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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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Presentation Transcript
terminology
Terminology
  • Upstream Processing
  • Downstream Processing
  • Lab Scale purification
      • g-mg
  • Large scale purification
      • kg
slide3

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
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
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
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
Contaminants: DNA

Problems:

Oncogenes (CHO, HeLa)

Solution:

Nucleases

endogenous or exogenous

Remove during chromatography

VALIDATE VALIDATE VALIDATE

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