Higher Order Structure Differences Among Insulin Crystalline Drugs Revealed by 2D heteronuclear NMR

During therapeutic protein development, two-dimensional (2D) heteronuclear NMR spectra can be a powerful analytical method for measuring protein higher order structure (HOS) in solution since the spectra exhibit much higher resolution than homonuclear ¹H spectra. However, 2D NMR capabilities for characterizing protein HOS in crystalline states remain to be assessed, given the low ¹³C natural abundance and intrinsically broader lines in solid-state NMR (SSNMR). Herein, high-resolution heteronuclear correlation (HETCOR) SSNMR was utilized to directly measure intact crystal drug products of insulin human, insulin analogs of insulin lispro and insulin aspart. The fingerprint regions in 2D ¹H-¹³C HETCOR spectra were identified, which distinguished the insulin crystals in their primary structure, HOS heterogeneity and dynamics, as well as the manufacturing processes. The HOS heterogeneity in insulin analogs is consistent with their therapeutic effect of rapid action; while insulin human crystals showed more structural homogeneity, consistent with their slower pharmacokinetics (PK) peak time than insulin analogs. Therefore, heteronuclear NMR could be broadly applicable to study protein drug dosage forms from liquid to solid, yielding improved molecular level structure data for assessing drug HOS in biosimilar drug development.