The document discusses the latest regulatory expectations and challenges for biosafety testing of gene therapies, including how to design a robust safety testing strategy to test for bacteria, fungi, mycoplasma, endotoxins, and adventitious viruses using methods such as sterility testing, PCR assays, and in vivo and in vitro testing. It also covers emerging areas like next generation sequencing, replication competent AAV detection, and characterization of residual DNA levels.

1. Merck KGaA
Darmstadt, Germany
Sarah Sheridan, Ph.D.
Principal Scientist, Field Development Services
Latest Updates in
Biosafety Testing for
Gene Therapy

2. The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.
2 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

3. Latest updates in biosafety testing for Gene Therapy
In this webinar you will find out:
• Current regulatory expectations for
Gene Therapies
• How to design a testing strategy
• How to overcome unique challenges
with Gene Therapies
3 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

4. Contents
3
2
1 Current regulatory expectations
What and when to test
Test methods
4 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

5. Safety
testing
strategy
Virus safety
risk
assessment
Regulations
& guidance
documents
Regulatory
updates
Building a robust safety testing strategy
5 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

6. European Consultation Document: Good Manufacturing Practice
for Advanced Therapy Medicinal Products
• Consultation ended on 26th Sept 2016
• Covers GMP for ATMPs for marketing authorisation and
investigational ATMPs
GMP for ATMPs
6 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

7. EP 5.2.12 Raw materials of biological origin for the production of cell-based and gene
therapy medicinal products, 01/2017:50212
• Risk assessment
• General requirements
• Origin
• Production
• General quality requirements
• Identification; Tests; Assay; Reference material/batch
• Storage
• Labelling
• Sera and serum replacements
• Proteins produced by recombinant DNA technology
• Proteins extracted from biological material
• Definition; production; identification; tests/assay
EP regulations for raw materials
7 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

8. • Reflection paper: Overview of current regulatory testing requirements
for medicinal products for human use and opportunities for
implementation of the 3Rs
• Opportunities for limited animal testing
• Rabbit pyrogen test
• Abnormal toxicity test
• Viral safety
• Other aspects reviewed: manufacture, characterisation and control of
the drug substance; cell and gene therapy products
EMA guidance on implementation of 3Rs
EMA/CHMP/CVMP/JEG-3Rs/742/466/2015
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9. Next Generation Sequencing
• WHO and European Pharmacopoeia documents have indicated the
potential usefulness of NGS
• Draft EP 5.2.14. Substitution of in vivo method(s) by in vitro
method(s) for the quality control of vaccines
• Novel, sensitive molecular techniques with broad detection
capabilities
• Use of new broad molecular methods has highlighted gaps with
the existing testing strategy
• The implementation of such new broad molecular methods
requires a comparison of the specificity and the sensitivity
9
9
Latest Updates in Biosafety Testing for Gene Therapy | May 2018

10. 21 CFR 610.12 revised 2012 by FDA - Effective 04 Jun 2012
• Greater flexibility and encourages use of the most appropriate and
state-of-the-art test methods
• Promotes improvement and innovation in the development of sterility
testing
• New methods can yield accurate and reliable test results in less time
and with less operator intervention
• ATP bioluminescence
• Chemiluminescence
• Carbon dioxide head space measurement
• Manufacturers may benefit from using such sterility test methods
• Sample must be appropriate to material tested
• No mandated method
Amendment to Sterility Rule 2012
10 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

11. Contents
3
2
1 Current regulatory expectations
What and when to test
Test methods
11 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

12. Case Studies of Microbial Contamination in Biologic Product Manufacturing
Suvarna, K., Lolas, A., Hughes, P., Friedman, R. Biotechnology Manufacturing Team, Division of Manufacturing and
Product Quality, Office of Compliance, Center for Drug Evaluation and Research, Food and Drug Administration
Facility
Equipment
Process
Materials
Utilities
Personnel
Each source
is a potential
entry point for
microbial
contamination
Acholeplasma laidlawii (<0.2um)
Leptospira species (>5 um)
Sources of Adventitious Agent Contamination
Minute virus of mice (MVM) ~18-24nm
12 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

