Protein adsorption to poly(2-aminoethyl methacrylate)-grafted Sepharose gel: Effects of chain length and charge density.

Abstract Grafting functional polymer chains onto porous resins has been found to drastically increase both adsorption capacity and uptake rate in protein chromatography. In this work, 2-aminoethyl methacrylate (AEM) was used for grafting onto Sepharose FF gel, and six anion-exchangers of different polyAEM (pAEM) chain lengths (ionic capacities, ICs), FF-pAEM, were obtained for protein adsorption and chromatography. It was found that protein adsorption capacity (qm) increased with increasing pAEM chain length, but the uptake rate, represented by the ratio of effective pore diffusivity to the free solution diffusivity (De/D0), showed an up-down trend, reaching a peak value (De/D0=0.55) at an IC of 313 mmol/L. Partial charge neutralization of the AEM-grafted resin of the highest IC (513 mmol/L) by reaction with sodium acetate produced three charge-reduced resins, FF-pAEM513-R. With reducing the charge density, the adsorption capacity kept unchanged and then decreased, but the uptake rate monotonically increased, reaching a maximum (about 2-fold increase) at a residual IC of 263 mmol/L. It is notable that, at the same IC, the charge-reduced resin (FF-pAEM513-R) presented similar or even higher values of qm and De/D0 than its FF-pAEM counterpart. Particularly, at the same IC of 263 mmol/L, a ~50% enhancement of De/D0 was observed. Both adsorption capacity and uptake rate in the charge-reduced resin with a residual IC of 339 mmo/L (FF-pAEM513-R339) decreased more sharply with increasing NaCl concentration by comparison with FF-pAEM513, indicating its increased salt-sensitivity than FF-pAEM513. That is, charge reduction on the AEM-grafted resin could accelerate protein uptake at 0 mmol/L NaCl but decrease salt tolerance. Column breakthrough experiments showed that FF-pAEM513-R339 was favorable for high flow rate protein chromatography at low NaCl concentration (0 mmol/L), whereas FF-pAEM513 was a good choice in a wide range of salt concentrations at low flow rate. This research proved the excellent protein chromatography performance of the AEM-based anion-exchangers.