Salt‐tolerant cation exchanger‐containing sulfate groups as a viable alternative for mixed‐mode type and heparin‐based affinity resins

Abstract

Ion‐exchange chromatography is still one of the most popular protein separation techniques. Before chromatographic separation, the high salt concentration in various samples necessitates additional steps. Therefore, low salt tolerance of ion‐exchange resins is a drawback that needs to be addressed. Herein, the differences in salt tolerance and hydrophobicity of strong cation‐exchange TOYOPEARL resins of sulfonium and sulfate‐types were investigated. Despite only a minor structural difference, differences in selectivity and salt tolerance between the sulfate and sulfonic groups were detected. In silico calculations were also carried out for model substances representing the sulfonium and sulfate groups, wherein significant differences in hydrophobicity was observed. These experiments confirmed the hypothesis that the salt tolerance, higher affinity, and selectivity for certain vitamin K dependent clotting factors are interrelated and dependent on the presence of the sulfate group. Separation of clotting factor IX from the prothrombin complex concentrate further to confirmed the affinity for these proteins. The results show that the use of only a resin with the sulfate ligand and not with the sulfonic acid ligand allows for a facile and rapid separation of clotting factor IX and other vitamin K dependent clotting factors.