Integrated Data Analysis of Oligonucleotide Therapeutics Using LC-MS, LC-MS/MS and MALDI techniques

Importance of the Topic

Therapeutic oligonucleotides such as siRNA and antisense oligonucleotides represent a key class of modern treatments for diseases with high unmet need. Their complex synthesis and modification process demands rigorous analytical techniques to confirm full length sequences and detect sequence related impurities. Integrated data analysis using multiple mass spectrometry modalities streamlines characterization and supports quality control and drug development.

Goals and Study Overview

The paper presents a unified software application LabSolutions Insight Biologics to process data from single quadrupole LC MS, high resolution QTOF LC MS MS and MALDI platforms applied to a phosphorothioate modified 20 mer oligonucleotide. Objectives include validating sequence identity, quantifying related impurities and demonstrating sequence coverage across techniques.

Methodology and Instrumentation

Solid phase phosphoramidite chemistry was used to produce the target antisense oligonucleotide. Analytical approaches included:

• Nominal mass single quadrupole LC MS in negative ion mode over m/z 600–2000 with a scan speed of 1429 u/s
• High resolution QTOF LC MS MS delivering TOF MS scans from m/z 550–2500 and ten data dependent MS/MS scans on charge states 2 to 9
• Nominal mass MALDI with in source decay MS/MS using THAP and HPA matrices on a MALDI 8030

Instrumentation Used

• Shimadzu Nexera XSTM inert LC with Shim pack Scepter Claris column
• Shimadzu LCMS 2050 single quadrupole
• Shimadzu LCMS 9050 QTOF
• Shimadzu MALDI 8030 with support for MALDI 8020 and MALDImini 1 formats

Main Results and Discussion

Nominal mass LC MS confirmed the monoisotopic mass of 7164.04 Da and identified shortmer and longmer impurities. High resolution QTOF LC MS MS achieved over 90 percent sequence coverage of the full length product in a single run and resolved structural details of impurities with MS/MS scores up to 1.00. MALDI analysis provided approximately 90 percent coverage using in source decay fragments. The integrated interface allowed direct comparison of abundance profiles and impurity assignments across all datasets.

Benefits and Practical Applications

The unified data processing workflow reduces analysis time and eliminates the need for multiple software tools. A one click procedure automatically detects the instrument type and applies optimized processing parameters. This solution enhances throughput and reproducibility in QC laboratories and accelerates oligonucleotide drug discovery and development.

Future Trends and Applications

Future developments may include automated reporting features, machine learning models for impurity classification, and expansion to novel modalities such as LNA and mRNA. Cloud integration and regulatory compliant workflows will further support adoption in pharmaceutical manufacturing and clinical research.

Conclusion

LabSolutions Insight Biologics enables comprehensive oligonucleotide characterization by integrating single quadrupole LC MS, high resolution LC MS MS and MALDI data within a single interface. The platform delivers robust sequence confirmation, impurity profiling and high sequence coverage across modalities, providing an efficient analytical solution for oligonucleotide therapeutic development.