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Clarity on GMP
Guidance Note No. 2

Assuring sterility of indirect

product contact surfaces.
 Clarity on GMP
Guidance Note No. 2

Published by the Pharmaceutical and Healthcare Sciences Society

6a Kingsdown Orchard, Swindon, Wiltshire, SN2 7RR
© Pharmaceutical and Healthcare Sciences Society 2020
Cover design and typeset by Originzone
Date of Publication: June 2020
 Ensuring sterility of indirect
product contacting surfaces
in Aseptic process filling.
Revised issue to cover
New and Existing Filling lines.
This guidance article is the second in a series of PHSS guidance
notes prepared by industry subject matter experts and sterile
product manufacturing specialists to facilitate understanding and
interpretation of aspects of GMP Regulatory (Health Authority)
requirements for aseptic manufacturing of sterile medicinal products.

Container Closure Vibrating

Feeder Bowls and Hoppers
 Introduction
The focus of this guidance is on the assurance
of sterility of indirect product contact surfaces
that contact product contacting parts e.g.
product container closures/ stoppers contact
associated vibratory feeder bowls, hoppers
and chute/ trackway surfaces. Indirect product
contact surfaces provide an indirect route to
sterile medicinal product contamination via a
surface to surface transfer mechanism during
Aseptic process filling.
This guidance considers the role VHP®/vH2O2
as the principle bio-decontamination method
for Isolator Barrier technology applied to aseptic
process filling and the application in assuring
sterility of indirect product contact surfaces on
process equipment installed in the barrier.
Regulatory Blogs and observations have
considered VHP®/vH2O2 as a ‘Fragile process’
to make clear the concern about the lack of and
‘Fragility of’ scientific and process knowledge in
applying VHP®/vH2O2 as a bio-decontamination
process in aseptic process filling and other
related applications. Although the VHP®/
vH2O2 process is capable of achieving surface
sterilisation that renders impermeable surfaces
free of microbial contaminants, reported
as zero colony forming units (CFU), there is
limited efficacy in penetration so the process
is not a full penetrative sterilization process.
GMP Regulatory inspections have exposed a
lack of scientific and process knowledge on
VHP®/vH2O2 bio-decontamination leading
to over claiming efficacy and over reliance
on inactivation of biological indicators (BIs)
although poor application and bad practice are
evident and do not assure surface sterility of
target surfaces adjacent to BI challenges.
This PHSS guidance considers how penetration
limitations of VHP®/vH2O2 can be managed
in the process of assuring sterility of indirect
product contact surfaces hence applying
the best attributes of VHP®/vH2O2 as a bio-
decontamination process whilst managing the
limitations based on scientific understanding
and risk mitigation.
The phenomenon of ‘Rogue BIs’ as unexpected
positive growth in an overkill cycle underlines
the ‘fragility’ of VHP®/vH2O2 to penetrate
Biological indicator microbial challenges that
either have mass spore clumps (typically at edge
confluence) or have extraneous contamination
4 | Clarity on GMP Guidance Note No. 2
because of poor BI manufacture. If the target
indirect product contact surfaces does not have
adequate bioburden control before the VHP®/
vH2O2 bio-decontamination cycle, although
adjacent Biological indicators that have well
distributed ‘clean spores may indicate 6log
efficacy and surface sterilisation with overkill
applied surface sterility on target surfaces
cannot be assured and claims of surface sterility
cannot be considered valid.
Although greatly extended VHP®/vH2O2 bio-
decontamination cycles have demonstrated a
greater degree of penetration of bioburden. In
principle the aseptic processing cycle time for
the Isolator barrier technology and the in-direct
product contact parts should be the same
and qualified with the same 6log challenge in
sporicidal reduction on biological indicators
(inoculated carriers in a Tyvek® primary
pack). To align the bio-decontamination cycle
efficacy /time between the barrier surfaces and
indirect product contact surfaces it is therefore
fundamental to focus on bioburden control
before the VHP®/vH2O2 cycle.
It should also be recognised the penetration
limitations of VHP®/vH2O2 are also impacted by
protective layering of microorganisms via fatty
acids (from human skin contact) and silicon oil
(used in stopper treatments). Therefore, cleaning
validation and appropriate handling of all items
and surfaces is essential before applying VHP®/
vH2O2 to indirect product contact surfaces if
sterility is to be assured.
Sterility must be assured for both direct
product contact surfaces e.g. filling needles/
sterile fluid pathways and for indirect product
contact surfaces e.g. stopper bowls/ track
ways that present an indirect route of
contamination transfer to sterile products.
Assurance of sterility requires application of
a qualified sterilization process that delivers
overkill levels of efficacy, together with
contamination control in aseptic process
operator interactions during manufacturing
of sterile medicinal products. Correct
application of GMP technical and operational
contamination control measures is essential.
In Aseptic process filling during manufacture
of sterile medicinal medicines a risk-based
contamination control strategy (CCS) is
mandatory in GMP (revised Annex 1 requirement)
and should be applied considering quality risk
management (QRM) principles.
Scope of guidance
During aseptic process filling within Isolator barrier
technology to assure sterility of indirect product
contact surfaces it is recommended as good
manufacturing practice to combine a recognized
sterilization process, bioburden control and a final
in-place VHP®/vH2O2 Bio-decontamination cycle.
