Large-scale purification of recombinant human angiostatin.
A process for the purification of recombinant human angiostatin (rhAngiostatin), produced by Pichia pastoris fermentation operated at the 2000-L scale, is reported. rhAngiostatin was recovered and purified directly from crude fermentation broth by cation exchange expanded bed adsorption chromatography. Anion exchange chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography were used for further purification. Full-length rhAngiostatin was separated from rhAngiostatin molecules fragmented by endoproteolysis. On average, 140 g of rhAngiostatin was produced per batch, with an overall yield of 59% (n = 9). The purification process was completed in approximately 48 h and used only inexpensive and nontoxic raw materials. Methods development, process synthesis, and process scale-up data are presented and discussed.
Shepard
S R
SR
Covance Biotechnology Services Inc., 3000 Weston Parkway, Cary, North Carolina 27513, USA. scot.shepard@covance.com
Boucher
R
R
Johnston
J
J
Boerner
R
R
Koch
G
G
Madsen
J W
JW
Grella
D
D
Sim
B K
BK
Schrimsher
J L
JL
D000645:Ammonium Sulfate / Q000378:metabolism; D043168:Angiostatins; D000970:Antineoplastic Agents / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D017886:Durapatite / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010710:Phosphates; D010843:Pichia / Q000235:genetics; D010958:Plasminogen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results
D000970:Antineoplastic Agents; D010446:Peptide Fragments; D010710:Phosphates; D011994:Recombinant Proteins; D043168:Angiostatins; D010958:Plasminogen; D017886:Durapatite; C018279:sodium phosphate; D000645:Ammonium Sulfate