Mass Spectrometric Characterization of Native Insulin Hexamers and Non‐Native Heptamers: Formation and Stability Across Insulin Analogs

Insulin aggregation and oligomerization present significant challenges in both therapeutic formulation and fundamental studies of amyloid formation, particularly due to the transient and heterogeneous nature of early-stage oligomers. Here, we employ ion mobility-mass spectrometry (IM-MS) to characterize the oligomeric distributions of human insulin and two clinically relevant analogs, aspart (rapid-acting) and glargine (long-acting), in excipient-containing solutions designed to stabilize native forms of insulin and under aggregation-inducing conditions. Comparison of insulin analogs revealed distinct aggregation propensities that correlate with each analog's therapeutic design. In general, stable zinc-coordinated hexamers formed most readily in the presence of excipients, and various oligomers formed under aggregation-inducing conditions, with a noticeable prevalence of heptamer formation. We further investigated the structure and stability of native hexamers and non-native heptamers using collision-induced dissociation and collision-induced unfolding experiments. Although the expected single-monomer ejection was the main dissociation pathway for both species, zinc-coordinated hexamers also dissociated into two zinc-adducted trimers, whereas heptamers dissociated into dimer/pentamer and trimer/tetramer pairs. Gas-phase unfolding indicated conservation of subunit tertiary structure in hexamers and no distinct folded structures in heptamer subunits. Overall, this work demonstrates the utility of IM-MS as a rapid, high-resolution platform for probing insulin aggregation pathways and evaluating current and future insulin analog formulations.