Assessing the Purity of an Antisense Oligonucleotide Sample by LC/MS

Significance of the Topic

Antisense oligonucleotides (ASOs) are a critical class of therapeutic biomolecules requiring rigorous purity assessment for safety and efficacy. Liquid chromatography–mass spectrometry (LC/MS) is the preferred approach to detect and quantify low-level impurities in synthetic oligonucleotides, supporting quality control in biopharmaceutical manufacturing.

Objectives and Study Overview

This application note presents a robust workflow for routine ASO purity testing using a unit mass detector. The study demonstrates how the Agilent InfinityLab Pro iQ Plus LC/MS system, combined with specialized software, enables full-scan mass analysis and impurity profiling without relying on high-resolution MS systems.

Methodology

Samples of a 20-mer ASO were prepared at 0.1 mg/mL and analyzed by reversed-phase ion-pair chromatography. The LC gradient and mobile phases were optimized to elute oligonucleotides in their 4– charge state. A full-scan MS range around m/z 1,750–2,150 captured the target ion distribution.

Used Instrumentation

• Agilent InfinityLab Pro iQ Plus LC/MS (G6170A single quadrupole)
• Agilent 1290 Infinity II bio binary pump, multisampler, column compartment
• Agilent 1260 Infinity II diode array detector HS

Main Results and Discussion

The system maintained stable ion transmission and mass axis performance over 24 hours at m/z 2,234, confirming robustness. Spectral overlays under “standard” and “harsh” electrospray conditions distinguished true product ions from adducts. Using the integrated Oligo Analysis Accelerator software, 25 ions exceeding a 0.2% threshold were classified, including full-length ASO, n–1 and n+1 species, depurination and alkylation by-products. Extracted ion chromatograms (EICs) quantified each impurity, yielding an MS purity of 86.9% versus 98.0% by UV.

Benefits and Practical Applications of the Method

• Routine QC testing without the need for high-resolution MS
• Automated peak integration and impurity classification
• Quantitative sensitivity down to 0.2% impurity levels
• Improved robustness for high-mass analytes

Future Trends and Opportunities

Advances may include higher-throughput workflows, integration with high-resolution detectors for confirmatory analysis, and further software automation for in-line QC. Expanding the mass range and enhancing ion transmission could support longer oligonucleotides and complex modifications.

Conclusion

The described LC/MS workflow using a unit mass detector provides reliable, sensitive purity and impurity profiling for antisense oligonucleotides. The Agilent InfinityLab Pro iQ Plus system coupled with dedicated software streamlines routine QC testing, balancing robustness and operational simplicity.

References

1. Rentel C., Gaus H., Bradley K., et al. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair–HPLC–MS. Nucleic Acid Ther. 2022;32(3):206–220. DOI: 10.1089/nat.2021.0056