Mechanistic Modeling of Reversed-PhaseChromatography of Insulinswith Potassium Chloride and Ethanol as Mobile-Phase Modulators

摘要

The purpose of this study was to investigate the adsorption mechanism in reversed-phase chromatography (RPC) of proteins and to develop a model for the effect of dual mobile phase modulators—a salt and an organic solvent—on this process. Two different adsorption mechanisms were considered: (1) pure association of a protein molecule and one or more ligands and (2) displacement of the organic modulator, with which the adsorbent is saturated, by the protein upon association with one or more ligands. One model was then derived from each of the two considered mechanisms, combining thermodynamic theories on salting-in, RPC, and the solubility of proteins. The model was then applied to chromatographic data from an earlier report as well as supplementary data for solubility and vapor–liquid equilibria, and case-specific simplifications were made. We found that an adaptation of Kirkwood’s electrostatic theories to hydrophobic interaction chromatography describes the observed effect of KCl well. Combining chromatographic and solubility data for one of the insulins, we concludedthat the variation in the activity coefficient of the insulin withrespect to the concentration of ethanol alone cannot describe itseffect on retention. Consequently, one or more other phenomena mustaffect the adsorption process. Our second model fits the retentiondata well, supporting the hypothesis that ethanol is directly involvedin the adsorption mechanism in this case. Using additional experimentsat a high-protein load, we extended the linear-range equilibrium modelinto a dynamic model for preparative conditions. This model showsgood agreement with the high-load data for one of the insulin variants,without any additional effects of the modulator concentrations onthe adsorption capacity.