Purification of messenger RNA directly from crude IVT using polyethylene glycol and NaCl precipitation

Abstract

The increasing demand for mRNA-based therapeutics requires scalable and cost-effective purification methods. Chromatography-based approaches, such as Oligo-dT affinity chromatography, require multiple processing steps, including buffer exchange and high-temperature treatments, which can lead to mRNA degradation and increased costs. Although precipitation is commonly used at the lab scale, its application in large-scale mRNA purification remains underexplored. In this study, we developed a rapid and efficient precipitation-based method for mRNA purification. The effects of different precipitation conditions, including salt type, co-precipitating agents, and time, were evaluated in terms of recovery yield and purity. Precipitation experiments were conducted for two different in vitro transcription crude materials containing mRNAs of varying lengths and concentrations. The optimal conditions, identified as a combination of NaCl and PEG 6000, achieved recovery yields of 80–93 % and purities of 80–83 %, with no detectable double-stranded RNA formation or fragmentation. The precipitated mRNAs were successfully transfected into 293 T and A549 cells, showing protein expression comparable to commercial and chromatography-purified mRNAs. These findings position PEG/NaCl precipitation as a rapid, scalable, and cost-effective alternative to traditional chromatography methods, significantly reducing process complexity and time for large-scale mRNA production.