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

doi:10.1016/j.cjche.2020.01.006

Abstract

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

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

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.

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.

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