Transcript POTENCY ASSAYS FOR PLASMID-BASED VACCINES AND THERAPEUTICS David C. Kaslow M.D.

POTENCY ASSAYS FOR
PLASMID-BASED VACCINES
AND THERAPEUTICS
David C. Kaslow M.D.
Chief Scientific Officer
Vical Incorporated
CTGTAC Meeting on Potency Assay
9 February 06
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
Plasmid Vaccines and Therapeutics:
Pre-biologic
PRE-BIOLOGIC
Gene sequence
plasmid
Transcription
mRNA
AAAAAAA
Translation
BIOLOGIC
Protein
Post-translational
modification
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 product…to
effect a given result
• In vitro or in vivo demonstration of
manufacturing and product consistency
Plasmid Vaccines and Therapeutics:
Pre-biologic Strength v Potency
PRE-BIOLOGIC
Strength
Gene sequence
plasmid
Transcription
mRNA
AAAAAAA
Translation
Potency
BIOLOGIC
Protein
Post-translational
modification
Plasmid Vaccines and Therapeutics:
Pre-biologic Strength v Potency
PRE-BIOLOGIC
Strength
plasmid
mRNA
A260
(Genetic Stability)
RT-PCR
AAAAAAA
Potency
BIOLOGIC
Protein
IP/WB or WB
ELISA
FACS
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
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
Key Assumptions-Part 2 of 3
• If the immediate biological activity of a prebiologic is to effect transcription of an immunogen
or therapeutic protein, then the immediate biologic
result of the product is mRNA.
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
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
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
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
Evolution
Potency Assay Development Stages & Timeline
Pre-clinical
Phase I
Phase II
Phase III
Commercial
IP/WB
Immunogenicity
Cell Proliferation
+
ELISA
FACS
Protein-based
% Relative Potency
Single point
or
mRNA-based
RT-PCR
Development
% Relative Potency
Single point
Qualification
Pre-Validation
Validation
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
®
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 (DMRIE:DOPE)
Development and Validation of a
Potency Assay for Lot Release
• Key regulatory issues:
• HLA-B7 detectable and distinguishable
• β2M complexed with HLA-B7
• HLA-B7 & β2M correct size
• Development strategy:
• In vitro expression
• Quantification
• FACS (Fluorescence Activated Cell Sorting) %RP (Relative Potency)
• Identity
• IP/WB (Immuno-Precipitation/Western Blot)
Overview
Allovectin-7® Potency AD Stages & Timeline
Pre-clinical
Phase I
Phase II
IP/WB
FACS
Single point
FACS
Single point
FACS
Single point
Phase III
IP/WB
FACS %RP
Dose
response
Assay Development
Assay Qualification
Assay Pre-validation
Assay Validation
Commercial
IP/WB
FACS %RP
Dose
response
FACS %RP Assay Qualification
Allovectin-7 FACS %RP
Assay Qualification
• 8 dose transfection curves for HLA-B7 and β2M each:
• HLA-B7: 0, 0.16, 0.31, 0.63, 1.25, 2.50, 3.75, 5.00 ug/mL
• β2M: 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
• β2M: 21 reference curves, 24 pairs
• Statistical analysis based on
• Finney, D.J. Statistical Method in Biological Assay. Second Edition.
1971. Griffin, London.
Typical Dose Response
Reference Curves
HLA-B7 FACS Dose Response
B2M FACS Dose Response
40
% B2M Positive Cells
% HLA-B7 Positive Cells
50
30
20
10
100
90
80
70
60
50
40
30
20
10
0
0.00
0
0.00
1.25
2.50
3.75
1.25
5.00
2.50
ug/mL pDNA
ug/mL pDNA
HLA-B7
β2M
Dose response
model fit
Slope ratio
Parallel line
Dose treatment
(x-axis)
Untransformed
Log-transformed
Relative
Potency formula
R=
ßT
ßR
T - R
R = 10

3.75
5.00
FACS %RP Assay Validation
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
Allovectin-7® CTM
%RP Results
Lot
HLA-B7 (%RP)
β2M (%RP)
A
88.6
99.8
B
79.3
93.6
C
90.0
88.2
D
81.3
91.2
E
87.4
91.1
Mean  SD
85.3  4.7
92.8  4.4
Allovectin-7® IP/WB Assay
β2M
HLA-B7
MW
kDa
1
2 MW 3
MW 1
kDa
2
MW
188
188
98
62
49
38
28
98
62
49
17
14
38
28
6
17
14
3
6
3
Lane #
Sample
Lane #
Sample
1
Allovectin-7® ref.
1
Allovectin-7® ref.
2
Positive control
2
Positive control
3
Negative control
Allovectin-7® CTM
IP/WB Assay Results
Lot
HLA-B7 (kDa)
ß2M (kDa)
B
50.3
9.2
C
50.7
9.5
D
49.2
9.8
E
50.4
9.1
F
49.7
10.2
Mean ± SD
50.1 ± 0.6
9.6 ± 0.5
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
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
Genetic Stability
What & 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
Genetic Stability
Protocol Overview
MCB
Predicted pDNA sequence
MWCB
Fermentation
Purify pDNA
Transform & select colonies
pDNA from colonies
Observed pDNA sequence
Genetic Stability
Example of Sample Size and Confidence Level
Sample
Size
Mutants
Probability
20
0
12.16%
30
0
4.24%
50
0
1.70%
60
0
0.90%
There is a >95% probability of detecting one or more
mutations in a sample of 30 independent clones if the actual
mutation prevalence is >10%.
