Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification

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Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification
Title:
Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification
Journal Title:
Bioresources and Bioprocessing
Publication Date:
13 December 2023
Citation:
Ingavat, N., Wang, X., Liew, J. M., Mahfut, F. B., But, K. P., Kok, Y. J., Bi, X., Yang, Y., Shintaro, K., Tsoumpra, M., & Zhang, W. (2023). Harnessing ceramic hydroxyapatite as an effective polishing strategy to remove product- and process-related impurities in bispecific antibody purification. Bioresources and Bioprocessing, 10(1). https://doi.org/10.1186/s40643-023-00713-9
Abstract:
AbstractBispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, 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. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size. Graphical abstract
License type:
Attribution 4.0 International (CC BY 4.0)
Funding Info:
This research is supported by core funding from: BTI
Grant Reference no. : NA

This research was supported by HOYA Technosurgical Corporation, Singapore Branch and the Agency for Science, Technology and Research, Singapore.
Description:
ISSN:
2197-4365