This approach should comprise of out-of-place
e.g. moist heat or dry heat sterilization of indirect
product contact parts with bioburden control to
prevent unacceptable recontamination in transfer
and set-up on the filling line followed by a VHP®/
vH2O2 bio-decontamination in-place within the
Isolator closed barrier. Set-up would be considered
with Isolator barrier door(s) open to the surrounding
Grade C/ ISO8 Cleanroom. Such an approach would
meet GMP requirements without further application
of QRM and alternative practice that would require
justification and supporting risk assessments.
It is appreciated that new filling lines at the
design phase can take on the full PHSS guidance
recommendations as design, software controls and
operating practices/ procedures can be adopted
from the project outset.
In contrast some existing filling lines may not be
possible to implement design changes and software
control changes so that full PHSS recommendations
can be implemented and other Quality Risk
Management aspects and approaches need to be
considered.
The focus of this guidance is not sterility assurance
for direct product contact parts e.g. filling
needles and sterile product fluid paths in aseptic
manufacturing, where sterilised surfaces should only
be exposed to Grade A environments inside barrier
systems (Isolators or Restricted Access Barrier
Systems [RABS]). However, due consideration
should be given to associated closed transfer
methods and protection via uni-directional airflow in
Grade A environments.
For example, product contact pre-sterilised filling
needles and associated product tubing should only
be exposed for aseptic process filling under Grade
A conditions inside the barrier Isolator zone. Entry
to the Grade A zone should be via a closed transfer
method with entry only after a qualified VHP®/vH2O2
bio-decontamination cycle when Grade A conditions
are established.
After entry into the barrier system protective uni-
directional airflow at Grade A conditions provide
a suitable environment for aseptic processing.
Alternatively, a product pathway with clean-in-place
(CIP) and sterilize-in-place (SIP) of the filling needles
may be applied in a barrier system, but only after
completion of a VHP®/vH2O2 cycle.
Examining the relative limitations and
advantages of hydrogen peroxide
vapor VHP®/vH2O2 in aseptic
processing – considering surface bio-
decontamination where zero CFU
recovery is required inside Grade A
environments
This guidance considers the strengths and
limitations of VHP®/vH2O2 relative to the penetration
limitations and impact on efficacy of high or
protected bio-burden on indirect product contact
surfaces. Guidance includes requirements for
contamination/ bioburden control after application
of a qualified sterilization process. Bioburden control
is required through subsequent sterilizer offload,
transfer in protective wrapping and staging in a
Grade C surrounding environment of an Isolator
barrier system together with aseptic assembly into
place before final closed barrier VHP®/vH2O2 bio-
decontamination in advance of aseptic processing.
Vibrating container closure feeder bowls, hoppers
and guide chutes within an aseptic processing
Isolator cannot be sterilized in place by a recognized
penetrative sterilization process typically applied to
equipment loads and porous loads.
VHP®/vH2O2 is a surface treatment process
and is not defined as a penetrative sterilization
process suitable for porous load sterilization so not
equivalent to recognised sterilization processes
such as moist heat, dry heat and gamma irradiation.
Other gaseous sterilization processes e.g. Ethylene
oxide (ETO) are applied with vacuum used in
the sterilization cycle to achieve the necessary
penetration into porous loads during the sterilization
cycle. VHP®/vH2O2 under well characterised and
controlled conditions can achieve an outcome with
surfaces free of CFU recovery required in aseptic
processing. Surface sterility qualified by zero CFU
recovery should not be confused with sterilization
and sterility assurance metrics that apply a much
higher level of penetrative sterilization.
Achieving surface sterility requires surfaces that are
impermeable hence not harbouring microorganisms
in material structure/ matrixes that may be protected
from VHP®/vH2O2 exposure and subsequent bio-
decontamination. Impermeable surfaces need to be
cleanable, both in terms of access e.g. crevice free
and ability to be cleaned with a recognised cleaning
agent and/or process. Residue removal is important.
For indirect product contact surface cleaning
needs to be qualified to reduce bioburden to
specified levels before the VHP®/vH2O2 bio-
decontamination process and also assure protective
residues from cleaning agents and extraneous
protective contaminants such as fatty acids and
Clarity on GMP Guidance Note No. 2 | 5
 silicon oil are removed. For reference non product
contact surfaces of the Isolator barrier and process
equipment also require cleaning before a VHP®/
vH2O2 cycle, but it is generally considered ‘visibly
clean’ is acceptable based on the surfaces have no
greater levels of microbial contamination than the
Grade C Cleanroom surround.
As stated for aseptic process filling within an Isolator
to provide the necessary assurance of sterility for
container closure e.g. stoppers contact surfaces
a contamination control strategy is required.
Regulatory expectation and best practice combines
an out-of-place sterilisation step, in-process
transfers with bioburden control and a final in-place
VHP®/vH2O2 cycle and it is recommended such an
approach applies to new filling lines.
Procedural in-process transfer steps require
contamination risk mitigation. Handling of sterilized
and wrapped equipment would include staging in
a Grade C cleanroom and aseptic assembly into
the Isolator barrier as a final set-up step through an
open barrier door before the closed barrier VHP®/
vH2O2 cycle.
