Automated, compliance-ready, intact mass confirmation of modified oligonucleotides using an LC-MS platform

Importance of the Topic

Therapeutic oligonucleotides are increasingly used in drug development and manufacturing and require robust methods for identity confirmation and purity assessment to meet quality control and regulatory requirements.
Intact mass analysis by LC-UV-MS provides a rapid and reliable approach to confirm the average mass of modified oligonucleotides in a compliance-ready environment.

Goals and Study Overview

This study demonstrates the deployment of an automated eWorkflow in Thermo Scientific Chromeleon CDS for streamlined, compliance-ready intact mass confirmation of phosphorothioated 20mer oligonucleotides using Vanquish Flex UHPLC and ISQ EM single quadrupole mass spectrometry.

Methodology and Instrumentation

A set of five 20 nucleotide oligonucleotides with one to five phosphorothioate modifications was reconstituted at ~2 pmol/µL and analyzed using ion-pairing reversed-phase UHPLC with a trap-and-elute technique.
Detailed instrumentation setup:

• Vanquish Flex UHPLC system with Binary Pump F, Split Sampler FT, Column Compartment H, and active pre-column heater
• Thermo Scientific DNAPac RP 2.1×50 mm, 4 µm column operating at 70 °C with HFIP/TEA ion-pairing mobile phases
• ISQ EM single quadrupole mass spectrometer in negative HESI mode, full scan 600–2000 m/z, 0.5 s scan time
• Chromeleon CDS v7.3.2 eWorkflow and Intact Protein Deconvolution using the ReSpect algorithm

Main Results and Discussion

All five oligonucleotides eluted within a narrow retention window, demonstrating the high-throughput trap-and-elute approach without separation of non-retaining impurities.
Raw data were averaged across the 0.7–0.9 min retention time, and the ReSpect algorithm deconvoluted the spectra to yield average masses.
Measured mass accuracies ranged from 0.4 to 1.6 Da (mean 0.77 Da), all within the predefined 2 Da tolerance, resulting in a pass/fail assessment for each sample.

Benefits and Practical Applications

• Fully automated sample-to-report workflow reduces manual intervention and potential errors.
• Compliance-ready eWorkflow can be shared and deployed across laboratories, ensuring consistency.
• Rapid mass confirmation supports quality control in oligonucleotide drug development and manufacturing.

Future Trends and Opportunities

Further integration with high-resolution mass spectrometry could enhance mass accuracy and support complex oligonucleotide modifications.
Cloud-based data processing and artificial intelligence–driven deconvolution algorithms may automate interpretation of larger and more diverse oligonucleotide libraries.
Expansion of eWorkflow capabilities to cover other biopolymer analyses could streamline broader QC assays in pharmaceutical laboratories.

Conclusion

The presented generic eWorkflow in Chromeleon CDS enables high-throughput, compliance-ready intact mass confirmation of modified oligonucleotides with minimal user input.
By automating data acquisition, processing, and reporting, laboratories can achieve rapid and reliable quality assessments of therapeutic oligonucleotides.

References

• Thermo Fisher Scientific. Application Note 000457: High-throughput analysis of oligonucleotides using a single quadrupole mass spectrometer for quality control.