Analysis of siRNA Duplexes at Non- Denaturing UPLC Conditions Using MaxPeak Premier Column Technology

Importance of the Topic

Small interfering RNAs (siRNAs) represent a critical class of nucleic acid therapeutics capable of regulating gene expression in vivo. Ensuring the integrity and purity of siRNA duplex formulations through non-denaturing analysis is essential for drug safety, efficacy, and regulatory compliance.

Objectives and Study Overview

This application note describes the development of a non-denaturing ion-pair reversed-phase UPLC method to:

• Separate and quantify two combined siRNA duplexes in a drug formulation.
• Resolve intact duplex species from single-stranded oligonucleotide contaminants.
• Evaluate method performance in terms of recovery, selectivity, repeatability, linearity, and carryover.

Methodology and Instrumentation

Sample Preparation:

• Reference solutions of two siRNA duplexes at 0.2 % to 130 % levels relative to a 2.0 mg/mL standard.
• Drug formulation prepared at 2.0 mg/mL in demineralized water.

Instrumentation:

• ACQUITY UPLC H-Class PLUS Bio System with Quaternary Solvent Manager, Column Heater, Active Preheater, Flow-Through Needle Sample Manager, and PDA detector.
• ACQUITY Premier Peptide BEH C18 Column (2.1 × 150 mm, 1.7 µm, 300 Å) maintained at 20 °C.
• Mobile phase A: 0.2 % hexylamine and 0.5 % HFIP in water; Mobile phase B: 80 % methanol/20 % acetonitrile.
• Flow rate 0.15 mL/min, injection volume 6 µL, detection at 260 nm. Data processed using Empower v3.0.

Main Results and Discussion

The advanced hybrid-silica MaxPeak Premier column technology effectively eliminated nonspecific adsorption on metal surfaces, yielding high sample recovery. Single-stranded oligonucleotides eluted early and were fully resolved from duplex peaks. The 150 mm column achieved baseline separation of the two siRNA duplexes, which appeared as multiple diastereomer peaks due to phosphorothioate linkages. Carryover tests confirmed negligible residual signal after high-level injections, validating system cleanliness.

Benefits and Practical Applications

• Accurate and reproducible separation of siRNA duplexes and single-stranded impurities.
• Elimination of on-column degradation and sample loss on metal surfaces.
• High chromatographic selectivity enabling confident identification and quantification of each duplex and its variants.
• Reliable determination of duplex molar ratios and excess single strands in drug formulations.

Future Trends and Possibilities

Continued innovations in column surface chemistries will further reduce analyte adsorption and expand applications to other metal-sensitive biomolecules. Coupling non-denaturing IP-RP UPLC with mass spectrometry will enable detailed structural characterization of complex oligonucleotide mixtures. These advances will streamline siRNA therapeutic development and quality control workflows.

Conclusion

The non-denaturing IP-RP UPLC method using the ACQUITY Premier Peptide BEH C18 column provides a robust, high-resolution platform for quantitative siRNA duplex analysis. Superior recovery, selectivity, and absence of carryover meet stringent requirements for therapeutic oligonucleotide characterization and support reliable quality control.

Reference

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