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Glucose-limited high cell density cultivations from small to pilot plant scale using an enzyme-controlled glucose delivery system

机译：使用酶控制的葡萄糖输送系统，从小规模到中试规模的葡萄糖有限的高细胞密度培养

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摘要

The enzyme controlled substrate delivery cultivation technology EnBase1 Flo allows a fed-batch-like growth in batch cultures. It has been previously shown that this technology can be applied in small cultivation vessels such as micro- and deep well plates and also shake flasks. In these scales high cell densities and improved protein production for Escherichia coli cultures were demonstrated. This current study aims to evaluate the scalability of the controlled glucose release technique to pilot scale bioreactors. Throughout all scales, that is, deep well plates, 3 L bioreactor and 150 L bioreactor cultivations, the growth was very similar and the model protein, a recombinant alcohol dehydrogenase (ADH) was produced with a high yield in soluble form. Moreover, EnBase Flo also was successfully used as a controlled starter culture in high cell density fedbatch cultivations with external glucose feeding. Here the external feeding pump was started after overnight cultivation with EnBase Flo. Final optical densities in these cultivations reached 120 (corresponding to about 40 g L-1 dry cell weight) and a high expression level of ADH was obtained. The EnBase cultivation technology ensures a controlled initial cultivation under fed-batch mode without the need for a feeding pump. Because of the linear cell growth under glucose limitation it provides optimal and robust starting conditions for traditional external feed-based processes.
机译：酶控制的底物递送培养技术EnBase1 Flo允许分批培养中的补料状生长。先前已证明，该技术可用于小型培养皿，例如微孔板和深孔板，以及摇瓶中。在这些规模下，证明了大肠杆菌培养的高细胞密度和改善的蛋白质产量。这项当前的研究旨在评估受控葡萄糖释放技术对中试规模生物反应器的可扩展性。在所有规模上，即深孔板，3 L生物反应器和150 L生物反应器培养中，生长非常相似，并且以可溶形式高产量地生产了模型蛋白，重组醇脱氢酶（ADH）。此外，EnBase Flo还成功地用作带有外部葡萄糖补料的高细胞密度补料分批培养中的受控发酵剂培养。在此处使用EnBase Flo过夜培养后，启动了外部进料泵。在这些培养物中的最终光密度达到120（对应于约40g L-1干细胞重量），并且获得了高表达水平的ADH。 EnBase栽培技术可确保在分批补料模式下进行受控的初始栽培，而无需加料泵。由于葡萄糖限制下线性细胞的生长，它为传统的基于外部饲料的工艺提供了最佳而稳定的起始条件。

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