title: Protein instability during HIC: describing the effects of mobile phase conditions on instability and chromatographic retention.
abstract: Hydrophobic interaction chromatography (HIC) is known to be potentially denaturing to proteins, but the effects of mobile phase conditions on chromatographic behavior are not well understood. In this study, we apply a model describing the effects of secondary protein unfolding equilibrium on chromatographic behavior, including the effects of salt concentration on both stability and adsorption. We use alpha-lactalbumin as a model protein that in the presence and absence of calcium, allows evaluation of adsorption parameters for folded and unfolded species independently. The HIC adsorption equilibrium under linear binding conditions and solution phase protein stability have been obtained from a combination of literature and new experiments. The effect of salt concentration on protein stability and the rate constant for unfolding on the chromatographic surface have been determined by fitting the model to isocratic chromatography data under marginally stable conditions. The model successfully describes the effects of added calcium and ammonium sulfate. The results demonstrate the importance of considering the effects on stability of mobile phase modifiers when applying HIC to marginally stable
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, 22904-4741, USA.
authors.forename: Yunzhi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Freed
authors.forename: Tara Tibbs
authors.initials: TT
authors.lastname: Jones
authors.forename: Kostantinos
authors.initials: K
authors.lastname: Makrodimitris
authors.forename: John P
authors.initials: JP
authors.lastname: O'Connell
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D000327:Adsorption; D000465:Algorithms; D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D003198:Computer Simulation; D017666:Deuterium Oxide / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007768:Lactalbumin / Q000737:chemistry; D013058:Mass Spectrometry; D008962:Models, Theoretical; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: D011506:Proteins; D014867:Water; C020927:phenyl-sepharose; D012685:Sepharose; D007768:Lactalbumin; D004492:Edetic Acid; D017666:Deuterium Oxide; D000645:Ammonium Sulfate; D002118:Calcium