Releasing Your AAV Therapy with Confidence: Regulatory Considerations and Key Assays

The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Releasing your AAV therapy
with confidence – Regulatory
considerations and key
assays
Steven McDade &
Alfonso Lavorgna Ph.D.

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

Agenda
Regulatory considerations throughout the AAV manufacturing process
Steven McDade
Review of the rcAAV and infectious titer assays
1
2

Regulatory
considerations
throughout the AAV
manufacturing process

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
Difficult for enveloped viral vectors – but possible for AAV
A virus safety risk assessment should be conducted
(process indicated in European Pharmacopoeia 5.1.7)
5

Increase in regulations for cell and gene therapy observed
in recent 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
PIC/S Annex 2A Draft guidance for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
FDA: Potency tests for cellular and
gene therapy products
FDA Draft guidance on CMC and Retroviral
testing guidance
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.
 Chemistry, manufacturing and control
(CMC) for human gene therapy INDs
ChP: General Chapter of Gene Therapy
Products for Human Use (draft)
2008 2011 2017 2018 2019 2020
6

Building a testing strategy – what and when to test
MCB
WCB /
CAL
bioreactor
purified drug
substance
final drug
product
raw
materials
plasmid
plasmid stock
Biosafety
Identity
Biosafety
Identity
Biosafety
Identity
Biosafety
Identity
Biosafety
Identity
Biosafety
Stability
Container
integrity
Potency
Identity
Identity
Biosafety
Potency
Identity
Biosafety
Potency
Residuals
Properties
Residuals
E.coli
cell banks
Identity
Biosafety

Questions & 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 (EMA 2021)
Principles of GMP apply to plasmid manufacturing
For certain starting materials of biological
origin, (such as e.g. linear DNA used as
template for ex vivo transcription into mRNA,
plasmids to generate viral vectors and/or
mRNA, and vectors) used to transfer genetic
material for the manufacturing of ATMPs it is,
however, mandatory to comply with the
principles of GMP.

• “Should be capable of maintaining a level of cell viability upon reconstitution which is both consistent
and adequate for production use”
• “The design and performance of specific tests for adventitious microbial agents and adventitious
cellular contaminants should take into account the properties of the banked cell, the likely
contaminants based upon scientific literature, source, methods and materials used for cultivation and
other organisms present in the lab”
• “Visual examination of the characteristics of well isolated colonies is suggested, using several
microbiological media”
• “Analysis of growth on selective media is usually adequate to confirm host cell identity at the species
level”
• “Either phenotypic or genotypic characteristics may be used in identity testing”
• “Consistency of the coding sequence of the expression construct should be verified”
• “Restriction endonuclease mapping or other suitable techniques should be used to analyse the
expression construct for copy number, for insertions and deletions”
• “For extrachromosomal expression systems, the percent of host cells retaining the construct should be
determined”
Key elements of E.coli cell bank characterization
Identity
Purity
Stability
Viability
ICH Q5B & Q5D plus Ph. Eur. 5.14 apply to address the characterization & genetic stability of E.coli cell
banks

Overview of E.coli characterization assays
Description
Viability Determination of cell viability in the sponsor’s cell bank
Purity
Qualification of purity testing for microbial cell banks
Purity testing of microbial cell banks: Presence of bacterial and fungal contaminants
Determination of the purity of the sponsors bacterial strain by Gram's staining
Detection for the presence of bacteriophage in material derived from a cell bank of E. coli or material used in the
propagation of E. coli cultures.
Detection of lysogenic bacteriophage in test material derived from Escherichia coli cell banks
Identity
Confirmation of the identity of an E. coli strain by RAPD analysis
Identification of Enterobacteriaceae and other Gram negative rods using the API® 20 Identification system
Determination of the partial genotype of the sponsor's E. coli;
Ampicillin Resistance
Kanamycin resistance
Chloramphenicol Resistance
Tetracycline Resistance
Nalidixic Acid Resistance
Streptomycin Resistance
Determination of the partial genotype of the sponsor's E. coli;
Determination of gal genotype
Determination of lac genotype
Determination of met genotype
Determination of thi genotype
Determination of recA-1 genotype
Determination of supE genotype
Determination of F genotype
Genetic
Stability
Retention of Expression Construct in a Bacterial Cell Bank
Nucleotide sequence analysis of recombinant plasmid expression vector
Genetic Stability and Plasmid Identity by restriction enzyme map analysis
Design and qualification of a droplet digital PCR assay (ddPCR) for the determination of plasmid copy number
Determination of Plasmid Copy Number in a Microbial Cell Bank by droplet digital PCR (ddPCR)

Plasmids used for vector production
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
cells used for the manufacture of plasmid vectors” section
Recommended testing for plasmids:
− Identity: Sequencing or PCR
− DNA concentration
− DNA forms: HPLC or CE
− Supercoiled, multimeric, relaxed monomer and linear forms
− Residual host cell DNA, RNA and host cell protein
− Sterility, endotoxin
− Appearance, pH, extractable volume, residual moisture (if freeze dried)
11

