12.5, all pipework connected to the process vessels is
designed to present minimum dead legs, with aspect ratio of ca. 1 : 1,
minimum 1 inch length, to ensure free draining and heat penetration.
• Bursting discs for overpressure relief. There is continued debate among
engineering authorities about the relative reliability of bursting discs and
relief valves. Some retain the view that only quick-discharge relief valves
can give the unimpeded flow required to reduce overpressure in a system
rapidly. The bursting disc solution was chosen because of its undoubted
superiority in preventing 'weeping', that is the slow leakage of vessel
contents at pressures below the design discharge value.
When the treatment cycle is completed, the effluent is allowed to cool
naturally to below 100°C. At this point, the drain is opened and ballast
pressure (0.3 bar g) is used to discharge the effluent via a throttling valve.
This allows the rate of discharge to be set to suit the ability of the site
drains to cool the outflowing effluent to near-ambient temperature within a
short distance of the release point.
The overpressure relief containment system (see detail in Figure 12.5)
uses a stainless steel vent stack (4 inch diameter pipe) to the roof of the
building. It is terminated by an oversize hydrophobic filter. Calculations
indicate that, in the event of a burst, the volume of liquid that will be
discharged can be accommodated in an extension (ca. 10 feet long) of the
vent stack below the discharge point. This accommodation is necessary to
avoid the build up of a hydrostatic head above the pressure vessel which
would impede further pressure-relief. The remainder of the discharge will
be gaseous, including significant amounts of steam and some air. At the
top of the stack, 30 feet or so above the discharge point, the discharged gas
volume required to pass the filter will be reduced by natural cooling and
thus condensation and contraction of the vent gases. All these factors
combine to reduce the pressure drop across the vent filter and so avoid the
otherwise irreconcilable requirements of containment of hazardous agents
and free discharge of pressure vessel contents. In the rare event of bursting
disc rupture, the stack and vent filter will be sterilised by the injection of
formaldehyde and steam at the bottom of the stack. The vent filter
cartridge will then, as a matter of course, be replaced.
This chapter has attempted to give an overview of some of the practical
problems and solutions in addressing waste management and disposal in
the bioindustries. No one worker in this field has a monopoly of all the
answers, and there are no 'wrong' solutions. At CAMR, hazardous
materials have been handled for many years, and the examples given here
help to illustrate how the requirements for containment generally become
more stringent with time. It must be emphasised, however, that they
represent the approach of a single Institution to solving part of its unique
waste disposal predicament.
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ing. In Separations for Biotechnology, Werrall, M.S. and Hudson, M.J. (Eds), Ellis-
Horwood/SCI, pp. 469-482.
11. Leaver, G., Salusbury, T.T., Stewart, I.W. (1988). Containment Monitoring Methods-
Micro-organisms and their Products. DTI: Industrial Biosafety Project: Confidential
State-of-the-Art Report No. CR 3009 (BS).
12. Acheson, E.D., Barnes, H.R., Gardner, M.J., Osmond, c., Pannett, B. and Taylor,
C.P. (1984). Formaldehyde in the British chemical industry. Lancet, 17 March.
13. Trujillo, R. and David, T.J. (1972). Appl. Microbiol., 23, 618--622.
14. Russell, A.D. (1982). The Destruction of Microbial Spores. London: Academic Press.
15. Gardner, J.F. and Peel, M.M. (1986). Introduction to Sterilization and Dinsinfection.
London: Churchill Livingstone.
16. Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J. (Eds) (1982). Principles and Practice of
Disinfection, Preservation and Sterilization. London: Blackwell Scientific.
17. Block, S.S. (Ed.) (1991). Disinfection, Sterilization and Preservation (4th edn)
Philadelphia: Lea and Febiger.
18. Smelt, J.P.P.M. and Mossell, D.A.A. (1982). Applications of thermal processes in the
food industry. In Russell, A.D., Hugo, W.B. and Ayliffe, G.A.J. (Eds) Principles and
Practice of Disinfection, Preservation and Sterilization. London: Blackwell Scientific.
19. Working Party on Pressure Steam Sterilizers (1959). Sterilization by Steam under
increased Pressure. A Report to the MRC, Lancet (i), 425-435.
20. McBride, R.J. (1985). The Fo Concept: The Parenteral Society.
21. Peto, S. and Maidment, B.J. (1969). Tables of the upper limit to the estimate of the
density of contaminating particles in a medium. 1. Hyg. Camb., 67, 533-583.
22. Safety Precautions: Notes for Guidance. Public Health Laboratory Service staff
handbook, 1988 edn.
23. Safety of Pressure Systems: Pressure Systems and Transferable Gas Container Regulations,
1989. Approved Code of Practice HSC: 1990. London: HMSO.
24. Grossell, S.S. (1990). An overview of equipment for containment and disposal of
emergency relief system effluent. 1. Loss Prevo Process Ind., 3, 112-124.
25. Kieffer, R.G., quoting Theodore Byers (1986). Why validation. In Validation of Aseptic
Pharmaceutical Processes, Carleton, F.J. and Agalloco, J.P. (Eds). New York: Marcel
Dekker.
26. Kemper, C.A. (1986). Design, installation and calibration of thermocouple measuring
systems. In Validation of Aseptic Pharmaceutical Processes, Carleton, F.J. and Agalloco,
J.P. (Eds) New York: Marcel Dekker.
13 Sampling methods for testing and
monitoring biosafety of biotechnology equipment
and activities
J.E. BENBOUGH
13.1 Introduction
A review by Hambleton et at.! recognised public concern about the inter-

related problems of the protection of operators to hazardous materials
generated by biotechnological activities and protection of the environment
from such substances. They considered that the hazard of aerosol
generation is probably the most significant posed by biotechnology
processes because inhalation is the most likely route whereby process
organisms, extrinsic antigens or other products might gain access to the
body. Such exposure is not limited to the immediate workers since aerosols
might not be confined to the laboratory or factory boundaries and hence
could cause illness in surrounding populations. Many process operations in
biotechnology facilities and laboratories may cause aerosols to be
generated and these have been extensively assessed by Chatigny2 and by
Darlow. 3 Work done by Harper4 showed that 27% of sealed centrifuge
buckets or rotors failed to contain aerosols generated by simulated
breakage of centrifuge pots containing viable spores of Bacillus subtilis as a
tracer. In a survey of accidents involving the operation of fermenters,
Ashcroft and Pomeroy5 showed that the failure of the anti foam system and
the breakdown of extract air filters caused the release of aerosols
containing micro-organisms. Cameron et al. 6 also showed that if incorrect
technique was used to sample suspensions from a fermenter then
significant aerosols containing the micro-orgamisms were produced.
Dimmick7 introduced the concept of the 'spray factor' to quantify the
magnitude of the aerosols generated by laboratory operations and its
significance is fully discussed in chapter 6 by Norris. Norris also emphasizes
the importance of the size, shape and density of the particle in relation to
the site of deposition in the body. Most aerosols generated by biotechnology
operations consist of particles of a wide range of size and it is necessary to
have an understanding of the particle size distribution before their
hazardous effect can be fully appreciated.
The characteristics of aerosols generated from suspensions containing
SAMPLING METHODS 269
micro-organisms (see chapter 6) must be carefully considered in developing

a strategy for sampling airborne microbes. Norris shows that the
composition and size distribution of the particles depend on the concentra-
tion of dissolved solids in the original suspension and the possibility of a
microbial cell occurring in each airborne particle will depend upon the
concentration of micro-organisms in the aqueous suspension.
13.2 Sampling microbial aerosols
Accurate measurement of an aerosol is dependent on obtaining a

representative sample from the air and also minimising or quantifying
losses from the sampling probe to the measuring instrument. This subject
itself is complex but a review of Fissan and Schwientek 8 provides a very
useful summary of the factors involved in sampling from moving air in
particular. Errors can arise when sampling from still air as some particles
fail to enter the probe inlet because their inertia prevents them from
accelerating, decelerating or changing direction sufficiently in response to
the air suction. Davies9 suggested that perfect aerosol sampling by an inlet
under calm air conditions can only be assumed if the suction rate is small
enough to make inertial forces small but high enough to make settling
negligible.
Very often air is required to be sampled from a cross-wind or from a
duct, e.g. the exhaust air-stream from the fermenter or downstream of a
filter to be tested. In this situation, errors can arise because the air flow in
the sampler does not match the wind flow rates, or the probe is aligned so
that the particles have to be deflected from the wind streamlines to the
probe streamlines. To address these problems the sampler would ideally be
arranged to operate under isokinetic sampling conditions, i.e. where the
velocity of the air in the sampler inlet equals that of the air being sampled
and also with the probe aligned for isoaxial sampling. If the wind velocity
and the tube suction velocities are mis-matched, curved streamlines around
the probe entrance are set up. Errors then arise because particles with
sufficient inertia do not follow these curved streamlines. In the case of
suction velocities less than the wind velocity, the sampling efficiency will be
greater than 100% since the larger particles are not deflected away from
the nozzle entrance and the sample will be biased with larger particles.
With suction velocities greater than the wind speed, efficiencies less than
100% occur with a collected sample biased with smaller particles (Figure
13.1).
This effect of mis-matched air velocities was shown by MaylO for a nozzle
of 1-2 cm diameter sampling at 5 mls (see Figure 13.2). This diagram
suggests there is a maximum loss of sampling efficiency at a wind suction
ratio of 0.4. Below 0.4 the efficiency increases as the still air situation is
(a)
,...,..,.....
>
(b)
""'-"
(c)
"""-'
Figure 13.1 Streamlines into sampling orifices. (a) Isokinetic sampling; (b) inlet velocity
into sampler higher than air velocity; (c) inlet velocity into sampler lower than air velocity.
approached. The reason that the efficiencies do not continue the

