Characterisation of Antisense Oligonucleotides by Ion-Pair Reversed-Phase UHPLC-HRMS: Method development using Design
Antonio Triolo1, Fabiana Tavani1, Prisca Barnini2
1Laboratory of Advanced Analytics, Menarini Ricerche Spa, Florence, Italy.
Journal of Mass Spectrometry : JMS
|March 19, 2026
Summary
Developing an ion-pair reversed-phase ultra-high-performance liquid chromatography-high-resolution mass spectrometry (IP-RP-UHPLC-HRMS) method optimizes therapeutic antisense oligonucleotide (ASO) quality control. This approach enhances the detection and characterization of impurities, ensuring API safety and efficacy.
Area of Science:
- Analytical Chemistry
- Pharmaceutical Analysis
- Biotechnology
Background:
- Therapeutic antisense oligonucleotides (ASOs) present analytical challenges in quality control due to complex synthesis and degradation.
- Ensuring the safety and efficacy of active pharmaceutical ingredients (APIs) requires robust analytical methods for ASOs.
Purpose of the Study:
- To develop and optimize an ion-pair reversed-phase ultra-high-performance liquid chromatography-high-resolution mass spectrometry (IP-RP-UHPLC-HRMS) method for therapeutic ASO analysis.
- To establish tailored analytical methods for specific ASOs, fomivirsen (FMV) and tofersen (TFR), using Design of Experiments (DoE).
Main Methods:
- Development of an IP-RP-UHPLC-HRMS method utilizing Orbitrap technology with fomivirsen and tofersen as model compounds.
- Preliminary scouting for optimal ion-pair reagents and MS parameters, followed by Design of Experiments (DoE) for in-depth factor evaluation.
- Multicriteria optimization using response surface methodology to define a 'sweet spot' for method performance.
Main Results:
- Two distinct IP-RP-UHPLC-MS methods were established for FMV and TFR characterization, sharing common mobile phase components and MS parameters.
- All four FMV impurities and 13 out of 15 detected TFR impurities were identified above 0.1% threshold.
- The DoE approach successfully balanced sensitivity and selectivity for ASO impurity analysis.
Conclusions:
- The developed IP-RP-UHPLC-HRMS method provides a structured approach for ASO quality control, meeting regulatory requirements.
- DoE is an effective tool for optimizing analytical procedures for oligonucleotides, tailored to specific compounds and ion-pair agents.
- This work paves the way for more efficient and reliable analytical method development in the pharmaceutical industry for ASOs.
Keywords:
antisense oligonucleotidesdesign of experimentsimpurities characterisationion‐pair reversed‐phase UHPLC‐HRMSresponse surface methodologyMore Related Videos
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