High Capacity Temperature-responsive Affinity Chromatography Designed For Antibody Separation

Antibody separation and purification methodologies with high separation efficiency,large adsorption capacity and high biological activity are challenging.The development of cost effective,efficient,and mild elution condition,high adsorption capacity,green and environment friendly methods for the separation and purification of antibodies is progressively becoming fundamental in the pharmaceutical industry.Owing to the rapid increase in the demand for monoclonal antibody product as well as the considerable challenges in antibody drug production,the development of new antibody drug production processes is significantly important for increasing production and reducing the production costs of monoclonal antibodies for bioengineering downstream.In the current work,a novel high capacity temperature-responsive affinity chromatography(TRAC)methodology was developed for antibody separation.The TRAC stationary phases based on the temperature-sensitive dendrimer polyamidoamine-poly(N-isopropylacrylamide)PAMAM-PNIPAAm as spacer arms and 4-mercapto ethyl pyridine MEP as ligand.The resulting TRAC stationary phases were employed to separate the mixture of BSA and γ-globulins by only adjusting the temperature of the aqueous mobile phase.The baseline separation of BSA and γ-globulins could be achieved successfully using TRAC stationary phases except for TRAC3.0.The effect of different temperatures on the separation performance of proteins was investigated in detail,and the adsorption capacity at different temperatures was compared.The results demonstrated that many more proteins were adsorbed at 40 °C than at 20 and 30 °C on the TRAC2.0 stationary phase.Therefore,40 °C was chosen as the adsorption temperature in the current study.Furthermore,the effect of different ligands and spacer arms on the separation performance and adsorption capacity of proteins were also investigated.The results demonstrated that the TRAC2.0 exhibit the highest binding and best separation due to more number of ligands with 2nd generation of PAMAM dendrimer spacer arm.The circular dichroism spectrum analysis indicated that the γ-globulins separated by TRAC2.0 could keep their natural structure.This new technology was employed for the separation of Ig G and Ig Y from human serum and egg yolk,and both the mass recoveries were found to above 90% with 98% purity.Moreover,the mechanism of antibody(BSA,γ-globulins)separation by TRAC was investigated in detail.Compared with traditional protein A-based affinity chromatography,the antibodies can be separated completely using TRAC only by adjusting the temperature of aqueous mobile phase,which overcomes the drawbacks of low p H elution leading the denaturation and/or deactivation of antibody in mixed mode chromatography and traditional affinity chromatography.The adsorption capacity of TRAC can be increased considerably using the temperature-responsive dendrimers PAMAM-PNIPAAm as the spacer arms,and solving the problem of low adsorption capacity of conventional affinity chromatography stationary phase.The novel methodology can overcome the previous negative effects of deactivation of antibody by traditional harsh elution with low p H,low adsorption capacity,and preserve its biological activity,and exhibits a great potential for antibody separation in antibody drugs production.Based on the above-mentioned research background and ideas,the main research work includes the following aspects:1.Literature Review: This chapter gives comprehensive overview of the characteristics and application of analytical method in HPLC for antibody separation and purification.Moreover,both the advantages and disadvantages of each method were discussed thoroughly.This chapter also provides the key method to solve the problems of existing methods.2.Preparation and characterization of thermo-responsive affinity chromatography stationary phase with linear spacer arms,and its application for antibody separation: A novel temperature-responsive affinity chromatography(TRAC0)stationary phase for antibody separation based on MEP as the ligands,the temperature-responsive linear polymer PNIPAAm as spacer arms and silica gel as the matrix was prepared and characterized by elemental analysis,XPS,NMR,and FTIR.The results confirmed that we successfully synthesized tempera-ture-sensitive affinity chromatography materials.The novel temperature-responsive affinity TRAC0 stationary phase can separate antibody by only changing the temperature of the aqueous mobile phase,which effectively avoids the traditional harsh acidic conditions.The effect of different temperatures on the separation performance and adsorption capacity were investigated in detail using standard proteins.The results indicated that the Qmax of the γ-globulins on TRAC0 with the linear spacer arm was 78.4 mg/g,and the baseline separation of γ-globulins from BSA could be achieved using TRAC0 stationary phase.In addition,the mass recovery was higher than 90%,and the purity of γ-globulins was 99%.The novel