Author: Katharina Schwaiger, acib GmbH
mRNA vaccines gained widespread notoriety for their revolutionary power in combating the Corona pandemic. But behind the scenes, a silent hero called pseudouridine plays a crucial role in maximising their efficacy and safety. Pseudouridine, a modified RNA building block, has the remarkable ability to increase RNA stability1 and fine-tune immune responses2, making it a valued component in the world of mRNA therapeutics3–5. However, with the increasing demand for these breakthrough vaccines, the question arises: how can we produce pseudouridine sustainably without compromising efficiency or environmental impact?
Meeting the Growing Demand
The demand for RNA-based therapies is skyrocketing. Just imagine, ~13 billion doses of mRNA vaccines have already been given to people worldwide, and there are over 60 new mRNA vaccines in clinical trials for treating cancer and infectious diseases3. But pseudouridine is not only essential for vaccines, it also shows great potential in enhancing the efficiency of gene-editing technologies like CRISPR6. As a result, the need for pseudouridine and its derivates is rapidly increasing3,6,7. These RNA building blocks hold the key to the future of cutting-edge therapies, and their production will play a crucial role in meeting the growing demand.
Since the first chemical synthesis of pseudouridine in 1961, scientists have explored various methods for its production. These methods involve reconstructing the uracil nucleobase8 or linking different molecules together9,10. The most advanced chemical synthesis produces pseudouridine with a yield of 40% over three steps10. Aside from not being the most efficient, these methods aren’t exactly the most sustainable or economical either. The use of extremely low temperatures (-78 °C) in cryogenic conditions entails a substantial energy demand, while the requirement for protective group chemistry reduces the efficiency of atom usage and involves the utilization of non-environmentally friendly chemicals. These factors collectively give rise to concerns regarding the eco-friendliness of the process. Consequently, there is a clear imperative for enhancing the sustainability of the current procedure.11. In this context, the field of biocatalysis emerges as a beacon of hope, presenting promising solutions that can replace such costly and ecologically unsustainable production processes.
A “Dream Reaction”
In a significant breakthrough, researchers from the Institute of Biotechnology and Biochemical Engineering at TU Graz and acib, namely Martin Pfeiffer, Andrej Ribar, and Bernd Nidetzky, have recently published an innovative approach to pseudouridine production. Their study, published in the prestigious journal “Nature Communications,” introduces a biocatalytic synthesis method that addresses the surging demand for mRNA therapeutics while prioritizing sustainability11. By harnessing the catalytic power of four enzymes in a cascade conversion, the researchers have devised a streamlined process to convert uridine into pseudouridine. This transformative production route consists of four steps that can be performed simultaneously in one-pot. The overall cascade achieves a remarkable 100% yield, coupled with an impressive productivity of ~40 grams pseudouridine per liter and hour. Notably, in this reaction all the atoms from the starting material can be found in the product, achieving a 100% atom-economy conversion. Moreover, it introduces a groundbreaking feature: the recycling of phosphate within the reaction itself. This ingenious design reduces the need for high phosphate concentrations, enhancing reaction efficiency, sustainability, and cost-effectiveness. From a process engineering point of view, another noteworthy aspect is the spontaneous formation of a pure pseudouridine powder directly from the reaction solution, yielding up to 25 g of isolated pseudouridine from 100 mL of reaction volume. This so-called in situ crystallization, represents a highly desirable technique of product isolation in process chemistry. Both, the efficient atom-economy and the impressive crystallization behavior of pseudouridine make this approach a true “dream reaction”.
The Power of Biocatalysis
The production process also boasts an exceptionally low waste factor, known as the E-factor, which measures waste per unit of product. With an E-factor of just 3, this process showcases remarkable sustainability. Furthermore, when considering the total mass of materials, including enzymes, used to produce a given mass of pseudouridine, named the process mass intensity (PMI), the number stands at around 4.5. These values are significantly lower than those typically observed for small-molecule processes in the pharmaceutical industry. For comparison, E-values usually hover around 25, while PMI values range from several tens to well over 100.12
The groundbreaking research of Pfeiffer, Ribar, and Nidetzky not only broadens the horizon of industrial biotechnology but also underscores the critical role of biocatalysis in advancing sustainable chemistry. Their pioneering work represents a novel and unprecedented achievement, and sheds light on the remarkable efficiency that multi-enzyme systems can have.
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- Pfeiffer, M., Ribar, A. & Nidetzky, B. A selective and atom-economic rearrangement of uridine by cascade biocatalysis for production of pseudouridine. Nat. Commun. 14, 2261 (2023).
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