Optimizing mRNA Manufacturing: Advanced Purification for High Purity and Yield

This presentation focuses on a streamlined approach to mRNA manufacturing, including the preparation of high-quality DNA templates needed to achieve high RNA purity. We introduce a scalable purification workflow featuring continuous in-line lysis and selective hydrophobic interaction chromatography (SHIC) for purification of supercoiled (sc) DNA. The process includes efficient sc pDNA linearization monitored by the PATfix analytical system. In vitro transcription (IVT) reaction, used to produce mRNA from DNA template, is monitored with rapid PATfix at-line monitoring which also tracks nucleoside triphosphate (NTP) consumption. Using a data-driven design-of-experiment (DOE) approach, we achieve a reaction yield of 25 g/L, and can transform the reaction from batch to fed-batch mode. mRNA is purified with Oligo dT affinity chromatography which is selective for polyadenylated mRNA. Residual dsRNA is removed either with reverse-phase chromatography or with aqueous denaturing chromatography also based on Oligo dT. The process is scalable from ug to multi-gram scale and suitable for clinical manufacturing.