Abstract: As the market for advanced therapy medicinal products (ATMP) matures the complexities of these molecules are evident and challenging when routine standard quality control (QC) testing is applied. Short shelf life from the point of manufacture to administration to the patient results in relatively low volumes for small scale clinical trials or small patient populations. Within a limited time period and with this low product volume, it is necessary to complete required regulatory QC testing, be that for early or late phase clinical trials, or for licensed drug product in a reduced timescale. So, the challenges with QC testing of cell and gene therapies using traditional test methods is time to results, due to short shelf-life, and availability of sufficient sample, due to low production volumes. Over the past years the application of rapid testing of short-life cell and gene therapies that may also help conserve limited product availability have been utilised. Regulatory expectations for using rapid test methods in place of classical or compendial test methods have been defined and this presentation will provide examples and data from our own experience of a range of alternate methods for application to ATMP products.

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Use of rapid quality control
test methods as alternatives
to traditional methods for
cell and gene therapies
Alison Armstrong, PhD
Global Head of Field Technology Management

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

3. Testing for Cell and Gene Therapy Products
Current testing considerations
Regulatory landscape and
future technologies
1
2
3
Agenda
AGENDA

4. Evolution of Biological Therapeutics
Plasma Components
Whole BLood
Monoclonal antibodies
Recombinant Proteins
Fusion Proteins
Transgenic Proteins
Cell
Therapies
Gene
Therapies
4

5. Viral Risk Mitigation Strategy for gene & cell
therapy
Safe sourcing and testing
of raw materials
Verify absence
of viral contaminants
at appropriate stages
Verify capacity of manufacturing
process to remove or inactivate
potential viral contaminants
Will this strategy,
that has worked so
well for traditional
biopharmaceuticals,
work for novel
biological therapies?
5

6. 6
Potential Sources of Viral Contaminants
Cell banks, virus/vector stocks, serum, trypsin, other animal-derived
process components
Improper training or gowning procedures, sick employee policy
Facility design and maintenance, ineffective cleaning/disinfection
procedures, open vs. closed processing, segregation between pre-and
post-viral inactivation, utilities
Cross contamination between batches/products, ineffective
cleaning/disinfection procedure
Virus contaminants may be present, but not detected in screening
assays
Raw Materials
Personnel
Facility
Equipment
Detection

7. Challenges with cell and gene therapies
• Regulatory landscape
• Limited shelf life
• Cell therapies: No terminal
sterilization process
• Small lot size/limited sample
volume
• Limited availability of starting
materials for process, product
and test method development
• Patient to patient variability and
cellular heterogeneity
Ex Vivo Gene Therapy–
Retro/Lentivirus
In Vivo Gene Therapy –
AAV/Adenovirus

8. • Materials needed for the collection, selection, culture and modification of cells and production of
viral vectors
• Each substance used in production should be clearly specified and evaluated as to its suitability
for the intended use
• Human source material
• Ensure serum is from approved blood bank and meets all blood donor criteria
• Identify other reagents as licensed product, clinical or research grade, provide COA or information about testing
of donor and/or reagent
• Test to show absence of relevant human viruses
• Porcine material
• Test to show freedom from porcine parvovirus, porcine circovirus and porcine hepatitis E virus (EMA guidance,
2014). Use irradiated materials if possible
• Bovine material
• Test to show absence of bovine viruses (EMA and US 9CFR requirements)
• Use irradiated materials
• Be aware of newly discovered bovine viruses not detected in normal screening
• Bovine herpes virus IV; bovine parvovirus 2,3,4; bovine AAV2; bovine Norwalk virus, kobuvirus
Reagents, Ancillary Materials, Excipients
Quality Control of Starting Materials
8

9. • Using ‘research’ grade reagents
− Verify the source, safety and performance of the reagent
− Establish a qualification programme
− Sterility, endotoxin, mycoplasma, viruses
− Virus clearance studies during validation of process for purification of ‘material’
− Functional analysis
− Purity testing, absence of potentially harmful substances
− Extent of testing will depend on how the specific reagent is used in the manufacturing
process
• US Pharmacopoeia <1043> Ancillary materials for cell, gene and tissue-engineered
products
− To help design qualification programmes, risk classification examples given
• Ph. Eur. 5.2.12 Raw materials of biological origin for the production of cell-based
and gene therapy medicinal products, 01/2017:50212
Reagents, Ancillary Materials, Excipients
9