Human cell line characterization
CMC Information for Human Gene Therapy IND applications (2020)
 “For biologically sourced reagents including human, bovine and porcine derived materials we
recommend that you also refer to FDAs Guidance for Industry “Characterization and Qualification of
Cell Substrates and other Biological Materials used in the Production of Viral Vaccines for Infectious
Disease indications (2010)”
 ICH Q5D is also referenced
− Ensure absence of microbial contamination
− For cell lines used for production of viral vectors, test for retroviral contamination (TEM and RT
assay)
− Adventitious agents including bovine/porcine viruses if exposed to serum/trypsin
− Species specific pathogens (CMV, HIV, HTLV, HHV 6,7,8, JC, BK, EBV, B19, HBV, Papillomavirus,
HCV, Circovirus)
− Confirm identity (Short Tandem Repeat, Karyology)
− Tumorigenicity
13

Cell line characterization (e.g. HEK293 cells)
MCB
Master cell bank
WCB
Working cell bank
CAL
Cells at limit of in vitro
cell age
 Identity: CO1 barcode,
STR analysis, karyology
 Microbiology: Bacteria,
fungi, mycoplasma,
mycobacterium
 Adventitious viruses: in
vitro, in vivo, PCR*
 Retroviruses: TEM,
reverse transcriptase,
retrovirus infectivity
 Genetic stability
(if stable transfection)
fungi, mycoplasma,
mycobacterium
vitro
fungi, mycoplasma,
mycobacterium
vitro, in vivo, PCR
 Retroviruses: TEM,
reverse transcriptase,
retrovirus infectivity
 Tumorigenicity: tumor
formation in nude mice
*HIV I & II, HTLV I & II, HAV, HBV, HCV, CMV, EBV, HHV 6, 7, 8, B19, HSV I & II,
human polyoma viruses, Bocavirus, Metapnuemovirus, etc.

16
Characterization of viral vectors
Identity Biosafety
Titer/Potency Residuals
Product
• Ph. Eur. 5.14 Gene Transfer Medicinal Products for Human use
• FDA CMC for Human Gene Therapy INDs (2020)
16

• Identity: Gene of interest
(GOI)
• Biosafety:
• Sterility or Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
and in vivo)
• Replication competent
AAV (rcAAV)
• Potency: Infectivity,
genomic titer
Testing bulk and final lots: AAV vectors
Unprocessed
bulk
Purified
bulk
• Identity: GOI, ELISA, vector genome
• Biosafety: Sterility, endotoxin, rcAAV
• Potency: infectivity, genomic titer,
expressed protein level/function
• Residuals:
• Residual host cell DNA
• Residual plasmid DNA
• Host cell protein, residual BSA,
residual Benzonase® endonuclease
• Purity by SDS-PAGE
• Empty:full capsid ratio
• Identity: GOI
• Biosafety: Sterility, endotoxin
• Potency: infectivity, genomic
titer, expressed protein
level/function
• Product properties:
• Vector aggregates
• Osmolality
• pH
• Extractable volume
• Appearance
• Particulates
Formulated
and vialed
final lots
17

18
BacT® Alert 3D rapid microbial detection system
Rapid sterility testing
Scan Load
Inoculate
 Samples are 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
 The BacT/ALERT® 3D system can detect most common
contaminants within 48 to 72 hours

Mycoplasma assay comparison: Cultivation vs QPCR
Historical compendial method Established alternative method
Method largely manual Automated, reproducible
Detection limit: ≤ 100 Colony Forming Units Detection limit: 10 Colony Forming Units
15 ml of UBH required 4 ml of UBH required
Assay turnaround time: 28 days Assay turnaround time: 3 - 7 days
The use of PCR for the detection of mycoplasma, supported by comparability data, has
been used within industry for several years. This has included the testing of starting
materials through to clinical material for a range of biopharmaceuticals and many
submissions have been made to the US and European regulatory authorities over this time.
Cultivation QPCR

Stability studies
• Demonstrate short and long-term stability of a
product after exposure to various environmental
conditions
− Temperature
− Humidity
− Light
• Analytical testing programs are designed by
industry-experienced scientists to suit your needs
• Accelerated stability and forced degradation studies
are available
• State-of-the-art instrumentation and cGMP
capabilities provide results you can trust
• Reliable service with fast turnaround delivers data on
time
Timepoint
Infectious
Titer

Viral clearance studies for AAV therapies
FDA CMC for Human Gene Therapy IND (2020)
references “In some instances, robust viral clearance
studies may be necessary to remove and inactivate
adventitious agents”. If safety can be built in to a
process, it should be.
Clearance steps should be evaluated and be aligned
with ICH Q5A expectations for study design. Draft of
ICH Q5A revision 2 expected to be issued in 2022.
Viral safety strategy includes prevention, detection,
and if possible, removal/inactivation of viruses
AAV are small, non-enveloped viruses
Focus on inactivation and filtration methods. Target
reduction of large, enveloped viruses and medium
sized non-enveloped viruses
21

