This document discusses regulatory expectations for characterization of single-use systems used in biopharmaceutical manufacturing. It summarizes guidance from USP <665> on characterizing components based on risk levels. A component's risk level depends on its materials of construction, contact temperature and duration, and chemical composition of the process stream. Higher risk components require a higher level of characterization, such as extractable and leachable profiling. The document provides an example of characterizing components in a typical monoclonal antibody process and assessing the total mass of potential leachables through the process. It notes that while standards like USP <665> provide guidance, individual organizations may have their own risk assessment templates. The overall goal of characterization is to ensure patient safety

1. The life science business of Merck KGaA,
Darmstadt, Germany operates as
MilliporeSigma in the U.S. and Canada.
Upcoming
USP <665>
Level of Characterization of Single-
Use Systems Today and Tomorrow
Dr. Simone Biel
July 15, 2021

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

3. Agenda
1
2
3
Regulatory Framework
Component Qualification
Likelihood of Leaching
4 Q&A

4. Regulatory
Framework

5. Boost Covid-19 vaccine production would not be possible without SUT
Fast Growing Biopharmaceutical Market Challenges
5
Business driver to implement SUT
 Speed to Market
 High flexibility
 Quick changeover of equipment
Ensure patient safety
 Identity
 Efficacy
 Purity

6. Health authorities and GMP inspectors will ask for E&L assessment
Regulatory Expectations on Single-Use Systems
6
Market
authorization
Manufacturing
authorization

7. 8.126 The extractable and leachable profile of the SUS and any
impact on the quality of the product especially where the system
is made from polymer-based materials should be evaluated. An
assessment should be carried out for each component to
evaluate the applicability of the extractable profile data. For
components considered to be at high risk, including those that
may absorb processed materials or those with extended material
contact times, an assessment of leachable profile studies,
including safety concerns, should be taken into consideration. If
applying simulated processing conditions, these should
accurately reflect the actual processing conditions and be
based on a scientific rationale.
EU GMP, Annex 1 draft, Feb 2020
“Specific risks associated with SUS”
EU GMP Annex 1 Draft – Manufacturing Authorization
7

8. Understand the nature and amount of potential leachables
Market Authorization Application
8
Guideline on process validation for the manufacture of biotechnology-derived
active substances and data to be provided in the regulatory submission,
EMA/CHMP/BWP/187338/2014
6.1.3. General issues related to single use equipment
Information should be provided on the nature and amount of potential
leachables, and the removal of such impurities. Besides data, this
normally includes a risk assessment. Data do not necessarily need to be
generated under actual process conditions, for example supplier data or
data generated under representative model conditions may be
suitable. During process evaluation, small scale studies are acceptable to
assess leachable profiles, leachable removal and the impact of such
impurities on cell culture performance. For verification studies, commercial
scale equipment should be used. Various batches of disposable
components should be used, as appropriate, in the manufacturing of
verification batches in order to assess their impact on the product quality.

9. Manufacture, process controls and characterisation
EMA Public Assessment Report
9
• “Based on the provided risk assessment it can also be agreed that leachables from the different
materials used in the AS manufacturing process pose a minimal safety risk.”
• “…in-process leachables (questionnaire-based evaluation, detailed risk assessment and safety
assessment). The overall strategy followed to characterise the FP manufacturing process is supported.”
• “A risk assessment concerning potential extractables and leachables from manufacturing
components and container closure systems has been performed according to the applicant in S.2.3 LNP.
However, no details are available on possible extractables”
• “In line with CPMP/QWP/4359/03, storage bags should be tested for extractables/leachables, a
respective commitment has been provided (REC).”
• “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.”
AS Active Substance, FP Finished Product
All references are published in today approved Covid-19 vaccine’s EMA Public Assessment Reports

10. Potential Leachables or Process
specific Extractables
• Only RELEVANT worst-case scenario
Extractables
• Technology/polymer specific
• ALL worst case conditions according to BioPhorum/USP<665>
Leachables
• Real Drug & Process
Representative Model Solvents
BioPhorum and USP <665> are driving the discussion
10

11. Lots of components, different materials of construction
The Challenge of Single-Use Systems
11

12. Component
Qualification

13. Proposed General Chapter USP <665>
Standardized Approach to Extractables Testing
Plastic Components and Systems Used to Manufacture Pharmaceutical Drug Products and
Biopharmaceutical Drug Substances and Products, Pharmacopeial Forum PF 46(5), 1st September 2020
 Chemically suited for intended use
− Appropriate chemical testing (e.g., extractables profiling)
− Interpretation (e.g., toxicological assessment)
 Higher risk → higher level of characterization
− Initial Assessment (component in scope?)
− Risk Assessment (low, moderate, high risk?)
 Standard extraction protocol
New informational chapter USP <1665> provides additional information
13

