Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring
Tina Vodopivec Seravalli, Janja Skok, Tjaša Marušič, and Rok Sekirnik
J. Vis. Exp. 2025 Apr 4:(218). doi: 10.3791/67503
The article presents a method for optimizing the in vitro transcription (IVT) reaction, which is a critical and costly step in mRNA production, particularly important given the recent advancements in mRNA technology. It introduces an at-line PATfix mRNA analytical paltform, utilizing chromatographic system that allows for near real-time monitoring of IVT reaction kinetics. This method precisely separates and quantifies key components like nucleoside triphosphates (NTPs), pDNA, and mRNA, providing crucial insights into their consumption and production. Such detailed monitoring enables effective optimization of IVT in both batch and fed-batch modes, ultimately aiming to increase mRNA yield and reduce overall production costs.