downward trend with lower wind speeds is presumed to be due to the lower
momentum of these largest particles.
If the probe is not aligned with the flow streamlines then there are losses
due to the inertia of particles which are required to follow a new direction
depending upon the angle of the problem relative to the wind direction.
This effect was also clearly illustrated by MaylO as shown in Figure 13.3.
13.3 Sampling methods
Samplers shown to be effective for collecting dust particles may not be

suitable for collecting microbial aerosols. Shear forces, static forces and
dessication effects have been mentioned by COX II to damage micro-
organisms during collection. COX I2 •13 also showed that during collection
into liquid the fraction of viable micro-organisms recovered depends if the
aerosol is pre-humidified before collection and on the constitution of the
collection fluid. Benbough and Hambleton l4 •15 showed that the damage to
Limit for very large particles
1.5
./ "./
()
"-
0
.... /.
.," /'
_......
() ..;./ __ - 20
---
>; .,:/' _-- _-5
,-- - --
0 ,~.....:::.---
c 1.0
.~
.9
.--.--:,: Very small particles
\', 5 - - /./.-.,
~
'"
.><
\ -- ;0-- / / .
os
.E '. '-'_._./' /
0.5 50 r--- Isokinetic sampling
t:--- .. Stili air
o 0.5 1.0 1.5
Ratio of wind speed to tube intake speed, Uol U
Figure 13.2 Effect of changing wind speed past a horizontal nozzle for particles of different
diameter (the sampling velocity is approximately 5 m S-I and the diameter of the nozzle is
between 10 and 15 mm) (Crown copyright reserved).
bacterial cells occurring during re-hydration is related to impairment of

membrane function leading to the inability of the cells to transport
nutrients into the cell. Hambleton 16,17 also found that aerosolised Gram-
negative bacteria became sensitive to hydrolytic enzymes such as lysozyme.
This is related to changes in the surface chemistry of the bacteria since
lysozyme conjugated to a fluorescent dye was found to bind to aerosolised
bacteria and not to unaerosolised cells. 18
Assessment of the viable microbial content of air is invariably carried out
by counting the visible colonies on solid nutrient medium after collection
and incubation. The selection of nutrient medium may be of significance
because for true assessment of viability of recovered microbial cells it is
essential that the media used contains all the ingredients that enable cell
repair to occur so that they multiply to form colonies. On the other hand
some media may contain ingredients to which stressed micro-organisms are
sensitised. This may prove significant where specific detection of particular
strains is done using particular media. For example, MacConkey agar is
used as a selective medium for the detection and differentiation of coliform
. . . . . __ -::::.ry
"---::::::.
~
.
small particles ,
~ ............... 5 microns
0.8
o
l)
~ ~
"-
l)
g 0.6 0- :\ ""
\
CD
·u
~
S
CD
\ 50 microns
0.4
.s \
C/C 0 = coso for \
0.2
o o o
o 20 40 80
Figure 13.3 Effect of probe angle relative to the wind direction for sampling velocities equal
to the wind speed using 1-20 mm diameter probe nozzles (Crown copyright reserved).
bacteria and other enterobacteriacea because these organisms are resistant

to bile salts. However, it has been shown (A.M. Bennett, personal
communication) that the recovery of coliforms from aerosols is significantly
less when assayed on MacConkey agar compared to nutrient agar. This is
related to the sensitivity of damaged bacterial cells to bile salts. In some
cases falsely low apparent recovery of micro-organisms from aerosols may
occur because essential nutrients which may promote repair of damaged
cells are missing. Differences in the stresses of collection by different
samplers explain the sometimes large differences obtained in the recovery
of viable micro-organisms from aerosols.
Therefore, the result of microbial recovery by air samplers should be
interpreted carefully. The efficiency of collection should be validated using
radio-labelled micro-organisms 18 or using methods which are independent
of using colony counts.
13.4 Air sampling devices
A wide range of existing devices have been used to sample viable airborne
micro-organisms. They fall into the following three broad classes: inertial
collectors, filters and precipitators (electrostatic and thermal). Precipitators
are complicated and bulky devices, are not widely used as collectors and
are not discussed here. Inertial collectors rely on inertia of airborne

droplets either moving under gravity or some other external influence such
as a pump. Many types of inertial collectors are considered here.
13.4.1 Inertial collectors
13.4.1.1 Settle plates

The technique of using settle plates is simply to expose a suitable medium
to ambient air for a known length of time. The usefulness of settle plates to
collect airborne micro-organisms is governed by the gravitational force on
the droplet. The velocity of the fall of the droplet depends on its mass and
it is possible to correlate the time taken for a droplet to fall with the
diameter of the droplet for assumed droplet densities.
This allows the following extrapolation to be made: if a column of air 1 m
high above a settle plate contains 15 !-tm diameter droplets it would have to
be exposed for 150 seconds for all the droplets in that column to be
collected. Similarly, for 1O!-tm droplets, an exposure time of 350 seconds is
necessary and for 5 !-tm droplets a time of 1500 seconds is required. For
droplets below about 2 !-tm the fall times are so long that they are assumed
not to settle. The effects of air currents on settling have to be considered
and problems arise by evaporation from the exposed plates during
prolonged sampling. Settle plates are most widely used as one of the
techniques for the qualitative assessment of clean rooms or clean zones
used for the manufacture of pharmaceutical products. For these purposes
settle plates are applied in defined positions in clean rooms according to
standard operating procedures. In this case subtle changes in air quality
can be detected using trend analysis on the monitoring data.
13.4.1.2 Impingers
The most widely used is the critical orifice impinger (Figure 13.4) (Porton
all glass impinger) in which the air flow rate is controlled by applying an
appropriate pressure drop across an orifice. 19 With a minimum pressure
drop of 0.5 bar, the air moving through the orifice achieves sonic velocity
which is limiting so that measured volumes of air can be sampled. The
sampler was designed to operate by drawing aerosols through a curved
inlet tube, to simulate the nasal passage, and then to a jet placed at 30 mm
above the impinger base. The jet consists of a short capillary tube so that
when the pressure drop across this attains a minimum of 0.5 bar the flow
through it becomes sonic and therefore rate limiting. This impinger usually
samples the air at 12.5 l/min and is very efficient for collecting particles
containing microbes in the respirable size range (0.8-15 !-tm). A large
proportion of the larger particles are trapped on the curved inlet and these
are subsequently recovered before assay by rinsing this region with the
Inlet
To pump
Inches
---
Jet
Figure 13.4 Porton all glass impinger.
collecting fluid. Usually about 10 ml of collecting fluid is used in the

impinger. May and Harper19 showed that the Porton raised impinger is
virtually 100% efficient for collecting aerosols where each particle contains
a single Bacillus globigii (B. subtiUs var. niger) spore when sprayed from a
spore suspension in distilled water. They confirmed this by using the
impinger to collect aerosolised radioactively-tagged bacterial cells as the
tracer. For this reason the Porton raised impinger is used as the standard
and the performances of other samplers can be compared with it. The main
disadvantage of this device is that there is evidence that impingement of
the more fragile vegetative bacteria or viruses on the bottom of the
impinger may inactivate or damage these organisms.
The three-stage glass impinger developed by May20 is intended to
correspond with the three deposition sites of the human respiratory system
(Figure 13.5). The top stage corresponds to the upper respiratory tract, the
middle stage to the bronchioles and the bottom stage to the alveoli. The
Inlet
Chamber 1
Access port
Sintered glass
Chamber 2
to pump
Chamber 3 Critical orilace
Inches
Figure 13.5 May three-stage liquid impinger (two cross-sectional views).
sampler is available in three sizes with corresponding flow rates of 55, 20

and 10 l/min. In operation, air is drawn through the intake tube into the
first stage containing a sintered glass impingement disc which is washed
continuously by agitating collecting fluid. The larger particles are impacted
at this stage and become dispersed in the collecting fluid. The air continues
through a narrower intake tube into a second stage where the larger of the
remaining particles are impacted on a similar impingement disc and
washed into the second fraction of collecting fluid. Particles which are too
small to impact on the middle stage continue in the air stream and are
drawn to the bottom stage through a gently tapered jet set tangentially to
another fraction of collecting fluid. This centrifugation causes the
collecting fluid to swirl around the chamber to allow efficient collection of
the smallest particles. The flow rate control is achieved by placing a critical
orifice between the vacuum and the impinger. These particles are collected
as gently as is practicable and is effective for recovery of fragile micro-
organisms from aerosols.
13.4.1.3 Multistage sieve impactor

