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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification
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Gradient elution behavior of proteins in hydrophobic interaction chromatography with a U-shaped retention factor

Arch Creasy1, Joseph Lomino2, Giorgio Carta1

  • 1Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA.

Journal of Chromatography. A
|October 15, 2018
PubMed

The gradient elution hydrophobic interaction chromatography of a monoclonal antibody that exhibits U-shaped retention as a function of the ammonium sulfate concentration is investigated for overloaded conditions at protein loads up to 30% of the column equilibrium binding capacity. Protein load and gradient slope affect both elution peak shape and protein recovery during the gradient. Higher protein loads result in tailing peaks with near 100% recovery that transition to fronting peaks and incomplete recovery as the protein load is reduced. The gradient slope also affects peak shape and recovery. Tailing peaks with lower recovery are obtained with sharper gradients and the most tailing peak and lowest recovery are obtained when step elution rather than gradient is implemented. Modeling the chromatographic elution based on independently determined adsorption isotherms as a function of protein and ammonium sulfate concentration predicts results in agreement with the experimental trends confirming that the unusual chromatographic behavior observed is due to the U-shaped protein binding as a function of the ammonium sulfate concentration. Although less pronounced than in the dilute limit, the U-shaped binding still produces peak shapes and recovery losses as a function of gradient slope that differ from those seen for systems where retention is a monotonic function of salt concentration.

Keywords:
AntibodiesGradient elutionHydrophobic interaction chromatographyOverloaded conditionsProteinsU-shaped retention factor curves

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