Getting IVT Right: The Power of Engineered Enzymes

August 14, 2024 by Kyle Studey

Enabling safer therapeutics

Part 1 of a two-part discussion on using IVT with mRNA therapeutics

Manufacturing mRNA, while technically simple, requires meticulous attention to detail to ensure a pure, safe and highly potent therapeutic. One particular challenge that arises during mRNA synthesis is the formation of unwanted impurities, such as double-stranded RNA (dsRNA). These dsRNA impurities can trigger immune responses within cells, leading to potential adverse effects and compromised therapeutic outcomes.

dsRNA is recognized by cells through activation of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), which initiate a cascade of immune responses. The activation of these receptors leads to the production of pro-inflammatory cytokines, interferons, and other immune mediators, resulting in inflammation and potentially harmful effects.

To address the challenge of dsRNA formation and its immunogenicity during mRNA synthesis, rigorous purification steps are typically implemented during the manufacturing process, including:

• Column chromatography
• Filtration techniques
• Enzymatic digestion

However, these purification methods can be time-consuming, costly, may not always eliminate dsRNA impurities, and could reduce overall RNA manufacturing yield.

Engineered enzymes provide an alternative
In recent years, mutant enzymes, such as mutant T7 RNA polymerases, have emerged as a solution to mitigate dsRNA formation in the first place. These engineered enzymes possess specific modifications that enhance their fidelity during transcription. By improving the fidelity of transcription, these enzymes minimize the occurrence of incomplete terminations and template switching, thereby reducing the formation of dsRNA impurities.

The use of engineered enzymes, particularly mutant T7 RNA polymerases such as Codex^® HiCap RNA Polymerase, not only reduces the need for extensive purification steps, reducing overall manufacturing costs, but also enhances the overall potency of the mRNA therapeutic, as the reduced immunogenic dsRNA results in less cytotoxicity and enhanced cellular translation.

The Takeaway
Minimizing dsRNA impurities in mRNA therapeutics is essential to ensure their safety and maximize their efficacy. The formation of dsRNA during mRNA synthesis poses challenges due to its immunogenicity; however, the use of mutant enzymes, such as mutant T7 RNA polymerases, offers a promising solution by reducing dsRNA formation. By incorporating these mutant enzymes into the manufacturing process, the need for extensive purification steps can be minimized, streamlining production and reducing costs.

By combining a robust mutant T7 RNA Polymerase with a well characterized, controlled manufacturing process, mRNA therapeutic developers and sponsors can spend less time and resources on ensuring a clean manufacturing product, and more on ensuring a highly potent, safe therapeutic with excellent patient outcomes.

In an upcoming post on Getting IVT Right, we’ll focus on mRNA capping and the impact of uncapped species, along with the strategies used to reduce them.

• Learn more about Aldevron’s RNA platform and services
• Have questions on this topic? ContactKyleStudey
• Learn more about Codex^® HiCap RNA Polymerase
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