Multi-specific antibody formats possess unique product-related species which have previously required development of new purification processes deviating from a traditional mAb platform. These novel formats also often produce high variability in levels of these product related forms among different constructs, straining traditional polishing chromatography methods. Therefore, interest remains to develop new purification strategies to improve “platformability,” productivity, and robustness for multispecific antibodies. Previous studies have demonstrated the potential of leveraging binding avidity with affinity chromatography to provide control for bispecific antibody product related impurities. This technique relies on differential binding to the media based on the number of binding motifs present on target products and impurities. However, the current literature lacks a comprehensive study of avidity-based separation using light chain affinity chromatography media with specificity for the constant regions of kappa or lambda antibody light chains. In this work, we evaluate the capability of available light chain affinity media to resolve asymmetric bispecific antibody heterodimers from mis-paired variants, aggregates, and low molecular weight species. Our results show complex separation behavior among different affinity media, and unexpected sensitivity of lambda affinity elution performance to low concentrations of mobile phase modifiers. We also observed that some low molecular weight impurities possessing a single light chain bind more strongly to light chain affinity media than intact heterodimer species. This finding may suggest that avidity-binding alone may not fully explain the separation behavior. Our investigation of the binding mechanisms and impacts on protein stability help further elucidate the selectivity observed.

Leveraging binding avidity in light chain affinity chromatography media for control of mis-paired byproducts during production of asymmetric bispecifics

ACS Spring 2022

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