Study on antibody adsorption and elution performance of carboxyl and hydrophobic groups on mixed-mode ligands.

Abstract

Molecular interactions between ligands and target biomolecules are crucial in the development of chromatographic techniques for the separation and purification of biotherapeutics. In this study, the role of functional moieties on a mixed-mode ligand (phenylalanine-tyrosine-glutamate-5-aminobenzimidazole) for human immunoglobulin G purification was investigated and a detailed mechanism was discussed. A similar ligand with glutamic acid substituted by glutamine (phenylalanine-tyrosine-glutamine-5-aminobenzimidazole) together with other resins including a commercial resin (CM Bestarose Fast Flow), phenylalanine-tyrosine-glutamate, glutamate-5-aminobenzimidazole, and 5-aminobenzimidazole resins were prepared for comparison. Molecular dynamics simulation and experimental studies were used to analyze the difference between these ligands. The results showed that the carboxyl group of phenylalanine-tyrosine-glutamate-5-aminobenzimidazole contributed 70% of the electrostatic interaction during human immunoglobulin G binding, and 5-aminobenzimidazole provided electrostatic repulsion for desorption, which showed low selectivity and binding capacities at pH 4.0 (dynamic binding capacities at 10% breakthrough of human immunoglobulin G=1.0mg/ml resin, dynamic binding capacities at 10% breakthrough of human serum albumin=1.2mg/ml resin) when used as an individual resin ligand. The results showed in this study demonstrated that it is possible to achieve optimal antibody separation and purification through reasonable ligand design by understanding the performance of key functional moieties in binding and elution processes.