BI Primary Pack
Considering the following evidence, it is
demonstrable that Biological indicator (BI)
inactivation at 6log sporicidal overkill levels of
efficacy via a VHP®/vH2O2 bio-decontamination
process applied in a barrier technology Isolator
cannot act as a proxy to assure surface sterility of
indirect product contact surfaces adjacent to the
BI. It has been shown that although BIs with a well
distributed monolayer of spores may be inactivated
at over 6log sporicidal reduction levels, and in
principle surface sterilisation may be claimed,
6 | Clarity on GMP Guidance Note No. 2
adjacent contaminated surfaces that are unsuitable
for penetration of VHP®/vH2O2 or have inadequate
exposure to process lethal conditions are not
necessarily rendered sterile – hence invalidating any
claims of surface sterility.
So-called Rogue BIs with mass spore layers, spore
clumps or contaminated BIs that demonstrate
unexpected positive growth in a qualified VHP®/
vH2O2 cycle demonstrate the penetration limitations
of VHP®/vH2O2 and emphasise the importance of
pre-cleaning surfaces (protective soiling removal)
and pre-cycle bioburden control.
Although qualification studies with no growth of
Geo Bacillus stearothermophilus spore BIs in TSB
media incubated at 55-60ºC for a 7-day duration
together with extrapolations from a death kinetics
survivor curve of 12 log sporicidal reduction indicates
sterilization conditions have been reached in the
sample tested, adjacent surfaces may not be sterile.
One important consideration is that indirect
product contact surfaces should be suitable for a
surface bio-decontamination treatment process
based on gas or vapor application e.g. non-porous
surfaces that do not occlude microorganisms in a
porous matrix-structure, with very low bioburden
and without mass microbial clumping that VHP®/
vH2O2 cannot or has limitations to fully penetrate.
Additionally, ensure there are no possible protective
contaminants like fatty acids from human skin
contact or silicon oil that can occlude VHP®/vH2O2
from effective bio-decontamination.
Claiming VHP®/vH2O2 as a sterilisation process
based on inactivation of BIs with well distributed
spores alone without an understanding of the
limitations of VHP®/vH2O2 has been challenged by
GMP Regulatory compliance as a ‘fragile process’
and can lead to failure to meet QRM principles where
process, science and risk knowledge are required.
VHP®/vH2O2 process lethality limitations as a result
of process variables needs to be managed based
on a good understanding of science and application
with allowance of variables in cycle development
and when applying overkill factors. Qualified
VHP®/vH2O2 cycles should support assurance of
sterility for in-direct product contact surfaces in the
same cycle used for bio-decontamination of the
Isolator barrier and enclosed process (non-product
contacting) equipment surfaces.
The impact on VHP®/vH2O2 cycle efficacy
from critical process variables such as starting
relative humidity and temperatures (surface and
environmental) should be based on science and
characterised as starting control parameters. VHP®/
vH2O2 is a condensable vapor and not a true gas
with flash evaporated molecules subject to hydrogen
bonding that can impact gas-molecule distribution,
thus water molecules in the environment at cycle
start can be a barrier to vH2O2 molecule distribution
via inherent localized bonding or incoming molecules
being attracted to preferential sites where surface
condensate layers have formed. It follows that
starting relative humidity has impact on both reaching
process lethal conditions and gas distribution.
Smoke visualisation aerosol droplets will not be
impacted by hydrogen bonding hence where
distribution is passive the kinetics between smoke
particle-droplets and VHP®/vH2O2 molecules will be
very different.
For active gas distribution achieved by distribution
devices e.g. injection nozzles of fans studies smoke
visualisation does play a role in study but full
gas distribution studies should include chemical
indicators that indicate the arrival of the vapor at
target locations.
Biological indicators are then used as a challenge in
the same risk assessed locations to verify process
lethal conditions are reached for cycle qualification.
Exposure of target surfaces to VHP®/vH2O2
is essential to any claim of surface bio-
decontamination. Exposure relates to prevention
of occlusion (touching surfaces) and process lethal
vapor reaching the target location/ surfaces. Dead-
legs, gaps and grooves may be at risk of VHP®/
vH2O2 exposure. Typically dead-legs for VHP®/
vH2O2 assured VHP®/vH2O2 efficacy are in the order
of one to two diameters in relative terms.
There are regulatory observations where BIs are
well exposed and bio-decontamination claimed at
6log sporicidal efficacy but target surfaces either
are not exposed e.g. hanging load items touching
or complex machine surfaces provide ‘dead leg’
challenges to gas distribution so lacking assurance
of VHP®/vH2O2 exposure.
It follows both bioburden control and control of
process variables are required to provide the
full assurance that the VHP®/vH2O2 cycle meets
specified claims of bio-decontamination and
supports assurance of sterility for indirect product
contact surfaces.
Other considerations where enzyme indicators (EIs)
are proposed as a proxy for biological indicators
would require correlation studies between BIs and
EIs for a given process, to provide comparability
data that demonstrates EI efficacy and inherent
variability are comparable or better than BIs.
However, VHP®/vH2O ‘fragility’ is about the
limitations and robustness of the process to bio-
decontaminate mass or protected contaminants
of process equipment surfaces and not just about
the challenges in qualification of VHP®/vH2O cycle
efficacy via studies using challenge indicators,
whether BIs or EIs.
Due consideration must be given to the penetration
limitations of VHP®/vH2O2 in bio-decontamination
of indirect product contact surfaces of processing
equipment e.g. stopper transport pathways, that are
required to be sterile and free from CFU recovery in
Grade A zones.
