Adsorption Selectivity And Separation Of Antibodies With Mixed-mode Resins

As the most important biopharmaceuticals,antibodies are in great demand for the medical and research uses.Traditional platform of antibody separation based on Protein A affinity chromatography has some limitations.Mixed mode chromatography(MMC)is a new technology for protein separation,which has showed a great potential in antibody purification.However,the understanding on the adsorption selectivity of MMC is still limited.Therefore,in this thesis,four resins,including commercial MEP HyperCel with ligand of 4-mercatoethyl-pyridine(MEP)and home-made resins with ligands of 2-mercapto-l-methylimidazole(MMI),5-aminobenzimidazole(ABI)and tryptophan-5-aminobenzimidazole(W-ABI),were tested as the typical MMC resins.The adsorption behaviors of varying antibodies from different sources were investigated.The separation performances from different feedstock were tested.Moreover,the isothermal calorimetry titration(ITC)and bioinformatics methods were used to study the adsorption mechanism and molecular interactions between antibody and MMC ligands.Firstly,the adsorption properties of IgG from different species and human IgG subclasses with four MMC resins and Protein A resins were studied.It was found that MMC resins could bind the IgGs that Protein A can bind strongly or weakly.The adsorption showed strong pH-dependency.High adsorption ratio was found at neutral pH,and the adsorption decreased significantly at acid pH.There were some differences for different IgG species and subclasses.At the pH range of 3.0?4.0,the adsorption ratios of rat IgG with MEP HyperCel,MMI-4FF and ABI-4FF were 10?30%higher than those of mouse and human IgG.The adsorption ratios of rabbit,cow,goat,donkey IgG with MMI-4FF were less than those of MEP HyperCel and ABI-4FF.MEP HyperCel,MMI-4FF and ABI-4FF adsorbed IgG3 much less,and adsorbed IgG2 15?20%higher than IgGl.Secondly,the adsorption behaviors of different avian IgYs(chicken,duck,turkey,ostrich)with four MMC resins were evaluated.The results showed that Protein A couldn't bind IgY,while MMC resins could adsorb IgY well.The adsorption of MMC resins was affected obviously by the pH.High adsorption ratio was found at pH 6.0-8.0 for MEP HyperCel,MMI-4FF and ABI-4FF and low adsorption ratio under acidic conditions.W-ABI-4FF resin was favor to more acidic conditions,high adsorption ratio at pH 3.0-6.0 and low adsorption ratio at pH 8.0-8.9.Then ABI-4FF resin was used to separate IgY from immunized chicken serum.After the optimization of loading and elution pH and injection volume,an efficient process was developed with IgY purity of 95%and the recovery of 90%.The biological activity of IgY purified was fully maintained.Thirdly,the adsorption properties of human IgM and IgA on four mixed-mode resins were investigated,which could be separated by Protein A affinity chromtography.The typical pH-dependent adsorption was found for four resins tested,and the trends were similar to IgG.High capacity was found at the pH range of 5.0?8.0,and the adsorption decreased obviously under acidic conditions.Compared with IgG,the adsorption of IgM and IgA showed a relatively broader pH range.The adsorption selectivity index was proposed to evaluate the separation efficiency of IgM,IgA and IgG.High selectivity of IgM/IgA and IgM/IgG was found by ABI-4FF and MMI-4FF at pH 4.0-5.0,which could be used to separate IgM,IgA and IgG by controlling the loading and elution pH values.Two application cases of IgM separation were investigated.The first one was the capture of monoclonal IgM from cell culture supernatant with ABI-4FF,and high purity(-99%)and good recovery(80.8%)were obtained.The second case was the separation of IgM directly from human serum with the combined two-step chromatography with ABI-4FF and MMI-4FF.After the optimization of separation conditions,IgM purity reached 65.2%with a purification factor of 28.3.Moreover,IgM separated could maintain the antibody activity after the processes.Fourthly,the binding selectivity of human hIgG,Fc and Fab fragments were evaluated with four mixed-mode resins.The obvious pH-dependent adsorption behaviors were also found.High separation specificity of hIgG/Fc was found at pH 8.9 for MEP HyperCel resin,and for W-ABI-4FF resin at pH 8.0 and 8.9.The separation of hIgG and Fc(1:1)was optimized with mixed-mode resins,and the best separation performance was obtained with W-ABI-4FF resin.Loading at pH 8.0 resulted in the flow through of Fc with purity of 90.4%and recovery of 98.8%,while elution at pH 3.6 provided hIgG with purity of 99.7%and recovery of 86.5%.In addition,isothermal titration calorimetry was used to evaluate ligand-protein interactions.It was found that the adsorption was dominated by hydrophobic interactions.The binding sites would be changed with the pH changing.The binding site might be at Fab or Fc fragment at neutral pH,while would be at Fab fragment at alkaline condition.Finally,in order to study the molecular mechanism of ligand-protein interactions,quantitative structure-activity relationship(QSAR)analysis was constructed with different algorithms.Human IgG1,IgG4,IgG1-Fc and IgG1-Fab were chosen as four model proteins.It was found that the QSAR models with support vector machine(SVM)algorithm showed high robustness,good fiting and predictable ability to describe the adsorption of MEP HyperCel,ABI-4FF and MMI-4FF resins under different pH values.The key descriptors for three resins were screened,which were related to the electrostatic potential and hydrophobility of protein.All key descriptors of three resins were combined together to obtain a new training set,and the general QSAR model was established successfully,which showed a good prediction of adsorption for MEP HyperCel,ABI-4FF and MMI-4FF resins.The general key descriptors selected were 2 ligand descriptors(ligand density and molar refractivity)and 5 protein descriptors(electrostatic potential and hydrophobic area).The results indicated that the charge and hydrophobic properties of proteins and ligands were essential for the adsorption of MMC resins.