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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.

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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.

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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.