title: Protein instability during HIC: hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects.
abstract: Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogen-deuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability.
authors.affiliation: Department of Chemical Engineering, University of Virginia, 102 Engineers' Way, Charlottesville, VA 22904-4741, USA.
authors.forename: Yunzhi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Tara T
authors.initials: TT
authors.lastname: Jones
authors.forename: Abigail H
authors.initials: AH
authors.lastname: Laurent
authors.forename: John P
authors.initials: JP
authors.lastname: O'Connell
authors.forename: Todd M
authors.initials: TM
authors.lastname: Przybycien
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
mesh_terms: D002847:Chromatography, Agarose / Q000379:methods*; D003198:Computer Simulation; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004355:Drug Stability; D008422:Materials Testing; D008956:Models, Chemical; D044367:Phase Transition; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry*
chemical_list: D011506:Proteins; D012685:Sepharose