Study On The Antibody Purification Of High Capacity Temperature-responsive Affinity Chromatography

Antibodies play an important role in cancer treatment and immune diagnosis,and antibody drugs have a broad market as a new generation of anti-tumor drugs.Nowadays,Protein A affinity chromatography is the most commonly used chromatographic technology platform for antibody purification,but it has the problems of high ligand cost,low adsorption capacity and harsh elution conditions,which to some extent restricts the development of antibody industry.Recently,polyamide-amine（PAMAM）dendritic macromolecules have been used more and more in the field of biomedicine and immunosorbent.It has been proved that using dendritic macromolecules as spacer arms is an effective ways to increase the ligand density and adsorption capacity.Temperature-responsive chromatography was developed base on themo-responsive polymer,can adjust the hydrophilic or hydrophobic property of the stationary phase surface in response to the temperature change to achieve separated and purified effectively of the target material under aqueous mobile phase.Base on this,this study combines the characteristics of dendritic polymer and temperature-responsive chromatography,a high capacity temperature-responsive affinity chromatography（TRAC）material was prepared.TRAC can not only used to separate the antibody by performing adsorption at 40℃and elution at 5℃in aqueous mobile phase at p H 8.0,but also increasing adsorption capacity evidently and separation process is environment friendly and safety.In addition,this novel chromatography method can overcome the present problem of acid condition elution easily lead to protein inactivation or aggregation in antibody purification progress.The effect of temperature,Na Cl concentration and p H of TRAC stationary phases on the antibody chromatographic separation and adsorption were investigated by using BSA andγ-globulin as model proteins,and the mechanism of antibody separation by TRAC was discussed.Meanwhile,the separation of immunoglobulin from real samples was investigated,and were obtained satisfactory results.The thesis is divided into the following three parts:1.Literature Review This chapter introduced the structure,function and the industrial production of antibody briefly,the comments focused on the chromatographic method for antibody purification at present.The advantages and applications of dendritic polymers,stimuli-responsive polymers and thermo-responsive chromatography in immunoabsorbent and protein purification were introduced.2.Preparation and characterization of high capacity temperature-responsive affinity chromatography stationary phaseThe high capacity temperature-responsive affinity chromatography stationary phase（Si O₂-G2.0@PNIPAM-MEP）was successfully prepared using silica gel as substrate,4-mercaptoethyl pyridine（MEP）as the ligand and temperature-responsive dendrimer polyami-doamine poly（N-isoprpylacrylamide）（PAMAM-PNIPAM）as the spacer arms.Chromatographic characterization was carry on TRAC stationary phase using BSA andγ-globulin as model proteins in aqueous mobile phase,by change temperature from 40℃to5℃to achieve one-step purification of mixed proteins,and the purity and mass recovery of Ig G reached 99%and 90%,respectively.As a results,the effect of temperature,Na Cl concentration and p H of TRAC stationary phase on the antibody chromatographic separation were investigated.The results indicated that temperature and elution p H affect protein separation effect significantly.As temperature and p H decrease,chromatographic peaks overlap and resolution becomes worse.With the increase of Na Cl concentration,the retention time of BSA increased,but the same treatment has little influence onγ-globulin,which reflected that the ligand was salt-independent.3.Adsorption properties and application of high capacity temperature-responsive affinity chromatography stationary phaseIn this work,the influence of Na Cl addition,p H and temperature change on the adsorption behavior ofγ-globulin in the TRAC stationary phase,as well as the dynamic binding,displacement behaviors,selectivity and reusability were investigated.Meanwhile,the saturation adsorption capacity Q_m and dissociation constant K_d of TRAC stationary phase under different conditions were calculated,optimized the adsorption conditions and discussed the adsorption mechanism.The results indicated that the affinity and hydrophobic interaction between antibody protein and ligand were enhanced with the temperature and p H value increasing,while the salt concentration effect was unobvious.In addition,the saturated adsorption capacity under optimized conditions could reach 140.8 mg/g finished in near two hours and the reusability of the TRAC stationary phase could reach to over 5 times.The TRAC stationary phase was employed for the separation of Ig G and Ig Y from human serum and egg yolk,and both the purity were found to above 98%.It means antibodies biological activity was preserve greatly and exhibit considerable potential for antibody separation in antibody drugs production.