Streamlining workflow from characterization to monitoring of therapeutic oligonucleotides impurities across IPRP-LC-HRAM-MS platforms

Significance of the Topic

Therapeutic oligonucleotides represent a rapidly evolving class of biopharmaceuticals. Their chemical modifications improve stability and efficacy but also introduce novel impurities. Robust analytical strategies are essential to ensure product identity, purity and consistent performance during development, manufacturing and quality control.

Objectives and Study Overview

This work aimed to develop and validate a unified workflow for both in-depth characterization and routine monitoring of modified oligonucleotides and their low-level impurities. Key goals included:

• Design of a system performance evaluation test (SPET) for IPRP-LC-HRAM-MS platforms.
• Integration of data-dependent MS2 for sequence confirmation.
• Seamless transfer of monitoring methods across multiple Orbitrap systems.

Methodology and Instruments

Sample preparation involved mixtures of synthetic oligonucleotides (10–55mer) and modified RNA standards. Chromatographic separation used a Thermo Scientific DNAPac RP column on a Vanquish Horizon UHPLC with a 42-minute gradient. Detection combined:

• High-resolution accurate mass (HRAM) full MS on Orbitrap Exploris 240.
• Data-dependent MS2 acquisition for base-level sequencing confirmation.
• Full MS only monitoring on Orbitrap Exploris MX detectors via Chromeleon eWorkflows.

Data processing leveraged BioPharma Finder 5.0 for deconvolution and fragment mapping.

Main Results and Discussion

System performance metrics such as retention time repeatability, mass accuracy and signal intensity were assessed over 10 SPET injections, meeting predefined acceptance criteria. MS/MS data provided clear fragmentation maps with average structural resolution (ASR) values close to 1.0 and mass deviations below 3 ppm. Relative abundance profiles of full-length product (FLP) and truncation impurities showed excellent concordance between LC-UV and LC-HRAM-MS quantitation. Method transfer to two Exploris MX instruments achieved reproducible impurity quantitation across platforms.

Benefits and Practical Applications of the Method

• Comprehensive identification and quantification of oligonucleotide impurities in a single workflow.
• High confidence in sequence confirmation and impurity profiling.
• Rapid system performance validation via SPET enhances laboratory throughput.
• Direct method transfer reduces requalification efforts across instruments.

Future Trends and Opportunities

Emerging opportunities include automation of SPET reporting, expansion to longer or more heavily modified oligonucleotides, integration with advanced data analytics and machine learning for impurity prediction, and broader deployment in regulated QC environments.

Conclusion

The described IPRP-LC-HRAM-MS workflow coupled with SPET offers a streamlined approach for both deep characterization and routine monitoring of therapeutic oligonucleotides. High mass accuracy, reliable sequence mapping and reproducible impurity quantitation support accelerated drug development and robust QC operations.

Reference

Yang H., Murphy K., Sutton J., Cook K., Rhodick C., Bashir T., Criscuolo A., Arrey T.N., Crone C., Du M. Streamlining workflow from characterization to monitoring of therapeutic oligonucleotides impurities across IPRP-LC-HRAM-MS platforms. Thermo Fisher Scientific Application Note. 2022.