Increasing yield of in vitro transcription reaction with at-line HPLC monitoring.

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 is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line HPLC monitoring of consumption of NTPs with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal 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 ARCA-capped mRNA construct, which requires an approximate 4:1 ARCA:GTP 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, thereby paving the way towards continuous manufacturing of mRNA. This article is protected by copyright. All rights reserved.

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

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. [ FROM AUTHOR] Copyright of Chemie Ingenieur Technik (CIT) is the property of John Wiley & Sons, Inc. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)