Performance Of Polymer-grafted Charged Media For Protein Adsorption And Facilitated Refolding

Polymer grafted resins for ion exchange chromatography (IEC) have been foundto possess high protein adsorption capacity (Q) as well as very high uptake rate (D),as compared to the traditional IEC resins with ion-exchange groups directly on thematrix surface. However, the mechanism origins of their advantages are not clearlyknown. Herein, we focused on the performance and application of the polymer graftedion exchangers, and the details of this work are summarized as follows.Firstly, the roles of the grafted dextran layer on the adsorption of γ globulin toIEC resins and mixed-mode chromatography (MMC) resins were investigated. It isshown that, in IEC, both the Q and D values increased with increasing dextran density.It is considered that the binding volume provided by the charged dextran layerbenefitted protein adsorption and the mechanisms (i.e. electrostatic coupling ofdiffusion fluxes) existed in the dextran layer facilitated mass transfer. However, inMMC, the Q value decreased with increasing dextran density, and the D value wasindependent of the dextran density. It is because that the hydrophobic interactionbetween the dextran chains caused the collapse of the dextran layer, which contributedlittle to mass transfer. Additionally, the collapsed dextran layer shield parts of theMMC ligands, giving rise to fewer available binding sites for protein.Considering the ionic groups in dextran-grafted resins existed in both thegrafting layer and on the matrix surface, poly(ethylenimine)(PEI) modifiedSepharose FF of10different ionic capacities (ICs,100–1220mmol/L) were preparedto investigate the role of the grafting layer on protein adsorption independently,excluding the influence of ligand distribution. It is found that there was a critical IC(cIC) or PEI density, above which both the Q and D values increased sharply. AtIC>cIC, the PEI chains were bonded to the gel surface at minimum numbers ofcoupling points and the polymers became extended to the pore space with greatflexibility. In addition, adjacent PEI chains became close enough to each other.Consequently, the extended three-dimensional PEI-layer provided greater accessibilityfor protein binding and the―chain delivery‖effect among the adjacent PEI chains bythe swings of the flexible chains facilitated transport of adsorbed protein molecules.The Q values of the PEI-Sepharose FF resins were less sensitive to salt concentrationthan both the commercial non-grafting resins and dextran-grafted ion exchangersreported in literature. It could be interpreted by the increase of pore accessibility with increasing IS. The D values of the PEI-Sepharose resins increased first and thendecreased with increasing IS. It was attributed to the dependencies of the―chaindelivery‖effect on protein capacity and binding strength, both of which are related toIS. Additionally, the PEI-Sepharose resins of ICs>cIC exhibited drastic changes of Dvalues with increasing IS, while those of ICs<cIC exhibited much mild changes,further proving that the―chain delivery‖effect did contribute significantly to theenhanced mass transfer at IC>cIC.In order to expand the application of PEI grafted resins, they were used in thefacilitated refolding of like-charged lysozyme. The effects of like-charged solid phaseproperties on refolding were investigated systematically. The results showed thatcharged group density of charged particles played the most important part in theenhancing effect. The higher the charged group density, the less the charged resinswere required to achieve a desired facilitating effect. As compared to charged groupdensity, the effects of particle size and pore size were secondary. At the same chargedensity, the refolding yield was independent of ligand chemistry. Besides, we haveobserved that positively charged proteins (lysozyme and cytochrome C) are adsorbedon Sepharose gel and Sepharose-based anion-exchangers, in a capacity of10–60μg/mL. Because the trace adsorption is usually ignored in IEC practices, it may tosome extent degrade the product purity and/or recovery in large scale processing. Theresults show that the trace adsorption was caused by the residual negatively chargedgroups, most probably sulfate groups, in the agarose material. The undesirable traceadsorption was reduced by increasing NaCl concentration and/or coupling cationgroups (i.e. PEI).This work will promote the devolopment of protein adsorption theories andbenefit the potential use of polymer-grated IEC resins in bioseparation processes.