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.