Protein adsorption to poly(allylamine)-modified Sepharose FF: Influences of polymer size and partial charge neutralization

Abstract

Recently, poly(allylamine) (PAA) of 17.5 and 900 kDa were grafted onto Sepharose FF gel to study protein adsorption and chromatography. In order to further investigate the influences of polymer size and partial charge neutralization, PAAs of 160 and 450 kDa were chosen to synthesize two more types of ion-exchangers (denoted as FF-PAA-M and FF-PAA-ML, respectively). Protein adsorption capacity (qm) of these resins showed a monotonic increasing trend till the maximum ionic capacity (IC) available (970–990 mmol/L), while the uptake rates (De/D0) showed a decreasing-then-increasing trend. The maximum qm and De/D0 reached 328 mg/mL and 0.715, respectively, with FF-PAA-M990 (IC = 990 mmol/L). Partial charge neutralization by modification of FF-PAA-M990 with acetate produced four charge-reduced resins (FF-PAA-Rs), of which the lowest IC reached 210 mmol/L. Both the qm and De/D0 of FF-PAA-Rs presented an increasing-then-decreasing trend with reducing the charge density. The maximum qm obtained with FF-PAA-R860 (IC = 860 mmol/L) was 40% higher than FF-PAA-M990, and all the charge-reduced resins presented 2–3 times higher uptake rates than FF-PAA-M990. Both the qm and De/D0 of FF-PAA-Rs decreased sharply with increasing salt concentration, indicating their increased salt-sensitivities. The FF-PAA-R860 and FF-PAA-R710 columns presented much higher dynamic binding capacities than the FF-PAA-M990 column. FF-PAA-M990 and the charge-reduced resins were suitable for protein chromatography handling feedstocks containing different salt concentrations due to their different sensitivities to ionic strength.