Manufacturing of Cellular

1
Manufacturing of Cellular Gene Therapies
Ensuring Product Safety and Quality

• Dan Takefman, Ph.D.
• Chief, Gene Therapy Branch
• Division of Cellular and Gene Therapies
• Center for Biologics Evaluation and Research
• FDA

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Before You Begin Manufacturing

• Guidance for Review Staff and Sponsors Content
  and Review of Chemistry, Manufacturing, and
  Control (CMC) Information for
• Human Gene Therapy Investigational New Drug
  Applications (INDs)
• Human Somatic Cell Therapy Investigational New
  Drug Applications (INDs)
• http//www.fda.gov/cber/genadmin/octgtprocess.htm

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Step-wise Approach to Application of Regulatory
Requirements

• For phase I submission, product safety is the
  focus of the CMC assessment
• Microbial contamination, freedom from
  adventitious agents, other assessments
• Characterization as directly related to safety
  will need to be documented
• Minimal characterization expected
• Assurance of product quality increases with
  clinical development
• Characterization (CFR 610)
• cGMPs (CFR 210, 211)

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Product Safety and Quality

• Components used in Product Manufacture
• Final Product Testing and Characterization
• Control of Manufacturing Process
• cGMP Practices
• In process controls

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CMC Information How Much is Enough?

• Important to describe your manufacturing process
  in enough detail for FDA to assess safety
• If safety tests done by contract lab, submit SOPs
  and available information on assay sensitivity
  specificity. If regulatory file available submit
  cross reference
• Flow charts with narrative
• When cross-referencing manufacturing information,
  be specific in what you are referencing.

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Components Used in Manufacture of Product

• Vector
• Cells
• Allogeneic autologous cell components
• Cell bank system
• Master cell bank/working cell bank
• Master viral bank/working viral bank
• Ancillary products/reagents
• Growth factors, cytokines, MoAb

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Vector

• Description, history and details on derivation of
  construct
• Vector diagram
• Sequence analysis (from MVB)
• Full sequence for vectors lt40KB
• Vectors gt40kb sequence inserts, flanking
  regions, modified regions
• Description of unexpected sequences
• Do not submit only raw data

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Cells

• Cell substrates for production of gene therapies
• History, source, general characteristics
• Autologous and allogeneic cells used for cell
  therapies or ex vivo modified gene therapies
• Source (tissue and cell type)
• Collection procedure (mobilization, surgery,
  leukapheresis, devices used)
• Donor Eligibility (infectious disease screening
  testing, 21 CFR 1271)

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Donor Eligibility Testing

• Allogeneic
• HIV 12, HBV, HCV, Treponema pallidum
• For viable, leukocyte-rich cells/tissues,
  additional test for HTLV I II
• For reproductive cells/tissues, additional tests
  for Chlamydia trachomatis and Neisseria gonorrhea
• FDA licensed or approved kits or description of
  test methods, controls, sensitivity
• Testing on donor mothers for cord blood or
  maternally derived tissue (not banked)

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Donor Eligibility Testing

• Autologous
• DE testing recommended, not required
• Determine if cell culture methods could propagate
  viruses

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Donor Eligibility Screening

• Allogeneic
• Risk-factors for, and clinical evidence of
  relevant communicable disease agents or diseases
• Communicable disease risk associated with
  xenotransplantation

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Master Cell Bank and Master Virus Bank

• Safety Testing
• Sterility
• Mycoplasma

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Master Cell Bank and Master Virus Bank

• Safety Testing (continued)
• Adventitious Virus
• In vitro and in vivo adventitious virus assays
• Bovine and porcine viruses (not needed if
  reagents tested)
• Human cell lines EBV, HBV, HCV, CMV, HIV 12,
  HTLV 1 2, B19, (others)
• Murine cell lines MAP, retroviruses
• RCV (GT products, typically on MVB)
• Refer to guidance for assays and limits

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Master Cell Bank Characterization

• Identity
• Cell therapies phenotype, genotype, other
  markers
• Gene therapies isoenzyme, others
• Purity
• Contaminating cells (GT products)
• Charactering cell types (CT products)
• Tumorigenicity
• May be considered a safety test for certain
  cellular products (e.g. stem cell products)

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Master Cell Bank Characterization

• Other- processes critical to safety
• Document culture conditions, medium,
  cryopreservation, storage, genetic phenotypic
  stability after multiple passages

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Master Virus Bank Characterization

• Identity
• Sequence of vector restriction map
• Activity/Expression
• Transgene specific protein expression
• Other
• Titer
• Stability

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Working Cell Bank and Working Viral Bank

• Safety
• Sterility
• Mycoplasma
• In vitro adventitious virus assay
• Replication competence virus (WVB)
• Characterization
• Identity
• Stability
• Others

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Reagents Used in Manufacturing

• Tabulation of reagents used
• Final concentration
• Vendor
• Source (human, bovine, etc.)
• Licensed product, clinical grade, reagent grade
• Certificates of Analysis
• Cross reference letter
• Qualification program
• Safety testing and quality assessment

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Product Manufacturing

• Vector production/purification
• Cell products/ex vivo transduced cells
• Method of collection/processing
• Culture/transduction procedures
• Other modifications (irradiation)
• Final harvest

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Product Manufacturing (cont.)

