Upstream development of Escherichia coli fermentation process with PhoA promoter using design of experiments (DoE)
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FERMENTATION, CELL CULTURE AND BIOENGINEERING - ORIGINAL PAPER
Upstream development of Escherichia coli fermentation process with PhoA promoter using design of experiments (DoE) Frank K. Agbogbo1 · Phil Ramsey2,3 · Renija George1 · Jobin Joy1 · Shikha Srivastava1 · Mian Huang4 · Jesse McCool1 Received: 8 April 2020 / Accepted: 14 August 2020 © The Author(s) 2020
Abstract In this work, a fed-batch fermentation development was performed with recombinant E. coli carrying the PhoA promoter system. The phosphate concentrations tested for this PhoA strain, 2.79 mM to 86.4 mM, were beyond the concentrations previously evaluated for cell growth and product titer. The results from the scouting work was used for design of experiments (DoE) where a range of phosphate levels from 27.1 mM to 86.4 mM was simultaneously evaluated with temperature, pH and DO set points. Definitive screening was used to evaluate these parameters simultaneously and the results indicate that fermentation temperature and phosphate content are the major contributors of product titer. The other factors tested such as pH had a minimal effect and DO had no impact on product titer. Keywords E. coli · Fermentation · DoE · PhoA · Model
Introduction For sponsors of Investigational New Drug Applications (IND) concerning cell-derived biological products, there is the need to develop process knowledge. The scientific understanding of the design space and process will make the scale-up and technology transfer a smooth and effective process. More recent guidelines from the Food and Drug administration emphasizes product and process understanding as well as process control, based on sound science and quality risk management [4, 9, 10]. Development of a robust, wellcharacterized platform drug substance (DS) upstream process (i.e., high-cell-density fermentation) can avoid costly Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02302-7) contains supplementary material, which is available to authorized users. * Frank K. Agbogbo [email protected] 1
Cytovance Biologics, 800 Research Parkway, Suite 200, Oklahoma City, OK 73104, USA
2
Predictum Inc., Austin, TX, USA
3
University of New Hampshire, Durham, NH, USA
4
BioMarin Pharmaceutical Inc., 770 Lindaro Street, San Rafael, CA 94901, USA
process development activities in the future. Given that the production in fermenters is a key upstream unit operation in pharmaceutical development, a good understanding of fermentation parameters and their impacts on cell growth [33] and final product yield is critical in defining the process. E. coli is a popular production host for commercializing biological products. Historically, promoters that are inducible by the allolactose analogue, Isopropyl β-D-1thiogalactopyranoside (IPTG) have been heavily used in the biologics industry. There are some industry-recognized disadvantages associated with IPTG-based induction processes that are related to stress on the cells [14, 15] and cost of goods [3, 7]
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