Gram-Scale mRNA Production Using a 250-mL Single-Use Bioreactor

The cost of mRNA production is driven by IVT reagents, particularly the co-transcriptional capping reagents. Optimization of mRNA yield is therefore crucial for lowering the cost of mRNA production. To monitor the IVT reaction over time, we implemented a rapid at-line HPLC monitoring of consumption of NTPs and production of mRNA, with a sub-3 min read-out. Use of CIMac PrimaS analytical column allowed us to determine and adjust key IVT components that influence the kinetics of mRNA production and are critical for optimization of continuous addition of reagents, i.e. fed-batch IVT.

Fed-batch reactions can also be performed by continuous feeding, requiring automated control system. We used Ambr® 250 bioreactor platform, demonstrating for the first time its potential for mRNA production. First we designed a fed-batch IVT reaction in a thermal shaker, sampled and analyzed at-line by CIMac PrimaS analytics. Based on NTP consumption kinetics, the Ambr® 250 protocol was then designed to feed a defined mixture of NTP-Mg 2+ continuously.