Protein Adsorption And Chromatography On Polyethylenimine-grafted Sepharose Gel

In the previous study on the ion exchange chromatography(IEC) of poly(ethylenimine)(PEI)-grafted Sepharose gel, a critical ionic capacity(c IC) was found for the uptake of bovine serum albumin(BSA), above which both protein capacity and uptake rate increased drastically. The improved protein capacity on PEI-grafted resins is attributed to the three-dimensional PEI-grafting layer, and the rapid protein transfer is facilitated by the ―chain delivery‖ effect, which is driven by the chemical potential toward the bead center as well as the interactions between neighboring flexible chains mediated by the bound proteins. Herein, we focused on the performance and application of the PEI-grafted Sepharose FF gels, and the details of this work are summarized as follows.Firstly, the breakthrough and linear gradient elution(LGE) behaviors of BSA were investigated on PEI-grafted IEC resins. It is shown that, in wide residence time and ionic strength(IS) ranges, the dynamic adsorption capacity(DBC) of PEI-grafted resin of IC>cIC could stay at higher values than the traditional non-grafted IEC resin. In addition, BSA was eluted as a single symmetrical peak from the PEI resin. This work has clearly shown that the PEI resin of IC>cIC exhibited high salt tolerance and could be used for high speed operation. In addition, protein could be eluted effectively by increasing salt concentration as the traditional IEC.To acquire more information on different protein adsorption behaviors on PEI-grafted IEC resins. The adsorption and uptake kinetics of BSA and γ-globulin, which were different in size and pI, were investigated on seven PEI-grafted Sepharose FF gels in the IC range of 270–1030 mmol/L. The results showed that the hopping of uptake rate of the larger protein(γ-globulin) was earlier than the smaller one(BSA), in other words, the cIC value for γ-globulin was smaller than that for BSA. It was considered that, bound protein of larger size and lower pI(less charges resulted in weaker binding strength) is easier to contact a neighboring chain and then to be transferred by the ―chain delivery‖ effect. The results indicate that both PEI layer and protein properties played important roles in protein adsorption to PEI-grafted IEC resins.To further deepen the understanding of PEI-grafted resins, the influence of counterions on the IEC performance of PEI-grafted resin of IC>cIC was investigated. The protein uptake rate on the PEI resin was much more sensitive to the counterions than the traditional non-grafted resins, DEAE Sepharose FF and Q Sepharose FF, and the counterion with higher preference with the PEI resin would result in higher protein uptake rate. It was considered that the ―chain delivery‖ effect was highly dependent on the interaction strength between the protein and the resin, which was related to the counterin preference.In order to expand the application of PEI-grafted resins, this work proposed to develop mixed-mode chromatographic(MMC) resins by modifying the grafted PEI chains with hydrophobic benzoyl groups, and then the protein adsorption and chromatographic behaviors were focused on the PEI-grafted MMC resins. It was found that resins with high PEI desities were not suitable for fabricating the MMC resins, because the ―CaSH‖ effect caused by the extension of the PEI chains under low pH value, resulted in the incomplete elutions on those MMC resins. However, the MMC resin with a low PEI density modified with high benzoyl density was favorable for protein binding and elution.This work will promote the development of protein adsorption theories on PEI-grafted gel and benefit the potential use of PEI-grafted resin in bioseparation processes.