Physiological capacities decline during induced bioprocesses leading to substrate accumulation

Reichelt, Wieland N.; Brillmann, Markus; Thurrold, Peter; Keil, Peter; Fricke, Jens; Herwig, Christoph

doi:10.1002/biot.201600547

Cited by 10 publications

(9 citation statements)

References 30 publications

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“…The interruption of feeding and overfeeding was assumed to be classic technical failures that occur in the late or final stages of the induction phase. Especially, overfeeding is usually a problem with increasing induction times, because cellular performance is usually decreasing over time [32]. When no automated closed-loop feed addition is performed, substrate overfeeding is usually present, as the specific substrate uptake rate of E. coli decreases [32].…”

Section: Methodsmentioning

confidence: 99%

“…Especially, overfeeding is usually a problem with increasing induction times, because cellular performance is usually decreasing over time [32]. When no automated closed-loop feed addition is performed, substrate overfeeding is usually present, as the specific substrate uptake rate of E. coli decreases [32]. Therefore, the technical failures in C9 and C10 were introduced at the usual end of the induction time.…”

Section: Methodsmentioning

confidence: 99%

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

Pekarsky

Konopek

Spadiut

2019

Bioprocess Biosyst Eng

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In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield.Electronic supplementary materialThe online version of this article (10.1007/s00449-019-02158-x) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Pekarsky

Konopek

Spadiut

2019

Bioprocess Biosyst Eng

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“…Knowledge about the state of IB QAs during a cultivation process is therefore of utmost importance. Three IB QAs are generally of importance: bead size, titer and purity, as those three quality attributes were already defined elsewhere [ 21 , 30 , 33 , 34 ]. It has been reported that inclusion body sizes can be measured with different methods, e.g.…”

Section: Introductionmentioning

confidence: 99%

“…The impact of single process parameters like pH on IB QAs has already been investigated in literature [ 36 , 37 ]. Reichelt et al [ 34 ] showed that alterations of (q s,glu ) influence the behavior of common IB-processes, using IPTG as an inducer. The impact of the feeding rate onto product formation in E. coli BL21(DE3) has been investigated recently, though lactose was used as inducer instead of IPTG [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

Slouka

Kopp

Hutwimmer³

et al. 2018

Microb Cell Fact

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BackgroundThe bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes.ResultsClassical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein.ConclusionsTime dependent monitoring of IB QA—titer, purity, IB bead size—showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate—time dependently—quality attributes in upstream processing to enable robust downstream processing.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0997-5) contains supplementary material, which is available to authorized users.

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“…It was shown within this study, that the physiological feeding rate in E. coli is highly dependent onto process parameters and consists of a rather “dynamic nature”. Physiological capacities decline during induced bioprocesses leading to substrate accumulation [35].…”

Section: Introductionmentioning

confidence: 99%

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

Kopp¹,

Slouka²,

Strohmer³

et al. 2018

Microorganisms

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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.

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Physiological capacities decline during induced bioprocesses leading to substrate accumulation

Cited by 10 publications

References 30 publications

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

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

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

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

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