TIDES: AN AUTOMATED WORKFLOW FOR INTACT MASS, PURITY AND SEQUENCE CONFIRMATION OF SYNTHETIC OLIGONUCLEOTIDES AND THEIR IMPURITIES

Significance of the topic

Synthetic oligonucleotides are gaining prominence as therapeutic agents due to their high selectivity and ability to modulate gene expression. Ensuring their quality and integrity demands analytical methods that can simultaneously confirm molecular mass, assess purity, and validate sequence identity in a compliance-ready format.

Objectives and study overview

This study presents an automated LC-MS workflow for intact mass confirmation, impurity profiling, and sequence verification of synthetic oligonucleotides, including a heavily modified 21-mer siRNA and its related impurities. The approach integrates two waters_connect applications: INTACT Mass for deconvolution and purity analysis, and CONFIRM Sequence for MS/MS-based sequence coverage.

Methodology

The 21-mer oligonucleotide containing nineteen 2′-OMe modifications was prepared at 1 µM in water, with 10 µL (10 pmol) injected on column. Separation used IP-RP chromatography with a gradient from 25% to 35% organic modifier over 25 min at 60 °C and 0.3 mL/min. INTACT Mass processed full-scan TOF and QTof data via the BayesSpray algorithm for charge deconvolution and impurity assignment. CONFIRM Sequence analyzed MS/MS and MSE spectra at optimized collision energies to deliver graphical dot-map sequence coverage and statistical fragment matching.

Used instrumentation

• BioAccord LC-MS System with ACQUITY Premier UPLC
• ACQUITY Premier OST column (2.1 × 100 mm)
• Xevo G2-XS QTof Mass Spectrometer
• waters_connect INTACT Mass and CONFIRM Sequence software modules

Results and discussion

Eleven impurities were resolved alongside the full-length product. INTACT Mass achieved intact mass accuracy better than 10 ppm down to 0.2% relative abundance. CONFIRM Sequence provided ≥75% MS/MS coverage for all analytes, including 100% coverage for the 21-mer and 95% for the lowest-abundance 11-mer impurity. The combined workflow enabled rapid, high-throughput identification and quantification of oligonucleotide impurities.

Benefits and practical applications

• Automated, compliance-ready data processing reduces manual intervention and supports regulatory requirements.
• High mass accuracy and low-level impurity detection enhance quality control in therapeutic oligonucleotide manufacturing.
• Comprehensive sequence coverage facilitates rapid validation of product identity and impurity characterization.

Future trends and opportunities

Integration of machine-learning algorithms for improved deconvolution and impurity prediction, expansion to multi-omics workflows, and real-time process monitoring represent potential developments. Continued enhancements in sensitivity and software automation will further streamline therapeutic oligonucleotide analytics.

Conclusion

The combined INTACT Mass and CONFIRM Sequence workflow on the BioAccord LC-MS platform delivers an efficient, high-accuracy solution for intact mass confirmation, purity assessment, and sequence validation of synthetic oligonucleotides and their impurities, supporting the evolving needs of oligonucleotide therapeutic development.

References

1. Sharma VK, Watts JK. Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem. 2015;7(16):2221–2242.
2. Waters Corporation. An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides. Application note P/N 720006820EN; 2020.
3. Waters Corporation. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Application note P/N 720007301EN; 2021.
4. Waters Corporation. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using the BioAccord System with ACQUITY Premier System and New Automated INTACT Mass Application. Application note P/N 720007546EN; 2022.
5. Waters Corporation. CONFIRM Sequence: a waters_connect Application for Sequencing of Synthetic Oligonucleotide and Their Impurities. Application note P/N 720007677EN; 2022.