Watch the recording of this presentation here: https://bit.ly/3SqOsoP Novel therapies, including cell and gene therapies, continue to be central to innovation in healthcare and represent the fastest growing area of therapeutic medicine. As a consequence, the number of gene therapies undergoing clinical trials has increased significantly in the last five years. Manufacturing processes for these novel therapeutics are very complex with a high risk of contamination. Regulatory agencies world-wide have responded by issuing guidance to outline their expectations for development and manufacture of cell and gene therapies. Currently, regulatory guidance is not harmonized globally and can often lead to confusion within industry and increased risk of non-compliance. In this webinar, we'll answer: • Which regulatory guidelines do you need to comply for your INDs? • When do you start implementing GMPs and validated assays? • How do you get your QC testing strategy ‘right the first time’? • How do you ensure testing is not your rate limiting step for the IND submission? Presented by: Manjula Aysola, Senior Regulatory Consultant Dr. Alison Armstrong, Sr. Director, Technical and Scientific Solutions

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Alison Armstrong, PhD
Global Head of Field Technology Management
Manjula Aysola
Senior Regulatory Consultant
Moving your gene
therapy from R&D to IND:
How to navigate the
regulatory landscape?

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

3. Which regulatory guidelines do you need
to comply for your INDs?
When do you start implementing GMPs
and validated assays?
How do you ensure testing is not your
rate limiting step for the IND
submission?
Agenda
AGENDA
How do you get your QC testing strategy
“right the first time?”

4. Which regulatory
guidelines do you
need to comply for
your INDs?

5. Gene Therapies Can Be Realized via In vivo or Ex vivo Effects
5
In vivo Ex vivo
Gene of
interest
Package
into virus
vector Directly
injected
into patient
Modified cell
injected back to
patient
Gene of
interest
Package
into virus
vector
Cell from
patient/donors
Virus vector
transfer gene
of interest into
cells
 Modifies or corrects abnormal genes by
administering a specific DNA or RNA.
 Viral vector system (mostly AAV) or non-viral system
is used to deliver gene of interest (GOI) into target
tissue.
− In most cases, GOI integrates into patient
chromosome.
 Healthy Donor or patients’ own cells are used to treat the
condition
 Genes are introduced into cells (autologous/allogeneic via
vectors or other gene-editing mechanisms
 For example: CAR-T produce receptors on the
surface of the T Cells called chimeric
antigen receptors (CARs)

6. Increase in Regulations for Cell and Gene Therapy
Observed in the Past 5 years
FDA: Content and review of CMC
information for
 Human gene therapy INDs
 Human somatic cell therapy INDs
EMA: Guideline on human cell-based,
gene therapy medicinal products
EMA: GMP for ATMPs
EU Draft : Annex 1 revision for
sterile medicinal products
EMA Guideline on quality, non-clinical
and clinical aspects of medicinal
products containing genetically
modified cells
FDA: Finalized guidance
Testing of retroviral vector-based
human gene therapy products for
replication competent retrovirus
during product manufacture and
patient follow-up.
CMC for human gene therapy INDs
ChP: General Chapter of Gene
Therapy Products for Human Use
FDA: Potency tests for cellular and
gene therapy products
 PIC/S Annex 2A Draft guidance for the
manufacture of ATMPs
 NIFDC. China: Quality Control of CAR-T
Cell Therapy Products and Consideration
for Non-clinical Research
 MHLW Japan: Ensuring the Quality and
Safety of Gene Therapy Medicinal
Products
2008 2011 2017 - 18 2019 2020 2021 - 22
6
 Questions and answers on the principles
of GMP for the manufacturing of starting
materials of biological origin used to
transfer genetic material for the
manufacturing of ATMPs
 FDA Draft guidance Human gene therapy
products incorporating genome editing
 FDA Draft guidance Considerations for
the development of
CAR-T cell products

7. 7
Standards Define Raw/Ancillary Material
Quality Requirements
Growth
factors
Microcarriers
/antibody
coated-beads
ISO 20399 General
requirements and best
practice guide for ancillary
materials used in cell
therapy manufacturing
USP <1043> Ancillary
materials for cell, gene and
tissue engineered products
Chinese Pharmacopoeia
2015. Requirements for
ancillary materials and
excipients used for
production of biologics
1 3
2 4
Ph. Eur. 5.2.12 Raw
materials of biological
origin for the production of
cell-based and gene
therapy medicinal
products, 01/2017:50212
Cell culture
medium

