Our previous studies on protein adsorption onto anion-exchangers of poly(ethylenimine) (PEI)-grafted Sepharose FF (PEI-Sepharose) proved their significantly improved performance over the commercial nongrafting anion-exchangers such as Q Sepharose FF, and it was found the protein adsorption behavior on PEI-Sepharose was more sensitive to counterions (Cl-, SCN-, HPO42- and SO42-). However, the complicated role of counterions has not been well interpreted due to their distinct chemical and physical characteristics. Thus, we have further studied the counterion effects by adding two halide ions (F- and Br-) to explore the effects of the three halide ions on bovine serum albumin adsorption and the results were compared with previous data. Furthermore, separation of recombinant human serum albumin (rHSA) from fermentation broth was studied. It was found that the counterion preference for PEI-Sepharose increased with the charge density of the counterions, demonstrating the favorable elution by choosing a proper counterion. Moreover, uptake kinetics onto PEI-Sepharose was very sensitive to counterions, even the three halide ions. In contrast, there is little difference among the halide ions for dynamic binding capacity of PEI-Sepharose, presenting a high value (104 ± 3 mg/mL) for the three halide ions. Furthermore, PEI-Sepharose exhibited much higher rHSA separation performance over Q Sepharose FF, characterized by sharper and more symmetrical elution peaks, higher recovery, enriched eluates, and reduced use of elution salt. The high recovery (>90 %) of rHSA from the Pichia pastoris culture supernatant proved the superiority of PEI-Sepharose column in downstream processing of therapeutic proteins.
Keywords: Counterion; Ion-exchange chromatography; Linear gradient elution; Pichia pastoris; Poly(ethylenimine); Recombinant human serum albumin.
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