Breaking Down RNA Stability: Accelerated Aging of Reference Standards with ASAP prime® Modeling

Significance of the Topic

Access to stable RNA reference materials is critical for reliable method qualification, system suitability testing, and regulatory compliance in oligonucleotide analysis. Lyophilized standards reduce hydrolytic degradation and aid analytical reproducibility across SEC, IP-RP, HILIC, and AEX platforms.

Objectives and Study Overview

This study applied an Accelerated Stability Assessment Program (ASAP) using ASAPprime® software to predict the shelf life of two lyophilized RNA standards—Waters Lipid-Conjugated ASO LC-MS and siRNA LC-MS. Samples were subjected to varied temperature and humidity stresses to model long-term stability and inform packaging and storage recommendations.

Methodology and Instrumentation

ASAP accelerated aging:

• Statistically designed exposure to multiple temperature (25–50 °C) and relative humidity (3–29 % RH) conditions for up to 21 days.
• Isoconversion time determination to reach predefined purity loss (10 % for ASO, 8 % for siRNA).
• ASAPprime® software (v7.0) for modified Arrhenius modeling to calculate activation energy, humidity sensitivity, and predict degradation rates at storage conditions.

HILIC analysis:

• ACQUITY™ UPLC H-Class System with Flow-Through Needle.
• GTxResolve™ Premier BEH Amide, 300 Å, 1.7 µm, 2.1 × 100 mm column at 5 °C sample temperature.
• Mobile phase: high organic content with ammonium acetate.
• Detection at 260 nm and data managed with Empower™ CDS.

Main Results and Discussion

Lipid-Conjugated ASO Standard:

• Initial purity 88 %; 10 % loss specification at 78 % purity.
• Strong temperature and moisture sensitivity observed—higher RH accelerated degradation.
• Predicted >10 % purity loss after six months at 5 °C/1 % RH; negligible degradation at –20 °C.

siRNA LC-MS Standard:

• Purity loss driven by rapid dehybridization under moisture stress, generating single-strand impurities.
• High humidity sensitivity necessitated milder stress conditions for modeling.
• Predicted two-year stability at 4 °C/10 % RH and –20 °C; real-time data confirmed <2 % purity loss after 15 months at –20 °C.

Benefits and Practical Applications

• Rapid prediction of oligonucleotide shelf life compared to traditional multi-year studies.
• Optimization of packaging (sealed foil pouches with molecular sieves) and storage conditions to minimize moisture-driven degradation.
• Enhanced method qualification and system performance monitoring across multiple chromatographic techniques.

Future Trends and Potential Applications

• Extension of accelerated stability modeling to diverse nucleic acid therapeutics and complex bioconjugates.
• Integration of AI-driven predictive analytics to refine degradation kinetics and packaging recommendations.
• Development of standardized stress protocols for regulatory submission and quality assurance of oligonucleotide standards.

Conclusion

ASAPprime® modeling combined with HILIC analysis provided robust, accelerated stability data for lipid-conjugated ASO and siRNA standards. The approach enables reliable shelf life predictions, informs optimal storage strategies, and supports sustained analytical performance in research and QC laboratories.

References

1. Waterman K.C., Swanson J.T., Lippold B.L. Scientific and Statistical Analysis of Accelerated Aging for Pharmaceuticals. Part 1: Accuracy of Fitting Methods. J Pharm Sci. 2014;103(10):3000–3006.
2. Li Y., Breaker R.R. Kinetics of RNA Degradation by Specific Base Catalysis of Transesterification Involving the 2’-Hydroxyl Group. J Am Chem Soc. 1999;121:5364–5372.
3. Finny A.S., Johnston T., Sauner B., Addepalli B., Lauber M.A. Reproducible Hydrophilic Interaction Chromatography for Denaturing and Non-Denaturing Analyses of Oligonucleotides Using GTxResolve Premier BEH Amide Columns. Waters Application Note. 2024 Aug.