10. • Cells
− Procured in compliance with US 21 CFR 1271 subpart C or EU Directive 2004/23/EC
− Follow detailed guidance from national authorised authority
− UK Human Tissue Authority: Guide to Quality and Safety Assurance for Human Tissues and Cells for
Patient Treatment, 12 Nov 2010
− US FDA Guidance for Industry: Eligibility Determination for donors of human cells. Tissues, and
cellular and tissue-based products (HCT/Ps) Aug 2007
− Tissue and cell type
− Collection or recovery method
− Autologous or allogeneic
− Cell bank system for allogeneic cells
− MCB and WCB
Components and materials
10

11. • Autologous cells
− Not required to make a donor eligibility determination or to perform donor screening
− Determine whether the manufacturing procedures increase the risk of propagation of pathogenic
agents that may be present in donor
− Take precautions to prevent spread of viruses to persons other than the autologous recipient
− Assess whether cell propagation process could lead to amplification of viruses
− Testing for some human viruses relevant:
− HIV 1 & 2, HTLV I & II, Hep B, Hep C, CMV
• Allogeneic cells
− Donor exclusion criteria
− Risk of transmission of prion diseases
− Donor screening
Donor selection
11

12. • All types of cells and tissues
− HIV-1 and 2, Hepatitis B virus [nucleic acid test (NAT) surface and core Ag], Hepatitis C virus,
Treponema pallidum (syphilis) and CJD
• Leukocyte-rich cells or tissues
− HTLV-1 and 2, Cytomegalovirus (CMV)
• US FDA published Draft Guidance for Industry: Use of nucleic acid tests to reduce the risk of
transmission of Hepatitis B virus from donors of human cells, tissues, and cellular and tissue-based
products. Jan 2016
Donor Screening – Allogeneic Cell Therapy
12

13. Vector and Cell Safety and Characterization
Plasmid or Virus
Master Cell Bank (MCB)
Working Cell Bank (WCB)
Process Development
(Growth/Production/Modification)
Plasmid Stock Master/Working Virus Bank
(MVB/WVB)
Drug Substance
Drug Product
Cell
Stability
Lot Release Testing
QA/QC
Identity
Safety
Purity
Identity
Safety
Purity
Identity
Safety
In-Process
Testing 过程
中检测
13

14. MCB and WCB should be produced to GMP
 Industry best practice
 EU GMP guidance Annex 2 revision
− Comes into operation 31 January 2013
− “Establishment of cell banks should be performed under circumstances which are demonstrably appropriate.”
− “During the establishment of the cell bank, no other living or infectious material (e.g. virus, cell lines or cell strains)
should be handled simultaneously in the same area or by the same persons.”
− “ Following the establishment of cell banks, quarantine and release procedures should be followed. This should
include adequate characterization and testing for contaminants.”
− “Cell banks should be stored and used in such a way as to minimise the risks of contamination (e.g. stored in the
vapour phase of liquid nitrogen in sealed containers)”
• US FDA Guidance for Industry: cGMP for Phase I Investigational Drugs (July 2008)
− Investigational New Drugs for Phase I trials are exempt from full compliance with cGMP as detailed in US 21 CFR
parts 210 and 211.
− Particular differences
− Air classification for rooms
− Equipment calibrated and maintained
Cell Bank Preparation
14

15. 15
HEK 293 Cell Line Characterization
Testing Assays MCB WCB CAL
Identity CO1 Barcode Analysis X X X
STR profiling X X X
Microbial Detection
Sterility X X X
Mycoplasma X X X
Mycobacterium X X X
Virus Detection
In vitro virus assay X X X
In vivo virus assay X X
TEM X X
QPERT X X
PCR for Human Viruses* X X
Bovine virus assay X (X)
Porcine viruses /PCV & HepE X (X)
PCR for Bovine Parvoviruses X (X)
*HIV 1 & 2, HTLC I & II, HBV, HCV, HAV, CMV, EBV, HHV 6, 7, 8, B19, HSV 1 & 2, human polyoma viruses,
Bocavirus, Metapnuemovirus, adenovirus and AAV.
15

