Disulfide Bond Mapping of Follitropin Delta, a Recombinant Follicle Stimulating Hormone (rFSH), at Atomic Resolution by X-Ray Crystallography

Background/Objectives: Follitropin delta is an approved recombinant follicle stimulating hormone (rFSH) expressed in a human cell line. The FSH protein is a heterodimer containing α and β subunits. The FSH α-subunit (FSHα) contains five intramolecular disulfide bonds: C7‑C31, C10‑C60, C28‑C82, C32‑C84 and C59‑C87. The α subunit has four closely spaced cysteine residues that form disulfide bonds. These clustered sites resist protease cleavage, making it difficult to accurately characterize the disulfide bond pairing in this type of protein therapeutic. Methods: The study uses an X-ray crystallography method to determine a high-resolution three-dimensional model structure of the recombinant FSH α-subunit. Results: The crystal structure of FSHα at 2.29 Å resolution, containing the disulfide bonds, is presented. This high-resolution structure provides definitive structural evidence that the disulfide bonds in rFSH are consistent with the expected native structural conformation. Conclusions: The rFSH structure validates the expected cysteine connectivity, overcoming limitations of prior mass spectrometry-based disulfide mapping attempts.