Toward an Understanding of Protein Conformation during Purification

The conformation of therapeutic proteins can be altered during manufacture, purification, and use, but the molecular mechanisms responsible are poorly understood. We are developing hydrogen-deuterium isotope exchange techniques to provide a residue-level view of conformational changes during protein purification.

In studies of lysozyme precipitation by salts, ammonium sulfate had minimal effects on protein structure while potassium thiocyanate led to partial unfolding and more complex precipitation behaviour. NMR measurements of hydrogen exchange indicated the unfolding occurs mainly in the beta sheet/loop-domain of the protein and increases with salt concentration. Further, NMR and mass spectrometry results show that the precipitate phase can contain a distribution of partially and completely unfolded forms of the protein.

In studies of protein adsorption to reversed phase chromatography (RPC) surfaces, we have analysed the resulting unfolded states of lysozyme and aprotinin. There is generally some protection from isotope exchange, suggesting that residual structure is often retained during RPC. Variations in degree and type of residual native structure may partially explain the inability of chromatographers to predict RPC retention of proteins as they can for small molecules. As a first step toward a theoretical description of these phenomena, a prospective general framework for understanding the effect of conformation on chromatographic performance will be discussed.