Development of a multi-technique analytical strategy for the thermal stability assessment of recombinant humanized...

To ensure the stability and efficacy of recombinant collagen during development, storage, and application, it is essential to establish systematic and reliable stability assessment methodologies. This study developed and validated an integrated multi-technique analytical strategy for the comprehensive evaluation of the thermal stability of recombinant humanized type III collagen (rhCol III). The strategy combines size exclusion chromatography-multi-angle light scattering (SEC-MALS), dynamic light scattering (DLS), circular dichroism spectroscopy (CD), SDS-PAGE, and µLC-MS/MS to perform correlative analyses of physical aggregation, secondary structure, chemical modifications, and biological functions in samples stored under accelerated degradation conditions (40 °C) and control conditions (4 °C) for three months. The results demonstrate that the established strategy can systematically differentiate and quantify degradation pathways under different temperature conditions. SEC-MALS, SDS-PAGE and DLS revealed that high-temperature storage significantly increased the formation of large aggregates (>100 nm), while circular dichroism spectroscopy confirmed that the secondary structure remained largely intact. The core of the strategy, µLC-MS/MS-based post-translational modification (PTM) analysis, quantitatively elucidated key chemical modifications: oxidation (up to 16.86%) and deamidation (up to 12.31%) levels were markedly elevated, whereas proline-4-hydroxylation, essential for structural stability, was substantially reduced (<6%). Key modification sites (e.g., N425, M131, and P144/P132) were successfully localized and found to be enriched in thermally sensitive regions N- and C-termini. This chemical modification profile was highly consistent with functional assay results: samples stored at 4 °C exhibited significantly superior promotion of cell adhesion (+42% vs. +21%) and migration (45.28% vs. 42.05%) compared to those stored at 40 °C. Moreover, the study found that a higher moisture content in the lyophilized samples not only significantly reduced aggregate formation but also affected the levels of PTMs. The analytical strategy developed in this study enables multidimensional and correlative assessment of protein stability. It not only provides a key stability-indicating methodology for rhCol III but also offers an analytical reference framework for stability studies, process optimization, and quality control of biomacromolecular materials.