# Presentation: 749349

## 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_

## 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)

- 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
- Validate by licensure
- 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!**

## 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