Annex 1 Environmental Monitoring

Common Questions and Answers

Mark Hallworth,

Confidential and proprietary

 EU GMP Annex 1 - 2023

• Previous two presentations covering:

• Annex 1 – What has changed / what’s new
• Deadline for operation: August 2023 – 1 Year from release
• Document is sectioned differently
▪ Certification (section 4) and Monitoring (section 9) are separated and differentiated.
• Contamination Control Strategy (CCS)
▪ Central holistic approach to how each aspect of contamination interacts with the facility as a whole – new paradigm
• Quality Risk Management (QRM)
▪ Central principle to defining processes, operations, limits and ties to CCS to balance process against risk
• Environmental Monitoring is essentially the same with a few enhanced descriptions to better align with QRM

• Annex 1 – Similarities / what’s not changed

• Fundamental principle of environmental monitoring
• Difference between Room certification and Monitoring
• Risk Assessment to determine locations and frequency
• Limits, especially 5µm in Grade A
• Tubing length requirements
 Focus sections of document

1. Scope 8. Production and specific technologies

Includes additional areas (other than sterile products) where the general Discusses the approaches to be taken with regards to aseptic and terminal
principles of the annex can be applied. sterilization processes. Discusses approaches to sterilization of products,
equipment and packaging components. Also discusses different technologies
2. Principle such as lyophilization and Form-Fill-Seal where specific requirements apply.
General principles as applied to the manufacture of sterile products.
9. Viable and non-viable environmental and
3. Pharmaceutical Quality System (PQS) process monitoring
Highlights the specific requirements of the PQS when applied to sterile This section differs from guidance given in section 4 in that the guidance
products. here applies to ongoing routine monitoring with regards to the design of
systems and setting of action limits alert levels and reviewing trend data.
4. Premises The section also gives guidance on the requirements of Aseptic Process
Simulation (APS).
General guidance regarding the specific needs for premises design and
guidance on the qualification of premises including the use of Barrier
Technology.
10. Quality control (QC)
Gives guidance on some of the specific Quality Control requirements relating
5. Equipment to sterile products.

General guidance on the design and operation of equipment. 11. Glossary

6. Utilities Explanation of specific terminology.
Guidance with regards to the special requirements of utilities such as water,
gas and vacuum.

7. Personnel
Guidance on the requirements for specific training, knowledge and skills.
Also gives guidance to the qualification of personnel.
 Top Questions From Webinars
• Classification and Monitoring Differences
• Which occupancy state – “at-rest” or “in-operation” to be defined for the activity not directly related to manufacturing (e.g., inventory of raw
materials)?
• Is it correct to understand that the EM sampling personnel is considered not “personnel” in the following paragraph from GMP? (e.g., personnel to
perform surface/air sampling)

• Maximum Tubing length and limitation

• How to justify/validate piping (hoses) longer than 1m for particle counter sampling points?

• Alert and action Levels/Limits

• How long should an EM team collect data, to revaluate sampling frequency?
• Annex 2008 stipulated 1 cubic meter sampling volume; Annex 2022 does not indicate a number. What is an acceptable number?
• For new builds or processes (no historical data) how do you set alert levels?

• Continuous Microbial Monitoring

• What is the best way of implementing a continuous monitoring strategy?
• Are both settle plates and active air samples required

• Other Applications
• How does this apply to Cell and Gene Therapy?
• Any need for continuous NVP sampling in Grade C/D rooms, where manufacturing takes place but are not critical areas or connected to Grade B
rooms/hoods?
• How does the annex 1 apply to non-sterile production?
 1. In Operation and At Rest Definition
• The questions associated with occupancy are more about an understanding of certification vs monitoring, than the operational state.

• There are two sources for what occupancy state requires. ISO14644-1 and Annex 1,

• ISO 14644-1
• 3.3 Occupancy states
• 3.3.1 as-built
▪ condition where the cleanroom or clean zone is complete with all services connected and functioning but with no equipment, furniture, materials or personnel present
• 3.3.2 at-rest
▪ condition where the cleanroom or clean zone is complete with equipment installed and operating in a manner agreed upon, but with no personnel present
• 3.3.3 operational
▪ agreed condition where the cleanroom or clean zone is functioning in the specified manner, with equipment operating and with the specified number of personnel
present

• EU ANNEX 1
• 4.29 Cleanroom classification should be carried out in the “at rest” and “in operation” states.
• i. The definition of “at rest” state is the condition whereby the installation of all the utilities is complete including any functioning HVAC, with the main manufacturing
equipment installed as specified but not operating and without personnel present in the room.

• ii. The definition of “in operation” state is the condition where the installation of the cleanroom is complete, the HVAC system fully operational, equipment installed and
functioning in the manufacturer’s defined operating mode with the maximum number of personnel present performing or simulating routine operational work.

