Quantifying protein release from hydrophobic ion pairs by cation exchange chromatography

Hydrophobic ion pairing is an established solubility engineering strategy for small molecules and peptides, but its extension to proteins remains comparatively underexplored, and quantitative methods for measuring protein release from these complexes under biorelevant conditions remain limited. Colorimetric and UV-based assays are subject to interference from co-formulated surfactants, bile salts and phospholipids. Here, we report a cation exchange chromatographic method, implemented on a fast protein liquid chromatography system, that separates lysozyme from these matrix components prior to UV detection. Validation in accordance with ICH Q2(R2) demonstrated linearity from 20 to 320 µg mL^-1 (R² of 0.9998), a detection limit of 7 µg mL^-1, a quantitation limit of 20 µg mL^-1, and recovery of 88.2% in high ionic-strength media. Recovery decreased to 42.6% in phospholipid-containing media, limiting absolute quantification under these conditions and supporting comparative assessment of protein release. Applied to lysozyme-sodium dodecyl sulphate and lysozyme-dioctyl sulfosuccinate complexes, the method resolved markedly different dissociation behaviour as a function of pH, ionic strength, and simulated intestinal fluid components. Lysozyme-sodium dodecyl sulphate released measurable protein under all conditions, whereas the more stable lysozyme-dioctyl sulfosuccinate complex resisted dissociation, with released lysozyme below the quantitation limit under most simulated intestinal conditions. Recovered lysozyme retained 17-40% of the catalytic activity of native lysozyme depending on the dissociation medium, demonstrating that chromatographic separation enables both quantification and functional assessment of released protein.