Requirements for
Environmental
Monitoring
Tracy Moore
2
• What are the GMP requirements?
– GMP Basics and monitoring methods
– Designing a monitoring programme
– Incubation strategies
– Excursion reporting/investigation
– Trending
– Weaknesses
• Find the right position in the isolator system
– Know your machine and isolator!
– Airflows; air visualization studies
COPYRIGHT © PDA 2023
3
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
4
Environmental Monitoring
GMP basics
• For the purposes of this training session current expectations will be
presented but quotes from the Annex 1 (August 2023) will be used.
• Traditionally referred to as “viable and non-viable” particles
• Viable = microbiological methods
• Non-viable = particle counting equipment
Non-viable is often thought of as only ‘inert’ particles but could also include
dead bacterial cells which wouldn’t be recovered by viable monitoring
methods…
The term has been updated to ‘total particle count’ in the 2023 Annex 1
5
Environmental Monitoring
GMP basics
• Section 9 of Annex 1 (August 2023)
• ‘Environmental and process monitoring programme’
• part of the overall CCS
• used to monitor the controls designed to minimize the risk of microbial
and particle contamination
i. Environmental monitoring – total particle.
ii. Environmental and personnel monitoring – viable particle
iii. Temperature, relative humidity and other specific characteristics.
iv. APS (aseptically manufactured product only)
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Environmental Monitoring
GMP basics
9.2 This programme is typically comprised of the following elements:
i. Environmental monitoring – total particle.
ii. Environmental and personnel monitoring – viable particle.
iii. Temperature, relative humidity and other specific characteristics.
iv. APS (aseptically manufactured product only).
9.3 The information from these systems should be used for routine batch
certification/release and for periodic assessment during process review or
investigation.
• Terminal sterilisation
• Aseptic processes
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Environmental Monitoring
GMP basics
Annex 1; 9.4
The purpose of the EM programme is….
i.
ii.
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Environmental Monitoring
GMP basics
Annex 1; 9.4
The purpose of the EM programme is….
i. Provide assurance that cleanrooms and clean air equipment continue to
provide an environment of appropriate air cleanliness, in accordance
with design and regulatory requirements.
ii. Effectively detect excursions from environmental limits triggering
investigation and assessment of risk to product quality
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Environmental Monitoring
GMP basics
Annex 1; 9.4
Risk assessments performed to establish [Comprehensive];
• Sampling locations,
• Frequency of monitoring,
• Monitoring methods and
• Incubation conditions
– (e.g. time, temperature(s), aerobic and/or anaerobic conditions)
Techniques to assist:
• Touchpoint analysis (perform the process and tag items touched each
time)
• Traffic flow analysis (aka heat maps)
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Environmental Monitoring
GMP basics
Annex 1; 9.4
Risk assessments based upon detailed knowledge of;
• The process inputs and final product,
• The facility,
• The equipment,
• The criticality of specific processes and steps,
• The operations involved,
• The routine monitoring data,
• The monitoring data obtained during qualification and
• The knowledge of typical microbial flora isolated from the environment.
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Environmental Monitoring
GMP basics
• Annex 1; 9.22
• Where aseptic operations 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 demonstrated not to have a detrimental impact on grade A
and B airflow patterns.
Cleanroom and equipment surfaces should be monitored at the end of
an operation
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Environmental Monitoring
GMP basics
• Continuous monitoring – should be exposed during all processing activity
in grade A (and often grade B) (or continuous active air sampling)
Continuous viable air monitoring in the grade A (e.g., air sampling or settle
plates) should be undertaken for the full duration of critical processing,
including equipment (aseptic set-up) assembly and filling operations.
A similar approach should be considered for grade B cleanrooms based on
the risk of impact on the aseptic processing.
The monitoring should be performed in such a way that all interventions,
transient events and any system deterioration would be captured, and any
risk caused by interventions of the monitoring operations is avoided -
Annex 1; 9.24
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Environmental Monitoring
GMP basics
First Air Principles
• First air refers to filtered air that has not been interrupted prior to
contacting exposed product and product contact surfaces with the
potential to add contamination to the air prior to reaching the critical
zone.
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Environmental Monitoring
methods - Viable
15
Environmental
Monitoring
• Annex 1; 9.22
• Where aseptic operations 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 demonstrated not to have a
detrimental impact on grade A and B
airflow patterns.
– Cleanroom and equipment surfaces
should be monitored at the end of an
operation.
16
Environmental Monitoring
methods - Viable
A1;9.23
Viable particle monitoring should also
be performed within the cleanrooms
when normal manufacturing
operations are not occurring (e.g., post
disinfection, prior to start of
manufacturing, on completion of the
batch and after a shutdown period),
and in associated rooms that have not
been used, in order to detect potential
incidents of contamination which may
affect the controls within the
cleanrooms.
