Comparison of the impact of anion and cation selection onto cation exchange chromatography of model proteins

For a further improvement of ion exchange chromatography for protein purification, the optimization of mobile phase composition is an important factor. The effects of salt type selection on cation exchange chromatography (CEC) have been investigated in this work. Hereby, the significance of co-ion and displacing ion selection were compared. Therefore, the suitability of a constant cationic or anionic strength for multivalent salts for the determination of salt impact were evaluated. The retention and separation behavior of the three model proteins bovine serum albumin, lysozyme and alpha-chymotrypsin were investigated with varying salts in the elution buffer. For this, five different cations and five different anions were used. Hereby, the effects of anion and cation selection were analyzed independently. It was shown that a constant cationic strength is a suitable setup for the determination of salt effects on protein retention in CEC, as the resulting retention factors did not show a clear trend regarding ion valence. It was observed that for the used process conditions the anion selection could change the separation factor by up to 30% and the cation selection by up to 16%. These changes were attributed to protein specific salt effects of citrate on bovine serum albumin and magnesium on alpha-chymotrypsin. It was demonstrated that at constant cationic strength the co-ion selection can be of the same or even of higher importance for protein separation as the selection of the displacing ion in CEC.(C) 2022 Elsevier B.V. All rights reserved.

Automated summary

This work investigated the effects of salt type selection on cation exchange chromatography (CEC) for protein purification. It was found that a constant cationic strength is more important than ion valence in determining protein retention in CEC. The selection of anions or cations can significantly impact the separation factor, with changes of up to 30% and 16% observed, respectively.