Binding mechanism of functional moieties of a mixed-mode ligand in antibody purification

•Carboxyl group showed a critical role in a mixed-mode ligand design.•A combination of carboxyl group and an elution moiety was suggested.•A model mixed-mode ligand was design by the principle proposed. Functional moieties are critical in mixed-mode ligand design for separation and purification of biomolecules. This study investigated the role of carboxyl group and a hydrophobic moiety on a recently developed mixed-mode ligand (Phenylalanine-Tyrosine-Glutamate-“5-aminobenzimidazole”, FYE-ABI) for hIgG purification. The effects of carboxyl group were focused for its appearance in several commercial mixed-mode ligands. A ligand with glutamic acid on FYE-ABI substituted by glutamine (Phenylalanine-Tyrosine-Glutamine-“5-aminobenzimidazole”, FYQ-ABI) was first prepared to explore the binding mechanism of the FYE-ABI ligand. The selectivity (52.1%) of the FYQ-ABI ligand was found inferior comparing to the original design, which was gradually recovered during the hydrolysis process of glutamine (Gln) to glutamic acid (Glu). Meanwhile, a cationic exchange resin CM Bestarose Fast Flow with carboxyl group was used for comparison and the results demonstrated that other moieties such as aminobenzimidazole were helpful for elution processes. A new model mixed-mode ligand with such a design principle (a carboxyl group for binding and aminobenzimidazole for elution) was thus synthesized and comparable separation performance was achieved using the prepared resins. The model ligand had much simplified chemical structure and this study can help for the design of related mixed-mode ligands for antibody purification.