viral vector analytical paradigms, platforms and …€¢ infectivity = report results (iu/ml) •...

Eric Pastor

Principal Scientist, Biopharmaceutics Development, Sanofi US

CASSS CMC Strategy Forum 2017

17 JUL 2017

Viral Vector Analytical Paradigms, Platforms and

Proposed Specifications

Agenda

1. How do we shape and manage a phase appropriate Gene Therapy

viral vector analytical paradigm to support Drug Substance/Drug

Product release and characterization?

• How do we select what tests are on release?

• What attributes do we escalate from characterization to release?

• How do evolve our paradigms throughout drug development?

• Are there platform gaps and how do we manage this?

2. How do we select phase appropriate specifications?

• How many lots do we need?

• What are the appropriate units and ranges for key quality attributes?

Not intending to promote what we do as “right” or “wrong” or

to champion any particular platform.

Objective is to share some strategies and start discussions….

Gene Therapy VV Analytical Paradigm

● What defines a viral vector analytical paradigm?● Critical Quality Attributes

• Most safety, efficacy and immunogenicity scores may be carried over from one AAV product to another if the manufacturing process is platformed

• Host-cell DNA

• PK/PD scores are likely to be product-specific based on change in therapeutic gene

• CQA’s may be introduced/removed based on changes to the purification process

● Regulatory Guidance*• Expectations are continuously increasing and evolving as more companies reach

later stage clinical trials

• Specifications are becoming more stringent, new attributes are emerging

• Specific gene therapy guidance documents are branching off from general biologics (i.e. Potency Assay recommendations)

● Overall goal is to release and characterize a safe and high quality product

Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Controls (CMC Information

for Human Gene Therapy Investigational New Drug Applications (INDs), USDHHS/FDA/CBER, 4/2008

Note for Guidance on the Quality, Non-Clinical and Clinical Aspects of Gene Transfer Medicinal Products,

CHMP/GTWP/671639/2008

Example Gene Therapy VV Analytical Paradigm(e.g. Adeno-Associated Virus)

Vector Genome Conc.

Infectivity

Potency

Total Capsids

P:I Ratio

Capsid Purity

Aggregation

% Empty Capsids

rcAAV

Non-transgene DNA

Infectious Helper Virus

Helper Virus Proteins

Helper Virus DNA

Oncogenic DNA

Host-Cell DNA

Host-Cell Proteins

Benzonase

Leachables

Purification Buffers

Transgene ID

Capsid ID

Mycoplasma

Endotoxin

Bioburden

Sterility

In vitro AVA

pH

Osmolality

Appearance

Strength / Content Product Variants and Impurities Identity

General Quality Safety

Process-Related Impurities

Release and Characterization*to be discussed later…

A Typical Gene Therapy VV Analytical Paradigm(e.g. Adeno-Associated Virus)

Vector Genome Conc.

Infectivity

Potency

Total Capsids

P:I Ratio

Capsid Purity

Aggregation

% Empty Capsids

rcAAV

Non-transgene DNA



Helper Virus DNA

Oncogenic DNA

Host-Cell DNA

Host-Cell Proteins

Benzonase

Leachables

Purification Buffers

Transgene ID

Capsid ID

Mycoplasma

Endotoxin

Bioburden

Sterility

In vitro AVA

pH

Osmolality

Appearance

Strength / Content Product Variants and Impurities Identity

General Quality Safety


Release and Characterization*to be discussed later…

Evolving Assays and ParadigmsEarly Phase to Late Phase

Current Release Methods* Current Characterization Methods

Vector Genome Concentration

Infectivity

Relative Potency

Capsid Purity

Transgene Identity

% Empty Capsids

rcAAV


Host-Cell DNA

Host-Cell Protein

Aggregation

Capsid Identity

Non-transgene DNA


Helper Virus DNA

Oncogenic DNA

Residual Benzonase

Residual Column Leachables

Residual Purification Buffers

NGS Sequencing

VP Modifications

Infection/Transduction Kinetics

HCP Profiles

*minus safety and compendials

Characterization methods focus on investigative attributes and/or CQA’s where

there are plans to put process controls around. May also include method

platforms that need additional development or are not yet “QC-friendly”

Release methods focus on high CQA’s and required

tests using validate-able method platforms

Current Release Methods* Current Characterization Methods


Infectivity

Relative Potency

Capsid Purity

Transgene Identity

% Empty Capsids

rcAAV


Host-Cell DNA

Host-Cell Protein

Aggregation

Capsid Identity

Non-transgene DNA


Helper Virus DNA

Oncogenic DNA

Residual Benzonase

Residual Column Leachables

Residual Purification Buffers

NGS Sequencing

VP Modifications

Infection/Transduction Kinetics

HCP Profiles

*minus safety and compendials

Evolving Assays and ParadigmsEarly Phase to Late Phase

Strength


(Dose-Defining Assay)

Prelim. Spec. = Dose ± 0.5 log

*qPCR on release, ddPCR on

charact. (not yet ready, need

suitable cross-over data and

system confidence, validation)

Infectivity (TCID50)

Plate A 1 2 3 4 5 6 7 8 9 10 11 Ctls.

