Enhancing <scp>mRNA</scp> vaccine production: Optimization of in vitro transcription for improved yield and purity

In vitro transcription (IVT) is a powerful method to generate RNA which not only facilitates RNA research but also plays a key role in the development and manufacture of RNA-based vaccines. mRNA is produced via the IVT process with a DNA template that contains information for the target antigen. However, as many disease-causing viruses mutate quickly and the cost of raw materials is high for the IVT reaction, there is a need for a system to develop a cost-effective and efficient IVT process platform. In this paper, we showed how total nucleoside-5'-triphosphates (NTPs) input, Mg²⁺ concentration and NTP preparation methods can influence IVT reaction yield and purity level of the final RNA constructs of different lengths and sequences. We propose an IVT design that will result in high RNA yield, high RNA integrity and low double-stranded RNA (dsRNA) concentrations for multiple RNA sequences. The approach presented here could significantly contribute to the development of a cost-effective, easy-to-adopt IVT process platform for RNA manufacturing.