Optimizing Hydrophobic Interaction Chromatography for Protein Elution

Forschungszentrum Jülich researchers led efforts to develop a new hydrophobic interaction chromatography (HIC) isotherm for better analysis of protein elution profiles. Their findings were published in the Journal of Chromatography A (1).

Hydrophobic interaction chromatography (HIC) is a technique that separates molecules based on their hydrophobicity (2). Typically, HIC is useful for purifying protein while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions. Overall, it has been useful in many bioprocesses, including biopharmaceutical manufacturing. As is the case with other types of chromatography, various parameters can be modified during HIC to achieve optimal process results, typically high product recovery and purity. These parameters include ligand types, pH, temperature, and flow rate, among others. That said, optimizing HIC steps is labor-, time-, and cost-intensive, even if small scale and high-throughput technologies are used.

The mechanisms underlying hydrophobic interaction can be difficult to capture in the form of isotherms, likely due to the mechanisms involved being of complex natures. Various HIC isotherms have been proposed, with one improving prediction accuracy by accounting for water molecules released upon protein binding; this was estimated based on the concentration of protein bound to the stationary phase. However, this isotherm also resulted in implausible predictions depending on selected chromatographic conditions.

In this study, the scientists investigated the assumptions made during isotherm formulation, then re-formulated the isotherm after identifying the root causes. They assumed the water molecules released upon protein binding were indistinguishable from other water molecules in the mobile phase. Upon using a salt-dependent water activity (SWA) isotherm, the researchers discovered the new isotherm substantially outperforming the original isotherm when applied on both synthetic in silico and experimental data of albumin and lysozyme proteins. When compared to other isotherms, the SWA isotherm also provided superior performance in most cases. Additionally, the scientists measured the precision of elution profile prediction, measuring the differences in elution peak height, skew, and position, with the measurements improving by an average of 2.8-fold and up to 5.6-fold.

Additionally, the isotherm was augmented into a unified form that could also account for pH effects, before being implemented into the CADET data simulation, which provides hierarchical models and computational engineering tools for various biotechnological processes, including chromatography (3). When implementing the new isotherm into the CADET modeling suit, the scientists unified it with previous parametrizations to facilitate fast and easy comparisons of different isotherm variants.

Expanding the isotherm for additional process parameters, such as temperature, could hold additional value when implementing HIC for in silico process optimization. The scientists also noted, however, that one must balance the benefits of additional parameters against the risks of overfitting. The new isotherm does not increase the number of fitting parameters. It only uses variables defined by the experimental conditions, such as salt concentration and activity. Future efforts will focus on verifying the new isotherm’s performance using additional proteins, ligands, and salt types, in addition to high loading densities, to reflect conditions during preparative protein purification.

References

(1) Jäpel, R.; Knödler, M.; von Lieres, E.; Buyel, J. F. Hydrophobic Interaction Chromatography (HIC) Isotherm Incorporating Salt-Dependent Water Activity Enhances Model-Based Analysis of Protein Elution Profiles. J. Chromatogr. A 2025, 1756, 466095. DOI: 10.1016/j.chroma.2025.466095

(2) Introduction to Hydrophobic Interaction Chromatography. Bio-Rad 2025. https://www.bio-rad.com/en-us/applications-technologies/introduction-hydrophobic-interaction-chromatography?ID=LUSN8F4VY (accessed 2025-6-11)

(3) CADET. Github 2025. https://github.com/cadet (accessed 2025-6-11)