High-throughput Screening For Human Serum Albumin And Antibody Separation With Mixed-mode Chromatography

Human serum albumin(HSA)and antibody are two important biotechnological drugs with huge market demand.The traditional HSA separation method based on ion-exchange chromatography has some disadvantages on poor salt-tolerance and low binding selectivity,etc.The conventional antibody separation process based on Protein A affinity chromatography has the limitations on high resin cost,difficulties of elution and regeneration,etc.Therefore,the development of novel separation methods is of great significance.Mixed-mode chromatography(MMC)showed great advantages on high adsorption capacity,good salt-tolerance,and excellent selectivity,which is suitable for target protein capture from complex feedstocks.High-throughput process development(HTPD)can quickly and efficiently screen resins and optimize the separation conditions to improve process development efficiency.Differential scanning fluorimetry(DSF)can investigate the affinity between low-molecular ligands and proteins,and improve the ligand screening efficiency.In this thesis,MMC was focused and recombinant HSA(rHSA)and human immunoglobulin G(hlgG)were used as the targets.HTPD and DSF were introduced to achieve high-throughput process development of protein separation,and to explore the new techniques on the application of HSA and antibody separation.Firstly,HTPD method based on microtiter filter plate was established.The operation conditions were optimized.The adsorption and elution performance of HSA on two ion-exchangers and four MMC resins were investigated,and the chromatographic separations were verified by lab-scaled column.The results showed that for two resins with anion-exchange groups(Q Sepharose FF and Capto adhere),pH 8.5 and pH 5.5 were suitable for HSA adsorption,and the elution conditions were pH 4.0,For four resins with cation-exchange groups(SP Sepharose FF,Capto MMC,MX-Trp-650M and Nuvia cPrime),the optimal loading conditions were pH 4.0,and the elution conditions were pH 7.0,pH 8.0 and pH 9.0,respectively.Under the optimized separation conditions based on high-throughput screening,the column chromatography performances were verified,and the resutls were consistent.All six resins had high DBC10%s and elution recoveries.Secondly,HTPD method was used to separate rHSA from Pichia pastoris fermentation broth,the adsorption selectivity and elution performance of two ion-exchangers and four MMC resins were investigated.It was found that four resins with cation-exchange groups had better adsorption capacity and selectivity than the resins with anion-exchange groups,especially for mixed-mode resins of Capto MMC and MX-Trp-650M.The elution optimization results showed that MX-Trp-650M resin had mild elution with high recovery and purity.Column chromatography results showed that MX-Trp-650M resin had high adsorption capacity,strong salt-tolerance,good selectivity,mild elution conditions,and high ability to remove host cell proteins and pigments.Under the optimal separation conditions,loading at pH 4.0 without dilution and elution at pH 8.0,MX-Trp-650M had the DBC10%of 49.8 mg/mL,rHSA recovery of 97.1%,the purity of 97.0%,as well as HCP of 581 ppm and A350/A280 of 0.12.The bed volume was scaled up to 20 mL,MX-Trp-650M still showed good performaces for rHSA capture from yeast broth,and the separation performance was consistent with that of small-scale chromatography.Thirdly,DSF was used to screen the suitable ligands for protein separation.It was found that SYPRO Orange dye could not be used for HSA,but could work with IgG.And the operation conditions were optimized.For human IgG and monoclonal antibody(mAb)as the target proteins,33 small molecules were selected as the potential MMC ligands,and the affinity of ligands to hIgG and mAb was investigated by DSF method.The results showed that for three ligands previously developed in our lab,ABI had the strongest affinity,followed by MEP and MMI was the weakest.With ?Tm of MMI,MEP and ABI as the low,general and high affinity indexes,respectively,the effects of pH and salt concentration were investigated for other 30 molecules.It was found that at pH range of 5.0-8.0,the melting temperature differences(?TM)were firstly increased and then decreased with the pH increase.With salt addition,?Tm remained basically constant,showing good salt-tolerant property.Based on the DSF lignad screening,it was found that six small molecules(PCA,6-ABT,BPA,BIA,IAAH and SFA)had higher affinity and showed great potential as the MMC ligands for antibody separation.Finally,six new MMC resins were prepared by coupling PCA,6-ABT,BPA,BIA,IAAH and SFA ligands on agarose matrix.The adsorption capacity of hIgG and pHSA was investigated with microtiter filter plate HTPD and compared with MEP and ABI resin.The results indicated that the BPA-B-6FF and SFA-B-6FF resin had high adsorption capacity of hIgG,high salt-tolerance and good selectivity.The dynamic adsorption results showed that ABI-B-6FF had high dynamic capacity and strong sal-tolerance at pH 7.4 and liner velocity of 102 cm/h,but the selectivity was general.BPA-B-6FF had high dynamic capacity and selectivity,but poor salt-tolerance,which could only used for hIgG capture at low conductivity.SFA-B-6FF had high dynamic capacity and selectivity,moderate salt-tolerance,which could capture antibodies at a wide range of conductivity.The results of chromatographic separation indicated that SFA-B-6FF could directly separate antibodies from human serum with the purity of 99.1%,the purity of hIgG was 87.5%.When adjusting serum dilution ratio to 4,the purity of antibodies and hIgG remained constant,but recovery increased to 83.8%.The results demonstrated that SFA-B-6FF resin had good selectivity for hIgG.wide elution pH range,moderate salt-tolerance,and the ability to specifically capture immunoglobulins from human serum.This thesis focused on new MMC methods for protein separation.The microtiter filter plate HTPD method was set up and used for resins and separation conditions screening for proteion separation.The DSF-based screening method was established for MMC ligand development,then new MMC resins were prepared.Novel separation process of rHSA and hIgG was obtained,and the efficient separation and purification from complex feedstock were achieved,which would be useful for further application of MMC.