PAMS: AUTOMATED WORKFLOWS FOR INTACT MASS, PURITY AND SEQUENCE CONFIRMATION OF OLIGONUCLEOTIDES

Significance of the Topic

The rise of synthetic oligonucleotides as therapeutic agents demands robust analytical workflows to guarantee product integrity and safety.
Accurate impurity profiling, mass confirmation and sequence verification underpin regulatory compliance and development of oligonucleotide medicines.

Study Objectives and Overview

This study presents a fully automated workflow combining two waters_connect applications (INTACT Mass and CONFIRM Sequence) for impurity analysis of a heavily modified 21-mer oligonucleotide.
The goal was to deliver rapid purity calculations, intact mass measurement and detailed sequence confirmation in both regulated and research environments.

Methodology and Instrumentation

Materials and Chromatography Conditions

• Analyte: 21-mer with 19 2′-OMe modifications, sequence GUA ACC AAG AGU AUU CCA UTT, elemental formula C229H306N76O143P20.
• Column: ACQUITY Premier OST, 2.1 × 100 mm; flow 300 µL/min; column temp. 60 °C.
• Mobile Phases: A) 7 mM TEA, 40 mM HFIP in water (pH 8.6); B) 3.5 mM TEA, 20 mM HFIP in 50 % MeOH; gradient 25→35 % B over 25 min; total run 40 min; UV detection at 260 nm.

Mass Spectrometry Conditions

• Instruments: Xevo G2-XS QTof and BioAccord UPLC-TOF systems in negative ESI mode.
• ESI Settings: capillary 2.5 kV; cone 45 V; source temp. 120 °C; desolvation 450 °C; m/z range 500–5,000; 1 Hz scan rate.
• Fragmentation: CID cone voltage ramp (10–70 V) and fixed collision energies (36–63 V) for precursors in MS/MS and DIA (MSE) experiments.

Data Processing

• waters_connect software with INTACT Mass for deconvolution and purity metrics, CONFIRM Sequence for MS/MS fragment matching and sequence coverage (dot-map visualization).

Main Results and Discussion

Impurity Profiling and Mass Accuracy

• Chromatography resolved 11 distinct impurities plus full-length product (FLP).
• INTACT Mass deconvolution achieved mass errors below 10 ppm for all species.

Sequence Verification

• CONFIRM Sequence delivered 100 % coverage for FLP and ≥75 % for eight impurities.
• Low-abundance (0.2 %) 11-mer impurity was confirmed with 95 % coverage.
• Software detected and localized a missing 2′-OMe cytidine in a 20-mer impurity, distinguishing isomeric possibilities.

Benefits and Practical Applications

• Fully automated impurity assignment, mass confirmation and sequence validation accelerate QC workflows.
• High sensitivity down to sub-percent impurity levels supports stringent regulatory requirements.
• Applicable to both research labs and GMP environments for oligonucleotide therapeutics.

Future Trends and Applications

Integration with laboratory information management systems and expansion to larger oligonucleotides (siRNA, sgRNA) and bioconjugates are anticipated.
Advances in fragmentation techniques and AI-driven data interpretation will further enhance sequence coverage and impurity characterization.

Conclusion

The combined waters_connect INTACT Mass and CONFIRM Sequence workflow delivers rapid, high-confidence purity assessments, accurate mass measurements and comprehensive sequence confirmation for synthetic oligonucleotides, even at low impurity levels.

References

1. Sharma VK, Watts JK. Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem. 2015;7(16):2221–2242.
2. McLukey SA, Van Berkel GJ, Glish GL. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. J Am Soc Mass Spectrom. 1992;3:60–70.
3. Waters Corporation. Automated Compliance-Ready LC-MS Workflow for Intact Mass Confirmation and Purity Analysis of Oligonucleotides. Application Note P/N 720006820EN; 2020.
4. Waters Corporation. Intact Mass Confirmation Analysis on BioAccord LC-MS System for Extensively Modified Oligonucleotides. Application Note P/N 720007028EN; 2020.
5. Waters Corporation. Analysis of Oligonucleotide Impurities on the BioAccord System with ACQUITY Premier. Application Note P/N 720007301EN; 2021.
6. Waters Corporation. LC-MS Analysis of siRNA, Single Guide RNA and Impurities using BioAccord with New Automated INTACT Mass Application. P/N 720007546EN; 2022.
7. Waters Corporation. CONFIRM Sequence: A waters_connect Application for Sequencing Synthetic Oligonucleotides and Their Impurities. P/N 720007677EN; 2022.