Characterization of Insulin Dimers by Top-Down Mass Spectrometry

High-molecular weight products (HMWP) are an important critical quality attribute in research and development of insulin biopharmaceuticals. We here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites. On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains. The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links. Identification of crosslink sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds. UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer. Thus, using varied multistage approaches, it was discovered that in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain. In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-. The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds. Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Automated summary

High-molecular weight products (HMWP) are critical quality attributes in insulin biopharmaceutical research and development. This study demonstrates the de novo characterization of cross-linked insulin dimers using top-down mass spectrometry workflows, including the efficient cleavage of interchain disulfide bonds and the identification of cross-link connectivities between chains. Various multistage approaches were utilized to identify the chemical composition and cross-link sites within the dimers, revealing specific cross-link patterns in different induced dimers.