[3P-62] SEC-SAXS法による溶液中の抗体変性構造の分離観測
Irreversible aggregation of biopharmaceutical monoclonal antibodies proceeds by collision and association of the non-native species whose aggregation-prone structures remain unknown. The non-native species are rarely populated at moderate temperatures and solution conditions. This study used a chemical denaturant to accumulate the non-native species and characterize their structures. A fluorescence spectroscopic analysis demonstrated that the unfolding proceeded for hours in the presence of 3 M guanidine hydrochloride (GdnHCl). A size exclusion chromatography (SEC) identified two elution peaks. The early elution peak (M1 peak) area increased, and the late elution peak (M2 peak) area decreased during the unfolding. A small-angle X-ray scattering coupled with SEC (SEC-SAXS) assigned both the elution peaks to the monomer. The SAXS at M1 and M2 peaks depicted the characteristic signals in the Kratky plots originating from the native-like ordered orientations and distances between Fab-Fc regions or Fab-Fab regions. The unfolding by 3 M GdnHCl does not involve a change to a flexible chain-like disordered structure but a local structural change.