Research On Preparation Of Dextran-Grafted Tetrapeptide Biomiietic Chromatographic Resins And Antibody Purification

Recently,protein A affinity chromatography as a platform technology for downstream antibody capture has limitations on application of large-scale antibody separation with the improvement of upstream cell expression and expansion of culture scale due to its high price,nsufficient elution low media utilization and other drawbacks.Short peptide biomirnetic affinity chromatography as a novel antibody separation chromatography technique is a potential akernative to protein A chromatography on prospective antibody purification with tow cost,high stability,fow immunogenicity,high selectivity and mild elutioa Dextran grafting was introduced to prepare a high-loading dextran-grafted short peptide affinity chromatography resin to increase both the equilibrium adsorption eapacity and mass transfer efficiency to further improve the dynamic binding capacity of corresponding resins.In this paper,we investigated the antibody adsorption performance and separation performance of feedstocks,and then analysized the influence of dextran grafting on short peptide biomimetic ehronatography,Providing a new perspective on modification of short peptide biomimetic chromatographic resins.The nain research contents and results obtained in this paper show as followsFirstly,dextran-grafted tetrapeptide biomimetic chromatography resins were prepared by HATU method with Ac-YFRH as firnctional ligand and Bestarose XL as matrix,and the static adsorption properties of resins for hlgG,BSA and Fc fusion proteins were investigated.The results showed that the adsorption of hlgG on Ac-YWRH-XL resin was dominated by hydrophobic interaction and electrostatic interaction.The adsorption eapacity eould reach 133 mg/g resin at pH 9.0 and no adsorption at pH 5.0,and Ac-YFRH-XL resin showed diferent salt tolenrance to sodium chlorkie and sodium caprylate.The adsorption of BSA on Ac-YFRH-XL resin was mainly based on electrostatic interaction and the highest adsorption performance was found at pH 5.0.Ac-YFRH-XL resin had higher adsorption capacity and binding ability to Fc fusion protein than hlgG,which was consistent with the conPuter simulation results based on the main binding sites between ligands and antibody Fc fragments.The introduction of dextran grafting can increase the ligand coupling density of the resin and increase its adsorption capacity and binding ability to antibody,which is superior to the non-grafted tetrapeptide chromatography resin.Then the dynamic adsorption performance of dextran-grafted Ac-YFRH-XL resin on hIgG and BSA was investigated.The results showed that the dynamic binding capacity(Q10%)of the resin reached to 36.8mg/mL at pH 9.0,about 2.5 times that of the non-grafted Ac-YFRH-4FF resin of 14.9 mg/mL,and BSA had no adsorption at this point.The Ac-YFRH-XL medium had good binding ability to antibodies under low salt conditions(NaC;concentration?0.2 M),alnost no adsorption under high salt conditions,wherer salt tolerance is weaker than non-grafted chromatography resin.In addition the binding capacity of the dextran-grafted Ac-YFRH-XL resin to antibody increased with a certain ligand density above the maximum eoaling density of the non-grafted resin showing aProtential application prospect in large-scale separation.Finally,the separation performance of dextran grafted Ac-YFRH-XL resin on different feedstocks was investigated.The results showed tirnt the purity and yield of WgG were 98.7%and 89%at pH 9.0 by separating WgG from the protein nixture(WgG:BSAF=1:4)systexm,respectively.The monoclonal antibody was isolated from the CHO culture supernatant at pH 9.0 to obtain the monoclonal antibody,and the purity and yield were 93.0%and 84.7%,respectively.Dextran-grafted Ac-YFRH-XL resin had comparable separation efficiency to the non-grafted Ac-YFRH-4FF resin but the former could achieve higher binding capacity,indicating its potential for separation in larger scale systems.