Advancements in mRNA Synthesis: Real Time Quantification and Characterization of In Vitro Transcription Reactions Using Modified Nucleotides

The recent push to make a viable mRNA based vaccine against COVID 19 highlighted the significance of modified nucleosides, as one of the candidate vaccines failed precisely because only regular NTPs were used to produce the mRNA 1 The groundbreaking discovery made by Karikó and Weissman in 2005 demonstrating that mRNA synthesized with ΨTP instead of UTP exhibits reduced immunogenicity, was recognized with the Nobel Prize in Chemistry in 2023 Modified nucleosides can change the structure, stability and even affect the rate of translation of the mRNA As more and more research is done in this field, we focused on developing a method enabling at line IVT reaction monitoring using two naturally occurring modified nucleotides 1 methylpseudouridin triphosphate (N1meΨTP and 5 methylcytidine triphosphate (m 5 CTP), on a multimodal CIMac PrimaS ® column using PATfix® analytical system The developed method facilitates the monitoring of in vitro transcription (reactions by accommodating the quantification of modified nucleotides (N1meΨTP and/or m 5 CTP), unmodified nucleotides and mRNA across varying ratios.