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Microorganisms 2018, 6(4), 116; https://doi.org/10.3390/microorganisms6040116

Inclusion Body Bead Size in E. coli Controlled by Physiological Feeding

1
Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria
2
Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria
*
Author to whom correspondence should be addressed.
Received: 1 October 2018 / Revised: 16 November 2018 / Accepted: 22 November 2018 / Published: 25 November 2018
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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Abstract

The Gram-negative bacterium E. coli is the host of choice for producing a multitude of recombinant proteins relevant in the pharmaceutical industry. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures combined with the usage of IPTG (isopropyl β-d-1 thiogalactopyranoside) as an inducer are often believed to cause stress reactions, leading to intracellular protein aggregates, which are so known as so-called inclusion bodies (IBs). Downstream applications in bacterial processes cause the bottleneck in overall process performance, as bacteria lack many post-translational modifications, resulting in time and cost-intensive approaches. Especially purification of inclusion bodies is notoriously known for its long processing times and low yields. In this contribution, we present screening strategies for determination of inclusion body bead size in an E. coli-based bioprocess producing exclusively inclusion bodies. Size can be seen as a critical quality attribute (CQA), as changes in inclusion body behavior have a major effect on subsequent downstream processing. A model-based approach was used, aiming to trigger a distinct inclusion body size: Physiological feeding control, using qs,C as a critical process parameter, has a high impact on inclusion body size and could be modelled using a hyperbolic saturation mechanism calculated in form of a cumulated substrate uptake rate. Within this model, the sugar uptake rate of the cells, in the form of the cumulated sugar uptake-value, was simulated and considered being a key performance indicator for determination of the desired size. We want to highlight that the usage of the mentioned screening strategy in combination with a model-based approach will allow tuning of the process towards a certain inclusion body size using a qs based control only. Optimized inclusion body size at the time-point of harvest should stabilize downstream processing and, therefore, increase the overall time-space yield. Furthermore, production of distinct inclusion body size may be interesting for application as a biocatalyst and nanoparticulate material. View Full-Text
Keywords: E. coli; recombinant proteins; bioprocess engineering; process control; inclusion body; size E. coli; recombinant proteins; bioprocess engineering; process control; inclusion body; size
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Kopp, J.; Slouka, C.; Strohmer, D.; Kager, J.; Spadiut, O.; Herwig, C. Inclusion Body Bead Size in E. coli Controlled by Physiological Feeding. Microorganisms 2018, 6, 116.

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