An Accurate\nand Fast <sup>31</sup>P qNMR Assay Method\nfor Oligonucleotide Therapeutics

Chemically modified nucleic acid\nmolecules have been developed\nas oligonucleotide therapeutics, and its assay is critical in quality\nassurance. The common DNA/RNA quantification method using UV-260 nm\ncan lack accuracy because of structure modifications and the possible\nformation of higher-order structure (HOS). Additionally, process-associated\nwater and counterions affect the accuracy in gravimetric analysis.\nThus, to improve accuracy, efficiency, and flexibility, in this work\na fast (<1 h) externally referenced <sup>31</sup>P quantitative-NMR\n(qNMR) method was developed. The qNMR assay results agreed within\n1–5% of the UV-260 nm results for the single-stranded DNA standards,\nconfirming the method accuracy. Next, an NMR and UV comparison study\nwas performed on intact oligonucleotide drug products. The <sup>31</sup>P qNMR method showed 7 ± 2%, 8 ± 1%, and 12 ± 1% lower\nconcentration values compared with drug product labels for eteplirsen,\ninotersen, and inclisiran, respectively. Meanwhile the UV-260 nm results\nshowed 28 ± 3%, 10 ± 3%, and 10 ± 1% lower concentrations\nthan the label for the same three drugs. The agreement between NMR\nand UV for phosphorothioate (PS)-based inotersen and mostly phosphodiester\n(PO)-based inclisiran suggest that the labeled concentration may have\nbeen obtained using different extinction coefficients. The underestimate\nof UV results for eteplirsen, which has a phosphorodiamidate morpholino\noligomer (PMO) structure, suggests that the UV-260 nm extinction coefficient\nmay need to be re-established for the PMO based oligonucleotide. Therefore,\nthe <sup>31</sup>P qNMR method could be a primary assay method for\nthe oligonucleotide drug and reference standard.