Effects Of Surface Ligand And Counterion On Protein Adsorption Onto Poly (Ethylenimine)-grafted Sepharose

Previous studies showed that poly(ethyleneimine)(PEI)-grafted Sepharose gel with the ionic capacity(IC)higher than critical ionic capacity(cIC,600 mmol/L),exhibited high adsorption capacity and fast uptake rate,because the grafted PEI chains at IC>cIC could provide three-dimensional binding volume and"chain delivery"effect.Herein,the effects of surface ligand and counterion on the protein adsorption behavior on PEI-grafted resins were studied.Details of this work are summarized below.Firstly,Sepharose FF was modified with diethtyllamioethyl(DEAE)and/or PEI,and three types of ion exchangers were prepared,namely FF-D(surface-ligand resin),FF-PEI(grafting-ligand resin),and FF-D-PEI(mixed-ligand resin),to explore the roles of surface ligand and grafting ligand on protein adsorption in FF-D-PEI resins.It was found that the effect of surface ligand on protein adsorption performance of FF-D-PEI resins varies with grafting ligand density.When the grafting ligand density was moderate,namely in the range of 400<IC?600mmol/L,the surface ligands could significantly enhance the uptake rate of protein.When the grafting ligand density was beyond the region,namely,too high or too low,there was little promoting effect on the uptake kinetics.The results indicate that surface ligand together with grafting ligand influenced protein transport in the mixed-ligand resins.This might be attributed that the grafting states of PEI chains as well as the distances between the adjacent PEI chains were different at different grafting ligand densities,making that the surface ligand between adjacent chains played different roles.When IC of the grafting ligand was close to cIC,the grafted PEI chains exhibited more extending state,and the distance between adjacent PEI chains was close.Then,the surface ligands could play a role of transfer stations between adjacent chains to significantly facilitate the uptake rate.Besides,previous studies also showed that the adsorption and elution performance of FF-PEI-R440,which was obtained by partial charge modification of the grafting-ligand resin FF-PEI-L680(IC>cIC),was greatly enhanced.So the influence of counterions on protein chromatographic behavior on FF-PEI-R440 was investigated systematically in this work.It was found that the counterions significantly affected the adsorption equibrium,uptake kinects,dynamic binding capacity of FF-PEI-R440,while there is little effect on elution behavior.Moreover,choosing Cl~-as the counterion in mobile phase was the most favorable for FF-PEI-R440 column chromatography.These results were different from FF-PEI-L680,indicated that the impact of counterions on protein binding and elution of grafted-PEI resin was related to not only the ligand chemistry of the ion exchange group,but also its charge density.The work will promote the development of adsorption theory and application technology of high efficient protein preparative chromatography.