Expanded bed protein A affinity chromatography of a recombinant humanized monoclonal antibody: process development, operation, and comparison with a packed bed method.
We show that expanded bed protein A affinity chromatography using Streamline rProtein A media is an efficient method for purifying a recombinant humanized monoclonal antibody from unclarified Chinese hamster ovary cell culture fluid and that it provides purification performance comparable to using a packed bed. We determined that the dynamic capacity of the expanded bed media is related to flow rate (measured in column volumes per hour) by a power function, which allows a high capacity at a low flow rate. At 250 cm h-1 with a 25 cm bed height (10 column volumes h-1), the dynamic capacity is 30 g l-1. The yield and purity (measured by the amount of host cell proteins, DNA, SDS-PAGE, and turbidity) of the antibody purified by expanded bed is comparable to the yield and purity obtained on a standard packed bed method using Prosep A media.
Fahrner
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Department of Recovery Sciences, Genentech, South San Francisco, CA 94080, USA. fahrner.robert@gene.com
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Zapata
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000378:metabolism; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D004247:DNA