Purification Of Biotherapeutics Research Articles

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

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.

Dye-ligand affinity adsorbents for enzyme purification.

Affinity chromatography is widely employed in laboratory and large-scale for the purification of biotherapeutics and diagnostics. Some of the most widely used ligands in affinity chromatography have been several reactive chlorotriazine dyes. In particular, immobilized anthraquinone dyes have found a plethora of applications in affinity chromatography because they are inexpensive, are resistant to chemical and biological degradation, are sterilizable and cleanable in situ, and are readily immobilized to generate affinity adsorbents which display high binding capacity for a broad spectrum of proteins. This article provides detailed protocols on the preparation of a dye-ligand affinity adsorbent. Also, detailed protocols for effective application of these media, emphasizing binding and elution conditions are presented.