Integration of mixed-mode chromatography and molecular imprinting technology for double recognition and selective separation of proteins

Abstract Adsorption selectivity is one of the most important parameters for chromatography. As a promising technology for protein purification, mixed-mode chromatography (MMC) has unique advantages; however, its selectivity needs to be improved further. In this study, one strategy for improving selectivity was tested. We combined MMC with molecular imprinting technology (MIT) to gain double-recognition capabilities. Molecularly imprinted polymer (MIP) enabled the enhancement of the selectivity of MMC. Cross-linked 4% agarose beads (4FF) with tryptamine (Try) as ligands were used to prepare the double-recognition resin (4FF-Try/MIPs), with bovine serum albumin (BSA) as the imprinting template protein. After removing the template protein from the imprinted layer, 3D cavities with double-recognition abilities toward BSA were obtained, owing to the combination of hydrophobic interaction and molecularly imprinted spatial matched cavities. The static and dynamic adsorption behaviors of BSA, bovine immunoglobulin G (bIgG), and bovine hemoglobin (BHb) on resins were investigated. The results showed 4FF-Try/MIPs retained the typical adsorption properties of pH dependence and salt tolerance of MMC. High and similar dynamic binding capacities for BSA were obtained for both 4FF-Try and 4FF-Try/MIPs. However, 4FF-Try/MIPs showed lower dynamic binding capacity for the competitive impurity bIgG. When 4FF-Try/MIPs was used to separate BSA from bovine serum, the purity reached 75.1%, which was higher than 4FF-Try by ∼20%. We conclude that MIT could be applied to enhance selectivity of MMC with double recognition capabilities, and this might be a straightforward strategy for improving selectivity in other modes of chromatography.