• It is important to understand that these “states” in BOTH documents are intended for classification purposes and reflect the two extremes of room
functionality – test the room with no personnel working, but equipment turned on – and test the room in the most severe case possibly, i.e., room
working, machine operating and product flowing (either real, or simulated). – they DO NOT represent the monitoring conditions of the room – only the
test parameters, controlled conditions to put two points on a graph.
 Occupancy State
• The only requirement for operational/at rest differentiation is that the room achieves the at rest conditions within 15 minutes;
which basically means the personnel leave, and the room air exchange rate can reestablish the defined at rest state – for
monitoring the client can state what that mean, as it is Risk based not classification based.
1. Which occupancy state – “at-rest” or “in-operation” to be defined for the activity not directly related to manufacturing (e.g.,
inventory of raw materials)?
If inventory is being performed, then the operational state is inventory performance using the required number of personnel and
appropriate alarms should be set. This is an operational state and different to the natural at rest state of the room.
2. Is it correct to understand that the EM sampling personnel is considered not “personnel” in the following paragraph from GMP? (e.g.,
personnel to perform surface/air sampling)
For room certification in the ‘at rest’ state the particle counter would be turned on and sufficient delay allowed for the operator to
leave the area and any disturbance due to their presence allowed to dissipate, typically 30-60 seconds. The tester should be outside
the room during performance. This is certification and as such tested only annually. For microbial testing – this is either monitoring,
so not applicable – or it is initial or ongoing room qualification and as such the presence f the testing personnel is noted in the record.
3. Should the occupancy state – “at-rest” and “in-operation” apply to the “room” or “area”? Multiple rooms
The occupancy state is tied to the normal maximum number of personnel present to perform routine operations, again for
classification only. It is a point of a curve to establish limits of operational performance. If different rooms have different occupancy
levels, this should be noted on the report – and should reflect the “normal maximum number of personnel required”.
4. Is it appropriate to define the condition as “at-rest” where the personnel still enter the production room after cleaning to take back
equipment/devices?
If the room has established an at rest state - <15 minutes, and then operators return into the area for any reason, it is now in
operational state – it may have different criteria for warnings and alarms etc., but it is no longer unoccupied. I would suggest that this
period be included in the operations phase of manufacture and ‘at rest’ begins when they leave.
 2. Maximum Tubing Length

• It is not always possible to locate the particle counter at the location of the ideal sample
point. In these cases, it is required to transport the facsimile of the environment to the
particle counter – using tubing
• Tubing causes a shift in the total number of particles and the distribution of particles within
a sample. Care should be taken to minimize these losses
• Misinterpretation of ISO14644-1 Appendix C, regarding macro particles; resulted in a 1m
maximum tubing requirement being initially quoted.
 Particle transport loss mechanisms

• Diffusion
• Sedimentation
• Turbulent inertial
deposition
• Inertial deposition
(bends)
• Inertial deposition
• Changes in tubing
diameter
• Other
 Particle transport loss mechanisms
• Annex 1 section 5.9 recommends ideally no
more than 1m tubing

• ISO TC209 is releasing a technical report

that discusses losses and recommends no
more than 2m length, no more than 2
bends (long radius)
• Manufacturers technical information on
losses
• Where outside these recommendations,
studies should be performed to determine
losses
• Edit alert and action levels to compensate
for monitoring
Available documents from PMS
New ISO 14644-21 Airborne Particle Sampling
Techniques
• Contents include
• Relationship between classification and monitoring
• Applications of particle counters
• Occupancy states
• Sample locations
▪ For classification
▪ For monitoring
• Sampling errors
• Sample tubing installation,
▪ tubing length
▪ Bends
▪ Valves and connectors
• Decision tree
 3. Alert and Action Limits
• B.3.1.3 When setting alert and action levels, it is important to be sensitive to the high variability of
airborne particle concentrations with time and at different locations. In particular, special care shall be
taken when considering alert and action levels for cleanliness classes ISO Class 5 and cleaner with low
concentrations of particles. In these circumstances, the occurrence of “nuisance alarms” due to false
counts and/or natural variability of particle concentration is more likely and should be avoided by
careful selection of alert and action levels. Frequent “nuisance” alarms should be avoided as they can
lead to alarms being ignored by users.
 Setting Action Limits
• Establish the normal operating range of cleanroom by observed measurements over a period of time
(Risk Assessment)
• Set the alarm, alert or action levels between the normal operating range and the cleanliness class limit.
• In most cases, a significant repositioning of the sample probe should be considered as the
establishment of a new location and should trigger a new series of observations to determine the
appropriate normal operating range, alarm or alert and action levels at the new location
• The duration of sample period needs to be considered in terms of allowable risk. Setting a longer
sample duration can smooth data and avoid potential “nuisance alarms” but may conceal an
unacceptably high level of airborne particle concentration over a short period caused by an unusual
contamination generating event.
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•1 event > 100 counts = alert

