POTENCY ASSAYS FOR PLASMID-BASED VACCINES AND THERAPEUTICS David C. Kaslow M.D. Chief Scientific Officer Vical Incorporated CTGTAC Meeting on Potency Assay

POTENCY ASSAYS FOR POTENCY ASSAYS FOR PLASMID-BASED VACCINES PLASMID-BASED VACCINES AND THERAPEUTICSAND THERAPEUTICS

POTENCY ASSAYS FOR POTENCY ASSAYS FOR PLASMID-BASED VACCINES PLASMID-BASED VACCINES AND THERAPEUTICSAND THERAPEUTICS

David C. Kaslow M.D.Chief Scientific OfficerVical Incorporated

David C. Kaslow M.D.Chief Scientific OfficerVical Incorporated

CTGTAC Meeting on Potency Assay 9 February 06CTGTAC Meeting on Potency Assay 9 February 06

OutlineOutline• 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: Plasmid Vaccines and Therapeutics: Pre-biologicPre-biologic

Gene sequence

Transcription

Translation

Post-translationalmodification

BIOLOGICProtein

plasmid

AAAAAAA

mRNA

PRE-BIOLOGIC

Plasmid Vaccines and Therapeutics: Plasmid Vaccines and Therapeutics: Pre-biologic Strength v PotencyPre-biologic Strength v PotencyStrength ≠ 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: Plasmid Vaccines and Therapeutics: Pre-biologic Strength v PotencyPre-biologic Strength v Potency

Gene sequence

Transcription

Translation

Post-translationalmodification

plasmid

AAAAAAA

mRNA

PRE-BIOLOGIC

Strength

Potency BIOLOGICProtein

Plasmid Vaccines and Therapeutics: Plasmid Vaccines and Therapeutics: Pre-biologic Strength v PotencyPre-biologic Strength v Potency

plasmid

AAAAAAA

mRNA

PRE-BIOLOGIC

Strength

Potency

RT-PCR

IP/WB or WBELISAFACS

A260

(Genetic Stability)

BIOLOGICProtein



• Potency assays of pre-biologics:• Key assumptions • Potency assay evolution• Protein-based potency assays


• 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 3Key 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 3Key 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.

Key Assumptions-Part 3 of 3Key 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





• 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 Evolution in Approach to Vaccine Regulation: Two ParadigmsVaccine 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 ReleaseTools 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

EvolutionEvolutionPotency Assay Development Stages & TimelinePotency Assay Development Stages & Timeline

Pre-clinical Phase I Phase II Phase III Commercial

Development Qualification

Pre-Validation Validation

RT-PCR Single point % Relative Potency

ELISAFACS Single point % Relative Potency

IP/WBImmunogenicityCell Proliferation

Protein-based

mRNA-based

+

or






• Summary

Sterile liquid, single-vial product, 2 mg/mL, stored at 2-8ºC:Bicistronic HLA-B7 + -2 microglobulinformulated with a cationic lipid (DMRIE:DOPE)

Treatment of chemo-naive metastatic melanoma patients who have at least one injectable cutaneous, subcutaneous, or nodal lesion

Allovectin-7Allovectin-7® ® Drug ProductDrug Product

Development and Validation of a Development and Validation of a Potency Assay for Lot ReleasePotency 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)

OverviewOverviewAllovectin-7Allovectin-7®® Potency AD Stages & Timeline Potency AD Stages & Timeline

Pre-clinical Phase I Phase II Phase III Commercial

Assay Development

Assay Qualification

Assay Pre-validation

Assay Validation

FACSSingle point

IP/WB

FACSSingle point

FACSSingle point

FACS %RP Dose response

IP/WBIP/WB

FACS %RP Dose response

FACS %RP Assay QualificationFACS %RP Assay Qualification

Allovectin-7Allovectin-7 FACS %RP FACS %RP Assay QualificationAssay 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 Typical Dose Response Reference CurvesReference Curves

HLA-B7 FACS Dose Response

0

10

20

30

40

50

0.00 1.25 2.50 3.75 5.00

ug/mL pDNA

% H

LA

-B7

Po

sit

ive

Ce

lls

B2M FACS Dose Response

0

10

20

30

40

50

60

70

80

90

100

0.00 1.25 2.50 3.75 5.00

ug/mL pDNA

% B

2M

Po

sit

ive C

ell

s

HLA-B7 β2MDose response

model fitSlope ratio Parallel line

Dose treatment (x-axis)

Untransformed Log-transformed

Relative Potency formula R = 10

T - R

R =ßT

ßR

FACS %RP Assay ValidationFACS %RP Assay Validation

FACS Validation FACS Validation All Parameters Met Acceptance CriteriaAll 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-7Allovectin-7®® CTM CTM%RP Results%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-7Allovectin-7®® IP/WB Assay IP/WB Assay

