关键词: 色谱, 蛋白质, 吸附, 动力学, 动态结合容量

Abstract:

Poly(methacrylate) (pMA)-grafted Sepharose FF with ionic capacity (IC) of 320mmol/L (FF-pMA-320) showed the high lysozyme and γ-globulin adsorption capacities, but its uptake rates for the two proteins were low. On the premise of maintaining polymer chain length, the charge density on pMA-grafted Sepharose FF was reduced by neutralization of the carboxyl groups on pMA chains with ethanolamine to improve protein uptake rate. FF-pMA-320 was partly neutralized to prepare the two charge-reduced cation exchangers with IC values (in mmol/L) of 230 and 170, which were respectively named as pMA-320-R230 and pMA-320-R170. Adsorption equilibrium, uptake kinetics and column breakthrough experiments were performed to study lysozyme and γ-globulin adsorption on to the two new resins, which were compared with the starting resin (FF-pMA-320). It was found that the adsorption capacities for the two proteins decreased with IC (charge density) decreasing from 320mmol/L to 170mmol/L, which was related to the decrease of adsorption site caused by the partial neutralization. With decreasing charge density of grafted polymer, there were weaker electrostatic repulsion between neighbouring polymer chains and lower protein adsorption capacity, thus increasing chain flexibility and reducing protein exclusion effect. Therefore, the uptake rates of lysozyme and γ-globulin on pMA-320-R170 were about 1.6 times and 5.5 times higher than those on FF-pMA-320, respectively. Column breakthrough results demonstrated that the dynamic binding capacity (DBC) of γ-globulin on pMA-320-R170 was higher than other two resins due to its high uptake rate, and kept over 10mg/mL in the flow rate range of 150—750cm/h. The findings in this work provided reference and direction to design and develop high-performance protein chromatography.

Key words: chromatography, protein, adsorption, kinetics, dynamic binding capacity