Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Abstract

Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50jC exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible. B 2004 Wiley Periodicals, Inc.

Manufacturing of recombinant therapeutic proteins in microbial systems

“…A wide variety of resins with different base matrices, ligands and ligand densities are available [176,177]. The nature of the matrix in combination with product properties and buffer composition [178][179][180][181] strongly influence yield and success of HIC steps for a given task. Lowering the conductivity in a stepwise and/or linear gradient mode triggers elution.…”

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography

“…The conformational behavior of α-chymotrypsinogen A on Butyl Sepharose ™ resin was previously investigated by Tibbs-Jones and Fernandez [38] using HX-MS. The solvent exposure of the adsorbed protein was observed to increase upon adsorption, although to a lesser extent than in the fully-labeled control experiment.…”

“…The stabilities of these latter two proteins are greatly reduced on the chromatographic surface relative to the solution phase. Another model protein, hen egg white lysozyme, has been shown to be conformationally stable on HIC resins in independent studies [23,38]. Though it is a structural homologue of α-lactalbumin [58], the reported structural stability of lysozyme (43.1 kJ/mol) [56] is substantially greater than that of holo α-lactalbumin.…”

“…Coupling hydrogen exchange with mass spectrometry (HX-MS) has allowed the efficient determination of conformation change of adsorbed protein, and insights into the effects of stationary and mobile phase properties on protein conformation during HIC have been gained [18,36–38]. Application of this technique to the quantitative description of adsorbed protein unfolding has been limited.…”

“…Application of this technique to the quantitative description of adsorbed protein unfolding has been limited. Tibbs-Jones et al [38] quantified the free energy of unfolding of a stable protein in the adsorbed phase, while Fogle et al [36] characterized unfolding kinetics via an empirical rate model.…”