Loading, stationary phase, and salt effects during hydrophobic interaction chromatography: α-Lactalbumin is stabilized at high loadings

• Published in: Journal of Chromatography A Vol. 1121; no. 2; pp. 209 - 218
• Main Authors: Fogle, Jace L., O’Connell, John P., Fernandez, Erik J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 21.07.2006; Elsevier
• Subjects: Adsorption; Amide hydrogen-deuterium exchange; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Liquid - methods; Fundamental and applied biological sciences. Psychology; Glycoproteins; Hydrophobic interaction chromatography; Lactalbumin - chemistry; Protein loading; Proteins; Salts; Spectrophotometry, Ultraviolet; Stability; α-Lactalbumin; Hydrophobic interaction chromatography; α-Lactalbumin; Stability; Amide hydrogen-deuterium exchange; Protein loading; Chemical stability; Adsorption isotherm; Adsorption capacity; Mobile phase; Lactalbumin; Ungulata; Stationary phase; Bovine; Phase composition; Isotope exchange; Glycoprotein; Hydrogen Isotopes; Langmuir isotherm; Protein; Vertebrata; Mammalia; Salt effect; Hydrophobic chromatography; Medium effect; Animal; Artiodactyla
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2006.04.015

Amide hydrogen-deuterium exchange labeling has been used to study the effects of salt and protein loading on α-lactalbumin (BLA) stability during hydrophobic interaction chromatography (HIC). Stability in the adsorbed phase increased dramatically with increasing loading, and unfolding was nearly undetectable close to the resin saturation capacity. We also found that a butyl surface destabilized BLA more than a phenyl surface, despite the fact that BLA was bound more strongly on the phenyl surface. These observations have important implications for HIC process design and indicate that in some cases column capacity does not have to be sacrificed to preserve protein stability.