On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column

This study documents the use of an integrated approach, involving on-line hydrophobic interaction chromatography interfaced with Small Angle X-ray Scattering (HIC–SAXS) measurements, to monitor the conformational status of proteins immediately upon elution from a chromatographic column operated at different temperatures. Moreover, this approach provides an additional avenue to interrogate the changes in protein shape that may occur across the eluted chromatographic peak. To this end, radii of gyration were extrapolated from the Guinier approximation with the HIC–SAXS data, whilst pair distribution functions and bead model simulations were generated by using the indirect transform program GNOM and ab initio reconstruction with GASBOR to provide further insight into protein conformational changes that occur during hydrophobic interaction chromatography.