sepharose and 5-aminobenzimidazole

Hydrophobic charge-induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge-induction resins, new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins with 5-aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)-grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 μmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non-grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)-grafted resins also reached the maximum at the moderate ligand density of 330 μmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)-grafted hydrophobic charge-induction resins have a promising potential for antibody purification applications.

Topics: Adsorption; Benzimidazoles; Chromatography; Humans; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Kinetics; Ligands; Polymethacrylic Acids; Resins, Synthetic; Sepharose

Expanded bed adsorption (EBA) can capture target proteins directly from unclarified feedstock without prior solid-liquid separation. Hydrophobic charge-induction chromatography (HCIC) is a promising technology for biomolecule separation with high capacity, good selectivity and relatively low cost without the pretreatment of dilution or salt addition. In this work, EBA and HCIC were combined to develop a new separation technology, hydrophobic charge-induction EBA. Two HCIC ligands, 4-mercapto-ethyl-pyridine (MEP) and 5-aminobenzimidazole (ABI), were coupled onto agarose beads containing tungsten carbide to prepare the resins for EBA, named T-MEP and T-ABI, respectively. The static adsorption and dynamic binding behaviors of bovine IgG (bIgG) were investigated. Two resins had similar saturated adsorption capacities and salt-tolerant properties, but T-ABI showed higher dynamic binding capacity than T-MEP, indicating that ABI ligand was more suitable for EBA. The performances in expanded bed were verified. With the protein mixture (2mg/ml bIgG and 10mg/ml bovine serum albumin) as the model feedstock, the effects of loading and elution pH, expansion factor and loading volume on the separation performance of bIgG were evaluated. Finally, T-ABI EBA was used to separate bIgG directly from bovine whey with optimized operation conditions. The purity and recovery of bIgG reached 90.6% and 78.2%, respectively. The purification factor was about 19.3. The results demonstrated that the combination of HCIC and EBA would be a potential platform for antibody capture with less feedstock pretreatments, high efficiency and relatively low cost.

Topics: Adsorption; Animals; Benzimidazoles; Cattle; Chemistry Techniques, Analytical; Chromatography; Hydrophobic and Hydrophilic Interactions; Immunoglobulin G; Ligands; Pyridines; Sepharose; Serum Albumin, Bovine; Sodium Chloride