Plate A A + + + + + + + +

B + + + + + + + +

C + + + + + + + +

D + + + + + + + + +

E + + + + + + + +

F + + + + + +

G + + + + + + +

H + + + + + +

Prelim. Spec.

• Infectivity = Report Results (IU/mL)

• Particle:Infectivity ratio = < X vg/IU*

*P:I will vary depending on serotype.

Arbitrary number, dependent on tropism

to assay cell line. Use for lot-to-lot

consistency and stability

Relative Potency

Prelim. Spec. = 50 – 200 % of Ref.

Measures both gene

expression and biological

activity of expressed gene (e.g.

enzymatic activity)

Reference Standard is from

Engineering Run, prior to GMP

CTM – full characterization of

RS including NGS!

Data courtesy of JD

Identity

Transgene

Prelim. Spec. = Conforms to Ref.

Capsid

Prelim. Spec. = AAVx Confirmed

Do we need both assays on Release?

What if one of the assay platforms does not lend itself well to a QC environment?

Viral vectors have both a nucleic acid and viral shell component• A multi-product facility could have the same capsid serotype, different transgene

AND/OR vice versa

Data courtesy of JD, LL and XJ

Product Variants and ImpuritiesAggregation / Particle Distribution

What is an ideal platform?• Quantifiable

• Monomeric AAV plus other (dimer,

trimer, higher order?)

• Validate-able (ICH Q2(R1))

• QC-friendly, low IVR

What other platforms can be used for

improved detection/quantitation?• Nanosight

• SEC-HPLC

• AUC

How do we report and what are the units for

specifications?• > X % monomer?

• < X % other?

• X – X nm?

• Comparable to Ref?

• No sig. HMWS?

Viral vectors (and their potential aggregates) exist in a nanoparticle to sub-visible particle range

(10 nm – 1 µm)• Faces similar challenges of traditional biologics

Data courtesy of BD


Is there a regulatory concern with hcDNA levels in rAAV products?

1 cellular genome = 15 pg DNA

~75 pg genomic DNA per 1 copy 18S

1 AAV genome = 4800 bp DNA

~0.0026 fg viral DNA per 1 copy 18S

Fold-difference = ~28,000

Genomic DNA STDs rAAV Test Article (DS)

If all we detect is encapsidated 18S (+/- DNase), can we convert genomic hcDNA to viral DNA?

5.6e5 pg per mL / 0.07519 pg genomic DNA per 18S copy = 7447799 copy per mL

7447799 copy per mL x 0.00000263 pg viral DNA per 18S copy = 19.6 pg/mL

560 ng/dose

(1 mL)

0.02 ng/dose

(1 mL)

WHO Guidance

= < 10 ng/dose


Empty Capsids / Full Capsids

Multiple platforms available to monitor % full and % empty particles (and % fragmented

genomes?)• Optical Density – Small range, need extinction coefficients, assumes all “full” particles are full length

• Capsid ELISA / qPCR - Combined variability of 2 platforms, “full” particles are those that you detect with

your primers/probes

• AUC – Difficult to validate, but discriminates between all species

• TEM - Difficult to quantify, but discriminates between empty and “other”

What will be the preferred platform?• Any of the ones listed above?

• Another platform? – e.g. HPLC? Cryo TEM?

What will be specifications in early development?• > 50 % full OR < 50 % empty

• Do we need to do in vivo experiments prior to IND-enabling studies?

• % full = 0%, 25%, 50%, 75%, 100%

• Use GLP Tox output as a benchmark? – or can we leverage other products?

• If GLP tox vector is 70% full, is the Phase I release spec = ≥ 70% full (< 30% empty?)

• But what about assay and process variability?

Data courtesy of BB

Questions and Discussion Points

1. What do phase-appropriate analytical paradigms look like for viral vector

gene therapy drug substance/drug product release and characterization?

2. How do we set phase appropriate specifications?

Thank you for your time!

Acknowledgments

Sanofi Biopharmaceutics Development

• Bioanalytics - Analytical Development

• Bioanalytics - Characterization

• Gene Therapy Skill Center

CASSS Organizers

viral vector analytical paradigms, platforms and …€¢ infectivity = report results (iu/ml) •...

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