• Formulation of final product
• Formulation buffer
• Excipients
• Vector/cell concentration
• Storage

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Final Product Testing

• Demonstration of product safety
• Assessment of product characterization
• Maintenance of product lot consistency
• Results available prior to patient administration
• Listing of tests, methods, acceptance criteria in
  IND

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Final Product Testing Safety

• Sterility (CFR 610.12)
• Mycoplasma (CFR 610.30)
• Endotoxin (CFR 610.13)
• Adventitious Virus (for gene therapy products)
• In vitro virus assay
• Replication competent virus

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Approaches to Release Testing for
Non-Cryopreserved Cells

• Sterility
• Regulation
• 14 day sterility assay on final product
• Approach
• Sample 72-48 hours or after last manipulation -
  release on no growth
• Results of Gram stain available-release on
  negative result
• Sample final product initiate 14 day sterility
  culture
• Action plan in the event of positive for
  microorganisms

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Approaches to Release Testing

• Mycoplasma
• Recommend testing at cell harvest
• Regulation 21-28 day culture, 5-7 day
  fluorescent
• Approach PCR (6-8 hour test)
• Pyrogenicity
• Regulation rabbit bioassay
• Approach LAL (1-2 hour test)
• Equivalent methods (CFR 610.9)
• Data demonstrating equivalence to methods in
  regulation needed for licensure

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Final Product Characterization

• Final product testing
• Purity (CFR 610.13)
• Identity (CFR 610.14)
• Potency (CFR 610.10)
• Stability
• Cell viability
• Development of test methods and acceptance
  criteria

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Final Product Characterization

• Identity
• Restriction map, structural characterization,
  cell phenotype
• Purity
• Residual contaminants (DNA, protein, culture
  reagents)
• Cell populations for cell therapies
• ParticleIU ratio for vectors
• Stability
• Should be tested throughout all clinical phases

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Final Product Characterization

• Potency
• Indicate biological activity (s)
  specific/relevant to the product
• Provide quantitative readout
• Required prior to initiation of phase III
• GT products transgene expression and titer
  acceptable in early phases
• Cell therapies, cell surface phenotype in early
  phases
• Guidance in development

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Approaches for Potency Measurements

• Direct measure of biological activity
• In vivo or in vitro assay
• Indirect measure of biological activity
• Analytical assay methods non-bioassay method
  directly correlated to a unique and specific
  activity of the product
• Multiple Assay Approach (Assay Matrix)
• May not be possible or feasible to develop a
  single assay that encompasses all elements of an
  acceptable potency assay

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Product Characterization Why?

• To demonstrate lot-to-lot consistency
• To show comparability after manufacturing changes
• To generate solid clinical data
• For pivotal trials characterization assays will
  need to be established with appropriate release
  limits

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Product Characterization

• Start collecting data early!
• Communicate progress with your CMC reviewer

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Current Good Manufacturing Practices (cGMP)

• Definition
• A set of current, scientifically sound methods,
  practices or principles that are implemented and
  documented during product development and
  production to ensure consistent manufacture of
  safe, pure and potent products
• Increase in control of the manufacturing process
  with clinical trial advancement

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cGMPs for Early Development

• For detailed guidance, please refer to Draft
  Guidance for Industry INDs Approaches to
  Complying with CGMP During Phase 1
• http//www.fda.gov/cber/gdlns/indcgmp.pdf

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GMPs Early Clinical Phase Critical Questions

• Is the process reproducible?
• Appropriate testing at critical steps?
• Quality of the raw and source materials
  adequately controlled?
• Are the records and record keeping systems
  adequate?

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Examples for Early Development

• Procedures to prevent contamination cross
  contamination
• Aseptic processing
• Tracking of autologous products (labeling system)
• Patient cell segregation
• Methods qualification
• Appropriate method specificity, sensitivity,
  reproducibility

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Examples for Early Development

• Process qualification
• Safety related process
• Irradiation of tumor cells/ cell lines, removal
  of toxic residuals, viral clearance
• Performance runs/development lots
• Equipment calibration
• Irradiators, other critical equipment

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Examples for Early Development

• Quality (QC) Program
• Ideal - separate unit with ultimate reporting to
  sponsor
• Responsibility and authority to accept or reject
  materials, procedures and specifications
• Designed to prevent, detect, and correct
  deviations and failures

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cGMPs that Develop with Clinical Studies
Examples

• Process validation
• Must first characterize your process
• Identify and control sources of variability and
  understand how variability affects your product
• Validate by licensure
• Methods validation
• Develop and refine
• Validate by licensure
• Process controls
• In-process testing, specifications
• SOP development

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Characterization and cGMPs

• cGMP are a set of current, scientifically sound
  methods, practices or principles that are
  implemented and documented during product
  development and production to ensure consistent
  manufacture of safe, pure and potent products
• Manufacturers often refer to their products as
  GMP grade, but arent assaying for potency or
  ensuring lot to lot consistency
• Need to characterize your product and process!

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Summary

• Step-wise Approach to Regulatory Requirements
• Safety testing
• Product characterization
• Control of Manufacturing Process
• cGMP Practices
• Ensure a Safe and Quality Product

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References and Contact Information

• References for the Regulatory Process for OCTGT
• http//www.fda.gov/cber/genadmin/octgtprocess.htm
• General questions for OCTGT
• 301-827-5102

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References and Contact Information

• CMC Questions for Cellular Therapies
• Keith Wonnacott
• keithwonnocot_at_fda.hhs.gov
• CMC Questions for Gene Therapies
• Dan Takefman
• dantakefman_at_fda.hhs.gov