Quantifying Oligonucleotide Deamination with Multi-Reflecting Time-of-Flight Mass Spectrometry

Importance of the topic

Deamination of cytosine and 5-methylcytosine in synthetic oligonucleotide therapeutics can compromise sequence integrity, reduce efficacy, and increase off-target effects. High-sensitivity methods capable of detecting low-level deamination impurities are essential for quality control, process development, and regulatory compliance in oligonucleotide drug manufacture.

Objectives and study overview

This study demonstrates the identification and quantification of deamination products in a 23-mer antisense oligonucleotide at levels below 1.5%. Using the Waters Xevo MRT high-resolution mass spectrometer, two complementary analytical approaches are applied: global isotopic distribution analysis and site-specific MS/MS fragmentation.

Methodology

Sample Preparation

• A fully phosphorothioated 23-mer antisense oligonucleotide (FLP) and its deaminated counterpart were synthesized and mixed at seven spike levels (0.10–10.00%) in a constant 0.1 mg/mL oligo concentration.
• Replicate injections (n=5) were performed for repeatability assessment.

Chromatography

• UPLC separation used a C18 OST column (2.1 × 50 mm, 1.7 µm) at 60 °C with a 6-minute gradient at 0.050 mL/min.
• Mobile phases consisted of aqueous TEA/HFIP buffer (A) and methanol buffer (B).

Instrumentation

• Waters ACQUITY Premier UPLC System
• Waters Xevo MRT Multi-Reflecting Time-of-Flight Mass Spectrometer operating at 100,000 FWHM resolution
• Negative-ion ESI, MS scan range 50–4000 m/z, and MS/MS fragmentation with collision energy ramp (10–30 eV).
• Data processed in UNIFI and CONFIRM Sequence applications.

Key results and discussion

MS/MS fragmentation localized the deamination site at the b2 ion, resolving the monoisotopic deaminated fragment (m/z 572.09) from the 13C isotopic peak (m/z 572.11) of the unmodified fragment. This allowed confident quantification at 0.5% and 1.5% impurity levels. The isotopic distribution factor (IDF) method measured subtle shifts in isotopic peak heights (P7–P12 vs. P1–P5), achieving excellent linearity (R² = 0.998) across 0.10–10.00% spikes. Repeatability was demonstrated with negligible standard deviation over five injections. The combined workflow reduced analysis time to 6 minutes per sample and enabled processing of 15 injections in under 2 hours.

Benefits and practical applications

• High sensitivity for deamination impurities below regulatory thresholds.
• Orthogonal confirmation via MS/MS and isotopic profiling enhances confidence in results.
• Rapid, high-throughput workflow suitable for QC and process development.
• Minimal sample consumption and reduced method complexity compared to previous approaches.

Future trends and potential applications

Advancements in ultrahigh-resolution instrumentation and real-time data processing will further lower detection limits and analysis times. Integration with automated sample handling and machine-learning-driven data interpretation could expand these methods to monitor a wider range of sequence modifications and impurities in oligonucleotide therapeutics and other biopolymer platforms.

Conclusion

Two complementary high-resolution mass spectrometry strategies—MS/MS fragment localization and isotopic distribution factor analysis—provide a fast, accurate, and robust solution for quantifying deamination in antisense oligonucleotides. This dual-approach workflow streamlines impurity assessment and supports the development and quality control of safe and effective oligonucleotide drugs.

References

• 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 Harmonisation. Guideline Q1A(R2): Stability Testing of New Drug Substances and Products. February 2003.
• Rentel C, DaCosta J, Roussis S, Chan J, Capaldi DC, Mai B. J Pharm Biomed Anal. 2019;173:56–61.