Andersen 21 described a six-stage impactor using sieve plates (see Table
13.1). Each sieve contain 400 precision drilled holes, and a Petri dish is
sited with the nutrient agar surface at a known distance below each plate.
Air is sampled at a constant rate of 28.3 l/min (1 cfm), thus with the hole
Table 13.1 Multistage sieve impactor
Hole diameter Air velocity Particle size

Stage number (mm) (m/s) (I-\m)
I 1.18 1.08 7.0

2 0.91 1.80 4.7-7.0
3 0.71 2.97 3.3-4.7
4 0.53 5.28 2.1-3.3
5 0.34 12.78 1.1-2.1
6 0.25 23.29 0.65-1.1
size decreasing from stage 1-6, the largest particles are collected in the first
stage and the smallest in the sixth.
After sampling a known volume of air and incubating the plates a count
can be made of the jets which delivered viable particles to the nutrient agar
surface. A correction factor can be applied to account for the possibility of
two particles containing viable organisms to fall in the same space to form a
colony. Colony counting in the Andersen sampler is relatively straight-
forward, because the patterns of colonies on microbial plates corresponds
to the pattern of the sieve plate holes (Figure 13.6).
Both the May and the Andersen samplers described above provide the
benefits of particle size distribution of a cloud containing micro-organisms.
Zimmerman et al. 22 compared the May three-stage sampler with the
Andersen sampler and showed that their efficiencies for collecting airborne
micro-organisms were very similar. They concluded that the Andersen
sampler was easier to use but is ineffective at high concentrations of
airborne microbes because it is readily overloaded. The May sampler
cannot, of course, be overloaded because the collecting fluid can be diluted
to a desired amount before assaying on nutrient agar. In practice if particle
size distribution of an aerosol cloud containing micro-organisms is required
the May sampler should be used nearest to the emission sources or points
of highest concentration and either of the samplers can be used furthest
from emission sources where low concentrations of airborne particles
containing microbes occur. The May sampler gives the concentration of
total airborne viable organisms rather than just the concentration of
airborne particles containing viable organisms. The May sampler provides
the additional flexibility to allow the airborne concentrations of certain
microbial constituents and products of biotechnology to be determined as
well as viable organisms.
13.4.1.4 Cascade impactor

The cascade impactor23 is a simple, robust, versatile device for collecting
and sizing of microbial aerosols. The principles involved are illustrated in
Figure 13.7. The air sample is split into a series of four progressively
Inlet
Air flow
Stage 1
Medium
Petri Dish
Stage 2
Stage 3 Gasket
Stage 4
Stage 5
Stage 6
To pump e:=;;;;;;;~
Figure 13.6 The Andersen microbial sampler.
Second stage
Third stage
Air in
Air out First stage

to pump
Fourth stage
Figure 13.7 The principles of the Cascade impactor. The broad lines represent the
microscope slides which are held in place against runners by springs. The jets are of
progressively smaller cross-section from the first to fourth stages.
smaller particle size ranges by decreasing the size of the jet at each stage.
The particles which are impinged on to the various microscopic slides may
either be examined under a microscope after staining or can be washed off
by a suitable buffer and plated out on a nutrient agar medium of
assessment. The cascade impactor designed at Porton allows the air to be
sampled at 17.5 IImin and the size fractions collected at the various stages
are as follows:
Stage 1 6-20 flm
Stage 1 2--6 flm
Stage 3 1-3 flm
Stage 4 0.5-1.5 flm
13.4.1.5 Slit impactors

Bourdillon et ai.24 developed the first slit sampler which consists of a slit
0.25 mm wide by 27.5 mm long arranged such that the sampled air is
impacted radially on a Petri dish containing nutrient agar. The height of
the plate is adjustable so that the slit to nutrient agar surface distance
ensures that all particles are impacted on the surface. In a recent model,
the Casella Airborne Sampler Mark II, the plate is rotated at a defined
speed for a fixed time by an electric motor and is designed to sample air
between 30 IImin and 700 IImin. This means the exposed plates following
incubation will provide a radial pattern of colony counts so that the
airborne concentration along radial segments can be correlated to specific
actions. The rotational speed of the sampler may be varied so that the
nutrient agar plate can be exposed for periods from 0.5 min to 15 min. This
means that the loading can be adjusted to correspond with the expected
concentration of airborne particles containing viable organisms by altering
the rotational speed.
The slit sampler is widely used in clean rooms for the quantitative
assessment of the airborne microbial content of clean rooms used in
pharmaceutical manufacture. The slit sampler is also commonly used to
test the integrity of HEP A filters and their seals in microbiological safety
cabinets and containment rooms when filters are challenged by aerosolised
micro-organisms. A probe linked to the sampler monitors the air
downwind of filters challenged by a concentration of about 5 X 107 cfu/m 3
of aerosolised B. subtilis var. niger spores. The sensitivity of the method is
very high with penetrations of less than 0.00002% being measured which is
100 times more sensitive than challenge methods using smoke or
aerosolised sodium chloride crystals. The probe linked to the slit sampler
can also be used to detect leaks around seals, windows and cable
penetrations in Class III microbiological safety cabinets during the
commissioning of these cabinets. This is done by injection of aerosolised B.
subtilis spores into the pressurised cabinets. The position of any leaks can
be located if the pattern of the colonies on the plates is correlated to the

position sampled.
In many instances where the naturally occurring aerosols containing
micro-organisms can be highly dilute, very large volumes of air may have
to be sampled in order to catch a few viable entities. This means that
monitoring by the slit sampler and other methods which depend on
impaction on gels such as the Andersen may have to be severely restricted
due to loss of water and consequent shrinkage of the gel which can prevent
colony growth. May2S showed that the incorporation of oxyethylene
docosanol (C18-22H37-4S0C2H40H) into the agar used in by these samplers
can suppress evaporation and enabled prolonged sampling to be done
without drying of the agar and enable colony growth to occur.
13.4.1.6 Cherwell surface to air sampler (SAS)

In the Cherwell sampler (SAS) (Figure 13.8) air is drawn by a pump
through a perforated plate and impacts any airborne particles containing
micro-organisms on a 55 mm diameter RODAC plate. The sampler is
relatively inefficient for collecting particles of less than 2 f-tm in diameter, 26
but it has the advantage of being portable, having are-chargeable
powerpack, and can easily be directed towards potential aerosol sources in
,4.--- Air flow
~~i~i~lli~~u:-
l_
Sieve plate
Rodac plate
Suction fan
Figure 13.8 Diagrammatic section of Cherwell SAS sampling head (Reproduced by

permission of Academic Press, London).
the workplace. This device is often used to measure aerosol generation in a

number of different locations. A multiple stage version, referred to as the
MTM3 model, is used in operating theatres, sterile areas and controlled
environments. This model is programmable to allow remote sampling of
the environment to occur and thus avoid intervention by the operator. The
three heads can also be programmed for time delay so that sequential
samples can be taken as required.
13.4.2 Air centrifuges

Centrifugal samplers cause the airstream entering the samplers to move in
a circular path which results in the effective mass of any droplets in the
airstream to increase compared to that under gravity. In the cyclone
sample shown in Figure 13.9, air enters tangentially to the cyclone body
which it strikes and thus achieves a tangential velocity component and
moves in a circular path. Droplets entrained in the air are impacted to the
Air to vacuum pump
Sampled air and

collecting fluid
injected
Pathway of particles
•
Collection fluid
Figure 13.9 All glass cyclone sampler.

