Upstream development of Escherichia coli fermentation process with PhoA promoter using design of experiments (DoE)

Frank K Agbogbo,Renija George,Mian Huang,Shikha Srivastava,Jobin Joy,Jesse Mccool,Phil Ramsey

doi:10.1007/s10295-020-02302-7

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.

Highlights

For sponsors of Investigational New Drug Applications (IND) concerning cell-derived biological products, there is the need to develop process knowledge
The observations from this study follow the trend reported in the literature where an increase in the phosphate concentration tested in phoA process (10.8 mM − 18.0 mM) [27] and (0.43 mM − 17.0 mM) [8] improved biomass and titers
The results from this work is consistent with previous studies except that higher range of phosphate concentration (2.79 mM − 86.4 mM) were tested resulting in higher biomass and product titers

Summary

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. 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

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