CONFIRM Sequence: A waters_connect Application for Sequencing of Synthetic Oligonucleotides and Their Impurities

Significance of the Topic

Synthetic oligonucleotides are emerging as key therapeutic modalities in modern medicine, offering targeted regulation of gene expression. Ensuring the correct sequence and purity of these molecules is critical for their biological activity and safety. Traditional manual interpretation of oligonucleotide fragmentation data is laborious and error-prone. This application note introduces an automated, compliance-ready liquid chromatography–mass spectrometry (LC-MS) workflow, paired with a novel software tool, CONFIRM Sequence, to streamline sequence confirmation of synthetic oligonucleotides and detect low-level impurities down to 0.2% abundance.

Objectives and Study Overview

The primary goal was to evaluate the performance of the CONFIRM Sequence waters_connect application for rapid, accurate sequencing of a heavily modified 21-mer oligonucleotide and its impurities. Key objectives included:

• Demonstrating complete sequence coverage (100%) of the full-length product (FLP) via targeted MS/MS and data-independent acquisition (MSE).
• Evaluating the ability to detect and sequence low-abundance truncated and modified impurities.
• Comparing results on high-performance (Xevo G2-XS QTof) and benchtop (BioAccord) LC-MS platforms.

Methodology and Instrumentation

A 21-mer with nineteen 2′-O-methyl and five 5-methyl modifications was analyzed by ion-pair reversed-phase UPLC coupled to either a Xevo G2-XS QTof or a BioAccord TOF system. Separations were performed on an ACQUITY Premier OST column (2.1×100 mm, 1.7 µm) at 60 °C with HFIP/TEA mobile phases. MS conditions included negative-mode electrospray, full-scan MS (m/z 500–5000), targeted MS/MS with optimized collision voltages, and high-energy MSE ramps. CONFIRM Sequence software modules—Synthetic Library, Monomer Library, Sequence Library, and Modifier Library—were used to define and process oligonucleotide structures and fragmentation data in an automated fashion.

Main Results and Discussion

• Full-Length Product: The 21-mer FLP achieved 100% sequence coverage via targeted MS/MS at an optimized collision energy (63 V) and via a single MSE run (40–60 V ramp).
• Low-Level Impurities: An 11-mer truncated impurity at 0.2% abundance was fully sequenced (100% coverage) using its triply charged precursor (39 V collision voltage).
• Sequence Variants: Isobaric 20-mer variants missing one modified cytidine were distinguished and assigned based on differential fragment coverage (80% vs. 50%).
• Platform Comparison: The benchtop BioAccord system yielded >70% coverage in a single MSE acquisition, demonstrating feasibility on smaller instruments.

Benefits and Practical Applications

• Automated Annotation: Rapid, high-confidence mapping of complex oligonucleotide fragmentation patterns.
• Complete Coverage: Robust sequence confirmation for full-length products and minor impurities without manual interpretation.
• Regulatory Compliance: Streamlined, audit-ready workflow suitable for therapeutic oligonucleotide quality control.
• Versatility: Applicability to both targeted MS/MS and data-independent MSE approaches.

Future Trends and Potential Applications

Automated sequencing tools like CONFIRM Sequence are anticipated to integrate machine-learning algorithms for improved fragment prediction and impurity classification. Expansion to other nucleic acid therapeutics—including siRNA, mRNA, and CRISPR guide RNAs—will enhance analytical throughput. Coupling with higher-resolution ion mobility separations and real-time database searching could further reduce analysis time and increase confidence in sequence assignments.

Conclusion

The CONFIRM Sequence waters_connect application, combined with LC-MS/MS or MSE data, delivers rapid, complete, and reliable sequencing of synthetic oligonucleotides and trace impurities. This automated workflow enhances analytical efficiency, supports quality-by-design, and meets the stringent requirements of therapeutic oligonucleotide development and QC.

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