This new, patented method for the production of the essential component of pseudouridine mRNA vaccines is more efficient, sustainable and cost-effective than previously used chemical synthesis.
Researchers from the Institute of Biotechnology and Biochemical Engineering at TU Graz and the Austrian Center for Industrial Biotechnology (acib) have developed a new method for the production of the main component of mRNA vaccines. They also applied for a patent.
In an article published in a specialist journal Nature CommunicationsNidetzky, Martin Pfeiffer and Andrej Ribar explain how they produced a pseudouridine vaccine base through biocatalytic synthesis and created an alternative to previous chemical synthesis methods.
One step process
The alternatives offer several distinct advantages. The chemical synthesis of pseudouridine not only involves toxic reagents and rare raw materials, but is also very energy and time consuming as it requires four to eight processing steps and cooling to minus 20 degrees.
Biocatalysis, on the other hand, requires only a single step process with four parallel reactions occurring at room temperature. In addition, only four enzymes are needed as a catalyst (uridine phosphorylase, phosphopentose mutase, pseudouridine monophosphate glycosidase and phosphatase). It can be produced easily with E. coli bacteria. Biocatalysis also produces no waste materials; the only waste is phosphate, but this is recycled during the catalytic process.
Another important advantage is efficiency. Because the chemical production of pseudouridine converts natural uridine, which is less efficient for vaccines, there is no 100% yield during the conversion process. However, due to the smaller number of process steps, biocatalytic synthesis achieves yields of 92 to 95%, compared to only 40 to 50% for chemical processes published so far.
Inspiration from nature
The researchers based the new process on one of their previous studies in which they found that the YeiN enzyme is a biocatalyst for C-nucleotide production. Since pseudouridine is the C-nucleoside of the RNA building block of uridine, they came up with the idea to take uridine, which can be produced in large quantities by bacterial fermentation, as a raw material and rebuild the bonds between its basic building blocks. Inspiration comes from nature.
Unlike pseudouridine, uridine has an N-glycosidic bond, which is cleaved into ribose-1-phosphate (sugar) and uracil during natural degradation in cells via the enzyme uridine phosphorylase. Then the enzyme phospentose mutase rearranges the ribose-1-phosphate to ribose-5-phosphate, which is metabolized in the cell. This is followed by the application of the enzyme YeiN, which is used to link ribose-5-phosphate and uracil to pseudouridine-5-phosphate. Finally, a phosphatase is used to separate the phosphate from the pseudouridine. Since pseudouridine is much less soluble in water than uridine, it only crystallizes during the reaction and can therefore easily be obtained by filtering the reaction supernatant.
Large scale production
“Our work shows that biocatalysis is a powerful alternative to the chemical synthesis of C nucleotides such as pseudouridine,” said Bernd Nidetzky, head of the Institute of Biotechnology and Biochemical Engineering at TU Graz.
“We hope to implement production on a larger scale soon and thereby make pseudouridine available sustainably and cheaply in larger quantities. It could also make production of mRNA vaccines cheaper in the medium term, as potential partners from the industry can deploy our applications fairly quickly.”