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

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

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)

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

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.

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

Vector Description, history and details on derivation of construct Vector diagram Sequence analysis (from MVB) Full sequence for vectors <40KB Vectors >40kb: sequence inserts, flanking regions, modified regions Description of unexpected sequences Do not submit only raw data

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)

Donor Eligibility – Testing Allogeneic HIV 1&2, 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)

Donor Eligibility – Testing Autologous DE testing recommended, not required Determine if cell culture methods could propagate viruses

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

Master Cell Bank and Master Virus Bank Safety Testing Sterility Mycoplasma

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 1&2, HTLV 1 & 2, B19, (others) Murine cell lines: MAP, retroviruses RCV (GT products, typically on MVB) Refer to guidance for assays and limits

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)

Master Cell Bank – Characterization Other- processes critical to safety Document culture conditions, medium, cryopreservation, storage, genetic & phenotypic stability after multiple passages

Master Virus Bank – Characterization Identity Sequence of vector & restriction map Activity/Expression Transgene specific protein expression Other Titer Stability

Working Cell Bank and Working Viral Bank Safety Sterility Mycoplasma In vitro adventitious virus assay Replication competence virus (WVB) Characterization Identity Stability Others

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

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

Product Manufacturing (cont.) Formulation of final product Formulation buffer Excipients Vector/cell concentration Storage

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

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

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

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

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

Final Product Characterization Identity Restriction map, structural characterization, cell phenotype Purity Residual contaminants (DNA, protein, culture reagents) Cell populations for cell therapies Particle:IU ratio for vectors Stability Should be tested throughout all clinical phases

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

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

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

Product Characterization Start collecting data early! Communicate progress with your CMC reviewer

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

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

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?

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

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

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

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 Process controls In-process testing, specifications SOP development

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 aren’t assaying for potency or ensuring lot to lot consistency Need to characterize your product and process!

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

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

References and Contact Information CMC Questions for Cellular Therapies Keith Wonnacott keithwonnocot@fda.hhs.gov CMC Questions for Gene Therapies Dan Takefman dantakefman@fda.hhs.gov