Poly(N,N-dimethylaminopropyl acrylamide)-grafted Sepharose FF: A new anion exchanger of very high capacity and uptake rate for protein chromatography.

Abstract

Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65-831\u2009mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (qm) increased rapidly from 91\u2009mg/mL at IC\u2009=\u200965\u2009mmol/L till reaching a peak value (368\u2009mg/mL) at IC\u2009=\u2009458\u2009mmol/L and then decreased slightly with further increasing IC (chain length) to 831\u2009mmol/L. The uptake rate, De/D0, defined as the ratio of effective diffusion coefficient (De) to free diffusivity of the protein (D0), also presented similar changes as a function of IC. Namely, De/D0 increased from 0.25 to 2.45 in the IC range of 65 to 458\u2009mmol/L, kept almost unchanged from 458 to 573\u2009mmol/L and then decreased slightly with further increase of IC to 831\u2009mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high qm and De/D0 values (368\u2009mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The qm and De/D0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230\u2009mg/mL and 2.3, respectively, at salt concentrations up to 100\u2009mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180-220\u2009mg/mL in the superficial flow velocity rang of 150-1350\u2009cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography.