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Annex 1 challenges in Life Science industry: Major topics of the new Annex 1

Technical Report · September 2023

DOI: 10.13140/RG.2.2.31248.17929

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 Annex 1 challenges
in Life Science industry

Major topics of the new Annex 1

and suggested solutions
by AM Instruments
Is your process
aligned to Annex 1?
The most challenging topics
of the new Annex 1,
according to Tim Sandle
Anyone working in drug manufacturing knows Dr. Tim Sandle. A microbiologist,
author, and science journalist, he is one of the leading experts and figurehead in
the field.

For this reason, we asked Dr. Sandle for his opinion on the effects of the new
ANNEX 1 in the strategies that, with due differences, the entire industry will have
to implement: from setting up new processes, to training personnel, to managing
non-conformities, this particular time requires the Life Sciences industry to pay
close attention.

AM Instruments solutions for new Annex 1 challenges

AM Instruments is focused on contamination control in cleanroom environments. Significant resources are
devoted to supporting pharmaceutical manufacturing with regards to regulatory news related to the final
release of the new Annex 1: from continuous microbiological monitoring to autoclave sterilisation processes,
from the transfer of materials to the new demands for biodecontamination.
Our product specialists support customers in aligning to the new regulatory requests.

CONSISTENT

2
summary
Sterilisation process

Routine monitoring

Transfer systems

Isolation technology

Cleaning & disinfection

Hands disinfection

Discover AM Instruments solutions

for your process alignment.

Is there something missing in the new Annex 1?

Future technologies

Ask for support

3
4
sterilisation process
Our interview opens with an as-
sessment of the overall impact of
the new Annex 1 on contamination
control strategies in the pharma-
ceutical industry. Dr. Tim Sandle,
the new Annex 1 revision is distin-
guished by a “holistic” view of
contamination control. What are
the most immediate consequen-
ces of this new approach?
Many facilities have developed ap-
proaches to contamination con-
trol, but the call within the Annex
1 is for pulling everything together
to provide a holistic overview of
the facility, the rationale, the gaps,
the level of risk for each gap, and
the remediation steps.
A contamination control strate-
gy is a system that considers all
the integral elements of pharma-
ceutical product manufacturing.
This is best achieved using quality
risk management principles and
supporting risk assessments for
contamination control and moni-
toring (the detectability of conta-
mination event).
As a governing ethos, processes,
equipment, facilities and manu-
facturing activities should be ma-
naged in accordance with risk ma-
nagement principles that provide
a proactive means of identifying,
scientifically evaluating and con-
trolling potential risks to quality.
The strategy at each facility will
vary, but common themes will in-
clude: microbial contamination,
cleaning and disinfection, sterility
assurance, facility design, chemi-
cal and particle contamination.
Outside of the microbial, other
The Annex 1 specifies that each item sterilised needs to be inspected for damage,
packaging material integrity and moisture on removal from the autoclave.
forms of contamination that can
arise from mix-ups, damaging
primary or secondary packaging,
distribution problems, and envi-
ronmental fluctuations. Overall,
a focus will need to be with redu-
cing contamination likelihood and
cross-contamination potentials.
This needs science and technical
procedures and controls
The format would be as one docu-
ment or series of connected do-
cuments developed to reflect the
site-wide strategy for minimising
contamination control.
Importantly, the strategy should
be a living document and be upda-
ted in relation to change controls,
process development, recurrent
deviations and in relation to other
quality records.
What are the challenges facing
the pharmaceutical industry in
sterilisation processes with re-
spect to the requirements of the
new Annex 1?
Perhaps the most significant
change is in relation to equip-
ment sterilisation when used for
aseptic processing. The 2017 draft
stated that critical surfaces with
direct impact need to be sterili-
sed (such as a filling manifold or
stopper bowl); however, with the
2020 revision this now requires
direct and indirect contact parts
to be sterilised. This could present
challenges to facilities with both
isolators and RABS devices.
There is a specific update for free-
ze-dried products in terms of fre-
eze-dryers (or lyophilisers). This is
for lyophilisers which are manual-
ly loaded or manually unloaded.
Here the freeze-dryers should be
sterilised before each load.
Other important Annex 1 points
include the requirement that the
feed water to a pure steam (cle-
an steam) generator should be
appropriately purified, to achieve
the required chemical and endo-
toxin levels, as assessed by the
Water-for-Injections pharmaco-
peial monitoring and testing re-
quirements. With the chemical re-
quirement, this is because steam
used as a direct sterilising agent
must not contain additives at a
level which could cause contami-
nation of product or equipment.
This part of the process needs to
be validated. The steam must also
consistently meet the parameters
for non-condensable gases, dry-
ness value (dryness fraction) and
superheat.
The Annex 1 also specifies that
each item sterilised needs be in-
spected for damage, packaging
material integrity and moisture
on removal from the autocla-
ve. Any item found not to be fit
for purpose, either damaged or
where there is evidence of a ‘wet
load’, should be removed from the
manufacturing area and an inve-
stigation performed.
On the subject of wet loads, this
is the most worrying factor. When
steam enters the autoclave cham-
ber and contacts with the pro-
duct, it is important that the ste-
am collapses (condenses) on the
product. This is in order for the
heat to be released to the load.
5
However, the formation of water
must be discharged through con-
densate management or re-vapo-
rised in order to prevent contami-
nation of the product. Removal
of the excess water is important
to prevent insulation of the load
from the steam.
We also need to be mindful of
pressure and its relationship to
temperature. Given that autocla-
ves destroy microorganisms by
direct steam contact at the requi-
red temperature and pressure for
a specified time, pressure control
is important. Poor pressure con-
trol can cause variations in steam
velocities.
What are the main critical issues
in autoclave sterilisation proces-
ses?
Some of the key issues are based
on the failure modes. Understan-
ding these modes is important for
controlling the critical issues. One
that stands out is load design.
Many companies will use a matrix
approach, whereby worst case
load configurations are validated
with a view to allowing other load
combinations to be used without
undergoing validation. Here care
must be taken with the design of
the matrix so that anything rela-
ting to the load that can affect the
distribution of the incoming ste-
am or which can affect uniformity
of temperature is identified and
accounted for. The assessment
should also take into account
anything that can take heat away
from the chamber can affect tem-
perature uniformity.
There are other factors that can affect sterilisation.
These include the shape of the equipment, the orientation and type of material.
6
Often mass is used for the matrix.
However, there are other factors
that can affect sterilisation. The-
se include the shape of the equi-
pment, where narrow pipework or
hoses presents a particular chal-
lenge in terms of steam penetra-
tion (here center of the length of
hose is the most difficult to sterili-
se); whereas with worst-case loca-
tion within a bottle, flask or cylin-
der the worst case is the center
near the bottom of the container.
The orientation of the equipment
is also important, and this affects
whether the item is capable of
free-draining. Where there is un-
certainty about what constitutes
worst case some initial work can
be undertaken thermometrical-
ly (heat penetration evaluation
tests); this allows data to be col-
lected about heat distribution,
and this ‘hard to heat’ study is
generally recommended. Account
should be taken, with liquid loads,
oft the viscosity of the liquid since
this can affect heat penetration.
A further factor is the type of
material, such as elastomeric ma-
terials and stainless steel. If the-
se types of materials are used in
combination, then a balance of
both may be needed for the worst
case. However, if in practice the
materials are sterilised separa-
tely, this may prompt two types of
worst case loads.
Let’s talk about materials: despite
the strong presence of cellulose,
many continue to use medical pa-
per. What are the risks and how,
with the new Annex 1 is it neces-
sary to deal with them?
Packaging materials, used to wrap
the items to be sterilised, are pro-
bably one of the most important
parts of the sterilisation process.
Packaging provides a protective
barrier for the sterile item and
prevents it from becoming re-con-
taminated. When used in aseptic
processing, the packaging should
be of low particle generation.
Cellulosic paper is not suitable
for higher grade cleanrooms,
especially due to the potential
for fibers to be released. Annex
1 emphasises that products need
to be free of visible particulates.
Non-autoclaved cellulosic paper is
also probe to microbial contami-
nation, given the range of micro-
bes that are capable of cellulose
hydrolysis.
An alternative is Tyvek® and Ty-
vek®/PET-PP. Tyvek® material is
made of continuous strong fibers
of pure high-density polyethylene,
is low linting and free of inherent
contaminants that could repre-
sent a risk in critical environmen-
ts. Tyvek® retains its dimensional
stability and integrity and main-
tains its tensile strength, micro-
bial barrier and the required air
permeability to enable effective
sterilisation.
In making the correct choice,
packaging needs to be well desi-
gned and of sufficient porosity
so that steam can pass through
packaging. In addition, packaging
should be water repellent to pro-
vide a sterile “field.” Furthermore,
the packaging material must not
change significantly during ste-
rilisation or release any substan-
ces that might interfere with the
action of the steam.
New Annex 1 requirements will
force pharmaceutical manufactu-
rers to revise their autoclave load
validation processes.
Validation needs to be designed
carefully to avoid failures (or fai-
lures with re-validation). The An-
nex 1 expects a greater level of
detail and investigation. Failure
to inactivate biological indicators
is normally due to inadequate air
removal rather than inadequate
conditions of temperature and
time.
Sometimes failures of re-valida-
tion can be attributed to using
biological indicators with much
higher D-values than those used
for the initial validation (where
the biological indicators used for
the re-validation have a far higher
resistance compared with those
used for the initial qualification.
This could occur, for example, if
the initial qualification was per-
formed using a biological indica-
tor with a D-value of 1.5 minutes
and the re-qualification executed
Tyvek® retains its dimensional stability and integrity and maintains its tensile strength,
microbial barrier and the required air permeability to enable effective sterilisation.
using a biological indicator with a
D-value of 3.0 minutes).
Issues can also arise due to chan-
ges to load patterns. How load
patterns are assessed such as
mass compared with item com-
plexity and configuration, as with
tubing.
One issue will be the requirement
to more carefully assess the qua-
lity of pure steam. There is also a
materials piece, in that before any
sterilisation process is adopted,
the suitability for the product or
equipment need to be assessed to
make sure it is suitable for the de-
sired sterilising conditions.
Another factor is with the desi-
gn of the load, as I’ve previously
mentioned. Other considerations
for the validation design include
handling items of a shape where
there is the potential for trapped
air (and thus difficulties with air
removal). There are also items
that may not allow steam to pe-
netrate and are thus hard to heat
(such as filters, check valves, and
small tubing). Plus, there are
items where there is potential for
condensate collection.
How do you evaluate the use of
washable products and what, if
any, critical issues do you see in
this type of choice?
The key issue with washable pro-
ducts is with the number of times
they can be processed and what
the impact of this is upon the li-
fe-time of the item. Integrity is-
sues and release of particles are
two areas of concern, along with
other forms of structural weak-
nesses and discoloration. This re-
lates not only to the washing pro-
cess but also to sterilisation – both
are aggressive processes that can
affect the material. The choice of
water and the type of sterilisation
process and the parameters sur-
rounding this represent important
choices to be made, in order to
avoid damage and to extend the
reprocessing cycle.
It is important to undertaken a ca-
reful evaluation, and to set an ap-
propriate maximum reprocessing
time.
7
challenges
choose the right packaging material
reduce particulate release
avoid risks of package breakage
reduce operating and handling times
maintain sterility at all stages of the process
be aligned with new Annex 1 requirements

