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

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 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.

Keywords

Bispecific antibody / Mixed-mode chromatography / Product- and process-related impurity removal / Chromatography-induced aggregation

References

Aoyama K, Chiba J. Separation of different molecular forms of mouse IgA and IgM monoclonal antibodies by high-performance liquid chromatography on spherical hydroxyapatite beads. J Immunol Methods, 1993, 162(2): 201-210.

Baek Y, Zydney AL. Intermolecular interactions in highly concentrated formulations of recombinant therapeutic proteins. Curr Opin Biotechnol, 2018, 53: 59-64.

BioRad. A Matrix with Unique Separation Properties and Unparalleled Selectivity and Resolution. https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_5667.pdf

Carta G (2018) Minimizing On-Column Monoclonal Antibody Aggregate Formation https://www.bioradiations.com/minimizing-on-column-monoclonal-antibody-aggregate-formation/#:~:text=The%20formation%20of%20aggregates%20is,%2C%20and%2For%20increased%20immunogenicity

Chen SW, Hoi KM, Mahfut FB, Yang Y, Zhang W. Effective flow-through polishing strategies for knob-into-hole bispecific antibodies. Bioresour Bioprocess, 2022, 9(1): 98.

Chen SW, Hoi KM, Mahfut FB, Yang Y, Zhang W. Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography. Bioresour Bioprocess, 2022, 9(1): 72.

Chen SW, Tan D, Yang YS, Zhang W. Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies. Mabs, 2020, 12(1): 1718440.

Chen SW, Zhang W. Current trends and challenges in the downstream purification of bispecific antibodies. Antib Ther, 2021, 4(2): 73-88.

Chollangi S, Parker R, Singh N, Li Y, Borys M, Li Z. Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies. Biotechnol Bioeng, 2015, 112(11): 2292-2304.

Cytiva (2020) Capto SP ImpRes, Capto Q ImpRes ION EXCHANGE CHROMATOGRAPHY. https://cdn.cytivalifesciences.com/api/public/content/digi-15744-pdf

Gagnon P (2006) Practical issues in the industrial use of hydroxyapatite for purification of monoclonal antibodies. 232nd Meeting of the American Chemical Society, San Francisco.

Gagnon P. Monoclonal antibody purification with hydroxyapatite. N Biotechnol, 2009, 25(5): 287-293.

Gagnon P, Cheung CW, Yazaki PJ. Cooperative multimodal retention of IgG, fragments, and aggregates on hydroxyapatite. J Sep Sci, 2009, 32(22): 3857-3865.

Gorbunoff MJ. The interaction of proteins with hydroxyapatite: I. Role of protein charge and structure. Analyt Biochem, 1984, 136(2): 425-432.

Gorbunoff MJ. The interaction of proteins with hydroxyapatite: II. Role of acidic and basic groups. Analyt Biochem, 1984, 136(2): 433-439.

Gorbunoff MJ, Timasheff SN. The interaction of proteins with hydroxyapatite: III. Mechanism. Analyt Biochem, 1984, 136(2): 440-445.

Hall T, Wilson JJ, Brownlee TJ, Swartling JR, Langan SE, Lambooy PK. Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process. J Chromatogr B Analyt Technol Biomed Life Sci, 2015, 975: 1-8.

Hilbrig F, Freitag R. Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography. Biotechnol J, 2012, 7(1): 90-102.

Hou Y, Morrison CJ, Cramer SM. Classification of protein binding in hydroxyapatite chromatography: synergistic interactions on the molecular scale. Anal Chem, 2011, 83(10): 3709-3716.

Itoh D, Yoshimoto N, Yamamoto S. Retention Mechanism of Proteins in Hydroxyapatite Chromatography - Multimodal Interaction Based Protein Separations: A Model Study. Curr Protein Pept Sci, 2019, 20(1): 75-81.

Jacob, L. R. (2000). Chapter 4 - Hydrophobic Interaction Chromatography. In M. Kastner (Ed.), Journal of Chromatography Library (Vol. 61, pp. 235–269). Elsevier. https://doi.org/10.1016/S0301-4770(08)60532-1

Kimerer LK, Pabst TM, Hunter AK, Carta G. Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. I. Experimental observations and phenomenological model. J Chromatogr A, 2019, 1601: 121-132.

