Harnessing Ceramic Hydroxyapatite as an Effective Polishing Strategy to Remove Product- and Process-related Impurities in Bispecific Antibody Purification

Abstract

Abstract Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to high diversity and complexity of bsAb structures and impurity profiles. The mixed-mode medium, ceramic hydroxyapatite (CHT), allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. CHT also yielded significantly less “chromatography-induced aggregation” for all aggregation-prone bsAb molecules. Developability of CHT for more efficient low molecular weight (LMW) impurity removal was further demonstrated via post-load-wash (PLW) optimization with the selected bsAbs, resulting in up to 48% additional LMW reduction. Furthermore, possible mechanistic explanation about CHT performance on both process- and product-related impurity removal in bsAb polishing was proposed. Positive CHT C-site cooperation mediated effective impurity removal and mitigated “chromatography-induced aggregation”, and domain composition and size of bsAbs molecules may determine the effectiveness of such C-site cooperation.