Investigation of Oligonucleotide Deamination Using High Resolution Mass Spectrometry

Significance of the Topic

Oligonucleotide deamination, a common degradation pathway affecting cytosine and 5-methylcytosine residues, leads to uracil and thymine formation. This reaction can compromise the stability and efficacy of therapeutic oligonucleotides, introducing impurities that may trigger off-target effects. Accurate quantification of deamination is therefore essential for quality control and the development of safe, effective nucleic acid therapies.

Objectives and Study Overview

This investigation aimed to establish a high-resolution LC-MS assay for quantifying deaminated oligonucleotide impurities. By spiking a synthetic antisense phosphorothioated oligonucleotide impurity into its non-deaminated counterpart at levels from 0.10% to 5.00%, the study evaluated the use of isotopic distribution factors (IDF) derived from overlapping isotope patterns to estimate relative deamination content.

Methodology and Instrumentation

Samples were prepared by adding defined amounts of deaminated impurity to the full-length product. Chromatographic separation employed an ACQUITY Premier UPLC Binary Solvent Manager coupled with an ACQUITY Premier OST column, using an ion-pairing reversed-phase gradient (5 mM tri-butyl ammonium acetate with EDTA in 10–80% acetonitrile) at 0.25 mL/min, 50 °C, and 3.7 min runtime. Mass spectrometry analysis was conducted on a Xevo G3 QTof in negative ESI mode (m/z 400–4000), with optimized voltages and temperatures. Data processing and centroiding utilized waters_connect software.

Instrumentation

• ACQUITY Premier UPLC Binary Solvent Manager
• ACQUITY Premier OST Column (2.1 × 50 mm)
• Xevo G3 QTof Mass Spectrometer

Key Results and Discussion

High-resolution spectra revealed overlapping isotopic distributions of full-length and deaminated molecules. The IDF, defined as the ratio of summed peak intensities (isotopic peaks 6–12 versus peaks 1–4), provided a reliable measure of deamination. The assay demonstrated excellent linearity across 0.10–5.00% impurity levels and highly reproducible IDF values over replicate injections and controls. Analysis of six spiked samples and their controls was completed in under 150 minutes, highlighting the method’s throughput and robustness.

Benefits and Practical Applications

The developed LC-MS assay offers precise quantification of oligonucleotide deamination, supporting stability studies, batch release testing, and stress-testing protocols in pharmaceutical development. Its rapid runtime and consistency make it suitable for routine quality assurance in research and industrial laboratories.

Future Trends and Applications

Advances may include multiplexed detection of multiple modification types, integration with automated sample handling and data analysis platforms, and application of machine-learning algorithms for enhanced spectral deconvolution. Expansion to other nucleic acid therapeutics and non-standard bases could further broaden the assay’s utility.

Conclusion

This work establishes a high-resolution mass spectrometry-based approach for accurate, reproducible measurement of oligonucleotide deamination. The IDF metric, coupled with robust chromatography and data processing, delivers a fast, sensitive tool for ensuring the quality of therapeutic oligonucleotides.

References

• Rentel C, DaCosta J, Roussis S, Chan J, Capaldi DC, Mai B. Journal of Pharmaceutical and Biomedical Analysis. 2019;173:56–61.
• Capaldi DC. Stress testing of oligonucleotides. In: Baertski SW, Alsante KM, Reed RA, editors. Pharmaceutical Stress Testing. 2nd ed. Informa Healthcare; 2011. p. 391–425.
• International Council for Harmonization (ICH). Q1A(R2): Stability Testing of New Drug Substances and Products. February 2003.