14. Component qualification versus selection as the chapter's focus
Comparison Current and Previous Draft
1. Clarify the scope with respect to
components, drug substances, and
drug products.
2. Decouple this chapter from Plastic
Materials of Construction〈661.1〉 and
focus the current proposal on
manufacturing components.
3. Clarify the component qualification
versus selection as the chapter's focus.
4. Delineate the hierarchical relationship
between component and materials
testing.
5. Provide for flexibility regarding the
design of the risk evaluation matrix.
6. Revise the risk-based chemical and
biological reactivity testing
requirements for components.
2019
2020
USP-PF 46(5)
USP-PF 45(2)
USP-PF 46(5)
14

15. Higher risk requires higher level of qualification
USP <665> versus BioPhorum Testing Requirements
15
Risk Level USP <665> BioPhorum
Low
50% Ethanol
(Non-volatile residue/UV absorbance)
Meets compendial requirements
(e.g. USP <88> Class VI, Ph. Eur. 3.1.x, etc.)
Medium
50% Ethanol
(Organic extractables profiling)
Low risk requirements plus Extractables data evaluation
50% Ethanol
0.5N NaOH
0.1M Phosphoric Acid
WFI
High
50% EtOH
0.2M KCl, HCl, pH 3
0.1M Phosphate buffer, pH 10
(Organic extractables profiling, Extractable
elements as necessary and appropriate)
Medium-risk requirements plus more complete
Extractables data involving different extraction
solvents/solutions.
Leachables studies may be needed.

16. Again: Lots of components, different materials of construction
Your Single-Use System Project
16

17. Family
Process
step
Component
name/
Supplier
Material of
construction
Certifications
Surface –
to-
volume
ratio
Max.
Product
Contact
Duration
Temper
ature
Compounding
bag
DS pooling
and mixing
SU mix Bag
Pall
Film: Inner layer
Ultra Low Density
Polyethylene
Gas barrier layer
EVOH
Outer layer Ultra
Low Density
Polyethylene
Ports Polyethylene
USP Class VI
USP 87 (cytotoxicity)
ISO 10993 (biological
compatibility)
USP 661
Ph.Eur. (Section
3.1.5); JP (Section 61
Part 1); European
directive 85/572/EEC
0,02
m^2/L
34h
Room
Temp
Connectors
and fittings
DS pooling
and mixing
MPC
connectors
Sartorius
Polycarbonate;
Silicone
USP class VI <0.001
m^2/L
2-10
Room
Temp
DP transfer
KPC Pall
connectors
Polycarbonate USP class VI <0.001
m^2/L
18h
Room
Temp
Filters
DP and
excipients
filtration
Millipak 200
filter
Filter membrane
PVDF
Structural
components
Polycarbonate
USP <88>,
USP class VI
0,03
m^2/L
3h
Room
Temp
DP filtration
Opticap XL5
Filter
Filter membrane
PVDF
Structural
components
polypropylene
USP <88>,
USP class VI
0,01
m^2/L
3h
Room
Temp
Tubing
DP transfer
tubes
Raumedic
Platinum-cured
silicone (SIK8694)
USP class VI
ISO 10993-5
0,02
m^2/L
18h
Room
Temp
DS/DP
transfer
PumpSil tube,
Watson
Marlow
Platinum-cured
silicone
USP Class VI
ISO 10993-4, 5, 6, 10,
11
EP 3.1.9
0,01
m^2/L
2-10 h
Room
Temp
Final fill
Peristaltic
pump tube,
B+S
Platinum-cured
silicone
FDA 21 CFR 177.2600,
EP 3.1.9, USP Class VI,
ISO 10993 (10&11),
NSF-51
<0.001
m^2/L
18h
Room
Temp
Process Materials Risk Management
17
Exemplified list (The Healthcare Business of
Merck KGaA, Darmstadt, Germany)

18. <665> focuses on component qualification versus selection
 Ph. Eur. 3.1.x, Materials used for the
manufacture of containers
 USP <661>, Plastic Packaging Systems and
Their Materials of Construction (obsolete in
2025)
 <661.1>, Plastic Materials of Construction
 <661.2>, Plastic Packaging Systems for
Pharmaceutical Use
 USP <87>, Biological Reactivity Test, In Vitro
 USP <88>, Biological Reactivity Test, In Vivo
 ISO 10993-xx, Biological evaluation of medical
devices
18
Level of characterization
Today
USP <665> doesn’t provide a pass/fail test

19. USP <88> Biological reactivity test, in vivo
Testing Matrix for Plastic Class Designation
19
USP <88>, 2013

20. <88>, in vivo test established since 1965
<87>, in vitro test published in 1990
− decision point in determining whether samples need to be tested in animals
<1031>, The Biocompatibility of Materials Used in Drug Containers, Medical Devices, and
Implants
− plastic classification based on route of administration duration of exposure
USP Stimuli Article, PF 46 (4), July 2020
“In practice, however, the most stringent classification, Class VI, has been utilized
as the default for all polymeric materials regardless of risk or material
type….This has led to excessive animal testing and resulted in a reconsideration of
the appropriateness of the tests in 〈87〉 and 〈88〉.”
“….to expand the number of in vitro tests to give end users more tools in the material
evaluation process.”
20
USP’s Approach for Future Revision of Biological Reactivity Chapters
3R – Reduce, Refine, Replace
Our 4th R: Responsibility for all animals in our reach
internally and among our business partners