A >99% probability of detection for a mutation prevalence of
>1% would require 459 independent clones
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
Plasmid Vaccines and Therapeutics:
Pre-biologic Potency
PRE-BIOLOGIC
plasmid
mRNA
RT-PCR
AAAAAAA
Potency
BIOLOGIC
Protein
IP/WB or WB
ELISA
FACS
Potency Assay
RT-PCR %RP Assay Rationale
mRNA
plasmid
Drug Product
• Pre-biologic
PROTEIN
AAAAAAA
•
• Prepro-biologic
•
•
•
•
RT-PCR Assay
Attributes
Measures the immediate
biological effect
(transcription)
•
“Quantitative”
• Large dynamic range
• Low inherent variability
“Specific”
“Validatable”
•
Reagents: invariable,
stable, readily available
•
•
•
WB, FACS, ELISA Assay
Attributes
Measures distant biological
effect (cell substrate
dependent)
“Quantitative”
• Narrow dynamic range
• High inherent variability
“Specific”
“Validatable” (w/ difficulty)
Reagents: lot-to-lot variability,
unstable, long development
time
Potency Assay
RT-PCR & Specificity of PCR Primer
In vivo
promoter
intron
Gene
plasmid
Transcription
mRNA
In vitro
Reverse Transcription (RT)
mRNA
Gene-specific reverse primer
TaqMan® PCR
cDNA-specific forward primer
Taqman Probe
R
cDNA
Q
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
Potency Assay
Poloxamer-formulated pDNA-based Vaccine
• DNA vaccine
• hCMV gB
Bivalent
• 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
Vaccine to protect against
CMV-associated disease
Potency Assay
Test Potency Samples
• Evaluate sample potencies
Sample Potencies of Test Lot
• Prepare samples of different
potencies based on
concentration
35
• Examined potency ranges
34
33
• Observation & model
• Dose range plots for various
potencies conform to parallel line
model
Expected
Potency
Observed
Potency
50%
54%
75%
88%
150%
135%
200%
194%
CT Value
• 50% to 200%
32
200%
100%
50%
31
30
29
28
27
26
0.01
0.1
Log Transfected Dose (ug)
1
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-γ ELISPOT
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
In Vitro / In Vivo Correlation
80C forced pDNA degradation (hrs of treatment)
pDNA type
Aggregates
Multimer
Open Circular
Linear
Supercoil
Fully
degraded
Ref 0
6
9 12
15 18
21 24 27 30 33 36 39
42 45 48 87 Ref
In Vitro / In Vivo Correlation
In Vivo Study Design: VCL-CB01
% Relative Potency
(formulation treatment)
Total Dose
(µg)
Total Volume
# of
(µL)
animals/group
~100% (0 hr)
10
100
11
~55% (12 hr)
10
100
11
~22% (24 hr)
10
100
11
~11% (39 hr)
10
100
11
~0% (87 hr)
10
100
5
Bilateral IM injection of rectus femoris administered on Days 0 and 14
Blood collected prior to 1st injection (Pre-bleed) and Day 26
In Vitro / In Vivo Correlation Study
Ab Results
Normalized Antibody and Relative Potency Response
In vivo & in vitro response normalized to untreated
0.00%
% Drop
-25.00%
Ab (EU/ml)
-50.00%
Relative Potency (%RP)
-75.00%
-100.00%
0
15
30
45
60
75
90
hrs treatment @ 80 degrees C
Time @ 80C
Relative Potency (%RP)
Anti-gB Ab (EU/ml)
0 hr
101.25
60,227 +/- 6,157
12 hr
55.1
30,402 +/- 3,504
24 hr
22.25
30,535 +/- 4,942
39 hr
11.25
4,081 +/- 2,737
87 hr
0
0
In Vitro / In Vivo Correlation Study
T-cell Results
• High variability
• Low responses
• Inconclusive results
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
(p=0.001)
• ELISPOT assay - inconclusive
• Variability too high
• Response lower than historical data
Summary: Potency Assay
Characterization & Lot Release
Nucleotide Structure
Transcription
Characterization
Lot Release
Characterization
Lot Release
Genetic
stability
Total size
Pre-clinical
“efficacy”
RT-PCR
% Relative
potency
Restriction
enzyme digest
In vitro / in vivo
correlation
HPLC analysis
(%SC, %OC, &
% linear)
Acknowledgements
• FACS %RP Assay
Development Team
• Basil Jones
• Lana Marjerison
• Beth Feher
• Robin Baptista
• Martha Till *
• Kris Carner
Alain Rolland, SVP Prod Dev
Keith Hall, Head QC/AD
Peggy Lalor Ph.D.
• RT-PCR %RP Assay
Development Team
• Beth Feher
• Lana Marjerison
• Basil Jones
• Mindy Sam
• Rama Ghatti
• Rohit Mahajan Ph.D.
Jukka Hartikka Ph.D.
Mary Wolch 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