Protein or peptide molecules fold into higher order structure (HOS) . Altered HOS could affect therapeutic efficacy and safety, therefore it needs to be characterized. In the process of biosimilar development and regulatory review, the HOS similarity should be demonstrated between a follow-on product and the reference listed drug (RLD). However, quantitative assessment of HOS similarity between the same drug substances (DS) in two different formulations is challenging. We present the Mahalanobis distance (DM) as a measure for quantifying the HOS differences among different brands of currently U.S.-marketed insulin drug products (DP) of insulin lispro and insulin glargine. The DM value was derived from principal component analysis (PCA) of 1H-NMR spectra acquired directly on insulin formulations. The calculated DM values between insulin analog RLDs and their recently approved follow-on products were less than 3.29. A larger DM value of 20.5 was obtained between two approved insulin human DPs by different vendors. To verify whether DS of insulin human is equivalent between the two DPs, the approach of mass-balanced and reversible dialysis to the same buffer were adopted, and the resulting DM value was reduced to 1.19 or less. Thus, the observed range of NMR-PCA derived DM values can be used as a robust and sensitive measure of HOS similarity. Overall, the DM values of 3.3 for DP and 1.2 for DS could be practically achievable similarity metrics to demonstrate a biosimilar’s HOS equivalence to a reference drug.
