Reverse-phase chromatography removes double-stranded RNA, fragments, and residual template to decrease immunogenicity and increase cell potency of mRNA and saRNA

mRNA is produced by <i>in vitro</i> transcription reaction, which also leads to formation of immuno-stimulatory impurities, such as double-stranded RNA (dsRNA). dsRNA leads to activation of innate immune response linked to inhibition of protein synthesis. Its removal from mRNA preparations increases efficiency of protein translation. Previous studies identified ion-pair reverse-phase high-performance liquid chromatography as a highly efficient approach for dsRNA removal. Here, we present a comprehensive study of IP-RP LC purification on monolith chromatographic supports for mRNA polishing, demonstrating its ability to remove dsRNA, as well as hybridized RNA fragments and residual DNA template, which are not fully removed by mRNA capture methods. We develop step elution methodology, including at microgram scale with novel spin columns operated by centrifugation. We demonstrate SDVB efficiency across a range of molecular sizes and explore the necessity for temperature control for effective dsRNA removal from self-amplifying RNA. SDVB-purified mRNA and saRNA showed significantly increased transgene expression in cell-based assays and reduced the activation of cell autonomous innate immunity in A549 at early time points. Our findings highlight the importance of IP-RP purification for high-quality mRNA production, while simplifying the technological requirements for its adoption in clinical mRNA and saRNA manufacturing processes.