Investigation of mobile phase salt type effects on protein retention and selectivity in cation-exchange systems using quantitative structure retention relationship models

In this paper, the effect of the salt counterion on protein retention in cation-exchange systems is investigated using experimental data and quantitative structure retention relationship (QSRR) models. Linear gradient experiments were carried out in the presence of three displacing counterions (sodium, ammonium, and calcium) on two cation-exchange stationary phase materials (Fast Flow Sepharose SP and Source 15S). QSRR models based on a support vector machine regression approach were developed using the experimental chromatographic data in concert with molecular descriptors computed from protein crystal structures. The resulting QSRR models were well correlated (r²=0.8452-0.9599), and the predictive power of these models was demonstrated with proteins not included in the derivation of the models. The key descriptors reflected in the models were then used to evaluate the phenomena responsible for protein retention in the presence of different salt counterions. In addition to the standard competitive binding effects of the cations, the results indicated that retention also depends on the relative ability of the cations to shield charges on the protein as well as the increased importance of hydrophobic interactions in the presence of a strong kosmotrope such as Ca²⁺. This work provides insight into the behavior of proteins in cation-exchange systems in the presence of various salt counterions and offers an efficient tool for the a priori prediction of protein retention.