Purifying circular RNA by ultrafiltration

Developing messenger RNA (mRNA) vaccines for COVID-19 renewed and intensified the interest in using mRNA for disease prevention and treatment. Despite their efficacy, linear mRNA molecules are short-lived in the human body, primarily due to enzymatic degradation at the free ends. In contrast, circular RNA (circRNA) exhibits enhanced stability and resistance to exonuclease degradation. However, this stability depends highly on purity. Unfortunately, the <i>in vitro</i> transcription (IVT)/self-splicing reaction to produce circRNA contains a mixture of circular and linear RNAs, and effective methods for purifying circRNA from solutions containing linear RNA contaminants do not exist. This study explored the feasibility of using ultrafiltration to purify protein-encoding circRNA produced by the self-splicing of a precursor RNA (preRNA) during IVT. We measured the sieving coefficients-a separation metric-of circRNA, linear precursor RNA, and nicked RNA conformers using polyethersulfone membranes with molecular weight cutoffs from 30 to 300 kDa, analyzing performance as a function of permeate flux. We also estimated the RNA critical fluxes and determined suitable operating conditions for purification. We achieved a purity of 86% with a yield above 50%. By comparison, the purity achieved by size-exclusion high-performance liquid chromatography (SE-HPLC), the leading alternative separation technology, was 41%, with a yield of 45%. These findings highlight ultrafiltration as a superior method for purifying circRNA at the research scale. Since ultrafiltration is already established as a scalable process in bioprocessing, this research may serve as a foundational step toward enabling large-scale manufacturing of circRNA-based therapeutics.