Scientific reportsMar 26, 2025

A new lab test using mass spectrometry to analyze multi-antigen mRNA vaccines

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 12, 2026

Abstract

The assay detected influenza hemagglutinin (HA) antigens from four distinct strains simultaneously.

The IVE-LC/MS/MS method provides an alternative to traditional flow cytometry for monitoring the expression of protein antigens from mRNA.
This technique is antibody-free and offers high sensitivity and selectivity, allowing for more detailed characterization of expressed proteins.
Capable of multiplexing, the IVE-LC/MS/MS assay can analyze multiple antigens at once, enhancing experimental efficiency.
Studies indicate that the method can evaluate dose-response relationships and assess mRNA-lipid nanoparticle stability under various conditions.
Findings suggest that IVE-LC/MS/MS may improve the performance and quality assessment of mRNA vaccines.

Simplified

The new era of messenger RNA (mRNA) vaccines has led to development of a novel, state-of-the-art characterization method for this class of molecules. Currently, flow cytometry-based assays with antigen-specific antibodies are utilized for monitoring in-vitro expression (IVE) of mRNA. Here we present development, optimization, and application of an in-vitro expression liquid chromatography tandem mass spectrometry () assay as an orthogonal method to IVE-flow cytometry that can be used for in-depth characterization of the expressed protein antigens and monitoring their relative expression levels in the cell post-mRNA transfection. The IVE-LC/MS/MS assessment accomplished the detection of influenza hemagglutinin (HA) antigens of four distinct strains simultaneously. The workflow is presented here, highlighting the optimization of all necessary steps required for protein purification and mass spectrometry method setup. The IVE-LC/MS/MS assay is a robust and versatile technique that complements the IVE-flow cytometry method and offers several advantages, such as being antibody-free, capable of multiplexing, and highly sensitive and selective. The various studies in this work, including evaluating dose-response relationships, refining transfection protocols, and examining mRNA-LNP stability under various conditions showcase the significant benefits of applying IVE-LC/MS/MS across different experimental settings. IVE-LC/MS/MS is a powerful tool for understanding and improving the performance and quality of mRNA LNPs.

Key numbers

10×

Increase in Expression Efficiency

Measured increase in in-vitro expression with 150 mM NaCl buffer.

Simultaneous Antigen Detection

Four distinct influenza hemagglutinin antigens detected simultaneously.

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A new lab test using mass spectrometry to analyze multi-antigen mRNA vaccines

Abstract

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