Hydrophobic interaction chromatography (HIC) isotherm incorporating salt-dependent water activity enhances...

Hydrophobic interaction chromatography (HIC) is a highly relevant separation technique that provides orthogonal selectivity to widely used ion exchange chromatography (IEC). However, in contrast to the latter, the mechanisms underlying hydrophobic interaction are difficult to capture in the form of an isotherm, probably due to the complex nature of the mechanisms involved. Of the several HIC isotherms that have been proposed, one improved the prediction accuracy by accounting for the water molecules released upon protein binding which was estimated based on the concentration of protein bound to the stationary phase. However, we found that this isotherm resulted in implausible predictions depending on the selected chromatographic conditions. For example, when altering the protein concentration of salt gradient elution experiments the location of the elution peak shifted drastically. Upon investigating the assumptions made during isotherm development, we replaced the previous estimate with a salt concentration-dependent water activity (SWA). Accordingly, our SWA isotherm utilizes the activity of the surrounding water molecules to describe the activity of the released bulk-like water molecules. We evaluated this new isotherm on in silico and experimental datasets and found that the unrealistic predictions disappeared. Additionally, the precision of elution profile prediction, measured as the differences in elution peak height, skew and position, improved by an average of 2.8-fold and up to 5.6-fold. We also augmented the isotherm into a unified form that can account for pH effects as well. Lastly, we implemented the isotherms in CADET, so they can easily be used from within the software suite.