Short oligonucleotides are an emerging platform for the development of biopharmaceuticals to treat various diseases, including those that are inaccessible to small molecule- and protein-based approaches. Despite the increasing focus on short oligonucleotide therapeutics, standardized methods to rigorously characterize the structure of these molecules, which is essential for drug development and quality control purposes, are not yet fully established. High-resolution NMR spectroscopy is increasingly utilized to characterize the structure of protein-based therapeutics, including monoclonal antibodies, yet this technology has only sparingly been applied to short oligonucleotides. Recent advances in NMR hardware and methodologies allow for the rapid acquisition of NMR spectra under full formulation conditions and at natural isotopic abundance. Furthermore, the non-native chemistries added to enhance the pharmacological stability of short oligonucleotide therapeutics afford additional NMR-active handles of 19F and 31P, which, at 100 % natural isotopic abundance, are more sensitive than 13C (1.1 %) and 15N (0.36 %). Here we demonstrate the applicability of NMR methods to structurally “fingerprint” short oligonucleotide therapeutics to aid the biopharmaceutical industry and regulators during multiple phases of the clinical pipeline, including discovery, manufacturing, and quality control. The demonstration of NMR fingerprinting methods for short oligonucleotide therapeutics allows for the assessment of quality attributes at the atomic level, providing greater confidence in the overall structural integrity of the drug. This benchmarking study provides a pathway for the implementation of NMR fingerprinting as a high resolution structural technique to complement other analytical approaches used during drug development and quality. 

Learning Objectives:

1. Describe how NMR spectra act as structural “fingerprints” of short oligonucleotide therapeutics.

2. Break down how NMR methods can be applied to a variety of structural features, including both native and non-native chemistries.