Influence of mixed salts on retention behavior of model proteins in cation exchange chromatography

Mobile phase composition is an important factor for a further improvement of ion exchange chromatography steps of proteins. In this work, the effects of mixed salts on the retention factors of the two model proteins lysozyme (LYZ) and bovine serum albumin (BSA) in cation exchange chromatography (CEC) were investigated and compared to effects previously observed in hydrophobic interaction chromatography (HIC). The model equation describing the effects in HIC was adjusted for linear gradient elution experiments in CEC. The investigated salts were sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. By varying binary salt mixtures as well as using pure salts, model parameters were determined. The normalized root mean square error (NRMSE) of the predicted retention factors for the calibration runs was 4.1% for BSA and 3.1% for LYZ. Additional validation experiments proved the ability of the model to describe and predict retention behavior of the proteins for further salt compositions. Hereby, the NRMSE values for BSA and LYZ were 2.0% and 1.5%, respectively. While the retention factors of LYZ changed linearly with the salt composition, non-linearities in the impact of the anion composition were found for BSA. This was contributed to an overlay of a synergetic salt effect on a protein-specific effect by sulfate on BSA with non-specific effects of the ions for CEC. However, the impact of the synergetic effects on protein separation is lower for CEC than for HIC, as mixed salts do not increase the separation of these proteins. The best salt composition for separating BSA and LYZ is pure ammonium sulfate. Thus, synergetic salt effects can also occur in CEC, but they have a lower impact than in HIC.