cyclone walls. Errington and Powe1l 27 described a cyclone for sampling

microbial 27 aerosols where a metered flow of collection fluid is injected
into the cyclone inlet where it forms a thin mist of liquid droplets in the air
stream. Sampled particles carried in the liquid mist are then trapped in the
coalescing liquid droplets and the liquid suspension accumulates at the
bottom of the cyclone from where it is collected into a sterile sample bottle.
The two cyclones developed by Errington and Powell are made of perspex
material and they sample 75 or 350 litres of air per minute. The buffer is
injected in the inlet airflow at a rate of 1 mllmin. A larger and simpler glass
cyclone (made by the Hampshire Glassware, 79 Dukes Road, Southampton
S02 OST (Figure 13.9), can sample up to 700 litres of air so allowing all
particles in this quantity of air to be concentrated into 1 ml of fluid.
Recent work carried out (A.M. Bennett, personal communication) in
an environmental chamber in Porton showed that significantly higher
recoveries of E. coli from aerosols were obtained using a cyclone sampler
compared to the all-glass impinger. These tests were done by generating
aerosols from suspensions containing a mixture of E. coli with B. subtilis
var. niger spores, acting as tracer organism. Assuming that the spores were
not inactivated by spraying, it was found that 87% of viable E. coli
organisms were recovered by the cyclone sampler compared to 49%
recovered by all-glass impinger. This may be related to the effects of pre-
humidification during collection by the cyclone sampler as discussed by
COX. 11 The main advantage of this sampler is that it can be used
continuously without dismantling for many hours. Samples of collected
fluid can be taken sequentially as required at the bottom of the cyclone in
sterile glass tubes for subsequent assay. The cyclone is also the most
convenient method for sampling and concentrating material for rapid
analysis of certain microbial constituents (e.g. endotoxin) released into
air. This system can also be used to determine the amount of microbial
products released with air (e.g. hormones, enzymes). The continuous
operation allows the sampling system to be easily linked to an automated
system for rapid detection of the collected organisms based on their
cellular constituents (e.g. ATP, haematin, endotoxin) or the analysis of
products of biotechnology.
Another device using a centrifugal technique is in the Biotest RCS Plus
Centrifugal Air Sampler (supplied in the UK by Biotest Ltd., Birmingham:
Figure 13 .10). Air is drawn towards a set of four impeller blades housed
within an open shallow drum in the device. The sampled air is subjected to
a centrifugal acceleration by the impeller rotating at 6000 rpm and
particles are impacted at high velocity on agar medium contained in a
plastic strip lining the interior surface of the drum. The Biotest RCS
samples air at about 50 I/min and the sampling time can be set from periods
of 30 s to 20 min. The exact volume is measured by regular calibration of
the instrument with an anemometer. As shown in Figure 13.11, the air
Figure 13.10 The biotest ReS plus centrifugal air sampler (Reproduced by permission of
Biotest (UK) Ltd).
enters the rotor from the front and is fed by the four blades over the agar
strip and the air is exhausted at the rear of the unit. After exposure, the
agar strip is incubated and the colony-forming units are enumerated. The
RCS centrifuge sampler is light (weighing less than 2.5 lb), compact, easy
to carry and is battery operated and can be used for 2 h continuously before
recharging the batteries. Specific organisms can be enumerated by using
strips containing selective agar medium.
The performance of an earlier Biotest RCS air sampler was studied by
Clark et al. 28 where they found that this device was inefficient for sampling
particles less than 5 !-tm and the effective sampling rate given by the
manufacturers was incorrect. For this reason the authors advised that
caution should be exercised in using this sampler for quantitative
assessment of micro-organisms in air.
The performance of the RCS Plus Sampler was compared with that of
the Casella slit sampler in a special facility providing a controlled
environment by Benbough et al. 29 Parallel samples were taken in the small
environmental room (about 28 m 3 ) into which test aerosols of controlled
Figure 13.11 Airflow patterns during operation of the Biotest ReS Plug sampler (Reproduced
by permission of Biotest (UK) Ltd).
sizes were generated by a spinning disc atomiser. 30 ,31 The atomiser disc
(25 mm in diameter) was driven at either 24 000 or 48 000 rslm and fed
with a suspension of B. subtilis var. niger spores in 80% ethyl alcohol
containing varying amounts of potassium iodide. Solutions containing 7%
of potassium iodide can produce 20 and 10 [.tm particles and solutions
containing 0.7, 0.07, 0.007% and no potassium iodide can be used to give
particle diameters of 4.9,2.3, 1.0 and 0.7 [.tm respectively. The size of the
larger particles were determined microscopically and the smaller particles
were determined by calculation; the 0.7 [.tm particle corresponds to a single
spore. The samplers were run simultaneously and the number of organisms
collected directly on nutrient agar or collecting fluid were determined.
The results of the tests carried out showed that the ReS Plus sampling
175
150
J-i-d
>- 125
(,)
c
Q)
·13 100
J
:E
w
~
0
75
/-1/
50
25
0
0.2 0.5 1.0 2.0 5.0 10 20 50
Equivalent particle diameter {JL m)

Figure 13.12 Efficiency of the Biotest RCS Plus sampler for collection of spores in various
size of particles compared to the Casella slit sampler (Reproduced by permission of Blackwell
Scientific Publications Ltd).
efficiency was similar to that of the Casella sampler for particles likely to be
encountered in the environment. For particles less than 4 !-tm down to sub-
microbial sizes the efficiency of sampling fell off only gradually so that the
efficiency of sampling for 1.0 !-tm particles was only reduced to about 50%
(Figure 13.12).
The ability to programme the RCS Plus sampler to pre-select any of 10
sampling volumes (1 I to 1000 I) depending on the expected loading should
allow it to be used in a variety of locations. It is well designed to operate in
biotechnology plants because it is battery operated, light and can be held
by hand to point towards vulnerable leakage points and aerosol-generating
sources. Specific agar-medium strips can be incorporated in the device
allowing, for example, for the specific detection of E. coli or yeast cells
(which are the most commonly used host organisms in genetic manipulation
techniques) so that the release of these organisms into the air can be
detected. This is particularly useful where there may be high or fluctuating
amounts of background airborne microbes present. As mentioned earlier
in this chapter, caution should be applied in the use of some solid media for
the collection of bacterial cells from aerosol.
13.4.3 Filtration
Filtration methods of capturing airborne micro-organisms by drawing
metered quantities of air through filters are probably the simplest methods
of sampling. Davids and O'Conne1l 32 compared the effectiveness of many

types of filter material for this purpose. Macher and First33 compared the
effectiveness of membrane and gelatin filters and Blomquist et al. 34 used
nuclepore filters (which contain uniform cylindrical pores) to capture
airborne micro-organisms. The captured organisms are enumerated by
either washing the organisms off the filter with a known quantity of sterile
buffer solution and transferring a portion of this fluid to nutrient agar or by
placing the filter directly onto nutrient agar. The former procedure would
allow the total number of viable organisms collected from particulates to
be determined and the latter would allow the number of particles
containing viable organisms to be determined. It was generally found that
filtration provides a very effective method of collecting aerosol stable
organisms such as spores of B. subtilis but the passage of large volumes of
air over the filter resulted in virtually no recovery of vegetative bacterial
cells such as E. coli.
Deans 35 reported the use of two commercially available filters fitted to a
plastic head linked by a tube to a Casella AFC 123 air sampler. The
sampler is a compact battery operated unit weighing about 100 g which can
be clipped to the belt of the user with sampling head clipped to the lapel. It
was found that a five-fold improvement in the recovery of vegetative
bacteria occurring using a Sartorius gelatin filter (pore size 3 [tm)
compared to the Millipore type AA cellulose mixture (pore size 0.8 [tm). It
was thought that the gelatin filters may prevent excessive dehydration of
the collected organisms before assay on nutrient agar plates.
A fairly compact microbial air sampler (MD-8 Isokinetic air sampler)
using the filtration principle has been manufactured by Sartorius, 3400
G6ttingen, Germany. It consists of a microprocessor-controlled pump
attached to sampling head via a flexible hose. Gelatin foam filters of a
diameter of 80 mm and a pore size of 3 [tm are fitted with this sampling
head and the air can be collected at any rate selected between 42 and 133
IImin. This sampler is well suited to monitor laminar flow installations and
air conditioned rooms since the rate of air intake of the sampler can be
adjusted to that of the laminar air flow and enables isokinetic sampling to
be achieved. The gelatin membrane filters are designed to collect bacteria
quantitatively and, being inherently moist, they should prevent the loss of
viability due to drying. After sampling the air the filter is placed on an agar
culture medium which is then incubated. The filter dissolves and merges
with the culture medium and the colonies which grow can be correlated to
the volume of air sampled. Extensive tests have been recently carried out
at CAMR (J.E. Benbough, A.M. Bennett, S.R. Parks unpublished data)
to evaluate the performance of this sampler. It was found to be very
effective for recovering micro-organisms in a wide variety of sizes of
airborne particles and also in recovering fragile forms such as vegetative
micro-organisms from the air.
13.5 Sample assessment
13.5.1 Background
All these sampling techniques are used in conjunction with microbial
culturing methods which means of course that the results are not obtained
for up to 18 hs. It would be desirable to have a method for the rapid
automatic assessment of microbial populations recovered from aerosols in
biotechnology plants. Strange 36 reviewed possible methods and observed
that the main problem that affects such systems is the presence of large and
highly variable background levels of microbial aerosol. Ordinary air
contains a mass of suspended biological particles consisting of fungal
spores, bacteria, skin flakes and pollen. 37 The content is highly variable
according to the season of year, the weather and the time of day. It is not
uncommon to find more than 107 fungal spores per cubic metre and the
number of pollen grains can be as high as 103 but the numbers are
extremely variable. Agricultural operations generate a massive natural
aerosol and the oceans make a significant contribution since bursting
bubbles generate an aerosol which is rich in protein-containing materials. 38
It is against this high and extremely variable background level (mean value
10-6 g/m3 of protein material) that the emissions from biotechnological
processes have to be detected and measured. Even in biotechnology plants
where the inlet air is filtered (using High Efficiency Particulate Air -
HEPA - filters) background problems can arise due to bacteria attached to
skin scales liberated in large and fluctuating amounts (between 200 and
17 000 bacteria per minute) and due to human activity in the plant.
13.5.2 Detection methods

