» MICROBIOLOGY »

Pharmaceutical Facility Introduction

Sanitization:
Cleanrooms and clean areas must be regularly cleaned and disinfected.
This is normally undertaken using a detergent step, followed by the
application of a disinfectant. It may be necessary to remove the residue

Best Practices Considered of the disinfectant using water. Cleaning and disinfection should also
extend to equipment. Furthermore, with personnel, sanitization is
important in relation to glove hands.

This article reviews the key points to consider for the practical application
of a cleaning and validation program within a pharmaceutical facility,
with a focus on disinfectant selection.

Dr. Tim Sandle

Bio Products Laboratory
Detergents
For cleanrooms, detergents are required to remove ‘soil’ (protein, grease
and so on). Detergents penetrate soiling and reducing the surface
tension (which fixes the soil to the surface) to allow its removal. This is
necessary in order for the disinfectant to work effectively. Furthermore,
microorganisms in suspension are easy to remove with rinsing or kill
with the disinfectant.

For this task it is important to select an effective detergent. Here it is

important that the detergent selected will:

• Work with different types of water (e.g. ‘hard’ and ‘soft’ water)

• Be compatible with the disinfectant

• Not damage the surfaces

• Non-foaming

• Be effective against different soils e.g. grease, dirt, oil,

protein, rust, skin

In general, neutral detergents are ideal. Depending on the area of use,

such as aseptic filling areas, detergents may need to be sterile.