21. <661>, <661.1>, <661.2> require biological reactivity <87>
USP update November 2020
21
USP <661>, May 2017, no longer official
USP <661>, official as of 01 November 2020
2017
2020

22. Up to individual organization to define the concept of well-characterized
Level of characterization
22
 USP <665>, component qualification
 USP <661.1> could be used for material
selection
− “Although chemical characterization of
materials may enable proper material
selection, chemical characterization of
materials is not a prerequisite for the
qualification of manufacturing components or
systems.” (USP <1665>)
 USP <87>,
− More quantitative results compared to <88>
− Additional cytotoxicity and genotoxicity tests
in the future (?)
Next
USP’s effort includes a definition of “pharmaceutical grade”

23. Toxicologists prefer E&L risk assessment over biological reactivity testing
Patient Safety
23
23
No
Yes
Quantity
above the
TTC/PDE?
Risk Mitigation
Leachables?
Dilution?
Material/ Process
Change?
Risk is acceptable
Quantity of Each
Extractable

24. Likelihood of
Leaching

25. Four Risk Dimensions per USP <1665>
1. The chemical composition of the process stream
2. The nature of the component's materials of
construction
3. The temperature of contact
4. The duration of contact
25
Risk Assessment

26. Filter, tube, connector
Use Conditions for Risk Assessment
26
Conditions Exposure
Contact Time 30 hours
Contact Temperature Room Temperature
Sterilization Method Gamma Irradiation
Process Stream Somewhat organic, pH 4

27. Risk Dimensions per USP <1665>
Process Stream
27
Organic
solvents (by
volume)
Surfactants
(by weight)
Blood/blood
-derived
substances
(by weight)
Lipids and
proteins (by
weight)
pH
Aqueous
Level 1
<5% <0.1% <1% <1% ≥ 3 and ≤ 9
Somewhat
organic
Level 2
5-40% 0.1-0.5% 1-25% 1-5%
Highly organic
Level 3
>40% >0.5% >25% >5% <3 or >9
If the process streams contain multiple solubilizers, e.g. protein and surfactant, the risk increases
Process
Stream

28. Risk Dimensions per USP <1665>
Material of Construction, Temperature and Duration
28
Additives
(by weight)
Treatment
for
sterilization
Processing
Inert
Level 1
<0.1%
Intermediate
Level 2
0.1-1%
chemical
adhesives/bond
-ing of
component's
materials
Reactive
Level 3
>1%
Irradiation
/chemical
treatment
chemical
adhesives/bond
-ing of
component's
materials
Temperature
(°C)
Duration
Level 1 Frozen (<-10) < 24 hrs
Level 2
Refrigerated
(2-8)
Ambient (15-
25)
1-7 days
Level 3
Elevated
(>30)
> 7 days
Duration
Material Tempera-
ture
28
Clearance and clinical mitigating factors should be taken into account when establishing the
characterization level
Flushing can be used to reduce the material reactivity terms by one level

29. Risk Evaluation Matrix per USP <1665>
Risk Score to Risk Level
29
Dimension Scores
of Level 3
Score Risk Level Remark
Four 3333 C (high risk)
Three 3332 C
3331 C
Two 3322 C
3321
B (medium risk) or
C
if Level 1 is in the Material Dimension, B,
otherwise C
3311
A (low risk)
or B
if one of the Level 1 scores is in the
Material Dimension, A, otherwise B
One 3222 B
3221 B
3211 B or A
if one of the Level 1 scores is in the
Material Dimension, A, otherwise B
3111 A
None 2222 B
XXXX A All other combinations
 Link the Risk Sequence
with a Level of risk
characterization
 Use mitigation factors to
adjust the result
(by -1 or -2)

30. Most unit operations consist of polymeric components
Typical Monoclonal Antibody Process
30
High Risk
Clearance
30

31. Distance along the production stream - considerations
Total Mass of Leachables through Process
31
0
50
100
150
200
250
Total
Mass
of
Leachables
[mg]
Note: 81L out of 150L of the day 13
Bioreactor fluid was carried into the DSP
1.7
1.0

32. End-user have already their risk assessment template in place
The Likelihood of Leaching
What is the
Risk
“In the end, 3 level of risk
are required: low,
medium, high”
 The chemical composition of
the process stream
 The nature of the
component's materials of
construction
 The temperature of contact
 The duration of contact
Risk Dimensions per <1665>
 Distance along the
production stream
 Exposure temperature
 Exposure duration
 Process fluid interaction
 Dilution ratio (exposure
surface area to process liquid
volume ratio)
BioPhorum Best Practice
Guide Leachables Assessment
32

33.  Speed to market
Better alignment on health authorities’
expectations
 Component qualification
Standard testing protocol USP <665>
 Material selection
To be discussed!
33
vaccines manufactured using SUT
Covid-19

34. Senior Regulatory Consultant
simone.biel@emdgroup.com
Dr. Simone Biel
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Detailed information on trademarks is available via publicly accessible resources.
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35. Q&A