8. Starting materials requiring high level characterization and GMP production
 Cell banks for vector production
 Plasmids used for transient transfection
− Produced using characterized bacterial cell bank
 Lentivirus for autologous cell transduction
Assuring the quality of cell substrates
 Recent US FDA guidance refers to FDA Guidance for Industry: Characterization and qualification of cell
substrates and other biological materials used in the production of viral vectors for infectious disease
indications 2020
 ICH Q5A, Q5B and Q5D
 European Pharmacopoeia 5.14 ‘Gene transfer medicinal products for human use’ references;
− Ph. Eur. 5.2.3 Cell substrates for the production of vaccines for human use
− Ph. Eur. 2.6.16 Tests for extraneous agents in viral vectors for human use
Existing Guidance for Characterization of Starting
Materials is Applicable
8

9. When do you start
implementing GMPs
and validated
assays?

10. Many Elements are Key to Successful Product
Development
 Product consistency
 GMP
 Ensure continued
production of quality
product
Manufacturing Control
 Safe
 Potent and Effective
 Pure
 Stable
Demonstrate Product is
 Build quality into product
throughout the process
 Appropriate testing and
characterization
Product Quality
10

11. Phase III /
Pivotal
Phase II
Phase I
11
FDA Recommends an Incremental Approach to
Manufacturing Controls
BLA/MAA
Preclinical
Assay Qualification for intended use Validation
Phase appropriate GMP
21CFR210, 211
21CFR610
Prior to Phase I :
need product safety
testing and basic
characterization info
Based on presentation by Denise Gavin, OTAT, CBER FDA
Guidance for Industry CGMP
for Phase 1 Investigational
Drugs

12. EMA Recommends GMP Principles for Manufacture of
Starting Materials
12
Example
Products
Application of GMP to manufacturing steps is shown in green (incl. inspection/certificate)
GMP Principles should be applied where shown in light green
Starting material Active substance Finished product
In vivo gene
therapy: viral
vectors
Plasmid
Manufacturing
Establishment of a cell bank (MCB, WCB)
and virus seeds when applicable
Vector
manufacturing
and purification
Formulation filling
Ex-vivo:
Genetically
modified cells
Donation
Procurement and testing
of tissues / cells
Establishment of a
cell bank (MCB,
WCB) for plasmid
and/or vector
expansion and viral
seeds when
applicable
Plasmid
Manufacturing
Vector
manufacturing
Genetically
modified cells
manufacturing
Formulation filling
• Eudralex volume 4, Guidelines on Good Manufacturing Practice specific to Advanced Therapy Medicinal Products 2017
• Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products EMA 2018
• Guideline on quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells EMA 2020
• EMA Question and answers on the principles of GMP for the manufacturing of starting materials of biological origin used to
transfer genetic material for the manufacturing of ATMPs 2021

13. 13
Risk Appropriate Characterization of Raw Materials is
Recommended
Avoid the use of animal-origin
raw materials if possible. If
unavoidable, ensure
 Sourcing from known safe
geographies/screened donors
 Testing for species relevant
contaminants
 Inactivation by validated
methods using heat,
irradiation, filtration etc.
 At least two orthogonal
mitigation methods for high-
risk materials
Monoclonal antibodies in product
contact during manufacturing
 Tested per FDA’s “Points to
Consider in the Manufacture
and Testing of Monoclonal
Antibody Products for Human
Use”
 Alternately include details of
antibody production and
characterization in a Master File
registered with the FDA
Good manufacturing practices
(GMP) under a quality
management system (QMS such
as ISO 9001) ensure high lot-
to-lot consistency and the
documentation to help users with
their risk assessments and
regulatory requirements
Well-characterized reagents Performance consistency
1 2 3
Animal-origin free
ICH Q9: Higher risk requires higher level of qualification. The level of effort, formality
and documentation of the quality risk management process should be commensurate
with the level of risk

14. How do you get your
QC testing strategy
“right the first time?”

15. 15
Biosafety Risk Mitigation Strategy
Martin Wisher (2018). Viral Risk Mitigation: A Global Regulatory Perspective. BioProcess International

16. 16
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

17. 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
In Vivo Gene Therapy –
AAV/Adenovirus
Ex Vivo Gene Therapy–
Retro/Lentivirus
17

