Protein cation exchangers derived by charge reversal from poly(ethylenimine)-Sepharose FF: Comparisons between two derivatization routes

Abstract

It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600 mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700 mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394 mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589 mmol/L) than PEI-L700-CRn (463 mmol/L) in which De/D0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192 mg/mL) is 33% higher than that of PEI-L700-CR680 (144 mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose.