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

BACKGROUND: The 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. RESULTS: Classical 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. CONCLUSIONS: Time 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.

Carbon utilization pattern as a potential quality control criterion for virulence of Beauveria brongniartii.

The registered entomopathogenic fungus Beauveria brongniartii (BIPESCO 2) was tested for its virulence after one, five and 10 times sub-culturing on four types of selective synthetic nutrient media. Bioassays with third instar Melolontha melolontha larvae showed that sub-culturing negatively affects the virulence of the fungus after 10 transfers. With the Biolog SF-P2 and Biolog SF-N2 microtiter plate systems the sub-cultivated B. brongniartii conidia were monitored for any change in the carbon utilization pattern of 128 carbon sources. With the help of Spearman's rank correlation, principal components analysis and canonical correspondence analysis, respectively, six carbon sources were identified as potential virulence indicators for BIPESCO 2 (pyruvic acid, maltose, glycyl-L-glutamic acid, malonic acid, glucuronamide and phenylethylamine). The Biolog microtiter plate system is suggested as a simple and inexpensive test-system for virulence determination of B. brongniartii strain BIPESCO 2 in routine quality control.

Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins.

Nutritional conditions causing droplet exudation by Metarhizium anisopliae var. anisopliae were studied. Exudation in droplets occurred only on media with more than one carbon source and was highly dependent on the ratio of a well metabolized sugar such as trehalose and a nonpreferred sugar, in particular arabinose. Exuded droplets contained destruxin A, B and E in concentrations similar to those on submerged culture on Czapek Dox medium with equivalent C:N ratios but was clearly less than previously reported on standard Czapek Dox or Sabouraud dextrose broth. Destruxins also were found in agar samples from directly below mycelium and from up to 2 cm from the colony edge. Exudates retrieved from different media were proven to have Pr1 protease-related enzyme activity. Additional HPLC analysis indicated that droplets from diverse media did not differ in their sugar and acid content. A hypothesis is presented regarding the trigger for guttation in Metarhizium during growth under these conditions.

Destruxin production of Metarhizium anisopliae under carbon and nitrogen exhaustion.

Destruxins (dtx) A, B, and E, showing a variety of biological activities, are the main toxic secondary metabolites of the entomopathogenous ascomycete Metarhizium anisopliae Bipesco 5, a widely used biocontrol production strain. Dynamics of dtx biosynthesis were monitored during liquid fermentation in a chemically defined medium. During shake flask cultivation with excess carbon, nitrogen and phosphate, approximately 50, 20, and 100 mg l(-1) dtx A, B, and E were produced after 12 d. Destruxins were produced during exponential growth phase and in the stationary phase. Carbon exhaustion in the culture broth was demonstrated to affect destruxin production to a minor degree: Absolute dtx amounts in the liquid increased also after glucose exhaustion; dtx amounts referred to biomass increased further evidently in shake flasks or slightly in bioreactor experiments after carbon limitation occurred. Contrarily, nitrogen exhaustion resulted in an evident decline in dtx amounts referred to biomass. Absolute amounts in the culture broth, however, still increased slightly the following four days in bioreactor experiments. From this we conclude that dtx production is highly influenced by nitrogen availability. Generally, dtx production in bioreactors with controlled aeration (1 vvm) was significantly lower than in shake flasks.