Natural Wood-Derived Macroporous Cellulose for Highly Efficient and Ultrafast Elimination of Double-Stranded RNA from In Vitro-Transcribed mRNA.

Abstract

Double-stranded RNA (dsRNA) is a major impurity that can induce innate immune responses and cause adverse drug reactions. Removing dsRNA is an essential and non-trivial process in manufacturing mRNA. Current methods for dsRNA elimination use either high-performance liquid chromatography or microcrystalline cellulose, rendering the process complex, expensive, toxic, and/or time-consuming. This study introduces a highly efficient and ultrafast method for dsRNA elimination using natural wood-derived macroporous cellulose (WMC). With a naturally formed large total pore area and low tortuosity, WMC removes up to 98% dsRNA within 5 min. This significantly shortens the time for mRNA purification and improves purification efficiency. WMC can also be filled into chromatographic columns of different sizes and integrates with fast-protein liquid chromatography for large-scale mRNA purification to meet the requirements of mRNA manufacture. This study further shows that WMC purification improves the enhanced green fluorescent protein mRNA expression efficiency by over 28% and significantly reduces cytokine secretion and innate immune responses in the cells. Successfully applying WMC provides an ultrafast and efficient platform for mRNA purification, enabling large-scale production with significant cost reduction.