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mRNA, IVT, immobilisation, magnetic beads, T7 RNA Polymerase

Abstract

Vaccinations play a vital role in stopping the spread of infectious diseases. An upcoming technology ismRNA vaccines, which have proved very effective during the COVID-19 pandemic. These vaccines are now being developed and tested for a panoply of diseases, including infectious diseases and  cancers, due to their manufacturing flexibility, safety and precision. Until now, mRNA is produced in cell-free system reactions from a linear DNA template, using RNA polymerase as a catalyst and nucleoside triphosphates (NTPs) as co-substrates, together with other reaction components. Typical  production titres are between 2 to 5 g·L−1 but recent studies have shown that titres can be increased to 12 g·L−1 in both batch and fed- batch mode. Despite the well-defined enzymatic manufacturing process and the tight reaction control, the cost of these vaccines is still prohibitive for LMICs, mainly  driven by the cost of goods.

In this work, we explore the use of magnetic beads to immobilize enzymes (e.g. T7 RNA Polymerase, T7RNAP) or substrates present in the IVT reaction. Magnetic beads offer the flexibility to operate IVT reactors in different modes of operation and/or reactor configurations, besides reducing  significantly purification steps and reaction components. Enzymes were immobilized on the magnetic beads exploring the biotin–streptavidin chemistry to simplify immobilization workflows. Reaction space was explored with optimum temperature and pH ranging from 41-44°C and pH ranging from  6.3-6.8, respectively. This is in contrast with the free enzyme system optimum conditions where high yields (12 g·L−1) are obtained at 44 °C and pH 6.8. Furthermore, T7 RNA polymerase activity could be maintained for 3 days at 4 °C without significant loss. The magnetic beads allowed for stable  operation over 5 cycles. Despite the impact of immobilisation on the kinetic parameters, e.g. km(app) and kcat, when compared to solution phase enzymes, the immobilisation proved highly robust. This work will contribute to achieving lower production costs, and ultimately making the processes  sustainable and affordable to all.

Acknowledgement

This research was funded by the UKRI Engineering and Physical Sciences Research Council (EPSRC).