Optimization of In vitro Transcription Reaction for mRNA Production Using Chromatographic At-Line Monitoring

Area of Science:

• Biotechnology
• Biochemistry
• Molecular Biology

Background:

• In vitro transcription (IVT) is crucial for mRNA drug substance production but is reagent-cost intensive.
• Optimizing IVT requires understanding reagent effects on reaction kinetics and yield.
• Traditional analytical methods for mRNA are low-throughput and not real-time.

Purpose of the Study:

• To develop and apply a near real-time analytical method for studying IVT reaction kinetics.
• To enable optimal reagent utilization and cost reduction in mRNA production.
• To facilitate the transition from batch to fed-batch IVT processes.

Main Methods:

• Development of a liquid chromatography (LC) method for separating nucleoside triphosphates (NTPs), plasmid DNA (pDNA), and mRNA.
• Application of the LC method for near real-time monitoring of IVT reactions.
• Analysis of reagent impacts on IVT kinetics and mRNA yield.

Main Results:

• The developed LC method successfully separates key IVT components in near real-time.
• The method provides insights into the influence of individual reagents on reaction kinetics.
• Demonstrated potential for optimizing IVT processes and increasing productivity.

Conclusions:

• Near real-time chromatographic analysis is essential for comprehensive IVT understanding.
• This analytical approach supports the optimization of IVT reactions for cost-effective mRNA production.
• The findings pave the way for converting batch IVT to more efficient fed-batch processes.