Insights into the Structural Coexistence of Hexameric States in Microcrystalline Insulin Formulations in the Solid State

The structural complexity and insoluble nature of solid-state therapeutic proteins pose major challenges for their pharmaceutical quality control. Many conventional analytical methods cannot be applied directly to fully formulated drug products, leaving important aspects of their higher-order structure (HOS) poorly understood. In this work, we use high-resolution, proton-detected solid-state NMR (ssNMR) spectroscopy under fast magic-angle spinning (MAS) to probe the atomic-level HOS of insulin directly in its native formulated state. Recombinant human insulin powder was compared with three commercial Neutral Protamine Hagedorn (NPH) crystalline suspensions: Humulin 70/30, Insugen 70/30, and NovoMix 30. Two-dimensional (2D) 13C–1H correlation spectra showed that the overall insulin fold is conserved across all samples. In contrast, the 2D 15N–1H spectra provided a much more sensitive fingerprint of HOS and revealed clear structural differences. The amorphous insulin powder exhibits a conformationally heterogeneous ensemble characterized by a complex interplay of static disorder, dynamic averaging, and potential partial unfolding reflecting a nonhexameric, T-like architectural state. In contrast, the formulated suspensions show significantly increased 15N chemical shift dispersion, consistent with more rigid, hexameric assemblies. Inspection of the allosteric reporter residues GlyB8 and ThrA8 shows that the NPH crystals are not locked into a single R6 conformation. Instead, multiple allosteric states are present, plausibly reflecting differences in hydration and phenolic ligand binding within the lattice. The NovoMix 30 formulation (insulin aspart) exhibits further line broadening, consistent with increased lattice disorder introduced by the ProB28 → Asp substitution. These observations illustrate how solid-state NMR can resolve formulation-dependent conformational heterogeneity and provide structural support for biosimilarity evaluation in solid-state protein therapeutics.