Unlike medical paper, which can release a significant number of particulates,

the unique continuous filament structure of Tyvek® has cleaning characteristics that minimise
the risk of contamination during the preparation and use of materials.

8
 solutions
TWO PHARMA-GRADE MANUFACTURING SITES
ONE COMPREHENSIVE GMP ANSWER

Covers & sterility

protection solutions
GRADE C OPERATIONAL CLEANROOM
GRADE A/B RABS
• Tyvek® covers and bags production
• repackaging service

Sterilisation &
packaging solutions
GRADE C OPERATIONAL CLEANROOM
• Tyvek®-PET/PP bags and reels production

HIGH CUSTOMISATION REPACKAGING SERVICE

Our product specialists intervene with targe-
ted proposals according to the critical issues
reported by the customer and carry out a fe-
asibility study in accordance with the speci-
fic requirements.
The Pharmaclean® team produces the sam-
ple allowing the customer to test the product
before placing the final order.
After any revision of the design according
to the results of the tests carried out, Phar-
maclean® arranges for batch production and
delivery within a short period of time.
Each batch is accompanied by complete do-
cumentation according to current regula-
tions.
Customised repacking in Grade A/B.
Transfer via Getinge DPTE® Alpha ports and
DPTE BetaBag®
Pharmaclean® performs:
• inspection and cleaning of incoming
materials
• packing in single, double, triple packaging
• packing materials in Getinge DPTE-
BetaBags®
• gamma sterilisation upon request through
qualified partners
• identification and traceability of batches
and process
• execution by qualified operators and with
GMP approach
• risk assessment

