Real-time control of antibody loading during protein A affinity chromatography using an on-line assay.
We show that an on-line chromatographic assay can reliably control antibody loading in real-time during protein A affinity chromatography purification of a recombinant antibody from clarified Chinese hamster ovary cell culture fluid. The on-line assay directly sampled preparative column effluent and provided real-time measurement of antibody breakthrough during loading. The on-line assay used protein A immobilized on perfusion chromatography media, equilibrated with phosphate-buffered saline at pH 7.2 and eluted with phosphate-buffered saline at pH 2.2. The assay reliably ended loading at 1% breakthrough with minimal yield loss. Reproducible yield and purity were obtained over 23 consecutive cycles. Yield remained constant while breakthrough capacity and the antibody concentration in the load changed.
Fahrner
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Department of Recovery Sciences, Genentech, Inc., South San Francisco, CA 94080, USA. fahrner.robert@gene.com
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D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D011994:Recombinant Proteins / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry
D000906:Antibodies; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A