The rapid identification of elution conditions for therapeutic antibodies from cation-exchange chromatography resins using high-throughput screening.

Cation-exchange chromatography is widely used in the purification of therapeutic antibodies, wherein parameters such as elution pH and counterion concentration require optimization for individual antibodies across different chromatography resins. With a growing number of antibodies in clinical trials and the pressure to expedite process development, we developed and automated a high-throughput batch-binding screen to more efficiently optimize elution conditions for cation-exchange chromatography resins. The screen maps the binding behavior of antibodies and impurities as a function of pH and counterion concentration in terms of a partition coefficient (Kp). Using this approach, the binding behavior of a library of antibodies was assessed on Poros 50HS and SP Sepharose Fast Flow resins. The diversity in binding behavior between antibodies and across resins translated to the requirement of a variable counterion concentration to elute each antibody. This requirement can be met through the use of a gradient elution. However, a gradient of increasing counterion concentration spans the transition from binding to non-binding for impurities as well as the antibody, resulting in the elution of impurities within the antibody elution peak. Step elution conditions that selectively elute the antibody while retaining impurities on the resin can now be rapidly identified using our high-throughput approach. We demonstrate that by correlating antibody Kp to elution pool volume and yield on packed-bed columns and through the calculation of a separation factor, we can efficiently optimize step elution conditions that maximize impurity clearance and yield for each antibody.