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Disinfection
Disinfection is either the inactivation or destruction of microorga-
nisms. Some disinfectants are bacteriostatic, others are bactericidal.
Importantly, disinfection is not the same as sterilization. Disinfection is
about a standardized reduction of microorganisms. There are several
types of disinfectants (or ‘sanitizers’) available, and they represent a
diverse group of chemical agents.
Types of Sanitizing Agents
A disinfectant is one of a diverse group of chemicals which reduces
the number of micro-organisms present (normally on an inanimate
object). Disinfectants kill vegetative micro-organisms but do not
necessarily kill bacterial spores. To be effective disinfectants must
meet either European standards (the CEN series) or US standards (the
AOAC standards). These standards involve challenging disinfectants
with high populations of a range of different microorganisms and
noting the log reduction over time. Such studies are undertaken for
the disinfectant solution (the ‘suspension test’), on surfaces and in ‘the
field’ (to develop appropriate cleaning frequencies).
Disinfectants vary in their spectrum of activity, modes of action and
efficacy. Some are bacteriostatic, where the ability of the bacterial
population to grow is halted. Here the disinfectant can cause
selective and reversible changes to cells by interacting with nucleic
acids, inhibiting enzymes or permeating into the cell wall. Once the
disinfectant is removed from contact with bacteria cells, the surviving
bacterial population could potentially grow. Other disinfectants
are bactericidal in that they destroy bacterial cells through different
mechanisms including causing structural damage to the cell; autolysis;
cell lysis and the leakage or coagulation of cytoplasm1.
There are many different types of disinfectants for use within the
pharmaceutical industry, with different spectrums of activity and
modes of action. The mechanisms of action are not always completely
known and continue to be investigated. A range of different factors
needs to be considered as part of the process of selection including
the mode of action, and also efficacy, compatibility, cost and with
reference to current health and safety standards2.
Surface disinfectants have varying modes of action against microbial
cells due to their chemical diversity. Different disinfectants target
different sites within the microbial cell. These include the cell wall,
the cytoplasmic membrane (where the matrix of phospholipids
and enzymes provide various targets) and the cytoplasm. Some
disinfectants, on entering the cell either by disruption of the
membrane or through diffusion, then proceed to act on intracellular
components. There are different approaches to the categorization and
sub-division of disinfectants, including grouping by chemical nature,
mode of activity or by bacteristatic and bactericidal effects on micro-
organisms3.
There are many different types of disinfectants and space does not
permit a list of all possible types. This guide describes some of the
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more commonly used types of disinfectants. Surface disinfectants can
be divided into:
Non-Oxidizing Disinfectants
Alcohols
The effectiveness of alcohols against vegetative bacteria and fungi
increases with their molecular weight (therefore ethanol is more
effective than methanol and in turn isopropyl alcohols are more
effective than ethanol). Alcohols act on the bacterial cell membrane
by making it permeable and efficacy is increased with the presence
of water leading to cytoplasm leakage, denaturation of protein and
eventual cell lysis The advantages of employing alcohols include a
relatively low cost, little odour and a quick evaporation4.
Aldehydes
Aldehydes include formaldehyde and glutaraldehyde. Aldehydes
have a non-specific effect in the denaturing of bacterial cell proteins
and can cause coagulation of cellular protein. There are some safety
concerns about the use of some aldehydes5.
Amphoterics
Amphoterics have both anionic and cationic character and possess
a relatively wide spectrum of activity, but they are limited by their
inability to damage endospores. Amphoterics are frequently used
as surface disinfectants. Examples include the alkyl di(aminoethyl)
glycine group of compounds.
Phenolics
Synthetic phenols are widely available such as the bis-phenols
(triclosan) and halophenols (chloroxylenol). Phenols are bactericidal
and antifungal, but are not effective against spores. Some phenols
cause bacterial cell damage through disruption of proton motive
force, while others attack the cell wall and cause leakage of cellular
components and protein denaturation.
Quaternary Ammonium Compounds (QACs)
QACs are cationic salts of organically substituted ammonium
compounds and have a fairly broad range of activity against micro-
organisms. They are ineffective against bacterial spores. QACs are
possibly the most widely used of the non-oxidizing disinfectants
within the pharmaceutical industry; examples include cetrimide and
benzalkonium chloride. Their mode of action is on the cell membrane
leading to cytoplasm leakage and cytoplasm coagulation through
interaction with phospholipids6.
Oxidizing Disinfectants
This group includes oxygen-releasing compounds (peroxygens) like
peracetic acid and hydrogen peroxide. They function by disrupting
the cell wall, causing cytoplasm leakage and denature bacterial cell
enzymes through oxidation. Oxidizing agents have advantages in that
they are clear and colorless, thereby avoiding surface staining.
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What Makes a Disinfectant Work Well
or Badly?
There are a number of factors that affect whether a selected
disinfectant works well or poorly7. These are briefly presented below:
1. Number of microorganisms: In general disinfectants are
more effective against a low number of microorganisms
than a higher number.
2. Types of microorganisms: Some microorganisms are
more resistant than others. Here Gram-positive bacteria
are generally easier to kill than Gram-negative bacteria;
vegetative bacteria are easier to kill than fungi; and
endospore forming bacteria are the hardest to kill (for
these a disinfectant classed as a sporicide is required)8.
3. Location of microorganisms: The key issue here is how
likely are microorganisms to be fixed to surfaces? The
degree of surface attachment can affect the removal and
destruction of organisms.
4. Contact time: This is the time taken for a disinfectant to kill
the microorganism and the time that the disinfectant must
be left in contact with the surface. This will typically be 1 to
