8 Buyers’ Guide 2021
suitable for more sensitive analysis. In addition, it was shown that the optimised parameters could be diverted to each other since a common platform was used in ion introduction, ion optics etc.
Figure 5: Quantitative analysis of Mipomersen-2’-deoxy using TQ-LC-MS: MRM chromatogram (left) and calibration curve (right).
The lower limit of quantifications of all analogues were 2 to 10 times higher than that of QTOF-LC-MS (Table 7). The sample volume used for injection in this measurement was 2 µL, smaller than that of QTOF- LC-MS. These results suggest that MRM measurement by TQ-LC-MS can achieve more sensitive determination.
Summary and conclusion
The results of charge state deconvolution using QTOF-LC-MS suggested that the molecular weight of the impurities generated by elimination or modification of therapeutic oligonucleotide could be confirmed with high mass accuracy of 1 ppm or less. Deconvolution with the ReSpect algorithm could achieve the multiple detection of a target oligonucleotide and the impurities even when they could not be separated by LC and were present in the same spectrum (data not shown). Therefore, more accurate purity determination of therapeutic oligonucleotides is expected even in samples that cannot be detected by conventional chromatographic methods such as LC-UV.
Phosphorothioate modifications have been used in many kinds of therapeutic oligonucleotides to confer resistance to nuclease activity. MRM measurement utilising a PSO2-fragment ion is therefore applicable to a wide range of oligonucleotide therapeutics other than Mipomersen. In this study, the Q-TOF system was shown to have higher resolution and mass accuracy, while the TQ system is more
Figure 6 shows a comparison between the precursor scan and the product ion scan of the LCMS-9030 and the LCMS-8060. Although there is a slight difference in intensity between the valence ions or between the product ions, the main ion species detected were almost the same for both. This makes it possible to use common analytical conditions in all stages of drug discovery, including quality characterisation of therapeutic oligonucleotide, quality control
in drug substance manufacturing, metabolite identification and its concentration measurement, and pharmacokinetic testing. This is expected to contribute to cost reduction in establishing analytical methods.
References
1.Stein C.A., Krieg A.M.
Applied Antisense Oligonucleotide Technology Wiley, ISBN: 978-0-471-17279-6 (1998)
2. Tozaki T., Karasawa K., Ishii H., Kikuchi M., Kakoi H., Hirota K., Kusano K., Nagata S. Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from moiety for doping control. BMC Res Notes, 11, 770 (2018)
3. Hughes J.A., Bennett C.F., Cook P.D., Guinosso C.J., Mirabelli C.K., Juliano R.L.
Lipid membrane permeability of 2′ modified derivatives of phosphorothioate oligonucleotides J Pharm Sci, 84(4), 597-600 (1993)
4. Norris D.A., Post N., Rosie Z Yu R.Z., Greenlee S., Wang Y.
Bioanalysis considerations on the pharmacokinetic evaluation of antisense therapeutics, Bioanalysis 11(21), 1909–1912 (2019)
Figure 6: Comparison of MS1 scan and product ion scan between QTOF-LC-MS (upper) and TQ-LC-MS (lower).
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