TIDES: INTACT MASS - A VERSATILE SOFTWARE FOR RAPID MASS CONFIRMATION OF OLIGONUCLEOTIDES AND THEIR IMPURITIES

Importance of the Topic

Recent advances in synthetic oligonucleotide therapeutics have heightened the need for robust analytical workflows capable of precise mass confirmation and impurity profiling. Accurate characterization supports quality control in pharmaceutical development, ensuring safety and efficacy of siRNA and single guide RNA molecules.

Objectives and Overview

This study demonstrates an automated, compliance-ready LC-MS workflow using Waters_connect INTACT Mass software embedded on the BioAccord platform. It aims to confirm intact mass of small interfering RNAs (21-mer) and larger single guide RNAs (100-mer), while identifying and quantifying low-level impurities down to 0.2% abundance.

Methodology and Instrumentation

All analyses were performed on a BioAccord LC-MS system coupled to an ACQUITY Premier UPLC with a 2.1×100 mm Premier OST column. Ion-pair reversed-phase separations employed volatile reagents (TEA/HFIP for 21-mers, DIPEA/HFIP for 100-mers) under optimized gradients. Key instrument settings included 0.3 mL/min flow, 60 °C column temperature, and negative ESI-MS detection. The INTACT Mass software applied Bayesian deconvolution across chromatograms for automated spectrum processing and impurity assignment.

Main Results and Discussion

Chromatographic separations achieved clear baseline resolution of major oligonucleotides and their impurities, with the Premier OST and CSH columns showing superior performance. The INTACT Mass workflow delivered mass accuracy better than 10 ppm for the 21-mer and better than 20 ppm for the 100-mer samples. Eleven impurities were identified for the 21-mer, the lowest at 0.2% abundance, while four closely related 100-mer impurities were detected down to ~1%. Comparison of three column chemistries highlighted the CSH phase as optimal for resolving single-modification variants.

Benefits and Practical Applications

• Automated, compliance-ready deconvolution accelerates impurity profiling across entire chromatograms.
• High mass accuracy supports reliable confirmation of modified oligonucleotides.
• Detection of low-abundance impurities enhances quality control in therapeutic oligo production.
• Seamless integration within standard LC-MS platforms facilitates routine implementation in QC labs.

Future Trends and Applications

Oligonucleotide analytics is expected to evolve with expanding molecular complexity, including longer guide RNAs and diverse chemical modifications. Future developments may focus on deeper impurity characterization via high-resolution MS, enhanced automation, and integration with data management systems to streamline regulatory compliance.

Conclusion

The INTACT Mass workflow on the BioAccord LC-MS platform enables rapid, accurate intact mass confirmation and impurity analysis of both small and large oligonucleotides. With mass accuracies <10–20 ppm and sensitivity to sub-percent impurities, this approach offers a robust solution for quality control in 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. An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides. Waters application note, P/N 720006820EN; 2020.
3. Intact Mass Confirmation Analysis on the BioAccord LC-MS System for Extensively Modified Oligonucleotides. Waters application note, P/N 720007028EN; 2020.
4. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Waters application note, P/N 720007301EN; 2021.
5. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using BioAccord and INTACT Mass Application. Waters application note, P/N 720007546EN; 2022.