Lane # Sample

1 Allovectin-7® ref.

2 Positive control

3 Negative control

β2MHLA-B7 MW 1 2 MW 3

kDa

188

98

6249

38

28

1714

6

3

kDa

188

9862493828

1714

6

3

Lane # Sample

1 Allovectin-7® ref.

2 Positive control

MW 1 2 MW

Allovectin-7Allovectin-7®® CTM CTM IP/WB Assay IP/WB Assay ResultsResults

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: Case study #1: Allovectin-7Allovectin-7®® & FACS Summary & 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






• Summary

Genetic StabilityGenetic StabilityWhat & HowWhat & 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

Observed pDNA sequence

Predicted pDNA sequence MCB

MWCB

Fermentation

Purify pDNA

Transform & select colonies

pDNA from colonies

Genetic StabilityGenetic StabilityProtocol OverviewProtocol Overview

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

Sample Size

Mutants Probability

20 0 12.16%

30 0 4.24%

50 0 1.70%

60 0 0.90%

Genetic StabilityGenetic StabilityExample of Sample Size and Confidence LevelExample of Sample Size and Confidence Level






• Summary

Plasmid Vaccines and Therapeutics: Plasmid Vaccines and Therapeutics: Pre-biologic PotencyPre-biologic Potency

plasmid

AAAAAAA

mRNA

PRE-BIOLOGIC

Potency

RT-PCR

IP/WB or WBELISAFACS

BIOLOGICProtein

PROTEINAAAAAAA

mRNA

WB, FACS, ELISA AssayRT-PCR AssayAttributes

• 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

Attributes• Measures the immediate

biological effect (transcription)

• “Quantitative” • Large dynamic range• Low inherent variability

• “Specific”• “Validatable”

• Reagents: invariable, stable, readily available

plasmid

Drug Product

• Pre-biologic

• Prepro-biologic

Potency AssayPotency AssayRT-PCR %RP Assay RationaleRT-PCR %RP Assay Rationale

mRNA

Reverse Transcription (RT)

Potency AssayPotency AssayRT-PCR & Specificity of PCR PrimerRT-PCR & Specificity of PCR Primer

intron Genepromoter

plasmid

TaqMan® PCR

Gene-specific reverse primer

mRNA

Transcription

In vivoIn vivo

In vitroIn vitro

R Q

cDNA

Taqman ProbecDNA-specific forward primer






• Summary

Potency AssayPotency AssayPoloxamer-formulated pDNA-based VaccinePoloxamer-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

Potency AssayPotency AssayTest Potency SamplesTest 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

Sample Potencies of Test Lot

26

27

28

29

30

31

32

33

34

35

0.01 0.1 1

Log Transfected Dose (ug)

CT V

alue

200%

100%

50%

Expected Potency

Observed Potency

50% 54%

75% 88%

150% 135%

200% 194%

In Vitro / In VivoIn Vitro / In Vivo Correlation 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


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


Ref 0 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 87 Ref pDNA type

Aggregates

Multimer

Open Circular

Linear

Supercoil

Fullydegraded

80C forced pDNA degradation (hrs of treatment)


% Relative Potency (formulation treatment)

Total Dose(µg)

Total Volume (µL)

# of 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

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

In Vitro / In VivoIn Vitro / In Vivo Correlation Study Correlation StudyAb ResultsAb Results

Time @ 80C 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

Normalized Antibody and Relative Potency Response

In vivo & in vitro response normalized to untreated

-100.00%

-75.00%

-50.00%

-25.00%

0.00%

0 15 30 45 60 75 90

hrs treatment @ 80 degrees C

% D

rop

Ab (EU/ml)

Relative Potency (%RP)

In Vitro / In VivoIn Vitro / In Vivo Correlation Study Correlation StudyT-cell ResultsT-cell Results

• High variability

• Low responses

• Inconclusive results

Case Study #2: Case Study #2: CMV & CMV & In VitroIn Vitro / / In VivoIn Vivo Correlation Summary 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 AssaySummary: Potency AssayCharacterization & Lot ReleaseCharacterization & 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)

AcknowledgementsAcknowledgements• 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.

Laureen Little Ph.D. – Consultant, BioAssaysJan Callahan Ph.D. - Consultant, Statistics

*formerly with Vical

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.

Documents

POTENCY ASSAYS FOR PLASMID-BASED VACCINES AND THERAPEUTICS David C. Kaslow M.D. Chief Scientific Officer Vical Incorporated CTGTAC Meeting on Potency Assay