16. Vector and Cell Safety and Characterization
Plasmid or Virus
Master Cell Bank (MCB)
Working Cell Bank (WCB)
Process Development
(Growth/Production/Modification)
Plasmid Stock Master/Working
Virus Bank
(MVB/WVB)
Drug Substance
Drug Product
Cell
Stability
Lot Release Testing
QA/QC
Identity
Safety
Purity
Identity
Safety
Purity
Identity
Safety
In-Process
Testing 过程
中检测
16

17. Ph. Eur. 5.14 Gene transfer medicinal products for human use
Plasmid intermediates used for vector production
Information needed
− Identification
− Source
− Means of isolation
− Nucleotide sequence
− Source and function of promoters, origin of replication, selection marker genes
 Bacterial cell bank used to generate plasmids must comply with the requirements of the “Bacterial cell used for the manufacture
of plasmid vectors” section.
• Plasmids tested for: Identity: sequencing or PCR DNA concentration
• DNA forms: HPLC or CE
− Supercoiled, multimeric, relaxed monomer and linear forms
− Residual host cell DNA
− Residual RNA
− Residual host cell protein
− Sterility, endotoxin
Plasmids used for vector production
17
17

18. 18
Gene Therapy Vector characterization (produced in 293 cells)
Testing Assays MSV WSV Bulk
Identity/Potency
Nucleotide Sequence Analysis X X X
Vector Specific PCR X X X
Infectious Titer X X X
Microbial Detection
Sterility X X X
Mycoplasma X X X
Mycobacterium X X X
Adventitious Virus
In vitro virus assay X X X
In vivo virus assay X X**
PCR for Human Viruses* X
Bovine virus assay X (X)
Porcine viruses X (X)
Detection of Replication Competent
Viruses
X X
QPERT X
*HIV 1 & 2, HTLC I & II, HBV, HCV, HAV, CMV, EBV, HHV 6, 7, 8, B19, HSV 1 & 2, human polyoma viruses,
Bocavirus, Metapnuemovirus, adenovirus and AAV.
**May not be needed if cells and vector stocks have been tested
18

19. 19
Characterization of Viral Vectors
Identity Purity
Titer/Potency Residuals
Product
19

20. • Identity: Gene of
Interest(GOI)
• Titer: TCID50 of viral vector
• Purity:
• Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
& in vivo)
• Replication competent AAV
(rcAAV)/Replication
competent lentivirus
Testing Bulk and Final Lots
Unpurified
bulk
Purified
bulk
• Identity: GOI, ELISA, Vector genome
• Titer: TCID50 of viral vector, Genomic
titer
• Potency: Expressed protein, function
• Purity: Sterility, Endotoxin, rcAAV or
rcLV
• Residuals:
• Residual host cell DNA
• Residual DNA size distribution
• Host cell protein, Residual BSA
• Residual AAV Affinity Ligand
• Purity by SDS PAGE
• Empty: Full Capsid
• Identity: GOI
• Titer: TCID50 of viral vector,
Genomic titer
• Potency: Expressed protein,
function
• Purity: Sterility, Endotoxin
• Product characteristics :
• Vector aggregates
• Osmolality
• pH
• Extractable volume
• Appearance
• Particulates
Formulated
& vialed
final lots
20

21. • Mycobacterium testing
• Vectors produced in Human cells.
• Spiroplasma Testing
• Vectors produced in insect cells
• Vectors produced in any cell grown using plant peptones (AOF media)
• Neutralizing antiserum is required for infectivity assays
• Should be prepared from a stock different that stock used for production and prepared using SPF
animals.
• Stock should not be grown in the same cell line as used for production
• Anti-serum should not be of human or simian origin
Additional Considerations for Vector Testing
21