During aseptic processing transfers of out-of-place
sterilised wrapped parts through Grade C areas and
associated in-place assembly set-up operations it
essential to consider sterile wrapped surfaces may
be at risk of bio-contamination through in-process
transfer steps and associated handling procedures.
Bio-contamination may in turn be protected by
subsequent process soiling which can act as a
barrier to hydrogen peroxide vapor efficacy e.g.
fatty acids from skin contact, including gloved hands
touching skin then contacting surfaces.
In addition, poor set-up of loads e.g. with surfaces
touching each (creating occluded surfaces) would
prevent exposure to VHP®/vH2O2 and together these
risks impact overall efficacy and robustness of the
bio-contamination control status.
Load hanging point contact support hooks that
are free to move in set-up procedures are not
considered as suitable for good contamination risk
management, as operator error could result in load
surfaces touching each other resulting in post VHP®/
vH2O cycle bio-contamination on occluded surfaces
and then contamination spread via gloved operator
handling in the Isolator Grade A zone.
Adjustable hooks must be fixed after set up so that
load items do not accidentally touch. Adjustable
hooks should be designed not to present un-
cleanable crevices or VHP®/vH2O2 dead-leg
pathways in fixing areas.
One of the greatest bio-contamination risks in
Isolator barrier technology is non exposed surfaces
to VHP®/vH2O2 bio-decontamination that as a result
present a risk of contamination during process
operations e.g. during contaminated load packaging
removal barrier gloves become contaminated with
contamination transfer to other surfaces that may
have a higher impact of contamination control and
sterile product contamination.
Vaporised molecules of hydrogen peroxide can be
delivered to target volumes for exposure and to
target surfaces in different environmental control
system configurations.
Application for surface bio-decontamination can
be achieved in different ways to achieve process
lethality where the oxidising potential and free
radical attack mechanisms apply. Hydrogen peroxide
vapour molecules that are generated by flash
evaporation are at Pico 10-12 size and small enough
to pass through HEPA filter media and Tyvek®
Clarity on GMP Guidance Note No. 2 | 7
 primary packaging of biological indicators. Such
a direct mechanism of vapour molecule transfer
through filter media is in variance to dry fog aerosol
droplets that are produced by pressurising a liquid
disinfectant through a nozzle producing droplets
that are orders of magnitude larger (micron size) than
vapour molecules hence micron size droplets cannot
pass directly through filter media.
As Dry fog aerosol droplets cannot pass directly
through filter media (HEPA or Tyvek) it may be
assumed Tyvek surfaces must become wetted
with a subsequent ‘wicking’ effect to pass H2O2
through to the biological indicator carrier for rapid
spore inactivation. Secondary in-direct evaporation
of wetted surfaces may assist Dry fog to become
effective but different vapour pressures between
water and H2O2 may impact as another process
challenge to consider – so not all processes that
apply H2O2 are equal or comparable.
Flash evaporated VHP®/vH2O2 molecules (pico size)
that directly pass through filter media also have
the ability to form around point contact supports to
demonstrate full load bio-decontamination. Note:
Point contact load support bio-decontamination is
typically qualified by swabbing after the VHP®/vH2O2
cycle with zero CFU recovery expected.
The contamination control strategy (CCS): EU GMP
Annex 1 requirement should include a justification
for the method of high level sporicidal disinfection
and bio-decontamination using H2O2 agents.
For processes that achieve saturated vapor
conditions, past dew point, the formation of H2O2
deposition layers (typically 2–6 micron is adequate
for 6log+ sporicidal reduction on surfaces) provides
a dynamic process with constant exchange of
molecules from the surface to gas volume.
The dynamic molecule exchange of VHP®/vH2O2
maintains efficacy on surfaces over a qualified
cycle phase time and varies from disinfection
processes that apply a static layer of disinfectant
for a contact time where other factors of varying
modes of action, reducing efficacy in evaporation
and reduction of contact time and possible residues
will need consideration.
The greatest strengths of VHP®/vH2O2 are its broad
spectrum efficacy, including sporicidal efficacy
and virus inactivation capability (via free radical
attack of virus DNA); plus, that it is safely broken
down to components of oxygen and water. This
means it is environmentally friendly with easy to
remove gas residuals via dilution or catalytic filter
cells. Such an agent also complies with biocide
directive regulations making it a widely applied bio-
decontamination process with years of development
knowledge and GMP compliance history in the
pharmaceutical industry.
8 | Clarity on GMP Guidance Note No. 2
Fragility of VHP®/vH2O2 should be put into context
as based on good science, knowledge and
management of penetration and exposure limitations
VHP®/vH2O2 efficacy can be robust for surface bio-
decontamination at 6log sporicidal efficacy.
Once the limitations are understood VHP®/
vH2O2 can be applied to many applications in
the pharmaceutical industry including: material
transfers, barrier system bio-decontamination with
associated indirect product contact surfaces bio-
decontamination in aseptic process filling. VHP®/
vH2O2 can also be applied to cleanroom and
process room gaseous disinfection in application
of bioburden control, rapid (and qualified with BIs)
return to control conditions after shutdown and in
applications of environmental virus clearance. For
room disinfection reduced efficacy, 3-4log may apply.
