Axial Distribution Behaviors In Expanded Bed And Separation Of Recombinant Human Serum Albumin

Expanded bed adsorption(EBA)is a novel bio-separation technology that can form a stable perfectly classified fluidized bed.It can capture the target protein directly from the biomass containing fermentation broths or homogenates.Mixed mode chromatography(MMC)is also new bio-separation technology with high adsorption selectivity and good salt tolerance,which is particularly suitable for the preliminary separation of proteins.In this thesis,the axial distribution characteristics in expanded bed were studied,and the mechanism of bed stability was discussed.Combining the advantages of EBA and MMC,a new resin was prepared and used for the separation of rHSA directly from yeast fermentation broths.(1)Axial distribution behaviors in expanded bed.With the quartz sand/agarose composite beads(Streamline quartz base)and the tungsten carbide/agarose composite beads(TC resin)as the model EBA resins,the in-bed sampling methods were used to measure the axial distribution characteristics of bead size,bead density,local bed voidage and liquid mixing in EBA.It is shown that the two resins have the similar rules of axial distribution.With the increase of bed height,the mean bead size and density decrease gradually,while the local bed voidage increases.At the same time,the number of theoretical plates(N)and increased and the height of equivalent theoretical plate(HETP)decreased gradually.In the zones near bed bottom,the effective axial dispersion coefficients(Dax_local)were obviously higher than those in the middle and top zones of bed,which meant that the bed stability improved with the bed height.It was found found that EBA operation has a suitable range of expansion factors as 1.8?2.4,smaller or larger expansion factors would hardly form the stable bed.Based on the comparison of two resins,TC resin has smaller bead size,higher bead density and wider distribution range of bead size and density,and could be used for higher operation velocity.(2)Preparation and adsorption property of MMC resins.With TC beads as matrix and tryptamine as the functional ligand,MMC resin of TC-TA was prepared,and the ligand density was 150?170 ?mol/ml resin.And the scale-up process was optimized.With human serum albumin(HSA)as the model protein,static adsorption,adsorption kinetics and dynamic binding capacity of TC-TA resin were investigated.It was found that the saturated adsorption capacity was up to 135 mg/ml with a certain salt tolerance.The adsorption rate and effective pore diflfusivity were highest at pH 5.0.The resin has high dynamic binding ability of 55.4 mg/ml,at the velocity of 100 cm/h,but the influence of the operation velocity on dynamic binding capacity(DBC)was obvious.TC-TA resin was used to capture rHSA directly from Pichia pastoris fermentation broth.For packed bed mode,the purity and recovery of rHSA monomer reached 98.2%and 73.4%.For EBA mode,rHSA could be separated directly from the yeast-containing fermentation broth without the pretreatments of centrifugation and dilution.The bed was quite stable during the whole separation process,and showed high separation and process efficiency.The results demonstrated that mixed-mode EBA process would be a promising new bio-separation technology to capture target protein directly from the complicated crude feedstock.