A novel in-vitro expression assay by LC/MS/MS enables multi-antigen mRNA vaccine characterization

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Messenger RNA (mRNA) vaccines represent a significant advancement in vaccinology.
• Current methods for monitoring in-vitro expression (IVE) of mRNA, such as flow cytometry, rely on antigen-specific antibodies.
• There is a need for orthogonal and more in-depth characterization methods for mRNA-based therapeutics.

Purpose of the Study:

• To develop and optimize an in-vitro expression liquid chromatography tandem mass spectrometry (IVE-LC/MS/MS) assay.
• To establish IVE-LC/MS/MS as an orthogonal method to IVE-flow cytometry for mRNA characterization.
• To demonstrate the application of IVE-LC/MS/MS for detailed analysis of expressed protein antigens.

Main Methods:

• Development and optimization of a novel IVE-LC/MS/MS workflow.
• Protein purification strategies tailored for mass spectrometry analysis.
• Mass spectrometry method setup for sensitive and selective detection of protein antigens.

Main Results:

• Simultaneous detection of influenza hemagglutinin (HA) antigens from four distinct strains.
• Demonstration of the assay's robustness, sensitivity, and selectivity.
• Successful application in evaluating dose-response, optimizing transfection, and assessing mRNA-lipid nanoparticle (LNP) stability.

Conclusions:

• The IVE-LC/MS/MS assay is a powerful, antibody-free, and multiplexing technique for mRNA characterization.
• This method serves as a valuable complement to IVE-flow cytometry, offering enhanced analytical capabilities.
• IVE-LC/MS/MS significantly aids in understanding and improving the performance and quality of mRNA LNPs.