Improved Purification Of Antibody By Novel Mixed-mode Chromatography

In this paper, aiming at the purification of γ-globulin from rabbit anti-humanT-lymphocyte(RATS) and anti-smallpox immune sera(ASRS), Sepharose Fast Flowwas activated withallyl bromide, and then the ligand, SLC-AN, was coupled onto theactivated beeds to fabricate the novel MMC beads AN Sepharose Fast Flow, with aseries of different ligand densities from15to65mmol/L.The influence of the ligand density for the adsorption behavior of γ-globulin andBSA on the MMC beads and the appropriate ligand density range for γ-globulinpurification were determined. The results of adsorption equilibria showed that ANSepharose Fast Flow had a minimum threshold value between20and25mmol/L andhad much higher binding amount and better affinity for γ-globulin at ligand densitiesof25mmol/L and above. AN Sepharose Fast Flow exhibited great selectivity toγ-globulin with ligand densities between25and30mmol/L whilst these beadsexhibited good tolerance to salt concentration. DBCs of proteins on the MMC beadswere investigated and the results showed DBC for antibody of AN Sepharose FastFlow was comparable to commercial MMC beads, MEP HyperCel, and Protein Abeads, MabSelect SuRe, and its good reuse performance was identified.With rabbit sera mentioned above as the raw materials, the purification ofγ-globulin by the MMC beads was compared with commercial MabSelect SuRe andMEP HyperCel beads. In the purification of γ-globulin from various sera, ANSepharose Fast Flow had the similar purity and yield of antibody to two commercialbeads. The results showed that the purity of RATS purification experiment couldreached88.9%while the yield reached80.4%with ligand density of25mmol/L. Onthis basis, a novel approach of multi-column continuous adsorption was proposed inthe antibody purification, purity and yield of γ-globulin products were significantlyimproved. The purity of RATS purification experiment with30mmol/L ANSepharose Fast Flow could be increased from61.2%to79.9%, while purity of MEPHyperCel was increased from85.6%to97.2%. Experimental results indicated thatmixed-mode chromatography bead AN Sepharose Fast Flow would have goodapplication prospects in the field of antibody purification.