100 Samples per Hour: High-Speed Oligonucleotide Analysis with BioAccord™ LC-MS and INTACT Mass Application

Importance of the Topic

Oligonucleotide-based therapeutics are gaining momentum across multiple disease areas thanks to their high target specificity and chemical versatility. Reliable, high-throughput analysis of intact oligonucleotides is essential to confirm molecular mass, assess purity, and detect impurities during early screening, synthesis optimization, and quality control workflows.

Objectives and Study Overview

This study evaluates a fully automated LC-MS workflow combining the Waters BioAccord System with the waters_connect INTACT Mass Application. The primary goals were to demonstrate mass confirmation and purity determination for oligonucleotides ranging from 10-mer to 60-mer, achieve sample throughputs exceeding 100 per hour, and minimize manual processing steps.

Used Instrumentation

• Waters ACQUITY Premier UPLC System (binary pump and Tunable UV detector at 260 nm)
• ACQUITY Premier Oligonucleotide C18 VanGuard FIT Cartridge
• Waters BioAccord Mass Spectrometer operating in LC-MSE negative mode
• waters_connect Informatics Platform with INTACT Mass Application for automated acquisition and processing

Methodology and Experimental Conditions

A trap-and-elute reversed-phase method was deployed with a 1 µL injection volume and a 1 mL/min flow rate. Mobile phases consisted of TEA/HFIP additives in water and in 50 percent methanol. The MS method used scheduled lockmass correction, a scan range of 400–5000 m/z, a 10 Hz scan rate, and a 70–110 eV MSE collision energy ramp. Lockmass scheduling reduced inter-sample checks, allowing LC-MS acquisition and processing in under 30 seconds per sample.

Main Results and Discussion

The integrated workflow achieved over 100 samples per hour throughput while maintaining high mass accuracy (better than 2 ppm) by using target chemical formulas in the deconvolution model. Automated impurity profiling flagged known adducts, n–1 products, and novel species via delta-mass reporting. A color-coded dashboard enabled rapid exception review. Representative case studies included accurate mass confirmation of nusinersen (–1.5 ppm error, lowest impurity at 0.18 percent), identification of novel impurities in GEM-91, and successful deconvolution of a co-eluting 10–60 mer ssDNA ladder with low-abundance species detected at 0.5 percent purity.

Benefits and Practical Applications

This automated LC-MS solution delivers:

• High throughput screening for early candidate selection
• Consistent mass confirmation and purity data across diverse chemistries
• Automated impurity detection and reporting to accelerate decision making
• Compliance-ready reporting and traceability for regulated environments

Future Trends and Potential Applications

Emerging developments may include integration with machine learning for predictive impurity profiling, expansion to even longer or more complex oligonucleotide constructs, and seamless coupling to robotic sample handlers. Such advances will further increase throughput and data reliability for both research and commercial production labs.

Conclusion

The BioAccord LC-MS System combined with the waters_connect INTACT Mass Application provides a fast, reliable, and fully automated workflow for intact oligonucleotide analysis. By achieving high sample throughput, robust mass accuracy, and streamlined data review, this platform accelerates screening and quality control in oligonucleotide development.

References

1. Shion H, Berger SJ, Yu YQ Application of a Mass Confirmation Workflow for Biotherapeutics Screening Waters Application Note 720007027 November 2020
2. Shion H, Boyce P, Berger SJ, Yu YQ INTACT Mass – a Versatile waters_connect Application for Rapid Mass Confirmation and Purity Assessment of Biotherapeutics Waters Application Note 720007547 February 2022