Affinity-reversed-phase liquid chromatography assay to quantitate recombinant antibodies and antibody fragments in fermentation broth

An automated dual-column liquid chromatography assay comprised of affinity and reversed-phase separations that quantifies the majority of antibody-rel ated protein species found in crude cell extracts of recombinant origin is described, Although potentially applicable to any antibody preparation, we here use samples of anti-CD18 (Fab'2LZ) and a full-length antibody, anti-ti ssue factor (anti-TF), from various stages throughout a biopharmaceutical. production process to describe the assay details. The targeted proteins wer e captured on an affinity column containing an and-light-chain (kappa) Fab antibody (AME5) immobilized on controlled pore glass. The affinity column w as placed in-line with a reversed-phase column and the captured components were transferred by elution with dilute acid and subsequently resolved by e luting the reversed-phase column with a shallow acetonitrile gradient. Char acterization of the resolved components showed that most antibody fragment preparations contained a light-chain fragment, free light chain, light-chai n dimer and multiple forms of Fab'. Analysis of full-length antibody prepar ations also resolved these fragments as well as a completely assembled form . Co-eluting with the full-length antibody were high-molecular-mass variant s that were missing one or both light chains. Resolved components were quan tified by comparison with peak areas of similarly treated standards. By com paring the two-dimensional polyacrylamide gel electrophoresis patterns of a n Escherichia coli blank run, a production run and the material affinity ca ptured (AME5) from a production run, it was determined that the AME5 antibo dy captured isoforms of light chain, light chain covalently attached to hea vy chain, and truncated light chain isoforms. These forms comprise the bulk of the soluble product-related fragments found in E. coli cell extracts of recombinantly produced antibody fragments. (C) Elsevier Science B.V. All r ights reserved.