Development Of Hybrid Chromatographic Ligands For Antibody Purification

With the development of biotechnology,antibodies are widely used as therapeutic drugs in disease treatment.However,downstream processes for antibody separation develops slowly in both industry and academy.Protein A affinity chromatography is the most widely used technique in antibody purification,which still has some limitations such as harsh antibody elution conditions and easy detachment of ligands.In this thesis,the interaction between protein A and antibody was studied by computer simulation,and the key amino acid residues in protein A were analyzed.Hybrid ligands with high selectivity and mild elution,which was constructed by biomimetic short peptide ligands and hydrophobic charge-inducing ligands for antibody purification was developed..The binding mode between protein A and antibody Fc fragment was studied by molecular dynamics simulation.The hotspot residues on the protein A ligand were screened and the interaction energy between residues and Fc fragment was analyzed.Moreover,a hybrid ligand FYQ-ABI(Phenylalanine-Tyrosine-Glutamine-"5-amino-benzimidazole")was designed based on the simulation results.Ligand-protein interactions and binding patterns were studied by molecular simulation.The ligand can stably bind to the Fc fragment under neutral conditions,and hydrophobic and electrostatic interactions played an important role in the binding process.The FYQ-ABI chromatographic resin was prepared and the static,dynamic and separation properties were investigated.The results showed that the FYQ-ABI resin had good hIgG selectivity,and the purity of hIgG isolated from hIgG/BSA mixed protein solutions can reach 99%.The coupling process for FYQ-ABI resin preparation was optimized to obtain higher ligand density.The resin showed high adsorption capacity for hIgG,which increased with the increase of ligand density.At pH 7.0,the dynamic binding capicity of the resin can reach 20.1 mg/mL.The FYQ-ABI resin had high adsorption capicity and good salt tolerance.The selectivity of the FYQ-ABI resin can be improved by adding NaCl.The stability of the ligand was measured in 0.1M NaOH solution.The amount of hIgG absorbed by the FYQ-ABI resin showed little changed when the treatment time increased,but the adsorption amount of HSA decreased sharply.Further verification found that it was caused by amide hydrolysis on glutamine.The FYE-ABI(Phenylalanine-Tyrosine-Glutamate-"5-amino-benzimidazole")ligand was synthesized and coupled to agarose matrix for characterization.The maximum saturated adsorption capacity was 91.5 mg/g resin at pH 7.0.With a retention time of 2 min,the dynamic binding capicity can reach 22.3 mg/mL.The effect of different elution pH on retention factor and elution yield was investigated,and hIgG can be effectively eluted under the conditions of pH 4.0?pH 5.0,which can be used as the elution condition for subsequent separation processes.The ability to separate hIgG from HSA/hIgG protein mixture and human serum was further investigated.The optimal elution pH of the human serum purification was 4.5,and the purity and yield of hIgG were 93.9%and 88.9%,respectively.