Because of the background problem, simply measuring the concentration
of protein nucleic acid or other microbial constituent in the air (e.g. A TP,
enzymes, co-factors and cell wall constituents) may not prove a measure of
that contributed by plant operation. Accordingly, highly specific assay
methods may be required. A rapid detection method based on the
universal presence of endotoxin in Gram-negative bacteria has been
developed at CAMR.39 Very low concentrations of endotoxin causes the
blood of the horseshoe crab, Limulus polyphemus, to clot. This reaction
has been developed and applied to detect the presence of endotoxins in
parenteral products and raw materials used in the preparation of these
products. Minute quantities of contaminating endotoxins have an un-
desirable pyrogenic effect if injected into the bloodstream of patients.
Because of the commercial opportunities, suppliers have been encouraged
to improve kits which can provide sensitive, reproducible and user-friendly
techniques to detect endotoxin. In one of the latest systems the endotoxin

activity is determined by measuring the time required to cleave a
chromaphore from a synthetic peptide which is analogous to the clottable
protein. Speight 39 has used such a system to determine the endotoxin
content of material collected by a depyrogenated all-glass cyclone using
pyrogen-free water as the collecting fluid. In a cyclone that collects
airborne particles in 700 l/min of air into 1 ml of collecting fluid, it was
found that 1 to 2 E. coli organisms/litre of air could be detected within 30
mins. Palchak et al. 40 sampled air for prolonged periods (35-45 mins) in a
facility where the industrial extraction of proteins from Gram-negative
bacteria was done. They used 0.45 ftm pore membrane filters mounted on
plastic cassettes linked to a pump operating at 2 IImin to collect any
airborne micro-organisms generated. The endotoxin content of the
material collected and extracted from the filter was determined colori-
metrically using a modified limulus assay technique. The results of this
survey are summarized later in this chapter.
In immuno-based assays, radio-linked, fluorescent-linked and enzyme-
linked homologous antibodies can also offer a practical approach to
detecting microbes and the use of DNA probes might also offer high
specificity and rapidity. The use of microscopic methods to examine
aerosols impacted onto adhesive surfaces could also be considered as a
rapid detection method. An experienced operator may be able to use the
shape of the particles as being characteristic to allow background material
to be differentiated from the material of interest. Naturally occurring
background aerosols tend to be solid particles, whereas aerosols generated
from biotechnology processes are not. The latter tend to consist of clusters
of separate bacterial cells bound together by the dried media constituents.
Therefore, when the latter are impacted and fixed on an adhesive surface
and an appropriate strain is added carefully, a very distinct pattern showing
a cluster of cells is obtained. The use of image analysers to determine the
number, size and shape of these patterns can help discriminate micro-
organisms of interest from the background. An automated system could be
devised if an effective air particle concentrator were linked to a moving
adhesive tape which could be subsequently continuously monitored by
image analysis.
In the 1960s and 1970s there was active development of sophisticated
continuous automatic sampling and detection devices capable of
providing early warnings of the presence of airborne biological agents to
enable personnel at risk to exposure to take necessary protective measures.
Such devices were able to discriminate very effectively between agent and
background aerosols and were linked to logic systems which analyse the
data on a continuous basis. This allows the sensitivity of the system to be
optimised against an acceptable false alarm rate. The space agencies have
also developed very sophisticated devices for detecting possible microbes
in space and have been described fully by Mitz.41 However, because of the
enormous capital and running costs involved with instant alarm systems for
microbiological aerosols it is unlikely that the biotechnology industry will
be interested in developing such systems in the near future.
13.6 Practical applications of sampling techniques
Bioprocessors have been monitored using many of the air sampling

techniques described above. Processes such as fermenter sampling,
centrifugation, homogenisation and cell disruption may all generate
significant aerosols of process organism or product. For example aerosols
were produced during operation of a disc bowl centrifuge and also during
subsequent removal of biomass from the centrifuge bowl 42 (Figure 13.13).
Similar results were achieved with both a slit sampler and SAS sampler
showing that there can be some flexibility in the choice of a monitoring
device. 37
Using the airborne sampling techniques for endotoxin used by Palchak et
al. 40 and described above, it was found that detectable amounts of
endotoxin were released during various stages of industrial scale extraction
(a)
25,----------------------------------------,
20
1000 cfu/m 3
15
10
Time after start of sampling (hr)

(b) 20 ,---~~---- -~~~~~---
15
Time after start of sampling (hr)
Figure 13.13 (a) Microbial aerosol produced by disc bowl centrifugation and subsequent
removal of cell paste measured by the Cherwell SAS sampler; (b) microbial aerosol produced
by disc bowl centrifugation and subsequent removal cell paste measured by the Casell slit
sampler operating over the same period as in Figure 13.13 (a).
of protein from Gram-negative bacteria. They found that the mean

values of the amounts of airborne endotoxin from a number of fermen-
tations, continuous centrifugation and mixing/homogenisation runs were
0.33 ng/m 3 (range 0.08-2.14), 139 ng/m 3 (0.08-12.8) and 0.86 ng/m 3
(0.07-4.52) respectively. When they took samples during experimental
batch harvesting when full containment was not considered due to the
experimental nature of the work they found that the mean airborne
endotoxin level reached 162 ng/m 3 (range 0.47-18.12).
Quantitative data obtained by air samplers need careful interpretation
and the choice of sampling methods used during biotechnological processes
depend on the circumstances. As far as background sampling is concerned
micro-organisms in ambient air tend to be very robust and are relatively
unaffected by environmental factors and by the trauma of collection by
various devices. Gram-positive micro-organisms generated from operators
are also robust and are not generally inactivated during collection. Both
these types of micro-organisms exist in particles of over 5 [.tm and micro-
organisms generated from humans are usually associated with skin scales.
Such microbes (i.e. robust and present in large particles) tend to be the
major source of contamination in pharmaceutical manufacturing plants.
Accordingly, the main attributes needed for air samples in such a facility is
ease of handling, minimal processing samples and minimal interference to
the manufacturing process and the movement of air in the room. For this
reason the Biotest Plus RCS and SAS samplers which are known to be
effective in collecting large particles are the most convenient. In such
critical operations the samplers are used in a routine way according to
standard operating procedures and any subtle changes in the air quality is
detected using trend analysis of the monitoring data. Validation of aseptic
pharmaceutical filling operations where the product, containers and
closures are momentarily exposed to air is demanded. This is done by
process simulation exercises where nutrient media is filled in the same
way as the product. The air monitoring data obtained by a sampling
technique can then be related to the rate of failure of media fills (i.e. the
number of vials per 10 000 where visible microbial growth has occurred
after incubation).
Recovery of vegetative organisms from the air depends greatly on the
sampling techniques used. These organisms (especially actively growing
organisms) are relatively labile to the stresses of aerosolisation. They are
also likely to exist in small particles (because they are generated into the air
by high energy activities such as being forced through filters and equipment
orifices by high pressures or by centrifugation) and are less likely to survive
environmental and collection stresses. Experience at CAMR Porton Down
has shown that as much as 50-fold differences in the recovery of viable E.
coli from aerosol has been shown when using different sampling devices.
The optimum survival values are obtained using the cyclone sampler or the
May 3-stage impinger and the worst are obtained using the cascade
impactor. The relative awkwardness of the cyclone and May samplers have
to be considered regarding their applicability in biotechnology plants.
Whereas the SAS and the RCS Biotest Plus samplers are easy to use and
can be pointed towards potential sources of release in biotechnology plants
their inability to collect particles efficiently in the respirable size range is a
significant disadvantage. The bulkiness of slit samplers may preclude their
widespread use in biotechnology plants.
It is not possible to make firm recommendations for sampling devices;
the user must take into consideration the performance characteristics of
the sampler and the nature of the information required, e.g. total quantity
of material released, time of release, particle size, and whether to use a
static sampler or a sampler attached to operators. However, if industry is
to operate to standards, then there must be a common approach to air
sampling/monitoring based on an agreed rationale.
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Index
abbreviated new drug application 37 cell disruption 288

