Development of Determination of mRNA Encapsulation in Lipid Nanoparticles of COVID-19 Vaccines using Fluorescence Assay
Development of Determination of mRNA Encapsulation in Lipid Nanoparticles of COVID-19 Vaccines
Keywords:
Covid-19 vaccine, mRNA vaccine, Lipid nanoparticles, Determination of mRNA, Fluorescence assayAbstract
The mRNA COVID-19 vaccine contains mRNA strands encapsulated in lipid nanoparticles to prevent degradation of the mRNA when injected into the body, and it can effectively induce immunity. The percentage of mRNA encapsulated in lipid nanoparticles is one of the quality control guidelines for vaccine quality established by the World Health Organization. During the pandemic, the manufacturer had not yet revealed the specifications for the relevant test method and standard substances. This study, therefore, explored development of the method using Ribogreen, a fluorescent dye available in commercial kits, to analyze the amount of mRNA in the lipid nanoparticle-encapsulated mRNA COVID-19 vaccine and examine the percentage of mRNA encapsulated. Triton X-100 was added to the vaccine to determine the total mRNA while quantifying the free mRNA; however, Triton X-100 was not necessary. The results showed that the method could be used to analyze the standard RNA amount and monitor the mRNA in the vaccine samples. The linear standard curve was obtained, and the detection ranges were established at 2.5–25 and 50–500 ng/mL for the amounts of low and high mRNA, respectively. All conditions had R2 values greater than 0.99, and %RE and %CV were less than 10%. The appropriate ranges to dilute the vaccine samples in determining the free mRNA and total mRNA were less than 1:100 and 1:1,000, respectively. The percentage of encapsulated mRNA calculated was found to be close to the results of the manufacturer's method. The method in this study has been proven to have the potential to determine the encapsulated mRNA COVID-19 vaccines, which may be useful for the mRNA vaccines under development, and in clinical studies in Thailand.
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