Comparing isotherm parameter determination methods for hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is a widely used separation method in biopharmaceutical downstream processing. For process development, mechanistic modeling can be used to reduce timelines by simulating protein transport and adsorption during chromatography. Accuracy of the parameters used in the model is essential for successful deployment. This work compares three isotherm parameter determination methods for a simplified linear HIC isotherm: the Parente and Wetlaufer method, the Yamamoto method, and the inverse method. These methods were tested for two proteins, using the same linear gradient elution (LGE) experiments. Accuracy of the obtained parameters was determined via cross-validation using three LGEs. Finally, the obtained parameters were tested for alternative linear gradients with varying initial and final salt concentrations. While all results were comparable, parameters obtained by the inverse method showed the greatest accuracy. This method requires high quality chromatograms, while the other methods only need retention volumes. Therefore, it is less suitable when signal quality is compromised. The Yamamoto method showed similar robustness as the inverse method while outperforming the Parente and Wetlaufer method. Therefore, the Yamamoto method is a good alternative for parameter determination. This comparison offers practical guidance for method selection for isotherm determination, thereby enabling reliable mechanistic modeling of HIC processes.