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, hydrogendeuterium 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. Biotechnol. Bioeng. 2007;96: 80-93.

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

“…Previously, deuterium labeling of adsorbed proteins had been used at single labeling times to qualitatively assess the effects of resin hydrophobicity, loading, and salt concentration (Fogle et al, 2006;Xiao et al, 2007b). By incorporating multiple labeling times, we are able to obtain a quantitative measure of both the free energy change and kinetics of unfolding on the surface.…”

“…Proteins may change conformation in the adsorbed state under strongly retaining conditions, as indicated by multiple or misshapen chromatographic peaks (Goheen and Engelhorn, 1984;Hahn et al, 2003;Jungbauer et al, 2005;Machold et al, 2002;To et al, 2007;Wu et al, 1986a,b), and increased solvent accessibility (Buijs et al, 1999;Fogle et al, 2006;Tibbs-Jones and Fernandez, 2003;Xiao et al, 2007b). This can reduce desired product recovery (Chen et al, 2007;Kepka et al, 2005;To et al, 2007), inhibiting the application of this purification method.…”

“…Jungbauer et al (2005) found that the area of a second peak observed upon step elution, which was presumed to be unfolded protein, increased with the salt concentration used in the first portion of the step elution. Also, using deuterium labeling methods, we found that the fraction of protein remaining in the native conformation on the resin decreased with both HIC media hydrophobicity and with increasing salt concentration (Fogle et al, 2006;Xiao et al, 2007b).…”

“…We have previously proposed a two-conformation adsorption model that includes the effects of salt concentration and temperature on both stability and adsorption to describe the effects of secondary protein unfolding on HIC behavior, specifically wild-type bovine a-lactalbumin adsorbed to Phenyl Sepharose TM 6FF low substitution resin (Xiao et al, 2006(Xiao et al, , 2007b. The choice of a-lactalbumin as a model protein was motivated by the ability to modify the solution phase protein stability by changing the available calcium for binding (Ikeguchi et al, 1986).…”

Unfolding and Aggregation of Monoclonal Antibodies during Cation Exchange Chromoatography

“…Fogle et al [39] and Xiao et al [55] extended this work to different HIC resins with varying hydrophobicity using whole protein HX-MS to determine the effects of protein load and kosmotrope concentration of the stability of bound apo-&-lactalbumin. Stability of this protein, as measured by differences in solvent accessibility, was found to increase dramatically with protein load and was nearly undetectable when the stationary phase was nearly completely saturated.…”