Workflow for Profiling Impurities in Synthetic Oligonucleotides Using the BioAccord™ LC- MS System with waters_connect™ Informatics

Significance of the topic

Synthetic oligonucleotides are emerging as key modalities in modern therapeutics. Accurate impurity profiling is critical to ensure safety, efficacy and regulatory compliance as the length and chemical complexity of oligonucleotides increase.

Objectives and overview of the workflow

This application note presents a streamlined workflow for profiling impurities in synthetic oligonucleotides. It introduces the Oligonucleotide Impurity Analysis Workflow Data Package implemented on the BioAccord LC-MS System with waters_connect informatics. The goal is to reduce data analysis time, minimize manual steps, and improve analytical confidence in a regulated environment.

Methodology and instrumentation used

The workflow combines UPLC separation on the ACQUITY Premier System with high-resolution mass detection via the BioAccord System (UPLC-Tof MS). Sample data are processed using the UNIFI App within waters_connect informatics, leveraging two parallel guided workflows:

• Targeted Workflow for known impurity quantification: UV and XIC integration, system suitability checks, calibration curve generation.
• Untargeted Workflow for unknown coeluting impurity screening: identity testing, adduct evaluation, comprehensive XIC summation.

Instrument conditions include an ACQUITY Premier Oligonucleotide C18 column, ESI negative ionization under soft (400 °C) and harsh (550 °C) desolvation, and intelligent data capture.

Main results and discussion

The UPLC-Tof approach halved run times and reduced solvent consumption compared with traditional HPLC–single quad methods, while resolving near-isobaric species (e.g., n-U vs. n-C) achieved by high mass resolution. Automated data review workflows cut analysis time from hours to minutes. The guided steps allowed precise UV dropline placement, repeatability assessment, calibration verification, and streamlined adduct discrimination, enhancing throughput and reliability.

Benefits and practical applications

• Automated, compliance-ready data processing reduces human error and manual workload.
• Rapid method execution lowers training burden and supports routine QC in regulated labs.
• Enhanced chromatographic and mass spectrometric performance improves impurity detection.
• Shorter gradients decrease solvent waste and hazardous byproducts.

Future trends and potential applications

As oligonucleotide therapeutics evolve in length and chemical modification, further integration of high-resolution MS workflows and AI-driven data analysis will be needed. Adaptation to emerging modalities, real-time monitoring, and automated regulatory reporting represent key development areas.

Conclusion

The BioAccord LC-MS workflow with waters_connect informatics provides a robust, efficient, and adaptable platform for impurity profiling of synthetic oligonucleotides. By automating critical data review steps and leveraging high-resolution detection, the workflow addresses the demands of modern therapeutic pipelines and regulatory requirements.

References

• Pourshashian S. Therapeutic Oligonucleotides, Impurities, Degradants, and Their Characterization by Mass Spectrometry. Mass Spectrom Rev. 2019 Dec;40(2):75–109.
• Doneanu CE et al. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Waters Application Note. 2021 Jul;720007301.
• Rentel C et al. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair-HPLC-MS. Nucleic Acid Therapeutics. 2022 Mar;32(3):206–220.