Application of VHP®/vH2O2 as a process of bio-
decontamination in Aseptic processing
It is important to understand the science behind the
bio-decontamination process applied in GMP aseptic
processing applications. Not all bio-decontamination
processes are the same and may be impacted by
different process variables, with specific limitations
that need to be understood and risks managed via
control measures. Deviations from the specified
control conditions via inherent and process variability
during process operations should be characterised
to enable overkill and detectability to be applied for
robust bio contamination control.
The qualified VHP®/vH2O2 cycle used to render
indirect product contact surfaces free of CFU
recovery in a Grade A aseptic processing
environment after a sterilization process e.g. moist
heat-autoclaving or dry heat sterilisation/ transfer/
staging/ aseptic assembly process into an Isolator
should be justified via a risk based approach with
consideration to science, process integration, impact
from process variables, inherent contamination
penetration limitations of VHP®/vH2O2 and surface
exposure for bio-decontamination.
Knowledge of VHP®/vH2O2 bio-decontamination
process and science is a key part of QRM with
training expected to cover:
Mode of action, characteristics of applied H2O2
bio-decontamination process, critical quality
attributes (CQAs) and critical process parameters
(CPPs), process variables and their impact on
efficacy, qualification of cycle via biological indicator
challenges (BIs) for the given application (and EIs if
correlated and accepted by regulatory authorities),
Bio-compatibility of VHP®/vH2O2 cycle residuals with
biological products (if filled), material compatibility
and absorption/ desorption effects, VHP®/vH2O2
cycle development and qualification/ periodic re-
qualification methodology.
For process operations training the required
practices and procedures for set-up, load
placements, cycle start and transition in production
modes of operation should be covered.
VHP®/vH2O2 safety in process operations should be
risk assessed with technical and procedural control
measures applied. Safety should be included in
training to make clear to operators what risks are
controlled by the system/technology and what risks
require procedural steps to apply risk mitigation.
Cycle records and cycle validity checks are required
for GMP compliance. Validity checking parameters
should be justified and specified with independent
review from operators.
Bio-contamination risks and risk
mitigation for assurance of sterility of
indirect product contacting surfaces
enclosed in an aseptic processing
Isolator – applies to new filling lines
The transfer of wrapped moist or dry-heat sterilized
parts through cleanrooms to a Grade C cleanroom
where an Isolator barrier is installed with continued
handling through set-up assembly into the Isolator
with an open barrier door aseptic assembly
procedure is not without contamination risks, so risk
mitigation is required.
Without the assurance that product contact sterilised
surfaces e.g. inside a feeder bowl or chute transport
surfaces remain sterile through all handling/ transfer
steps it is necessary that VHP®/vH2O2 is applied as
a final complementary bio-decontamination step
with 6log sporicidal activity. Such a step provides an
overkill relative to low levels of bio-burden to render
surfaces free of CFU recovery inside the Grade A
controlled aseptic processing environment.
Target surfaces should be exposed to the full
process lethality of the qualified VHP®/vH2O2 cycle;
it is recommended any Tyvek®/ bio-barrier covering
used to protect sterilized surfaces in transfer and
assembly through an open barrier door should be
removed before the VHP®/vH2O2 cycle.
Also, with the hydrogen bonding characteristics of
VHP®/vH2O2 molecules combine once inside (under)
the Tyvek® cover, full aeration will be compromised
in removing bonded molecules and deposition layers
through the filter media. VHP®/vH2O2 residuals
will remain under the Tyvek® until the covering is
removed.
The removal of the protective covering through the
open barrier door should be the final set-up step
before the barrier door is closed and the VHP®/
vH2O2 cycle is run.
Contamination risk mitigation measures, technical
and procedural, would be required as soon as the
protective covering is removed to exclude as far
as possible extraneous contamination on sterilized
surfaces.
Unavoidable bio-contamination before the VHP®/
vH2O2 cycle should be maintained at very low
levels of bioburden to assure process lethality is not
compromised and post-VHP®/vH2O2 cycle indirect
product contact surfaces are free of CFU recovery.
Such protective bio-shield/ bio-barrier packaging
can also create particle generation in handling
(to reduce size for waste removal through closed
transfer pathways e.g. Rapid transfer α-β port)
so the recommended practice is to remove the
covering via an open barrier door where subsequent
compression of the packaging for waste handling is
outside the Grade A environment.
Once the protective Tyvek® covering is removed
via the open Isolator barrier door the sterilized
surfaces are open to contamination exposure (both
particle and microbial) so the barrier door should be
closed as soon as the set-up process steps permit
after protective covering removal – set-up steps
should be risk assessed, monitored and subjected to
process simulation/ media fill studies.
Contamination risk mitigation steps are required
to protect as far as possible sterilized surfaces and
maintain unavoidable extraneous bioburden, as a
result of handling procedures, at an extremely low
level before the pre-aseptic processing VHP®/vH2O2
cycle:
PHSS guidance suggests;
• Less than 10 CFU bio-burden and without atypical
microflora.
• Elimination of protective contaminants through
good handling practices and good aseptic
technique.
Typical microflora should be characterised
in qualification studies and trended through
environmental monitoring.
