A Xevo™ G3-based Workflow for Purity Determination, Intact Mass Measurement, and MS/MS Sequencing of Impurities Detected in Synthetic Oligonucleotides

Importance of the Topic

Accurate characterization of synthetic oligonucleotides and their impurities is crucial for ensuring the safety, efficacy, and regulatory compliance of emerging therapeutic agents such as antisense oligonucleotides (ASO) and siRNA. Advanced analytical workflows that combine high-resolution intact mass measurement with detailed MS/MS sequencing can detect trace-level synthesis artefacts and degradation products, providing comprehensive impurity profiles that inform process development and quality control.

Objectives and Study Overview

This application note demonstrates a unified workflow based on the Xevo G3 QTof mass spectrometer coupled to an ACQUITY Premier UPLC System and integrated waters_connect informatics. The goals are to measure intact oligonucleotide masses, detect and quantify low-abundance impurities, and confirm their sequences via both data-independent (MSE) and targeted MS/MS approaches.

Methodology and Instrumentation

A 21-mer synthetic siRNA with multiple 2’-O-MOE and 5-methyl modifications was analyzed at 10 pmol injection. Reversed-phase UPLC separation used an ACQUITY Premier OST column with TEA/HFIP buffers and a gradient to resolve the full-length product (FLP) and 16 related impurities. Electrospray ionization in negative mode and high-resolution QTof detection captured intact spectra, while both targeted MS/MS and MSE fragmentation modes provided sequence-specific fragment ions.

Used Instrumentation

• ACQUITY Premier UPLC System with OST 2.1 × 150 mm, 1.7 µm CSH C18 column at 60 °C.
• Xevo G3 QTof mass spectrometer operating in ESI(-) mode with capillary 2.8 kV, desolvation at 450 °C, and CE ramp 15–45 V for MSE.
• waters_connect software: INTACT Mass App v1.8.0.10 and CONFIRM Sequence App v1.4.0.13.

Main Results and Discussion

The ACQUITY Premier column delivered robust separation of 16 low-level impurities down to 0.05% relative UV response. The INTACT Mass App performed automated deconvolution of intact spectra, assigning monoisotopic masses with <10 ppm error. Targeted MS/MS sequencing of the FLP achieved 100% coverage, and analysis of six impurity ions provided sequence confirmation with 83–96% coverage. The lowest-abundance 6-mer phosphorylated impurity (0.05%) was confidently identified through combined intact mass and MS/MS dot-map visualization.

Benefits and Practical Applications

By integrating high-resolution MS, automated deconvolution, and sequence annotation:

• Low-level synthesis impurities can be detected and quantified automatically.
• Full-length and truncated oligonucleotides are confirmed with high sequence coverage.
• Workflow is suitable for both regulated and non-regulated labs for development and routine quality assays.

Future Trends and Opportunities

Ongoing innovation in chromatographic surfaces and informatics algorithms will further enhance sensitivity for diverse modifications and even larger oligonucleotides. Deploying AI-based peak annotation and cloud-enabled data sharing promises faster characterization and streamlined regulatory submissions. The trend toward multiplexed or high-throughput oligo profiling could support next-generation oligonucleotide therapeutics pipelines.

Conclusion

The Xevo G3 QTof–UPLC workflow with waters_connect INTACT Mass and CONFIRM Sequence Apps provides a comprehensive platform for intact mass measurement, impurity quantitation, and sequence confirmation of synthetic oligonucleotides. This approach delivers robust, automated, and high-confidence impurity profiling, supporting therapeutic oligonucleotide development and quality control.

Reference

• Sharma VK, Watts JK. Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem. 2015;7(16):2221–2242.
• Roberts TK, Langer R, Wood MJA. Advances in oligonucleotide drug delivery. Nat Rev. 2020;19:673–694.
• Doneanu CE, Fox J, Williams BJ, Knowles C, Yu YQ. An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides. Waters Application Note. 2020.
• Doneanu CE, Knowles C, Fox J, et al. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Waters Application Note. 2021.
• Doneanu CE, Boyce P, Shion H, et al. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using the BioAccord System. Waters Application Note. 2022.
• Doneanu CE, Knowles C, Gorton M, et al. CONFIRM Sequence: A waters_connect Application for Sequencing of Synthetic Oligonucleotides and Their Impurities. Waters Application Note. 2022.