Functionality and translation fidelity characterization of mRNA vaccines using platform based mass spectrometry detection

Figures

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  Fig. 1. CFT-SW, CFT-MS, and CBT-MS approaches to characterizing mRNA functionality. Scheme outlining the cell-free and cell-based translation workflows coupled to Simple Western™ (CFT-SW) or reversed-phase liquid chromatography tandem mass spectrometry (CFT-MS and CBT-MS) workflows for detection of proteins translated from mRNA drug substances or drug products.

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  Fig. 2. Sequence coverage map from CFT-MS analysis of SARS-CoV-2 Delta (B.1.617.2) spike protein demonstrating the ability of the assay to confirm translated protein identity. Amino acids confirmed with MS/MS matches are represented in green and amino acids identified with mass only matches are represented in blue. Black amino acids indicate no mass only or MS/MS matches were identified. Overall, 94% sequence coverage was obtained from CFT-MS analysis with 13.1 µg/mL mRNA and considering only MS/MS matches. Both the N-terminus and C-terminus were covered with MS/MS matches, allowing verification of correct translation through the entire protein sequence. Peptide mapping results were obtained by combining results from CFT-MS analysis of Delta (B.1.617.2) spike protein using the enzymes trypsin, chymotrypsin, and alpha lytic protease. Additional details regarding which peptides were identified by each enzyme are included in Supplementary Fig. 1.

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  Fig. 3. Dose–response curve showing the relative peak area response for CFT-SW (red) and relative MS1 area response for CFT-MS (blue) detecting translated SARS-CoV-2 Delta (B.1.617.2) spike protein as a function of mRNA concentration. Normalization to calculate relative abundance values as shown was performed using the sample with the maximum translated protein as 100%. Insets show the linear range for each workflow as highlighted by the gray shaded box, demonstrating strong linearity (R2 > 0.97) in the region of 0 to 13.1 µg/mL mRNA. Plots that show the actual (not normalized) peak area (CFT-SW) and MS1 area (CFT-MS) of translated Delta (B.1.617.2) spike protein are included in the Supplementary Information (Supplementary Fig. 3). CFT-MS data was corrected and normalized with the spiked-in ADH protein to account for potential sample loss during sample preparation and analysis. Results from each assay are shown as duplicate analyses (data points) and the corresponding average (solid line).

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  Fig. 4. Demonstration of the functional readout from stability-indicating CFT-SW and CFT-MS assays. Following incubation of SARS-CoV-2 Delta (B.1.617.2) mRNA at 50 °C for varying time points to degrade the mRNA, wheat germ CFT was performed on 2 µg/mL mRNA from each time point. Translated protein was detected by either Simple Western™ (“SW”, red squares) or LC–MS/MS (“MS”, blue circles). The mRNA itself was also analyzed by microchip capillary electrophoresis as a direct measurement of mRNA integrity (“MCE”, green triangles, electropherograms provided in Supplementary Fig. 4). The y-axis shows relative intensity in percent corresponding to: mRNA integrity normalized to t = 0 for MCE, chemiluminescence signal relative to t = 0 for SW, and summed MS1 area relative to t = 0 for MS. CFT-MS data was corrected and normalized with the spiked-in ADH protein. Results from each assay are shown as duplicate analyses (data points) and the corresponding average (solid line).

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  Fig. 5. CFT-MS overcomes analytical challenges facing characterization of multivalent mRNA mixtures using antibody-based analysis approaches. A Resulting profiles from CFT-SW of a mixture of SARS-CoV-2 spike protein Delta (B.1.617.2, blue) and Omicron (B.1.1.529, green) strain mRNA using an antibody that should be specific to Omicron (B.1.1.529) only (A) or specific to Delta (B.1.617.2) only (B), demonstrating the cross-reactivity of antibodies that can occur when proteins have high sequence homology (in this case, >97% sequence similarity). Alternatively, analyzing multiple mixtures of Delta (B.1.617.2) and Omicron (B.1.1.529) spike protein mRNA with CFT-MS demonstrates the utility in using mass spectrometry for such a scenario (C). Relative abundance (%) of detected Delta (B.1.617.2) and Omicron (B.1.1.529) spike protein following CFT of mRNA mixtures with varying ratios (solid data points) agrees well with the theoretical values based on mixing ratios (dashed lines).

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  Fig. 6. CBT-MS detected translated proteins from multivalent mRNA drug product in cells. Resulting protein translation from hexavalent mRNA vaccine incubated with Huh7 cells at a dose of 6.67 ng per mRNA construct (blue) or 4.44 ng per mRNA construct. Translated proteins were detected with the LC–MS/MS platform workflow as described here and protein translation is shown as relative abundance (%) compared to the most abundant protein derived from mRNA1. Results show that the proteins are translated in an mRNA construct and dose-dependent manner.

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  Fig. 7. Ribonucleotide sequence and corresponding translated protein amino acid sequence of in-frame and + 1 ribosomal frameshifted mRNA. Segment of SARS-CoV-2 Delta (B.1.617.2) spike protein mRNA sequence and corresponding translated protein amino acid sequence for in-frame and two possible +1 ribosomal frameshifted products that result in the same off-target amino acid sequence. “XXX” codon motifs are used to obscure proprietary mRNA sequence information. “+1 FS” corresponds to +1 ribosomal frameshift.

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  Fig. 8. Mass spectrometry enabled confirmation of +1 ribosomal frameshiting following CFT of SARS-CoV-2 Delta (B.1.61.2) spike protein mRNA. CFT-MS analysis confirms the presence of the GYHLMSFPR peptide that proves translation of the frameshifted protein via comparing the experimentally-observed MS/MS spectrum (black) with theoretical y- (red), b- (blue), and a-type (green) fragments. Additional confirmed fragments such as internal fragments are not labeled. The location of all identified peptide fragments from MS/MS is shown in the diagram on top of the mass spectrum, demonstrating thorough coverage of the C-terminus where the +1 frameshifted amino acid sequence begins.