Short oligonucleotide therapeutics are an emerging class of biopharmaceuticals to treat a wide variety of diseases, many of which are inaccessible to small molecule- and protein-based approaches. An increase in FDA approvals over the past few years has fueled interest in this molecular class, yet there remains uncertainty on how to define them structurally, since they fall into a regulatory gray area in between small molecules and biologics. Although high-resolution NMR spectroscopy is increasingly being used to characterize the structure of protein-based biologics, to date this technology is only sparingly applied to short oligonucleotide therapeutics, despite the capability of NMR to rigorously evaluate many quality attributes. Here, we demonstrate NMR structural fingerprinting methods using the antisense therapeutic inotersen as our model system. This drug substance is a 20mer with 19 phosphorothioate linkages, leading to a theoretical diastereomer composition of over 500,000 isomers. A shift in this quality attribute has been shown to impact product efficacy. With this case study, we present the sensitivity of the NMR methods to pick up subtle changes to the spectral fingerprint due to shifts in the diastereomer composition. In addition to 1D and 2D NMR experiments that correlate 1H, 13C, 15N, and 31P, the non-native chemistries of this molecular class allowed for more diverse fingerprinting strategies. Taken together, these benchmarking studies provide a pathway for implementation of 2D NMR fingerprinting as a powerful method for the characterization of oligonucleotide therapeutic in the biopharmaceutical laboratory to support assessment of quality attributes at the atomic level, providing greater confidence in the overall chemical and structural integrity of the drug substance.
