Design And Protein Adsorption Mechanism Of Polymer-grafted Resins For Hydrophobic Charge-induction Chromatography

Hydrophobic charge-induction chromatography(HCIC)is a novel technique for antibody separation.HCIC ligands combine multimodal interactions,including hydrophobic interaction,electrostatic interaction,hydrogen bonding.HCIC has been used for antibody purification with the advantages of good selectivity,high salt-tolerant adsorption,mild elution condition and low cost.However,the binding force between HCIC ligands and antibody was relatively weak,so the dynamic binding capacity(DBC)of antibody on HCIC resin was relatively low at high operation velocity.The polymer-grafted ion-exchange chromatographic(IEC)resins had been developed with higher adsorption capacity,mass transfer rate and DBC than traditional non-grafted resins.Therefore,two kinds of polymer-grafted HCIC resins were designed for high-performance antibody separation in this research,and the effects of solid phase conditions(polymer-grafting,ligand density and grafting density)and liquid phase conditions(pH and additive salt)on protein adsorption and transport were analyzed.In addition,the microscopic mechanism was analyzed with inverse size exclusion chromatography(ISEC),isothermal titration calorimetry(ITC)and confocal laser scanning microscopy(CLSM),which would be useful for the design of novel HCIC resins.Main results are summarized as follows.Firstly,with dextran-grafted agarose gels as the media and 2-mercapto-methylimidazole(MMI)as the typical HCIC ligand,dextran-grafted MMI resins with different ligand densities were prepared by controlling ligand coupling conditions.The ligand density of dextran-grafted MMI resin was improved about 50%compared to that of non-grafted MMI resin,reaching 200?mol/g.The adsorption isotherm,batch uptake kinetics and column breakthrough of dextran-grafted MMI resins were investigated using human immunoglobulin G(hIgG)as the model protein.The results showed the adsorption capacity,binding affinity,adsorption rate and DBC of hIgG on dextran-grafted MMI resins increased with the increase of ligand density,and dextran-grafted MMI resin with high ligand density had higher adsorption capacity,adsorption rate and DBC than non-grafted HCIC resins.Adsorption capacity of hIgG on dextran-grafted MMI resin with high ligand density kept at 107.5?110.0 mg/g at pH 7.0?8.9 and was less affected by pH.DBC of hIgG reached 38.3 mg/g at linear velocity 100 cm/h.Adsorption capacity of hIgG on dextran-grafted MMI resin with high ligand density decreased more significantly at weakly acidic pH conditions,which was helpful for protein desorption.Besides,adsorption capacity of hIgG on dextran-grafted MMI resin with high ligand density was less affected by salt concentration,but its adsorption rate was more significantly affected by salt concentration.Secondly,novel poly(glycidyl methacrylate,GMA)-grafted MMI resins with different grafting densities and ligand densities were prepared by surface-initiated activator generated by electron transfer(AGET)atom transfer radical polymerization(ATRP).The results showed adsorption capacity and adsorption rate of hIgG reached the maximum on the poly(GMA)-grafted MMI resins with high grafting density and intermediate ligand density.Compared to dextran-grafted MMI resins and commercialized HCIC resin MEP HyperCel,the optimized poly(GMA)-grafted MMI resins had higher DBC,which was less affected by the operation velocity and reached 34.6 mg/g at linear velocity 300 cm/h.Besides,the optimized poly(GMA)-grafted MMI resin had good pH-dependence and salt-tolerance adsorption properties.The results demonstrated good selectivity and re-usability for antibody separation from protein mixture(hIgG/HSA=l:4)and CHO cell culture supernatant.The antibody purity and recovery kept at 98.5?99.8%and 90.2?96.9%during the use of 50 cycles,respectively.Finally,ISEC was used to investigate the effects of pore structure of polymer-grafted MMI resins;ITC was used to study the adsorption thermodynamic of different proteins(hIgG and BSA)on MMI resins;CLSM was used to observe the adsorption,transport and elution process of protein in single resin particle for the real-time.ISEC results indicated that dextran-grafted and poly(GMA)-grafted chain would form the three-dimensional space distribution on the surface of pores under no salt condition,which helped to improve the protein adsorption capacity.The grafted chains would collapse with the increased salt concentration,and dextran chains collapsed most severely,and thus decreasing chain-delivery effect and protein transport rate.ITC results indicated that AH and ?S values increased with the increasing ligand density and salt concentration for dextran-grafted MMI resins,which indicated that the hydrophobic interaction between dextran-grafted MMI resins and hIgG would improve significantly with the increasing ligand density and salt concentration.When pH decreased to 5.0,AH and AS values decreased dramatically for all MMI resins tested,which indicated that the electrostatic repulsion dominated the charge-induced desorption of hIgG on MMI resins.For BSA,?H and AS values were negative for all MMI resins and adsorption capacity decreased significantly with the increased salt concentration,which indicated that the electrostatic attraction dominated the adsorption of BSA on MMI resins.CLSM results showed that mass transport of hIgG and BSA in all MMI resins was dominated by homogeneous diffusion-controlled mechanism.The fluorescent intensity was higher for dextran-grafted MMI resin with high ligand density and poly(GMA)-grafted MMI resin with intermediate ligand density,and BSA reached the core of particle more quickly than hIgG.The fluorescent intensity decreased most significantly for poly(GMA)-grafted MMI resin with intermediate ligand density at pH 4.0.These results were consistent with the macroscopic adsorption,transport and separation performance.In this thesis,two kinds of novel polymer-grafted HCIC resins,dextran-grafted and poly(GMA)-grafted MMI resins,were prepared to improve the protein adsorption capacity of HCIC resins.The effects of polymer-grafting,ligand density,grafting density,pH,additive salt and protein characteristic on protein adsorption and transport performance of polymer-grafted HCIC resins were discussed.ISEC,ITC and CLSM were used to reveal the protein adsorption and transport mechanism from the microscopic scale.The results showed that the grafting polymer in the pore could improve the adsorption capacity,transport rate and dynamic binding capacity of antibody onto the HCIC resins,which indicated good application potential of polymer-grafted HCIC resins for antibody purification.