Qualification of a quantitative stability indicating potency assay for mRNA-LNP vaccine candidates

Figures

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  Figure 1.. Antibody screening data by ELISA. SARS CoV-2 purified S trimer recombinant proteins (SARS-CoV-2 WT and Omicron XBB1.5; Sino Biologicals) were coated on an ELISA plate at 1, 0.125, 0.0625, and 0.0078 μg/mL for the SARS-CoV-2 WT variant or 1 and 0.5 μg/mL for the Omicron XBB 1.5 variant. Rabbit (40592-R493, Sino Biologicals) and mouse (Ms 175, MHRA) antibodies against the SARS CoV-2 S antigen were tested across all coating concentrations in 2-fold dilutions starting at 1 μg/mL.

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  Figure 2.. Sandwich assay parameters and the effect of cell line and extraction buffer on protein concentration. The amount of protein recovered after overnight transfection with mRNA-LNP into A) HEPG2 cells and B) HEK293 cells using different extraction buffers (1% Triton X (T); RIPA, protease inhibitor (PI), freeze thaw cycle (FzT)) was measured by Sandwich ELISA.

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  Figure 3.. FFA and Direct ELISA development methods. A) FFA cell images of HEPG2 and HEK293 cells after mRNA-LNP exposure and measurement of protein expression, B) poly-D-lysine (PDL) and polyethylenimine (PEI) media additives effect on FFA protein measurement in HEK293 cells after mRNA and mRNA-LNP exposure, C) representative data from the FFA method testing mRNA-LNP at two different concentrations (0.25 μg and 0.125 μg) and after heat treatment at 75°C for 10 minutes, D) representative data from the direct ELISA method testing mRNA-LNP at two different concentrations (0.25 μg and 0.125 μg) and after heat treatment at 75°C for 10 minutes.

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  Figure 4.. Effect of ApoE3 on detectable protein by Sandwich ELISA. Effect of addition of 4 μg of ApoE3 on the amount of detectable protein generated in two different cell concentrations (1 x10 6 and 5 x10 5 cells/mL) by sandwich ELISA.

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  Figure 5.. Sandwich ELISA parameters. Final assay parameters tested for qualification (A) and Representative sandwich ELISA curves of a concentration range and heat stressed for SARS-CoV-2 WT and Omicron mRNA-LNP (B).

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  Figure 6.. Qualification testing flow chart. As shown testing by FFA, sandwich ELISA, direct ELISA and western blot were conducted for two operators using the same panel of test samples.

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  Figure 7.. Representative potency data from SARS-CoV-2 WT mRNA-LNP qualification testing. Test samples of SARS-CoV-2 WT mRNA-LNP at 0.25 μg, 0.5 μg and 1 μg were compared to a 0.5 μg standard in all test methods. A) FFA, B) Direct ELISA, C) Sandwich ELISA.

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  Figure 8.. Qualification western blot. A) SARS-CoV-2 WT mRNA-LNP B) Omicron mRNA-LNP extracts with a 0.5 μg 2-fold standard dilution curve and a high and low sample load for each of the transfection amounts of 1 μg, 0.5 μg and 0.25 μg.

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  Figure 9.. Representative potency data from Omicron mRNA-LNP qualification testing Test samples at starting concentrations of 0.25 μg, 0.5 μg and 1 μg were compared to a 0.5 μg standard. Representative data from all test methods A) FFA, B) Direct ELISA, C) Sandwich ELISA.

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  Figure 10.. ln (Measured RP) versus ln (Expected RP) across levels; with the unit line.

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  Figure 11.. Relationships of ln (Observed RP) versus ln (Expected RP) for data obtained in the qualified ELISA, a Direct ELISA, and FFA.

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  Figure 12.. Linear fits to ln (Observed RP) versus ln (Expected RP) by operator and overall.