Increasing Yield of in Vitro Transcription Reaction With at‐Line High Pressure Liquid Chromatography Monitoring

Domen Pregeljc, Janja Skok, Tina Vodopivec, Nina Mencin, Andreja Krušič, Jure Ličen, Kristina Šprinzar Nemec, Aleš Štrancar & Rok Sekirnik

Biotechnology and Bioengineering, December 2022

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.

  • What is the impact of each reagent on in vitro transcription yield and kinetics?
  • How can at-line analytics be implemented to monitor capping reaction?
  • Does feeding NTPs to the IVT reaction affect capping efficiency?
  • How to accurately quantify mRNA in a crude IVT reaction?
Individual IVT reaction components were optimised by understanding IVT reaction kinetics. The paper shows that development of fed-batch IVT requires more than just NTP addition, with Mg2+ being a critical component. Batch and fed-batch IVT were evaluated in terms of capping efficiency.

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