Based on an integrated approach of genetic engineering, fermentation process development, and downstream processing, a fermentative chymotrypsinogen B production process using recombinant Pichia pastoris is presented. Making use of the P. pastoris AOX1-promotor, the demand for methanol as the single carbon source as well as an inducer of protein secretion enforced the use of an optimized feeding strategy by help of on-line analysis and an advanced controller algorithm. By using an experimental system of six parallel sparged column bioreactors, proteolytic product degradation could be minimized while also optimizing starting conditions for the following downstream processing. This optimization of process conditions resulted in the production of authentic chymotrypsinogen at a final concentration level of 480 mg‚L -1 in the whole broth and a biomass concentration of 150 g‚L -1 cell dry weight, thus comprising a space-time yield of 5.2 mg‚L -1 ‚h -1 . Alternatively to the high cell density fermentation approach, a continuous fermentation process was developed to study the effects of reduced cell density toward oxygen demand, cooling energy, and biomass separation. This development led to a process with a highly increased spacetime yield of 25 mg‚L -1 ‚h -1 while reducing the cell dry weight concentration from 150 g‚L -1 in fed-batch to 65 g‚L -1 in continuous cultivation.
Expanded bed adsorption is an integrated technology that allows the introduction of particle-containing feedstock without the risk of blocking the bed. The biomass particles contained in the feedstock have to be treated as an integral part of the process and potential interactions between suspended biomass and the adsorbent must be excluded during process design. Because the electrostatic forces dominate the interactions between the biomass and adsorbent, the zeta potential has been studied as a tool to characterize biomass/adsorbent electrostatic interactions. The zeta potentials of four types of biomass (yeast intact cells, yeast homogenate, Escherichia coli intact cells, and E. coli homogenate) and two types of ion exchanger were measured systematically at varying process conditions. Using the cell transmission index from biomass pulse-response experiments as a parameter, the relations between zeta potential and the biomass/adsorbent interaction were evaluated. Combining the influences from zeta potential of adsorbent (zeta(a)), zeta potential of biomass (zeta(b)), and biomass size (d), parameter (-zeta(a)zeta(b)d) was found to be an appropriate indicator of the biomass/adsorbent interactions in expanded beds under various liquid-phase conditions for different types of biomass. The threshold value of parameter (-zeta(a)zeta(b)d) can be defined as 120 mV(2) microm for cell transmission of >90%, which means that systems with (-zeta(a)zeta(b)d) < 120 may have a considerable probability of forming stable expanded beds in a biomass suspension under the particular experimental conditions.
Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity.
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