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•3 consecutive events (alerts) = alarm

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Real Data – Capping point

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•1 event > 35 counts = alert

•Also, in ISO14644-2 (2015)

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•2 consecutive events (alerts) = alarm

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Real Data – Point of Fill

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Real Data – Capping 5.0um

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 Setting Appropriate Action Limits

• The performance of the monitoring system, the data gathered, norms established, and
trends should be reviewed periodically. Revision of alert and action alarm levels (relaxation
or tightening) should be considered based on performance evidence
• A trigger threshold value based on a series of consecutive higher readings. The higher
readings trigger a warning based upon the occurrence of a higher level of counts being
maintained over a period of time (for example, three consecutive 1-min readings all above
a specified level).
• A threshold value trigger based on a high frequency of elevated readings. Sometimes
referred to as “x out of y”, this strategy records readings that are above a specified
threshold; if a sufficient number of readings in a series are above the specified values, then
an alert or action alarm is triggered. For example, if 3 out of the last 10 readings are above
a threshold then an alert or action alarm will be triggered.
• Allow for any system – TUBING – losses in the setting of action levels.
 Alert and Action Limits

• How long should an EM team collect data, to revaluate sampling frequency?

• Most long-term trends refer to 3 months of data to be able to review and set appropriate changes to action levels, as
the systems may not be in continuous use, and many times the zeros generated in clean environments would skew
normal trended data to a lower value than appropriate.
• However, if the initial values used are obviously out of synchronization with actual findings this timeline can be reduced.
• One of the measures I like to use is the number of alert excursions encountered, rather than looking at a huge swath of
data the trending and frequency of alert events is a much smaller frequency and will offer quick guidance on whether
your limits are set too loose or too tightly to control levels that show a loss of environment conditions.

• For new builds or processes (no historical data) how do you set alert levels?
• The EMPQ and data gathered during APS will give a good set of baseline data to establish some operational alert levels.
• Historically alerts can be set at 50-75% of the action limit, but even with a small amount of ‘live’ data more appropriate
alert and action levels can be defined.
• For grade A the ≥5.0µm data is more difficult to establish numerical values for as it is more a question of frequency of
occurrence than number alone.
• For more information, we have a technical paper regarding establishing alarm levels for GMP applications on our
website for download.
 Alert and Action Limits cont.

• Annex 2008 stipulated 1 cubic meter sampling volume; Annex 2023 does not indicate a
number. What is an acceptable number?
• The 1m3 sample was for classification of a cleanroom based upon the number of ≥5.0µm particles
present in very small numbers (<20).
• The suitable sample volume for classification can now be calculated using the available formula in
ISO14644-1 (2015), along with the number of sample points required.
• However, for monitoring this is now risk based, the locations are based on a risk assessment and the
sample volume based on the number of particles present, the history of the sample point etc.
typically 3 x 1-minute sample will give sufficient data when portable monitoring is performed.
• In Grade A, and where continuous monitoring is required, the sample is a function of the
instrument flowrate and samples should be each minute, stored and trended accordingly
• Rolling cubic meter data is not desired as it smooths peaks and reduced the ability to respond in a
‘timely’ manner.
 4. How to perform Continuous Microbial Monitoring?
New instrumentation can ensure a faster and safer process with real time or
near real time data
Two primary options
• Single Use devices
• Rapid and Alternative
Microbiological Methods
 BioCapt Single Use
Traditional technology
Tested up to 4-hours in continuous use
Continuous vs Frequent Monitoring
 Improved Traditional - Validation of 4-Hour use
• GROWTH PROMOTION TEST
• Inoculation of microorganisms on:
• Stress BioCapt Single Use
• Non-Stress – BioCapt Single Use
(Negative Control)
• Incubation of the plates
• Recovery efficiency:
• was been calculated according to the
following formula: Recovery rate (%)
= “Test (stressed)BCSU - CFU” /
“Positive control- CFU”×100
Table of Microorganisms ATCC used for GPT
Pseudomonas aeruginosa ATCC 9027
Staphylococcus aureus ATCC 6538
Bacillus subtilis ATCC 6633
Candida albicans ATCC 10231
Aspergillus brasiliensis ATCC 16404
Clostridium sporogenes (in anaerobiosi) ATCC 11437
Environmental Isolated Microorganisms
Environmental Isolated Microorganisms 1
Environmental Isolated Microorganisms 2
• The Dehydration:
• This value is defined according to the feasibility study
performed by Particle Measuring Systems, and it is
defined according to the initial quantity of the agar)
• The Dehydration (%) was been calculated according to
the following formula: Dehydration (%) = W^pre-
W^post/ W^pre×100
• Dehydration limit is ≤ 40%
 AVERAGE RECOVERY - BCSU-CP% Value
Average - Recovery BCSU-
Microorganims Batch N.
CP %
1
B.Subtilis 2 112
3
1
Aspergillus Brasiliensis 2 104
3
1
Pseudomonas Aeruginosa 2 112
3
1
Recovery Efficency ≥ 70%
Staphylococcus Aureus 2 126
3
1
Staphylococcus Epidermidis 2 78
3
1
Clostridium Sporogenes 2 117
3
1
Candida Albicans 2 91
3
1
Stenotrophomonas Maltophila 2 99
3
 AVERAGE DEHYDRATATION

