Comprehensive, Automated, and Integrated Software for Oligonucleotide Characterization and Sequence Confirmation

Significance of the Topic

Oligonucleotide-based therapeutics such as siRNA, antisense oligonucleotides, aptamers, and CRISPR guides are rapidly advancing in research and clinical applications. Accurate identification of the target sequence, confirmation of its integrity, and detection of related impurities are critical for quality control and regulatory compliance. Manual data analysis of high-resolution mass spectrometry (HRMS) results can be laborious and error-prone, highlighting the need for automated, integrated software solutions.

Objectives and Study Overview

This study introduces a comprehensive software platform that automates both mass spectrometry (MS1) and tandem MS (MS/MS) data analysis for oligonucleotide characterization. The key aims were to streamline target plus impurities (TPI) workflows and sequence confirmation processes, improve throughput, and ensure reliable results with minimal manual intervention.

Methodology and Used Instrumentation

Oligonucleotide samples—including DNA and RNA standards (21-mer, 40-mer, 100-mer), a synthetic 21-mer and its base-swap variant, and a heavily modified oligonucleotide—were prepared at 0.50 mg/mL in deionized water. Analytical workflows were performed using high-resolution LC/Q-TOF and dedicated bioinformatics software.

• Liquid Chromatography: Agilent 1290 Infinity II LC system with AdvanceBio Oligonucleotides column.
• Mass Spectrometry: Agilent 6545XT AdvanceBio LC/Q-TOF operated in HRAM mode for both MS1 and MS/MS.
• Chromatography Conditions: Ion-pairing mobile phases containing triethylamine and hexafluoroisopropanol to achieve optimal separation.
• Software: Agilent MassHunter BioConfirm version 12.0 with dedicated Target Plus Impurities and Sequence Confirmation workflows.

Main Results and Discussion

Comprehensive evaluations demonstrated high chromatographic resolution of oligonucleotide ladders and synthetic constructs. Monoisotopic and average masses matched expected values within sub-ppm accuracy using the Find-by-Formula and Maximum Entropy deconvolution algorithms. MS/MS fragmentation yielded 100% sequence coverage for all tested oligonucleotides, including heavily modified backbones. Impurity profiling of a synthetic 21-mer revealed multiple N-1 and N-2 truncated species, quantified with relative standard deviations below 3% across 12 replicates.

Benefits and Practical Applications

The integrated software enables:

• High-accuracy mass assignment (sub-ppm) for both intact oligonucleotides and impurities.
• Fully automated sequence confirmation with complete fragmentation coverage.
• Robust relative quantification of impurities, supporting stringent quality control.
• Streamlined data processing that reduces manual effort and potential for human error.

Future Trends and Opportunities

Advancements may include cloud-based data sharing, AI-driven fragment annotation, and integration with automated sample preparation platforms. Expansion to multiplexed assays and multiomic workflows will further enhance throughput and applicability in biopharmaceutical development.

Conclusion

The presented software delivers a robust, end-to-end solution for oligonucleotide characterization, combining automated workflows with high-resolution mass spectrometry. It achieves precise mass accuracy, comprehensive sequence validation, and reproducible impurity quantification, making it an invaluable tool for research, quality assurance, and regulatory compliance.

References

1. Wong D; Rye P; Madden S; Slysz G; Cody C. Comprehensive, Automated, and Integrated Software for Oligonucleotide Characterization and Sequence Confirmation; ASMS 2022 Poster WP386; Agilent Technologies; 2022.