Watch the presentation of this webinar here: https://bit.ly/3kk0Qs1 In this webinar, you will learn: - About the GMP Annex 1 draft regulatory overview - How to incorporate the integrity testing & PUPSIT in the filtration systems validation - How to design a bacterial retention test in terms of organism selection and single vs multiple use validation Detailed description: In this webinar we will discuss the implications of the EU GMP Annex 1 draft on the filtration of medicinal products and how this impacts the validation studies. Bacterial Retention Testing is a critical part of the manufacturing validation process and is required by all regulatory bodies worldwide. Using case studies, our experts will explain how the Annex 1 draft is incorporated into the filtration systems validation exercise, specifically for integrity testing & PUPSIT (Pre-Use Post Sterilization Integrity Testing), the selection and justification of the appropriate test organism, and validation implications of single versus multiple use.

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Dr Simone Biel
Senior Regulatory Consultant
Pascale Richert
Senior Validation Consultant
16 September 2021
EU GMP
Annex 1 draft:
Implications on sterilizing
grade filter validation

2. The life science business
of Merck KGaA, Darmstadt,
Germany operates as
MilliporeSigma in the U.S.
and Canada

3. Agenda
Regulatory framework
Validation study # 1
Integrity test & PUPSIT
Validation study # 2
Appropriate test organism
1
2
3
4
Validation study #3
Single versus multiple use

4. Regulatory
Framework

5. Sterile Medicinal Product
 Subject to regulatory guidelines
 Compliant filter qualification
EU GMP Annex 1 – guidance on sterile medicinal product manufacturing
Contamination Risks Throughout the Process
Sterile media filtration
 Not subject to regulatory guidelines
 BUT should require aspects of sterilizing filter
qualification
5

6. 6
Prevent any contamination in the final product
QRM and CCS Throughout Annex 1 Draft
Current draft as of 2020
 16 targeted stakeholder organisations to consult
 WHO and PIC/S to maintain global alignment
Key Changes
 Introduction of new sections
 Restructured to give more logical flow
 Added detail to a number of the previous sections to
provide further clarity
 Quality Risk Management (QRM) Principles
 CCS = Contamination Control Strategy
Annex 1 draft, 2020

7. 7
Filter sterilization if terminal sterilization is not possible
Patient Safety
Sterility Assurance
 Sterile filter
 Filter compatibility
 Bacterial retention
 Integrity at every stage of use
Quality and Efficacy
 No adsorption (API, excipients)
 No leachables
 No particles

8. EU GMP, Annex 1 draft, Feb 2020
“Sterilizing grade filter – A filter that, when
appropriately validated, will remove a defined
microbial challenge from a fluid or gas producing a
sterile effluent. Usually, such filters have a pore size
equal or less than 0.22 μm”
PDA TR 26, revised 2008
A filter that reproducibly removes test
microorganisms from the process stream,
producing a sterile filtrate.
FDA cGMP, Guidance for Industry, 2004
“A sterilizing grade filter should be validated to
reproducibly remove viable microorganisms from
the process stream, producing a sterile effluent.
Currently, such filters usually have a rated pore size of
0.2 μm or smaller.”
8
More than a defined pore size and integrity test
Sterilizing Grade Designation is a Functional Definition

9.  Validation of maximum filtration
time/total time filter is in contact
with fluid
 Discard filter after processing of a
single lot
 Don’t use filter for more than one
working day (unless validated)
 Conduct and document effective
validation and qualification studies
to demonstrate that the duration of
filter use does not compromise
performance of the sterilizing filter
or filtrate quality
 Sterile filtration should be
validated
− validation can be grouped
− but done under worst case
conditions
− rationale for grouping justified
and documented
 Filter validation wherever possible
with product
 Justify challenge organism used in
bacterial retention test
 Establish appropriate integrity test
value specification
 Filter selection
− minimize generation of
fibers and particulates
− no unacceptable levels of
impurities
− compatible with the fluid
− evaluate adsorption and
extraction/leaching
 Filtration concept (serial,
redundant, bioburden
reduction)
 Process parameters
− pressure, wetting, flushing,
hold-time, flow rate,
maximum volume
− Allow operation within
validated process
parameters
 In-place integrity testing
pre- and post-use
9
Much more details compared to current Annex 1
Annex 1 Draft: Specific Expectations for Filter Sterilization

10. 10
Filter data to be provided (EMA)
Market Authorization
“Filter validation
Acceptable information has been provided
during the procedure for filter validation on
the filters used for sterile filtration,
describing the material, pore size and
surface area. All study results met the
predetermined acceptance criteria and the
studies for microbial retention, membrane
compatibility, extractable substances and
integrity test determination have shown
that the filters are appropriate for sterile
filtration of the finished product.”
EMA/CHMP/CVMP/QWP/850374/2015 (6 March 2019), Guideline on the sterilisation
of the medicinal product, active substance, excipient and primary container
Public Assessment Report, Comirnaty,
COVID-19 mRNA vaccine, EMA/707383/2020

11. Validation
study # 1
Integrity test &
PUPSIT

12. Check correct installation
 Detects system leaks due to o-rings, gaskets, faulty seals
Confirms manufacturers specifications
 Assures the correct pore size filter
Check for damages
 Assures integrity pre-use
 Assures integrity post use
Regulatory requirements
 GMP Requirement
 Link between validation and actual processing conditions
12
Why perform integrity test ?

