Preparation Of HCIC Affinity Ligand-Functionalized Graphene Based On "Thiol-ene" Click Chemistry For Selective Separation And Purification Of Antibody

Therapeutic antibody drugs are a very important class of drugs in the global pharmaceutical industry,and they own wide application potentials in the fields of disease diagnosis and treatment.The acquirement of antibody products generally requires several steps including cell culture,antibody expression,separation and purification,among which the separation and purification of antibodies has become a key step that restricts the large-scale production of antibodies.Hydrophobic charge induction chromatography（HCIC）is a new type of mixed-mode chromatography that can take into account both cost and selectivity.It is considered as an attractive alternative of traditional protein A chromatography for the separation and purification of antibodies.However,the antibody purification process has problems such as relatively cumbersome steps,time-consuming,and high cost,which might restrict production of antibodies.In contrast,solid phase extraction technology is quite simple and easy to be operation,good selectivity and high enrichment efficiency,resulting in the good application prospects in the field of biological separation.Therefore,in this paper,we combine solid-phase extraction technology and the principle of HCIC to design and synthesize a series of new types of solid-phase extraction adsorbents that exhibit high affinity for antibodies by using graphene oxide（GO）as the substrate and thiol-ene click chemistry as the functionalization method.We employ human immunoglobulin（Ig G）as the antibody model to establish new methods for antibody separation and purification.This thesis is divided into three parts:Chapter 1:This chapter briefly summarizes the structure,function and production of antibodies;commonly used methods for antibody separation and purification;the properties,synthesis,functionalization of graphene and its application in solid-phase extraction field;the principles,features of thiol–ene click chemistry,and its application in surface modification of materials.Chapter 2:In this chapter,3-（methacryloyloxy）propyltrimethoxysilane（MPS）was selected to modify the surface of graphene oxide（GO）for introduction of alkene groups onto the GO-MPS composites.The alkene groups serve as clickable sites to react with2-mercaptoimidazole（MI）,2-mercapto-1-methylimidazole（MMI）,and4-pyridineethanethiol hydrochloride（MEP）via thiol–ene click reaction,respectively.The obtained three HCIC affinity ligand functionalized graphene composites were named as GO-MPS-MI,GO-MPS-MMI and GO-MPS-MEP composites,respectively.The three composites were characterized by FT-IR and TGA analysis.The adsorption behaviors of Ig G on the three composites,as well as the influence of p H,salt concentration and contact time on the adsorption performances were carefully investigated.The results show that the three composites all exhibit the typical characteristics of hydrophobic charge-inducing ligands,i.e.,p H-dependent and salt-independent,and can reach adsorption equilibrium in a short time（20 min）.By comparing the adsorption selectivity of three composites to Ig G,it is found that GO-MPS-MEP containing MEP ligand displays better selectivity,indicating that MEP ligand is more suitable for the selective separation and purification of antibodies.However,due to the low bonding density of the affinity ligand on the surface of the composites,the non-specific adsorption cannot be effectively inhibited.Chapter 3:In this chapter,for overcoming the drawbacks of low affinity ligand bonding density and strong non-specific adsorption of substrate,octamercaptopropyl oligomeric silsesquioxane（POSS-SH₈）was selected to modify the surface of GO to obtain GO-POSS-SH composites which contains a large number of sulfhydryl groups.Then,4-vinylpyridine（VP）was covalently bonded to the surface of GO-POSS-SH through thiol–ene click reaction to obtain GO-POSS-S-VP composites with high-density MEP-like affinity ligand.The GO-POSS-S-VP composites was characterized by FT-IR,TGA,TEM and XPS.Based on the specific affinity between MEP-like ligand and Ig G,as well as the high bonding density of the affinity ligand,GO-POSS-S-VP shows high selective adsorption performance toward Ig G.At the same time,the non-specific adsorption of other proteins is effectively inhibited（less adsorption efficiency than 27%）.The adsorbed Ig G molecules can be effectively recovered with acetate buffer(0.05 mol L^-1,p H 3.5),giving rise to a desorption efficiency of ca.55%.The GO-POSS-S-VP was applied for the separation and purification of antibody from human serum samples.SDS-PAGE assay and MALDI-TOF-MS analysis results demonstrated that antibodies can be highly selectively isolated from complex sample matrices with high purity.