Protein adsorption to poly(ethylenimine)-modified Sepharose FF. IV. Dynamic adsorption and elution behaviors

We have previously investigated bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose FF. It was found that there was a critical ionic capacity (cIC; 600 mmol/L) for BSA, above which the protein adsorption capacity and uptake kinetics increased drastically. In this work, two poly(ethylenimine) (PEI)-grafted resins with IC values of 271 mmol/L (FF-PEI-L270) and 683 mmol/L (FF-PEI-L680), which were below and above the cIC, respectively, were chosen to investigate the breakthrough and linear gradient elution (LGE) behaviors of BSA. Commercially available anion exchanger, Q Sepharose FF, was used for comparison. The DBC values of FF-PEI-L680 were much higher in the entire residence time range (2−10 min) than the other two resins due to its high static adsorption capacity and uptake kinetics. At a residence time of 5.0 min, the DBC of FF-PEI-L680 (104 mg/mL) was about seven times that of FF-PEI-L270 and three times that of Q Sepharose FF. A rise-fall trend of the DBCs with increasing ionic strength (IS) was found for all the three resins studied, indicating the presence of electrostatic exclusion for protein uptake at low IS. With increasing NaCl concentration from 20 to 200 mmol/L, FF-PEI-L680 kept very high DBC values (64−114 mg/mL). In addition, FF-PEI-L270 showed more favorable adsorption properties than Q Sepharose FF at 100-300 mmol/L NaCl. These results proved that the three-dimensional grafting ion exchange layer on the PEI resins enhanced their tolerance to IS. In the study of LGE, the three resins showed similar elution behaviors and no distinct peak tailings were observed. The salt concentrations at the elution peaks (IR) were in the order of FF-PEI-L680 > FF-PEI-L270 > Q Sepharose FF, indicating that the elution for the PEI resins needed higher salt concentrations, which was also an appearance of the salt-tolerant feature of the PEI resins. When protein loading amount was increased to the value equivalent to the DBC at 10% breakthrough, the adsorbed BSA could be eluted at lower salt concentrations. The chromatographic study has provided new insights into the practical application of the PEI-based anion exchangers.