Bispecific antibodies (bsAbs) are becoming of increasing interest in a wide range of clinical applications. A host of product related impurities and light chain mis-pairs are inevitably produced during the manufacturing process which poses a significant challenge to downstream processing. Multimodal (MM) chromatography has been shown to remove some of these mis-pairs, but there is a lack of fundamental understanding as to what drives these MM separations which makes optimization a challenge. The objective of this work is to understand what drives the separation of the bsAb from the mis-pairs at the molecular level. First, we conducted linear salt gradient experiments with the two parent mAbs and the bsAb at various mobile phase conditions on a set of homologous MM cation exchange (CEX) resins. In order to understand how each of the parent mAbs contributes to the observed behavior for the bsAb, we conducted similar linear salt gradient experiments on the Fab, Fc and (Fab)2 fragments of the parent mAbs that were prepared using enzymatic digestion. The observed retention and selectivity behavior from these experiments was then tied to electrostatic potential (EP) and surface aggregation propensity (SAP) surface property maps as well as novel surface property clusters recently developed in our lab. Finally, the selectivity of bsAb as compared to other challenging product related impurities was investigated. The fundamental understanding of parent mAb domain contributions in MM resins and the retention behavior of an array of product related impurities will help guide the selection of mobile and stationary phase process conditions for the purification of the target bsAb from parent mAbs, mis-paired variants, and other product related impurities. The domain contribution framework presented in this work has important implications for the development of a workflow for the purification of bsAbs and also for the design of bsAbs for biomanufacturability.

Investigating selectivity trends of a bispecific antibody from key impurities on multimodal cation exchange systems

2019 Spring National Meeting

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