Method considerations for therapeutic ASO RNA analysis. Adducts and insource impurity generation

Significance of the Topic

Antisense oligonucleotides (ASOs) and siRNA have emerged as powerful therapeutic modalities, driving the need for precise impurity characterization during manufacturing.
These biomolecules present analytical challenges due to their charged backbone and phosphorothioate modifications, which can lead to chromatographic complexity, metal and amine adduct formation, and in-source impurity artifacts.
Addressing these issues is crucial for reliable quality control in drug development.

Objectives and Study Overview

The study aimed to develop a high-resolution mass spectrometry (HRMS) workflow that:

• Eliminates amine, HFIP, and metal ion adducts in oligonucleotide analysis.
• Avoids generation of in-source impurities during ionization.
• Maintains chromatographic resolution for full-length product (FLP) and related N-1 and N+1 impurities.
• Implements a fully automated, GLP-compliant process for routine quality control.

Methodology and Instrumental Setup

Sample Preparation and Chromatography:

• Synthetic ASO RNA and DNA oligonucleotides diluted to 1 mg/mL in water.
• IPRP separation on a DNAPac RP column (4 μm, 2.1 × 100 mm) using a Vanquish Flex UHPLC system.
• Eluent optimized with perfluorinated ion pairing reagents (HFIP and a small hydrophobic amine), high-purity solvents, and avoidance of silicate glass to suppress adduct formation.

Mass Spectrometry and Data Analysis:

• Orbitrap Exploris 240 and Thermo Scientific MX mass spectrometers for high sensitivity and resolution.
• Optimized source conditions to remove amine adducts at 30 eV collision energy without inducing fragmentation artifacts.
• Isotopic sliding windows Xtract deconvolution in BioPharma Finder v5.2 for impurity identification and relative quantitation.
• Chromeleon 7.3.2 for GLP-compliant sequence control, data acquisition, deconvolution, and reporting.

Key Results and Discussion

The optimized workflow effectively removed metal and amine adducts, yielding clean mass spectra devoid of extraneous peaks.
Under mild source conditions, no in-source impurity signals were observed; harsher settings increased fragmentation artifacts such as single-base loss.
Relative standard deviations (RSD) for the FLP were below 0.4%, and low-level N-1/N+1 impurities showed RSDs under 6%.
Linearity studies in water and FLP matrix confirmed the absence of ion suppression.
Custom Chromeleon reports enabled annotation and quantitation of both expected and unknown impurities through mass comparison.

Benefits and Practical Applications

• Robust, high-throughput impurity profiling for ASO RNA and siRNA therapeutics.
• Fully automated, GLP-compliant workflow compatible with QC and R&D environments.
• Improved data quality and quantitation accuracy compared to low-resolution MS methods.
• Flexible reporting framework for routine and investigative analyses.

Future Trends and Potential Applications

• Integration of ion mobility separation to further resolve isobaric species.
• Real-time data analysis and AI-driven impurity identification.
• Extension to native mass spectrometry for higher-order structure assessment.
• Broader adoption in biopharmaceutical characterization and regulatory submissions.

Conclusion

The developed UHPLC–HRMS method on the Exploris 240 or MX platform offers a simple, fast, and automatable solution for accurate impurity analysis of therapeutic oligonucleotides.
By eliminating adducts and avoiding in-source artifacts, it significantly enhances quantitation reliability under GLP conditions.
This approach outperforms traditional low-resolution systems and provides a versatile tool for quality control in oligonucleotide drug development.

References

1. Vanhinsbergh C.J., Criscuolo A., Sutton J.N., Murphy K., Williamson A.J.K., Cook K., Dickman M.J. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.2c00765
2. Rentel C., Gaus H., Bradley K., Luu N., Kolkey K., Mai B., Madsen M., Pearce M., Bock B., Capaldi D. Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair–High-Performance Liquid Chromatography–Mass Spectrometry. Nucleic Acid Therapeutics. https://doi.org/10.1089/nat.2021.0056