1. Food and Drug Administration Center for Biologics Evaluation and Research The Office of Cellular, Tissue, and Gene Therapies Web Seminar Series presents: The Office of Cellular, Tissue, and Gene Therapies Web Seminar Series presents: The chemistry, manufacturing and controls (CMC) section of a gene therapy IND Andrew P. Byrnes, Ph.D. Chief, Gene Transfer and Immunogenicity Branch Division of Cellular and Gene Therapies

2. What are gene therapy products? Gene therapy products: –Are administered as nucleic acids, viruses or genetically-engineered microorganisms, and –Mediate effects via: Transcription or translation of transferred genetic material, or Integration into the genome How are gene therapy products used? –To modify cells directly in patients, or –To modify cells in vitro that are then administered to patients

3. Examples of gene therapy products 1.Plasmid expressing an enzyme 2.AAV expressing a coagulation factor 3.T cells modified with a retrovirus to express a novel receptor 4.Bacterium expressing a tumor antigen 5.Oncolytic adenovirus expressing a cytokine

4. Complexity of a gene therapy manufacturing process Growth factors Donor- derived PBMCs CD34+ cells Retroviral vector Transduced CD34+ cells CD34+ selection Fibronectin coated flask

5. Before you begin manufacturing… 2008 Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs)

6. Presentation outline Components used in product manufacture Final product testing and characterization Good manufacturing practices (GMPs)

7. Components used to manufacture the product Vector Cells Banking system –Master cell bank (MCB) –Master viral bank (MVB) Reagents

8. Vector Description, history and details on derivation of construct Vector diagram Sequence analysis (from MVB) –Full sequence for vectors <40 kb –Vectors >40 kb: sequence inserts, flanking regions, modified regions –Description of unexpected sequences Raw sequence data is not sufficient

9. Cells Cell substrate for production of vector –History, source, general characteristics Cells used as cell therapies –Source tissue and cell type –Collection procedure mobilization, surgery, leukapheresis, devices used –Donor Eligibility infectious disease screening & testing, 21 CFR 1271

10. MCB and MVB safety testing Sterility Mycoplasma Adventitious Virus –In vitro and in vivo adventitious virus assays –Bovine and porcine viruses Not needed if reagents tested –For human cell lines: Typically EBV, HBV, HCV, CMV, HIV 1&2, HTLV 1 & 2, B19 –For murine cell lines: Typically mouse antibody production test, retroviruses –Replication-competent virus (for MVB)

11. Master cell bank characterization Identity –Examples: Isoenzyme Karyotype Short tandem repeat (STR) profiling Viability Stability

12. Master virus bank characterization Identity – Sequence of vector & restriction map Activity / expression –Transgene-specific protein expression Titer Stability

13. Reagents used in manufacturing Tabulation of reagents –Final concentration –Vendor –Source (human, bovine, etc.) –Grade e.g. licensed product, clinical grade, reagent grade May need to provide details on reagent manufacturing Certificates of Analysis Cross reference letters Qualification program –Safety testing and quality assessment

14. Product Manufacturing Vector production / purification –Describe all steps e.g. cell growth, infection, harvest, purification, formulation, vialing, storage –Flowchart Describe the formulation –Buffer components –Excipients –Product concentration –Storage

15. Final Product Testing Goals: –Safety –Product characterization –Product lot consistency List all of your: –Release tests –Test methods –Acceptance criteria (specifications)

16. Final product testing: safety Sterility Mycoplasma Endotoxin Adventitious Virus –In vitro virus –Replication competent virus

17. Final product testing: characterization Final product lot release testing –Concentration –Purity e.g. residual cellular DNA, empty viral particles –Identity e.g. restriction digest –Activity e.g. infectious titer –Potency e.g. transgene-specific protein expression –Cell viability (if a cell-based product) Stability –Storage –Shipping –Compatibility with delivery devices

18. Product characterization: why? To demonstrate lot-to-lot consistency To generate solid clinical data –For pivotal trials, characterization assays will need to be established with appropriate release limits To show comparability after manufacturing changes

19. Current Good Manufacturing Practices (cGMP) Goals: –A product with defined and reproducible quality –Increased control of the manufacturing process as clinical trials advance 2008 Guidance for Industry: CGMP for Phase 1 Investigational Drugs

20. cGMPs Quality control Quality (QC) Program –QC independent of production unit –Authority to accept or reject materials, lots, procedures and specifications –Prevent, detect, and correct deviations and failures

21. cGMPs Questions for phase I Is the manufacturing process reproducible? Do you have appropriate testing at critical steps? Is there adequate control of the quality of the raw and source materials? Are the records and record keeping systems adequate?

22. cGMPs Examples for early development Procedures to prevent contamination & cross-contamination –Aseptic processing –Facility and equipment cleaning and changeover –Tracking and segregation of patient-specific products Methods qualification –Appropriate method specificity, sensitivity, reproducibility –Lack of interference Process qualification of safety related processes –Removal of potentially dangerous impurities

23. For a phase I submission, product safety is the focus of the CMC assessment –Freedom from microbes and adventitious agents –Safety-related characterization –Appropriate GMPs Gene therapies and other complex biologics can be a challenge to characterize, and often require unique assays Product control and process control should increase with clinical development Summary

24. Further information CBER guidance documents: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplian ceRegulatoryInformation/default.htm If the webinar series and referenced websites leave you with unanswered questions: CBEROCTGTRMS@fda.hhs.gov or 301-827-5102