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Differential Protein Precipitation-Based GalNAc-siRNA Sample Preparation with LC/MS Method Development Workflow in
Youngjae Kim1, Ting Ting Zhang1, Hiroshi Sugimoto1
1Department of Drug Metabolism and Pharmacokinetics, and Modeling, Takeda Development Center Americas, Incorporation, 35 Landsdowne Street, Cambridge, Massachusetts 02139, United States.
Analytical Chemistry
|May 26, 2025
Summary
A new differential protein precipitation method enhances the extraction of liver-targeted N-acetylgalactosamine (GalNAc)-siRNA conjugates for accurate quantification using liquid chromatography-mass spectrometry (LC/MS). This cost-effective approach improves sensitivity for siRNA bioanalysis.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Pharmacology
Background:
- Quantification of small interfering RNAs (siRNAs) using liquid chromatography-mass spectrometry (LC/MS) is vital for therapeutic development.
- Current extraction methods like solid-phase extraction (SPE) and hybridization for N-acetylgalactosamine (GalNAc)-siRNA conjugates present challenges in recovery, cost, and applicability.
- Efficient recovery of GalNAc-siRNA from complex biological matrices like plasma is crucial for accurate bioanalysis.
Purpose of the Study:
- To develop a novel, efficient, and cost-effective extraction method for the LC/MS bioanalysis of liver-targeted GalNAc-siRNA conjugates.
- To overcome the limitations of existing SPE and hybridization methods for siRNA extraction.
- To achieve highly sensitive quantification of specific GalNAc-siRNAs and their metabolites in biological samples.
Main Methods:
- Developed a differential protein precipitation method utilizing an optimized organic solvent mix to selectively remove plasma proteins.
- Optimized the workflow for intense MS/MS transitions and fine-tuned LC-MS/MS parameters on high-resolution Orbitrap and QTRAP mass spectrometers.
- Validated the method using four FDA-approved GalNAc-siRNAs (Givosiran, Lumasiran, Inclisiran, Vutrisiran) and a Givosiran metabolite (AS(N-1)3') in plasma samples from an in vivo rat study.
Main Results:
- Achieved a lower limit of quantification in the single-digit ng/mL range for targeted GalNAc-siRNAs and a major metabolite.
- Successfully demonstrated the method's applicability by analyzing plasma samples from rats treated with Givosiran, Givosiran AS(N-1)3', and Inclisiran.
- The novel extraction method proved to be straightforward, robust, highly sensitive, and cost-effective.
Conclusions:
- The developed differential protein precipitation method offers a significant advancement for the LC/MS bioanalysis of GalNAc-siRNAs.
- This approach provides a sensitive, robust, and cost-effective alternative to existing extraction techniques.
- The method is readily adaptable for the bioanalysis of diverse GalNAc-siRNAs and suitable for late-stage sample analysis.
