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The prevalence of diabetes is steadily increasing, prompting the widespread use of various commercially available insulin analogs products in clinical practice. Assessing the chemical and structural stability of these pharmaceuticals should be carefully addressed, as their long-term efficacy and safety are intrinsically linked to chemical degradation pathways, such as deamidation [1]. Currently, it is unknown exactly which positions and to what extent deamidation occurs in the various insulin analogues. Therefore, identifying the most frequently occurring deamidated forms and thoroughly investigating their potential physiological effects is of particular importance [2].
Our research group successfully separated the deamidated forms of various insulin analogs, as well as distinguishing them from the native active pharmaceutical ingredient, using capillary zone electrophoresis [3]. We employed collision-induced dissociation (CID) to fragment the deamidated forms of human insulin during their separation. Although CID is a widely used technique, its efficiency is limited in this context: fragmentation events occurring in the region enclosed by the disulfide bridges do not result in a change in m/z, as the disulfide bonds hold the structure together. Terminal fragments outside of this region allow the straightforward identification of some deamidation sites. Appropriate low-abundant internal fragments had to be selected to identify the rest of the deamidation sites. Overall, we were able to separate human insulin from four singly deamidated forms and identified the site of deamidation for each component.
[1] G. Wilcox, Clin. Biochem. Rev. 2005, 26, 19.
[2] D. Gervais, J. Chem. Technol. Biotechnol. 2016, 91, 569-575.
[3] M. Andrasi, B. Pajaziti, B. Sipos et al. J. Chromatogr. A. 2020, 1626, 461344.
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