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 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 How do we keep it clean? 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 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 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 Problems: Oncogenes (CHO, HeLa) Solution: Nucleases endogenous or exogenous Remove during chromatography VALIDATE VALIDATE VALIDATE