Review of the rcAAV and
infectious titer assays

Assays
Replication competent AAV (rcAAV) assay
AAV Infectivity TCID50 assay
1
2

Detection of replication competent AAV (rcAAV)
• Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and
Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs),
dated April 2008
“…to determine the amount of replication competent AAV present in the final vector product and
report the results in your IND”
• Guidance for Industry: Chemistry, Manufacturing, and Control (CMC) Information for Human Gene
Therapy Investigational New Drug Applications (INDs), dated January 2020
“We recommend that you test for rcAAV, which could potentially replicate in the presence of helper
virus, and report these results.”
24

• To detect replication competent AAV, if present,
in unprocessed bulk/purified bulk and report the
results.
• Test material includes: AAV drug substance or
cell banks modified with AAV vector.
• What subtype/serotype of AAV can be tested?
HEK293 cell line is used for the amplification
phase and it can support replication subtype 1, 2,
3, 6, 8 and 9. Other subtypes will need to be
evaluated case by case.
• Share Rep2 sequence to confirm qPCR target
compatibility.
Assay Purpose Key Questions
1 2
25
• Cell culture allows amplification of rcAAV present
in the test material at low level.
• Endpoint qPCR is targeting Rep2 gene, which
should be present in rcAAV viruses but not in the
replication incompetent AAV.
• Limit of detection of the assay is 20 IU/inoculum.
• Qualification required? Y
• Sample type: drug substance or cell bank
• Sample Volume: 0.5-1.0mL
• Compliance: GMP
Key Assay Info
4
Method / Technology
3

Cell transduction efficiency
Test System
 HEK 293
Ellis et al. Virology Journal 2013, 10:74
26

27
Detection of replication competent
AAV (rcAAV) – How the assay is
performed
TEST CONDITIONS
PSQ
• Cell Control
• Negative Control
• Test Article D1*
• Positive Control 20 IU
• Test Article D1+ PC 20 IU
GMP
• Cell Control
• Negative Control
• Test Article
• Positive Control 20 IU
• Test Article + PC 20 IU
* - dilutions (doses) of TA
3
days
Detection
(Rep2 qPCR)
3
days
3
days
Sampling
Inoculation
(max 1.0E11 vg)
Cell Passages (HEK293)
P2 P3
P1
Sampling
R2 R3
R1

Assay essentials: Positive Control, Test System and Assay End-Point
Test system
End-point
rep2-qPCR
Positive
Control
Virus
Client-specific assay Development
Test system
Platform-based validation:
➢ Simplified validation protocol and report
➢ Document templates
➢ Limited number of tests for validation
Assay platform
Generic assay
Test system
HEK293
Ad5
End-point
Rep2-qPCR
Positive
Control
Virus
AAV2
From generic to client-specific assays
28

AAV Infectivity: TCID50
 Content/Potency (ICH Q2(R1))
Parameters included in validation: accuracy, precision, specificity, linearity and range
AVV-based gene therapies require high concentrations of viral vectors to be effective. That is because such
treatments often need to be administered in low volumes in smaller areas of the body. An accurate titration is
critical for the success if the product.
Assay essentials: Reference Material, Test System and Assay End-Point
Reference Standard: universal AAV Reference Standard Material (AAV RSM), which is an AAV
sample that has been quantified by 16 labs around the world and can be used to validate their qPCR assay.
HeLaRC32: modified cell line that can amplify many AAV subtypes.
Assay endpoint: qPCR for CMV promoter which is applicable to many vectors.
29

• The generic assay has been fully validated
• The Product Specific Qualification (PSQ) is
recommended:
a. to establish a serial dilution range for testing
b. to assess some of the parameter assessed in
validation
• GMP testing
Deliverables: Technical specification, Certificate of
Analysis, Laboratory Records (QA Audited)
• NRT testing
Deliverables: Unaudited Final Report, Raw data
summary if required.
30
AAV Infectivity: TCID50

31
AAV infectivity: TCID50
1 ml
etc…
H
G
F
E
D
C
B
A
N
1
-0.7
2
-1.4
3
-2.1
4
-2.8
5
-3.5
6
etc.
7
8
9
10
11
12
1 ml 1 ml 1 ml 1 ml
4 ml 4 ml 4 ml 4 ml
Virus
Dilution
Proportion of
positive wells
1
1
0.75
0.5
0.375
0.125
0
0
0
0
0
0
Wells are scored as + or -
50% Infection
< 50% Infection
> 50% Infection
Infect replicate wells with serial dilutions of sample
Virus Detection by PCR

Assay essentials: Positive Control, Test System and Assay End-Point
Client-specific assay Development
Platform based validation:
➢ Simplified validation protocol and report
➢ Document templates
➢ Limited number of tests for validation
Assay platform
Generic assay
Test system
HeLaRC32
Ad5
End-point
CMV-qPCR
Positive
Control
Virus
AAV2
From generic to client-specific assays
Test system
End-point
GOI-qPCR
Positive
Control
Virus
provided by
Sponsor
32

Summary
33
Holistic approach
Assay versatility
Risk assessment
Regulatory considerations
1
2
3
4

Operations Manager, Virology Services
Senior Technical Specialist, Field
Technology Management
Steven McDade
Thank you!
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.
© 2021 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

Releasing Your AAV Therapy with Confidence: Regulatory Considerations and Key Assays

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