Evaluation of global conformational changes in peptides and proteins following purification by supercritical fluid chromatography.

Abstract Supercritical fluid chromatography (SFC) has become the fastest growing analytical tool for chiral and achiral small-molecule pharmaceutical separations. The benefits from savings in cost (as a result of lower solvent and energy consumption), and time have made SFC a proven effective tool for solving many analytical problems for small-molecules over the years. There is, however, a gap in the application of SFC for larger biomolecules, proteins and peptides. There has been a notable increase of protein- and peptide-based drug therapies that contain a higher-order structure important to their efficacy. These studies leverage the use of size exclusion chromatography coupled with hydrogen–deuterium exchange (SEC-HDX) methodology and circular dichroism (CD) spectroscopy to probe global conformational structures of model peptides and proteins following purification by preparative SFC. It was demonstrated that bradykinin and insulin can be used in SFC purification, and moreover, insulin was able to recover its original higher-order structure when compared to pre-purification insulin by three orthogonal techniques: 1) calculated percent alpha-helicity based on CD spectra, 2) alpha-helix – temperature hysteresis analysis by CD and 3) SEC-HDX-MS at different temperatures. However, it was shown that the higher order structures of the other three model proteins used in the study (ubiquitin, cytochrome C, and apomyoglobin) were significantly modified during SFC purification and were unable to re-fold to their original conformations. The present workflow was applied successfully to several peptide therapeutic programs at our comp any and in addition can be applied for small proteins. Highlights • We studied whether higher order structures of the proteins were affected by SFC purification. • We used a preparative SFC method that uses an organic-aqueous blended modifier. • We used a set of model peptides and proteins prepared by this SFC method. • We used combined SEC-HDX and CD to compare proteins' higher order structures before and after purification. • We demonstrated that some small proteins could be recovered to their original structure after purification. [ABSTRACT FROM AUTHOR]