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

doi:10.1021/acs.analchem.2c00026

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

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

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 language	English
Pages (from-to)	5875-5882
Number of pages	8
Journal	Analytical chemistry
Volume	94
Issue number	15
DOIs	https://doi.org/10.1021/acs.analchem.2c00026
Publication status	Published - 2022 Apr 19

Bibliographical note

Funding Information:
This work was supported by the Samsung Research Funding and Incubation Center of Samsung Electronics under Project Number SRFC-MA1801-07 (S.K.) and SRFC-MA1501-51 (S.J.). We thank Samsung Bioepis for kindly providing industrial harvest feedstock, Prof. Gyun Min Lee at Korea Advanced Institute of Science and Technology for providing cell culture medium containing trastuzumab, and Dr. Yunhee Seo at Korea Institute of Science and Technology for providing support for TEM observations.

Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.

ASJC Scopus subject areas

Analytical Chemistry

Access to Document

10.1021/acs.analchem.2c00026

Cite this

@article{6ff15bf55f1245359f7f2b4540ba78d7,

title = "Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin",

abstract = "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.",

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

note = "Funding Information: This work was supported by the Samsung Research Funding and Incubation Center of Samsung Electronics under Project Number SRFC-MA1801-07 (S.K.) and SRFC-MA1501-51 (S.J.). We thank Samsung Bioepis for kindly providing industrial harvest feedstock, Prof. Gyun Min Lee at Korea Advanced Institute of Science and Technology for providing cell culture medium containing trastuzumab, and Dr. Yunhee Seo at Korea Institute of Science and Technology for providing support for TEM observations. Publisher Copyright: {\textcopyright} 2022 American Chemical Society. All rights reserved.",

year = "2022",

month = apr,

day = "19",

doi = "10.1021/acs.analchem.2c00026",

language = "English",

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T1 - Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin

AU - Park, Heesun

AU - Jeon, Hyungsu

AU - Cha, Hyung Jin

AU - Bang, Jinho

AU - Song, Youngwoo

AU - Choi, Mihyun

AU - Sung, Daekyung

AU - Choi, Won Il

AU - Lee, Jin Hyung

AU - Woo, Jae Sung

AU - Jon, Sangyong

AU - Kim, Sunghyun

N1 - Funding Information: This work was supported by the Samsung Research Funding and Incubation Center of Samsung Electronics under Project Number SRFC-MA1801-07 (S.K.) and SRFC-MA1501-51 (S.J.). We thank Samsung Bioepis for kindly providing industrial harvest feedstock, Prof. Gyun Min Lee at Korea Advanced Institute of Science and Technology for providing cell culture medium containing trastuzumab, and Dr. Yunhee Seo at Korea Institute of Science and Technology for providing support for TEM observations. Publisher Copyright: © 2022 American Chemical Society. All rights reserved.

PY - 2022/4/19

Y1 - 2022/4/19

N2 - 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.

AB - 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.

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