Fig. 1: Thiol–disulphide reaction and stability of insulin icodec.

a Solvent exposure of disulphide bonds in insulin icodec. Colour scheme: light blue, insulin A-chain; dark blue, insulin B-chain; yellow, sulphur atoms forming disulfide bonds. Relative solvent exposure for sulphur atoms in each disulphide bond is indicated. b Schematic representation of thiol–disulphide exchange reaction leading to insulin chain-splitting. RSH represents a thiol group reacting with a disulphide bond in insulin. c High-performance liquid chromatography chromatograms showing human insulin after 4-h incubations at 37 °C under different redox conditions as described in “Methods”. Top panel represents the incubation of human insulin without added glutathione, the middle panel shows human insulin incubated with 0.625 mM GSH and 1 mM GSSG representing a state where ~50% of HI is degraded and the bottom panel shows human insulin incubated with 6.3 mM GSH and 1 mM GSSG. A corresponds to A-chain isoforms containing two internal disulfide bonds; B corresponds to B-chain containing an internal disulphide bond; 2B corresponds to two insulin B-chains forming a dimer via two disulfide bonds; HI corresponds to human insulin. Additional peak assignment and MS spectra of the corresponding species are shown in Supplementary Fig. 2. d Stability of selected insulin analogues exposed to varying redox potential as described in the Methods section, showing mean \(\pm \) SD, n = 3. See the text for a definition of insulin substitutions. The two dashed lines represent a glutathione redox potential in rat and human plasma, respectively. e Stability of selected insulin analogues exposed to varying concentrations of guanidinium hydrochloride as described in “Methods”, n = 1. Source data are provided as a Source Data file.