22. Identity Testing Regulatory
Guidelines
− Identity must be demonstrated
using :
− Immunochemical methods –
ELISA
− PCR based assays
− Sequencing
− Ph. Eur. and FDA guidelines
require sequencing of entire
genome.
− Ph. Eur. 5.14 – entire
genome of vector is
sequenced for a suitable
number of production runs
− FDA
− If genome <40 kb
sequence whole genome
− If vector is >40 kb
sequence gene insert,
flanking regions and
modified regions of the
vector
22
Vector Identity
 PCR Identity Assay
− Gene of Interest Target
− Product Specific Development
− Validation
− Client specific assay
PCR
Identity
 AAV Capsid ELISA
− Serotype specific
− 2, 8, 5, 9
− Detects viral particles
− Quantitative (Titer)
 LV/RV ELISA
− Detects p24/p30
− Quantitative (Titer)
A
ELISA
 NGS
− Virus identity test
− Confirm Identity
− Whole genome sequencing
− Comparison to known
reference
Sequencing
Confirm identity
of Viral Vector
Confirm identity
of Transgene
Confirm
Patient Cell ID

23. Regulatory Updates for Alternative
Methods
Sterility
• US FDA Amendments to sterility test
requirements for biological products
• Provides flexibility of methodology and
encourages the use of state-of-the-art
methods
• Sample size requirements appropriate to
the material tested
• Must meet or exceed compendial assay
specifications
Mycoplasma
• European Pharmacopoeia, 2.6.7.
Mycoplasmas
• Allows PCR assays as long as the assays
are of equivalent sensitivity and
specificity as the broth/agar/indicator
cell assay
• US FDA Guidance for Industry (2010)
• “PCR-based assays may be used to
detect mycoplasma, provided that such
an assay can be shown to be comparable
to the agar and broth procedure and the
indicator cell culture procedure.”
23
Purity
Novel Methods
 Sterility
 Mycoplasma
 In vitro adventitious virus
 In vivo adventitious virus
 BacT/ALERT® – Sterility
− Detects changes in pH due to bacterial growth
− Real time sample monitoring
− Objective readout
 Mycoplasma PCR
− Equivalent sensitivity and specificity to compendial method
− GMP and EP
 NGS Adventitious Agent Testing
− Detection and identification of adventitious agents
− Circumvents toxicity and neutralization issues

24. Virus
Characterization
 Confirm identity
 Assess variants /
sub-populations
Cell Line AAT
 Identify unknown
contaminants
 Troubleshoot
contaminations
Gene Editing
 On and off target
affects
 Cell/gene therapy &
CRISPR applications
Virus AAT
 No need for
neutralizing Ab
 Assure stock
purity
Genetic Integration
& Stability
 Integration site
assessment
 Genetic stability of
production cells
NGS applications are focused across cell and gene
therapy evaluations
24

25. 25
NGS application for Gene Therapy testing
Testing Adenovirus AAV1 Retro/Lentivirus
MSV Identity Y N N
Vector Identity Y Y Y
Plasmid Identity2
N Y Y
Sample volume
2x1mL @>2e8
particles/mL
2x1mL @>2e8
particles/mL
or
5ug plasmid
2x1mL @>2e8 particles/mL or
5ug plasmid
1 Only if AAV is produced by plasmid transfection
2 This may be 1-4 plasmids and several vector batches. Repeat testing for vector and plasmid depends on client process and supply needs.
Sequencing Regulatory Guidelines for CGT
Viral Vectors
EP and FDA guidelines require sequencing of
entire vector genome.
 EP 5.14 – entire genome of vector is
sequenced for a suitable number of
production runs
 FDA
− If genome <40 kb sequence whole
genome
− If vector is >40 kb sequence gene insert,
flanking regions and modified regions of
the vector
Plasmids
25