13. Diffusion + Bubble point
Integrity test types
13
Viscous flow
Bubble point
Pressure
Diffusion
Bubble
point
Gas
Flow
Bubble
Point
Pressure
Diffusion
Viscous flow

14. Which wetting fluid?
When is integrity test performed ?
14
time
Post use IT
Pressure
Pre-use IT
Pre/post sterilization (PUPSIT)
Water Buffer
Product
Filter CoQ Product based
specification is
required
Wetting fluid:

15. Step 1: Laboratory determination
How to determine a Product based specification ?
15
1
Diffusion pressure
2
Water integrity test
3
Product integrity test
4
Product IT specification
 Water & Product BP
1 membrane disc
 Diffusion pressure
calculated
 3 -9 filters from 1-3
lots tested
 Water BP & diffusion
determined
 Filter devices dried
 Product BP & diffusion
determined for each
filters
 BP ratio (BPR) &
Diffusion ratio (DR)
calculated
 Minimum product
BP calculated:
 Maximum diffusion
rate calculated:
Example for a hydrophilic filter

16. Step 1: Laboratory determination
How to determine a Product based specification ?
16
Water BP
(mbar)
Product BP
(mbar)
BPR
Water
diffusion
at 2760 mbar
(ml/min)
Product
Diffusion at
2100 mbar
(ml/min)
DR
Filter 1 3790 2971 0.78391 2.75 1.11 0.40364
Filter 2 3735 2885 0.77242 3.09 1.15 0.37217
Filter 3 3759 2907 0.77334 2.84 1.12 0.39437
Average 0.77656 0.39006
Product BP specification
BPwater x BPR
3450 mbar x 0.77656 -> 2680 mbar
Diffusion specification
Diffwater x DR
5.0 ml/min x 0.39006 -> 2.0 ml/min at 2100 mbar
Example of a 4’’ hydrophilic filter

17. Step 2: In process confirmation
How to determine a Product based specification ?
17
Lab Product BP specification
≥ 2680 mbar
BP 1
2990
mbar
In process BP confirmation
3 devices data points
Data within
expected
range
BP 2
3105
mbar
BP 3
3110
mbar
Ongoing product attribute
monitoring

18. Step 2: In process confirmation
How to determine a Product based specification ?
18
Lab Product BP specification
≥ 2680 mbar
BP 1
2560
mbar
In process BP confirmation
3 devices data points
Data NOT
in expected
range
BP 2
2685
mbar
BP 3
2710
mbar
In process BPR validation
9 devices data points
Ongoing product attribute
monitoring
If results not statistically valid
validate with standard fluid
flush after product filtration

19. Bacterial retention validation
How to validate a process including PUPSIT ?
19
Actual
Drug
Actual
Filter
Actual
Process
Challenge
organism
Demonstrate that the filter produces a
sterile filtrate under
product & process specific conditions
 Product: formulation, pH
 Filter: membrane and device
 Process parameters:
o Temperature
o Differential Pressure/ flow rate
o Filtration & Contact time
o Batch size → volume/cm²/time unit
o Flow dynamics: continuous, intermittent flow
o Challenge organism → Size , shape , Quantity

20. Bacterial retention validation
How to validate a process including PUPSIT ?
20
Process conditions
Laboratory
conditions
1 Simulates the process sequence
2 Respect PUPSIT type:
Bubble point and/or diffusion

21. Bacterial retention validation
How to validate a process including PUPSIT ?
21
Process & lab
parameters
Product
flush
PUPSIT
Diffusion
Product
batch
Filter
Blow down*
FIT
Bubble point*
Volume (ml/cm²) 0.4 NA 80 NA NA
Duration 20 min 10 min 48 hrs 15 NA
∆P (mbar) 1500 2100 1500 3000 3500
Filter
wetting
product
PUPSIT -
diffusion
simulation
Product
filtration
Filter blow
down
FIT –
bubble
point
Bacteria inoculation 107 cfu/cm²
* Downstream line still connected for product recovery
Bacteria load

