An Oligonucleotide Impurity Analysis Workflow Using LabSolutions Insight Biologics Software

Importance of the Topic

Oligonucleotide therapeutics are emerging as a powerful modality in drug discovery, but their safety and efficacy require rigorous impurity profiling. Detecting and characterizing trace-level impurities helps ensure product quality, supports regulatory compliance, and accelerates development of nucleic acid–based pharmaceuticals.

Objectives and Study Overview

This work demonstrates a streamlined workflow for comprehensive impurity analysis of a phosphorothioate–modified 20-mer oligonucleotide (sequence CTG CTA GCC TCT GGA TTT GA) using the Shimadzu LCMS-9050 quadrupole time-of-flight system coupled with LabSolutions Insight Biologics software. The goal was to identify and sequence both the main component and low-abundance impurities down to 0.5% relative abundance.

Methodology and Instrumentation

Sample: Unpurified phosphorothioate-modified 20-mer oligonucleotide.

Chromatography:

• System: Nexera XS inert UHPLC
• Column: Shim-pack Scepter Claris C18-120 (50×2.1 mm, 1.9 μm) at 60 °C
• Mobile phases: A) 100 mM HFIP/10 mM TEA in water; B) 50% methanol with 50 mM HFIP/5 mM TEA
• Gradient: 5%→40% B over 26 min, hold, then re-equilibrate
• Flow rate: 0.3 mL/min; injection: 2 µL

Mass Spectrometry:

• Instrument: Shimadzu LCMS-9050 Q-TOF
• Ionization: ESI negative mode; MS scan m/z 550–2500, DDA
• Interface voltage: –3.0 kV; nebulizing gas 3 L/min; drying/heating gas 10 L/min
• Interface temperature: 350 °C; DL: 250 °C; block heater: 400 °C

Data Analysis:

• Software: LabSolutions Insight Biologics
• Sequence entry: Nucleobases, linkers, ribose and modifications configured in real time
• Target modifications tab: Defines expected impurity classes (strand truncations, base losses, depurination/depyrimidination, deamination, protecting groups, custom modifications)
• Component chromatogram: Aggregates signals over charge states and isotopes into a single extracted chromatogram
• Fragment coverage: Visualized in fill and branch modes to assess MS2‐driven sequence confirmation

Main Results and Discussion

PDA and MS chromatograms were aligned to compare UV and mass signals. The software generated a deconvoluted mass spectrum for the main PS 20-mer and identified over 30 distinct impurities including truncated strands, missing bases, and modified residues. MS2 fragmentation achieved full sequence coverage of the 20-mer and an impurity present at 0.5% relative abundance, as confirmed by high coverage percentages and graphical tick marks.

The fill mode displayed intensity distribution and completeness of aligned fragments, while the branch mode traced individual fragment sequences along the oligonucleotide backbone. This dual‐mode visualization accelerated error checking and impurity localization.

Benefits and Practical Applications of the Method

• High sensitivity detection of low‐abundance oligonucleotide impurities
• Comprehensive sequence confirmation for main and impurity components
• Graphical fragment coverage simplifies assessment of missing or modified nucleotides
• Flexible target modification definitions allow detection of known and unknown variants
• Streamlined workflow integrates sequence entry, data acquisition, deconvolution, and visualization

Future Trends and Opportunities

Advancements in mass spectrometry sensitivity, higher-resolution fragmentation, and integration of machine learning for automated impurity prediction will further enhance oligonucleotide quality control. Cloud-based data platforms and real-time analytics could enable rapid method development, while expanding software libraries of chemical modifications will broaden applicability to novel therapeutic constructs.

Conclusion

The combined LCMS-9050 and LabSolutions Insight Biologics workflow delivers robust, end-to-end impurity profiling for oligonucleotide therapeutics. It achieves complete sequence coverage of both main and trace impurity species, supports flexible modification searches, and provides intuitive visualization for accelerated decision making.

Instrumentation Used

• Shimadzu Nexera XS inert UHPLC system
• Shimadzu LCMS-9050 quadrupole time-of-flight mass spectrometer
• LabSolutions Insight Biologics data analysis software