Clarity on GMP Guidance Note No. 2 | 9
 Contamination risk mitigation steps
for the aseptic assembly procedure
on installation of wrapped sterilized
indirect product contact parts into the
Isolator barrier are recommended as:
o Additional gowning: mask and eye covering (or full
face covering) and sterile Tyvek® long sleeves to
slip-on over grade C gowning to reduce risks of
bioburden contamination into the Isolator Grade A
zone during open barrier door set-up procedures
where operators have to reach and lean into the
Isolator Grade A process zone. After the barrier
door is closed for the VHP®/vH2O2 cycle additional
gowning applied in set-up of the Isolators can be
removed and gowning reverted to that required
for Grade C cleanrooms and Isolator aseptic
processing operations.
o At each entry into the open barrier door during
aseptic assembly set-up procedures gloved hands
should be applied with a disinfectant to control
bio-burden and extraneous contamination.
o Isolator down-flow uni-directional airflow in
operation with open-door outward flow to
surround. Airflow should be characterised by
visualisation studies to verify protective attributes
are provided. Airflow protection in the Isolator
is only required to be characterised by smoke
studies over the parts being installed with lower
level airflow patterns not necessarily providing
full protection from exchange with the Grade C
Cleanroom environment.
The approach for set-up of sterilized parts
in Isolators is in difference to expectations
required for Open-RABS where aseptic assembly
of sterilized parts requires full aerodynamic
protection of sterilized parts and the Grade A
process environment as there is typically no
subsequent VHP®/vH2O2 in Open RABS. In the
case of Open-RABS outward flowing protective
airflow from the Grade A zone through the open
barrier door would be expected together with a
Localised Uni-directional airflow protection zone
outside the barrier to cover the open barrier door
and where the operator stands for the set-up
aseptic assembly procedure. In the case of RABS
sterility of in-direct product contact parts should
be maintained and assured without compromise.
o Door access control, only barrier door(s) that is/are
defined for the procedure are open.
o Bioburden characterisation and qualification
studies are completed on indirect product contact
sterilized surfaces to verify less than 10 CFU
bioburden before the VHP®/vH2O2 cycle. Any
bioburden comprises typical microflora in the
cleanroom transfer pathway from the sterilizer
and does not include atypical microflora or
10 | Clarity on GMP Guidance Note No. 2
objectionable or harmful microorganisms e.g.
Gram negatives/ positives (that are endotoxin risk),
fungi/moulds/yeast and bacterial spores.
o The operation of the Isolator HVAC Air-flow
management systems to provide protective air
during open barrier door set-up procedures
will require a software configuration to disable
pressure alarms when the barrier door is open.
o If more than one barrier door is required to be
open during the set-up procedure protective
airflow characterization studies should cover open
doors or alternatively doors opened in sequence,
reflecting the normal/ worst-case operating
procedure.
Case study New filling lines: (not
mandatory) the following recommended
process steps should be applied for
assurance of sterility of indirect product
contact surfaces of container closure
transport systems: Feeder bowls,
hoppers, chutes, trackways.
For a given application QRM would apply together
with specifying assurance of sterility control
measures in an associated contamination control
strategy (CCS).
1. Clean indirect product contact parts out-of-place,
ideally using an automated cleaning process.
2. Autoclave/ moist heat or dry heat sterilize
container closure contact surfaces e.g. stopper
feed surfaces in protective wrapping e.g. Tyvek®
coverings for moist heat sterilization. The moist
heat sterilization cycle should result in dry
surfaces after the full sterilization and cooling
cycle. The applied protective covering should
as a minimum cover the surfaces that should be
sterile in aseptic processing operations e.g. inside
of feeder bowl, chute container closure transfer
surfaces.
Applied coverings should allow in-place
mechanical assembly/ fixing to process machines
inside the Isolator Grade A zone without full
protective covering removal until all the set-
up assembly procedures are completed. Such
wrapping may include a bowl ‘bonnet’ and over-
wrap.
If the sterilized-wrapped parts hold or staging
time in the Grade C area or in-process transfer
paths require movements through a number of
areas with extended pathways there is a greater
risk of contamination and further risk mitigation
should be considered. It may be necessary to
consider double wrapping of parts for moist
heat-autoclave or dry heat sterilization with the
 secondary wrapping removed after hold/staging
and before the primary wrapped/covered parts
are assembled into the Isolator (pre-VHP®/vH2O2
cycle set-up procedure). The validation of the
seals of the double wrapped items should be
confirmed during the qualification of the loads to
ensure all packaging remains integral.
3. Transfer sterilised/ wrapped equipment to the
Grade C cleanroom where the Isolator is installed.
The transfer path, time and any hold conditions
or staging time (pre-entry to Isolator) should be
qualified so that sterilised parts remain protected
as far as possible from extraneous contamination.
Staging/ holding of wrapped-sterilized parts
should not be at floor level or in areas where
contamination levels put the items at risk of
extraneous contamination.
4. Assemble single wrapped/ covered sterilised
parts into the Isolator onto the process machine
with an open barrier door(s) intervention and
apply contamination risk mitigation control
measures.
5. At the last open barrier door set-up procedure
remove the primary Tyvek®/ protective coverings
through the open barrier door and close the
barrier door ready for a VHP®/vH2O2 cycle.
6. Operate a qualified and repeatable 6log+ overkill
sporicidal VHP®/vH2O2 bio-decontamination cycle
with aeration to target cycle endpoint (typically
less than 1ppm if processing biological products
that are highly sensitive to free radical attack and
oxidation at low levels of gaseous residuals) as an
automatic bio-decontamination cycle with cycle/
batch record.