5 minutes, although the time can only be assessed through
disinfectant efficacy testing9.
5. Disinfection concentration: Disinfectants are manufactured
or validated to be most effective at a set concentration
range (the proportion of the chemical to water). Over- or
under-dilution will lead to a loss of efficacy.
6. Temperature: The temperature at which the disinfectant
is used at influences the rate of reaction. In general, lower
temperatures, especially those below the threshold at
which the disinfectant has been assessed; mean that the
disinfectant may not work. This means that if disinfectants
are used in cold storage areas, they should be assessed to
see if they remain effective.
7. pH: Like temperature, extremes of pH can influence
disinfectant efficacy.
8. Soil: As discussed above, if soil is not effectively removed
this can either interfere with the disinfectant or prevent
the disinfectant from making contact with the microbial
cell wall.
9. Type of water: Hard water can be a problem. The water
used to prepare disinfectants in the production facility
should be incorporated into disinfectant efficacy studies.
How to Select the
Appropriate Disinfectant?
Taking the above ‘influencing factors’ into account, there are some key
criteria that should be considered when selecting a disinfectant. It is
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good practice to use these as a check-list when making a selection10.
The criteria are:
1. Wide spectrum of activity: This is the ability of the
disinfectant to kill different types of microorganisms.
2. Possibly a ‘sporicide’: This will depend on the application
and a review of the microorganisms recovered from the
environmental monitoring program.
3. Rapid action: Ideally a contact time of <5 minutes.
4. Compatible with a second disinfectant for rotation.
5. Work across a wide pH and temperature range
6. Detergent compatibility.
7. Compatibility with different surface types: Disinfectants
must not damage the material and problems of corrosion
and discoloration can arise. For more aggressive
disinfectants, a wipe down using water or an alcohol is
recommended11.
8. An assessment should be made to see if the disinfectant
leaves surface residues. This could lead to surface damage
or disinfectant/detergent computability issues. To avoid
this, a surface rinse may be required (such as with Water-
for-Injections or alcohol).
9. Sterility: If the disinfectant is to be used in aseptic filling
areas it must be capable of being rendered sterile without
loss of efficacy. Methods of sterilization include gamma
irradiation and filtration.
10. Format: The presentation of the disinfectant should be
considered. Is a ready-to-use concentrate, trigger spray or
saturated wipe format required?
11. Other considerations include operator health and safety
and the environmental impact.
Sanitization Regime
There are a number of important steps involved with respect to
cleaning and disinfection. These are:
Cleaning
Cleaning, in the context of pharmaceutical manufacturing, is the
process of removing residues and soil from surfaces to the extent that
they are visually clean. This involves defined methods of application
and often the use of a detergent. Detergents generally work by
penetrating soiling and reducing the surface tension (which fixes the
soil to the surface) to allow its removal.
For cleanrooms such cleaning steps are necessary prior to the
application of a disinfectant. It is essential that a surface or item of
equipment has been properly cleaned before the application of a
disinfectant in order for the disinfectant to work efficiently.
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Disinfection
Disinfectants are applied to surfaces which have been cleaned. When
applying a disinfectant, as previously discussed, the critical aspect is
the contact time. The disinfectant is only effective when left in contact
with the surface for the validated time. This can be achieved more
easily when the disinfectant is applied in overlapping strokes. When
rotation of disinfectants is required, a water rinse (normally employing
WFI) is employed between the change-over of disinfectants. This is in
order to remove traces of disinfectant and detergent residue (such as
anions) which may act to reduce the efficacy of the new disinfectant.
Cleaning and Disinfection Techniques
When cleaning rooms the equipment used (mops and buckets)
should be of an appropriate design for the grade of cleanroom. When
undertaking cleaning, a strict cleaning regime should be followed.
Cleaning and disinfection using cloths and mop heads is ideally
performed by saturating the cleaning item and wiping the area using a
series of parallel, overlapping strokes (with an approximate one quarter
overlap) and never in circular motions. The direction of the cleaning
should be towards the operator (from top to bottom, from back to
front). Only one application of the disinfectant or detergent should be
applied to avoid over concentration. Cleaning and disinfection should
begin with the visually ‘cleanest’ area first and towards the ‘dirtiest’ area
last. Cleaning is normally undertaken in each process area before use.
In general, the frequency of cleaning should be established through
risk assessment.
Rotation of Disinfectants
The scientific basis for disinfectant rotation, in terms of the build-
up of resistant strains is questionable on the basis that no reliable
evidence has been provided about the acquisition of resistance by
microorganisms. A more plausible argument for rotation comes from
the spectrum of activity that different biocides provide. Given, as
indicated above, that different disinfectants have different modes of
action and some are more effective against different microorganisms
than others, then the use of two disinfectants to ensure a greater range
of effectiveness against microorganisms is more logical. Whatever the
merits of the argument, the use of two disinfectants in rotation is a
common regulatory question and in Europe it is stated in Annex 1 of
EU GMP. The guidance states that “where disinfectants are used, more
than one type should be employed”.
Thus in selecting disinfectants many pharmaceutical manufacturers
will opt to have two ‘in-use’ disinfectants and sometimes to have a
third disinfectant as a reserve in case a major contamination incident
arises, such as a bioburden contamination build up, which appears
resistant or difficult to eliminate using the routinely used disinfectants.
The reserve disinfectant will often be more powerful and sporicidal,
such as an oxidizing agent, the routine use of which is restricted
