Development of analytical methodology for quality control of messenger RNA vaccine

Introduction: Vaccines using messenger RNA technology have gained prominence in recent years due to their efficiency, speed of development and flexibility.Therefore, to obtain vaccines with maximum efficacy, it is necessary to carry out Quality Control (QC) of the development stages to ensure the safety, efficacy, and consistency of the product.In this way, one of the stages of the QC for the development of the mRNA vaccine involves the analysis of chemical modifications present in the mRNA, such as capping and the poly-A tail, located in the 5' and 3' regions, which are essential for the stability of the mRNA as they prevent the action of ribonucleases. Objectives:The general goal of the work is to analyze by capillary electrophoresis the chemical modifications of capping and poly-A tail of conventional Covid-19 mRNA. Methodology:The capping analysis used a hybridization step with RNA/DNA hybrid probes (probes 41 and 57) complementary to the mRNA molecule and cleavage with ribonuclease H.The 41 and 57 probes were made to anneal to the regions corresponding to 41 and 57 nucleotides, counting from the 5' region, to obtain fragments that could later be analyzed by LC/MS.For the assay to verify the length of the poly-A tail, initial cleavage was carried out with ribonuclease T1, followed by affinity purification with oligo(dt) magnetic beads.The samples obtained from both processes were analyzed by capillary electrophoresis. Results:The results obtained from the capillary electrophoresis showed that both probes 41 and 57 were annealed to the respective expected regions of the target mRNA.Finally, the analysis of the length of the poly-A tail used an internal control with the same number of adenines as the poly-A tail sequence of the Bio-Manguinhos mRNA vaccine.Therefore, we observed a majority band at the expected height of the poly-A tail, showing that the tail is being synthesized properly in the in vitro transcription (IVT) process. Conclusion:In conclusion, the capillary electrophoresis technique was able to provide preliminary information on the success of the processing of the mRNA samples, although it is necessary to confirm this result using LC/MS.