Efficient Method Development of Small Interfering RNA by Reversed-Phase Ion-Pair Chromatograph

Significance of the Topic

Small interfering RNA (siRNA) has emerged as a key modality for gene silencing therapies, offering precise control over protein expression. Reliable separation of sense and antisense strands along with related impurities is critical for therapeutic efficacy, safety, and regulatory compliance.

Objectives and Study Overview

This work aimed to establish a rapid, robust reversed-phase ion-pair chromatography method for siRNA analysis. Using LabSolutions MD software, the study explored various ion-pair reagents, fluoroalcohol concentrations, column chemistries, and temperatures to identify optimal separation conditions.

Methodology and Instrumentation

The analytical approach employed denaturing ion-pair reversed-phase liquid chromatography. Key experimental variables included:

• Ion-pair reagents: triethylamine (TEA), diisopropylethylamine (DIPEA), dibutylamine (DBA)
• Fluoroalcohol (HFIP) levels: 100 and 200 mmol/L
• Column temperatures: 45, 55, and 65 °C
• Stationary phases: C18 (12 nm and 30 nm pores) and C4 (30 nm pores)

Instrumentation

An inert Nexera XS UHPLC system (Method Scouting configuration) was paired with Shim-pack Scepter Claris columns to prevent sample adsorption. Detection was performed at 260 nm using an inert‐cell UV detector.

Main Results and Discussion

DIPEA outperformed TEA and DBA in resolving sense/antisense strands and impurities. A fluoroalcohol concentration of 200 mmol/L HFIP provided slightly better peak separation. Design space mapping revealed that higher column temperatures combined with optimal DIPEA concentrations maximized resolution. Among tested columns, inert C18 phases delivered sharp peak shapes, while C4 exhibited distinctive selectivity for certain impurities.

Benefits and Practical Applications

The integration of LabSolutions MD streamlined method development by automating design‐of‐experiments and design-space evaluation. This workflow reduces manual effort and dependence on user expertise, ensuring reproducible high-resolution analysis for siRNA quality control and research applications.

Future Trends and Potential Applications

Continued advancements in automated method platforms and novel stationary phases will further accelerate oligonucleotide analysis. Coupling with high-resolution mass spectrometry and microfluidic chromatography could enhance sensitivity and throughput for complex therapeutic candidates.

Conclusion

By leveraging design space evaluation and advanced software tools, the optimized IP-RP LC method achieves efficient, reproducible separation of siRNA strands and impurities. This approach strengthens analytical robustness and expedites development timelines for oligonucleotide therapeutics.

Reference

1. Sean M. McCarthy, Martin Gilar, John Gebler; Analytical Biochemistry, Volume 390, Issue 2, 15 July 2009, Pages 181–188.