Protein adsorption to poly(ethylenimine)-modified sepharose FF: VII. Complicated effects of pH.

Highlights • pH showed complicated effects on protein uptake to two PEI-grafted Sepharose. • Both capacity and uptake rate on high-charge resin showed up-down-up trends with pH. • Charge-reduced resin gave both maximal capacity (257 mg/mL) and uptake rate at pH 5.5. • The two resins showed high dynamic capacities at different pH or salt concentrations. • Dynamic capacities over 120 mg/mL were observed on charge-reduced resin at pH 5.5–8. Abstract Previously, we have studied protein adsorption behaviors on a series of poly(ethylenimine) (PEI)-grafted Sepharose FF, and a critical ionic capacity of PEI-grafted resins was observed, above which both protein adsorption capacity (q) and effective pore diffusivity (D e) increased drastically. Moreover, reducing the charge density of the PEI-grafted resins from an ionic capacity of 740 mmol/L (FF-PEI-L740) to 440 mmol/L (FF-PEI-R440) by neutralization of the amino groups of PEI chains with sodium acetate brought out a three-fold increase of D e value at pH 8. In this work, FF-PEI-L740 and FF-PEI-R440 were selected to investigate the complicated effects of pH on protein adsorption behavior using bovine serum albumin (BSA) as the model protein. It was found that, for FF-PEI-L740, both the q and D e values decreased significantly when pH decreased from 9 to 5.5, and then increased dramatically at pH 5, and finally decreased remarkably at pH 4.5. The results were considered due to the following causes: The decrease of pH from 9 to 5.5 led to a greatly increased electrostatic repulsion between the PEI chains due to the increased dissociation degree of PEI, which caused the increase in steric hindrance effects and decrease in the bound protein transport by chain delivery. At pH 5, which was near the p I of BSA (∼4.9), the presence of few protein charges greatly decreased the electrostatic hindrance effect on protein transport and the high charge density of FF-PEI-L740 provided extensively accessible binding sites and facilitated the happening of chain delivery as well. At pH 4.5, the net charge of BSA shifted to positive, likely charged with PEI, so the overall electrostatic repulsion greatly hindered protein uptake. For FF-PEI-R440, whose charge density was much lower than FF-PEI-L740, its q and D e values even increased mildly when pH decreased from 8 to 5.5, because its slightly-increased charge density did not cause much increased electrostatic and steric hindrance effects but provided more binding sites for protein adsorption. At pH 5, the few charges of BSA and the low charge density of FF-PEI-R440 could not afford enough electrostatic interaction for protein binding, resulting in significantly decreased q and D e values. Besides, column breakthrough experiments revealed that FF-PEI-R440 kept high dynamic binding capacity (DBC) (>120 mg/mL) at pH 5.5–8, while FF-PEI-L740 offered high DBC (>80 mg/mL) at pH 5, 7 and 8 with a higher salt concentration (100 mmol/L NaCl). These findings demonstrated the excellence of FF-PEI-L740 and FF-PEI-R440 in different conditions, and would help in the design and selection of suitable resins for high-performance protein chromatography. [ABSTRACT FROM AUTHOR]