Maximizing the mRNA productivity for in vitro transcription by optimization of fed-batch strategy

In vitro transcription (IVT) is the main manufacturing method to produce mRNA vaccines. In this study, a fed-batch strategy was systematically optimized for IVT process with dual goals of achieving a high reaction rate and maximizing the final mRNA yield simultaneously. Initially various experimental conditions were investigated including Mg²⁺, nucleotide triphosphates (NTP), dithiothreitol (DTT), spermidine, as well as the temperature and ionic strength. It was found that the concentrations of Mg²⁺ and NTP had a significant impact on IVT process. Subsequently, under the optimized conditions, dividing the IVT reaction into three distinct phases was proposed to enable more efficient transcription. By optimizing the concentrations of Mg²⁺ and NTP in two replenishment processes, our fed-batch strategy resulted in the production of 367.8 μg of mRNA with reduced dsRNA byproducts within 180 min. This was achieved under conditions of a final volume of 30 μL, 250 U T7 RNA polymerase (RNAP), and 2 μg DNA template. In conclusion, the IVT process using a fed-batch approach, rather than an excessive one-time NTP input, is an efficient method to improve productivity given a fixed amount of T7 RNAP and DNA template.