Measurement of Oligonucleotide Impurities Using Shimadzu Nexera XS Inert Coupled To LCMS-9030 QTOF

Importance of the Topic

Oligonucleotide therapeutics, including antisense and siRNA agents, are gaining prominence in precision medicine. Characterizing their purity and identifying synthesis-related impurities is critical to ensuring safety, efficacy, and regulatory compliance. High-resolution mass spectrometry (HRMS) offers the sensitivity and accuracy required to profile the main product and minor by-products in these complex biomolecules.

Objectives and Study Overview

This study demonstrates an end-to-end workflow for quantifying oligonucleotide impurities. By spiking known short-mer impurities into a 20-mer antisense oligonucleotide (Tofersen) as a Full-Length Product (FLP), the approach evaluates assay performance in terms of repeatability, intermediate precision, and accuracy according to ICH Q2 (R2) guidelines.

Methodology and Instrumentation

Sample Preparation:

• A 5 % n-1 (19-mer) impurity was spiked into the FLP and diluted to 20 µM in nuclease-free water.
• Samples were loaded into Shimadzu TORAST-H Bio vials for injection.

Chromatography and Mass Spectrometry:

• UHPLC: Shimadzu Nexera XS Inert system with Shim-pack Scepter Claris C18-300 column (2.1×100 mm, 1.9 µm).
• Mobile phase: 100 mM HFIP/10 mM TEA in water (A) and methanol (B), gradient elution over 20 min at 0.3 mL/min, 60 °C.
• MS: LCMS-9030 QTOF in negative DDA mode, m/z 700–2200, heated ESI source.
• Data processing: LabSolutions Insight Biologics software for deconvolution, impurity quantification, and sequence coverage mapping.

Results and Discussion

Over three days, six replicates per day were analyzed. Recovery rates ranged from 84 % to 93 %, meeting the 80–120 % criterion. Repeatability (intra-day RSD) was below 1 % and intermediate precision (inter-day RSD) was under 1.2 %. Mass score consistency (0.91–1.00) confirmed reliable spectral quality. Chromatograms displayed clear separation of FLP and n-1 impurities, while deconvoluted spectra and MS/MS sequence coverage (fill and branch modes) verified product identity and minor species.

Benefits and Practical Applications

This integrated UHPLC-QTOF workflow enables:

• Rapid, sensitive detection of short-mer and other impurities in oligonucleotides.
• Regulatory-grade quantification aligned with ICH Q2 (R2) requirements.
• Automated data analysis and reporting via specialized software, reducing manual interpretation.

Future Trends and Potential Applications

Advances may include higher-throughput columns, enhanced bioinert coatings, and ion mobility integration for improved isomer separation. AI-driven spectral interpretation could accelerate impurity profiling, while multiplexed HRMS platforms may support simultaneous analysis of multiple oligonucleotide candidates in drug development pipelines.

Conclusion

The combination of Shimadzu Nexera XS Inert UHPLC, Shim-pack Scepter Claris C18-300 column, LCMS-9030 QTOF, and LabSolutions Insight Biologics software delivers a robust, precise, and compliant solution for oligonucleotide impurity analysis, supporting pharmaceutical research and quality control.

Reference

1. Shimadzu Application Note 01-00595-EN: Oligonucleotide Impurity Analysis Workflow Using LabSolutions Insight Biologics Software.
2. Sutton JM, Guimaraes GJ, Annavarapu V, van Dongen WD, Bartlet MG. Current State of Oligonucleotide Characterization Using Liquid Chromatography–Mass Spectrometry: Insight into Critical Issues. J Am Soc Mass Spectrom. 2020;31:1775–1782.