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.
Highlights
The gram-negative bacterium E. coli might be one of the best-characterized organisms and has found its place in many different industrial applications [1,2], because E. coli shows very fast replication rates [3,4] on comparatively cheap media [5]
We investigated size dependence based on physiological feeding in E. coli during cultivation
A correlation of the inclusion bodies (IBs) size based on the fed substrate could clearly be indicated
Summary
The gram-negative bacterium E. coli might be one of the best-characterized organisms and has found its place in many different industrial applications [1,2], because E. coli shows very fast replication rates [3,4] on comparatively cheap media [5]. The missing glycosylation pattern [1,6,7], as well as intracellular protein production, implies major drawbacks when compared to mammalian cells and yeasts as hosts [8]. Moffatt back in 1986 [9] is often used on an industrial scale because of very low acetate formation, high replication rates and high production as an effect of the integrated T7-polymerase [9,10,11,12,13,14]. Repressing the lac operon with isopropyl β-D-1 thiogalactopyranoside (IPTG) is still state of the art in most industrial applications [15,16], as the pET-expression-system is known for high replication rates, leading to high amounts of the desired protein [3,12,16]
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