Optimization of In Vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

mRNA synthesis by in vitro transcription reaction (IVT) is a critical step in the production of mRNA therapeutics. IVT is a complex, multi-component enzymatic reaction where mRNA is synthesized from a DNA template using RNA polymerase, typically T7 RNA polymerase, and nucleoside triphosphates (NTPs) in a buffered solution containing magnesium ions. The concentrations and ratios between reaction components need to be carefully balanced to maximize mRNA yield while minimizing the formation of product-related impurities and the cost of the reaction. This protocol describes the use of a chromatographic analytical method for separation of NTPs, pDNA, and mRNA with a 4-minute read-out. At-line analysis which allows to monitor mRNA production and NTP depletion can be used to study the influence of reaction parameters on reaction kinetics and yield as well as for reaction optimization and transfer to fed-batch mode.