Levers To Optimize The IVT Reaction For Increased mRNA Yield

By Alexander Latta, Neera Sammeta, Stefan Kutter, Monika Seller, Bhawana Poudel-Bochmann, Peter Rasor, and Birgit Rogell

Optimizing in vitro transcription (IVT) is essential for scaling mRNA production from research to therapeutic manufacturing. While off-the-shelf kits offer convenience, maximizing yield requires fine-tuning chemical levers—most notably the concentration and ratio of magnesium ions and ribonucleoside triphosphates (rNTPs). Because hydrogen ions are released during nucleotide incorporation, maintaining a stable pH through robust buffer systems like HEPES or Tris is critical for sustained enzymatic activity.

The reaction environment also benefits significantly from the addition of pyrophosphatase. This enzyme degrades pyrophosphate, a reaction byproduct that otherwise inhibits synthesis by precipitating magnesium ions. Furthermore, data indicates that replacing UTP with modified nucleotides like N1-methyl-pseudo-UTP does not compromise yield, allowing for the production of highly effective mRNA. By balancing DNA template amounts and polymerase concentrations, developers can achieve high-quality output while managing production costs. Explore the full report to master these critical reaction parameters.