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June 01, 2009 (Vol. 29, No. 11)

Controlling Nutrient Release in Cell Cultivation

EnBase Designed to Optimize Feeding in the Production System

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    Figure 1. Fed-batch cultivation in a bioreactor, an EnBase cultivation vessel, and cell growth of E. coli

    With the arrival of the post-genomic era, functional genomics and proteomics have been fields of intense research. Extensive investigations on thousands of novel proteins to understand their function and interactions in biological systems have increased the need for the development of high-throughput methods. The need for reliable and efficient technologies suitable for parallelization or automation is urgent.

    BioSilta’s EnBase™ has applicability for high-throughput protein production as well as scale-up of industrial bioprocesses.

    Small-scale cultivation in shake flasks and microwell plates is usually performed as a batch culture, which may lead to oxygen limitation, medium acidification, and overflow metabolism. As a result, only low cell densities with OD600 from 1 to 10 are obtained.

    High cell density cultivation of microorganisms in a bioreactor is an efficient method for achieving a high specific productivity where final cell densities up to an OD600 of 400 can be obtained in the cultivation of E. coli.

    One problem that has to be addressed in  high cell density cultivation is the increasing demand for oxygen, this is problematic as oxygen’s solubility decreases with growing cell densities. To avoid the problems that can occur with the use of pure oxygen, cultivation can be carried out with lower specific growth rates.

    The most exploited technique used to achieve high cell densities in E. coli cultivations is fed-batch fermentation. Once the growth-limiting substrate has been consumed, a feeding solution containing the concentrated substrate is continuously added. During the fed-batch process, it is critical to control the specific growth rate as the formation of inhibitory by-products, cell productivity, and plasmid stability are all related.

    Adept control of reaction rates avoids problems associated with cooling and oxygen transfer. Furthermore, careful oversight of metabolic processes avoids osmotic effects, catabolite repression, and overflow metabolism. Scale-down is a challenging task when one views reactor configuration in relation to sensor technique, high viscosity of the feeding solution, continuous substrate delivery, mixing on small scale, and precise feed control in parallel cultures.

    EnBase technology enables the feeding of glucose in closed systems for small-scale cultivation under fed-batch conditions. The slow release of glucose into the cultivation medium supplies the bacterial culture with limiting substrate. A biocatalytic reaction controls the supply of glucose, which is derived from a polymeric substrate embedded in a gel and diffused into the medium or dissolved directly in the medium.

    The enzyme functions like a pump in the bioreactor (Figure 1A) to control glucose supply and turns a shake flask, microwell plate, or deep-well plate into an efficient bioreactor system (Figure 1B). The cell growth rate can be controlled by using different enzyme concentrations (Figure 1C).

    Recombinant protein expression with EnBase is performed in a two-phase cultivation (Figure 2). In the first step, cells grow in a fed-batch mode controlled by the addition of an amylase. Balanced growth and favorable pH after overnight cultivation make the bacteria ready for efficient recombinant protein production.

    In the second phase, medium optimized for protein expression is supplied after the addition of boosters. It is possible to induce recombinant protein production at high cell densities without sacrificing the protein productivity per cell because medium conditions and the physiological state of the cells can be kept optimal.

    EnBase works well with induction cell densities of OD600 of 5 to 15 and expression times of up to 24 hours. The balanced addition of carbon and nitrogen during induction increases the final volumetric product yield.

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    Figure 2. Two-phase cultivation with EnBase

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