J. Puc, N. Mencin, A. Krušič, E. Nett, M. Perković, U. Sahin, A. Štrancar, and R. Sekirnik,
Separation and Purification Technology, 2026
Abstract:
Double-stranded (ds) RNA is an immunogenic byproduct of the in-vitro transcription (IVT) reaction. It poses a real risk in mRNA-based therapeutics as it can elicit an immune response in the human body and must therefore be meticulously removed to ensure patient safety.
This publication explores how to exploit the very basic physico-chemical stabilizer of dsRNA formation – the hydrogen (H) bonding. The authors explained how a brief incubation of an Oligo-dT purified mRNA sample in a buffer with low pH results in denaturation of H-bonds in the dsRNA. These denatured fragments do not contain a poly(A) tail and cannot bind on the Oligo dT monolith during the second Oligo dT purification. In this way, the final mRNA sample is purified, its integrity preserved, and the mRNA-based drug does not put the patient in danger.
This simple, aqueous, and scalable method establishes a new purification approach for producing high-quality mRNA drug substance with high recovery, compatible with clinically validated affinity Oligo dT technology.
