Deciphering Protein-Protein Interactions in Suspension Formulation of Neutral Protamine Hagedorn Insulin Using Hydrogen/Deuterium Exchange-Mass Spectrometry.

Recombinant insulin and insulin analogues have a range of time-action profiles, which are modulated either by incorporating mutations into the native insulin sequence or through modification of formulation compositions. Neutral protamine Hagedorn insulin suspension is an intermediate-acting insulin in which the time-action profile is prolonged by formulation components, driven by insulin-protamine interactions. Precise details of the dynamic interactions of insulin with protamine that influence its absorption kinetics and product stability have not been completely understood. Here, we used hydrogen/deuterium exchange (HDX) mass spectrometry of the crystalline suspension state to assess the structural features governing insulin-protamine interactions. Bottom-up and middle-down HDX revealed that the N-terminal residues B1-11 are critical for interactions, with residues LeuB6 and GlyB8 being predominantly involved in the binding. Additionally, we identified that protamine also protects the molecule from AsnB3 deamidation. Overall, our findings support the unique understanding of insulin-protamine interactions at the molecular level and their role in product-stability mechanisms.