Messenger RNA (mRNA) technology came of age during COVID-19 with several vaccines and therapeutics coming to market. However, since the height of the pandemic, the flow of new mRNA-based products has slowed.

In part, the slowdown reflects efficacy and safety issues some developers have encountered. For others, manufacturing mRNA at scale is the issue, according to researchers at the Institute for Separation and Process Technology, Clausthal University of Technology in Germany, who suggest digital process optimization is a potential solution.

The team focused on in vitro transcription (IVT)—a template-directed, enzymatic process in which mRNA molecules are formed from nucleotide building blocks—which has proven to be challenging to carry out at commercial scale.

IVT challenges

In current IVT methods, nucleotides and other regents are added before the process starts. There is no real option to replace compounds used up as transcription progresses.

While this approach works for smaller, lab-scale runs, for longer commercial processes nucleotide concentration soon becomes a limiting factor, resulting in the formation of truncated mRNA molecules.

Some firms—notably Moderna and BioNTech—have sought to solve this by developing fed-batch IVT processes where nucleotides and regents are on a continuous basis via bolus feeding. However, wider commercial use of these methods has been limited.

Digital twins

So instead, the Clausthal team set out to optimize IVT to maximize mRNA yields, developing a digital model to better understand the interaction between the reagents and reaction conditions.

Key kinetic parameters—determined through experimentation—were used to construct a digital twin of IVT. The researchers used this model to determine the optimal reagent concentrations and reaction conditions.

“With detailed process comprehension of the IVT fundamentals, the conditions for the operation of continuous in vitro transcription could be optimized in this work to produce 55% more mRNA with 33% less truncated mRNA, compared to our initial starting point.”

The authors suggest their approach could be the basis for a fully continuous, bottleneck-free production process of mRNA that can be applied more widely.

“The feasibility of a segmented flow approach allowed for high-throughput screening (HTS), enabling the production of 20 vaccine candidates within a short time frame, representing a 10-fold increase in productivity,” they write. “The findings presented for the first time here contribute to the development of a fully continuous and efficient manufacturing process for mRNA and other cell and gene therapy drugs and or vaccine candidates.”

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