Scalable Cell‐Free Production of Active T7 RNA Polymerase

ABSTRACT The SARS‐CoV‐2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell‐free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a 1‐L cell‐free gene expression (CFE) reaction that achieves over 90% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell‐free reaction conditions, such as temperature, additives, purification tags, and agitation, to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than four orders of magnitude in reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.