Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography
Li Li1, Joe P Foley2, Roy Helmy3
1Analytical Sciences, Merck & Co., Inc, West Point, PA, 19486, USA; Drexel University, Department of Chemistry, Philadelphia, PA, 19104, USA.
Journal of Chromatography. A
|May 11, 2019
Summary
A new ultra-high-performance liquid chromatography method enables simultaneous analysis of small interfering RNA (siRNA) and lipids in nanocarrier drug delivery systems. This advances the characterization of lipid nanoparticle (LNP) formulations for novel therapeutics.
Area of Science:
- Pharmaceutical Sciences
- Analytical Chemistry
- Biotechnology
Background:
- RNA interference (RNAi) therapeutics, particularly small interfering RNA (siRNA), offer potential for treating diseases with
- undruggable
- targets.
- Effective systemic delivery of siRNA relies on nanocarrier systems, such as lipid nanoparticles (LNPs), which are complex formulations.
- Robust analytical methods are crucial for characterizing LNP composition and ensuring drug product stability for manufacturing and quality control.
Purpose of the Study:
- To develop and validate a novel ion-pair reversed-phase ultra-high-performance liquid chromatography (UHPLC) method.
- To achieve simultaneous separation and analysis of both siRNA and functional lipids within LNP formulations.
- To provide a critical tool for the characterization and quality control of LNP-based drug products.
Main Methods:
- Development of an ion-pair reversed-phase UHPLC method.
- Investigation of critical chromatographic parameters: ion-pair agent structure, stationary phase chemistry, column temperature, and organic additive.
- Application of the method to analyze an experimental LNP formulation, including potential degradation products.
Main Results:
- The UHPLC method successfully achieved simultaneous separation of siRNA and functional lipids in LNPs.
- siRNA retention time was tunable via ion-pair reagent selection, showing a relationship with alkyl chain length.
- Phospholipid separation was not significantly impacted by the type of ion-pair reagent used.
- A BEH phenyl column with dibutylammonium acetate demonstrated satisfactory selectivity for siRNAs and phospholipids.
Conclusions:
- The developed ion-pair UHPLC method provides a robust and selective approach for characterizing complex LNP formulations.
- This analytical advancement supports the development and quality control of siRNA-based nanomedicines.
- The method facilitates the assessment of LNP stability and the identification of critical components and potential impurities.
Keywords:
Ion-pair reversed-phase liquid chromatographyLipid nanoparticlePhospholipidSmall interfering RNAMore Related Videos
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