Microscale to manufacturing scale‐up of cell‐free cytokine production—a new approach for shortening protein production development timelines
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Open Access
- 17 February 2011
- journal article
- research article
- Published by Wiley in Biotechnology & Bioengineering
- Vol. 108 (7), 1570-1578
- https://doi.org/10.1002/bit.23103
Abstract
Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell‐based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli‐based open cell‐free synthesis (OCFS) system that is optimized for predictable high‐yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high‐level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte‐macrophage colony‐stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide‐bonded biotherapeutic proteins. Biotechnol. Bioeng. 2011; 108:1570–1578.Keywords
This publication has 41 references indexed in Scilit:
- A Critical Examination of Escherichia coli Esterase ActivityPublished by Elsevier BV ,2009
- Design Parameters to Control Synthetic Gene Expression in Escherichia coliPLOS ONE, 2009
- Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell-Free Protein SynthesisBiotechnology Progress, 2008
- Large‐Scale Batch Reactions for Cell‐Free Protein SynthesisPublished by Wiley ,2007
- Cell‐free synthesis of proteins that require disulfide bonds using glucose as an energy sourceBiotechnology & Bioengineering, 2007
- Production and purification of recombinant human granulocyte–macrophage colony stimulating factor (GM-CSF) from high cell density cultures of Pichia pastorisBioprocess and Biosystems Engineering, 2007
- A cradle for new proteins: trigger factor at the ribosomeCurrent Opinion in Structural Biology, 2005
- Maintaining rapid growth in moderate‐density Escherichia coli fermentationsBiotechnology & Bioengineering, 2005
- Expression of human granulocyte‐macrophage colony‐stimulating factor gene in insect cells by a baculovirus vectorFEBS Letters, 1990
- IN VITRO SYNTHESIS OF PROTEIN IN MICROBIAL SYSTEMSAnnual Review of Genetics, 1973