9
routine monitoring
The new Section 9 of Annex 1 de-
als with the monitoring of pro-
duction processes and classified
environments with regard to the
counting of viable and non-viable
particles.
“Spot” monitoring is no longer
acceptable. Frequent (even conti-
nuous) monitoring should be con-
sidered in the Contamination Con-
trol Strategy (CCS). In particular,
the focus is on routine monitoring
that must be carried out in opera-
tions at all critical stages.
What can be considered critical
phases?
The reference in the new Annex
1 refers to sterile products manu-
facturing. Here each stage of ma-
nufacturing should be captured,
especially for aseptic processing.
This includes line set-up; periods
between set-up and the start of
filling; during filling; during any
lyophilisation; and during over-
sealing. The product and direct
and indirect product components
must be protected from contami-
nation at all times.
With how long monitoring should
last for, with aseptic processing,
then typically settle plates are
exposed for the duration of a fill
(additional settle plates may need
to be used if the fill exceeds the
validated plate exposure time).
Active air samples can be taken
continuously using the new ge-
neration of air-samplers that take
a sample over a four-period to
match the settle plate. For other
types of samplers, these should,
as a minimum, be taken at the
(near) start; (near) middle; and
Each stage of manufacturing should be captured, especially for aseptic processing.
10
(near) end of the fill.
Finger plates will be taken imme-
diately after connection activity,
for any persons present during
the fill at a random time during
the fill. Plus, after a Grade A zone
intervention and after any item of
critical equipment is inadvertently
touched or where another activity
has taken place by which aseptic
technique or practice could be
compromised.
Surface monitoring (direct con-
tact samples) will take place im-
mediately at the end of the fill.
This should not be performed
during filling due to the invasive
and disruptive nature of the tech-
niques. Contact plates of gowns
should be taken from all person-
nel immediately before they exit
the Grade B areas.
In addition, for Grade C and D
areas the monitoring should be
representative and meaningful,
which includes targeting actual
activities. For this, a risk fra-
mework should be developed
whch we can discuss later.
In the new Annex 1 the QRM princi-
ples provide a tool to scientifically
identify and assess potential qua-
lity risks. In microbiological moni-
toring, they mention system desi-
gn, setting action limit and alert
levels and reviewing data trends.
Can you give us a correct approa-
ch to these three activities?
These are three important areas.
The first is with the design of the
environmental monitoring pro-
gramme. Here it is important that
the programme is based on quali-
ty risk management and it takes
into account the different types
of microbial hazards that may be
present and the potential vectors
for contamination to end up in the
product. Vectors include water,
air, surface-to-surface transmis-
sion and via people.
Further to design, each organisa-
tion should have a documented
environmental monitoring pro-
gramme, and be able to answer
the following questions:
• How frequent should the moni-
toring be?
• How long should the monitoring
last for?
• How should the locations for
monitoring be selected?
• How should the monitoring pro-
gramme react to cleaning and di-
sinfection practices?
• Describe the sampling procedu-
re and sample handling
• Describe the sample incubation
regime
• Outline the methods for data
analysis (statistical data trending)
• Outline the investigative respon-
ses to exceeded action levels (and
upward trends)
• Describe the execution respon-
sibilities
Alert and action levels are the
main way of signalling that so-
mething unusual has happened.
These can be the numerical va-
lues, as described in the Annex.
But we also need to look at the
frequency of events – especially
non-zero counts – and be mindful
of the detection of different types
of microorganisms.
With setting alert and action le-
vels, for newly built cleanrooms
regulatory guidance values can
be used. However, after a certain
time (such as six months) or when
a certain number of samples have
been taken (say more than100),
each user must set alert and le-
vels appropriate to the facility.
This should use some type of
statistical technique, such as per-
centile cut-off or negative expo-
nential distribution. Generally,
the percentile cut-off approach is
easiest to use and this approach
supports data that is not normally
distributed.
Interestingly, the revised Annex
places particular emphasis upon
alert levels as indicators of poten-
tial drift, especially of where there
are atypical patterns.
The mention of atypical patterns
brings us to the third area and
trend analysis. I’ve always main-
tained that environmental moni-
toring becomes a wasteful and
meaningless exercise unless the
monitoring has been thoughtfully
targeted and the data correctly
interpreted to ascertain the ‘true’
trends. Hence, trend analysis is
an important aspect of the moni-
toring programme since data ori-
ginating from single samples are
often not significant.
Data graphs, histograms and sta-
tistical process control charts are
examples of the tools that can be
used and should be applied. En-
vironmental monitoring results
which exceed the action level; or
where there is an upward trend re-
lating to excursions of the alert le-
Quality risk management is very useful for determining sampling points
and helping to set out the frequency of monitoring.
vel; or where the frequency of in-
cidents exceeds a predetermined
cut-off value, represent scenarios
which should be investigated.
When putting data together vi-
sually it is important to include ap-
propriate information with tables
and graphs. This helps to identify
patterns and possible reasons for
a given trend. Such information
includes:
• Locations;
• Dates;
• Times;
• Identification results;
• Changes to room design;
• Operation of new equipment;
• Shift or personnel changes;
• Seasons;
• HVAC problems (e.g. an increase
in temperature).
Essentially we need such infor-
mation to help us to interpret the
trend.
Risk assessment can be suppor-
tive in one of the most critical
issues: the selection of sampling
points and related frequency for
monitoring. How might his be
achieved?
Quality risk management is very
useful for determining sampling
points and helping to set out the
frequency of monitoring. Althou-
gh there are different risk tools
available, for sample site selection
my preference is with hazard
analysis critical control points
(HACCP). This approach is based
on understanding and visualising
product, people and waste flows.
Through HACCP, the process be-
gins with conducting a hazard
analysis, which means identifying
what the hazards that can genera-
te a risk are from a process flow.
This enables us to determine cri-
tical control points, which is about
where risk is greatest and seeking
to reduce the risk. In areas where
risks are higher, the process pro-
ceeds to establish critical limits
and to establish a system to moni-
tor each critical control point. It is
also useful to think about the cor-
rective actions to be taken when
each critical control point has an
excursion or goes out of control.
The HACCP needs to be documen-
ted.
In the case of environmental mo-
nitoring, we can use a HACCP risk
assessment to consider the main
areas of risk such as where is per-
sonnel interaction is greatest, ac-
cording to the highest level of acti-
vity and perhaps where activities
are variable. We need to consider
what could lead to contamination
entering the airstream and whe-
re contact with critical surfaces
could occur. This includes under-
standing if the activity in the area
or the item itself contribute to the
spread of contamination. As well
as the flow of people, materials
and waste there may be other
indicators of contamination tran-
sfer that we especially want to
focus on such as airlocks, transfer
hatches and locations that are po-
tentially difficult to clean.
An important passage in the new
Annex 1 is Section 9.24, in which
several sampling methods are
suggested, aimed at avoiding in-
terference between continuous
11
monitoring and pharmaceutical
operations: Continuous viable air
monitoring in grade A (e.g. air
sampling or settle plates) should
be undertaken for the full dura-
tion of critical processing, inclu-
ding equipment (aseptic set-up)
assembly and critical processing.
A similar approach should be con-
sidered for grade B cleanrooms
based on the risk of impact on the
aseptic processing. The monito-
ring should be performed in such
a way that all interventions, tran-
sient events and any system dete-
rioration would be captured and
any risk caused by interventions
of the monitoring operations is
avoided.
With the frequency of monito-
ring, the use of risk filtering can
be useful. For cleanrooms outside
of the aseptic core, these can be
grouped into different frequency
patterns based on the risk presen-
ted by different sources and vec-
tors. For instance, we can consider
room activity and ask ourselves
‘What is going on?’, ‘When is it ta-
king place?’, ‘What type of equip-
ment is involved?’ and ‘How many
personnel are involved?’ We can
also considered product exposu-
re risks where we can account for
open processing being a higher
risk than closed processing, and
longer exposure times being at a
higher risk than shorter exposure
times.
Other factors to consider include
room temperature. Is the room
cold, warm or ambient? General-
ly, the risks are lower for cold ro-
Settle plates are an important monitoring tool and they are seeking to achieve
something different to active air sampling.
12
oms. Similarly, wet areas will be at
a greater risk to dry areas given
the greater chance of microbial
proliferation in wet areas. We may
also want to increase monitoring
downstream where there are
fewer microbial reduction steps.
In essence, we are seeking to mo-
nitor where controls are more
challenging or where the impact
to product is greatest.
Another important point in Annex
1: “In developing the APS plan,
consideration should be given to
the following: the method of de-
tection of microbial contamina-
tion should be scientifically justi-
fied to ensure that contamination
is reliably detected.”
Grade A: do you consider the
use of exposed plates still a valid
method or only active sampling
should be considered?
Settle plates are an important mo-
nitoring tool and they are seeking
to achieve something different to
active air sampling. With active
air, we are assessing the popula-
tion of organisms within a given
volume of air (one cubic metre) wi-
thin the vicinity of our work area.
With settle plates we are looking
at the potential for microbial car-
rying particles to settle out of the
air, which can occur due to air cur-
rent, air slowing down, eddying
and so on, and being deposited
into the product. Settle plates are
particularly useful at working hei-
ght under unidirectional airflow
environments with their locations
informed by airflow visualisation
patterns. In short, both forms of
air sampling are useful, especially
within Grade A environments.
What about methods and justifi-
cation within the CCS? As Annex
1 suggests: Where aseptic opera-
tions are performed, microbial
monitoring should be frequent
using a combination of methods
such as settle plates, volumetric
air sampling, glove, gown and
surface sampling (e.g. swabs and
contact plates). The method of
sampling used should be justified
within the CCS and should be de-
monstrated not to have a detri-
mental impact on grade A and B
airflow patterns. Cleanroom and
equipment surfaces should be
monitored at the end of an ope-
ration.
New technology can help the con-
tinuous monitoring activity: mem-
brane is one of them.
What’s your opinion about it?
With environmental monitoring,
environmental control must come
first. Without good control, the ri-
sks of product contamination are
high so we should seek to redu-
ce control weaknesses wherever
possible. The range of methods
used for monitoring should be
based on an understanding of ri-
sks and activities, but generally a
comprehensive array of methods
should be used to assess air, peo-
ple, and surfaces. These methods
are somewhat limited in terms of
sample size, the metrology of the
methods, and the inability of cul-
ture media to grow all organisms
(and with many organisms being
too stressed to grow). To a degree
we can address this by trending,
however a new generation of
methods offers improved detecta-
bility.
It is pleasing to see Annex 1 em-
brace alternative and rapid mi-
crobiological methods and there
are a number emerging on the
market. This includes technology
for bioburden testing, looking for
biological markers, and flow cyto-
metry techniques for continuous
water system assessments.
With environmental monitoring
specifically, there are some emer-
ging systems that can detect colo-
Without good control, the risks of product contamination are high
so we should seek to reduce control weaknesses wherever possible.
nial growth earlier than is possible issues with the technology, but it
with the human eye, scanning for has a powerful potential.
microcolonies using techniques
like light excitation. Another inte- Overall, rapid microbiological
resting area is with spectropho- methods are to be encouraged.
tometric counters. These are par- When appropriately selected and
ticle collection devices that use validated they provide data that
advances in light scattering, opti- is more sensitive, accurate and
cs and special software, providing faster, when compared with con-
real-time data about particles and ventional, growth-based methods.
biological activity in air. With the
system, air is passed through a la-
ser and the instrument counts the
numbers of particles in a sample
of air (inert and biological) throu-
gh two detectors. Biological parti-
cles are detected using a fluore-
scence detector, looking for three
biological markers. These are the
metabolites: NADH, riboflavin,
and DPA. There are some teething
13
challenges
what do we need to monitor

The contamination in air

• It can move contamination around
as a vector
The contamination that can settle out of air
onto surfaces
• If it can settle onto a surface in the
facility it could settle onto product
or product-contact components
• People are also supposed to keep Room activity:
these surfaces clean and “germ- What is going on?
free” and can be negligent in doing When is it taking place?
this What type of equipment is involved?
The people who shed contamination into the How many personnel are involved?
air
• People are mainly subject to procedural
controls Exposure risk:
How long is the product exposed?
Is open processing involved?
Room temperature:
Cold, warm or ambient?

Process stage e.g.:

how do we monitor
Raw material processing, interme-
Viable counting methods: diate manufacturing or final formula-
tion?
• Settle plates
• Active air-samplers
• Contact plates Duration of process activities.
• Swabs

Particle counting
Water exposure/wet area?

• Rapid microbiological methods

The classic techniques are well established,

but what do we need to consider for
an effective environmental monitoring
programme?

14
 solutions
CONDITION OF MONITORING
Monitoring during ‘in use’ (or ‘in operation’ or
‘dynamic’ state) is essential for routine assessment.
Monitoring during the ‘at rest’ (or ‘static’ state) can be
useful for benchmarking

DURATION OF MONITORING
Aseptic processing

• Settle plates are exposed for the duration of a fill.

• Active air samples will be taken at the (near) start;
(near) middle; and (near) end of the fill.
• Finger plates will be taken immediately after a:
• Connection activity, for any persons present
during the fill at a random time during the fill;
• After a Grade A/ISO Class 5 zone intervention;
• After any item of critical equipment is
inadvertently touched;
• When some activity has taken place by
which aseptic technique or practice could be
compromised.
• Plates should also be taken at any other time a
microbiologist believes it to be necessary.
• Surface monitoring (direct contact samples) will
take place immediately at the end of the fill.
• Contact plates of gowns will be taken from all
personnel immediately before they exit the Grade B
/ ISO Class 7 area (Aseptic Filling Suite).
TIME OF MONITORING
For routine monitoring, provided the room is in
use, any time is probably ‘equal case’.
There are points when time matters:
• Filling line set-up;
• Aseptic filling;
• Filling line strip down;
• Loading freeze driers;
• Loading autoclaves and Isolators;
• Operation of centrifuges and C-I-P units
(especially in relation to particle count
generation).

