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)

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

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

18. 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

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

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

21. 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

22. Final Product Testing – Safety
Sterility (CFR )
Mycoplasma (CFR )
Endotoxin (CFR )
Adventitious Virus (for gene therapy products)
In vitro virus assay
Replication competent virus

23. Approaches to Release Testing for Non-Cryopreserved Cells
Sterility
Regulation
14 day sterility assay on final product
Approach
Sample 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

24. Approaches to Release Testing
Mycoplasma
Recommend testing at cell harvest
Regulation: 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

25. Final Product Characterization
Final product testing
Purity (CFR )
Identity (CFR )
Potency (CFR )
Stability
Cell viability
Development of test methods and acceptance criteria

26. 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

27. 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

28. 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

29. 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

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

31. 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

32. cGMPs for Early Development
For detailed guidance, please refer to: “Draft Guidance for Industry: INDs – Approaches to Complying with CGMP During Phase 1”

33. 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?

34. 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

35. 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

36. 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

37. 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

38. 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!

39. 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

40. References and Contact Information
References for the Regulatory Process for OCTGT
General questions for OCTGT

41. References and Contact Information
CMC Questions for Cellular Therapies
Keith Wonnacott
CMC Questions for Gene Therapies
Dan Takefman