18. • 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
18

19. 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
Quality Control of Starting Materials
19
Cells

20. − 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
Donor Selection:
− Donor exclusion criteria
− Risk of transmission of prion diseases
Donor screening
− 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
20
Donor Selection and Screening
Autologous cells Allogeneic cells

21. 21
Test Methods Applied for Allogeneic and Autologous therapies
Microbiology
Adventitious
Viruses
Expressed
Retroviruses
• Sterility
• Mycoplasma
• Mycobacterium
• in vitro
• in vivo
• NGS
• QPERT
• TEM
• Detection or enumeration, and identification
of microbial contaminants
• Normal culture based assay by direct
inoculation
• Rapid testing assay for terminal sterilization
of living cells (BacT/Myco PCR)*
• in vitro co-culture with 3 detect cell lines
within 28 days
• in vivo testing with suckling mice, adult mice
and embryonated eggs
• NGS may be used to reduce consumption of
limited MCB material
• Quantitative Fluorescent Product Enhanced
Reverse Transcriptase (QPERT), Test RT
activity of retroviruses in biologics
• Visible virus particle will be counted in
Transmission electron microscopy
• Specific pathogens including human agents
and animal origin derived pathogens
• In vitro culture testing and /or QPCR
• Human
• Animal
Specific
pathogens
Ensure sufficient
materials are
prepared
and available for
each test method

22. 22
Testing for Allogeneic and Autologous Therapies
*Tumorigenicity or TPD50 in adult nude
mice
Autologous
Allogeneic
WCB
MCB
Cells at Limit of In
Vitro Cell Age (LIVCA) DP
Identity
Adventitious
Viruses
Expressed
Retroviruses
Bovine and
Porcine Viruses
Specific Human
Pathogens
*
Cell properties
Microbiology

23. 23
Unpurified and Purified Bulk Testing
Virus Production
Purification
Unpurified Bulk Purified Bulk
Identity
• GOI
Titer
• TCID50 of viral vector
Purity
• Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
& in vivo)
• Replication competent virus
(RCV)
Identity
• GOI
• ELISA
• Vector genome
Titer
• TCID50 of viral vector
• Genomic Titer
Potency
• Expressed protein
• Function
Purity
• Sterility
• Endotoxin
• RCV
Residuals
• Residual host cell DNA
• Residual DNA size
distribution
• Host cell protein, Residual
BSA
• Purity by SDS PAGE
• Empty: Full Capsid ( for AAV)

24. Formulated Final Lots
Virus Production
24
Product
Characteristics
Identity
Purity
Potency
GOI
Sterility,
Endotoxin
TCID50 of viral vector
Genomic titer
Expressing protein
Vector aggregates
Osmolality
pH
Extractable volume
Appearance
Particulates
Format Endotoxin Upper
Limit*
intrathecally 0.2EU/kg/hour
intraocularly 2EU/dose/eye
ophthalmic 0.5EU/ml
Others 5 EU/Kg/hour
Endotoxin claim in different format*
* CMC Information for Human Gene INDs 2020 FDA
• Dosing units
• Genotypic or phenotypic variation
• Particle number and size
• Aggregation state
• Infectivity
• Specific activity (ratio of infectious to
non-infectious particles or full to
empty particles)
• Biological activity
• Potency
• Immunological activity
*Parameters relevant to the
performance of the DP

25. Regulatory Updates for Alternative Methods
Sterility (21CFR 610,611, 2012)
• 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.”
25
Safety testing
Use of Alternative Technologies
 BacT/ALERT® rapid sterility system
− 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

26. Regulatory Guidance on Replication Competent Virus Testing
26
Guidance for Industry: Testing of Retroviral Vector-Based
Human Gene Therapy Products for Replication Competent
Retrovirus During Product Manufacture and Patient Follow-up.
FDA 2020
FDA-2020
• Recommendation that in-house standard virus stocks
are available that represents your clinical vector attributes,
including, the genetic background, envelop protein, and
deletion of accessory proteins.
EMA-2005 GUIDELINE ON DEVELOPMENT AND
MANUFACTURE OF LENTIVIRAL VECTORS
• For HIV-based LV, an attenuated HIV lacking all
accessory genes as a positive control for RCL assays
could be of value
26

