Impurity Profiling and Characterization of Therapeutic Oligonucleotides using Nominal Mass Spectrometry on a Single Quadrupole LC-UV-MS system

Significance of the Topic

Therapeutic oligonucleotides offer precise modulation of gene expression and protein function, driving novel treatments for conditions unmet by traditional small molecules. Robust characterization of product and product-related impurities is crucial for ensuring safety, efficacy, and regulatory compliance during development and manufacturing.

Aims and Study Overview

This study demonstrates the application of nominal mass spectrometry on a single quadrupole LC-UV-MS system to profile and characterize impurities in a model phosphorothioate-modified oligonucleotide.

• Assess chromatographic separation of full-length oligonucleotide and shortmer impurities.
• Evaluate the performance of flow injection analysis for rapid mass confirmation under co-elution conditions.
• Demonstrate data processing using LabSolutions Insight Biologics software for impurity ratio and purity calculations.

Methodology and Instrumentation

An oligonucleotide standard (20-mer anti-sense, monoisotopic mass 7164.04) was spiked with 10% of two shortmer impurities (N-1 and N-3) and analyzed by ion-pair reversed-phase LC coupled to UV and single quadrupole mass detection.

• Chromatography: Shimadzu Nexera XS inert LC using Shim-pack Scepter Claris column with HFIP/DBA additives.
• UV Detection: PDA at 260 nm, 4 nm bandwidth.
• Mass Spectrometry: Shimadzu LCMS-2050, m/z 600–2000, negative ion mode, profile acquisition.
• Software: LabSolutions Insight Biologics for peak integration, charge-state deconvolution, and impurity profiling.

Main Results and Discussion

Optimized LC conditions achieved chromatographic resolution of the full-length product and both N-1 and N-3 impurities, enabling precise purity and impurity ratio determination. Calibration curves for each component showed linear response over 10–50 pmol/µL. Under modified conditions simulating co-elution, flow injection analysis combined with nominal mass detection successfully distinguished and quantified all three species, with abundance ratios closely matching those from resolved LC separations.

Key observations:

• Chromatographic separation yielded 85–83% full-length product abundance and 6–8% for each impurity.
• Charge-state deconvolution enabled rapid molecular weight confirmation without high-resolution instrumentation.
• Flow injection approach provided a streamlined workflow for impurity screening when chromatographic separation is limited.

Benefits and Practical Applications

The workflow offers:

• Rapid sequence confirmation and impurity quantification in compliance-ready environments.
• Reduced method development time by leveraging nominal mass detection.
• Streamlined data processing with Insight Biologics for routine QA/QC of oligonucleotide therapeutics.

Future Trends and Potential Applications

Emerging directions include integration of higher-throughput flow injection protocols with automated data analysis, adoption of high-resolution mass analyzers for more complex impurity profiling, and in-process monitoring of oligonucleotide synthesis. Advances in software algorithms may further improve deconvolution accuracy and expand capabilities to longer or more heavily modified sequences.

Conclusion

Nominal mass single quadrupole LC-UV-MS combined with Insight Biologics software provides a practical, compliance-ready platform for oligonucleotide impurity profiling. Both chromatographic and flow injection approaches enable sensitive detection and quantification of full-length products and related shortmers, supporting rapid decision-making in therapeutic development.