CONFIRM Sequence - A NEW SOFTWARE TOOL FOR SEQUENCE CONFIRMATION AND IMPURITY ANALYSIS OF SYNTHETIC OLIGONUCLEOTIDES

Significance of the Topic

Oligonucleotide therapeutics have become a powerful class of precision medicines, necessitating robust analytical approaches for quality control and impurity profiling. Accurate sequence confirmation and the detection of minute impurities are critical for ensuring safety and efficacy in both regulated and research laboratories.

Objectives and Study Overview

This work presents CONFIRM Sequence, a novel software tool designed for rapid sequence confirmation and impurity analysis of synthetic oligonucleotides. The key goals were to automate data processing for both MS/MS and MSE spectra, achieve high sequence coverage for full-length products and low-abundance impurities, and support streamlined workflows suitable for compliance-driven environments.

Methodology

The analytical workflow combined ion-pair reversed-phase UPLC with high-resolution mass spectrometry. A heavily modified 21-mer oligonucleotide (2′-OMe on 19 nucleotides) was separated on an ACQUITY Premier OST column using TEA/HFIP mobile phases under a 25-to-35 % gradient over 25 min. Mass spectra were acquired in negative ESI mode on a Xevo G2-XS QTof across m/z 500–5 000. MS/MS fragmentation employed fixed collision energies optimized per precursor, while MSE (data-independent fragmentation) used a stepped cone voltage ramp.

Used Instrumentation

• ACQUITY H-Class Bio UPLC with Premier OST column (2.1×100 mm)
• Xevo G2-XS QTof mass spectrometer
• Waters waters_connect software suite and CONFIRM Sequence application
• Milli-Q water purification

Main Results and Discussion

CONFIRM Sequence processed MS/MS and MSE datasets to deliver dot-map based sequence coverage for full-length oligonucleotides (100 %) and for low-abundance impurities (<0.2 % relative) with coverage up to 95 %. The software identified and localized single-nucleotide deletions (e.g., loss of a 2′-OMe cytidine at position 5) and provided statistical metrics for each fragment match. Automated matching of fragment ions facilitated rapid confirmation of eight distinct impurities and the full-length product in a single 40-min run.

Benefits and Practical Applications

• High throughput sequence confirmation in regulated QC environments
• Sensitive detection of low-level impurities and sequence variants
• Support for both targeted MS/MS and untargeted MSE workflows
• Graphical and tabular outputs for streamlined data review

Future Trends and Opportunities

Advancements may include integration of machine learning for automated fragment annotation, expansion to longer and more complex oligonucleotides (e.g., siRNA and sgRNA), real-time data acquisition, and broader support for diverse chemical modifications. Enhanced DIA methods and cloud-based processing could further accelerate workflows and regulatory compliance.

Conclusion

CONFIRM Sequence provides an efficient, automated solution for sequence verification and impurity profiling of synthetic oligonucleotides. It achieves high sequence coverage across a range of product variants and low-abundance impurities, meeting the needs of both research and regulated laboratories.

References

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4. Waters Corporation. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Application note, 2021; P/N 720007301EN.
5. 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. 2022; P/N 720007546EN.
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