(T1530-08-53) The Effects of Aberrant Trisulfide Bond Formation in Therapeutic Proteins for Product Quality and Comparative Analytical Assessments

Purpose: Post translational modifications 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 as some can lead to impurities that negatively impact protein function. One example of a post translational modification is trisulfide formation, which has been identified as a product related impurity in multiple biologic drug products. However, the impact of trisulfide formation on protein aggregation, stability, potential immunogenicity, and overall safety remains undefined.

Methods: Using erythropoietin products for a pilot study, trisulfide levels were monitored via liquid chromatography coupled to mass spectrometry. Size exclusion chromatography and circular dichroism were utilized to detect changes in purity and structure of proteins induced with higher trisulfide levels. Additionally, protein quality was monitored via a THP-1 immunogenicity assay, a stability assay utilizing circular dichroism to monitor protein melting, and current work is focused on utilizing Bio-Layer Interferometry to measure possible changes in receptor binding mediated by increased levels of trisulfide formation.

Results: One erythropoietin product has a detectable level of trisulfide in its formulation and shows increasing trisulfide formation correlated with higher levels of H₂S present. In contrast, a biosimilar erythropoietin has no detectable trisulfide in its formulation; however, trisulfide formation can be induced to a lower level than its biosimilar product with no added affect with increasing H₂S concentration. Overall protein structure is observed to be highly similar with and without H₂S present via circular dichroism. Changes in high and low molecular weight species are detectable in all products with the addition of H₂S via size exclusion chromatography. A 1.45°C decrease in protein melting temperature was observed when treated with H₂S via circular dichroism. No change was observed via a THP-1 Blue NF-κB reporter cell assay as a screening tool for potential immunogenicity.

Conclusion: Our data show that the presence of H₂S can induce higher levels of trisulfides in biologics and differences in formulation may influence those overall levels. When increases in trisulfide species are observed, the overall structure of the protein remains highly similar but a drop in stability of those structures can be detected. At the trisulfide levels we tested, there was not a detectable indication of immunogenicity response in THP-1 cells. Additionally, studies are being conducted to investigate trisulfide content in other pairs of biosimilars to determine trends in trisulfide formation in various therapeutic proteins. Together, these data will provide evidence on the criticality of aberrant trisulfide formation have on overall product quality and comparability assessments.

Acknowledgements: This project was supported in part by an appointment to the Research Participation Program at the Office of Biotechnology Products in the Center for Drug Evaluation and Research at the U.S. Food and Drug Administration (FDA), administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA. The funding source had no part in the study design, data collection/analysis, decision to publish, or preparation of the manuscript, the authors have no conflicts of interests. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the United States Food and Drug Administration and the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.