Upstream Manufacturing Platforms for Gene Therapy Viral Vectors

Christopher Murphy, Chief Manufacturing Officer, Brammer Bio chris.murphy@brammerbio.comCMC Forum- “Next Generation” Biotechnology Product Development, Manufacturing and Control StrategiesGaithersburg, MD 16 July 2018

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• Background on Gene Therapy

• Viral Vectors

• Manufacturing Processes and Technology

• Critical Raw Materials and Analytics

• Considerations for Process Performance Qualification

Presentation Outline

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What is Viral Vector Gene Therapy?

Viral Vector Gene Therapy

in vivo delivery of a viral vector into a patient's cells as a treatment for disease. The nucleic acid sequence replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight the disease. It may fix a genetic problem at its source or address the symptoms of the disease. The nucleic acid sequence (“payload”) is either expressed as a protein, interferes with protein expression, or

possibly corrects genetic mutation.

Example product: LuxturnaTM

Gene Modified Cell Therapy

Growing a patient’s or a donor’s cells and modifying them ex vivo, using a viral vector, to provide therapies to patients

Example products: Strimvelis®, KymriahTM, YescartaTM

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Decades of clinical development and scientific advances

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Gene Therapy Viral Vectors

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Adeno-associated Virus (AAV)

AAV2 Genome4.7 kb

• Member of the Parvoviridaefamily and the Dependoparvovirus genus

• Rep and cap genes are required to replicate and build the virus.

• AAV requires a helper virus for lytic replication.

• Wild type AAV can insert nucleic acid at a specific site on chromosome 19; engineered vectors remain episomal.

• 11 naturally occurring serotypes with hundreds of “pseudotypes” developed by researchers.

Key Ingredients for Producing AAV

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Upstream Manufacturing Platforms for AAV

Transfection Sf9/Baculo Producer Cell Line Herpesvirus System

Attribute Transfection Sf9/Baculovirus Producer Cell Line HSV Co-Infection

Specialized Raw Materials

Plasmids, cell banks (E. coli and mammalian), Serum (for adherent

processes)

MBIIC (baculovirus infected insect cells), cell bank

Helper virus (e.g. Adenovirus), producer cell bank, helper virus

cell bank

Cell banks (HSV banks and AAV production), HSV viral

banks

Scalability Considerations

Efficiency/time sensitivity of transfection can limit

scale

Suspension process; Size of MBIIC (MOI strategy)

Suspension process; Size of helper virus bank (MOI strategy)

Suspension process; Size of the viral banks

Impurity ProfileConsiderations

Residual Plasmid DNASerum components

Endogenous virusBaculovirus

Wild-type AdenovirusCell Line Components

HSV and genetic components

Cell Culture System

Adherent or Suspension Suspension Adherent or Suspension Adherent or Suspension

Time Consideration

Generation of PlasmidsCloning of baculovirus and generation/qualification) of

MBIICs

Cloning of producer cell line and generation/qualification of banks

Cloning of HSV vector and viral banking; Generation and

Qualification©2018 Brammer Bio Page 7

Diversity of Platforms and Processes

Mammalian Cells Mammalian Cells Transient Transfection Mammalian Cells Infection Insect Cells

AAVProducer cell line + Ad

Adherent + Suspension

Suspension + HSV

Suspension + Baculovirus

Adenoviral Adherent + Suspension

Herpesviral Adherent + Suspension

Lentiviral Packaging/producer cell line Adherent + Suspension

Retroviral Packaging/producer cell line Adherent + Suspension

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Upstream Technology- Anchorage Dependent Systems

Corning® HYPERStack®

Pall iCellis® 500

• Limited surface area (HS 36=18,000 cm2)

• Low CAPEX• Labor Intense

Flat-stock

Fixed Bed

• Up to 5,000,000 cm2 (~280 HS 36)

• High CAPEX• Media Intense

Courtesy of Pall Life Sciences

Cells attached to microcarrier

matrix

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Complex Critical Raw Materials Required

Animal Derived Components

Cell Banks Plasmid Viral Banks(BAC, HSV, Ad)

Single Use Bioreactor

Cell Culture

Variability and/or demand may impact:

• Viral clearance

• Impurity profile

• Analytics

• Process robustness/consistency

• Supply Chain

• Yield

HyperstackShortage Caused by Gene Therapy Surge

Headline on Thursday, July 5, 2018 in Bioprocess InternationalTM

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Upstream Manufacturing Process Drives the Analytics

Nucleic acid based

Protein based

Analytical chemistry

Cell based

Example Analytics

Infectious titer (TCID50)Vector genome titer (qPCR)Capsid titer (ELISA)P24 ConcentrationResidual Plasmid DNAResidual Genomic DNAInfectious Helper Virus (TCID50 or Plaque assay)Host cell Protein Host Cell DNA AUC (empty to full capsid ratio)Adventitious agentsMycoplasma and endotoxin

Bioprotocol Vol 4, Iss 22, November 20, 2014

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FDA Issues Six New Draft Guidance Documents in July 2018

Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs); Draft Guidance for Industry (PDF - 486KB)July 2018 Long Term Follow-up After Administration of Human Gene Therapy Products; Draft Guidance for Industry (PDF - 294KB)July 2018 Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up; Draft Guidance for Industry (PDF - 124KB)July 2018 Human Gene Therapy for Hemophilia; Draft Guidance for Industry (PDF - 371KB)July 2018 Human Gene Therapy for Rare Diseases; Draft Guidance for Industry (PDF - 136KB)July 2018 Human Gene Therapy for Retinal Disorders; Draft Guidance for Industry (PDF - 172KB)July 2018

“These factors make it even more critical that a sponsor of a GT product for a rare disease establish a well-controlled manufacturing process along with suitable analytical assays to assess product CQA as early in development as possible, optimally before administration of the GT product to the first subject.”

“GT products may have CQA with higher variability than drugs or well characterized biologics, which can add to CQA uncertainty……. However, demonstrating process control to ensure a consistent product with predefined CQA for potency, identity and purity isrequired to demonstrate compliance with licensure and regulatory requirements.”

“Sponsors should consider designing their first-in-human study to be an adequate and well-controlled investigation that has the potential, depending on the study results, to provide evidence of effectiveness to support a marketing application.”

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Viral Vector Process Validation

Lifecycle approach 3 stages

1. Process characterization (PC) with scaled-down models l Control strategy

2. Process performance qualification (PPQ) Process consistency l performance qualification l comparability

3. Continued process verification (CPV)Monitoring plan at short-term and long-term Process Design l

CharacterizationPPQ CPV

1 2 3

Stages

Licensure

Small scale runsEngineering runs

PPQ runs and support runs

Stage 3AShort-term(data collection)

Stage 3BLong-term(statistical controls)

• Cost per batch is high and critical raw materials can be limiting; this can limit the amount of data collected in Phase I

• For some rare diseases, first in human trials are being designed to demonstrate efficacy. This has the potential of accelerating clinical development and compressing CMC development timelines.

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Thank You

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