Labrijn AF, Janmaat ML, Reichert JM, Parren P. Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov, 2019, 18(8): 585-608.

Li W, Prabakaran P, Chen W, Zhu Z, Feng Y, Dimitrov DS. Antibody Aggregation: Insights from Sequence and Structure. Antibodies, 2016, 5(3): 19.

Li Y. Effective strategies for host cell protein clearance in downstream processing of monoclonal antibodies and Fc-fusion proteins. Protein Expression Purification, 2017, 134: 96-103.

Li Y, Wang Y, Shen P, Zhou W. Matte A. Chapter 8 - A roadmap for IgG-like bispecific antibody purification. Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics, 2020, New York: Elsevier, 167-179.

Luellau E, von Stockar U, Vogt S, Freitag R. Development of a downstream process for the isolation and separation of monoclonal immunoglobulin A monomers, dimers and polymers from cell culture supernatant. J Chromatogr A, 1998, 796(1): 165-175.

Lüllau E, Marison IW, von Stockar U. Carrondo MJT, Griffiths B, Moreira JLP. Ceramic Hydroxyapatite: A New Tool for Separation and Analysis of IGA Monoclonal Antibodies. Animal Cell Technology: From Vaccines to Genetic Medicine, 1997, Netherlands: Springer, 265-269.

Luo H, Macapagal N, Newell K, Man A, Parupudi A, Li Y, Li Y. Effects of salt-induced reversible self-association on the elution behavior of a monoclonal antibody in cation exchange chromatography. J Chromatogr A, 2014, 1362: 186-193.

Merchant AM, Zhu Z, Yuan JQ, Goddard A, Adams CW, Presta LG, Carter P. An efficient route to human bispecific IgG. Nat Biotechnol, 1998, 16(7): 677-681.

Pham NB, Meng WS. Protein aggregation and immunogenicity of biotherapeutics. Int J Pharm, 2020, 585.

Ridgway JBB, Presta LG, Carter P. ‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng Des Sel, 1996, 9(7): 617-621.

Rouet R, Christ D. Bispecific antibodies with native chain structure. Nat Biotechnol, 2014, 32(2): 136-137.

Sharkey B, Pudi S, Wallace Moyer I, Zhong L, Prinz B, Baruah H, Lynaugh H, Kumar S, Wittrup KD, Nett JH. Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients. Mabs, 2017, 9(2): 257-268.

Song M, Yang H, Cao W, Liu Q. Separation of bispecific antibody related impurities with mixed-mode chromatography. Process Biochem, 2023, 132: 110-120.

Surowka M, Schaefer W, Klein C. Ten years in the making: application of CrossMab technology for the development of therapeutic bispecific antibodies and antibody fusion proteins. Mabs, 2021, 13(1): 1967714.

Tustian AD, Endicott C, Adams B, Mattila J, Bak H. Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity. Mabs, 2016, 8(4): 828-838.

Wang X, Mathieu M, Brezski RJ. IgG Fc engineering to modulate antibody effector functions. Protein Cell, 2018, 9(1): 63-73.

Wang Y, Carta G. Competitive binding of monoclonal antibody monomer-dimer mixtures on ceramic hydroxyapatite. J Chromatogr A, 2019, 1587: 136-145.

Wang Y, Carta G. Dynamics of competitive binding and separation of monoclonal antibody monomer-dimer mixtures in ceramic hydroxyapatite columns. J Chromatogr A, 2020, 1609: 460504.

Wang Y, Carta G. Separation of monoclonal antibody monomer-dimer mixtures by gradient elution with ceramic hydroxyapatite. J Chromatography A, 2020, 1629: 461465.

Wei J, Yang Y, Wang G, Liu M. Current landscape and future directions of bispecific antibodies in cancer immunotherapy. Front Immunol, 2022, 13: 1035276.

Wu Z, Cheung NV. T cell engaging bispecific antibody (T-BsAb): From technology to therapeutics. Pharmacol Ther, 2018, 182: 161-175.