An Accurate and Fast P qNMR Assay Method for Oligonucleotide Therapeutics.

Chemically modified nucleic acid molecules have been developed as oligonucleotide therapeutics, and its assay is critical in quality assurance. The common DNA/RNA quantification method using UV-260 nm can lack accuracy because of structure modifications and the possible formation of higher-order structure (HOS). Additionally, process-associated water and counterions affect the accuracy in gravimetric analysis. Thus, to improve accuracy, efficiency, and flexibility, in this work a fast (<1 h) externally referenced P quantitative-NMR (qNMR) method was developed. The qNMR assay results agreed within 1-5% of the UV-260 nm results for the single-stranded DNA standards, confirming the method accuracy. Next, an NMR and UV comparison study was performed on intact oligonucleotide drug products. The P qNMR method showed 7 ± 2%, 8 ± 1%, and 12 ± 1% lower concentration values compared with drug product labels for eteplirsen, inotersen, and inclisiran, respectively. Meanwhile the UV-260 nm results showed 28 ± 3%, 10 ± 3%, and 10 ± 1% lower concentrations than the label for the same three drugs. The agreement between NMR and UV for phosphorothioate (PS)-based inotersen and mostly phosphodiester (PO)-based inclisiran suggest that the labeled concentration may have been obtained using different extinction coefficients. The underestimate of UV results for eteplirsen, which has a phosphorodiamidate morpholino oligomer (PMO) structure, suggests that the UV-260 nm extinction coefficient may need to be re-established for the PMO based oligonucleotide. Therefore, the P qNMR method could be a primary assay method for the oligonucleotide drug and reference standard.