For Grade C and D process areas and in non-sterile

manufacturing:
FREQUENCY OF
• Viable monitoring should be for a defined for periods
of time during an event or a process. MONITORING
• Active air samples should be run so that one cubic For sterile manufacturing:
metre of air is sampled. • Each operation during batch filling.
• Settle plates must be exposed for four hours where • A set frequency for other areas in relation to the
possible. manufacturing process.
• Surface monitoring should be performed at some • Based on risk assessment
point during the dynamic state. • Grade B, C and D (plus CNC?)
• Following maintenance work.
• Pre and post shutdowns

15
transfer systems
New Annex 1 elaborates on air-
locks and states that they must
be designed and used to ensure
physical separation and to minimi-
se microbial and particle contami-
nation of different areas. The last
step of the airlock must be the
same grade as the area to which
it gives access.
4.10 The transfer of equipment and
materials into and out of the cle-
anrooms and critical zones is one
of the greatest potential sources
of contamination. Any activities
with the potential to compromise
the cleanliness of cleanrooms or
the critical zone should be asses-
sed and if they cannot be elimina-
ted, appropriate controls should
be implemented. Annex 1
The transfer of materials as well
as the associated contamination
risks are given great attention in
the new Annex 1. The CCS leaves
nothing to chance.
What is new compared to past re-
visions of the document?
In the new Annex, microbiological
contamination from materials en-
tering the cleanroom is provided
with a far greater emphasis. This
has been identified as one of the
major routes of contamination
for items coming into the facility.
The optimal means of transfer is
via a double-ended autoclave and
using moist heat. Where this is
not possible, of where single-use
sterile items are required, then a
disinfection process is required.
This can be automated (as with a
decontamination chamber using a
vapour like hydrogen peroxide or
Microbiological contamination from materials entering the cleanroom has been identified
as one of the major routes of contamination for items coming into the facility.
16
chlorine dioxide) or a manual pro-
cess using a transfer hatch equip-
ped with a localised HEPA filter air
supply.
The Annex places emphasis upon
both cleaning and disinfection.
Most disinfectants are very poor
at penetrating ‘soil’ and therefore
items need to be cleaned prior to
the application of a disinfectant.
The Annex also indicates that
the disinfectant should be ideally
sporicidal and that a sporicide is
required for the transfer of mate-
rials from Grade C to Grade B. It
is also important that items being
transferred in are multi-wrapped
and that a layer of wrapping can
be removed at each stage of the
transfer process as the item mo-
ves from the external area and
through the cleanroom cascade.
Critical steps in the material tran-
sfer process
What are the most critical steps in
the materials transfer process?
Bacterial spores in the Grade A
environment present a significant
risk of contamination in aseptical-
ly prepared products and poten-
tial patient harm. Ensuring that
any spores (plus vegetative orga-
nisms are not transferred in from
Grade C to Grade B is critical. This
is to ultimately protect Grade A
environments.
In assessing this, an understan-
ding of how goods arrive into
a warehouse and end up in the
aseptic processing area.
To carry out this task correctly, a
quality risk assessment is requi-
red. The risk assessment will need
to address:
The packaging: Are there suffi-
cient layers? Is the packing resi-
stant to the disinfectant?
Detergent: Is the detergent sui-
table for cleaning? Is the deter-
gent compatible with the disin-
fectant?
Disinfectants: Is the disinfectant
sporicidal? Has the disinfectant
been validated? Is there a risk of
residuals?
Environmental monitoring: Has
the transfer disinfection process
been qualified? (it is normal to
carry out at least three simula-
tions of wort-case load transfers
and to conduct surface environ-
mental monitoring). In addition,
at what frequency will this be ve-
rified? Here any manual process
is subject to variations in techni-
que and there needs to be regular
assessment in place. Furthermo-
re, ongoing monitoring of the bio-
burden associated with incoming
items should be undertaken and
understood in terms of microbial
numbers and species variation.
Non-routine items: How will atypi-
cal or non-routine items be hand-
led? What is this risk assessment
process in place for these?
Procedure: It is important that the
entire process is documented and
that the procedures are used to
facilitate personnel training.
With the disinfection step, becau-
se there is a high risk of contami-
nation therefore the disinfection
process must be carefully con-
trolled. Maintaining a wet contact
time is critical for effective disin-
fection. If the validated time is not
being achieved, contamination
may persist on the surface of the
items.
Material transfer with biodeconta-
mination system
4.11 The transfer of materials,
equipment, and components into
an aseptic processing area should
be carried out via a unidirectional
process. Where possible, items
should be sterilised and passed
into the area through double-en-
ded sterilisers (e.g. through a dou-
ble-door autoclave or depyroge-
nation oven/tunnel) sealed into
the wall. Where sterilisation on
transfer of the items is not pos-
sible, a procedure which achieves
the same objective of not intro-
ducing contaminant should be
validated and implemented, (e.g.
using an effective transfer disin-
fection, rapid transfer systems for
isolators or, for gaseous or liquid
materials, a bacteria-retentive fil-
ter). Annex 1
The pass-box should have an in-
tegrated bio-decontamination sy-
stem. What are the specific requi-
rements of this integration?
The optimal approach is to auto-
mate the decontamination pro-
cess. This can be put in place using
a biodecontamination chamber,
for multi-wrapped items. These
items must have been sterilised
using a proven technology (all
items entering Grade A must be
sterile). Typically items are sterili-
sed by radiation (such as gamma,
X-rays or electron beam) or some-
times by a penetrative gas (such
as ethylene oxide).
The most common technology for
The optimal approach is to automate the decontamination process.
this is hydrogen peroxide vapour,
although there are alternatives.
The advantage is that, through
development, the process can be
qualified and demonstrated to be
consistent. In addition, the level of
microbial inactivation is far grea-
ter than can be achieved with a
manual process. To demonstrate
this, biological indicators can be
used. It is important that the bio-
logical indicators selected have
proven resistance to the deconta-
mination agent (such as Geobacil-
lus stearothermophilus for hydro-
gen peroxide vapour) and are of a
suitable population to demonstra-
te a six-log reduction.
With cycle development, control-
ling key parameters like tempe-
rature, humidity, concentration
of the biocide, and the contact
time are each essential. Attention
should be paid to load configura-
tion and for this all items should
be hung so that the decontami-
nation agent can circulate around
each item.
Items should also be multi-wrap-
ped, enabling a layer of wrapping
to be removed for the eventual
transfer into Grade A. This is re-
quired because the vapours used
for decontamination are only ef-
fective on the outer surfaces of
the items placed within the cham-
bers, they have no penetrative
ability.
Activities to reduce contamina-
tion
And, if they do not have a bio-de-
contamination system, what are
the main activities that can redu-
ce the risk of contamination?
The most important steps for the
manual process are to have a
well-built transfer hatch (fashio-
ned from good quality stainless
steel) and or the hatch to have a
local HEPA supply capable of deli-
vering Grade A air.
Prior to use all surfaces of the
hatch must be wiped with a spo-
ricide, and then each item going
in must also be treated with spo-
ricide. Items should then be pla-
ced inside for the duration of the
contact time under the localised
HEPA airflow. The wiping of items
with sporicide is essential, simply
spraying is not effective.
Upon removal, the outer layer of
each item must be removed.
Transfer methods
The science-based philosophy
behind the new Annex 1 also af-
fects material transfer activities,
for which it is explicitly required:
4.13 Where materials, equipment,
components and ancillary items
are sterilised in sealed packa-
ging and then transferred into
the Grade A zone, this should be
done using appropriate, validated
methods (for example, airlocks or
pass-through hatches) with ac-
companying disinfection of the
exterior of the sealed packaging.
The use of rapid transfer port
technology should also be con-
sidered. These methods should
be demonstrated to effectively
control the potential risk of con-
tamination of the Grade A zone
and Grade B area and, likewise,
the disinfection procedure should
17
be demonstrated to be effective
in reducing any contamination on
the packaging to acceptable le-
vels for entry of the item into the
Grade B and Grade A areas.
How might this be achieved?
In summary to what we have co-
vered, there is a hierarchy of tran-
sfer methods:
1. Autoclaving/depyrogenaton
tunnels
2. Automated cycle decontami-
nation chambers
3. ‘Dynamic’ pass-through ha-
tches with HEPA filtered air
supply
With the Annex 1 updated, ‘static’ pass through chambers with no air supply are no longer permitted.
18
The important message is that all Other, old-fashioned means of
items that are intended for Grade getting items in, such as person-
A need to take this route, including nel transferring items (e.g., via
culture media to be used for en- changing rooms) must be prohi-
vironmental monitoring. Drawing bited.
up a list of approved items is es-
sential to avoid anything for being
missed. This includes everything
from tools to cables. For each
item the route to be taken must
be understood.
With the Annex 1 updated, ‘sta-
tic’ pass through chambers with
no air supply are no longer per-
mitted. Organisations have until
August 2023 to phase these out.
Using risk assessment to mitigate
this would not be acceptable.
19
challenges
Annex 1

4.10 - The transfer of equipment and ma-

terials into and out of the cleanrooms
and critical zones is one of the greatest
potential sources of contamination. Any
activities with the potential to compro-
mise the cleanliness of cleanrooms or
the critical zone should be assessed and
if they cannot be eliminated, appropriate
controls should be implemented.

MyBox®

20
 solutions
A NEW GENERATIONOF FLUXED BIODECONTAMINATION
PASS-BOXES
AM Instruments has applied the most innovative
technologies to develop a new pass-box model.

