LC-MS Analysis of siRNA, Single Guide RNA and Impurities Using the BioAccord™ System with ACQUITY™ Premier and New Automated INTACT Mass Application

Significance of the Topic

Oligonucleotide therapeutics including siRNA and sgRNA play a pivotal role in gene modulation and editing, offering alternatives to small-molecule and protein drugs. Reliable characterization of these molecules and their low-level impurities is critical for manufacturing, quality control, and regulatory compliance.

Objectives and Study Overview

This study presents an automated, compliance-ready LC–MS workflow for intact mass confirmation and purity assessment of small (20-mer) siRNA and larger (100-mer) single guide RNA using the BioAccord System with ACQUITY Premier and the INTACT Mass Application. The work aims to address chromatographic and spectral challenges associated with very long oligonucleotides.

Methodology and Instrumentation

A reversed-phase ion-pairing LC–MS method was developed with the following key components:

• Instrumentation: BioAccord System integrating ACQUITY Premier UPLC, Tunable UV detector, and ACQUITY RDa ESI-TOF mass detector
• Software: waters_connect platform with INTACT Mass Application for automated peak detection, spectral summation, deconvolution (MaxEnt1 and BayesSpray), and impurity searching
• Chromatography: ACQUITY Premier CSH C18 column (1.7 µm, 2.1 × 100 mm) at 50 °C, 300 µL/min; mobile phases containing HFIP/DIPEA in water and acetonitrile
• MS Conditions: negative-ion ESI, 400–5000 m/z range, 2 Hz acquisition, optimized source parameters

Main Results and Discussion

For a 21-mer modified siRNA, the workflow resolved and identified eleven low-level impurities (0.2%–8%) with mass accuracies < 15 ppm using BayesSpray deconvolution. For a 100-mer sgRNA, column screening revealed the Premier CSH column provided superior selectivity, enabling chromatographic separation of a PS to PO desulfurization impurity (Δ16 Da). Automated deconvolution of coeluting charge states allowed detection of four major impurities down to ~ 1% abundance with mass accuracies < 20 ppm. ESI-MS artifact formation was ruled out by observing distinct retention of the desulfurized species.

Benefits and Practical Applications

• Compliance-ready automation reduces manual intervention and supports regulatory requirements
• High mass accuracy (< 20 ppm) ensures reliable confirmation of full-length products and impurities
• Detection of low-level impurities to 0.2% (short oligos) and 1% (long oligos) enhances quality control
• Flexibility to analyze a broad range of oligonucleotide sizes and modifications

Future Trends and Opportunities

Advancements in column chemistries and ion-pair reagents will further improve resolution of long oligonucleotides. Enhanced informatics including machine-learning deconvolution, expanded modification libraries, and higher-throughput LC–MS platforms will streamline QC workflows. Integration with genome-editing applications may drive demand for real-time impurity monitoring and inline analysis.

Conclusion

The BioAccord System with ACQUITY Premier and INTACT Mass Application offers a robust, automated LC–MS solution for intact mass confirmation and impurity profiling of therapeutic oligonucleotides, achieving high mass accuracy and sensitivity for siRNA and sgRNA analyses.

Reference

Doneanu CE, Boyce P, Shion H, et al. LC-MS Analysis of siRNA, Single Guide RNA and Impurities Using the BioAccord System with ACQUITY Premier and New Automated INTACT Mass Application. Waters Corporation, Application Note 720007546, March 2022.