Enabling functionality and translation fidelity characterization of mRNA-based vaccines with a platform-based, antibody-free mass spectrometry detection approach

The success of mRNA-based therapeutics and vaccines can be attributed to their rapid development, adaptability to new disease variants, and scalable production. Modified ribonucleotides are often used in mRNA-based vaccines or therapeutics to enhance stability and reduce immunogenicity. However, substituting uridine with N[1][1]-methylpseudouridine has recently been shown to result in +1 ribosomal frameshifting that induces cellular immunity to the translated off-target protein. To accelerate vaccine development, it is critical to have analytical methods that can be rapidly brought online to assess the functionality and translation fidelity of mRNA constructs. Here, a platform-based, antibody-free method was developed using cell-free translation (CFT) and liquid chromatography-tandem mass spectrometry (MS) that can detect, characterize, and provide relative quantification of antigen proteins translated from mRNA vaccine drug substance. This workflow enabled the evaluation of mRNA subjected to thermal stress as well as bivalent (i.e., two mRNA encoding different antigen variants) drug substance. Additionally, the MS detection approach exhibited high sensitivity and specificity by accurately identifying all six translated proteins and their relative abundances in a dose-dependent manner following transfection of human cells with a hexavalent mRNA mixture encapsulated in lipid nanoparticles (LNPs), despite significant protein sequence homology. Expanding on these efforts, we show the utility of the CFT-MS approach in identifying the presence and junction of +1 ribosomal frameshifting resulting from N[1][1]-methylpseudouridation. Overall, this CFT-MS methodology offers a valuable analytical tool for the development and production of mRNA-based vaccines by facilitating the evaluation of mRNA quality and functionality while ensuring accurate translation of antigen proteins. ![Figure][2] ### Competing Interest Statement All authors are current employees of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. The authors declare a competing interest of a PCT international patent application PCT/US2023/033931F titled AN ANALYTICAL METHOD USING LC-MS/MS PROTEOMICS TO CHARACTERIZE PROTEINS TRANSLATED FROM MRNA. This patent application is filed by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA with AQS, BWR, MH, CT, and XL named as inventors. [1]: #ref-1 [2]: pending:yes