Insulin is digested under non-reducing conditions and analysed by LC/ES-MS using a Q-TOF mass spectrometer. Bridging patterns can be assessed in MS mode and signals verified by fragment ions generated by MSe
An assessment of disulfide bridge mis-matching or scrambling is particularly important for products manufactured using E. coli cell systems where the disulfide bridging has to be created post translationally using a chemical process, rather than happening co-translationally (as is the case in mammalian cells).
With all Cysteine containing biopharmaceuticals, disulfide bridge analysis should also be used to assess for other Cysteine containing peptide forms such as mismatched disulfide bridged peptides, trisulfide bridges and thioether linkages. The regulatory guidelines define product containing these forms as product-related impurities that must be identified.