Manufacturing of Cellular & Gene Therapies: Ensuring Product Safety and Quality

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

2. 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

3. 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)

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

5. 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.

6. 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

7. 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

8. 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)

9. 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)

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

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

12. Master Cell Bank and Master Virus Bank • Safety Testing • Sterility • Mycoplasma

13. 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

14. 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)

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

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

17. 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

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

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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

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

30. 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

31. 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

32. 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?

33. 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

34. 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

35. 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

36. 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

37. 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!

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

39. 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

40. 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