• fluxed pass-box and pass-boxes, stand-alone or integrated

into existing ventilation systems
• material transfer chambers within adaptable shapes that
can be adjusted to suit every need
• solutions also proposed with HMI touch screen operator
panels for the traceability of transfer cycles and door
opening times
• possibility of option to integrate UV lamps and hydrogen
peroxide decontamination systems.
Using integrated low-concentration hydrogen peroxide
systems (<8%) with HPE technology, our decontamination
chambers guarantee a degree of bacterial reduction
equal to log 6 in extremely short times. Ideal for the
decontamination of thermolabile materials and electronic
components.
MyBox® is produced in accordance with the GMP standards
for pharmaceutical environments, with high-quality
and high-finish materials. It can perform automatic
biodecontamination cycles through an integrated PLC,
adapting to the specifications of the individual load.
During the validation phase, chemical and
microbiological mapping with bioindicators (Geobacillus
Stearothermophilus) ensures the correct distribution and
effectiveness of the hydrogen peroxide in guaranteeing
the desired level of biodecontamination. MyBox® can be
easily installed for the passage of material from classified
C/D areas into sterile Grade B areas, guaranteeing the
sterilisation of external surfaces. This device is essential
when the products/components to be sterilised are not
autoclavable.

GENERATOR WITH HPE TECHNOLOGY

HPE technology is based on a hydrogen peroxide solution
with a concentration lower than 8% which, passing
through a high voltage electromagnetic field, undergoes
an ionisation process allowing a high and effective
diffusion with validated biodecontamination cycles and a
reduction of the surface microbial load of log 6, including
viruses, bacteria, moulds and microbial spores. The system
is equipped with a control panel that can be connected to a
supervision system for complete automation of the cycle.
In the environment, the ionised mist behaves with the
dispersion characteristics of a gas.
• highly compatible with all materials used in controlled
environments
• utilities required: compressed air, connections to an
electrical supply and to an external exhaust system
• simple and comprehensive design suitable for any need

21
isolation technology
A fundamental consideration in
Annex 1 is the risk posed by peo-
ple and the necessity to exclude
people from Grade A environmen-
ts. Any personnel interaction with
Grade A poses a risk. The primary
means to address this is trough
the use of separative devices –
isolators and forms of RABS.
What challenges the pharmaceu-
tical industry is facing in adapting
the use of separative devices to
the Annex 1 requirements?
Already in the “Principles”
section, the new Annex 1 clearly
expresses the need for the use of
appropriate containment techno-
logies:
“The use of appropriate techno-
logies (e.g. Restricted Access Bar-
riers Systems (RABS), isolators,
robotic systems, rapid microbial
testing and continuous monito-
ring systems) should be conside-
red to increase the protection of
the product from potential extra-
neous sources of endotoxin/pyro-
gen, particulate and microbial
contamination such as personnel,
materials and the surrounding en-
vironment, and assist in the rapid
detection of potential contami-
nants in the environment and the
product.”
Not only that, but the new Annex
also explicitly calls for justifica-
tion for their non-use. There use
should be considered in the CCS.
Any alternative approaches to the
use of RABS or isolators should be
justified. This last step represents
The choice between RABS and isolators has to be made accordingly to a risk based process
with a wide meaning of “risk”, embracing technical, economic and environmental considerations.
22
a major difference from past ver-
sions.
A fundamental consideration in
Annex 1 is the risk posed by peo-
ple and the necessity to exclude
people from Grade A environmen-
ts. Any personnel interaction with
Grade A poses a risk. The primary
means to address this is trough
the use of separative devices –
isolators and forms of RABS.
The choice between RABS and
isolators has to be made accor-
dingly to a risk based process with
a wide meaning of “risk”, embra-
cing technical, economic and en-
vironmental considerations.
Isolators are devices that provide
for total separation between one
environment and another. An iso-
lator does not directly exchange
air with the surrounding environ-
ment and all air must enter throu-
gh a HEPA filtration system. All
transfer of material into the iso-
lator must be accomplished while
maintaining complete environ-
mental separation. The interior
of the isolator, and all equipment
contained therein, must be able to
be subject to a decontamination
process in a highly reproducible
manner. An alternative to an iso-
lator or to a conventional cleanro-
om is a Rapid Access Barrier Sy-
stem (RABS). The debate between
isolators and RABS is driven by se-
veral factors: patient needs, per-
sonnel capabilities, finance. The
major technical factor in favour of
the isolator is, of course, the total
segregation of the operator from
the process. This separation ser-
ves to protect the product during
aseptic production: the environ-
ment / operator in containment
applications, and the patient, pro-
duct, operator and environment
when used for sterile or cytotoxic
products. With aseptic filling the-
re is a far greater chance of achie-
ving a zero growth with the me-
dia filling trial on a repeated basis
that there is within a conventional
cleanroom or with a RABS.
As RABS become more sophisti-
cated and in some cases look very
similar to isolators there is a risk
that they are considered to be the
same, or that a RABS is a lower
grade of isolator. RABS are an ad-
vancement on the Grade A zone
with Grade B surrounding clean-
room concept whereas isolators
are designed to totally isolate the
aseptic process and therefore do
not need to be located in a Grade
B zone. Misunderstanding the fun-
damental differences in operating
principles has led to requests for
isolators to be located in Grade B
areas and RABS gloves to be te-
sted in the same way as isolator
gloves.
Design according to CCS
Key considerations when perfor-
ming the risk assessment for the
CCS of an isolator should inclu-
de (but are not limited to); the
bio-decontamination programme,
the extent of automation, the
impact of glove manipulations
that may potentially compromi-
se ‘first air’ protection of critical
process points, the impact of po-
tential loss of barrier/glove inte-
grity, transfer mechanisms used
and activities such as set-up or
maintenance that may require the
doors to be opened prior to the
final bio-decontamination of the
isolator. Where additional process
risks are identified, a higher grade
of background should be conside-
red unless appropriately justified
in the CCS. Airflow pattern studies
should be performed at the inter-
faces of open isolators to demon-
strate the absence of air ingress.
The processes of compliance with
the new Annex 1 and especially
the Contamination Control Stra-
tegy, which is a pillar of it, seem
to involve automation in an extre-
mely important way. The ultimate
goal seems to be to eliminate the
human factor in Grade A.
These are important points and
central to the CCS. It is very im-
portant to focus on the design of
the isolator or RABS and to build
in as much quality as possible at
this stage. When designing an iso-
lator for a particular process, the
following should be considered:
• Knowledge of the process steps
• Choosing between positive and
negative pressure type isolators
• Choosing between open
(continuous process) or closed
(batch process) isolators
• Deciding upon whether
unidirectional or turbulent flow
is required
• Selecting the appropriate
continuous or batch transfer
systems for the ingress and/or
The ultimate goal seems to be to eliminate the human factor in Grade A.
egress of components
• Fabrication of a essentially
identical mock-up of the finished
isolator
• Simulating the process with full
consideration of fatigue factors
• Identifying the materials of
construction
• Preparation of a detailed
functional description include
detailed drawings
An important additional aspect
is the selection of realistic leak
tightness criteria for the isolator,
consistent with process require-
ments and fabrication capability.
Isolators rely on the integrity of
the physical barrier to protect the
aseptic process and so frequent
integrity testing is required.
Another important consideration
is with the air and airflow. First air
must not be interrupted and there
should be no turbulence created
which could prevent the effective
removal of any particles genera-
ted by the intervention or which
could entrain extraneous particles
and carry them towards the pro-
cess. This is usually carried out
by airflow visualisation studies –
(smoke studies).
Requirements vary between dif-
ferent applications such as small
scale sterility test and pharmacy
isolators, negative pressure iso-
lators used to contain hazardous
materials, and larger scale com-
plex systems for pharmaceutical
manufacturing processes. The
test pressures and limits used for
initial set up and those used for
routine monitoring may also vary
to account for variations in tem-
perature between tests.
Ideal background of isolator pro-
cesses
Let us deepen the processes in
isolator. The unwrapping, assem-
bly and preparation of sterilised
equipment, components and an-
cillary items with direct or indi-
rect product contact should be
treated as an aseptic process
and performed in a grade A with
a grade B background. The filling
line set-up and filling of the ste-
rile product should be treated as
an aseptic process and performed
in a grade A with a grade B back-
ground. Where an isolator is used,
the background should be in ac-
cordance with paragraph 4.20
What is the ideal background for
a process in isolator? And what
must a proper risk analysis cover
to avoid contamination risks?
Whilst the interior of the isolator
is required to meet Grade A requi-
rements, there is no requirement
for the isolator be located in a
Grade B area. EU Annex 1 specifies
at least a Grade D background al-
though many companies use Gra-
de C. This also means that opera-
tors are not gowned to Grade A/B
standards.
The isolator is therefore not ful-
ly protected by the classical cle-
anroom continuum of Grade D to
Grade A zones and so there are
a number of additional require-
23
ments which are key to successful
operation.
During set up isolators may be
open to the environment (Grade
D or Grade C) and so a validated
bio-decontamination of all surfa-
ces is required. This must include
the surfaces of the isolator itself
and also of any materials and
equipment placed into the isola-
tor before closing up the system.
Bio-decontamination is usually
carried out by a gaseous bio-de-
contamination process such as
with hydrogen peroxide vapour. A
6 log reduction in a spore challen-
ge is considered to demonstrate
effective biodecontamination.
Packaging for isolator processes
Materials in and out of isolators.
Preparing material for its inser-
tion into isolator is a lengthy and
extremely delicate process. Many
pharmaceutical companies out-
source the process, requiring pro-
per packaging in appropriately
packaged BetaBags.
What are the indispensable ele-
ments that determine the quality
of a packaging service provider?
Transfer of materials into and out
of the isolator is a major risk area.
During set up isolators may be open to the environment (Grade D or Grade C) and so
a validated biodecontamination of all surfaces is required.
24
In order to meet the requirements tor doors seal together ensuring
that: a contamination free connection.
RTPs can be designed so that ma-
• all materials entering the Grade terials such as stoppers can be
A zone are sterilised and sterilised in the RTP.
• the isolator remains physically
closed during operation A similar concept is with sterili-
For small batch operations it mi- sation bags with docking system.
ght be possible to wrap and steri- Items such as vial stoppers are
lise all the items required. These sterilised in flexible bags which
can then be loaded into the iso- have an alpha beta port which al-
lator before close up and bio-de- lows the bag to be docked directly
contamination. For larger volume to the wall of an isolator or RABS.
processes materials are required The alpha beta system is designed
to be sterilised and transferred in such a way that the outer non-
across the isolator’s physical bar- sterile surfaces of both units lock
rier, maintaining the sterility of together and then open to provide
the items and the integrity of the a sterile pathway for the dischar-
isolator. This is generally achieved ge of the stoppers.
by the use of transfer isolators or
rapid transfer ports (RTPs).
Transfer isolators are fixed to
the processing isolator. Sterilised
wrapped materials are loaded into
the transfer isolator and surfa-
ce decontaminated as described
above. Following biodecontamina-
tion the connection between the
transfer isolator and the proces-
sing isolator can be opened.
RTP’s utilise alpha-beta con-
nections which ensure that when
docked together the contamina-
ted sides of the RTP and the isola-
25
challenges
The drive towards the implementation of more modern, safer technologies in
aseptic pharmaceutical production encourages production sites and processes to
change rapidly to adapt to the increasingly stringent demands of regulatory bo-
dies. The use of physical separations (Restricted Access Barrier Systems) betwe-
en the operator and the product must be considered a primary solution in the
design of new filling lines or the renovation of existing ones

