Purification Of Recombinant Human Serum Albumin With Mixed-mode Chromatography And Expanded-bed Adsorption

Human serum albumin(HSA)is one of the most important proteins in human plasma.Plasma-derived HSA(pHSA)is used widely in many fields but often in ashortage of supply in China due to the limited blood resources.Recombinant HSA(rHSA)could avoid the risk of virus infection and resolve the problem of blood shortage.However,high purity of rHSA for clinical practice leads to a great challenge of rHSA purification.Cation ion-exchange chromatography is used normally for rHSA capture and some pretreatments of fermentation broth such as dilution and pH adjustment are needed to ensure high adsorption capacity,which certainly results in low separation efficiency.Therefore,alternative separation methods with high selectivity and salt-tolerant adsorption are expected for rHSA separation.Currently mixed mode chromatography(MMC)and expanded bed adsorption(EBA)are two innovative bioseparation technologies.MMC has the advantages of high adsorption capacity,good selectivity,salt tolerance and facile elution.EBA can capture target proteins directly from the crude feedstock containing bio-particles.In this thesis,based on the ligands screening new MMC resins would be prepared,and rHSA separation with MMC andEBA would be investigated.Combining the advantages of MMC for effective protein separation and EBA for high production,new separation technology of MMC-EBA would be developed and used for rHSA capture from Pichia pastoris culture broth.The main results are summarized as follows:(1)MMC ligands screening based on tryptophan analogues.On the basis of tryptophan binding specifically with HSA,some tryptophan analogues were tested as functional ligands and 10 resins were prepared for MMC.The adsorption behaviors with series of MMC resins were investigated.It was found that pH had a great influence on HSA adsorption and the highest adsorption capacity was around the isoelectric point(pI)of HSA for all resins tested.TA-B resin with tryptamine as ligand showed the highest saturated adsorption capacity Qm(143.54 mg/ml).Both of Qm and association constant(Ka)decreased obviously at acidic condition or salt addition.However,TA-B resin could remain a relative high adsorption capacity(95 mg/ml)under high salt condition.The results indicated that TA-B with tryptamine ligand has a strong affinity,good salt tolerance and high capacity in wide-range pH for HSA adsorption.(2)Preparation and adsorption property of MMC resin with tryptamine ligand.The preparation process of MMC resin was investigated with tryptamine as the ligand and three kinds of agarose gels as the matrix.After the optimization of gel activation and ligand coupling,series of MMC resins with different ligand density were obtained.It was found that ligand density influenced HSA adsorption and high adsorption capacity was obtained for proper matrix and medium ligand density.Comparing with some commercial MMC resins,TA-B-6FF showed higher adsorption capacity of HSA.HSA adsorption rate and effective pore diffusivity were highest at pH 5.0 and diffusion rate decreased with salt addition.Protein breakthrough results showed that dynamic binding ability of TA-B-6FF was strong and flow velocity had less influence on dynamic binding capacity(DBC)of HSA.DBC reached 54.5 mg/ml at the flow velocity of 700 cm/h,which would be suitable for high velocity operation.(3)Separating rHSA with mixed mode chromatography.TA-B-6F was used to separate rHSA from Pichia pastoris culture broth.Similar to pHSA,the best adsorption performance of rHSA was also found at pH 5 and Qm reached 54.60 mg/ml.In addition,Qm varied slightly in the pH range tested(6.0?8.0)and the adsorption capacity dropped significantly at pH 4.0.Due to slight effect on rHSA adsorption with dilution,Pichia pastoris culture broth could be loaded directly without dilution and pH adjustment.DBCs of rHSA decreased with the increase of flow velocity.At the flow velocity of 100?300 cm/h,Q10%was in the range of 19.7?30.3 mg/ml resin.The appropriate loading amount was 2.0?3.5 ml yeast broth/ml resin.The separation process was optimized and two-step elution method was proposed.Recovery and purity of rHSA monomer were 98.5%and 87.8%with the purification factor of 11.The removal of pigment and host cell protein reached about 90%.Moreover,the separation process was stable during 20 cycles of chromatographic separation.(4)rHSA separation by expanded bed adsorption.Two MMC resins for EBA(TA-S and TA-T)with tryptamine as functional ligand were prepared with quartz-densified agarose beads and tungsten carbide/agarose composite beads as matrix,respectively.The best adsorption of two resins were found at pH 5.0.Qm of TA-S and TA-T were 164.18 and 72.06 mg/ml,respectively.Moreover,TA-S and TA-T both showed a relative good salt tolerance.For packed bed adsorption,flow velocity had an obvious influence on HSA adsorption.With expanded bed adsorption mode,DBC decreased with the increasing expansion rate.The optimized expansion factor for EBA was 1.8 and the DBCs for TA-S and TA-T were 27.54 and 18.04 mg/ml settled resin,respectively.The Pichia pastoris culture broth could be loaded directly without dilution after pH adjustment to 5.0.TA-S resin showed better separation performance with two-step elution method.The recovery and purity of rHSA monomer were 91.6%and 83.3%.(5)Analysis of the interactions between tryptamine ligand and HSA.The molecular simulation methods were used.Four potential binding sites of tryptamine ligand on the surface of HSA molecule were identified by molecular docking and one site is located on the indole-binding site of HSA.The results of molecular dynamics simulation showed futher that the binding of tryptamine ligand was stable.Around the binding sites,most of residues were hydrophobic,but some negative or polar residues could contribute higher electrostatic interaction energy.It indicated that electrostatic interactions might dominate HSA adsorption at pH 5.0,while hydrogen bond and hydrophobic interactions played the assistant roles.Isothermal Titration Calorimetryanalysis was also used and the results showed that tryptamine-based resin adsorb HSAmainly through the electrostatic interaction in the pH range of 4.0?7.0 and the hydrophobic interaction might be the main driving force for HSA binding at pH 8.0.In addition,the tryptamine-HSA interaction shifted from electrostatic interaction to hydrophobic interaction with the increasing NaCl concentration.This thesis aimed to rHSA separation and focused on the two innovative bioseparation technologies,MMC and EBA.Based on ligand screening,resin preparation and adsorption evaluation,rHSA was separated efficiently from yeast culture broth after process optimization.The results demonstrate that new MMC-EBA technology has a great potential for rHSA capture with high process capacity and efficiency.