Analytical characterization of aberrant trisulfide bond formation in therapeutic proteins and their impact on product quality.

Post translational modifications (PTMs) of proteins play an integral role in maintaining the overall structure and function of proteins including their proper folding, binding, and potency. However, not all PTMs play a positive role in protein drugs as some can lead to product-related impurities that negatively impact protein function. One example of a PTM is trisulfide formation, which appears as a product related species in multiple biologic drug products. The impacts of trisulfide formation on protein structure, stability, potency, and safety remains under investigation. Herein, we investigated and report the impact of aberrant trisulfides on erythropoietin (EPO) and somatropin (growth hormone/GH) therapeutic proteins. Utilizing LC-MS we show that one EPO product contains measurable basal levels of trisulfide bonds in its formulation and exposure to HS induced aberrant trisulfides in all products investigated. We report that exposure to HS produces moderate effects on protein stability via thermal melting monitored by circular dichroism, protein purity utilizing size exclusion chromatography, and particle content using micro-flow imaging. No changes were observed in protein folding via circular dichroism, immunogenicity screening via a THP1-blue assay, or receptor binding activity via biolayer interferometry. Together, these data provide evidence on the effects of aberrant trisulfide formation on overall product quality.