Preparation Of 5-aminobenzimidazole-based Hydrophobic Charge-induction Chromatographic Resins And Protein Adsorption

Hydrophobic charge induction chromatography (HCIC) is a new method for antibody separation, and the functional ligands normally combine hydrophobic and electrostatic interactions. The target protein could be adsorbed by the hydrophobic interaction at neutral pH, and desorbed by the electrostatic repulsion interaction with adjusting the pH value. However, the adsorption ability of HCIC resins should be improved. In this thesis,5-aminobenzimidazole (ABI) ligand was chosen as the functional ligand and series of HCIC resins were prepared using three coupling methods. The effects of ligand density and coupling methods on protein adsorption were studied. Main results are summarized as follows.Firstly, with agarose gel as the matrix, ABI-B-6FF resin with different ligand density was prepared by controlling ligand coupling conditions. The equilibrium adsorption, adsorption kinetics and column breakthrough behaviors were determined using human immunoglobulin G (hIgG) and bovine serum albumin (BSA) as model proteins. With the increase of ligand density, the adsorption capacity and effective diffusion coefficient increased firstly and then tended to saturation. Moreover, high dynamic binding capacity (DBC) was found for the resins with high ligand density had and was hardly affected by the flow rate. The adsorption capacity of hlgG is higher than that of BSA, and the selectivity could be enhanced with the increase of ligand density. The pore surface ligand density (PSLD, ligand number/nm2) was introduced and good linear relationships could be found between PSLD and adsorption parameters.Secondly, with dextran-grafted agarose gel as the matrix, ABI-B-XL resin with different ligand density was prepared. With hIgG as the model protein, the influences of pH, salt addition and ligand density on protein adsorption was studied. It was found that pH had a significant effect on protein adsorption, and high adsorption capacity was found at the pH range from 7.0 to 8.5. Moreover, salt addition had little effect on adsorption, showing good salt-tolerance of HCIC resin. With the increase of ligand density, the adsorption capacity, effective diffusion coefficient and DBC increased firstly and then decreased. The optimized ligand density was 220 μmol/g, and DBC at 100 cm/h reached 66.70 mg/g. The results indicated that the adsorption ability of IgG could be improved obviously by dextran-grafted ABI-B-XL resins compared to non-grated ABI-B-6FF.Thirdly, with glycidyl methacrylate (GMA) as the grafting monomer and agarose gel as the matrix, G-ABI-4FF resin with different ligand density was prepared by controlling the addition of GMA and ligand. The ligand density of G-ABI-4FF resins was significantly higher than that of ABI-B-XL. The influence of ligand density on protein adsorption was focused. With the increase of ligand density, the adsorption capacity, effective diffusion coefficient and DBC increased firstly and then decreased. The optimized ligand density was 330 μmol/g. DBC at 100 cm/h could reach 82.10 mg/g, which was hardly affected by the flow rate. The results demonstrated that the antibody adsorption ability could be further enhanced by GMA-grafted HCIC resin G-ABI-4FF compared to dextran-grafted ABI-B-XL.This thesis focused on the preparation of new HCIC resin. The influence of ligand density and coupling methods on antibody adsorption was studied systematically. It was found that the adsorption capacity, effective diffusion coefficient and DBC could be improved by the grafting in the pores of HCIC resins, especially for GMA-grafted resins. For G-ABI-4FF resin, the ligand density was enhanced and the ligand distribution would be more uniform, which improved the dynamic binding ability of HCIC resins and showed a good application potential for antibody purification.