Protein adsorption to poly(ethylenimine)-modified Sepharose FF: VI. Partial charge neutralization drastically increases uptake rate

Abstract

The adsorption and elution behaviors of bovine serum albumin (BSA) on poly(ethylenimine) (PEI)-grafted Sepharose FF resins were recently studied and a critical ionic capacity (cIC; 600mmol/L) was found, above which the uptake rate increased drastically due to the occurrence of significant “chain delivery” effect. Moreover, above the cIC value, higher salt concentrations were required for protein elution due to the high charge density of the resins. In this work, we have reduced the charge density on the PEI chains of a PEI-grafted resin by neutralization of the amine groups with sodium acetate. PEI-modified resin with IC of 740mmol/L (FF-PEI-L740, IC>cIC) was chosen as the starting material, and three resins with residual IC values of 660, 560 and 440mmol/L (FF-PEI-R440) were obtained. The adsorption and chromatographic behaviors of these resins for BSA were investigated. It was found that, with IC decreasing from 740 to 440mmol/L, the adsorption capacity kept almost unchanged; the effective protein diffusivity (De) also showed negligible variations as IC decreased from 740 to 560mmol/L (De/D0=0.38±0.04). However, it was interesting to observe a three-fold increase of the De value for FF-PEI-R440 (De/D0=1.23±0.08). It is considered that the occurrence of the drastic uptake rate increase in FF-PEI-R440 was attributed to the decreased available binding sites for protein molecule, which led to the decrease of binding strength, thus facilitated the happenings of “chain delivery” effect of bound proteins. Besides, a study on the effect of ionic strength clarified that the lower the IC value, the higher the sensitivity of protein binding to salt concentration due to the easily screened electrostatic interactions at low surface charge densities. The ionic strength at the elution peak also decreased with decreasing IC in accordance with the salt sensitivity order. Column breakthrough studies demonstrated that the dynamic adsorption capacity of FF-PEI-R440 was much higher than the other three resins at flow rates higher than 30cm/h because of its high uptake rate. The findings in this work provided new insights into the effects of the interactions between proteins and grafted polymers on adsorption equilibria and uptake kinetics, which would help the selection and design of suitable media for high-performance protein chromatography.