Development of poly(methacrylate)-grafted Sepharose FF for cation-exchange chromatography of proteins

Abstract

Polymer-grafted media have been a focus of recent development for ion exchange chromatography (IEC) because of their capacity and uptake kinetics that can lead to high dynamic capacity in protein purification. This work is devoted to developing novel cation exchangers of high adsorption performance by grafting polymerization of sodium methacrylate (MA) onto a commercial agarose gel Sepharose FF (FF). Five polyMA (pMA)-grafted FF gels were prepared with the same grafting density but different chain lengths (i.e., different ionic capacities, ICs), and named as FF-pMA-IC (IC denotes IC value in mmol/L). The effects of chain length (IC) and ionic strength (IS) on protein adsorption and chromatographic behaviors were examined using lysozyme (at pH 8.0) and γ-globulin (at pH 5.0) as model proteins. It was found that lysozyme adsorption capacity increased with increasing IC till reaching a plateau (390 mg/mL) over IC=540 mmol/L (FF-pMA-540), while there was an optimum IC (320 mmol/L, FF-pMA-320) at which γ-globulin adsorption capacity reached the highest (208 mg/mL). With increasing chain length (IC), the uptake rates of both the proteins presented decreasing trends due to the steric hindrance caused by the polymer chains. At the same IC, however, the uptake rate of lysozyme was much higher than that of γ-globulin because of the different sizes of the two proteins. Increasing salt concentration obviously promoted the uptake rates of the proteins, which led to the increase of dynamic binding capacities (DBCs) in different salt concentration ranges. The DBC value of lysozyme on FF-pMA-540 kept as high as 108-198 mg/mL in the salt concentration range of 0-150 mmol/L, and the DBC of γ-globulin on FF-pMA-320 increased to 27 mg/mL with increasing salt concentration from 100 mmol/L. This work clearly indicated the presence of optimal IC values (chain lengths) for different sized proteins, and IS was also crucial for reaching a high DBC for a specific protein. The findings provided insight into the selection of FF-pMA-n gels and operational conditions (e.g., IS) for the purification of a target protein of defined size.