# Presentation: 856214

## 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

**Plasmid Vaccines and Therapeutics: Pre-biologic**

- Gene sequence
- Transcription
- Translation
- Post-translational
- modification

**AAAAAAA**

***mRNA***

**PRE-BIOLOGIC**

## 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

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

- Gene sequence
- Transcription
- Translation
- Post-translational
- modification

**AAAAAAA**

***mRNA***

**PRE-BIOLOGIC**

***Strength***

***Potency***

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

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

**AAAAAAA**

***mRNA***

**PRE-BIOLOGIC**

***Strength***

***Potency***

- RT-PCR

- IP/WB or WB
- ELISA
- FACS

- A260
- (Genetic Stability)

## 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 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 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**

**Development **

**Qualification**

**Pre-Validation**

**Validation**

***Protein-based***

***mRNA-based***

**+**

***or***

## 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

- 2 mg/mL, stored at 2-8oC:
- Bicistronic HLA-B7 + -2 microglobulin
- formulated 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-7****® ****Drug Product**

## 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**

**Phase III**

**Commercial**

**Assay Development **

**Assay Qualification**

**Assay Pre-validation**

**Assay Validation**

**FACS**
**Single point**

**IP/WB**

**FACS**
**Single point**

**FACS**
**Single point**

**FACS %RP Dose response**

**IP/WB**

**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 | β2M |
| --- | --- | --- |
| Dose response model fit | Slope ratio | Parallel line |
| Dose treatment (x-axis) | Untransformed | Log-transformed |
| Relative Potency formula |  |  |

*R* = 10

***T **- ****R*
**

## 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

| Lane # | Sample |
| --- | --- |
| 1 | Allovectin-7® ref. |
| 2 | Positive control |
| 3 | Negative control |

**β****2M**

**HLA-B7**

**         ****MW      1         2   MW  3**

- _kDa_
- 188
- 98
- 62
- 49
- 38
- 28
- 17
- 14
- 6
- 3

| Lane # | Sample |
| --- | --- |
| 1 | Allovectin-7® ref. |
| 2 | Positive control |

**MW   1       2      MW**

## 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

**Notes:**
Genetic Stability
201

## Genetic StabilityProtocol Overview

**Observed pDNA sequence**

**Predicted pDNA sequence**

** ****MCB**
**MWCB**
**Fermentation**
**Purify pDNA**
**Transform & select colonies**
**pDNA from colonies**

**Genetic Stability****Protocol 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  

- 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 Stability****Example of Sample Size and Confidence Level**

**Notes:**
Genetic stability
207

## 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

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

**AAAAAAA**

***mRNA***

**PRE-BIOLOGIC**

***Potency***

- RT-PCR

- IP/WB or WB
- ELISA
- FACS

## Potency AssayRT-PCR %RP Assay Rationale

***plasmid***

- _Drug Product_

-  Pre-biologic
-  Prepro-biologic

**Potency Assay****RT-PCR %RP Assay Rationale**

**Notes:**
The FACS potency assay case study provided a rationale and strategy for developing a protein based potency assay for a plasmid based therapeutic.
FACS, ELISAs, and western blot assays all require a reliable source of sufficiently pure and stable assay reagents, such as Abs and/or standards. Development and optimization of an appropriate Ab based assay can take quite a long, especially if initial reagents are not commercially available.   
Using Reverse Transcriptase Polymerase Chain Reaction (RTPCR) as an quantitative assay, the upstream product from the trans-gene protein, the mRNA, can be quantitated as an indirect measure of protein expression. In addition the mRNA represents the primary product of the plasmid or pro-drug.  
Like the FACS assay, a reference control can be run in the RTPCR , from which relative potency can be determined.  
The primers and probes for the RTPCR assay can be readily obtained from multiple sources. Reference curves providing for primarily quantiation are usually ready within a couple weeks.
In addition western blot analysis can be used to verify mol wt of therapeutic protein (no deletion or insertions) 

## Potency AssayRT-PCR & Specificity of PCR Primer

**Reverse Transcription  (RT)**

**Potency Assay****RT-PCR & Specificity of PCR Primer**

**TaqMan****®**** PCR**

**Gene-specific reverse primer**

**mRNA**

**Transcription **

***In vivo***** **

***In vitro***** **

**Notes:**
A brief overview of the RTPCR assay 
Cells are first transfected with the CMV vaccine. (1st figure) 
The cells are then harvested and lyzed and the RNA isolated. (2nd figure)
TaqMan® PCR reactions using the isolated RNA are performed in 96 well plates using an ABI HT7900 SDS automated thermocycler with a fiber optic detection system. (3rd and 4th figures)
Intron spanning forward primer makes assay specific for mRNA  (4nd figure)
The forward primer is designed to recognize sites flanking both ends of the intron.  
Productive binding will only occur when the intron is spliced out during messenger RNA processing.
The reverse primer is designed to recognize the gene of interest.
The probe, along with providing the signal for quantitation, also provides an additional level of specificity for the cDNA amplicon.

## 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
- 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

**Notes:**
DNA vaccines function through induction of the immune response by introducing genes encoding antigens for specific pathogens.
Vical’s CMV vaccine consists of two plasmids each encoding for one transgene protein, pp65 (a phosphoprotein) and gB (a glycoprotein).
The two plasmids are formulated with poloaxmer, BAK and PBS to yield the final drug product.

## Potency AssayTest 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

| Expected    Potency | Observed Potency |
| --- | --- |
| 50% | 54% |
| 75% | 88% |
| 150% | 135% |
| 200% | 194% |

**Notes:**
This is example of data generated during development of the assay that were used to determine the range, initial accuracy and dose response model fit.

##  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

##  In Vitro / In Vivo Correlation

|  % 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 Vivo Correlation StudyAb Results

| 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 |

**Normalized Antibody and Relative Potency Response**** **

## 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

**Notes:**
We’re in the process of completing the qualification work, primarily the robustness studies

We’re currently conducting in vitro in vivo pilot studies potency correlation studies using forced degraded final product.

## 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

**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, BioAssays
- Jan 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.