Boosting Recombinant Inclusion Body Production—From Classical Fed-Batch Approach to Continuous Cultivation

Abstract

State of the art microbial recombinant protein production is regularly performed in fed-batch based cultivations. However, these cultivations suffer from highly time-dependent changes in productivity and product quality, leading to high variations in the downstream process. Continuous biomanufacturing offers the possibility of a time independent process, boosting the time-space-yield of the recombinantly produced protein and further reducing costs for production, also as downstream gets more predictive. In the current work, the continuous production of a pharmaceutically relevant protein in form of an inclusion body in E. coli BL21(DE3) was investigated in single vessel cultivations by varying dilution rates using glycerol as carbon source, inducer (lactose or IPTG) and respective inducer concentrations. Attempts to increase low specific productivities observed in single vessel continuous cultivations, led to the establishment of a continuously operated cascade of two stirred tank reactors to spatially separate biomass formation from recombinant protein production. Process performance was substantially improved compared to a single vessel chemostat culture, as specific productivity and space-time yield were boosted using an optimized cascaded process by about a factor of 100. This study shows the potential of a two-stage continuous process as promising alternative to benchmark fed-batch processes achieving constant inclusion body production at a time-independent level.

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

“…Therefore, diauxic growth can be excluded as a possible explanation. Even though IPTG transport was found to be highly dependent on lactose permease ( Fernández-Castané et al, 2012 ), chemostat cultivations with Bl21(DE3) and IPTG induction also resulted in an irreversible drop of productivity ( Kopp et al, 2019a ). Only galactose accumulation was found to alter when comparing glucose and glycerol cultivations on Bl21(DE3), which will be discussed in more detail.…”

“…Several efforts have been made to enable continuous processes in E. coli ( Kopp et al, 2019a ), but we are still far away from application of such systems. Long-term cultivations with E. coli showed enhanced cell burden using IPTG induction and clearly favored feeding of the disaccharide lactose ( Dvorak et al, 2015 ).…”

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

“…Product per Substrate Yield at the beginning of the continuous cultivation might compete with repetitive fed-batch cultivations (Tables 4, 5, and Supplementary Table 1). However, a severe decrease in productivity over time was monitored for chemostat cultivations, as microbial chemostat cultivations are known to result in fluctuating productivity (Peebo and Neubauer, 2018;Kopp et al, 2019a).…”

“…In this study we performed repetitive fed-batch cultivations for recombinant protein production using the production host E. coli in combination with lactose induction. In previous studies, the negative side effects of IPTG induction onto recombinant protein production in long-term fermentations were shown, whereas lactose was found to have a beneficial effect on productivity (Malakar and Venkatesh, 2012;Dvorak et al, 2015;Kopp et al, 2019a). We believe that no studies on repetitive fed-batch cultivation with E. coli for recombinant protein production have been published yet, either due to toxic effects of IPTG and the consequent decreasing productivity over time (Dvorak et al, 2015;Kopp et al, 2019b) or due to the absence of an induction strategy comparable to the established yeast system (Fricke et al, 2013).…”

Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

“…In fact, bioprocess design during recombinant protein production has been shown to impact IB size and physicochemical quality of recombinant protein, achieving constant IB production [22][23][24]. In addition, aggregation propensity of recombinant proteins in expression systems may be enhanced by the addition of aggregation-prone peptides (APPs) in the design of the coding DNA sequence of the gene [25].…”