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Large Scale Issue for Purification. BIT 230. Terminology. Upstream Processing Downstream Processing Lab Scale purification  g-mg Large scale purification kg. Concerns particular to Large Scale. Regulatory: FDA Purity: Higher Purity of protein for in vivo use

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Terminology l.jpg
Terminology

  • Upstream Processing

  • Downstream Processing

  • Lab Scale purification

    • g-mg

  • Large scale purification

    • kg


  • Slide3 l.jpg

    Concerns particular to Large Scale

    Regulatory: FDA

    Purity: Higher Purity of protein for in vivo use

    Expense: purification typically gets less costly with scale

    leave most expensive step till last

    Scalability: Steps may not be amenable to scale (homogenization)

    Increase column diameter

    Consider column capacity

    check expression level


    Parenteral preps l.jpg
    Parenteral Preps

    Sterile products intended for administration by

    injection, infusion, or implantation

    PURITY IS ESSENTIAL! Sterility IS ESSENTIAL!

    How do we keep it clean?

    Clean rooms (environmentally controlled areas)

    HEPA filter

    High efficiency particulate air filters


    Table 4 2 possible contaminants l.jpg
    Table 4.2 Possible contaminants

    Viral

    HIV, Hep B

    test prior to growing cells

    kill by elevated temps, irradiation

    remove with chromatography

    test end product

    Microbial

    may cause septicemia (widespread damage of tissues)


    Contaminants cont d pyrogens l.jpg
    Contaminants Cont’d: Pyrogens

    • cause fever

    • may be endotoxins (derived from bacteria)

    • Mechanism of Pyrogenic Effect

    • endotoxin stimulates IL1 prod./activates complement system

    • causes septic shock

    • Endotoxin Assays

    • horseshoe crab (Limulus polyphemus)

    • bacterial infection cause coagulation of blood

    • Limulus ameobocyte lysate (LAL) assay

    • lyse blood cells (ameobocytes)

    • add sample

    • if clot forms in tube…

    • … positive for endotoxin


    Contaminants dna l.jpg
    Contaminants: DNA

    Problems:

    Oncogenes (CHO, HeLa)

    Solution:

    Nucleases

    endogenous or exogenous

    Remove during chromatography

    VALIDATE VALIDATE VALIDATE


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