Comparing isotherm parameter determination methods for hydrophobic interaction chromatography.

Area of Science:

• Biopharmaceutical Downstream Processing
• Chromatographic Separation Science
• Mechanistic Modeling in Bioprocessing

Background:

• Hydrophobic interaction chromatography (HIC) is crucial for biopharmaceutical purification.
• Mechanistic modeling accelerates process development by simulating protein behavior.
• Accurate isotherm parameters are vital for reliable HIC model predictions.

Purpose of the Study:

• To compare the accuracy of three methods (Parente and Wetlaufer, Yamamoto, inverse) for determining linear HIC isotherm parameters.
• To evaluate the performance of these methods under varying linear gradient elution conditions.
• To provide guidance on selecting appropriate isotherm determination methods for HIC process development.

Main Methods:

• Linear gradient elution (LGE) experiments were conducted for two proteins using HIC.
• Isotherm parameters were determined using the Parente and Wetlaufer, Yamamoto, and inverse methods.
• Parameter accuracy was assessed via cross-validation and testing with alternative linear gradients.

Main Results:

• The inverse method yielded the most accurate isotherm parameters, but requires high-quality chromatograms.
• The Yamamoto method demonstrated comparable robustness to the inverse method and outperformed the Parente and Wetlaufer method.
• All tested methods provided comparable results, but accuracy varied depending on method requirements and experimental conditions.

Conclusions:

• The Yamamoto method is a robust and accurate alternative for HIC isotherm parameter determination, especially when high-quality chromatograms are unavailable.
• Accurate isotherm parameters are essential for reliable mechanistic modeling of HIC processes.
• This comparative study offers practical insights for selecting the optimal method for isotherm determination in biopharmaceutical HIC development.