Department of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Biologics Development: Microbial Dev't, Sanofi-Aventis Deutschland GmbH, Frankfurt, Germany.
Biotechnol Bioeng. 2019 Nov;116(11):2906-2918. doi: 10.1002/bit.27116. Epub 2019 Jul 30.
Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.
由于传质限制，在大型工业规模生物反应器中会出现浓度梯度，这对工艺效率有重大影响。因此，研究菌株对这种非均质性的反应，以降低工艺放大过程中失败的风险是很有必要的。尽管有各种缩小规模的技术可以研究这些影响，但在生物工艺的早期开发阶段，很少应用缩小规模的策略，因为它们尚未在小规模平行培养设备上实施。在这项研究中，我们将机理生长模型与平行微型生物反应器系统相结合，创建了一个高通量平台，用于研究大肠杆菌菌株对浓度梯度的反应。作为一种缩小规模的方法，应用了基于模型的葡萄糖脉冲进料方案，并与连续进料曲线进行了比较，以研究葡萄糖和溶解氧梯度对细胞生理学和非典型氨基酸掺入重组胰岛素前体的影响。结果表明，在具有葡萄糖/氧气波动的培养物中，可溶性细胞内提取物和重组产物中，非典型氨基酸正缬氨酸的掺入量显著增加。有趣的是，正缬氨酸的量取决于脉冲频率，连续进料时可忽略不计，这证实了大规模培养的观察结果。最重要的是，与参考培养相比，结果还表明，与参考培养相比，更多的模型参数受到缩小规模方案的显著影响。在这个例子中，有可能在单个平行实验中描述波动的影响。该平台提供了将菌株筛选与缩小规模研究相结合的机会，以选择最适合生物工艺放大的稳健菌株。