methodology can overcome the previous negative effects of deactivation of antibody by traditional harsh elution with low p H,and preserve its biological activity,and exhibits a great potential for antibody separation in antibody drugs production.3.Preparation and Characterization of High Capacity Thermo-responsive Affinity Chromatography Stationary Phases with Different Generation of PAMAM Dendritic Spacer Arms: In this chapter,a novel high capacity thermo-responsive affinity chromatography stationary phases for antibody separation based on thermo-responsive dendritic polymer PAMAM-PNIPAAm as spacer arms,small functional molecule MEP as the ligands and silica gel as the matrix was prepared.First,the activated silica gel surface was aminated using a silane coupling agent,APES,then the dendritic polymer PAMAM was grafted via the Michael addition of MA and an amidation reaction of ethylenediamine onto the amino modified silica gel.Second,a linear temperature-responsive polymer,PNIPAAm-DDTTC,with a carboxyl group was prepared by reversible addition-fragmentation chain transfer polymerization(RAFT).Finally,a high capacity thermo-sensitive affinity chromatographic stationary phases were prepared by the amidation reaction of amine group of PAMAM with the carboxyl group of PNIPAAm-MEP.The prepared high capacity thermo-responsive stationary phases were characterized by elemental analysis,XPS,NMR,and FTIR.The results confirmed that we successfully prepared TRAC stationary phases by grafting PAMAM-PNIPAAm and the MEP ligand on the silica gel surface.The number of ligands was successfully increased by the incorporation of different generation of PAMAM dendrimer as spacer arms,and it will increase the adsorption capacity of thermo-responsive affinity chromatography materials.4.Antibody separation Via High Capacity Thermo-responsive Affinity Chromatography Stationary Phase with PAMAM Dendritic Spacer Arms: In this chapter,the prepared TRAC stationary phases with different spacer arms were packed into the chromatographic columns.The packed thermo-responsive stationary phases were applied for the separation of proteins from standard and real sample.The results showed that the baseline separation of BSA and γ-globulins can be achieved successfully using TRAC stationary phases except TRAC3.0.The SDS-PAGE analysis demonstrated that the TRAC stationary phase is able to capture BSA and γ-globulins from mixture.The CD spectrum analysis indicated that the γ-globulins separated by TRAC2.0 could keep their natural structure.The effect of different temperatures on the separation performance of proteins was investigated in detail,and the adsorption capacity at different temperatures was compared.The selectivity of thermo-responsive stationary phase TRAC was determined and the results indicated that the silica-PAMAM(G2.0)column have no interaction and incapable of binding proteins.Furthermore,the effect of different ligands and spacer arms on the separation performance and adsorption capacity of proteins were also investigated.This new technology was employed for the separation of Ig G and Ig Y from human serum and egg yolk,and both the mass recoveries were found to above 90% with 98% purity.The results demonstrated that the TRAC2.0 exhibit the highest binding and best separation due to more number of ligands with 2nd generation of PAMAM dendrimer spacer arm.Compared with traditional protein A-based affinity chromatography,the antibodies can be separated completely using TRAC only by adjusting the temperature of aqueous mobile phase,which overcomes the drawbacks of low p H elution leading the denaturation and/or deactivation of antibody in mixed mode chromatography and traditional affinity chromatography.The adsorption capacity of TRAC can be increased considerably using the temperature-responsive dendrimers PAMAM-PNIPAAm as the spacer arms,and solving the problem of low adsorption capacity of conventional affinity chromatography stationary phase.5.Mechanism of Antibody Separation by High Capacity Temperature-responsive Affinity Chromatography Stationary Phases: The main purpose of this chapter is to investigate the mechanism of antibody separation(BSA and γ-globulins)by the prepared TRAC stationary phases.Stoichiometric displacement model SDM-A was applied to investigate the mechanism of antibody(BSA and γ-globulins)separation on high capacity temperature-responsive affinity chromatography stationary phases(TRAC)by employing adsorption isotherms of γ-globulins at various temperatures.In addition,the mechanism of antibody separation was also demonstrated by performing separation of BSA and γ-globulins on temperature-responsive stationary phases,i.e.,silica-PNIPAAm and silica-PAMAM-PNIPAAm without the MEP ligand.Moreover,the retention of γ-globulins on the TRAC stationary phases was verified,when PNIPAAm was end capped by MEP as ligand and γ-globulins can be captured by the TRAC stationary phases.These disparate interactions of BSA and γ-globulins with the TRAC stationary phases led to their selective retention and separation on the TRAC stationary phases.