Streamlining characterization and monitoring of oligonucleotide impurities using an Orbitrap-based LC-HRAM-MS platform

Significance of the Topic

Therapeutic oligonucleotides such as antisense and siRNA are increasingly important in treating rare diseases. Their complex chemical modifications enhance stability and reduce immunogenicity but create analytical challenges. Robust methods are needed to characterize full length products and impurities to ensure safety and quality throughout development and manufacturing.

Objectives and Study Overview

The study aimed to demonstrate how an Orbitrap based LC-HRAM-MS platform combined with BioPharma Finder and Chromeleon software can streamline sequence mapping, impurity identification, and relative quantitation for modified RNA samples. A Chromeleon eWorkflow procedure was also evaluated to enable direct method transfer across multiple instruments.

Methodology and Instrumentation

Modified and unmodified RNA samples were desalted, HPLC purified, and analyzed at 10 pmol per injection using ion-pairing reversed-phase UHPLC with TEA/HFIP mobile phases. Data-dependent MS2 acquisition on the Orbitrap Exploris 240 was optimized with stepped collision energies, while full MS scans were collected on both the Exploris 240 and Exploris MX detectors. Sequence creation and mapping utilized BioPharma Finder 5.1, and quantitative processing employed Chromeleon CDS 7.3.1.

Used Instrumentation

• Vanquish Horizon and Flex UHPLC systems
• DNAPac RP 2.1×250 mm, 4 μm column
• Orbitrap Exploris 240 mass spectrometer
• Orbitrap Exploris MX mass detector
• BioPharma Finder 5.1 software
• Chromeleon CDS 7.3.1 and eWorkflow module

Main Results and Discussion

Sequence mapping via ddMS2 provided confident base by base confirmation of full length RNA and truncated or modified impurities with delta mass accuracy under 4 ppm and average structural resolution of 1.0. Site specific modifications such as 2′ fluorination, O-methoxyethylation and biotinylation were localized. Relative quantitation by high resolution MS closely matched UV based measurements for well resolved impurities. The Chromeleon eWorkflow enabled seamless transfer of methods between Exploris 240 and MX instruments, yielding consistent impurity profiles with under 10 percent variation.

Benefits and Practical Applications

• Comprehensive characterization of therapeutic oligonucleotides and low level impurities
• High confidence sequence confirmation and modification localization
• Accurate relative quantitation by LC-HRAM-MS without requiring complete chromatographic resolution
• Efficient method deployment and reproducibility across multiple Orbitrap platforms
• Valuable tool for quality control and process development in biopharma laboratories

Future Trends and Applications

Integration of automated MS2 driven workflows may enable real time impurity monitoring. Higher throughput instruments and advanced data processing could extend characterization to novel chemistries and larger oligonucleotide constructs. Adoption of eWorkflow procedures will support regulatory compliance and efficient method transfer across sites.

Conclusion

An Orbitrap based LC-HRAM-MS platform combined with BioPharma Finder 5.1 and Chromeleon CDS delivers a streamlined solution for sequence mapping, impurity identification, and reliable quantitation of therapeutic oligonucleotides. The approach supports consistent method transfer and robust analytical performance essential for drug development and quality assurance.

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