Scientific Abstract

Characterization of Trisulfide in Insulin-like Growth Factor Binding Protein-3 and Structure/Function Assessment (WCBP)

Scientific Event

January 28-31, 2019
Washington D.C.


Chen Li1, Serah Liu1, Paul A. Salinas2, Justin Prien2, Jennifer S. Chadwick1, Chris Barton2, Andrew O’Keefe2, Bernice Yeung2, Shiaw-Lin Wu1
1BioAnalytix Inc., Cambridge, MA; 2Shire, Lexington, MA.


Disulfide bonds in protein biopharmaceuticals can be susceptible to incorporation of a third sulfur atom to form a trisulfide bond during bioproduction or when otherwise exposed to hydrogen sulfide (H2S). Disulfide bonds are important for ensuring the physical stability and/or bioactivity of protein drugs and, if altered, there is potential to impair interactions with binding partners or the pharmacological target. As such, accurate characterization of disulfide linkages in a biotherapeutic is critical to ensuring product quality during clinical development.  Mecasermin rinfabate is a two-protein drug being developed to treat developmental complications of prematurity and is comprised of the pharmaceutically active component recombinant human Insulin-like Growth Factor-1 (rhIGF-1) plus its carrier protein recombinant human Insulin-like Growth Factor Binding Protein-3 (rhIGFBP-3). We previously developed a novel LC-MS/MS-based method to elucidate for the first time the disulfide-bonded pairs in rhIGFBP-3, and comprehensive analysis revealed the presence of a trisulfide in the N-terminal region. Here, detailed characterization, including quantitation, of the trisulfide bond in rhIGFBP-3 using the developed LC-MS method is reported. To understand potential impact of trisulfide in rhIGFBP-3 on biological activity of rhIGF-1, mecasermin rinfabate was treated with increasing levels of H2S to increase the level of trisulfide variants. The treated materials, along with a non-treated control, were examined by chromatographic- and activity-based release and characterization methods, and the assay results analyzed to identify correlations between the assays and demonstrate the impact of the trisulfide variant on structure and function.  No diminution in bioassay activity, binding affinity to rhIGF-1 or dissociation of the complex was observed with elevation of trisulfide levels. This exemplifies how detailed structural characterization and subsequent functional studies can then inform biotherapeutic product development.

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