17
Environmental Monitoring
methods
• Viable monitoring methods
• Settle plates
• Contact plates
• Swabs
• Active air sampling
• Rapid methods
18
Settle plates
• Typically 90mm plates
• Tryptone Soya Agar (TSA) – general use, can recover both
bacteria/moulds
• Sabaraud Dextrose Agar (SDA) – specifically for yeasts and moulds
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Settle Plates
• May need to include neutralisers depending on application (e.g.
monitoring of antibiotic powder-filling processes)
• Passive monitoring method
• Quantitative
• Exposure time four-hour maximum or duration of session if less than four
hours (may need to be shorter time / more frequent changes to avoid
drying out depending on location – but same limits still apply)
20
Contact plates
• Typically 55mm plates (~25cm2)
• Typically need to include neutralisers (and validate – Annex 15, 9.3)
• Media is raised in the centre to allow full contact with the flat surface
being monitored
• Quantitative
• Easy to use but only good for flat surfaces
• Residues need to be carefully removed
• NOT to be used for finger dabs…
21
Swabs
• Good for uneven surfaces and nooks & crannies
• Need to be moistened prior to use (or pre-moistened)
• May or may not be quantitative
• Can be placed directly in media (qualitative)
• Can be plated out (quantitative – but need to validate recovery
efficiency)
22
Active air sampling
• Sample a known volume of air (equipment needs routine calibration)
• Quantitative, but only a relatively small ‘snapshot’ of the overall volume
• Mobile samplers difficult to sanitise
• Need to sanitise (or sterilise for critical zones) the heads before use
• Variation in efficiency between different types (d50 value)
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Personnel monitoring
• 9.25 A risk assessment should evaluate the locations, type and frequency of personnel
monitoring based on the activities performed and the proximity to critical zones
• Monitoring should include
– sampling of personnel at periodic intervals during the process.
– Sampling of personnel should be performed in such a way that it will not compromise the
process.
– Particular consideration should be given to monitoring personnel following involvement in
critical interventions (at a minimum, gloves, but may require monitoring of areas of gown
as applicable to the process) and on each exit from the grade B cleanroom (gloves and
gown).
– Where monitoring of gloves is performed after critical interventions, the outer gloves
should be replaced prior to continuation of activity.
– Where monitoring of gowns is required after critical interventions, the gown should be
replaced before further activity in the cleanroom.
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Personnel monitoring
• 9.25 A risk assessment should evaluate the locations, type and frequency of personnel
monitoring based on the activities performed and the proximity to critical zones
• Monitoring should include
– sampling of personnel at periodic intervals during the process.
– Sampling of personnel should be performed in such a way that it will not compromise the
process.
– Particular consideration should be given to monitoring personnel following involvement in
critical interventions (at a minimum, gloves, but may require monitoring of areas of gown
as applicable to the process) and on each exit from the grade B cleanroom (gloves and
gown).
– Where monitoring of gloves is performed after critical interventions, the outer gloves
should be replaced prior to continuation of activity.
– Where monitoring of gowns is required after critical interventions, the gown should be
replaced before further activity in the cleanroom.
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Personnel monitoring
– Particular consideration should be given to monitoring personnel following involvement in
critical interventions (at a minimum, gloves, but may require monitoring of areas of gown
as applicable to the process) and on each exit from the grade B cleanroom (gloves and
gown).
– Finger dabs (all fingers plus thumb on each hand)
• Limits per glove (5 digits), not all 10 (no averages)
• Same limits for the Isolator gloves
• Need to understand which direction glove is used.
• Left hand / right hand considerations – 90 mm/ 150mm settle plate?
• In the grooves - swabs?
• A swab for all? Recovery?
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Personnel – finger dabs
and gowns
• Finger dabs (all fingers plus thumb on each hand)
• Should be taken after direct interventions to grade A zone (without
spraying hands immediately before)
• Also taken on exit from the cleanroom
• Gloves should be changed if work session continues
• Limits per glove (5 digits), not all 10 (no averages)
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Personnel – finger dabs
and gowns
• Gown monitoring
• Routine monitoring of gowns should be
performed
• Typically on exit from the cleanroom –
don’t want residues of growth media left
on gowns during a working session
• Typically forearms, chest and head for
routine monitoring
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Rapid microbiological
methods
• Quicker time to a result but need robust validation
• Not widely adopted – fears of regulatory non-acceptance…
• Examples include:
• ATP-bioluminescence
» Luciferin/luciferase reaction in growing cells
» Amount of light equates to the number of cells present
» Can also detect non-microbial ATP
• Aurofluorescence detection
» All living cells fluoresce under blue light
» Regular scanning of plates during incubation for early colony detection
• Colourimetric growth detection
» Limited range of organisms detected but good where specific absence is important
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Methods – total
particle monitoring
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Methods – total
particle monitoring
• Laser particle counters used for total particles
• Tubing length should typically be less than 1m
• No. bends minimised
• Bend radii per manufacturer’s specifications
• Isokinetic sampling probes
• Continuous monitoring required in grade A
• Consider contamination risks from mobile counters
31
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
32
Monitoring locations
• All EM and the associated locations should be based on an
appropriate risk assessment:
• Initial facility qualification
• Ongoing monitoring, trending and knowledge development
• Assessment should include:
• Knowledge of process and risks – e.g. review of number of
manipulations in each zone, use of process heat maps etc.
