Purification of Double-Stranded RNA Impurities From In Vitro-Transcribed mRNA Using Size-Exclusion Chromatography.

dsRNA is a highly immunogenic byproduct generated during in vitro transcription of mRNA, leading to unwanted immunostimulatory responses and reduced vaccine efficacy. Removal of dsRNA impurities from mRNA is challenging, as they share similar physicochemical properties. Here, we present a lab-scale size-exclusion chromatography method that effectively separates dsRNA impurities from mRNA. This method achieves baseline resolution between dsRNA impurities and mRNA (resolution factor of 1.6) while maintaining high recovery of the target mRNA (81.8%). This work introduces a novel chromatographic strategy for the removal of dsRNA from mRNA therapeutics.