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

## TL;DR

This study used X-ray crystallography to map disulfide bonds in Follitropin delta, a hormone, confirming its correct structure.

## Contribution

A novel X-ray crystallography approach was used to map disulfide bonds in Follitropin delta, overcoming limitations of mass spectrometry.

## Key findings

- The structure of recombinant FSH was determined at 2.29 Å resolution.
- Electron density clearly defined disulfide bonds in both α and β subunits.
- Cysteine connectivity matches known FSH structures, confirming correct folding.

## Abstract

Background/Objectives: Follitropin delta is an approved recombinant follicle-stimulating hormone (rFSH) expressed in a human cell line. Correct disulfide connectivity is a critical quality attribute for rFSH, a heterodimeric glycoprotein composed of noncovalently associated α and β subunits and stabilized by an extensive network of intramolecular disulfide bonds. Disulfide characterization is typically performed by mass spectrometry (MS). However, the closely spaced disulfide bonds within the FSH α-subunit are particularly resistant to proteolytic cleavage, complicating conventional MS-based disulfide mapping. Methods: To overcome limitations of MS-based methods, an X-ray crystallography strategy was employed using a ternary complex of the recombinant FSH heterodimer with an anti-FSHα Fab and a stabilizing anti-kappa VHH. Crystals of the desialylated rFSH/Fab/VHH complex were obtained and diffraction data were collected. Results: The structure of recombinant FSH was determined at 2.29 Å resolution. Electron density surrounding cysteine residues in both the α and β subunits was well defined, allowing unambiguous assignment of all intramolecular disulfide bonds in the crystallized protein. The observed cysteine connectivity is fully consistent with the disulfide architecture of FSH from other sources and supports correct folding of the recombinant Follitropin delta.