Purifying circRNA by ultrafiltration with membranes having well-defined pores

Circular RNA (circRNA) is a promising therapeutic modality owing to its enhanced stability conferred by exonuclease resistance. However, large-scale production remains limited by inefficient purification methods, as the standard approach using size-exclusion chromatography suffers from peak overlap and low yield. Here, we present an ultrafiltration strategy for purifying circRNA using polycarbonate track-etched membranes with precisely controlled pore diameters. Membrane pore sizes were fine-tuned by gold plating to separate circular and linear RNA conformers. Sieving coefficients of purified RNA conformers were measured to establish operating conditions, and continuous diafiltration experiments were conducted within the range of flux values predicted to achieve both high purity and yield. This approach resulted in 94% purity and 59% yield of circRNA from in vitro transcription mixtures after six diavolumes. Transfection studies in HEK293T cells confirmed that diafiltered circRNA exhibited gene expression levels comparable to enzymatically purified circRNA, albeit with modestly reduced transfection efficiency. These results establish membrane-based ultrafiltration with tailored pore sizes as a scalable, non-chromatographic platform for purifying circRNA and provide a framework for further development toward therapeutic circRNA manufacturing.