Batch N. BCSU ID Average Dehydration %

BCSU1 38

1 BCSU 2 39

BCSU 3 35 Dehydratation ≤ 40%

BCSU1 38

2 BCSU2 40

BCSU3 36

BCSU1 38

3 BCSU2 35

BCSU3 40
 RMM - Cell Fluorescence
Endogenous Fluorophore Absorption and Fluorescence Maxima

Absorption Fluorescence
Fluorophore (nm) (nm)

Tryptophan Essential amino acid 220, 280, 288 320 - 350

Thyrosin Protein 220, 275 305

Collagen Animal protein 300 - 340 420 - 460

Elastin Connective tissue protein 300 - 340 420 - 460

NADH Co-enzyme for metabolic reactions 260, 340 470

NADPH Co-enzyme for anabolic reactions 260, 340 470

Flavins
(Riboflavin) Vitamins 260, 370, 450 530

404 - 568,
Porphyrins Active component of blood cells 620 580 - 622
Active component of vegetative
Chlorophyll cells 425, 455, 670 660 - 685

• Cell fluorescence in the 400 – 700nm wavelength band would detect the presence of:
• NADH
• Riboflavin
• Key triggers in determining the activity of bacterial cells
 Optical Fundamentals

Particle Counter Size Channels

Particle Pulses
Fluorescence
5 µm Pulses

Volts
2 µm

0.5 Noise
µm
Time

• The sample is pulled through the optical chamber and illuminated by the laser source.
• Elastic light scatter occurs when a particle enters a laser beam.
• The trigger from the elastic light scatter activates the fluorescence circuit to detect any
fluorescence from that particle at a wavelength shift.
• Filters ensure only the active component of fluorescence is detected and the scattered light
does not interfere with the signal
 Performance Qualification

Particle Count
Microbial
Data
Sample Data

Real-Time
Microbial Data

• 5.0μm particle counts & Real-time Bio-counts & Microbial growth

• Allows for a triangulation of data points to demonstrate control
• Testing is to demonstrate the statistical validity of zero (0) CFU – the most appropriate,
quantifiable method / methods should be applied.
 5. Other Applications

• How does this apply to Cell and Gene Therapy

• ATMP facilities are included in Annex 1, as there is
very useful information and guidance in how the
thinking of environmental monitoring and controls vs
risk related to the product.
• Significant differences in batch size and cross
contamination factors mean that the final selection of
instrumentation and location will be different, but
this is aligned with the CCS and the risk assessment
performed.
• The use of RMM in conjunction with traditional
particle counting and traditional microbiology will
allow for informed decisions to be made in a timely
manner, but good aseptic practice is at the core of
what is being monitored.
• We have a new Poster, presentation for the Annual
PDA conference, and a new technical paper that
discusses ATMP facilities directly.
 Other Applications

• Applicability to medical device manufacturers

• The GMP standards for both medical devices and terminally sterilized products are a grey area, and we try to use relevant standards
to overlap coverage in an aim to demonstrate cleanroom contamination control.
• For medical devices, the terminal sterilization rate ensures that a known number of surface microbes will be reduced to an
acceptable level, and that if manufactured in a clean controlled space the additional cleaning required is reduced.
• As the standards change then secondary users of the standard will also need to shift their goal post to maintain the original
alignment argument.
• The reason for the original webinar is to show that it has not really changed very much, and the limits and interpretation of guidance
is very similar to previously released.
• The only additional challenge may be in the certification mapping of the microbiological burden in the room, and the potential for a
CCS, but reviewing how each of the contamination control elements fit together is not a bad practice to perform.

• Any need for continuous NVP sampling in Grade C/D rooms, where manufacturing
takes place but are not critical areas or connected to Grade B rooms/hoods?
• There is no specific drive from regulation to put continuous sensors in these areas, although we do have applications where they
have been installed. These areas are normally monitored by operators moving from location to location using portable
instrumentation.
• This exercise has a cost, labour is difficult to find and train, when many of these rooms/locations can be covered by 1 or 2 fixed
sensors, allowing operators to perform more value-added functions.
Global Partnership
EU GMP Annex 1

QUESTIONS?
 THANK YOU

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