Development And Application Of Functional Ligands And Matrix Materials For Antibody Separation

Antibodies are the fastest-growing and dominant category of biomacromolecules in treatment of autoimmune diseases,tumors,cardiovascular and cerebrovascular diseases.Traditional antibody purification process is based on the three-step method using protein A chromatography as a platform technology.However,its high cost and low binding capacity limit large scale antibody production.Conventional chromatographic separation resin is composed of functional ligand and matrix.Functional ligand can provide high antibody selectivity.Matrix is the framework of separation materials,and its morphology,structure,specific surface area,pore size distribution and other properties will affect separation performance.In this thesis,the relationship between ligand structure and function was analyzed and the feasibility of non-porous separation materials in the application of antibody separation was explored.Firstly,the binding mode between antibody and the previously developed ligand FYE-ABI(Phenylalanine-Tyrosine-Glutamate-"5-aminobenzimidazole")was explored.Using molecular docking and molecular simulation techniques as auxiliary tools,the analysis of binding region showed that the binding site of antibody was dominated by positive potential,and regionalized hydrophilic distribution provided binding environment for ligands.The simulation results showed that the binding site on the antibody could be divided into three regions.Region 1 contained some polar residues and charged residues,which provided pi-pi stacking sites.Region 1 provided the most binding energy(-85.9 kJ/mol).Region 2 provided a strong hydrophobic and positive potential environment.Region 3 was composed of polar,non-polar,charged residues,providing a heterozygous environment and hydrogen bonding sites for the ligand.The functional groups of FYE-ABI ligand were analyzed,and the results showed that the binding of FYE-ABI and antibody was owing to synergistic effect of multiple functional groups.Among them,ABI structure served as the "head" of FYE-ABI and was anchored in the surface cavity.By optimizing the orientation of benzimidazole to narrow the distance between FYE-ABI and antibody,the fixation on antibody surface was realized.Glu structure(Glutamic acid)of tripeptide with a carboxyl group contributed 52%electrostatic energy.Tyr structure(Tyrosine)provided the van der Waals interaction,but the binding effect was limited by steric hindrance effect.Phe structure(Phenylalanine)provided strong hydrophobic,effective hydrogen bonding and pi-alkyl interaction.Meanwhile,under acidic conditions,ABI structure provided electrostatic repulsion with antibody through protonation,which increased the distance between FYE-ABI and antibody,resulting in the separation of FYE-ABI and antibody.The binding and elute mechanism of FYE-ABI ligand and antibody was elucidated,and functions of each functional groups were discussed,which provided a theoretical basis for design of new functional ligands.Based on molecular dynamics simulation results,FYE-ABI resin was prepared for mAb(monoclonal antibody)separation from CHO cell culture to investigate mAb separation performance and host cell protein removal ability.The results showed that FYE-ABI performed excellent selectivity and host cell protein removal ability at pH 3.5,pH 4.0 and pH 4.5.The purity of mAb was 99.1%,98.8%and 98.9%,respectively,and log reduction value was 1.54,1.41 and 1.49,respectively.FYE-ABI showed a good prospect of mAb purification.In addition,in order to verify the simulation results of ligand functional groups,FYE and ABI resins were prepared.The results showed that these two resins were both inferior to FYE-ABI resin in antibody selectivity and elution performance.Among them,the performance of FYE resin is consistent with that of CM Bestarose FF(a weak cation exchange resin),indicating that carboxyl group played a key role in FYE resin.ABI resin showed bad hIgG selectivity,but was able to achieve elution at pH 4.0.Model ligands,E-ABI1 and E-ABI2,were designed to further verify the effect of carboxyl group and ABI.It was found that E-ABI1 containing carboxyl groups showed better hIgG selectivity,while E-ABI2 without carboxyl groups showed no specific adsorption.And E-ABI1 showed great hIgG selectivity from protein mixture and human serum mixture,and hlgG purity was 99%and 96%,respectively.In addition to functional ligands,matrix is another essential component of the separation material.The agarose-porous matrix and non-porous silica matrices of different sizes were taken as the research objects,and these two matrices were analyzed and compared from the aspects of morphology,particle size and specific surface area.The results showed that the particle size range of agarose-porous matrix is wider(50165 μm),and the specific surface area of non-porous silica matrix can reach more than 200 m2/g.SiO2-COOH nonporous cation separation material was prepared.When pH<7.0,carboxyl groups attracted hIgG by electrostatic attraction,and the maximum adsorption capacity reached 89.8 mg/g.Moreover,magnetic nonporous cation separation material,Fe3O4@SiO2-COOH,was prepared and the adsorption performance for hIgG was measured.The adsorption equilibrium was reached within 5 min,and the adsorption capacity was up to 204.2 mg/g.The entire hIgG batch separation process took only 24 min with Fe3O4@SiO2-COOH,which was 66%more efficient than the agarose-porous resin under the corresponding conditions.The magnetic non-porous separation material showed the advantages of high binding capacity,high efficiency and low consumption.In this thesis,functional ligand and matrix were studied.On the one hand,the binding mode of ligands and antibody was explored through molecular dynamics simulation,and the structure-function relationship of functional groups was analyzed in combination with experimental results,providing a theoretical basis for the design of new ligands.On the other hand,the separation performance and efficiency of nonporous material were explored.Combined with magnetic separation technology,magnetic non-porous separation material was prepared,which showed high efficiency and low consumption.This study can provide reference for the development of new separation material and process.