22. Validation
study # 2
Appropriate test
organism

23. Bacterial retention validation
How to justify the challenge organism ?
23
In draft Annex 1 : Point 8.86
‘…. The challenge organism used
in the bacterial retention test
should be justified.’
1 Assess product bioburden
2 Assess if any process isolate might be as
small as B. diminuta
Consider process isolates
3 Assess if the process isolate is an appropriate
candidate for the retention test
4 Bacterial retention validation with process
isolate & bacterial retention with B. diminuta

24. Culture Conditions & standardization
How to justify the challenge organism ?
24
B. diminuta
 Used in 99% of filter bacterial challenges
 Smallest organism, consistently cultured, non-pathogenic
ASTM F838 method
SLB Frozen paste
Length [µm] Width [µm] Length [µm] Width [µm]
Average 0.68 0.31 0.89 0.34
σ 0.14 0.04 0.13 0.03
Max 1.07 0.38 1.17 0.43
Min 0.40 0.22 0.66 0.28

25. “small size” organisms
How to justify the challenge organism ?
25
Organism Source Medium Size µm
B. diminuta ATCC 19146 SLB 0.68 0.31
P. luteola Process isolate SLB 0.72 0.39
P. luteola H2O 0.86 0.33
S. maltophilia H2O 0.88 0.44
P. fluorescens SLB 0.90 0.53
C. testoseroni SLB 0.99 0.38
B. cepacia Saline 1.00 0.43
P. fluorescens H2O 1.02 0.22
P. pseudoalcaligenes RPMI 1.06 0.32
B. cepacia ATCC 35254 DI H2O 1.11 0.46
B. cepacia ATCC 25416 SLB 1.15 0.42
P. fluorescens Process isolate SLB 1.17 0.46
B. cereus Media Fill 1.19 0.36
P. stutzeri SLB 1.22 0.50
S. maltophilia DI H2O 1.28 0.37
R. pickettii CDC/Anderson DI H2O 1.37 0.48
S. maltophilia Process isolate Product 1.40 0.52

26. “small size” organisms
How to justify the challenge organism ?
26
Organism Source Medium Size µm
C. freundii Process Isolate 0.9% saline 1.12 0.61
C. violaceum Product 0.90 0.50
C. violaceum SLB 1.20 0.60
P. agglomerans Product 1.70 0.60
P. agglomerans SLB 1.30 0.50
P. putida Product 1.10 0.60
P. putida SLB 1.00 0.50
S. marcescens ATCC SLB 0.76 0.43
All these “Small size” organisms are micro-organisms retained by
sterilizing-grade 0.22µm filter membrane.

27. Is it an appropriate candidate for the validation?
How to justify the challenge organism ?
27
Retention test with a
0.45 µm filter
Process isolate in the
filtrate
Sterile filtrate
Process isolate
‘bigger’ than
B. diminuta
Process isolate as
‘small’ as
B. diminuta
0.45µm PVDF filter
Candidate
for validation
NOT a candidate
for validation
Size assessment with the identified ‘small’ process isolate
Process isolate

28. Cultivation
 Develop the process isolate cultivation method
 Target concentration ~108 cfu/ml
 Size controls:
retention on 0.22µm filter and passage on 0.45µm filter
Recovery & viability
 Demonstrate adequate bacteria recovery on the assay filter used
to assess the bacterial retention filtrate sterility.
 Demonstrates bacteria viability in product over the process time
28
Preliminary studies
Bacterial retention with process isolate

29. Cultivation & size controls
 Gam negative bacteria, rod shaped soil bacterium
Example: R. pickettii
Bacterial retention with process isolate
29
Slant
48 hours
30 ± 2°C
TSB
48 hours
30 ± 2°C
SLB
1.2 108 cfu/ml
24 hours
30 ± 2°C
0.45 µm PVDF
filter
0.22 µm PVDF
filter
Retention
Pass
0.45 µm
filter
Size
controls

30. Recovery
 Demonstrate adequate bacteria recovery on 0.45µm mixed cellulose esters (MCE) membrane disc
Example: R. pickettii
Bacterial retention with process isolate
30
Product
R. Pickettii
~10-100 cfu
Water control
R. Pickettii
~10-100 cfu
0.45µm MCE
membrane disc
R2A plate
36 hours
30 ± 2°C
Plate count (cfu)
Water
control
22 ± 4°C
Product
22 ± 4°C
49 37
37 53
46 48
Average 44 46
% recovery
(≥ 70%)
96 %
R. Pickettii is recoverable

31. Viability
 Demonstrates bacteria viability in product over the process time
Example: R. pickettii
Bacterial retention with process isolate
31
Product
R. Pickettii
~ 105 cfu/ml
Water control
R. Pickettii
~ 105 cfu/ml
Assess concentration
process duration
/temperature
R2A plate
36 hours 30 ± 2°C
Time Points
(hours)
Concentration (cfu/ml)
Water
control
22 ± 4°C
Product
22 ± 4°C
0 4.2 x 105 2.8 x 105
2 4.5 x 105 4.9 x 105
4 3.4 x 105 3.3 x 105
8 5.4 x 105 3.9 x 105
25 1.4 x 106 1.1 x 106
Viable in product for 25h
at ambient temperature
No
LRV

