Oligonucleotide mapping using BioPharma Finder software

Significance of the topic

Oligonucleotide drugs represent a rapidly growing class of therapeutics with applications in rare genetic, neurological, hepatic, muscle and cardiovascular diseases. Precise characterization of their sequence and chemical modifications at the single–base level is essential for development, quality control and regulatory compliance. High-resolution accurate mass (HRAM) MS coupled with data-dependent tandem MS (ddMS2) and advanced software tools addresses the need for confident oligonucleotide mapping without requiring predefined mass lists or targeted methods.

Goals and overview of the study

This work describes the development and optimization of an untargeted liquid chromatography ddMS2 workflow for oligonucleotides on an Orbitrap-based HRAM instrument. Using Thermo Scientific BioPharma Finder 4.0 software, the study aimed to achieve accurate mass measurement, comprehensive sequence coverage and unambiguous localization of modifications. Key objectives included method parameter optimization (S-lens RF level and collision energy), comparative analysis of multiple oligo sizes and demonstration of isomer discrimination and mixed sample identification.

Methodology

Ion-pairing reversed-phase chromatography was performed on a DNAPac RP column using HFIP/DIPEA buffers with a gradient from 20 to 80 % organic at 0.4 mL/min. A Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer operated in negative mode acquired Full MS at 70,000 resolution and Top5 ddMS2 at 35,000 resolution. Data were processed in BioPharma Finder using the Oligonucleotide Analysis workflow, which supports manual sequence input, custom modifications and multi-consensus comparative review.

Used instrumentation

• Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer
• Thermo Scientific Vanquish Horizon UHPLC System
• Thermo Scientific DNAPac RP Column (4 μm, 2.1×50 mm)
• BioPharma Finder 4.0 software

Main results and discussion

Optimization of the S-lens RF level revealed an optimal range of RF60-65 to maximize signal intensity across multiple charge states. Collision energy (CE) was tuned using stepped normalized CE (NCE) ranges and evaluated by average structural resolution (ASR), a metric reflecting fragment coverage completeness. Ideal stepped NCEs varied by oligo length: for 20-mers, NCE 15-17-19 achieved full coverage (ASR≈1.0); for 25-mers, stepped NCEs in the 12-20 range provided complete mapping of high charge states; and for longer 45-mers and RNA oligos, similar CE windows delivered comprehensive fragmentation. The workflow distinguished isomeric sequences (20merAC vs 20merCA) via diagnostic fragment ions and successfully identified three oligos in a single mixture using a common stepped NCE setting.

Benefits and practical applications

• Accurate mass and high-confidence sequence mapping of DNA and RNA oligonucleotides without predefined targets
• Automated handling of custom modifications and variable building blocks
• Comparative analysis across multiple method variations for rapid parameter optimization
• Capability to resolve isomeric sequences and analyze complex mixtures
• Integration into a single software platform supports accelerated oligonucleotide therapeutic development and QC workflows

Future trends and opportunities

Continued advances in software algorithms will further automate de novo sequencing and modification localization. Integration with high-throughput and real-time quality control systems can support large-scale oligonucleotide manufacturing. Expanding HRAM ddMS2 approaches to conjugated, branched and more heavily modified oligos will extend this workflow to next-generation nucleic acid modalities.

Conclusion

The optimized HRAM ddMS2 method combined with BioPharma Finder 4.0 offers a robust, flexible and user-friendly platform for comprehensive oligonucleotide characterization. Tailored instrument parameters and advanced data analysis deliver accurate mass measurement, full sequence coverage and confident modification mapping, facilitating therapeutic discovery, development and regulatory support.

Reference

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