ablation 191 centrifuga I 281, 290
Academy of Sciences (USSR) 70, 74 centrifugation 288
access 215 continuous automatic 287
accidents, fermenters 94 continuous detection 287
ACDP see advisory committees cyclone sampler 280, 290
ACGM see advisory committees devices 272-280
ACRE see advisory committees efficiency 284
addition systems 227 filtration 284
adhesion 96 homogenisation 288
adsorption 99 impingers 273-275, 290
advisory committees isokinetic 285
Advisory Committee on Dangerous multistage sieve impactor 275-276
Pathogens 24,25,151,241,246,258 settle plates 273
Advisory Committee on Genetic slit impactor 278-279
Modification 24,25,214,233,241, slit sampler 282
246 surface to air sampler 279
Advisory Committee on Releases into airborne allergens 103
the Environment 25 airborne particles see also aerosols,
Dangerous Pathogens Advisory particle size, particle distribution
Committee 24 biological behaviour 100
Genetic Manipulation Advisory Group allergen 103-104, 181
24, 151, 174 inhaled 112
Howie Code 24 allergic reaction 81, 112
aerosols 122 allergy 109 see also allergic reaction
centrifuges 268 biological products 113
challenge 263 extrinsic allergic alveolitis 114
conductivity measurement 230 incidence 85
freeze drying 181, 182 micro-organisms 115
generation 92, 268 alveolitis 104, 114
generation on centrifugation 156, amino acids 57, 61, 78
167, 169 ampoules 184
in filtration 153 heat scaling 186
infection 91 ANDA see abbreviated new drug
persistence 95-96, 98 application
protective agents 103 Andersen sampler see air sampling
release 230 Animal & Plant Health Protection Service
respiratory tract 99 50
sampling 269 animal food additives 39
transmission 110 antibiotics 119
agricultural chemicals 61 antitoxin 38
air analysis 77 APHIS, see Animal & Plant Health
air centrifuges see air sampling Protection Service
air filtration 134 application of sampling techniques 288
air monitoring 229 artificial chromosomes
air sampling yeast 35
air centrifuges 280 Asilomar Conference 34, 67
Andersen sampler 276 Aspergillus 83, 112, 115, 116, 119
cascade impactor 276--278, 290 asthma 82,85, 104, 112, 115
294 INDEX
autoclave 72 Centers for Disease Control 151

auxotrophic 233 centrifugation 122, 135,289
air sampling 288
bacilli 233 centrifuges 92
Bacillus anthracis 101 centritech separator 155
Bacillus globigii 274 containment 155
Bacillus subtilis 60,92, 113, 268, 274, continuous flow 139
27S,2SI disc stack 158
Bacillus thuringiensis S5, 116 production scale ISS
ball mills 166 scroll decanter 155, 158-160
barrier fluid 222 solid bowl 154, 158
BATNEEC 21 tubular bowl 154, 155-158
bead mill homogenisers 139, 166 cGMP see current good manufacturing
biocides 199-203 practice
ethylene oxide 202 chemical hazards see hazards
formaldehyde 203, 249 chronic bronchitis see bronchitis
hydrogen peroxide 203 chronic lung disease see lung disease
propiolactone 20 I c1adosporidium 103
biohazardous materials 181 classification
biohazards 129 group I 216
biological containment 60 group II 216
biological products 3S clean room 96, 182
bioreactor exhaust gas 145, 224 clearance 99
bioreactors see fermenters Clostridium botulinum 139
boundary layer 98 Codes of Practice 110
Brenner classification Howie code 24
access 215 collagen 61
damage 215 colony stimulating factor 57
expression 215 Commission of the European Communities
British Standards Institute 25, 132 CEN activities 152
bronchitis S2 regulation in biotechnology
bronchopulmonary complaints 85 commissioning 24S
Brucella abortus 112 Committee for European Standardisation
Brucella suis 101 152,174,231,236
BSI see British Standards Institute Competent Authorities 6
bund walls 22S condensate lines 144
bursting disc 156,223,265 condensate traps 145
byssinosis 116 condenser 198
conductivity measurement 230
Canada 32, 52 conjunctivitis 85, 118
carcinogens 110 contained usc directive 3,6, 15, 235
cascade impactor 276--278, 290 containment 5
CDC see Centers for Disease Control inspectors 27
CEC see Commission of the European notification requirements 4
Communities contained usc regulations 22, 213, 232,
cell disruption 122 234
air sampling container breakage 190
liquid extrusion 167 containment 25, 70, 80
non physical 172 categories 60,216,220,222,225,
physical methods 166 235,240,244
sonic 170--172 centrifuges 155
cell separation cost 146
centriguation 154-166 design engineering 144
filtration 152-153 design principles 218
membrane filtration 153 devices 245
CEN see Committee for Standardisation effluent 264
Central Regulatory Commissions 76 effluent management 244
Center for Veterinary Medici ne 40 fermentation 137. 144,220,224
INDEX 295
in downstream processing 149-177 dispensing 183

measuring and monitoring 228 dispersed oil particle penetration 134
negative pressure 183 DNA probes 287
personal physical protection 131 DoE see Department of the
primary containment 131,151,155, Environment
229 DOP penetration see dispersed oil
principles 217 particle penetration
secondary containment 131,144,155 DPAG see advisory committees
temporal 132 drains 263
tertiary containment 131 DRD see deliberate release directive
containment levels 73,216 drugs 62 see also animal food additives
see also containment categories dust 84
continuous release 98 duties
contraceptives 119 of employees 18, 19
control of substances hazardous to health of manufacturers and suppliers 20
21,214,230,241
convection cell 264
cooling water 243 EC see European Community
coordinated framework 32, 34, 43, 48 Educational National Interest Group 26
COSHH see control of substances EEA see European Economic Area
hazardous to health effluent 240
coughing 118 autoclave 252
cross-flow filtration see filtration containment 264
CUD see contained use directive costs 263
culture medium 243 dedicated plant 252
current good manufacturing practice 38 downstream processing equipment
CVM see Center for Veterinary 263
Medicine fermenters 263
cyclodextri n 61 handling 146
cyclone sampler 280, 290 plant design 252
cyclopentadecane 57 plant qualification 256
retention 249
D-value 250 sterility 251
damage 215 testing 251
dead legs 246 treatment 257
decimal reduction time 250 Err A see European Free Trade
decontamination 138, 199 Association
deliberate release 61,65, 76,79-80 electricity at work regulations 24
risks 65 electrostatic precipitation 195
deliberate release directive 5, 6 endotoxic reactions 116-118
deliberate release regulations 22, 213 endotoxin 116, 117,231, 286, 289
Department of Agriculture 32, 49 inhalation 116
Department of the Environment 27,214 enforcement see also safety
dermatitis 82, 85 improvement notice 28
detection methods 286 prohibition notice 28
detergent 57, 113, 231 Enterobacter agglomerans 116
diagnostics 62 entry ports 222
diaphragm valves 227 environment 6,45,65,81
diarrhoea 111 Environment Agency (Japan) 65
dip tube 227 environmental
directive see European Community monitoring 230
discharge centrifuges see centrifuge, pollution 67
scroll decanter Protection Act 21, 22, 241
diseases Protection Agency 32, 43, 44, 48
occupational 218 risk assessment 218, 234
disinfectant fluid locks 139 safety 232
disinfectants 139 enzyme 61, 82, 85, 114
disinfection 25, 249 erythropoietin 57, 62
296 INDEX
Escherichia coli 60, 73, 116, 120,231, Fungicide & Rodenticide Act
233,243, 281, 287 filters see also filtration
ethylene oxide see biocides effluent management 245
EUP see experimental usc permit frame membrane 154
European Commission 15.217 freeze drying 197
European Communities Act 22 HEPA 60, 156,224,230,278,286
European Community membrane 287
clearance procedures 6 pilot scale 154
directives 2.7.15,21,22,109-110 plate membrane 154
national legislation 15 press 153
product legislation 7 sterilisation 264
European Community Directives tubular membrane 154
biological agents 15, 214 vent 263
contained usc 3, 15 filtration 123, 134, 196 see also cell
deliberate use 5. 15 separation
development of I airborne sampling 284
genetic modification 2 cross flow 139, 153
European Economic Area 11-12 effluent treatment 248-249
European Free Trade Association II exhaust gases 224-5
experimental use permit 44, 46 fine chemicals 57
exposure limits 231 flexible film isolators 143 see also
expression 215 isolators
fluid handling 173-174
Factory & Workshop Act 16 FMEA see failure mode and effect
Factory Act 16 analysis
factory inspector 26 F" concept 251, 254
failure mode and effect analysis 242 foam breakers 220
FAP see food additive petition 40 Food & Drug Administration 32, 36, 40
fast protein liquid chromatography 139 food 64
FD&C act see Federal Food Drug & additive petition 40
Cosmetic Act additives 64
Federal Food Drug & Cosmetic Act 36, general requirements 42
42 safety 61
"Federal Insecticide Fungicide & foot and mouth virus 101
Rodenticide Act 43, 44 forced expiratory volume 231
federal register 40 formaldehyde
federal regulatory structure 33 decontamination 138 see also
fermentation 122 biocides
airborne endotoxin 289 formulation
containment 137 freeze drying 189
open pan 115 general concepts 188
fcrmenters prefreezing 189
accidents see accidents FPLC see fast protein liquid
additions 227 chromatography
bottom drive 220 fragrances 57
containment 137, 144 Francisella tularensis 91
harvest 227 freeze drier
magnetic drive 222 decontamination 199
pressure hold test 227 design and operation 192
pressure relief 223 drying chamber 197
sampling 2S8 fabrication 194
top plates 222 positioning of filters 196
top drive 222 protective devices 195
valves 227 freeze dryi ng 178
FEY see forced expiratory volume aerosols 181, 182
fibrosis 114 cxcipients 189
field testing 45, 63 formulation 188
FIFRA see Federal Insecticide good manufacturing practice 182
INDEX 297
hazards 181 Monbusho, STA, MAFF, Russia

