A WORKFLOW FOR PURITY DETERMINATION, INTACT MASS MEASUREMENT AND MS/MS SEQUENCING OF OLIGONUCLEOTIDE IMPURITIES DETECTED IN SYNTHETIC OLIGOS

Importance of the Topic

The accurate measurement of oligonucleotide purity and precise sequencing of low-level impurities are critical in research and quality control of synthetic oligos. Advanced workflows that combine intact mass analysis with MS/MS fragmentation enable rapid identification and characterization of impurity species, supporting regulatory compliance and robust analytical performance.

Aims and Overview of the Study

This study presents an automated workflow for purity determination, intact mass measurement, and MS/MS sequencing of impurities in a heavily modified 21-mer synthetic oligonucleotide. The objectives were to demonstrate fast, accurate impurity profiling and high sequence coverage of both the full-length product and minor impurity species down to 0.05% relative abundance.

Methodology and Instrumentation

The 21-mer target, modified with multiple 2’-MOE and 5-Me groups, was analyzed by UPLC-UV coupled to a Xevo G3 QTof mass spectrometer using negative-mode ESI. Chromatographic separation used an ACQUITY Premier OST column with TEA/HFIP ion-pairing reagents over a 25-min gradient.

Used Instrumentation

• UPLC System: ACQUITY Premier UPLC with TUV detector (260 nm)
• Column: ACQUITY Premier OST, 2.1×150 mm
• Eluents: A - 7 mM TEA/40 mM HFIP in water (pH 8.6); B - 3.5 mM TEA/20 mM HFIP in 50% methanol
• Flow rate: 300 µL/min; Gradients: 13→23% B over 25 min
• Mass Spectrometer: Xevo G3 QTof; acquisition mass range 500–5000 amu; ionization negative ESI

Main Results and Discussion

Intact mass deconvolution using the INTACT Mass App identified 16 impurities with mass accuracies below 10 ppm. The full-length product (FLP) showed 94.51% purity by UV and 100% MS/MS sequence coverage. Six impurities were selected for targeted MS/MS; the lowest-abundance species at 0.05% relative area was sequenced with 83.3% coverage. The CONFIRM Sequence App efficiently processed CID and MSE data, confirming sequence integrity and localizing modifications.

Benefits and Practical Applications of the Method

• Automated deconvolution and rapid impurity assignment accelerate analytical throughput.
• High mass accuracy (<10 ppm) ensures confident identification of minor variants.
• Comprehensive sequence coverage supports rigorous QC in regulated environments.
• Software-driven workflows reduce manual interpretation and improve reproducibility.

Future Trends and Potential Applications

Integration of high-throughput intact mass workflows with advanced data-independent acquisition will broaden impurity profiling capabilities. Enhanced software features for real-time monitoring and cloud-based collaborative analysis are expected. The approach may extend to larger nucleic acid therapeutics, including mRNA and CRISPR guide RNAs.

Conclusion

The presented workflow combining INTACT Mass App and CONFIRM Sequence App on a Xevo G3 QTof platform delivers rapid, accurate impurity profiling and high sequence coverage for synthetic oligonucleotides. It is well suited for both research and regulated QC laboratories requiring trace-level impurity characterization.

References

• 1. An Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides, 2020, Waters application note, P/N 720006820EN.
• 2. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier System and New Automated INTACT Mass Application, 2022, Waters application note, P/N 720007546EN.
• 3. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using the BioAccord System with ACQUITY Premier System and New Automated INTACT Mass Application, 2021, Waters application note, P/N 720007301EN.
• 4. CONFIRM Sequence: A waters_connect Application for Sequencing of Synthetic Oligonucleotides and Their Impurities, 2022, Waters application note, P/N 720007677EN.