Annex 1 Annex 1

The use of appropriate technologies Isolators or RABS, which are different

(e.g. Restricted Access Barriers Systems
(RABS), isolators, robotic systems, rapid
microbial testing and continuous moni-
toring systems) should be considered to
increase the protection of the product
from potential extraneous sources of en-
dotoxin/pyrogen, particulate and micro-
bial contamination such as personnel,
materials and the surrounding environ-
ment, and assist in the rapid detection
of potential contaminants in the environ-
ment and the product.
technologies, and the associated proces-
ses, should be designed to provide pro-
tection through separation of the grade
A environment from the environment of
the surrounding room. The hazards in-
troduced from entry or removal of items
during processing should be minimized
and supported by high capability tran-
sfer technologies or validated systems
that robustly prevent contamination and
are appropriate for the respective tech-
nology.

26
solutions
AMTech® offers feasibility studies to align
production lines with the most innovative
design solutions. There is the option to
design custom-made RABS with glove
doors positioned in accordance with the
specific needs of each process.
Transfer ports are used to eliminate
contamination due to the transfer of
critical process materials through Grade
B areas into Grade A areas.

27
cleaning&disinfection
“For disinfection to be effective,
prior cleaning to remove surfa-
ce contamination should be per-
formed. Cleaning programmes
should effectively remove disin-
fectant residues. More than one
type of disinfecting agent should
be employed to ensure that whe-
re they have different modes of
action, their combined usage is
effective against bacteria and
fungi. Disinfection should include
the periodic use of a sporicidal
agent.”
Rotation of disinfectants
The latest version of Annex 1 adds
in paragraph 4.33: “more than one
type of disinfecting agent should
be employed to ensure that whe-
re they have different modes of
action, their combined usage is
effective against bacteria and
fungi”. Thus, it seems that more
products effective against bacte-
ria and fungi should be used in ad-
dition to a sporicidal agent. Is the
two-product rotation (one bacteri-
cide/fungicide and one sporicide)
– already in use by many pharma-
ceutical companies – acceptable?
When cleanroom disinfectants are
selected many users opt to select
two or more disinfectants. If a spo-
ricide is a regularly used agent,
then two disinfectants should be
sufficient.
Rotation is important for several
reasons:
• Most disinfectants do not have
a complete spectrum of activity
effective against all microorgani-
sms (spectrum of activity is the
More than one type of disinfecting agent should be employed to ensure that where they have different
modes of action, their combined usage is effective against bacteria and fungi.
28
ability of the disinfectant to kill
different types of microorgani-
sms and microorganisms which
are in different physiological sta-
tes). The disinfectants commonly
used are often effective against
vegetative cells but are not spo-
ricidal. To maintain an effective
contamination control, the elimi-
nation of bacterial endospores
through a sporicidal disinfectant
is recommended (these are so-
metimes referred to as high-level
disinfectants). Here a sporicide
would be used in rotation with a
non-sporicidal disinfectant.
• The disinfectants with the for-
mulations which are effective
against the greatest range of mi-
croorganisms are often expensi-
ve. With this, many manufacturers
use a general broad-spectrum di-
sinfectant daily or weekly with a
sporicidal disinfectant used we-
ekly or monthly (a decision often
based on the results of microbio-
logical environmental monitoring
and the characterisation of the
isolated microorganisms).
• Some disinfectants, such as spo-
ricides, are corrosive. While the
risk to surfaces can be reduced
through rinsing, rotation is so-
metimes undertaken in order to
reduce the risk of damage to cle-
anroom equipment and working
benches.
• Rotating two disinfectants can
also reduce the possibility of re-
sistant strains of microorgani-
sms developing. Whilst the phe-
nomenon of microbial resistance
is an issue of major concern for
antibiotics there are few data to
support development of resistan-
ce to disinfectant. Nonetheless, it
remains a regulatory expectation.
Thus, the reasons for rotation are
approached from either a desire
to widen the mode of action or
to address anticipated regulatory
concerns.
Effective cleaning & disinfection
Cleaning and disinfection affect
floors, walls, ceilings, but also ma-
chinery and equipment, and often
hard-to-reach surfaces. Which cle-
aning and disinfection methods
are most effective in this respect?
These depends on the circum-
stances, however good applica-
tion techniques are important, for
example with floor cleaning and
disinfection:
Either roll all of the floor surface
with a tacky roller to remove any
loose debris and fibers, moving
any equipment to one half of the
room nearest exit door, or wipe
the floor using a neutral detergent
solution and mop and bucket.
Use a use disinfectant impregna-
ted mop wipes and mops (alterna-
tively a mop with a ready prepa-
red disinfectant solution could be
used).
Using overlapping mop strokes,
wipe down half the floor area,
working methodically around the
area starting at furthest reach
point and working towards your-
self in a straight line. Replace the
mop wipe if the wipe becomes vi-
sibly contaminated or dry.
NOTE: Particular attention should
be paid to corners and edges.
Leave the disinfectant solution
on the surface for the manufactu-
rer’s recommended contact time
to allow the disinfectant to be ef-
fective.
Remove the mop head and discard
to waste.
Fit a second mop head with etha-
nol impregnated mop wipe (alter-
natively a mop with a ready pre-
pared alcohol solution could be
used).
After the allotted time, using the
fresh mop head wipe down using
overlapping mop strokes to remo-
ve any residue, working methodi-
cally around the area starting at
furthest reach point and working
towards yourself in a straight line.
Replace the mop wipe if the wipe
becomes visibly contaminated or
dry.
Use of fumigation
Where disinfection is difficult, is it
advisable to use fumigation?
Yes, and this is always advisable
following events that can lead to
high rates of contamination, such
as maintenance works or a facili-
ty shutdown. Fumigation-based
approaches are advantageous for
decontaminating the inside of bu-
ildings because they are easily di-
spersed, penetrate into difficult to
access areas, decontaminate the
interior volume of the space (not
only surfaces) and are less labour
intensive than many spray based
approaches. Optimal agents are
hydrogen peroxide and chlorine
dioxide.
Attention needs to be paid to the
concentration and dwell time, and
with controlling temperature and
humidity, as well as the mecha-
nism of dispersing the fumigant.
Each of these factors contributes
to the overall efficacy.
Fumigation-based approaches are advantageous because they are easily dispersed, penetrate into dif-
ficult to access areas, decontaminate the interior volume of the space (not only surfaces).
“The movement of material or
equipment from lower grade or
unclassified area to
higher-grade clean areas should
be subject to cleaning and disin-
fection commensurate with the
risk and in line with the CCS.”
The Contamination Control Stra-
tegy refers to risk-commensurate
cleaning and disinfection.
Validation
The validation of detergent and
disinfectant products, as well as
the validation of cleaning is a very
important step: what are the man-
datory activities?
Validation studies are broken
down into three sections: suspen-
sion tests (phase 1 and 2) to eva-
luate the reduction of a known
organism population inoculated
directly into a sample of the liquid
disinfectant, surface tests (phase
2) that assess a disinfectant’s abi-
lity to reduce the number of chal-
lenge organisms on an inoculated
surface, and field trials (phase 3)
a final assessment of the environ-
mental monitoring data to valida-
te the approach.
Suspension methods evaluate the
reduction= of a known organism
population inoculated directly
into a sample of the liquid disin-
fectant. Following inoculation and
the observation of a predetermi-
ned contact time, samples of the
inoculated substance are remo-
ved, neutralised and evaluated for
survivors as compared to an un-
treated control suspension. Since
the simulation of organism films
on the specific environmental sur-
face types are not accounted for
in this method, it is recommended
that suspension-based tests be
used only for initial disinfectant
screening purposes.
Surface testing involves aliquo-
ting onto a surface coupon a mix
of the challenge organism and,
where required, an interfering
substance (such a protein, to simu-
late dirty conditions). The surface
coupon will be fashioned from a
representative surface in the cle-
anroom (here several different
materials will require testing in or-
der to show how the disinfectant
performs). To this an amount of
the test disinfectant is added. The
solutions are left for the required
contact time. Once the contact
time has elapsed, the coupon is
transferred to a neutraliser solu-
tion. Then, as with the suspension
test once sufficient time has been
given for neutralisation, microbial
survivors are assessed by plating
out or filtering the disinfectant
neutraliser solution using a micro-
bial culture method.
A possible array of surfaces to
consider (this will be facility de-
pendent) are:
Stainless steel
Glass
Aluminum
Epoxy
Enamel
Acrylic
Mipolam
Vinyl
Hardwood
Plastic
29
Plexiglas
Chromium
Once the testing has been per-
formed, a report should be ge-
