Accelerating Usp

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Considerations for upstream process development

Considerations for upstream process development

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  • 1. Accelerating Your Success: Regulatory Considerations in Upstream Process Development Barry Rosenblatt, PhD
  • 2. Biotech Process of Creating a Drug Target Identification Candidate Selection Candidate Optimization Pre-IND Evaluation and Clinical Trials Time Conc t 1/2 Target Validation 
  • 3. Characterization of Continuous Cell Lines
    • What do the ICH/EU guidelines and “Points to Consider” suggest?
    • Definitions of cell banks
    • Test descriptions
    • A typical cell testing approach
  • 4. Guideline Documents
      • ICH:
        • VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN Q5A(Rl)
        • DERIVATION AND CHARACTERISATION OF CELL SUBSTRATES USED FOR PRODUCTION OF BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS Q5D
      • FDA:
        • Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
      • EU:
        • GUIDELINE ON VIRUS SAFETY EVALUATION OF BIOTECHNOLOGICALINVESTIGATIONAL MEDICINAL PRODUCTS Doc. Ref. EMEA/CHMP/BWP/398498/2005 (FEB 2009)
  • 5. ICH Guidelines And Points To Consider
        • Cell Lines
          • History & genealogy
          • Cell seed systems (banks)
          • Culture medium
          • Growth in vitro
          • Time of sampling and testing
          • Production and testing facilities
  • 6. Master Cell Bank (Definition)
    • The Master Cell Bank (MCB) is a quantity of cells derived from a single tissue and stored frozen at -70°C or below in aliquots, one or more of which would be used for the production of the Working Cell Bank (WCB)
  • 7. Working Cell Bank (Definition)
    • The Working Cell Bank (WCB) is a quantity of cells derived from one or more of the ampoules of the cell seed and of uniform composition stored as -70°C or below in aliquots, one or more of which would be used for the production of a biological product.
  • 8. End of Production or Cells at In-Vitro Limit
    • Cells obtained at or several generations past the stage at which crude product was harvested.
    • End of Production Cells (EoP)or “Cells at the Limit of in vitro Cell Age Used for Production” (CAL) are typically derived from the expansion of the WCB
  • 9. Cell Line History & Genealogy
    • Age, sex and species of the donor
    • Individual and family medical history
    • Culture history of the line including methods used for the isolation of the tissues from which the line was derived, split ratio used in passaging, media, etc.
  • 10. MCB & WCB Storage
    • Stored in liquid or vapor phase of liquid nitrogen
    • Documented location, identity, and inventory of ampoules
    • Stored in multiple locations/sites (minimum of 2)
  • 11. Culture Medium
    • Raw materials testing, i.e., serum adventitious agents
    • Accurate records on composition and sources
    • Testing for residual amount in final product
    • Absence of antibiotics
  • 12. Growth Characteristics In Vitro
    • Stable Pattern and morphological appearance
      • Population doubling time (PDL) to post-production
      • Determine PDL’s through senescence
      • Determine number of generations
  • 13. Time of Sampling and Testing
    • Tests should be performed on cell suspensions (lysates)/fluids derived from the WCB propagated to or beyond the level at which they are to be used in production.
  • 14. Production and Testing Facilities
    • Absence of contamination with transmissible agents
      • Cross-contamination with other cell lines
      • i.e., Good Manufacturing Practices
        • 21CFR parts 210-211
        • ICH Q7
  • 15. Cell Line Characterization
    • Mammalian (general)
      • Mycoplasma & Sterility
      • In Vivo adventitious agent detection
      • In Vitro adventitious agent detection
      • Karyology & Isoenzymes
      • Transmission Electron Microscopy
      • Reverse Transcriptase Activity
      • Tumorigenicity
  • 16. Test Description
      • Mycoplasma & Sterility
      • Mycoplasma
        • Cultivable and non-cultivable
        • Aerobic and anaerobic
        • Culture and DNA fluorochrome methods
      • Sterility
        • Bacteria and fungi
