The COVID‐19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low‐throughput, end‐point analytics. We implemented a workflow based on rapid at‐line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub‐3 min read‐out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed‐batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti‐reverse cap analog (ARCA)‐capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed‐batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed‐batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

One of the major challenges of mRNA based vaccines has been their requirement for distribution and storage at extremely low temperatures, indicating that exposure of mRNA to suboptimal physico-chemical conditions can result in degradation and loss of potency; it is unclear whether this is due to instability of mRNA drug substance, or LNP-encapsulated mRNA, or both. In this study we compare the stability of model mRNA drug substance (eGFP, 995 nt) prepared by affinity chromatography with the stability of mRNA purified by precipitation. We show that both purification methods lead to highly pure mRNA drug substance, however, mRNA purified by chromatography remains stable for 28 days at 37°C, whereas mRNA purified by precipitation is subject to significant degradation under the same storage conditions. We conclude that chromatography eliminates elements and/or conditions with adverse impact on the quality of mRNA to a greater extent than precipitation method and that choosing appropriate purification strategy is crucial not only to achieve target purity but also to obtain a stable product with retained integrity.

The COVID‐19 pandemic triggered an unprecedented surge in development of mRNA‐based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single‐use bioreactors are traditionally used for cell‐based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell‐free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed‐batch IVT approach, and demonstrate the feasibility of continuous fed‐batch production, paving the way towards continuous manufacturing of mRNA.

The COVID-19 pandemic triggered an unprecedented rate of development of mRNA vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of IVT depends on understanding of the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which