Jiayi Li, Fu Chen, Deyi Zhang, Yan Wang, Darby Kozak, Kang Chen
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引用次数: 0
Abstract
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 31P 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 31P 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 31P qNMR method could be a primary assay method for the oligonucleotide drug and reference standard.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.