7. Respect Isolator ‘First air’ principles and good
aseptic technique in barrier aseptic process
operations without any barrier glove contact
to surfaces that have requirements of assured
sterility – both product contacting parts e.g. filling
needles, containers closures and in-direct product
contact surfaces e.g. Container closure transport
surfaces, inside stopper bowls etc.
During aseptic processing barrier operations
if barrier gloves come into contact with direct
or indirect product contact surfaces the event
should be reported, documented and assessed
with appropriate actions taken relative to
contamination risks and product impact.
Product contact surfaces including product fluid
path and filling needles should be subjected to
a recognised and qualified sterilization process
and only exposed to Grade A aseptic processing
environments e.g. after a subsequent qualified
VHP®/vH2O2 bio-decontamination cycle.
Sterilized parts such as single-use filling needles
should only be introduced into the Grade A
controlled environment after the completion of a
valid VHP®/vH2O2 cycle.
Non-product contact surfaces of process machinery
inside the Grade A zone of the Isolator, including
outside surfaces of stopper bowls, are bio-
decontaminated together with the internal Isolator
barrier surfaces and indirect product contact parts
via an automated VHP®/vH2O2 bio-decontamination
cycle qualified to meet greater than 6log sporicidal
efficacy.
For non-product contact surfaces pre-VHP®/vH2O2
cycle bioburden control is applied as a function of
pre-cleaning. Such surfaces do not need assurance
of sterility in transfer or during set-up and assembly
procedures, but as the surfaces are to be enclosed in
the Grade A environment during aseptic processing
any environmental monitoring surface sampling
should result in zero (0) CFU recovery.
Recommended contamination risk
mitigation steps for bioburden control
before operation of a VHP®/vH2O2
cycle applied to case study for new
filling lines
Bioburden should be characterized and controlled
throughout the in-process transfer and aseptic
assembly steps. Before the VHP®/vH2O2 cycle is
operated and directly after the Tyvek/ protective
covering is removed, and barrier door(s) closed
bioburden studies should be completed on the
container closure contact surfaces e.g. inside
surfaces of stopper bowl, on trackways and chute
(upper) transport surfaces.
1. The Isolator HVAC; Airflow management system
delivering uni-directional down-flow air at
velocities around 0.45 m/s (± 20%) should be
active during the open barrier door intervention.
Pressure control alarms need disabling during the
open-door intervention with airflow operational.
Outward airflow from the open barrier door to
the surrounding Grade C cleanroom should be
qualified and maintained during the inherent set-
up intervention aseptic assembly procedure.
2. Barrier doors should have access control, with
open doors recorded during aseptic processing
set-up operations. Only the door(s) that are
required to complete the qualified aseptic
assembly process should be opened to carry out
the procedure.
Clarity on GMP Guidance Note No. 2 | 11
 3. For the open barrier door aseptic assembly
procedure additional gowning should be applied
including (but not limited to);
• Face mask, beard covering, goggles or full face
covering
• Long disposable sterile Tyvek® sleeves added
to gowning or change to full sterile suit.
4. Hand-Glove disinfection procedures should be
applied at each entry into the open barrier door
Grade A zone during the aseptic assembly set-up
steps before the closed barrier VHP®/vH2O2 cycle.
5. The open barrier door assembly procedure
should follow good aseptic technique practices
to minimise the risk of bioburden contamination
transfer that may compromise assurance of
sterility of the container closure indirect product
contact surfaces.
New filling lines are considered those that are at the
start of the design phase where barrier and process
equipment design together with control software
can be specified to accommodate the requirements
for a combined Out-of-Place sterilization, Bioburden
control and VHP®/vH2O2 cycle.
Filling line projects can take a number of years to
execute so a line not through qualification but where
the design was completed much earlier in the project
may not be considered a new filling line suitable to
meet the full PHSS recommendations.
Point to consider for Existing filling lines
Existing filling lines may not be able to follow the full
guidance provided for new filling lines as process
equipment and barrier design change may not be
possible and associated software changes also
not practical with adverse impact or production
operations (continuity of medicines supply) or added
(not reduced) risk.
The detailed barrier and equipment design and/or
procedural steps recommended for new filling lines
should be risk assessed on impact of assurance
of sterility of indirect product contact parts if an
alternative approach with risk mitigations is applied
that follows QRM principles.
Points to consider where VHP®/vH2O2 is considered
the sole method of surface sterilization of indirect
product contact surfaces
Some existing filling lines have been specified and
designed from the outset that the method of surface
sterilisation of the indirect product contact parts is
VHP®/vH2O2 only without application of an out-of-
place sterilization process. Following QRM principles
such an approach is not considered best practice
(as applied to new filling lines) but it is recognised
12 | Clarity on GMP Guidance Note No. 2
alternative QRM approaches with supporting
rationale and where necessary data based evidence
with supporting risks assessments may need to be
applied to facilitate continued filling line use. In all
cases where alternative approaches are applied
based on QRM principles they should have the
outcome without added risk to sterile product
quality, efficacy and patient safety whilst meeting or
exceeding GMP requirements.
Where possible if an out-of-place sterilization
process cannot be applied parts cleaning should
applied in a controlled cleaning environment, using
qualified procedures and where practical with
automated cleaning equipment (parts washers). Out
of place ‘Deep’ cleaning may be applied periodically
with in-place cleaning applied in batch production if
removal of parts is not possible (by design) or impact
on production operations by removal of parts is
considered an added risk.