        • USP, 21 CFR 610.12, EP 2.6.1
  • 17. Test Description
        • In Vivo Safety Tests
        • Adult and suckling mice
        • Embryonated hen’s eggs
        • 21 CFR 630.35
        • Simian cell lines may require
          • Rabbits
          • Guinea pigs
  • 18. Test Description
      • Presence of Viruses
      • In vitro cell culture (Cell Line dependant)
        • Cell line being characterized or one from the same species
        • A normal human embryo cell line
        • A monkey kidney cell line
          • Minimum 14 day culture
          • Normal morphology
          • Hemadsorbing viruses
  • 19. Test Description
    • Karyology/Isoenzyme
    • Gross abnormalities in chromosome number or morphology
      • Abnormalities intrinsic to original fetal material
      • Exposure to chemical or physical mutagens
      • Mislabeling and/or cross contamination
  • 20. Test Description
    • Electron Microscopy
      • TEM sections at 50,000 X
      • Pelleted cell supernatant
      • Examine for viruses or other microbial agents
  • 21. Test Description
    • Specific Tests for Retroviruses
    • Reverse transcriptase conventional
    • PBRT (PCR based reverse transcriptase)
    • Inoculation of RV-supporting cell lines with supernatants from production cultures
  • 22. Test Description
    • Tumorigenicity Testing
    • In Vivo
      • Nude Mice
        • Immunosuppressed newborn hamsters, mice or rats
        • Thymectomized and irradiated mice or rats
    • In Vitro
      • Colony formation in soft agarose
      • (Must be shown to be at least as sensitive as in vivo )
  • 23. Murine Cell Line Characterization (Master Cell Bank)
    • In vitro (3 cell line, 28 day)
    • Mouse Antibody Production (MAP)
    • In vivo (adult & suckling mice, eggs)
    • Transmission Electron Microscopy
    • Mycoplasma
    • Reverse Transcriptase
    • Sterility
    • Extended XC
    • Extended ERV
    • Extended S+L-
    • Karyology & Isoenzymes
    • Bovine & Porcine Viruses (optional)
  • 24. Murine Cell Line Characterization (Working Cell Bank)
        • Sterility
        • Mycoplasma
        • In vitro ( 3 cell line, 28 day - optional)
        • Isoenzyme (optional)
  • 25. Murine Cell Line Characterization (End of Production Cells)
    • In vitro (3 cell line, 28 day)
    • In vivo (adult & suckling mice, eggs)
    • Transmission Electron Microscopy
    • Mycoplasma
    • Reverse Transcriptase
    • Sterility
    • Extended XC
    • Extended ERV
    • Extended S+L-
    • Karyology & Isoenzymes
    • Bovine & Porcine Viruses (optional)
  • 26. ICH Summary Virus Testing
  • 27. EU Guidance (Cell Harvest) * Where possible, test material should contain cells or cellular fragments in order to detect cell associated viruses. For perfusion cell cultures, manufacturers should determine and justify the most appropriate stage at which to derive samples containing cells for testing. It is also acceptable to derive test material from cells that have been cultured beyond the scale used to generate the batch of product; in these circumstances, the approach taken should be justified. Testing for infectious retroviruses may be omitted when more sensitive tests have shown negative results. § Quantification of retroviruses or retroviral-like particles need only be performed for the first three bulks for a specific stage of development (or less, if less than three bulks are prepared). Yes, once for given scale Yes, once for given scale Yes, all bulks§ All other cell lines No Yes, once for given scale Yes, all bulks§ NS0 and Sp2/0 No No Yes, all bulks§ CHO In vivo testing* Tests for infectious retroviruses* In vitro testing
  • 28. Optimize cell line Media Subclone Expression system The Cycle
  • 29. Comparability & Setting Specifications Develop Apply Analyze Specs?
  • 30. Comparability & Setting Specifications Specs ? Comparability? Comparability? Develop Apply Analyze
  • 31. Conclusions
    • Cell Line optimization can occur at multiple points during development
    • Media optimization goes hand-in-hand with cell line optimization
    • Customized media may require customized testing
    • Re-testing is required for every new cell line (Establishment of bank)
    • Comparability testing should be planned in advance of cell line/process changes