27. 1. 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.
2. Guidelines have threshold levels of how many
virus particles/dose are tolerated
3.Examples of Replication competent virus testing
− Retrovirus (RCR)
− Lentivirus (RCL)
− AAV (rcAAV)
− HSV (rcHSV)
− Adenovirus (RCA)
27
Replication Competent Virus Detection
1If data on a sufficient number of batches can be submitted to US FDA
then this routine testing may be omitted
21% or 108 cells (whichever is less)
RCL
Lentiviral
vector
Infection of
C8166: 2-5 days
8 passages
PERT on culture
supernatant

28. Supernatant
testing
1
2
3
4
Determine volume in which a single
RCR can be detected.
Total test volume should be divided
into replicate samples, each
containing the volume demonstrated
to detect a single RCR
Sufficient supernatant should be
tested to ensure a 95%
probability of detection of RCR if
present at a concentration of 1
RCR/dose equivalent
In all cases, recommend
testing at least 5% of the total
supernatant by amplification on
a permissive cell line
Vector supernatant assays should
include culture of supernatant on a
permissive cell line
Amplified material may then be
detected in an appropriate endpoint
assay
5
US FDA: Replication Competent Retrovirus /
Lentivirus (RCR/RCL) Testing
28

29. Cell product
testing
1
2
3
4
Infectivity assay based on
co-cultivation with detector
cell lines and PERT
endpoint takes ≥ 4 weeks
Global regulations: Not
required by EMA and China
Required by US FDA
Patient monitoring for
Replication competent virus
(RCV) at 3,6 and 12 months
Expedited infectivity assay with end-
point analysis at 10 days or PCR assay
US FDA: Replication Competent Retrovirus / Lentivirus
(RCR/RCL) Testing

30. 30
Typical Characterization and release packages
Blazar® panels, as directed PCR based alternative to
broad spectrum adventitious agent testing
Choosing Advanced Technologies
Maintaining biosafety of cell and gene therapy products
NGS replacement to
address AAT and
novel agents

31. 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 Applicable Across Cell and Gene
Therapy Evaluations
31

32. HeLaRC32
➢Genomic copies
− Measurement of the
number of virus
genomes in a
preparation
− PCR based assay
➢Number of particles
− ELISA based assays to
measure virus capsid
proteins
➢Concentration of
viral particles that
can transduce cells
− Infectivity assays
− Require appropriate
cell substrate for
propagation
− Virus measurement
using PCR, ELISA, flow
cytometry, plaque/foci
formation.
Total Intact Viral
Particles
Infectious Titer Transgene Expression Functional Activity
➢ Flow cytometry
➢ Can also be used to
deduce transduction
efficiency
➢ ELISA
➢ Other (e.g.HPLC)
➢Biological effect
related to MOA in
physiologically
relevant system
➢Start development
of potency
tests early
➢Relevant controls
and appropriate
data analysis
methods
A Multi-faceted Approach is Recommended to Assess
Biological Activity
32

33. How do you ensure
testing is not the
rate limiting step for
the IND submission?

34.  Principles of testing are constant for both process and product even though these are novel
products
− Reduction in time, cost, and improved quality through standardization
 Forward planning and preparation of typical sample requirements for all cell substrate
characterisation and vector based testing
 Use a platform assay approach to minimise assay qualification requirements and improve overall
testing timelines
 Apply alternate testing methods to maintain specificity and sensitivity, save on use of
limited materials and obtain results in a timely manner
34
Key Steps in Manufacturing and Planning QC testing

35. 35
Multi step approach
Cell and Gene Therapy Testing
Virus Seed* Cell Banks Plasmids
Unprocessed
Bulk Harvest
Purified Bulk
Harvest
Final Lot
Sterility*
Identity*
Titer
Adventitious Agents*
Cell Properties
Vector Concentration*
Expression of Gene
Residuals
Product Characteristics*
Endotoxin*
* Rapid and alternate technologies may be appropriate

36. 36
Summary
 Global regulatory agencies continue to issue guidance
for development of cell and gene therapies in
response to an increasing number of clinical trials
 A stage appropriate implementation of GMP is
acceptable to regulatory agencies
 Emerging methods can provide better
characterization and biosafety methods
 Package approaches are expected and supported
 State of the art technologies are being applied at an
expansive rate for these novel therapies
 Regulatory agencies specify that AAV particle
composition should be measured, monitored and
reported
 Industry and regulators are working together
to build the knowledge-base for new
therapeutic modalities

37. Q&A

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