Abstract
The bacterium Escherichia 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, which often leads to protein aggregates inside of the cytoplasm, forming so the called inclusion bodies (IBs). When compared to other protein expression strategies, inclusion body formation allows high product titers and also the possibility of expressing proteins being toxic for the host. In the past years, the comprehension of inclusion bodies being only inactive protein aggregates changed, and the new term of non-classical inclusion bodies emerged. These inclusion bodies are believed to contain a reasonable amount of active protein within their structure. However, subsequent downstream processing, such as homogenisation of cells, centrifugation or solubilisation of IBs, is prone to variable process performance and is often known to result in low extraction yields. It is hypothesised that variations in IB quality attributes are responsible for those effects and that such attributes can be controlled by upstream process conditions. In this review, we address the impact of process design (process parameters) in the upstream on defined inclusion body quality attributes. The following topics are therefore addressed: (i) an overview of the range of inclusion body applications (including emerging technologies); (ii) analytical methods to determine quality attributes; and (iii) screws in process engineering to achieve the desired quality attributes for different inclusion body–based applications. Process parameters in the upstream can be used to trigger different quality attributes including protein activity, but are not exploited to a satisfying content yet. Design by quality approaches in the upstream are already considered for a multitude of existing processes. Further intensifying this approach may pave the industrial application for new IB-based products and improves IB processing, as discussed within this review.
Highlights
The first steps for recombinant protein expression have been made back in 1973, where Stanley Cohen and Herbert Boyer invented the possibility of in vitro DNAcloning (Cohen et al 1973, Baeshen et al 2014)
Like temperature, pH and physiological feeding, in combination with different induction mechanism, are powerful tools to trigger the properties of inclusion bodies (IBs) in order to fit the desired quality
IBs are still widely exploited for the production of pharmaceutical processes for high product titer and expression of toxic proteins, where no posttranslational modifications are required, e.g. fragmented antibodies (Fabs)
Summary
The first steps for recombinant protein expression have been made back in 1973, where Stanley Cohen and Herbert Boyer invented the possibility of in vitro DNAcloning (Cohen et al 1973, Baeshen et al 2014). Results show that IBs are strongly dependent on the amount of the specific lactose uptake rate in the mixed feed and on the specific uptake rate of the primary carbon source These cultivation techniques help to overcome the problem of cell stress caused by harsh induction using IPTG and render the possibility for tuning between soluble and IB production. Performed fed-batch-based cultivations using an industrial protein fused to NPro exclusively expressing IBs (Slouka Christoph 2018) showed high impact on IB size based on classical process parameters tested in a design of experiments (DoE) approach. Different process parameters were recently identified to increase the expression of active structures within produced IBs. Castellanos-Mendoza et al (2014) showed that changes in the culture pH changed the IB characteristics of sphingomyelinase-D between the classical and active form. Several analytical methods presented in the literature were summarised in this review and represent the most important pillar for process understanding and control
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