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

Abstract

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 (IVE-LC/MS/MS) 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.

Fig. 1

Overview of In-Vitro Expression (IVE)-Flow Cytometry and IVE-LC/MS/MS Workflow. IVE begins with the production of approximately 200—400 k viable HEK293T cells. These are transfected with mRNA encapsulated in LNPs such that the total dose of mRNA is controlled, usually from 3 to 500 ng. After 24 h the transfected cells are harvested and split for flow cytometry and LC/MS/MS PRM analysis. For LC/MS/MS PRM analysis, pelleted cells are fully solubilized in sodium dodecyl sulfate with 90 °C heating and sonication, then protein disulfides are reduced, and cysteine thiols are alkylated. Proteins and nucleic acids are acetonitrile-precipitated, and the pellet is resolubilized in a nuclease digestion reaction solution. Finally, proteins are Lys-C/Trypsin-proteolyzed in a 2 h incubation. No further sample handling is required prior to LC/MS/MS injection. Figure created with Biorender.com.

Fig. 2

Spiking Study of Four Recombinant Flu Hemagglutinin and Covid Spike Antigens Using Various MS Parameters. (A) LC-PRM profile of four recombinant influenza hemagglutinin (HA) proteins (Wisconsin, Darwin, Austria, and Phuket) and recombinant SARS-CoV-2 S1 domain of spike protein at 2 pmol each spiked into negative control HEK293T cell pellets. (B) Quantitation results of spike study at high resolution (method 1). Antigen peptide signal (%) for each unique antigen relative to specific HEK293T peptide signal at each spike level were plotted and fit with linear curve. The fit slope and R² value for each antigen are provided in the table below. Orange: Covid; Black: Austria; Blue: Wisconsin; Magenta: Phuket; Red: Darwin. (C) Representative PRM profile of Wisconsin HA peptide EQLSSVSSFER at 0.13 and 2 pmol spike-in levels using high (method 1), medium (method 2), and low (method 3) resolution settings. (D) Quantitation results of spike study at medium resolution (method 2). Antigen peptide signal (%) for each unique antigen relative to specific HEK293T peptide signal at each spike level were plotted and fit with linear curve. The fit slope and R² value for each antigen are provided in the table below. Orange: Covid; Black: Austria; Blue: Wisconsin; Magenta: Phuket; Red: Darwin.

Fig. 3

mRNA Vaccine Dose Response. The mRNA vaccine dose response, encapsulating the efficiency of mRNA transfection into HEK293T cells and subsequent expression of the encoded antigen proteins, was measured by IVE-LC/MS/MS and IVE-flow cytometry. HEK293T cells were dosed with a quadrivalent mRNA vaccine containing an equal theoretical load of each mRNA construct. The resulting data from the IVE-LC/MS/MS assay are graphed as % antigen peptide signal for each unique antigen relative to specific HEK293T peptide signal vs. total mRNA dosed (ng) per plate well (R² for Wisconsin, Darwin, Austria and Phuket are: 0.963, 0.985, 0.99 and 0.980 respectively) (A). A linear regression of these same data in pmol antigen per pellet is plotted in B (R² for Wisconsin, Darwin, Austria and Phuket are: 0.992, 0.970, 0.997 and 0.995 respectively). IVE-flow cytometry assay results are graphed as % cells positively expressing the antigen protein of interest (C) and mean fluorescence intensity (MFI) ratio (D) vs. the total mRNA (ng) per plate well.

Fig. 4

Dose–response and Time-course Study of mRNA Transfection. The mRNA vaccine in-vitro expression levels over different doses and time points were measured using IVE-LC/MS/MS. HEK293T cells were dosed with a quadrivalent mRNA vaccine. Panel A presents the raw PRM data for the IVVDYMVQK peptide in response to varying doses. Panels B and C show the antigen peptide signal overlays at 24 h (in red) and 48 h (in blue) relative to specific HEK peptide signals. This is plotted against the total mRNA (in pmol) dosed per well for two representative HA antigens—Wisconsin HA in Panel B and Darwin HA in Panel C. Panels D and E illustrate the absolute amounts of Wisconsin and Darwin HA antigens in cell pellets relative to the total mRNA (in pmol) dosed per well. Panels F (R² = 0.77) and G (R² = 0.936) display the correlation between total protein per sample, measured by UV280 absorbance, and the average HEK peptide peak area at 24 and 48 h, respectively.

Fig. 5

Stability Study of Quadrivalent Flu LNPs: Relationship between %Antigen/HEK, % Antigen Positive Cells and MFI. The mRNA vaccine stability was measured by IVE-LC/MS/MS and IVE-flow cytometry. HEK293T cells were dosed with a quadrivalent mRNA vaccine. Overlays of the change in expressed antigen by IVE-LC/MS/MS and the percentage of antigen positive cells and MFI ratio by flow cytometry for up to 12 months at -80 °C (Panels A-D) and at 5 °C (Panels E–H) for four HA antigens. Each data point measured by IVE-LC/MS/MS is representing the average biological replicates (n = 3, %RSD ≤ 6%) and each IVE-Flow cytometry is representing the average of 2 biological replicates.

Fig. 6

Effect of Various Buffers on Phuket Monovalent Flu LNP Expression. Antigen expression in different buffers was measured by IVE-LC/MS/MS. HEK293T cells were dosed with a monovalent mRNA vaccine at four different doses. The figure depicts overlays of % antigen peptides signal to HEK293T peptides signal vs. total mRNA (ng) dosed per well for three buffer combinations. n = 2 for each transfection dose. R² for Buffers 1, 2 and 3 are 0.993, 0.981 and 0.996 respectively.

Fig. 7

Antigen Expression is diminished after LNP shaking. Antigen expression of LNPs containing monovalent mRNA was measured after shaking for 30 and 240 min at mRNA concentrations 0.12, 0.06, and 0.01 mg/mL using IVE-flow cytometry (% HA positive cell, panels A, B, and C, and MFI ratio, panels D, E, and D) and IVE-LC/MS/MS (% Antigen/HEK, panels G, H, and I). Black squares depict non-shaken, green triangles and red diamonds depict 30- and 240-min shaking, respectively. R² for NS, 30 min and 240 min conditions respectively are as follows: D, 0.98, 0.99, 0.99; E, 0,95, 0,95, 0,99; F, 0.98, 0.98, 0.98; G, 0.98, 0.99, 0.98; H, 0.99, 0.96, 0.99; I, 0.99, 0.98, 0.99. n = 2 for IVE-flow cytometry dose–response data.

Fig. 8

Relative levels of antigen compared to non-shaking. Antigen expression of LNPs containing monovalent mRNA was measured after shaking for 30 and 240 min at mRNA concentrations 0.12, 0.06, and 0.01 mg/mL using IVE-flow cytometry (% HA positive cell, panel A, and MFI ratio, panel B) and IVE-LC/MS/MS (% Antigen/HEK, panel C). Data represented as % change from non-shaking condition (NS). Change from NS is calculated based on average of 3 doses (IVE-MS: 50,100,150 ng/well; Flow cytometry: 8, 16, 32 ng/well).