Increasing Productivity of IVT Reaction Using At-Line Monitoring by HPLC Chromatography

This webcast features: Rok Sekirnik, PhD, Head of Process Development mRNA/pDNA, BIA Separations, now a Sartorius company.

In Vitro Transcription (IVT) reactions are normally performed as batch processes. Considering their catalytic basis, it is possible to extend reaction times and yields by continuous addition of consumed reagents to the reaction mixture. However, fed-batch strategies reported to date have only managed to achieve a 40 – 100% increase in the production of mRNA. One of the main limitations of development has been low throughput of analytics for quantification of mRNA and NTP precursors; productivity is typically determined as an end-point measurement of mRNA concentration.

In this webinar, we will discuss an HPLC-based analytical method using a multimodal ligand based on anion exchange/hydrogen-bonding ligand (PrimaS) to monitor the IVT reaction, which allows for simultaneous quantification of NTPs, capping reagent, plasmid, and mRNA within 3.5 min.

When applied to monitoring IVT reaction a batch approach with an average productivity of 3-5 mg/mL can be converted to fed-batch yielding 10-12 mg/mL. Two approaches for controlling IVT reaction will be demonstrated, one focusing on productivity of uncapped mRNA, another on productivity of capped mRNA, thereby requiring a precise control of NPT:capping reagent ratio.

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