nerated that must conclude the
disinfectant efficacy test outco-
me in relation to the acceptance
criteria. If a disinfectant passes
the test, deeming it suitable for
use, cleanroom procedures must
reflect the practices adopted du-
ring the qualification, such as di-
sinfectant concentration, contact
time and method of application to
surfaces. The disinfectant’s final
adoption must then be based on
a follow-up assessment or field
trial, which includes an evaluation
of microbial counts and species
recovered.
Use of sterile concentrates
“Disinfectants and detergents
used in grade A and grade B are-
as should be sterile prior to use.
Disinfectants used in grade C and
D may also be required to be ste-
rile where determined in the CCS.
Where the disinfectants and de-
tergents are diluted / prepared by
the sterile product manufacturer,
this should be done in a manner to
prevent contamination and they
should be monitored for microbial
contamination. Dilutions should
be kept in previously cleaned con-
tainers (and sterilised where ap-
plicable) and should only be sto-
red for the defined period. If the
disinfectants and detergents are
supplied “ready-made” then re-
sults from certificates of analysis
or conformance can be accepted
subject to successful completion
of the appropriate vendor qualifi-
cation.”
Appropriate training for all personnel dealing with disinfectants is vital.
30
In-house prep: in the latest Annex
1 version there is more focus on
both in-house preparation of de-
tergents and disinfectants and on
the use of WFI water.
What do you think about the use
of sterile concentrates in cleaning
and disinfection procedures?
The use of a concentrate that is
made up or the use of ready-to-u-
se solutions, is a choice of each
user. Both methods are effective.
In particular, many companies,
switching to under-isolator pro-
duction, are considering the use
of concentrated products for Gra-
de C/D outdoor environments.
Would you recommend their use?
If so, with which procedure?
Yes, there are no concerns, espe-
cially within lower grades of cle-
anrooms. The important point is
to ensure the dilution is correct
and that the correct grade of wa-
ter is used.
Training of personnel
Personnel training: with the la-
test Annex 1, and the increasingly
stringent rules on cleaning and di-
sinfection, personnel training is a
key point. Are there also changes
in training methods with the new
Annex 1?
The Annex could go further with
training. Appropriate training
for all personnel dealing with di-
sinfectants is vital. This training
should include all relevant staff
including contract personnel wor-
king within the facility. Documen-
tation of this training is essential.
All personnel should understand
the importance of cGMP. For a new
employee, this training could be
part of their initial GMP induction.
A pre-determined program of trai-
ning should be in place and do-
cumented as well as a system to
measure the effectiveness of the
training. Personnel should all be
trained in good cleaning techni-
ques using the appropriate equi-
pment. All staff should be provi-
ded with appropriate clothing in
order to perform this operation,
e.g., cleanroom clothing in ma-
nufacturing areas with adequate
PPE. Procedures should also be in
place if spillage occurs with any of
these agents.
Training programs could include:
The importance of disinfection
in relation to GMP, its necessity
in preventing microbial prolifera-
tion, cross contamination and the
importance and significance of
good disinfection routines.
The importance of the correct
handling of disinfectants, whether
it is during the disinfectant’s pre-
paration, testing or use.
Basic microbiology and how con-
tamination is transferred in the
workplace.
An understanding of the proper-
ties of disinfectants and their cor-
rect application.
A refresher program should also
be in place, ensuring that once
personnel are trained, their know-
ledge is kept up to date. Many
companies now incorporate this
into regular GMP refresher trai-
ning, recommended to be under-
taken on a periodic basis. Ade-
quate documentation of these
updates in an employee training
record is essential.
Cleaning & Disinfection in Grade C
& D areas
Finally, the CCS in the new Annex 1
extends to all areas at risk of con-
tamination.
Also in paragraph 4.35 it is stated
that “disinfectants used in grade
C and D may also be required to
be sterile where determined in
the CCS”. This implies increased
attention also for non-sterile are-
as and for producers of cosmetics,
ointments and biological interme-
diates with low bacterial load etc.
For the latter, the challenge will
certainly be significant. What are
the essential activities to adapt?
Some good practices for the
adoption and use of disinfectants,
in accordance with GMP, are:
Written procedures should be in
place.
Responsibilities for cleaning
should be assigned. Often this is
interpreted as the need to have
independent cleaning staff sepa-
rate from those involved in pro-
duct manufacture.
Staff must be trained in cleaning
In compliance with GMP, cleaning and disinfection procedures should be included in any audit program
as it maintains assurance that contamination control procedures are adequate and in control.
techniques and have a training re-
cord.
Details of cleaning frequencies,
methods, equipment, and mate-
rials must be recorded in written
procedures. This may relate to an
approved supplier specification.
The cleaning of equipment and
materials must take place at regu-
lar intervals.
Inspection of equipment for cle-
anliness before use should be part
of routine operations.
A cleaning log should be kept. The
purpose is to keep a record of the
areas cleaned, agents used, and
the identity of the operator.
The microorganisms isolated (the
microbiota) from environmental
monitoring programs should be
examined for resistant strains.
Some isolates from these reviews
should be incorporated into disin-
fectant efficacy studies.
The monitoring for microbial con-
tamination in disinfectant and de-
tergent solutions should be perio-
dically undertaken.
The storing of disinfectant and
detergent solutions should be for
defined (and short) periods.
Room use should be recorded
after each operation.
There should be a technical agre-
ement with the company who sup-
plies the disinfectant. Ideally the
disinfectants purchased should be
lot tracked.
SOPs containing references to
disinfectants, cleaning agents,
materials and equipment used
and calibration of equipment are
accessible. Here the cleaning and
disinfection methods are clearly
defined.
The SOP should include cleaning
method details e.g., wiping front
to back or top to bottom with
overlapping strokes.
Cleaning method, e.g., double
bucket, rinsing wipe action, con-
tact time and cleaning of cleaning
materials must also be included in
the
Documentation of rotation, ratio-
nale, and frequency, needs to be
set out.
Cleaning logs also need to be in
place and available.
In compliance with GMP, clea-
ning and disinfection procedures
should be included in any audit
program as it maintains assuran-
ce that contamination control
procedures are adequate and in
control.
31
challenges
Regulatory authorities set limits for mi-
crobial contamination by class of clean-
room in order to provide the necessary
end product protection and ensure these
standards are achieved.
Cleaning programs should be ef-
fective in the removal of disinfectant
residues.
Disinfectant Residues Impact
The occurrence of residues may af-
fect the integrity of materials and
products used in the manufacturing
areas.
The cleaning process should be va-
lidated so that it can be demonstra-
ted that it prevents chemical and
particulate contamination of the
product during the process and pri-
or to disinfection.

Where microbial testing of surfaces

in cleanrooms is carried out, vali-
dation should be performed on the
test method to confirm that saniti-
zing agents do not influence the re-
covery of microorganisms.

Residues require a removal step during the contamination con-

trol management process resulting in lost production time.
Should residues be ignored, a deep clean may be needed or even
a shutdown, resulting in a serious impact in productivity.

32
 solutions
CLEAN
SMALL SURFACES

DISINFECT
AREAS

REMOVE
LARGE SURFACES

DISINFECT

33
hands disinfection
The highest risk within a cleanro-
om is personnel. Despite this, the
new Annex 1 seems to give not
enough attention to such an extre-
mely delicate operation as gloved
hand disinfection. Human error,
its imponderability, and especially
the absence of traceability make
this a high-risk operation. What is
your opinion about this “lack” wi-
thin the new Annex 1?
Hand disinfection is essential for
aseptic working. The essential
elements are:
1. Frequency
2. Coverage
3. Time
4. The correct disinfectant
15 to 30 seconds is necessary to
reduce the typically maximum le-
vels of bioburden found in a clean-
room environment.
In gloved hand disinfection, the
risk of cross-contamination can
be averted through the use of
automated devices with specific
characteristics. What are they?
There are devices on the mar-
ket that can make the hand di-
sinfection process more reliable.
Such devices, which can also digi-
tally capture data, can control the
quantity of disinfectant dispensed
and time how long the employee
spends undertaking the glove sa-
nitisation step.
Traceability can be useful for the
quality assurance assessment
and to provide reassurance for
the production manager. This is
another area where digital tech-
nology can be advantageous. De-
vices can record how often an in-
dividual operator accessed a hand
sanitisation station. This can be
important to verify, for example,
that hands were disinfected prior
to performing interventions, as an
example.

