Commercial-scale production of mRNA products is limited by their expense, complexity, and the high level of technical expertise required, which make them unsuitable for much of the world. A new one-pot method, however, simplifies the processes and reduces costs, making the production of mRNA vaccines and therapeutics feasible even in resource-constrained environments.

“Our platform… uses a co-transcriptional capping strategy that utilizes T7 RNA polymerase and Vaccinia capping enzymes to synthesize capped mRNA in one pot,” Alison Obinna Nwokeoji, PhD, a research fellow, department of chemical and biological Engineering, University of Sheffield, tells GEN. “This platform obviates the need for expensive synthetic cap analogues (significantly reducing mRNA production costs) while gaining the advantageous co-transcriptional capping feature that previously was exclusive to synthetic caps.”

This approach, developed by Nwokeoji and colleagues at the University of Sheffield and All First Technologies, hinges upon an integrated reaction buffer that simultaneously caps enzymes for both catalytic and in vitro transcription processes. Its primary elements, detailed in a recent paper, are T7 polymerase, Vaccinia capping enzyme (D1 and D2 subunits), nucleotide triphosphates (NTPs), S-adenosyl methionine (SAM), and an optimized buffer compatible with both enzymes. This method side steps the need for multiple purification steps between transcription and mRNA capping. “The buffer can be adapted or modified to suit the needs of any setting,” he says.

Streamlining mRNA production

Streamlining mRNA production processes would, logically, also reduce production costs but, Nwokeoji cautions, “a quantitative, peer-reviewed cost analysis comparing one-step and two-step production options is yet to be performed.”

The researchers also developed “a rapid detection method for mRNA and capped structures from crude in vitro transcription (IVT) products without prior mRNA purification,” he says. That method uses “inexpensive DNA primers or probe to protect the 5’ end of mRNA before digestion… and is followed by analysis by denaturing urea-PAGE,” the researchers wrote. Not only are the components inexpensive, but the technique also streamlines the workflow by not requiring biotin tagging, autoradiography, or purification of the transcription material before it is analyzed.

Nwokeoji says the next step is to scale mRNA vaccine production in a variety of settings. “We aim to make all production aspects low-cost. We also are looking at sustainable methods for nucleotide triphosphates production, mRNA delivery and product characterization.”

To do this, “Collaborative research and knowledge exchange programs often are crucial in establishing new platforms in any setting, and especially for large-scale production,” he says. Such partnerships help adapt production and analytics to high-throughput environments and ensure compatibility with labs and production facility workflows.

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