Comprehensive characterization of higher-order structure (HOS) is a regulatory prerequisite for monoclonal antibody (mAb) therapeutics, particularly in establishing biosimilarity. A significant analytical challenge lies in detecting the early onset of structural heterogeneity within bulk drug substances, where signal dilution often masks precursor states of degradation. This study proposes a novel, multitechnique spectroscopy platform consisting of UV-vis, Far-UV circular dichroism, intrinsic and extrinsic fluorescence, and Fourier transform infrared (FTIR) spectroscopy coupled with chemometrics for monitoring heterogeneities in HOS during the thermal degradation of trastuzumab, an IgG1mAb therapeutic. While size exclusion chromatography (SEC) confirmed a progressive, monotonic accumulation of soluble aggregates, the proposed multitechnique spectroscopic platform yielded richer information. Intrinsic and extrinsic fluorescence identified an early conformational drift characterized by tertiary core destabilization and hydrophobic exposure immediately upon thermal stress (<1 h), well before the onset of global backbone unfolding detected by circular dichroism. Data fusion via principal component analysis (PCA), biplot, and dendrogram allowed for a more nuanced understanding of the HOS evolution through the thermal stress timeline. The data-fusion approach explained in this study offers a robust, high-resolution framework for monitoring structural consistency through direct HOS mapping, offering a powerful solution for monitoring the quality of biotherapeutic products during stability studies as well as for biosimilarity assessments.
Agrawal et al. (Sat,) studied this question.