Protein adsorption on DEAE ion‐exchange resins with different ligand densities and pore sizes

Abstract

Ion exchange chromatography (IEC) is a common and powerful technique for the purification of proteins. The ligand density and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In the present work, the adsorption isotherms of bovine serum albumin (BSA) and human serum albumin (HSA) were investigated systematically with series of diethylaminoethyl (DEAE) ion-exchange resins, which have different ligand densities and pore sizes. The Langmuir equation was used to fit the experimental data and the influences of ligand density and pore size on the saturated adsorption capacity and the dissociation constant were discussed. The zeta potentials and hydrodynamic diameters of proteins at different pHs were also measured, and the surface charge characteristics of proteins and the adsorption mechanism were discussed. The results demonstrated that the ligand density, pore size, and protein properties affect the protein adsorption capacities in an integrative way. An integrative parameter was introduced to describe the complicated effects of ligand density and pore size on the protein adsorption. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.

Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion‐exchange resins

“…S1. It was found that two properties of the resins, ligand density and pore size, had an integrative impact on the adsorption of bovine IgG, which is similar to our previous work with bovine serum albumin (BSA) [26]. Therefore, the influences of ligand density, pore size, and protein characteristics were investigated in detail as following.…”

“…For bovine IgG, a and b are 48.37 and 204.84, respectively. For BSA, the parameters of a and b are 47.08 and 136.52, respectively [26]. It can be found that the slopes of correlation lines are nearly same for two proteins, indicating that the integrated effects of ligand density and pore size on the adsorption capacity of bovine IgG and BSA are similar.…”

“…When N reaches around 0.2, the protein binding might reach saturation and the q m tends to a constant or even a slight drop due to some steric hindrance effects. Based on our previous work [26], the adsorption of BSA with series of DEAE resins as the function of parameter N is also shown in Fig. 3A.…”

“…In order to combine the influences of ligand density and pore size, an integrative parameter N was introduced as the previous work [26], which was defined as the ratio of ligand density to the square of mean pore radius. The saturated adsorption capacities as the function of parameter N is shown in Fig.…”

“…Although the tendencies of q m s with the parameter N are quite similar for bovine IgG and BSA, the q m s for bovine IgG are relatively higher than those of BSA, and the differences are about 70∼80 mg/g. In order to obtain a quantitative relationship between q m and the parameter N, the following empirical equation was used as our previous work [26]:…”