Scalable Cell‐Free Production of Active T7 RNA Polymerase

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.

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Publication Details

Journal
Biotechnology and Bioengineering
Published
2025-04-29
DOI
https://doi.org/10.1002/bit.28993
Citations
4
Primary Topic
Viral Infectious Diseases and Gene Expression in Insects
Type
article
Field-Weighted Citation Impact
3.22

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article

Scalable Cell‐Free Production of Active T7 RNA Polymerase

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4 citations
Biotechnology and Bioengineering
Viral Infectious Diseases and Gene Expression in Insects
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Scalable Cell‐Free Production of Active T7 RNA Polymerase

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article en
4 citations

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.

Biotechnology and BioengineeringVol. 122(8)
Northwestern University (US), University of Maryland, Baltimore (US), Cornell University (US), GE Global Research (United States) (US), Stanford University (US)
Defense Advanced Research Projects Agency
Good health and well-being
Openalex Percentile: Top 9%
Viral Infectious Diseases and Gene Expression in Insects
3.22
DOISourceOpenAlex
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