J. Puc, N. Mencin, A. Krušič, E. Nett, M. Perković, U. Sahin, A. Štrancar, and R. Sekirnik,
Separation and Purification Technology, 2026
Abstract:
Removal of double-stranded RNA (dsRNA) is a critical part of production processes for mRNA vaccines and therapeutics, due to the high immunogenicity of this impurity. dsRNA contaminants are produced during in vitro transcription reaction, and typically removed using ion-pair reverse-phase or cellulose based chromatography. However, removal is challenging because of dsRNA’s intrinsically similar physicochemical properties with single stranded RNA (ssRNA).
We report a novel approach to remove dsRNA which targets hydrogen bonding as the physico-chemical stabiliser of dsRNA: breaking these hydrogen bonds increases chromatographic differentiation, and can be achieved with pH modulation in a cheap, non-hazardous and scalable technique. We demonstrate that incubation of mRNA at pH ≤3.5 denatures dsRNA within seconds for mRNA/saRNA constructs spanning 1–10 kb, without compromising mRNA integrity. Coupling in-line pH 3 treatment with Oligo dT capture reduced dsRNA from various mRNAs from 0.14 to 0.77% to 0.02–0.09% while achieving ≥90% recovery irrespective of column loading, and improving integrity by concomitantly removing short RNA fragments.
This purification approach increased mRNA cellular potency 5-fold in A549 cells and markedly reduced type I interferon signalling, while maintaining cellular viability. This simple, aqueous, and scalable method establishes a new purification paradigm for producing high-quality mRNA drug substance with high recovery, compatible with clinically validated Oligo dT technology.
