Analysis of siRNA Drugs at Denaturing UPLC Conditions Using MaxPeak Premier Column Technology

Importance of the Topic

In recent years, small interfering RNA (siRNA) therapeutics have emerged as a powerful modality for treating a broad range of diseases. Robust analytical methods are essential throughout development and quality control to quantify and characterize siRNA duplexes and their single-strand components under denaturing conditions.

Objectives and Study Overview

This study evaluated the performance of two UPLC columns—conventional stainless-steel ACQUITY UPLC Peptide BEH C18 (300 Å) and hybrid-surface ACQUITY Premier Peptide BEH C18 (300 Å)—for denaturing ion-pair reversed-phase liquid chromatography of siRNA duplexes. The primary objectives were to:

• Ensure complete denaturation of the siRNA duplex into single strands at elevated temperature.
• Compare recovery, repeatability and calibration linearity between column technologies.
• Demonstrate method suitability for quantitative analysis in drug formulations.

Methodology

siRNA duplex standards were prepared in Milli-Q water at concentrations ranging from 0.2 % to 200 % relative to a 0.40 mg/mL reference solution. Denaturing separations were performed at 75 °C using a gradient of 5 mM TEA/60 mM HFIP (aqueous) against 70/30 % methanol/acetonitrile. UV detection at 260 nm quantified the two main single-strand peaks (approx. 15.9 min and 17.5 min). Peak areas were summed and evaluated for recovery, repeatability (%RSD) and calibration linearity.

Used Instrumentation

• ACQUITY UPLC H-Class PLUS Bio System with quaternary solvent manager, column heater, active preheater, flow-through needle sample manager and 5 mm titanium PDA cell.
• Columns: conventional ACQUITY UPLC Peptide BEH C18, 300 Å, 1.7 µm, 2.1×150 mm and ACQUITY Premier Peptide BEH C18, 300 Å, 1.7 µm, 2.1×150 mm.
• Empower 3.0 software for data integration and quantitation.

Main Results and Discussion

On the conventional stainless-steel column, initial injections exhibited low recovery and required multiple conditioning runs to saturate metal-surface adsorption sites. In contrast, the Premier column with hybrid-silica hardware delivered consistent peak areas from the first injection, indicating elimination of non-specific oligonucleotide adsorption. Calibration curves on the Premier column achieved linearity down to 0.2 % nominal concentration, whereas the conventional column showed zero response at low levels. Repeatability studies on the Premier column yielded %RSD well below acceptance criteria (≤15 % at 0.2 %, ≤2 % at 100 %).

Benefits and Practical Applications

• Elimination of lengthy column conditioning and sample adsorption artifacts.
• Improved quantitation accuracy, repeatability and dynamic range for siRNA analysis.
• Streamlined workflows for early-stage development, clinical testing and quality control in pharmaceutical laboratories.

Future Trends and Potential Applications

Advances in hybrid-surface column technologies may extend to other metal-sensitive biomolecules such as antisense oligonucleotides and modified RNAs. Integration with mass spectrometry workflows promises enhanced sensitivity and characterization. Automated conditioning protocols and further hardware innovations are expected to simplify high-throughput nucleic acid analytics.

Conclusion

The ACQUITY Premier Peptide BEH C18 (300 Å) column with MaxPeak High Performance Surfaces offers superior analytical performance for denaturing UPLC of siRNA drugs. It provides reliable single-strand recovery, excellent repeatability and robust linearity without non-specific adsorption issues, making it ideally suited for quantitative quality-control applications.

References

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