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

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.

The impact of technical failures during cultivation of an inclusion body process

“…Nevertheless, the found IB content of around 30% in the biomass stood in good agreement with recent findings in our research group. We have shown that the maximum intracellular IB size varied between 500 and 700 nm [16], which resembles a ratio around 30% of IB per cell given the rough E. coli size estimation of 2 µm. Furthermore, the basic and cheap refolding with glycerol as single additive in deionized water resulted in a refolding yield of around 30% compared to ~ 49% [39] or 32.3% [35] for similar proteins in more complex buffers.…”

“…IBs are formed in the cytosol of E. coli and they usually consist of aggregated, insoluble target protein that is misfolded or partially unfolded, leading to no or reduced activity [12, 13]. Their formation is mostly dependent on the used promoter system and strength [14], the target protein class [12], and the process conditions [15, 16]. However, the misfolded/unfolded character of IBs makes formation kinetics, size distributions in the cytosol, and IB purity comparable between similar protein classes (e.g., [15, 17]) until the initial IB solubilisation procedure, in which protein specific conditions have to be considered.…”

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

“…During the induction period, samples were taken in a maximum of 120 min intervals and analyzed subsequently. Details on the process analytics can be found elsewhere ( Kopp et al, 2018 ; Slouka et al, 2018 ). For chemostat cultivations samples were collected after batch and afterward once or, if necessary, twice a day.…”

“…Soluble protein samples were filtered (0.2 μm mesh) and directly used in the HPLC. Inclusion Body (IB) pellet samples were prepared according to Kopp et al (2018) , and subsequently solubilized using following buffer: 7.5 M guanidine hydrochloride, 62 mM Tris at pH = 8 and 125 mM DTT was added right before use. The filtered IB samples were quantified by HPLC analysis (UltiMate 3000; Thermo Fisher, Waltham, MA, United States).…”

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

“…Therefore, CatIB size might be an important parameter for CatIB application. As shown by Kopp et al (2018) for conventional, inactive IBs, the size of IBs can be adjusted by nutrient feeding. This strategy might also be applicable for CatIB production to optimize the specific activity of CatIBs.…”