Understanding the Impact of In Vitro Transcribed mRNA Impurities on Cellular Responses

Advances in mRNA technology have enabled mRNA-based therapies to enter a new era of medicine. Such therapies benefit from a single, standardized in vitro transcription (IVT) manufacturing process, applicable to a wide range of targets. This process includes several downstream purification steps to remove impurities, yielding safe and effective mRNA-based medicines. The impact of various impurities on translation efficiency and cellular responses, however, has not been investigated in detail yet. This talk will present a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. For this study, mRNAs with similar sequences by using several bacteriophage T7 RNA polymerases were employed to obtain different purity profiles. Important attributes such as purity, integrity, and functional activity were then measured using advanced physicochemical and cellular assays. Impurities, including abortive transcripts, mRNA with a short poly(A) tail, and double-stranded RNA by-products, were characterized along with their impact on cellular response using different cell models. We have developed IP-RP-HPLC and CGE methods to determine the purity of mRNA samples. Mass photometry showed differences in the amount of dimers between mRNA samples, identified as sense-antisense dsRNA by-products whose impact was investigated using bone marrow-derived dendritic cells from mice. In addition, we demonstrated that native mass spectrometry is a robust analytical tool to characterize short (i.e., <20 nucleotides) 3'-loopback dsRNA by-products, sensitive enough to highlight differences among different T7 RNAP, which are not detected by other conventional assays. Overall, this study exhibits a strong correlation between mRNA purity profiles and cellular functionality (i.e., translation efficiency and reactogenicity), underlining the value of physicochemical characterization tools to ensure quality control of RNA-based therapeutics.