ingestion 181 ACDP 241
inhalation 181 ACGM 241
laceration 181
pathogenic materials 181 harvest systems 227
prefreezing 179 hazard and operability study 232, 234,
primary drying 179 242
reconstitution 188 hazards 30 see also heath hazards
secondary drying 179 animal cell culture 120--121
stoppering 180 bioprocessing equipment 122-124
storage and reconstitution 180 cell disruption 122
technical features 180 centrifugation 122
fungal spores 82 chemical 110
definition 240
gas containers fermentation 122
regulations 23 filtration 123
gastrointestinal complaints 85 freeze drying 181
generally recognised as safe 35, 43 genetically modified micro-organisms
genetic modification 3 119-120
genetically modified micro-organisms 3 ingestion 90
see also good industrial large scale inhalation 90, 96
practice micro-organisms 136
group I 4,216,234 plant cell culture 121-122
group II, 4,216 quantification 97
guidelines for downstream skin contact 90
containment 174 HAZOP see hazard and operability
hazards 119, 129 study
low risk 4 Health & Morals Apprentices Act 16
prevention of release 129 health and safety
small-scale 4 genetic manipulation regulations 1
type A operations 4,216 Health & Safety at Work Act 17, 18,22,
type B operations 4, 216 241
genetically modified organisms 1, guidelines 25
109-110,213,240 local involvement 26
Contained Use Regulations (1992) 22, regulations 20
213 Health & Safety Commission 17,214
Deliberate Release Regulations (1992) Health & Safety Executive 15, 17, 124,
22,213 132,260
guidelines for release 55,61 Technology & Health Sciences Division
GENHAZ 234 27
genital infection 84 health hazards 109-128 see also
GILSP see good industrial large scale hazards
practice heat exchanger design 254
glass bead disintegrator 138 heat treatment 250
GMAG see advisory committees HEPA see filters
GMMOs see genetically modified hepatitis 111
micro-organisms hepatitis B vaccine 39,57,62
GMOs see genetically modified high pressure vessels 110
organisms holding tanks 259
good industrial large scale practice 2, 61, hollow fibres 154
65, 144,217,222,223,232, 235 homogenisation 118, 289
good occupational safety and hygiene air sampling 288
216,217,232,235 homogeniser 169
GOSH see good occupational safety and hormones 119, 121
hygiene host-vector systems 60
gram positive micro-organisms 289 HSC see Health & Safety Commission
GRAS see generally recognised as safe human genome project 35
guidelines see also NIH, OECD, human growth hormone 57
298 INDEX
human health 45,215 JHSF see Japan Human Science

hyaluronic acid 57,61 Foundation
hybridoma 6
hydrogen peroxide see biocides Keidanren see Japan Federation of
Economic Organisations
TBC see Institutional Biosafety kill tanks 144, 221,224
Committee
TBP see Industrial Biosafety Project laboratory infection see infection
IFD see International Dairy Federation laboratory scale 59, 153
nCA see Inter-American Institute for laboratory scale containment 216
Cooperation in Agriculture labour safety see worker safety
ILC see in-line cleaning laminar flow work stations 137
image analysers 287 large scale containment 216
immuno-based assays 287 large scale production 35
impingers 273 large-scale 144
three stage 274, 290 large-scale use 234
in-line cleaning 223 laryngeal complaints 85
INAD see investigational new animal Latin America 32, 54
drug leak detector spray 247
incineration 195 leak location 228
IND see Investigational Exemption New leak rates 246
Drug leak testing 188, 246
Industrial Biosafety Project 213,218,230 Legionnaires Disease 101
industrial safety 67 legislation: see also regulation
industrial scale 61 legislation: Austria 12
industrial scale processes 58 legislation: Belgium
infection 81 Department of Agriculture 11
laboratory associated 111 Department of Public Health 11
infectious viruses 121 legislation: Denmark
infectivity of bacteria 97 Environment Ministry 11
inhalation 268 Environmental & Gene Technology Act
inlet lines 220 10
inoculation 92 Labour Inspectorate 11
inspection aims 27 National Agency of Environmental
inspectors 27 Protection 10
instantaneous release 98 legislation: Finland 12
Institutional Biosafety Committee 35 legislation: France
insulin 57, 62, 224 AFNOR 10
integrity testing 145, 228, 246, 261 Commission de Genie Biomoleculaire
Inter-American Institute for Cooperation 10
in Agriculture 32 Genetic Engineering Commission 10
interferon 57,62,63,119, 121 Ministry for Research & Technology
interleukins 121 10
International Dairy Federation 223 Ministry of Agriculture 10
Investigational Exemption New Drug 37 legislation: Germany
Investigational New Animal Drug 39--40 Federal Biological Office 9
isoki{letic air sampling 285 Federal Environment Office 9
isokinetic sampling 269 Federal Health Ministry 9
isolators 25 Federal Nuisance Act 9
isopropanol 139 recombinant DNA guidelines 8
legislation: Greece 11
legislation: Iceland 12
Japan 57 legislation: Ireland 11
Japan Bioindustry Association 59 legislation: Italy 11
Japan Federation of Economic legislation: Liechtenstein 12
Organisations 59 legislation: Luxembourg 11
Japan Human Science Foundation 59 legislation: Norway 12
JBA see Japan Bioindustry Association legislation: Portugal 11
INDEX 299
legislation: Spain 11 Ministry of Health & Welfare 58-59,61,

legislation: Sweden 12 fi4
legislation: Switzerland 12 Ministry of International Trade & Industry
legislation: The Netherlands 58-59,61
Chemical Substances Act 9 MITI see Ministry of International
Ministry of Housing Planning & Trade & Industry
Environmental Protection 9 MMD see Mass Medium Diameter
Nuisance Act 9 MONBUSHO see Ministry of Education
Provisional Committee on Genetic Science & Culture
Modification 9 monitoring of biosafety equipment
legislation: United Kingdom 268-292
Department of the Environment 8
Environmental Protection Act 8
Health & Safety at Work Act 8 NADA see New Animal Drug
Health & Safety Executive 8 Application
legislation: USA 12 nasal symptoms 104
Environmental Protection Agency 12 National Competent Authorities 5
Food & Drugs Administration 12 National Institutes of Health 34, 151
US Office of Science & Technology 12 guidelines 34, 55, 69, 244
lipopolysaccharide 116 Recombinant Advisory Committee 34
liquid effluent 242 NDA see New Drug Application
local genetic modification safety needles, dispensing 183
committee 26 negative pressure containment 183
lung disease 114 New Animal Drug Application 36, 39
lung infection tests 124 New Drug Application 36
1yophilisation see freeze drying NIG see Educational National Interest
Group
NIH see National Institutes of Health
MAFF (Japan) see Ministry of 34
Agriculture Forestry & Fisheries notification 50
magnetic coupling 220 nucleic acids 61
magnetic drives 222
maintenance and training 232
management systems 20 O-ring 171, 222
Management of Health and Safety at double 222
Work Regulations 20 sealed 265
marine organisms 65 seals 144
Mass Medium Diameter 93 OAF see open air factor
maximum permissible concentrations OAS see Organisation of American
78-79 States
Medical Device Amendments (1976) 41 occupational
medical devices 36,41 allergy 109
medical surveillance 115 asthma 115
melon 63 diseases 218
Methylophilus methylotrophus 117 exposure limit 231
MHW see Ministry of Health & Welfare hazard 84
microbial pesticides 45 OECD see Organisation for Economic
microbial toxins 73 Cooperation and. Development
microbiological safety cabinets see safety OEL see occupational exposure limit
cabinets oncogenic 121
microorganisms, safe use 80 open air factor 10 1
microscopic methods 287 open pan fermentation 115
microthreads 101 operational safety 206
Ministry of Agriculture Forestry & Organisation for Economic Cooperation
Fisheries 58-59,61,63 and Development
Ministry of Education Science & Culture Guidelines 1, 63, 64-65
58-59 Organisation of American States 32, 55
guidelines 59-61 oxygen, effect on micro-organisms 102
300 INDEX
PAHO see Pan American Hcalth propiolactone see biocides

