Overcoming solubility limits in overloaded gradient hydrophobic interaction chromatography

• Solubility limitation in gradient protein chromatography on HIC media was studied.
• Equilibrium and kinetics of crystallization were determined for model protein.
• Supersaturation region where crystallization was inhibited was identified.
• Dynamic model was used for the prediction of protein crystallization in the column.
• SLE data of a target protein can be used to design economic chromatographic process.

The impact of the solubility limits on the performance of gradient protein chromatography has been studied. As a case study elution of model protein, i.e., lysozyme, on hydrophobic interaction media has been selected. A dependence of the protein solubility and crystallization kinetics on the content of cosmotropic salt in the mobile phase has been determined. Moreover, adsorption properties of the protein versus the mobile phase composition have been quantified. A model of chromatographic column dynamics has been developed which incorporated the mass transport kinetics accompanying both adsorption and crystallization processes. The model was used to study the influence of operating parameters such as flowrate and concentration loading on the solubility pattern inside the column and the separation performance. The analysis performed indicated existence of supersaturation regions for which, due to slow kinetics of crystallization, chromatographic process could be performed under conditions of strong concentration overloading while avoiding undesirable effects of flow blockage in chromatographic systems.