• Smoke studies
• Historical review
• Common sense (often missed!)
33
Monitoring locations
• Review of vectors that breach the ‘layers’ and other risks, and need
to monitor accordingly:
• Process flows
• Equipment
• Materials
• People
• Important to remember that monitoring must however not
compromise the process
34
34
Monitoring locations
and Contamination
Control Strategy
• Will connect
– all SOPs
– Risk assessments
– Discuss the ’approach’ to EM
– The HVAC arrangements
– The filling environment
35
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
36
Incubation strategies
• Not a one size fits all
• Baseline expectation where only one media is used (TSA):
• Aerobic
• Incubate at 20-25°C (typically 3-5 days)
• Followed by 30-35°C (typically 2-4 days)
• If not, needs to be a suitable scientific rationale, based on
statistically valid data from the site’s own facility
• If using separate media (not common) then respective incubation
conditions apply – TSA 30-35°C; SDA 20-25°C
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Incubation strategies
• But monitoring and incubation strategy needs to be based on
process knowledge and risk
• Anaerobes?
• Psychrophiles or cryophiles?
• Wet processes?
• Specific organisms?
• Also remember the incubators should be qualified and subject to
temperature mapping
38
Reading the plates
• Magnifying colony counters should be used – expecting low
counts, so need the best opportunity to see them
• Manual process and potential for DI issues:
• Has this been identified as a risk within the site’s DI review?
• e.g. are second checks performed on at least zero count
plates?
• How are the results recorded – paper / direct data entry?
39
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
40
Identification
• 9.31 Microorganisms detected in
the grade A and grade B areas
should be identified to species
level and the potential impact of
such microorganisms on product
quality (for each batch
implicated) and overall state of
control should be evaluated.
• Consideration should also be given to the identification of microorganisms
detected in grade C and D areas (for example where action limits or alert levels
are exceeded) or following the isolation of organisms that may indicate a loss of
control, deterioration in cleanliness or that may be difficult to control such as
spore-forming microorganisms and moulds and at a sufficient frequency to
maintain a current understanding of the typical flora of these areas.
41
Excursion reporting /
investigations
• Principles should be the same for all investigations:
• Timely
• Appropriate investigation to determine root cause
• Documented
• Commensurate to risk
• Appropriate CAPA
42
Excursion reporting /
investigations
• Common issues in general:
• Risk assessment doesn’t include an appropriate ‘bracket’
• Inspectors routinely see “there was an excursion but all other data ok”
• “EM the day before and day after OK so must be OK” (but don’t know
root cause)
• Initial risk assessment to understand the possible or actual risk to
other products not done quickly
• Not considering the wider picture and trends appropriately
43
Excursion reporting /
investigations
• Common issues for smaller sites:
• Contract labs – receipt of initial report can be slow
• Generally not timely – time to sub culture for ID
• IDs often also outsourced which adds to the timelines
• Documented investigations weak
• Risk assessments often based on literature (e.g. Bergey’s
Manual) with insufficient microbiology expertise in-house
• All too often “isolated excursion and all other data from that
session were ok”…
44
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
45
Limits and trending
• Limits for monitoring
– Action limits
– Annex 1 limits are used
– Tighter action limits can be set for lower grades based on
historical performance
– Alert limits
– Should be set based on historical data – science
– Do not just set at 50% action limit
– Limits should be regularly reviewed based on site data
– If the trend review suggests the proposed alert limits are
widened, this should trigger an investigation as it indicates a
decline in performance
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Trending
Should include….
i. Increasing numbers of excursions from action limits or alert levels.
ii. Consecutive excursions from alert levels.
iii. Regular but isolated excursion from action limits that may have a
common cause, (e.g. single excursions that always follow planned
preventative maintenance).
iv. Changes in microbial flora type and numbers and predominance of
specific organisms.