13. Viral Safety Strategy for Traditional Biological Products
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
13 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

14. Challenges with gene therapies
• Regulatory landscape
• FDA and EMA
• No terminal sterilisation process
• Small lot size/limited sample volume
• Limited availability of starting materials for process, product and test
method development
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15. Vector/Cell
Master Cell Bank (MCB)
Working Cell Bank (WCB)
Process Development
(Growth/Production/Modification)
Master/Working Virus Bank
(MVB/WVB)
Drug Substance Drug Product
Identity
Purity
Safety
Identity
Safety
QA/QC
In-process
testing
In-process
testing
Container Closure
Stability
Lot Release Testing
Shipping
Identity
Purity
Safety
Expression
In-process
testing
Vector and Cell Safety and Characterisation
Raw
materials
Safety
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16. • Identity: GOI
• Titre: TCID50 of viral vector
• Purity:
• Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
& in vivo)
• Replication competent AAV
Testing AAV bulk and final lots
Unpurified
bulk
Purified
bulk
• Identity: Serotype, GOI, Vector
genome
• Titre: TCID50 of viral vector, Genomic
titre
• Potency: r-AAV expressed protein
• Purity: Sterility, Endotoxin, Replication
competent AAV
• Residuals:
• Residual host cell DNA, Human DNA
• Residual DNA size distribution
• Host cell protein, Residual BSA
• Detection and measurement of AAV
affinity ligand
• Purity, Empty versus full capsid ratio
• Identity: GOI
• Titre: TCID50 of viral vector,
Genomic titre
• Potency: r-AAV expressed
protein
• Purity: Sterility, Endotoxin
• Product characteristics:
• Vector aggregates
• Osmolality
• pH
• Extractable volume
• Appearance and Particulates
Formulated
& vialed
final lots
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17. Contents
3
2
1 Current regulatory expectations
What and when to test
Test methods
17 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

18. Bacteria & Fungi

19. Direct or Filtration methods
 Direct: Test article inoculated directly into media
 Membrane Filtration: Test article filtered through
a membrane, which is then rinsed. Media added
to membrane canister
 Two media types used for each
TSB media – Incubated at 20-25°C
THIO media – Incubated at 30-35°C
• Media is observed for microbial growth on day 3,
4 or 5, day 7 or 8, and day 14.
• Sampling plan is dependent on Product type
Sterility Test – compendial method
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20. Inoculate test article into
aerobic and anaerobic
media
Scan samples onto system
using LIMS barcode labels
Load samples into
incubator
Sterility test
 Samples automatically monitored and read every 10 minutes
 Sensor in broth bottle monitors CO2 production, indicative of the
presence of microbial growth
 Presence of turbidity caused by debris/cellular material does not
interfere with interpretation of results
 Non-destructive technology allows for subculture of positive broths
for identification
BacT/ALERT 3D Rapid Microbial Detection System
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21. Mycobacterium

22. Tests for Mycobacterium species
• Genus Mycobacteria (gram-positive, rod-shaped bacteria of the
Family Actinomycete) > 40 recognised species
• Mycobacteria (M.tuberculosis, M.bovis, M.africanum, M.microti)
considered the most pathogenic to man
• M tuberculosis sub-clinically infects a third of the world's population,
killing 1.3 million people each year (WHO, Global tuberculosis report
2013)
• Regulatory guidance:
• US FDA Guidance, WHO & EP
• Cell banks and virus batches
• in vitro culture method, PCR or guinea pigs
Test in guinea pigs
PCR assay
Cultivation assay
22
Latest Updates in Biosafety Testing for Gene Therapy | May 2018

23. Mycoplasma

24. Test for the presence of Mycoplasma (USP, EP, 1993 PTC)
24
Mycoplasma hyorhinis
Latest Updates in Biosafety Testing for Gene Therapy | May 2018

