High Resolution LC/MS Analysis of Therapeutic Oligonucleotides on a New Porous Polymer-Based Reversed Phase Column

Significance of the Topic

Therapeutic oligonucleotides including antisense ONs, siRNAs, aptamers and immunostimulatory RNAs offer high target specificity and modular modification capabilities. Regulatory bodies require detailed characterization of sequence integrity and chemical modifications to ensure efficacy and safety.

Objectives and Study Overview

This study aimed to develop a rapid, high resolution LC/MS method for simultaneous analysis of oligonucleotide products, n-1 failure sequences and common modifications such as phosphorothioate linkages, 2’-O-methylation and CpG methylation.

Methodology

Ion-pair reversed phase separation was performed on a new porous polymer DNAPac RP column (2.1×50 mm, 4 μm) using TEA/HFIP mobile phases at controlled pH and temperature. Short gradients of 3 to 5 minutes enabled efficient elution of target ONs and related species.

Used Instrumentation

• Thermo Scientific Dionex UltiMate 3000 BioRS HPLC system
• Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (negative ion mode)

Key Results and Discussion

• Baseline separation of ON products and n-1 failure sequences within minutes, with high mass accuracy (<2 ppm).
• Distinct resolution of phosphorothioate diastereomers on siRNA strands, and separation of four possible 2’-O-methyl PS isomers.
• Clear mass shift detection for CpG methylated ONs, confirming one methyl addition via −3 charge state analysis.

Benefits and Practical Applications

This LC/MS approach facilitates rapid quality control of therapeutic oligonucleotides, enabling precise identification of impurities and modification isomers. Direct MS coupling improves throughput and supports regulatory compliance for ON-based drug development.

Future Trends and Opportunities

• Exploration of advanced stationary phases and alternative ion-pair reagents to further reduce analysis time.
• Integration with tandem MS and data analysis pipelines for automated characterization.
• Extension to more complex ON architectures and emerging chemical modifications.

Conclusion

The combination of a novel porous polymer RP column with TEA/HFIP ion-pair mobile phases and high-resolution Orbitrap MS delivers a fast, robust platform for comprehensive analysis of therapeutic oligonucleotides and their modifications.

References

1. Dias N. et al. Molecular Cancer Therapeutics (2002) 1, 347-355.
2. Resnier P. et al. Biomaterials (2013) 34, 6429-6443.
3. Jones P.A. et al. Nature Reviews Genetics (2002) 3, 415-428.