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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
        • Growthin 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 -70C 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 -70C 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 ofin vitroCell 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 CharacteristicsIn Vitro

  • Stable Pattern and morphological appearance
    • Population doubling time (PDL) to post-production
    • Determine PDLs 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 Vivoadventitious agent detection
    • In Vitroadventitious 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 VivoSafety Tests
      • Adult and suckling mice
      • Embryonated hens eggs
      • 21 CFR 630.35
      • Simian cell lines may require
        • Rabbits
        • Guinea pigs

18. Test Description

    • Presence of Viruses
    • In vitrocell 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 asin 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 & su

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