32. R. Pickettii and B. diminuta
Bacterial retention with process isolate
32
Filtrate analysis
ENTIRE filtrate volume
2 bacterial retention performed - Robust and Complete validation
Product inoculated
with R. pickettii
Product inoculated
with B. diminuta
FULL retention

33. Validation
study # 3
single versus
multiple use

34. Definitions - What are the differences ?
Single use versus multiple use
34
In draft Annex 1 : Point 8.95
‘Liquid sterilizing filters should be
discarded after processing of a single
lot and the same filter should not be used
for more than one working day unless
such use has been validated’
& NEW Point 8.96
‘Conduct and document effective validation
and qualification studies to
demonstrate that the
duration of filter use for a given
sterile filtration process and for a given
fluid does not compromise
performance of the
sterilizing filter or filtrate quality.’
1 Single use : discard of the filter after one batch
2 Multiple use # A: filter is re-used for multiple batches with
no cleaning and/or sterilization between batches
3 Multiple use # B: filter is re-used for multiple batches with
a cleaning cycle but no sterilization processes between
batches
4 Multiple use # C: filter is re-used for multiple batches
with sterilization processes between batches

35. Integrity/sterility failure
 Additional hydraulic stress over long process duration & multiple filter blow downs.
 Additional thermal stress when multiple sterilization cycles.
 Chemical incompatibility; filter material degradation accelerated by product residual compounds heated
during multiple sterilization cycles.
 Bacteria concerns during long product wet hold periods between batches
Product quality concerns (addressed by filter user)
 Influence of cleaning solutions on product quality
 Product contamination / carry over
 Increase of endotoxin level
Potential risks
Single use versus multiple use
35
These risks could be mitigated by a well-designed filter validation strategy

36. Extractables & leachables
Multiple use validation
36
Batch 1 Batch 2 Batch 3
Filter
SIP
Process
conditions
Filter
SIP
Filter
SIP
➢ After first use, majority of extractables have been removed
➢ First batch is worst case
➢ Multiple sterilization prior to first use are modelled
Per batch
Process
Parameters
Test
Parameters
Sterilization SIP
Sterilization
Conditions
135°C
30 minutes
3 X
Contact Time 30 hours
Temperature 25°C
Lab
conditions

37. Compatibility evaluation
Multiple use validation
37
Batch 1 Batch 2 Batch 3
Filter
SIP
Process
conditions
Filter
SIP
Filter
SIP
Undergo the cumulated batch process conditions
Process
Parameters
Test
Parameters
Sterilization SIP
Sterilization
Conditions
135°C
(30 minutes x 3) 3 X
Contact Time 30 hours 3 X
Temperature Ambient
Lab
conditions

38. Bacterial retention validation
Multiple use validation
38
Batch 1 Batch 2 Batch 3
Filter
SIP
Process
conditions
Filter
SIP
Filter
SIP
FIT FIT
(Filter Integrity test)
FIT
1
2
Test with membranes
Test with process ‘used’
filter device
Both tests with a sterile
effluent outcome
Validates the filter multiple
use

39. Retention test- Membrane scaled down testing
Multiple use validation
39
Batch 1 Batch 2 Batch 3
Filter
SIP
Process
conditions
Filter
SIP
Filter
SIP
All process steps between 2 sterilization cycles
Per batch
Process
Parameters
Test
Parameters
volume/cm² 80 L
Contact time 30 hours
Pressure 1500 mbar
Temperature Ambient
Blow-down
& FIT
15 min / 3000mbar
Diffusion 10 min / 2100 mbar
& Bubble point up to 3500 mbar
Lab
conditions

40. Retention test- Filter device testing
Multiple use validation
40
Batch 1 Batch 2 Batch 3
Filter
SIP
Process
conditions
Filter
SIP
Filter
SIP
FIT FIT
FIT
Challenge test on a used filter devices
ASTM F838 methodology
Process Parameters
volume/cm² 80 x 3
Contact time 30 hours x 3
Pressure 1500 mbar
Temperature Ambient
Blow-down
& FIT
15 min / 3000mbar x 3
Diffusion 10 min / 2100 mbar x 3
& Bubble point up to 3500 mbar x 3
FULL retention

41. 41
Filtration systems
validation is a key element
of sterility assurance
 Include the Integrity Test in your filter
validation
 Bacterial retention is not always a
Brevundimonas diminuta retention test
only
 Multiple uses can be validated (but better
to keep your inspector in the loop)

42. Dr. Simone Biel
Senior Regulatory Consultant
Darmstadt, Germany
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Pascale Richert
Senior Validation Consultant
Molsheim, France