With frequency, gloves need to be In addition, devices can also indi-

disinfected before and after each
aseptic operation (with the addi-
tional requirement in Annex 1 to
change gloves after an interven-
tion, one finger dabs have been
taken).
It is also important that personnel
perform regular glove sanitisa-
tion, such as every 10 to 15 minu-
tes even when idle.
With coverage, the application
technique is important. The vo-
lume should be standardised and
it should be sufficient to cover all
parts of the glove. The technique
used to rub the disinfectant (whi-
ch is normally 70% IPA) across
the palm and fingers is important
and this should be well practiced.
In terms of time, as with other
forms of disinfection, the contact
time is important. Studies show
cate the expiry of the disinfectant
and warn when the solution is
running out.
Another advantage is that de-
vices that be position close to
where they are needed, to make
access convenient for personnel
and avoid the need to criss-cross
too often, helping with the logical
flow which is needed to underpin
aseptic operations.
However, even with sophisticated
automated devices, personnel still
need to be trained in the impor-
tance of glove sanitisation and
with sanitising their hands at the
appropriate time point.
Traceability of hand disinfection
is not required in any regulations.
Doesn’t this seem a contradiction
to the very meaning of the Conta-
mination Control Strategy?

Traceability can be useful for the quality assurance assessment

and to provide reassurance for the production manager.

34
35
challenges
7.16 Gloves should be re-
gularly disinfected du-
ring operations.

ANNEX 1

The number of inspections by the authorities regarding the

accuracy and adequacy of data is continuously increasing. The
regulatory body requires data integrity fully in compliance with
GMP rules and regulations that companies have to guarantee
with continuous process improvement.

Prevent risk of cross contamination

Trace activity of gloved hand sanitisation
Improve automation avoiding human error

36
solutions
My&Clean+ is a system that tra-
ces every gloved hand sanitisa-
tion, their correct execution and
frequency, also notifying the ne-
cessity of repeating the opera-
tion in case a failure occurs.
AM Instruments has created the
ultimate system that simulta-
neously solves the problems of
consistency and reliability of the
data and the risk of cross-conta-
mination.

TOTAL ELIMINATION
OF CROSS
CONTAMINATION RISK
The liquid is released directly
from the original bottle housed
in the device, an innovation that
cuts down the need for bottle
handling, eliminating the risk of
cross contamination. Sterility of
sanitisation product is guarante-
ed by the bottle itself, which ori-
ginal packaging is not compromi-
sed at all during the housing and
use of the device.

TOTAL TRACEABILITY
My&Clean+ can be installed in-
dividually or as multiple inter-
connected devices. The device
identifies the operator thanks to
a silicon RFID bracelet, then vali-
dates and records the hand sani-
tisation, with no need for direct
contact with the machine.
It is also possible to check the le-
vel of liquid available in the trig-
ger directly on the display.
From a central remote console
My&Clean+ Station, you can ma-
nage multiple devices, register
new operators, check the level of
liquid, view and generate repor-
ts.

37
missing in Annex 1
We asked Tim Sandle, In the light of his especially with isolators. The importance of separa-
tive devices and exclusion personnel should be given
experience, if there are any topics that
more focus in my opinion.
the new Annex1 has left out or dealt with
ambiguously but that he considers es- It was also disappointing that the emphasis upon
sential for maintaining sterility in drug rapid microbiological methods was less strong. As
an industry we need to be pursuing better ways to
manufacturing. verify the robustness of our contamination control
strategy (and in real time).
In terms things that are missing, the Annex makes
many references to ‘risk’ but very few to hazards.
There is also some ambiguity, such as with many re-
Hazards are the elements that if they are sufficient-
ferences to “ampoules and vials“. I think the term
ly likely and sufficiently severe, they will lead to
“primary packaging containers” is better, with a defi-
unacceptable risks. I think this places the Annex out
nition. This would acknowledge forms of processing
of alignment with ICH Q9, where ‘hazard identifica-
like bow-fill-seal and include any other containers in
tion’ is the important step in order to undertake a
addition to ampoules and vials, as well.
risk assessment. This is especially so with ICH Q9
having recently undergone an update to introduce
In the personnel section I questioned the portion of
more formality into the risk management process.
the text that states shoes should be disinfected – I
do not think the disinfection of shoes would pass a
With environmental monitoring, while finger dabs
disinfectant efficacy test; instead I’d recommended
and gown plates are now included, swabs remain
the use of captive shoes.
missing from the text. This makes little sense as
swabs are required for narrow and irregular surface
Another ambiguity is with process validation; this
monitoring and for sampling critical surfaces like fil-
could be scoped out. The term process validation is
ling needles.
often understood as manufacturing process valida-
tion but should be enlarged to all relevant processes
Annex 1 states that environmental monitoring sam-
in this context. I’d recommend: “process validation
pling methods and equipment used should be fully
including manufacturing, cleaning, decontamination
understood, with the recovery efficiency of the sam-
sterilisation, transport.”
pling methods chosen qualified. I prefer: “The reco-
very efficiency of the sampling methods should be
For personnel movement, with “The use of separa-
understood.” This is because ‘qualified’ brings with it
te change rooms for entering and leaving the grade
a level of analytical method validation that becomes
B area is desirable.” I support separate routes, but
challenging for imprecise methods.
I felt that ‘desirable’ was too vague a term. There
were other areas where I think we need tighter lan-
For cleanrooms, the minimal differential pressure
guage.
is defined for adjacent cleanrooms of different gra-
des. However, a minimal air exchange rate for clean
With disinfection, the confusing reference to ‘resi-
rooms is no longer quoted in the text. While this is
stant strains’ remains. Here the phrase ‘’develop-
connected to energy saving, I think that a minimal
ment of resistant strains “is often misinterpreted as
standard should continue to be set. The old guidance
development of acquired resistance (a theory which
of ~20 air changes provided a point of guidance.
has largely been discredited). We assume the inten-
tion of this sentence is to draw attention to recovery
There are some issues that have been downplayed
of organisms with innate resistance. I’d proposed
trough the different drafts and towards the publica-
that the word development be deleted “develop-
tion of the final version of the Annex. For example,
ment”, so that the text changes to read: ‘‘Monitoring
there was a greater focus on separative devices,
should be undertaken regularly in order to show the

38
effectiveness of the disinfection program and to de-
tect the recovery of resistant and/or spore forming
strains.” ANNEX 1
IS THERE ANYTHING MISSING?
In terms of water sampling, the Annex states that
users should ensure that at least one representati-
ve sample is included every day of the water that
TRACEABILITY OH HANDS
is used for manufacturing processes. This could be DISINFECTION
tightened. I felt that for WFI systems a sample at the
end of the distribution loop each day that the water RISK & HAZARD
is used should also be sampled and tested.
ENVIRONMENTAL
MONITORING: MISSING
Although I have made some points of criticism, it re- SWABS!
mains that Annex 1 is a good document and it is a
step forwards. The Annex directs us to embrace a CLEANROOM AIR
EXCHANGE
holistic, proactive and risk based methodology ba-
sed on process understanding and risk knowledge
and a Quality Risk Management (QRM) approach to SEPARATIVE DEVICES
contamination control that requires documenting in
a CCS.
RAPID MICROBIOLOGY

AMPOULES AND VIALS

RAPID MICROBIOLOGY

PROCESS VALIDATION

DEVELOPMENT OF
RESISTANT STRAINS

WATER SAMPLING

39
future technologies
There are so many technologies that are set to transform life sciences. I’ll just pick three examples: artifi-
cial intelligence (AI) and machine learning; robotic process automation; and edge computing. With AI, this
is being used with drug discovery and this has allowed the screening of compounds to be accelerated and
for drug interactions to be examined at a far faster scale. AI has also helped with resupposing medicines,
that is looking for established medicines that have been used to treat one type of diseases and seeing if
the medicine can be used against something different entirely. AI has also assisted with the personalised
medicine initiative, which is concerned with tailoring individual medicines for specific patients. Anti-cancer
medications is a prime example.
With robotic process automation, this refers to the use of software with artificial intelligence and machine
learning capabilities to handle high-volume, repeatable tasks that previously required humans to perform.
Such technology can help to simplify some production processes, especially where there are high data vo-
lumes and the potential for high error rates.
For the third area, edge computing, this is about processing data as close to the source as possible, which
reduces both latency and bandwidth use. This concept is seen as critical for furthering the Internet of Thin-
gs and for driving the development of autonomous vehicles. Edge computing is especially useful in cases
where a lot of data is generated. The approach allows for the successful triage of data locally so that some
of it is processed locally, leading to faster response times. This type of technology is useful in biotech, where
rapid adjustments are required. Edge computing can provide insight to bioengineers about the status of a
machine, with the ability to action data in real-time and providing “always on” availability.

Tim Sandle

40
41
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