Green ion pair and HFIP free method for ASO RNA analysis with GLP compliant automated data handling

Importance of the Topic

Recent advances in therapeutic oligonucleotides, including antisense oligonucleotides (ASOs) and mRNA, have created a pressing need for robust analytical workflows. Accurate impurity profiling and characterization are essential to ensure safety, efficacy and regulatory compliance. Traditional ion-pairing reagents and HFIP pose challenges for method development, instrument maintenance and GLP compliance. A streamlined, ion-pair-free LC-HRMS approach offers significant advantages for pharmaceutical quality control and research.

Objectives and Study Overview

This work demonstrates a fully automated, GLP-compliant method to analyze ASO purity without using HFIP or amine-based ion-pairing agents. Using Spinraza (Nusinersen) as a model, the study aims to achieve high sensitivity, reliable impurity identification and quantitative performance by combining high-pH reversed-phase UHPLC, high-resolution mass spectrometry and advanced data handling in a single chromatography control platform.

Methodology and Instrumentation

The analytical workflow comprises:

• Reversed-phase UHPLC on a DNAPac RP column (4 μm, 2.1 × 100 mm) using a Thermo Scientific Vanquish Flex Binary UHPLC system with an ammonium acetate mobile phase at elevated pH.
• High-resolution MS detection on a Thermo Scientific Orbitrap Exploris instrument, controlled by Chromeleon 7.3.2 software.
• Data processing with Chromeleon’s 64-bit reporting engine, featuring automated deconvolution, extracted ion chromatogram (XIC) generation, impurity annotation and flexible report generation.

Key Results and Discussion

• Source optimization minimized adduct formation and in-source fragmentation, leading to cleaner mass spectra and reliable impurity assignments.
• Chromatographic separation delivered sharp peaks for the full-length product and related impurities, with high signal-to-noise ratios.
• Charge state profiling enabled discrimination between true impurities and spectral artefacts; deconvoluted spectra and targeted XIC quantitation produced comparable purity results.
• Elimination of HFIP and ion-pair reagents allowed gentler source conditions, reducing maintenance burdens and improving reproducibility.

Benefits and Practical Applications

• Comprehensive ASO purity assessment in a single run, supporting method development and QC release.
• Fully GLP-compliant data handling and reporting streamline regulatory submissions.
• Removal of toxic or corrosive mobile-phase additives enhances laboratory safety and instrument longevity.
• Automated workflows reduce user intervention and turnaround times in routine analysis.

Future Trends and Opportunities

• Broader adoption of HFIP-free, high-pH reversed-phase methods for diverse oligonucleotide modalities.
• Integration of triple quadrupole and high-resolution MS platforms for targeted quantitation strategies.
• Advanced data analytics, machine learning and collaborative laboratory informatics to improve impurity prediction and method transfer.
• Expansion of automated, GLP-compliant reporting frameworks to support large-scale oligonucleotide production and quality assurance.

Conclusion

The presented HFIP-free, ion-pair-free RP-LC HRAM MS workflow, combined with Chromeleon’s advanced deconvolution and XIC capabilities, offers a powerful, fully automated solution for ASO purity analysis. The method meets GLP requirements, simplifies instrument operation and delivers reliable, high-confidence impurity data critical for therapeutic oligonucleotide development and quality control.