Organisation prosecution 29
Pan American Health Organisation 32 protease 113
particle counters 230 protection factor 135
particle size 101, 103-104 protective agents see aerosols
particle size distribution 93 pseudomonas 112
pass-boxes 139 Pseudomonas aeruginosa 118
pathogenic organisms 90, 112, 129, 138, Pseudomonas syringae 120
257 public opinion 58, 119
categories 134 pumps 262, 265
freeze drying 178, 181 centrifugal 173
penicillium 116 dispensing 183
perfume bases 61 peristaltic 173
personal physical protection 131-132 progressive cavity 173
personal protection 206 rotary, positive displacement 173
pesticide registration 47 seals 220
pesticides 45 sliding vane 173
pharmaceutical manufacture 137
pharmaceutical sector 62 qualification 256
pharmaceuticals 61
physical containment 70 r-DNA see recombinant DNA
physiological state and toxicity 101 rabies vaccine 52
pigments 57,61 RAC see National Institutes of Health
pilot plant 118,144 rashes 85
pilot scale 153 Recombinant Advisory Committee see
pipetting 92 National Institutes of Health
pipework 246, 263 recombinant DNA 1,34,40,51,57,73,
planned preventative maintenance 145, 213
248 agricultural applications 63-64
plant cell culture 121 cell fusion 3
plume dispersion model 224 conjugation 3
PMA see pre market approval guidelines 58
application hybridisation 3
PMN see premanufacturing notification hybridomas 3
POMEC see Porton mobile enclosed in vitro fertilisation 3
chemostat micro-injection 3
Port on glass impinger 273 mutagenesis 3
Port on mobile enclosed chemostat 137 natural processes 3
positive pressure 183 polyploidy induction 3
PPM see planned preventative safety 70
maintenance 145 self cloning 3
pregnancy see worker safety techniques 3
premanufacturing notification 48 transduction 3
premarket approval application 36 transformation 3
pressure vector systems 3
decay 247 recombinant plants
hold test 228 melon 63
relief 144, 223 release 61
relief system 243, 255, 263 rice 63
steam sterilisers 250 tomato 63-64
system regulations 23 reconstitution 180, 188
probe entry ports 145 regulation see also legislation
probes 220, 222 Canada 32, 53-54
process fluids 243 Europe 1-13
product dispensing 183 former Soviet Republics 67-89
product handling 123, 183 Good Manufacturing Practice 182
product removal 186 Japan 57-66
product storage 187 Latin America 32, 54-56
INDEX 301
Russia 67-89 physical containment 60

United Kingdom 8 see COSHH, seal 144, 151, 156, 170, 218
Contained Use Regulations, doublc mechanical 154, 220, 262
Deliberate Release Regulations, dynamic 172, 174, 219
Electricity at Work Regulations, failure 166
management systems, gas containcrs mechanical 156. 167
United States 32-52 primary 246
relative humidity, effect on pumps 245
micro-organisms 101-102 rotating 220, 245
Report on National Biotechnology Policies secondary 219. 246
33 static 172.219,222.265
respiratory allergy 109 stirrers 245
respiratory tract 99 self cloned 6
retention 99 sensitisation 81. 114
retrovi rus 63 Serratia marcescens 117
rhinitis 85, 118 serum 38
rice 63 settle plates 273
risk 30, 76 significant new use rule 49
risk assessment 6, 105, 123, 181,214, single cell protein 67, 78-79. 81. 83, 117
218,234,235,241 sinks 263
Robens Committee 17 skin reactions 115
rotary vacuum drum 153 skin tests 115, 124
Royal Commission on Environmental slit impactor 278-279
Protection 234 slit sampler 282
rupture discs 223 sludge 243
smallpox 24
Saccharomyces cerevisiae 60 small scale containment 216
safety SNUR see significant new use rule
enforcement 26 sonication 170
environmental 14 SOP see standard operating
human health 215 procedures
inspection 26 South America see Latin America
local committees 26,35,76 Soviet regulations 67
occupational 14 spillage 186
UK 14 spinning disc atomiser 283
safety cabinets 25, 132-137 spray drying 118
application to process containment spray factor 93,97, 123,268
137 spring loaded safety relief valves 223
centrifugation 155, 167 ST A see Science & Technology Agency
class I 60, 132, 135-136 stability of micro-organisms 100
class II 60, 132-137 standard operating procedure 232, 247
class III 60, 132, 137, 146,252 state regulatory bodies 51
laminar flow 137 steam 204, 250, 262
microbiological 132 autoclave 252
product dispensing 207 barrier 144, 246
safety standards 77 condensate 243, 262
sample assessment 286 sterilisation 138
sample valve 145 trace 222, 246
sampling devices 272 sterilisation 264
sampling efficiency 269, 284 dry heat 204
sampling methods 272 ga~eous biocides 200
biosafety equipment 268-292 steam 204
sampling systems 225 sterilisers 250
sanitising agents 199 stirred bioreactor 144
SAS see surface to air sampler stirrer shaft 144
scalding 110 stirrers 262
Science & Technology Agency 58-59 mechanical 265
guidelines 59-61 stoppering 186
302 INDEX
storage 180 vacine 38,62, 121, 182

freeze dried materials 190 vaccum pump 1<)8
sump tank 263 validation 145,248,256
sunlight, effect on micro-organisms 102 valves 245, 246, 265
surface contamination 96 butterfly 227-228
surface to air sampler 279, 290 diaphragm 227,263,265
survival 97 vegetative organisms 290
vent filter 263
tank venting 254, 265 vent stack 265
temperature, effect on micro-organisms ventilation 72
101-102 systems 84
terminal bronchioles 104 theory 97
thermal death 250 veterinary biologics 53
thermocouple 264 vials 185
tissue plasminogen activator 57, 62 oversealing 187
tomato 63-64 vibrating reed aerosol generator 92
toxic chemicals 110 viral insecticides 86
Toxic Substances Control Act 43, 48 viral vaccines 39
toxins 38,73,11<) virulence 105
tracer gas analysis 247 virus serum toxin act 3<)
transportable gas container regulations vitamins 61
23
Treaty of Rome 3, 5
trial releases 6 waste 72
triclover 223 liquid 234, 242
TSCA see Toxic Substances Control Act waste streams 6
turbulent diffusion 98 waste water 83, 95
type A operations 216 as fertiliser 95
type B operations 2 I 6 worker protection 2
worker related diseases 84 see also
ultrasonic 247 worker safety
ultrasonic disruption 170 worker safety 72, 77, 215 see also
ultraviolet irradiators 1<)5 worker related diseases
uncontrolled release 80,264 personnel 84
unit processes 130 pregnancy 84
United States 32 workplace release 78
urokinase 57
US Federal Register 32
USDA see Department of Agriculture Y ACs see also artificial chromosomes

Biosafety in Industrial Biotechnology

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Biosafety in Industrial Biotechnology

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

Typeset in 1O/12pt Times by Cambrian Typesetters, Frimley, Surrey

@ Printed on acid-free text paper, manufactured in accordance

As an industry, biotechnology may be likened to the Hymn Book, being

G.D.J. Adams Freeze Drying Research and Development Section,

A. Rimmington Centre for Russian and East European Studies, The

1 The development of European legislation on genetically

2 Occupational and environmental safety: the UK legislative

2.7 Advisory committees 24

3 Regulation of biotechnology in the United States, Canada,

4 The legal and regulatory framework for biotechnology in

4.1 An overview ofthe Japanese biotechnology industry 57

5 Biotechnology and industrial microbiology regulations

6 Physical aspects of the uncontrolled release of material in

7 Health hazards in biotechnology 109

7.1 Introduction 109

8 Containment of unit processes 129

8.1 Introduction 129

9 Containment in downstream processing 149

9.1 Introduction 149

10 Freeze-drying of biohazardous products 178

10.1 Introduction 178

10.3 Risk assessment 181

11 Interpretation of regulatory requirements to large scale

11.1 Introduction 213

12 Managing the emuent from bio-industrial processes 240

12.1 Introduction 240

13 Sampling methods for testing and monitoring biosafety of

13.1 Introduction 268

13.4.3 Filtration 284

1.2 The development of the EC directives

Biotechnology' in which it set out proposals for Community regulations.

1.3 The EC Directives on genetic modification

On 23 April 1990, the Council of Ministers adopted legislation in the form

States of the EC who were obliged to implement this Community

1.3.1 Directive 9012191 EEC - Contained use of genetically-modified

Table 1.1 Notification requirements of the Contained Use Directive

First use of installation

Subsequent uses of GMMOs

1.3.2 Directive 9012201 EEC - Deliberate release of genetically-modified

sites. Subsequent releases of a GMO are still to be subjected to separate

Should any disagreement take place, which cannot be resolved within

1.4.1 United Kingdom

1.4.2 Federal Republic of Germany

Handling of r-DNA'. These were binding on publicly-funded work and

Inspectorate for health and safety and the Environment Ministry.

1.4.7 Remaining EC Member States

1.5 Situation in the EFT A nations (as of February 1993)

(EEA), the principal benefit being an extension of the single trading

1.6 Europe vs. the USA

1993 was a watershed in the development of regulatory controls for

Biotechnology is an age-old process with its origins in processes such as

2.2 International influences

The approach taken to the assessment and control of industrial hazards is

biotechnology not being generally developed. The introduction of genetic

A third Directive on 'Biological Agents' covers work with all organisms,

2.2.1 The UK approach

1. Promote high standards of safety.

Figure 2.1 UK framework of legislation and guidance.

2.3 Occupational health and safety legislation

and provided a comprehensive piece of legislation where all the previous

2.4 The Health and Safety at Work Act

The aim of the Act is to: