POTENCY ASSAYS FOR PLASMIDBASED VACCINES AND THERAPEUTICS

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POTENCY ASSAYS FOR PLASMID-BASED VACCINES AND
THERAPEUTICS
David C. Kaslow M.D.Chief Scientific
OfficerVical Incorporated
CTGTAC Meeting on Potency Assay 9 February 06
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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro In vivo correlate
• Summary

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Plasmid Vaccines and Therapeutics Pre-biologic
PRE-BIOLOGIC
Gene sequence Transcription Translation
Post-translational modification
mRNA
AAAAAAA
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Plasmid Vaccines and Therapeutics Pre-biologic
Strength v Potency

• Strength ? Potency
• Strength
• Determines dose
• Based on DNA concentration
• A260 (or equivalent)
• Potency
• Specific ability or capacity of the productto
  effect a given result
• In vitro or in vivo demonstration of
  manufacturing and product consistency

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Plasmid Vaccines and Therapeutics Pre-biologic
Strength v Potency
PRE-BIOLOGIC
Gene sequence Transcription Translation
Post-translational modification
Strength
mRNA
AAAAAAA
Potency
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Plasmid Vaccines and Therapeutics Pre-biologic
Strength v Potency
PRE-BIOLOGIC
Strength
A260 (Genetic Stability)
mRNA
RT-PCR
AAAAAAA
Potency
IP/WB or WB ELISA FACS
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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro In vivo correlate
• Summary

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Key Assumptions-Part 1 of 3

• The assay development focus should be on in vitro
  assays
• In vitro responses are less variable than in vivo
  assays
• In vitro responses have a greater dynamic range
  than in vivo assays

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Key Assumptions-Part 2 of 3

• If the immediate biological activity of a
  pre-biologic is to effect transcription of an
  immunogen or therapeutic protein, then the
  immediate biologic result of the product is mRNA.

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Key Assumptions-Part 3 of 3

• If a pre-biologic product is genetically stable,
  then
• there will be no lot-to-lot variability of
  primary nucleotide sequence
• there will be no lot-to-lot variability of
  primary, secondary or tertiary protein structure
• the only potential lot-to-lot variability of the
  drug substance is
• Strength
• Higher order DNA structure

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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro in vivo correlate
• Summary

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Evolution in Approach to Vaccine Regulation Two
Paradigms

• OLD Vaccine potency, as measured in the
  laboratory, is the most important characteristic
  to ensure human efficacy
• NEW Vaccine potency is only one of the tools
  used to ensure that a manufacturing process
  yields immunobiologicals of quality consistent
  with that of lots proven efficacious
• From Assays and laboratory markers of
  immunological importance
• Bruce D. Meade Juan L. Arciniega
• Laboratory of Methods Development and Quality
  Control
• Office of Vaccines Research and Review, CBER,
  FDA
• February 2001

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Tools for Characterization and Release

• Strength
• Nucleic acid concentration (A260)
• Identity
• Total pDNA size
• Restriction fragment length
• Potency
• Single point expression
• Relative potency
• RT-PCR
• ELISA
• FACS
• IP/WB
• Cell proliferation
• In vivo immunogenicity

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EvolutionPotency Assay Development Stages
Timeline
Pre-clinical
Phase I
Phase II
Phase III
Commercial

Protein-based
or
mRNA-based
Development
Qualification
Pre-Validation
Validation
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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro in vivo correlate
• Summary

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Allovectin-7 Drug Product
Treatment of chemo-naive metastatic melanoma
patients who have at least one injectable
cutaneous, subcutaneous, or nodal lesion
Sterile liquid, single-vial product, 2 mg/mL,
stored at 2-8ºC Bicistronic HLA-B7 ?-2
microglobulin formulated with a cationic lipid
(DMRIEDOPE)
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Development and Validation of a Potency Assay
for Lot Release

• Key regulatory issues
• HLA-B7 detectable and distinguishable
• ß2M complexed with HLA-B7
• HLA-B7 ß2M correct size
• Development strategy
• In vitro expression
• Quantification
• FACS (Fluorescence Activated Cell Sorting) RP
  (Relative Potency)
• Identity
• IP/WB (Immuno-Precipitation/Western Blot)

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OverviewAllovectin-7 Potency AD Stages
Timeline
Pre-clinical
Phase I
Phase II
Phase III
Commercial
IP/WB
IP/WB
IP/WB
FACS Single point
FACS Single point
FACS Single point
FACS RP Dose response
FACS RP Dose response
Assay Development
Assay Qualification
Assay Pre-validation
Assay Validation
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FACS RP Assay Qualification
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Allovectin-7? FACS RP Assay Qualification

• 8 dose transfection curves for HLA-B7 and ß2M
  each
• HLA-B7 0, 0.16, 0.31, 0.63, 1.25, 2.50, 3.75,
  5.00 ug/mL
• ß2M 0, 0.16, 0.31, 0.40, 0.63, 1.25, 2.50, 5.00
  ug/mL
• Data used for statistical analysis
• HLA-B7 43 reference curves, 32 pairs
• ß2M 21 reference curves, 24 pairs
• Statistical analysis based on
• Finney, D.J. Statistical Method in Biological
  Assay. Second Edition. 1971. Griffin, London.

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Typical Dose Response Reference Curves
?T - ?R ?
R 10
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FACS RP Assay Validation
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FACS Validation All Parameters Met Acceptance
Criteria

• Accuracy
• RP levels /- 35 of the expected recovery
• Precision
• Intra-assay 35 CV
• Inter-assay 35 CV
• Range
• Measured vs expected slope for all five RP
  levels 0.70 Slope 1.30
• Linearity
• Meets pre-determined suitability criteria for
  slope, intercept, RMSE
• Specificity
• Data from precision runs meets suitability
  criteria for /- controls

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Allovectin-7 CTMRP Results
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Allovectin-7 IP/WB Assay
ß2M
HLA-B7
MW 1 2 MW 3
MW 1 2 MW
kDa 188 98 62 49 38 28 17 14 6 3
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Allovectin-7 CTM IP/WB Assay Results
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Case study 1 Allovectin-7 FACS Summary

• FACS RP and IP/WB assays developed with FDA
  input
• Both assays successfully validated and used to
  evaluate Phase 2 CTM retains
• Phase 3 CTM to be evaluated against reference
  standard /- statistically determined reference
  variability
• Phase 3 CTM and validation lots will be used to
  determine commercial specifications

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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro in vivo correlate
• Summary

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Genetic StabilityWhat How

• Characterization (not release) assay
• Determined once for a MCB/WCB
• Stepwise approach completed as part of
  commercial-scale process validation
• At IND
• Sequence of MCB/WCB
• Restriction fragment size pattern on drug
  substance
• During clinical development
• Intermediate analysis to identify risk
• By commercial filing
• Complete analysis of plasmid backbone at
  full-scale
• Statistically significant GXP analysis of
  expression cassette at full-scale

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Genetic StabilityProtocol Overview
Predicted pDNA sequence
MCB MWCB Fermentation Purify pDNA Transform
select colonies pDNA from colonies
Observed pDNA sequence
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Genetic StabilityExample of Sample Size and
Confidence Level

• There is a gt95 probability of detecting one or
  more mutations in a sample of 30 independent
  clones if the actual mutation prevalence is gt10.
• A gt99 probability of detection for a mutation
  prevalence of gt1 would require 459 independent
  clones

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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro in vivo correlate
• Summary

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Plasmid Vaccines and Therapeutics Pre-biologic
Potency
PRE-BIOLOGIC
mRNA
RT-PCR
AAAAAAA
Potency
IP/WB or WB ELISA FACS
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Potency AssayRT-PCR RP Assay Rationale
PROTEIN
plasmid
Drug Product

• Pre-biologic
• Prepro-biologic

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Potency AssayRT-PCR Specificity of PCR Primer
In vivo
Transcription
In vitro
Reverse Transcription (RT)
mRNA
Gene-specific reverse primer
TaqMan PCR
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Outline

• Context
• Pre-biologics pDNA vaccines and therapeutics
• Potency v strength
• Potency assays of pre-biologics
• Key assumptions
• Potency assay evolution
• Protein-based potency assays
• Case study 1 Allovectin-7 FACS
• Polynucleotide-based potency assays
• Genetic stability
• mRNA the immediate given result
• RT-PCR
• Case study 2 CMV In vitro in vivo correlate
• Summary

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Potency AssayPoloxamer-formulated pDNA-based
Vaccine

• DNA vaccine
• hCMV gB
• hCMV pp65
• Plasmid backbone
• Optimized Vical design
• Tested in prior clinical trials
• Formulation
• 2 pDNAs (5mg/mL)
• CRL1005 poloxamer (7.5mg/mL)
• BAK (0.11 mg/mL)
• PBS

Bivalent
Vaccine to protect against CMV-associated disease
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Potency AssayTest Potency Samples

• Evaluate sample potencies
• Prepare samples of different potencies based on
  concentration
• Examined potency ranges
• 50 to 200
• Observation model
• Dose range plots for various potencies conform to
  parallel line model

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In Vitro / In Vivo Correlation

• Goal
• Determine whether in vitro relative potency
  correlates to changes in the CMV pDNA
  vaccine-mediated immune response
• In vitro Relative Potency (RP)
• response relative to a reference, using RT-PCR
• In vivo immune response in mice
• Anti-gB antibodies by ELISA
• pp65 T-cell responses using IFN-? ELISPOT

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In Vitro / In Vivo Correlation

• Method
• Evaluate hypo-potent CMV vaccine (80?C
  heat-degraded) versus the 100 potent CMV vaccine
  within the linear range of the in vitro and in
  vivo assays dose-response curves

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In Vitro / In Vivo Correlation
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In Vitro / In Vivo Correlation
In Vivo Study Design VCL-CB01
Bilateral IM injection of rectus femoris
administered on Days 0 and 14 Blood collected
prior to 1st injection (Pre-bleed) and Day 26
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In Vitro / In Vivo Correlation StudyAb Results
Normalized Antibody and Relative Potency Response

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In Vitro / In Vivo Correlation StudyT-cell
Results

• High variability
• Low responses
• Inconclusive results

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Case Study 2 CMV In Vitro / In Vivo
Correlation Summary

• Conclusions
• Forced degradation of pDNA correlates with a drop
  in relative potency (RP) by RT-PCR
• Drop in RP correlates to drop in CMV-mediated
  immune response
• Antibody data appears to correlate best with RP
  and degradation
• Slope analysis of downward trend statistically
  significant (p0.001)
• ELISPOT assay - inconclusive
• Variability too high
• Response lower than historical data

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Summary Potency AssayCharacterization Lot
Release
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Acknowledgements

• FACS RP Assay Development Team
• Basil Jones
• Lana Marjerison
• Beth Feher
• Robin Baptista
• Martha Till
• Kris Carner

• RT-PCR RP Assay Development Team
• Beth Feher
• Lana Marjerison
• Basil Jones
• Mindy Sam
• Rama Ghatti
• Rohit Mahajan Ph.D.

Alain Rolland, SVP Prod Dev Jukka
Hartikka Ph.D. Keith Hall, Head QC/AD Mary
Wolch Ph.D. Peggy Lalor Ph.D. Andy Geall
Ph.D. Gretchen Jimenez Ph.D.
Laureen Little Ph.D. Consultant, BioAssays Jan
Callahan Ph.D. - Consultant, Statistics
formerly with Vical