Brammer Bio Capabilities

Upstream Manufacturing Platforms for Gene Therapy Viral Vectors

Christopher Murphy, Chief Manufacturing Officer, Brammer Bio [email protected] CMC Forum- “Next Generation” Biotechnology Product Development, Manufacturing and Control Strategies Gaithersburg, MD 16 July 2018 ©2018 Brammer Bio Page 1 Presentation Outline

• Background on Gene Therapy

• Viral Vectors

• Manufacturing Processes and Technology

• Critical Raw Materials and Analytics

• Considerations for Process Performance Qualification

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 © Brammer Bio

AAV2 Genome • Member of the Parvoviridae Key Ingredients for Producing AAV 4.7 kb family 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.

Transfection Sf9/Baculo Producer Cell Line Herpesvirus System

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

Plasmids, cell banks (E. Helper virus (e.g. Adenovirus), Cell banks (HSV banks and Specialized Raw coli and mammalian), MBIIC (baculovirus infected producer cell bank, helper virus AAV production), HSV viral Materials Serum (for adherent insect cells), cell bank cell bank banks processes) Efficiency/time sensitivity Scalability Suspension process; Size of Suspension process; Size of Suspension process; Size of of transfection can limit Considerations MBIIC (MOI strategy) helper virus bank (MOI strategy) the viral banks scale Impurity Profile Residual Plasmid DNA Endogenous virus Wild-type Adenovirus HSV and genetic components Considerations Serum components Baculovirus Cell Line Components Cell Culture Adherent or Suspension Suspension Adherent or Suspension Adherent or Suspension System Cloning of baculovirus and Cloning of HSV vector and viral Time Cloning of producer cell line and Generation of Plasmids generation/qualification) of banking; Generation and Consideration generation/qualification of banks MBIICs Qualification ©2018 Brammer Bio Page 7 Diversity of Platforms and Processes

Mammalian Cells Mammalian Cells Transient Transfection Mammalian Cells Infection Insect Cells

Producer cell line + Ad Suspension + HSV AAV Adherent + Suspension Suspension + Baculovirus

Adenoviral Adherent + Suspension

Herpesviral Adherent + Suspension

Lentiviral Packaging/producer cell line Adherent + Suspension

Retroviral Packaging/producer cell line Adherent + Suspension

Flat-stock

• Limited surface area (HS 36=18,000 cm2) • Low CAPEX • Labor Intense

Corning® HYPERStack®

Fixed Bed

• Up to 5,000,000 cm2 (~280 HS 36) • High CAPEX • Media Intense

Animal Derived Cell Culture Cell Banks Plasmid Viral Banks Single Use Components (BAC, HSV, Ad) Bioreactor

Variability and/or demand may impact: • Viral clearance Hyperstack Shortage • Impurity profile Caused by Gene Therapy • Analytics Surge • Process robustness/consistency Headline on Thursday, July • Supply Chain 5, 2018 in Bioprocess • Yield InternationalTM

Nucleic acid Protein Cell Analytical based based based chemistry

Example Analytics

Infectious titer (TCID50) Vector genome titer (qPCR) Bioprotocol Vol Capsid titer (ELISA) 4, Iss 22, P24 Concentration November 20, 2014 Residual Plasmid DNA Residual Genomic DNA Infectious Helper Virus (TCID50 or Plaque assay) Host cell Protein Host Cell DNA AUC (empty to full capsid ratio) Adventitious agents Mycoplasma and endotoxin

Chemistry, Manufacturing, and Control (CMC) Information for Human “Sponsors should consider designing their first-in- Gene Therapy Investigational New Drug Applications (INDs); Draft human study to be an adequate and well-controlled Guidance for Industry (PDF - 486KB) investigation that has the potential, depending on the study results, to provide evidence of July 2018 effectiveness to support a marketing application.” Long Term Follow-up After Administration of Human Gene Therapy Products; Draft Guidance for Industry (PDF - 294KB) July 2018 “GT products may have CQA with higher variability Testing of Retroviral Vector-Based Human Gene Therapy Products for than drugs or well characterized biologics, which can Replication Competent Retrovirus During Product Manufacture and add to CQA uncertainty……. However, demonstrating Patient Follow-up; Draft Guidance for Industry (PDF - 124KB) process control to ensure a consistent product with July 2018 predefined CQA for potency, identity and purity is Human Gene Therapy for Hemophilia; Draft Guidance for Industry (PDF required to demonstrate compliance with licensure - 371KB) and regulatory requirements.” July 2018 Human Gene Therapy for Rare Diseases; Draft Guidance for Industry (PDF - 136KB) “These factors make it even more critical that a July 2018 sponsor of a GT product for a rare disease establish a well-controlled manufacturing process along with Human Gene Therapy for Retinal Disorders; Draft Guidance for suitable analytical assays to assess product CQA as Industry (PDF - 172KB) early in development as possible, optimally before July 2018 administration of the GT product to the first subject.”

Lifecycle approach 3 stages 1. Process characterization (PC) with Stages scaled-down models l Control strategy 2. Process performance qualification (PPQ) Process consistency l performance 1 2 3 qualification l comparability

3. Continued process verification (CPV) Licensure Monitoring plan at short-term and long-term Process Design l PPQ CPV Characterization • Cost per batch is high and critical raw materials Small scale PPQ runs Stage 3A can be limiting; this can limit the amount of runs and support Short-term data collected in Phase I Engineering runs (data collection) runs Stage 3B • For some rare diseases, first in human trials are Long-term being designed to demonstrate efficacy. This (statistical has the potential of accelerating clinical controls) development and compressing CMC development timelines.