Routine manufacture of recombinant proteins using expanded bed adsorption chromatography.
Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30-44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-1 nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.
Shepard
S R
SR
Covance Biotechnology Services, Inc., Cary, NC 27513, USA. scot.shepard@covance.com
Boyd
G A
GA
Schrimsher
J L
JL
D000327:Adsorption; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D011994:Recombinant Proteins