because of likely damage to the equipment and premises. Typically
the two primary disinfectants are rotated.
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Cleaning and Disinfection Procedures
Cleaning and disinfection must be detailed in a Standard Operating
Procedure (SOP) to ensure consistency of practice. Furthermore,
sufficient detail in SOPs is important because detergents and
disinfectants are only partially effective if they are not applied
correctly12. An SOP should describe:
• The type of detergents and disinfectants to be used (which
are compatible).
• The frequency of rotation of disinfectants.
• A list of suitable cleaning materials.
• Cleaning techniques.
• Contact times.
• Rinsing.
• Frequency of cleaning and disinfection.
• Procedure for the transfer of cleaning agents and
disinfectants into and out of clean areas (including the
procedure for sterilization of disinfectants).
• Holding times for detergents and disinfectants
Including these essential standard operating procedures will ensure
that a cleaning and disinfection procedure can be applied consistently.
Equipment Sanitization
Effective cleaning and sanitization of equipment is important because
equipment may not be amenable to visual inspection and it may be
prone to biofilm formation.
The main method for cleaning industrial equipment is by making the
mechanism for cleaning integral to the equipment itself. This can be
achieved by use of pressure, heat, steam sterilization, mechanical
removal or chemicals, and is termed Clean-in-Place (CIP) or Steam-
in-Place (SIP). Prior to chemical or heat treatment attempts must be
made to remove process residues and particles using steam or high
pressure water cleaning. Alkali-based disinfectants and detergents
are commonly used for CIP systems, with sodium hydroxide among
the most widely used. Such caustic alkalis can readily remove organic
deposits without affecting the equipment. It is important that
equipment cleaning is validated.
Glove Sanitization
For staff undertaking critical activities gloved hands should be
sanitized on a frequent basis using an effective hand sanitizer.
Disinfected glove hands can aid staff who need to carry out aseptic
practices although the sanitizing agent itself is not a replacement for
poor aseptic technique.
There are many commercially available hand sanitizers with the
most commonly used types being alcohol-based gels. To ensure
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that the hand sanitizer selected is effective, within Europe there is a
standard describing the approach for their validation (EN 149913 and
EN 150025A14). The test determines if a hand sanitizer can reduce the
number of transient microflora under simulated practical conditions.
The standard outlines the approach for the evaluation of hygienic
handrubs. Most hand sanitizers are ethanol or iso-propanol alcohol
based15.
There are three key variables which affect the use of hand sanitizers.
These are the act of agitation and rubbing the hand sanitizer into the
glove, the frequency of application and the quantity applied16.
Summary
This article has considered sanitization in pharmaceutical facilities
and has sought to present best practices. The article has not so much
centered on the science of disinfection, but rather to offer practical
advice for those who need to use and select disinfectants and to put
them into effective use.
Ensuring that contamination is controlled in cleanrooms, on and
in equipment, and in relation to personnel is an important step in
maintaining microbial control of the production process and a focus
on the fabric and structure in which pharmaceutical products are
prepared is as important as bioburden reduction steps within the
product itself.
References
1. Sandle, T. (2014) A Practical Approach to the Selection of Cleanroom Disinfectants, Pharma
Focus Asia, Issue 21, pp27-30
2. Block, S. (1977) Disinfection, Sterilisation and Preservation, Third Edition, Lea and Febiger,
Philadelphia, pp10-15
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3. Denyer, S.P. and Stewart, G. (1998) Mechanisms of action of disinfectants, International
Biodeteriroration and Biodegradation, 41: 261-268
4. McDonnell, G. and Russell, A. (1999) Antiseptics and Disinfectants: Activity, Action and
Resistance, Clinical Microbiology Reviews, 147–179
5. Angelillo, I.F., Bianco, A., Nobile, C. and Pavia, M. (1998) Evaluation of the efficacy of
glutaraldehyde and peroxygen for disinfection of dental instruments, Letters in Applied
Microbiology, 27: 292–296
6. Bergan, T. and Lystad, A. (1972) Evaluation of Disinfectant Inactivators, Acta Path Microbiol
Scand Section B, 80: 507–510.
7. Sandle, T. (2012). ‘Cleaning and Disinfection’. In Sandle, T. (2012). The CDC Handbook: A
Guide to Cleaning and Disinfecting Cleanrooms, Grosvenor House Publishing: Surrey, UK,
pp1-31
8. Sandle, T. (2012). A new wave of sporicidal disinfectants, Clean Air and Containment
Review, Issue 10, pp10-13
9. Vina, P., Rubio, S. and Sandle, T. (2011): ‘Selection and Validation of Disinfectants’, in Saghee,
M.R., Sandle, T. and Tidswell, E.C. (Eds.) (2011): Microbiology and Sterility Assurance in
Pharmaceuticals and Medical Devices, New Delhi: Business Horizons, pp219-236
10. Sandle, T. (2012). ‘Application of Disinfectants and Detergents in the Pharmaceutical
Sector’. In Sandle, T. (2012). The CDC Handbook: A Guide to Cleaning and Disinfecting
Cleanrooms, Grosvenor House Publishing: Surrey, UK, pp168-197
11. Sandle, T. (2012). Practical Selection of Cleanroom Disinfectants, Hospital Pharmacy
Europe, Issue 63, pp39-41
12. Sandle, T. (2014) Selection and use of Cleaning and Disinfection Agents in Pharmaceutical
Manufacturing. In Handlon, G. and Sandle, T. (Eds.) Industrial Pharmaceutical Microbiology:
Standards & Controls, Euromed Communications, Passfield, UK, 9.1–9.32
13. EN 1499. 2015; Chemical disinfectants and antiseptics. Hygienic handwash. Test method
and requirements (phase 2/step 2)
14. EN 1500. 2015; Chemical Disinfectants – Quantitative Carrier Test to Evaluate the
Bactericidial Activity of a Hygenic Handrub Solution (Phase 2/2). Chemical disinfectants
and antiseptics. Hygienic handrub. Test method and requirements (phase 2/step 2)
15. Best, M. and Kennedy, M.E. (1992) Effectiveness of handwashing agents in eliminating
Staphylococcus aureus from gloved hands, Journal of Applied Bacteriology, 73: 63–66.
16. Larson E, Mayur K and Laughon B. (1989) Influence of two handwashing frequencies
on reduction in colonizing flora with three handwashing products used by health care
personnel, American Journal of Infection Control, 17(2): 83–88.
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