26. • Guidelines recommend specific testing to detect replication
competent viruses.
• Potential for vectors to recombine and revert to wild type.
• Occurs at a low frequency
• Testing method sensitivity is important
• Guidelines have threshold levels of how many virus
particles/dose are tolerated
• Replication competent virus testing
• Retrovirus (RCR)
• Lentivirus (RCL)
• AAV (rcAAV)
• HSV (rcHSV)
• Adenovirus (RCA)
26
Replication Competent Virus Detection
RCL
Lentiviral
vector
Infection of
C8166: 2-5 days
8 passages
PERT on culture
supernatant
26

27. • Identity: Gene of Interest
(GOI)
• Titer: TCID50 of viral vector
• Purity:
• Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
& in vivo)
• Replication competent AAV
(rcAAV)/Replication
competent lentivirus
Testing Bulk and Final Lots
Unpurified
bulk
Purified
bulk
• Identity: GOI, ELISA, Vector genome
• Titer: TCID50 of viral vector, Genomic
titer
• Potency: Expressed protein, function
• Purity: Sterility, Endotoxin, rcAAV or
rcLV
• Residuals:
• Residual host cell DNA
• Residual DNA size distribution
• Host cell protein, Residual BSA
• Residual AAV Affinity Ligand
• Purity by SDS PAGE
• Empty: Full Capsid
• Identity: GOI
• Titer: TCID50 of viral vector,
Genomic titer
• Potency: Expressed protein,
function
• Purity: Sterility, Endotoxin
• Product characteristics :
• Vector aggregates
• Osmolality
• pH
• Extractable volume
• Appearance
• Particulates
Formulated
& vialed
final lots
27

28. Key Elements:
• Sensitive
• Enough to discriminate small differences in biological activity and stability indicating
• Quantitative readout over a range of treatment concentrations
• Endpoint analysis is suited to consistently and accurately measure the biological effect
• Easy to use & robust
– Relevant controls and appropriate data analysis methods
Potency
28
28

29. 29
Product Related Impurity Nature of Lot Release Test Used
Empty Capsids Chromatography (IEX), Ultracentrifugation; EM
Nuclease resistant Host cell
DNA (encapsidated)
qPCR to target generic host cell sequences or
specific sequences of concern e.g. AdE1
qPCR
Nuclease resistant helper DNA
(plasmid)
qPCR to target generic helper virus sequence
Replication competent Virus Various depending on Vector system
Non-infectious particles Total viral particle (VP): infectious unit (IU)
Aggregated, oxidised,
degraded vector
Size exclusion chromatography, electrophoresis,
DLS and others.
Purity
29

30. 30
Residuals
Process and product-related impurities
 Manufacturing processes should be designed to minimize or remove impurities.
 Appropriate tests must be used to measure levels of process or product-related impurities.
 Host Cell DNA
 PCR detection
 Species or cell line specific
 Host cell proteins
 ELISA based detection
 Kit based
 Species specific
 Benzonase® endonuclease
ELISA
 BSA ELISA
Host Cell Impurities Process Related
Impurities
30

31. 31
Host Cell DNA
Presence of residual host cell DNA may impact
product quality and safety.
− Cellular DNA may contain tumorigenic
sequences.
− Limit the amount of residual DNA to <10
ng/dose.
− Reduce DNA size to below 200 base pairs
HEK 293T cells
− Contain transforming sequences E1A and SV40
− Need to determine levels of these sequences in
your product.
 Residual DNA PCR based assays
 Species specific
 Gene specific
 Process Residuals: Benzonase, BSA
 DNA Sizing
 Universal DNA assay
 Size distribution for all double stranded DNA present in
sample
 Small base pair to kilobase sized fragments
 Can size picogram levels of DNA
Residual DNA Guideline Methods
31

32.  Scientific innovation has supported a number of different treatment options under the
banner of cell and gene therapy
 Global regulatory agencies continues to issue guidance for development of cell and gene
therapies in response to an increasing number of clinical trials
 Emerging methods can provide better characterization and biosafety methods
 State of the art technologies are being applied at an expansive rate for these novel therapies
 Industry and regulators are working together to build the knowledge-base for new
therapeutic modalities
Conclusions
32
32

33. The vibrant M, Blazar, Benzonase, and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates. All other trademarks are the
property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.