Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin

Heesun Park, Hyungsu Jeon, Hyung Jin Cha, Jinho Bang, Youngwoo Song, Mihyun Choi, Daekyung Sung, Won Il Choi, Jin Hyung Lee, Jae Sung Woo, Sangyong Jon, Sunghyun Kim

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)


Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.

Original languageEnglish
Pages (from-to)5875-5882
Number of pages8
JournalAnalytical chemistry
Issue number15
Publication statusPublished - 2022 Apr 19

Bibliographical note

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© 2022 American Chemical Society. All rights reserved.

ASJC Scopus subject areas

  • Analytical Chemistry


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