25. • Classical mycoplasma assay often rate-limiting step
• PCR assays developed with equivalent or better sensitivity and
specificity
European Pharmacopoeia, 2.6.7. Mycoplasmas
 Allows PCR assays with equivalent sensitivity and specificity
US FDA Guidance for Industry: Characterisation and Qualification
of Cell Substrates and Other Biological Starting Materials used in
Production of Viral Vaccines for the Prevention and Treatment of
Infectious Diseases, 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”
• Produce comparability data (PCR and culture methods) for testing
Mycoplasma PCR
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26. Endotoxins

27. Tests for Endotoxins
27 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

28. Monocyte activation test (MAT)
28 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

29. Adventitious viruses

30. Cells inoculated with Test Article
Monitor for infection
Cytopathic Effect Hemagglutination
Guinea pig, chicken and
human type O erythrocytes
Negative for
Hemadsorption
Positive for
Hemadsorption
Hemadsorption
Uninfected
Control Cells
virus-infected cell Negative Well Positive Well
In vitro assay
30
Latest Updates in Biosafety Testing for Gene Therapy | May 2018

31. In vivo assay
Inoculate 20 suckling mice
per os (0.01ml)
i.p. (0.1ml)
i.c. (0.01ml)
Sacrifice and pool organs
Inoculate homogenate
into 10 suckling mice
Observe
suckling
mice for 14
days
Observe suckling
mice for 14 days
Sacrifice
Inoculate 10 adult mice
per os (0.05ml)
i.p. (0.5ml)
i.c. (0.03ml)
i.n. (0.05ml)
Observe
mice
for 28 days
Sacrifice
Suckling mice Adult mice
31 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

32. 10-11 day old embryos inoculated
via the allantoic cavity
6-7 day old embryos inoculated
via the yolk sac
Incubate for 3 days
Assess embryos for viability
Haemagglutination using chicken,
guinea pig & human type ‘O’ rbcs
Assess embryos for viability
Haemagglutination using chicken,
guinea pig & human type ‘O’ rbcs
10-11 day old
embryos
Inoculated via the
allantoic cavity
Incubate for 3 days
Assess embryos
for viability
6-7 day old
embryos inoculated
via the yolk sac
Incubate for 9 days
Incubate for 9 days
Assess embryos
for viability
In vivo assay
Embryonated eggs
32 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

33. Next Generation Sequencing
• Agnostic tool to detect contaminants
• No selection of nucleic acid prior to sequencing
• Depth of sequencing may allow construction of whole virus genome
Sample Processing Sequencing Bioinformatics
Next Generation Sequencing (NGS)
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34. Titre, rcAAV & residual DNA

35. Infectivity
Infectivity assay - TCID50
• only one cell substrate available-
HeLa-RC32
• limited number of reference standards
- AAV2 and AAV8
• GOI-specific end-point assay
35 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

36. Replication Competent AAV Assay design
Amplification in HEK-293 cells
Negative CTRL Test article (rAAVN) Positive Control Spiked test article
rcAAV2: 20IU 40IU 20IU 40IU
Inoculation R1 R2 R3
3
days
Detection: rep2 qPCR
3
days
3
days
Sampling:
Three rounds (R) of amplification in HEK-293 cells with monitoring rep2 presence by qPCR
36 Latest Updates in Biosafety Testing for Gene Therapy | May 2018

37. Regulatory
Guidance
Residual DNA Sizing
1 Residual DNA Assays are required by WHO
and FDA guidance for Industry
Agencies provide guidance on both quantity
and size distribution of DNA
2 Quantity is guided as 10 ng/dose (FDA &
WHO)
Size is guided by the FDA as a “median
DNA size of 200 bp or lower” for Vaccines
and related Biological products
3 Size distribution is important because of
potential Oncogenic effects of residual DNA
greater than 200 bp in length
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38. End Repair/ dA-Tailing
Adapter Ligation
PCR Amplification
BioAnalyzer
Sizing Residual DNA Assay
Work Flow
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39. Conclusion
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40. Principal Scientist,
BioReliance® Services
Email: sarah.sheridan@sial.com
Tel.: + 44 (0) 7515 975450
Sarah Sheridan, PhD
The vibrant M 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.
© 2018 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.