Downstream
Introduction Downstream Processing overview
1. Cell concentration/removal
a. cell removal → extracellular
b. cell concentration → intracellular
c. stabilization e.g. protease inhibitors
2. (Cell disruption)
a. only for intracellular
3. Product concentration
a. Centrifugation
b. Precipitation
c. Extraction
d. Filtration
4. Product purification → chromatography
a. Size exclusion
b. Ion exchange
c. Hydrophobic interaction
d. Reverse phase
e. Affinity
5. Formulation
a. solid → free drying
b. liquid → filter sterilization
c. suspension → e.g. ammonium sulfate
proteins are much smaller than
bacteria and yeast
, Organism
Growth Media Expression Secretion Glycosylation Other
PTMs
Bacteria rapid low cost high no no no
(E. coli) (30-50% tot)
Yeast rapid low cost high- yes high mannose only limited
moderate
Filamentou rapid low cost yes high mannose only limited
s fungi
Insect slow expensive moderate - low yes complex yes
(baculo)
Mammalian slow expensive moderate - low yes complex yes
& complex
Bacteria
+ simple genetics
- inclusion bodies
- methionine at N-terminal
- reducing conditions in cytoplasm can give improper folding
- presence of endotoxin = a toxin present inside a bacterial cell that is released when it
disintegrates.
Yeast
+ Well known genetics
+ Generally Recognized As Save (GRAS) at FDA
Filamentous fungi
+ GRAS at FDA (A. niger)
- genetic manipulation is complex
Insect (baculo)
+ most protein processing mechanism available for higher eukaryotes
+ nonpathogenic
- very sensitive to shear forces
Mammalian
- very sensitive to shear forces
Introduction Downstream Processing overview
1. Cell concentration/removal
a. cell removal → extracellular
b. cell concentration → intracellular
c. stabilization e.g. protease inhibitors
2. (Cell disruption)
a. only for intracellular
3. Product concentration
a. Centrifugation
b. Precipitation
c. Extraction
d. Filtration
4. Product purification → chromatography
a. Size exclusion
b. Ion exchange
c. Hydrophobic interaction
d. Reverse phase
e. Affinity
5. Formulation
a. solid → free drying
b. liquid → filter sterilization
c. suspension → e.g. ammonium sulfate
proteins are much smaller than
bacteria and yeast
, Organism
Growth Media Expression Secretion Glycosylation Other
PTMs
Bacteria rapid low cost high no no no
(E. coli) (30-50% tot)
Yeast rapid low cost high- yes high mannose only limited
moderate
Filamentou rapid low cost yes high mannose only limited
s fungi
Insect slow expensive moderate - low yes complex yes
(baculo)
Mammalian slow expensive moderate - low yes complex yes
& complex
Bacteria
+ simple genetics
- inclusion bodies
- methionine at N-terminal
- reducing conditions in cytoplasm can give improper folding
- presence of endotoxin = a toxin present inside a bacterial cell that is released when it
disintegrates.
Yeast
+ Well known genetics
+ Generally Recognized As Save (GRAS) at FDA
Filamentous fungi
+ GRAS at FDA (A. niger)
- genetic manipulation is complex
Insect (baculo)
+ most protein processing mechanism available for higher eukaryotes
+ nonpathogenic
- very sensitive to shear forces
Mammalian
- very sensitive to shear forces
