Rezvani Ryan N, Aw Rochelle, Chan Wei, Satish Krishnathreya, Chen Han, Lavy Adi, Rimal Swechha, Patel Divyesh A, Rao Govind, Swartz James R, DeLisa Matthew P, Kvam Erik, Karim Ashty S, Krüger Antje, Kightlinger Weston, Jewett Michael C
Cell-free Protein Synthesis and Microbial Process Development, National Resilience Inc., San Diego, California, USA.
Department of Bioengineering, Stanford University, Stanford, California, USA.
Biotechnol Bioeng. 2025 Aug;122(8):2241-2250. doi: 10.1002/bit.28993. Epub 2025 Apr 29.
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.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）大流行凸显了对强大且快速的生物制造模式的迫切需求。在此，我们展示了T7 RNA聚合酶的快速工艺开发和可扩展的无细胞生产，T7 RNA聚合酶是mRNA疫苗合成中的关键组件。我们进行了1升无细胞基因表达（CFE）反应，相对于商业T7 RNA聚合酶，该反应实现了超过90%的纯度、低内毒素水平和增强的活性。为实现这一成果，我们采用滚环扩增来规避DNA模板生成中的困难，并调整无细胞反应条件，如温度、添加剂、纯化标签和搅拌，以提高产量。我们在超过四个数量级的反应体积中实现了类似质量和效价的T7 RNA聚合酶的生产。这一原理证明将CFE定位为分散式生物治疗制造的可行解决方案，增强了应对未来公共卫生危机或突发威胁的准备能力。
