optimization-vitro-transcription-reaction-for-mrna-production-using

Optimization of <em>In vitro</em> Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

In vitro transcription reaction (IVT) is a complex, multi-component enzymatic synthesis of mRNA from a linear DNA template, catalyzed by an RNA ...

Sartorius BIA Separations d.o.o., a Sartorius company

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.

Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring.

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.

Article	Year
Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring. Biotechnology and bioengineering\| PubMed ID: 36471904	2023
Determination of linearized pDNA template in mRNA production process using HPLC. Analytical and bioanalytical chemistry\| PubMed ID: 38438547	2024

Rok Sekirnik

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