Scalable Cell-Free Production of Active T7 RNA Polymerase

Area of Science:

• Biotechnology
• Molecular Biology
• Bioprocess Engineering

Background:

• The SARS-CoV-2 pandemic underscored the need for rapid and resilient biomanufacturing processes.
• T7 RNA polymerase is a key enzyme for synthesizing messenger RNA (mRNA), essential for vaccine development.

Purpose of the Study:

• To demonstrate rapid process development and scalable cell-free production of T7 RNA polymerase.
• To establish cell-free gene expression (CFE) as a viable method for producing critical biotherapeutic components.

Main Methods:

• Utilized rolling circle amplification for efficient DNA template generation.
• Optimized cell-free reaction conditions, including temperature, additives, purification tags, and agitation.
• Performed 1-L cell-free gene expression reactions to produce T7 RNA polymerase.

Main Results:

• Achieved over 90% purity and low endotoxin levels for cell-free produced T7 RNA polymerase.
• Demonstrated enhanced activity compared to commercially available T7 RNA polymerase.
• Successfully scaled production across four orders of magnitude in reaction volume with consistent quality and titer.

Conclusions:

• Cell-free gene expression (CFE) is a powerful and scalable technology for producing high-quality T7 RNA polymerase.
• This approach offers a flexible and rapid solution for decentralized biomanufacturing.
• The findings enhance preparedness for future public health emergencies and emergent threats requiring rapid vaccine component production.