Separation of Impurities from a Fully Phosphothioated and 2’-O-methylated RNA Using Ion-Pair Reversed- Phase Chromatography

Importance of the Topic

Oligonucleotides with phosphorothioate (PS) linkages and 2’-O-methyl modifications are widely investigated as therapeutics for viral infections, neurodegenerative and cardiovascular diseases. Quality control of such molecules requires efficient chromatographic methods to detect oxidation by-products (phosphodiester, PO) and chain-length truncations for safety and efficacy assurance.

Objectives and Study Overview

This work evaluates ion-pair reversed-phase (IPRP) chromatography to separate a fully phosphorothioated, 2’-O-methylated 40mer RNA (REP 2139) from PO and n-1, n-3, n-5 truncation impurities. Key parameters—ion-pair reagents, temperature, pH, organic modifier and EDTA additive—were systematically investigated on a polymer-based reversed-phase column.

Methodology and Instrumentation

• Sample: REP 2139 (40mer RNA, alternating adenosine and 5-methylcytidine, fully PS, all riboses 2’-O-methyl) and its 1–3 PO and n-1, n-3, n-5 impurities.
• HPLC system: Thermo Scientific Vanquish Quaternary Pump F, Split Sampler FT, Column Compartment H with pre-column heater and post-column cooler, Diode Array Detector HL, Chromeleon CDS.
• Column: DNAPac RP, 4 µm, 3.0×100 mm, capable of high pH/temperature.
• Mobile phases: A = water, B = acetonitrile (or methanol), C = 0.2 M TEA-acetate pH 7.0 (or alternative ion-pair buffers), D = 50 mM EDTA pH 7.3.
• Variables: ion-pair reagents (triethylamine, hexylamine, dibutylamine, diethylmethylamine), temperature (80–110 °C), pH (5.9–9.4), organic modifier, 5 mM EDTA.

Main Results and Discussion

Triethylamine-acetate at pH 7.0, 100 °C, acetonitrile and 5 mM EDTA delivered baseline separation of n-1 and 1 PO from the full-length product, enabling accurate impurity quantitation. Stronger hydrophobic ion-pair reagents reduced resolution, while weaker amines induced diastereomer splitting, hindering quantitation. Elevated temperature sharpened peaks up to 100 °C; at 110 °C resolution declined and elution order shifted. High pH caused partial diastereomer separation, and methanol as organic modifier reversed impurity elution and decreased overall resolution. EDTA addition collapsed metal-stabilized conformers, improving peak shape and impurity integration.

Benefits and Practical Applications

• Reliable quantification of oxidation and truncation impurities in PS-modified RNA therapeutics.
• Rapid method development using quaternary pump to screen mobile phases and additives.
• Enhanced peak sharpness and resolution under elevated temperature and EDTA conditions.
• Applicable to quality control in oligonucleotide manufacturing and stability studies.

Future Trends and Opportunities

• Integration of mass spectrometry detection to confirm impurity identities and improve sensitivity.
• Exploration of novel stationary phases tailored for large nucleic acid polymers.
• Use of automated method scouting software for multi-parameter optimization.
• Development of one-run multi-analyte assays combining PS diastereomers, PO species and chain-length variants.

Conclusion

Optimal separation of PS RNA impurities was achieved with triethylamine-acetate at pH 7, 100 °C, acetonitrile and 5 mM EDTA. This protocol offers robust impurity quantitation in fully phosphorothioated, 2’-O-methylated RNA but does not resolve all diastereomeric PO species individually. The method can be adapted for broader oligonucleotide QC workflows.

References

• J.R. Thayer, Y. Wu, E. Hansen, M.D. Angelino, S. Rao, Journal of Chromatography A, 1218, 802–808 (2011).
• L. Li, T. Leone, J.P. Foley, C.J. Welch, Journal of Chromatography A, 1500, 84–88 (2017).
• A. Vaillant, Antiviral Research, 133, 32–40 (2016).
• Thermo Fisher Scientific Application Note AN21476 (2016).
• L. Gong, J.S.O. McCullagh, Rapid Communications in Mass Spectrometry, 28, 339–350 (2014).