- Particular attention should be given to organisms recovered that
may indicate a loss of control, deterioration in cleanliness or
organisms that may be difficult to control such as spore-forming
microorganisms and moulds.
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Trending
• Ideally data should be reviewed in different ways, appropriate to
what is trying to be understood from the data
• Should include a microbiologist or appropriately experienced
person
• e.g. by area / workstation / activity / operator
• All provide different but meaningful data and sometimes issues
may be missed by the way the data are presented
• e.g.:
“0.1% of finger dab monitoring shows recovery so that’s a low
level”
If 90% of those recoveries were from one individual operator this
would be missed without appropriate review
48
What are the GMP
requirements?
GMP Basics and monitoring methods
Designing a monitoring programme
Incubation strategies
Excursion reporting/investigation
Trending
Weaknesses
COPYRIGHT © PDA 2023
49
Weaknesses
• Microbiological monitoring is actually very inefficient…
– Only a small fraction of air and surfaces that can impact product can
be sampled
– Monitoring techniques have low recovery efficiency
– Limited range of organisms can be isolated using common media and
incubation conditions
– A positive recovery is therefore a significant event
– A negative result may therefore be misleading…
50
Find the right position in the
isolator system
Know your machine and isolator!!
Airflows; air visualization studies
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51
Know your machine
and isolator
• Location, Location, Location!!!
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Know your machine and isolator
• All EM and the associated locations should be based on an
appropriate risk assessment:
• Initial facility qualification
• Ongoing monitoring, trending and knowledge development
• Assessment should include:
• Knowledge of process and risks – e.g. review of a number of
manipulations in each zone, use of process heat maps, traffic
maps, touch point analysis etc.
• Smoke studies
• Historical review
• Common sense
53
Know your machine and isolator
• Review of vectors that breach the ‘layers’ and other risks, and need
to monitor accordingly:
• Process flows
• Equipment
• Materials
• People
• Important to remember that monitoring must however not
compromise the process
54
Know your machine and isolator
Annex 1;4.19
a. The design of open isolators should ensure Grade A conditions with
first air protection in the critical zone and unidirectional airflow that
sweeps over and away from exposed products during processing
b. The design of closed isolators should ensure Grade A conditions
with adequate protection for exposed products during processing.
4.20 The background environment for isolators should ensure the risk
of transfer of contamination is minimized.
open isolators - Grade C
closed isolators - Grade D
Based on RA & justified in CCS.
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2/29/2024
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Know your machine
and isolator
• Zones
Bowl Bowl
Glass
accumulation
zone
Filling Stoppering Over-seal
56
Know your machine
and isolator – 3D This Photo by Unknown Author is licensed under CC BY-NC-ND
Machine bed
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57
Find the right position in the
isolator system
Know your machine and isolator!!
Airflows; air visualisation studies
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58
Air visualisation studies
• WFI generators; natural buoyancy
• Nozzles
– Size & Shape
– Direction
• Needs a carefully thought-out protocol
– Performed by experienced personnel
– Multiple cameras or multiple shots
• A protocol & report that captures the
activity accurately
– Includes the test scripts
– Includes review of the video.
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Air visualisation studies
Isolator door opened as part of
set up to load indirect product
Is the UDAF
contact parts. Open isolator in
on when the
Grade C
Smoke door is open
studies – to Grade C?
air ingress
and egress
‘Mouse hole’
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‘First Air’ and VHP
considerations
Filtered air that has not been interrupted by items such as operators with
the potential to add contamination to the air prior to reaching the critical
zone
• Hanging items in isolator for VHP considerations – fixed to avoid
contact with each other but…
– Is the item over open vials during filling activities?
– Over the stopper bowl and stoppers?
61
Summary
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62
Summary
• Different monitoring methods form part of the overall tool kit
• EM should be based on good knowledge and understanding of
both the process and the facility and the isolator!
• Any recovery, particularly from grade A should be unusual and
investigated appropriately
• Monitoring methods are not very efficient so understanding the
limitations is important
• Just because there was a zero count isn’t the end of the sterility
assurance story…
• Each zone should be monitored V + TP
• The location should be 3D and be capable of detecting risk to
open units
• Air visualisation studies should be used in assessing location
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Summary
What How When Where Batch
related?
Facility; Active air, RA frequency Air, walls, Not usually
corridors, settle, (inc. 9.23 floors
PAL, MAL rodacs points)
Filling Room All +TP To cover the Air, RA Yes*
duration of the locations
Filling Line All +TP fill (inc. set-up) Air, RA Yes *
+ RA locations
frequency
Operators Contacts, After critical Gloves and Yes*
Engineers finger dab intervention gown Yes*
plates On each Exit
Micro staff Yes*
QA staff On each Exit Not usually
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