Monovalent bispecific antibodies (bsAbs), a class of biological product that enables the binding of multiple targets while retaining the conventional monoclonal antibody (mAb) structure, are becoming of increasing importance in a wide range of clinical applications. During cell culture, incorrect pairing of the heavy and light chains of the bsAb lead to the formation of various ‘mis-paired species’ that have very similar surface properties as the target bsAb and pose significant purification challenges. While multimodal (MM) chromatography is often used as a polishing step after Protein A capture, there is limited fundamental understanding of the resin and protein surface properties that drive the separation of the bsAb product from the mis-paired variants. In this work, the retention behavior of the bsAb, the parental mAbs, and two key mis-paired variants (coeluting with the bsAb in the Protein A step) were studied on a set of homologous MM cation exchange (CEX) resins under various conditions to identify selectivity trends. To understand how the constituent domains contributed to the observed behavior on the multimodal systems, chromatographic evaluation was carried out on Fab, Fab2 and Fc fragments obtained from these species by enzymatic digestion. The observed behavior from these experiments were then tied to surface property maps such as electrostatic potential (EP) and surface aggregation propensity (SAP) maps as well as novel surface property clusters to identify key surface patches that are important binding sites for MM systems. Experimental techniques were then used to confirm the surface patches on the parental mAbs that are important for binding on mixed-mode systems found using <i>in silico</i> surface property mapping tools. The techniques presented in this work have important implications for the development of a workflow for the purification of bsAbs, for the design of bsAbs for biomanufacturability as well as for understanding MM ligand design and applications.

Fundamental approach to understanding selectivity trends for bispecific antibody purification by multimodal chromatography

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.