(412c) Salt Concentration and Loading Effects on Protein Unfolding during Hydrophobic Interaction Chromatography
AIChE Annual Meeting
2005
2005 Annual Meeting
Bioseparations
Molecular Aspects of Bioseparations
Wednesday, November 2, 2005 - 3:55pm to 4:15pm
Hydrophobic interaction chromatography (HIC) is a separation method often used to separate aggregates and misfolded proteins from their native counterparts. Operating with hydrophobic chromatographic surfaces is desirable to minimize use of salt and maximize capacity. However, these same conditions can lead to protein instability and conformational changes that can compromise product yields.
We are investigating the adsorption and stability of a model protein, α-lactalbumin, during adsorption to HIC surfaces with a variety of experimental and theoretical approaches. Studies are being carried out as a function of (NH4)2SO4 concentration, protein concentration, and stationary phase. The stationary phases being examined include Phenyl Sepharose 6 FF (hi sub and low sub), and Butyl Sepharose 4 FF. Adsorption thermodynamics are being determined from adsorption isotherms obtained in the presence and absence of calcium, a metal ion that stabilizes the native state. Kinetic and thermodynamic parameters for protein folding in solution and intrinsic hydrogen exchange labeling rates have been determined from independent experiments and the literature. The unfolding behavior of α-lactalbumin on the surface is being investigated by hydrogen-deuterium isotope exchange and Raman spectroscopy.
Our experimental results show that increasing salt concentration or surface hydrophobicity lead to more protein unfolding. Notably, protein unfolding decreases markedly with increases in the concentration of adsorbed protein. These results are being analyzed with a model of the HX-MS experiment that incorporates rate and thermodynamic parameters for protein adsorption, unfolding, and isotope labeling. Insights into kinetic and thermodynamic mechanisms of adsorbed protein unfolding behavior as well as implications for protein purification by HIC will be discussed.