How Acidic Denaturation Facilitates dsRNA Removal
In vitro transcription (IVT) reaction is a widely established mRNA production reaction, essential for creating biopharmaceuticals and vaccines that protect and improve human health. However, the process can accidentallygenerate immune-triggering double-stranded (ds) RNA constructs.
Why is dsRNA removal important?
These ds-impurities need to be meticulously eliminated from the final mRNA sample to ensure safety of biopharmaceuticals and vaccines for patients.
How to remove dsRNA?
One possibility to remove the stubborn dsRNA by using reversed-phase chromatography. It uses organic solvents and can effectively fractionate DNA, dsRNA, and ssRNA based on their lengths.
As some facilities are not equipped to work with organic solvents, we have developed a brand-new technique to remove dsRNA from mRNA samples, which utilizes basic chemical principles of hydrogen (H) bonding and the monolithic column, often employed for affinity-based capture of mRNA directly following IVT reaction.
How does the new Acidic Oligo dT method work?
dsRNA is made up of deoxyribonucleotides, which are bound by H-bonds. The basic principle of this approach is a brief acidic denaturation of the Oligo dT-purified mRNA sample (e.g., in pH 3), followed by another Oligo dT chromatographic purification. The denatured double-strands are removed during the second Oligo dT-purification in the flow through.
The acidic Oligo dT approach is a reliable and simple method to efficiently purify mRNA, eliminating dsRNA impurities, without the need for organic solvents or special and complicated multi-step methods. The user also benefits from using a monolithic column ensuring high binding capacity, exceptional mRNA recovery (>95 %), and scalability from milligram to multi-gram scale.
Do you want to learn more about this topic? Read our brand new paper , take a look at this insightful column published by GenEngNews and listen to our webinar. Explore all our CIM® chromatographic solutions for the entire production process, from DNA template to purified mRNA.
