1. Large Scale Issue for Purification
BIT 230

2. Terminology Upstream Processing Downstream Processing
Lab Scale purification
g-mg
Large scale purification kg

3. 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

4. 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

5. 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)

6. 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

7. Contaminants: DNA Problems: Oncogenes (CHO, HeLa) Solution: Nucleases
endogenous or exogenous
Remove during chromatography
VALIDATE VALIDATE VALIDATE