For a VHP®/vH2O2 only approach cleaning should
be considered a separate qualification to bioburden
reduction (disinfection in-place) and VHP®/vH2O2
bio-decontamination qualification.
Cleaning of indirect product contact surfaces
should have the objective of physical removal of
contaminants on surfaces including environmental,
human commensals and process contaminants e.g.
silicon oil. In addition the final cleaning step should
be one of qualified residue removal to remove any
residues of cleaning detergent/ disinfection agents
applied.
If cleaning has to be completed in-place because
process equipment indirect product contact parts
cannot be removed from the filling line a full risk
assessment is required to justify this practice with
consideration given to cleaning accessibility of all
in-direct product contact surfaces and subsequent
qualification of residue removal.
Following cleaning the next step is bioburden
reduction to below the target of CFU recovery
specified for new filling lines ahead of a VHP®/
vH2O2 cycle e.g. below 10 CFU. Although cleaning
will reduce bioburden this step is should only
be qualified for physical clearance of surface
contaminants that include viable and non-viable
contamination including residues from cleaning
agents.
Bioburden reduction in-place on the filling line would
be considered a manual disinfection step applied
to the indirect product contact surfaces e.g. inside
surfaces of a stopper/ syringe plug feeder bowl
or container closure transfer track way to point of
insertion with an alcohol based disinfectant suitable
(as the subsequent VHP®/vH2O2 cycle is sporicidal).
The application of an alcohol based disinfectant
supports the residue free requirement before the
VHP®/vH2O2 cycle.
Qualification of bioburden reduction should consider
surface sampling of indirect product contact surfaces
just before a VHP®/vH2O2 cycle would be applied
and all open barrier door interventions and set-up
operations completed.
Data based evidence is required that bioburden
reduction routinely meets specified requirements
and the surfaces are free of protective residues that
would impact the VHP®/vH2O2 cycle efficacy and
assured surface sterility.
As cleaning and disinfection (bioburden reduction)
are qualified separately and the combined process
steps are primarily based on manual application
of cleaning and disinfectant agents it will be
necessary to provide data based evidence that such
procedures are repeatable and deliver the required
residue/ contaminant free surfaces and bioburden
level (below 10 CFU) to assure the VHP®/vH2O2
cycle supports the assure of zero CFU recovery
(surface sterility) for the indirect product contact
parts.
It is recommended that Regulators/ GMP Inspectors
and company GMP auditors who assess current
GMP compliance of the Filling line are consulted
with to discuss the application of VHP®/vH2O2
only as the method of surface sterilization of
indirect product contact parts with associated
contamination control and risk mitigations as
such an approach may be considered a deviation
from regulatory expectation and QRM requires
both interpretation and supporting science based
rationales and risk assessments.
Recommended application of this
guidance
This guidance applies only to Aseptic processing
Isolator operations with VHP®/vH2O2 bio-
decontamination and with a surrounding
environment for the Isolator of Grade C.
RABS: Restricted Access Barrier systems used
in aseptic process filling are subject to separate
guidance with contamination risk mitigation and risk
control measures applied relative to contamination
risks in a Grade B surrounding area, but in principle
the following applies:
Open design RABS may be specified and qualified
with manual disinfection of the RABS barrier and
non-product contact surfaces of enclosed process
equipment where VHP®/vH2O2 is not applied.
In this case the surrounding environment is required
to be Grade B for GMP compliance and sterility
assurance is applied via out-of-place sterilization
processes for direct and indirect product contact
parts. Assurance of sterility is maintained through
qualified open-door set-up operator interventions
including contamination risk mitigation and control
measures. Such operator interventions should be
the subject of study to verify contamination control
measures are effective in process simulation trials
and media fills.
Closed design RABS that employ VHP®/vH2O2 bio-
decontamination of the RABS barrier and enclosed
process equipment would also require a Grade B
surrounding environment hence bioburden is already
low. In this case QRM can be applied with VHP®/
vH2O2 bio-decontamination of indirect product
contact parts as a sole method to achieve surface
conditions with zero CFU recovery, if risks are higher
with sterilization out-of-place and transfers through
Grade C/B areas into place. Such an approach
must be justified, risk assessed and defined in the
contamination control strategy. Not all Closed design
RABS employ VHP®/vH2O2 bio-decontamination
and in these cases the same requirements as Open
design RABS apply.
Considering Quality risk management RABS should
be operated as a closed operation with no open-
door barrier operator interventions after the last
bio-decontamination step inside the barrier and
through aseptic processing. Any justified and risk
assessed open-door interventions should be rare
and subjected to qualification studies in process
simulation trials for example PSTs and Media fills.
PHSS guidance is not mandatory but represents a
consensus view from the PHSS aseptic processing
and bio-contamination special interest group
on good manufacturing practice and has been
reviewed by the MHRA before publication from
a regulatory compliance perspective. However,
MHRA would point out that the user should not
try to implement this guidance without a proper
application of knowledge and true understanding of
the science behind the strengths and weaknesses
of VHP®/vH2O2. Each case will be reviewed on
its own merits and early engagement with your
regulator is strongly encouraged.
Guidance prepared by PHSS aseptic processing
and Bio-contamination special interest group with
regulatory review before open access publication,
via the PHSS and BPOG (Biophorum)
Clarity on GMP Guidance Note No. 2 | 13
PHSS.co.uk