Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure

doi:10.1016/j.cjche.2020.01.006

中国化学工程学报 ›› 2020, Vol. 28 ›› Issue (7): 1904-1910.DOI: 10.1016/j.cjche.2020.01.006

• Biotechnology and Bioengineering • 上一篇下一篇

Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure

Run Liu¹, Qinghong Shi^1,2

1 Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China;
2 Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, China

收稿日期:2019-11-18 修回日期:2020-01-13 出版日期:2020-07-28 发布日期:2020-08-31
通讯作者: Qinghong Shi
基金资助:
This work was supported by the National Natural Science Foundation of China (Nos. 21476166 and 21878221), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004).

Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure

Run Liu¹, Qinghong Shi^1,2

1 Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China;
2 Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, China

Received:2019-11-18 Revised:2020-01-13 Online:2020-07-28 Published:2020-08-31
Contact: Qinghong Shi
Supported by:
This work was supported by the National Natural Science Foundation of China (Nos. 21476166 and 21878221), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (No. 21621004).

摘要/Abstract

摘要： Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields. In this work, protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl (SP) ligand, SP Sepharose XL and Capto S, and non-grafted cation exchange adsorbent, SP Sepharose FF, using five proteins. With an increase of buffer pHs, retention factors of proteins decreased among all the adsorbents, demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents. The evidences further revealed that the scattered positive charges on the surface of protein molecules, rather than net charge of protein molecule, determined protein retention on cation exchange adsorbent. Likely, counterions including NH₄⁺, K⁺, Na⁺ and Mg²⁺ exhibited distinct influence on protein retention. It was well ascribed to solvent-mediated indirect ion-macromolecule interactions and direct ion-macromolecule interactions. Compared with SP Sepharose FF, polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes, thereby regulating protein retention in cation exchange chromatography. By comparing the retention of myoglobin and β-lactoglobulinB in SP Sepharose XL and Capto S, we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S. The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.

关键词: Protein retention, Linear gradient elution, Dextran-grafted cation exchange adsorbents, Counterion, pH

Abstract: Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields. In this work, protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl (SP) ligand, SP Sepharose XL and Capto S, and non-grafted cation exchange adsorbent, SP Sepharose FF, using five proteins. With an increase of buffer pHs, retention factors of proteins decreased among all the adsorbents, demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents. The evidences further revealed that the scattered positive charges on the surface of protein molecules, rather than net charge of protein molecule, determined protein retention on cation exchange adsorbent. Likely, counterions including NH₄⁺, K⁺, Na⁺ and Mg²⁺ exhibited distinct influence on protein retention. It was well ascribed to solvent-mediated indirect ion-macromolecule interactions and direct ion-macromolecule interactions. Compared with SP Sepharose FF, polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes, thereby regulating protein retention in cation exchange chromatography. By comparing the retention of myoglobin and β-lactoglobulinB in SP Sepharose XL and Capto S, we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S. The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.

Key words: Protein retention, Linear gradient elution, Dextran-grafted cation exchange adsorbents, Counterion, pH

Run Liu, Qinghong Shi. Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure[J]. 中国化学工程学报, 2020, 28(7): 1904-1910.

Run Liu, Qinghong Shi. Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure[J]. Chinese Journal of Chemical Engineering, 2020, 28(7): 1904-1910.

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Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure

Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs, counterions and polymer structure

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