Research Area Biochemical Engineering, Institute of Chemical, Environmental and Bioscience Engineering, Vienna University of Technology, Gumpendorferstrasse 1a/166, 1060, Vienna, Austria.
Competence Center CHASE GmbH, Altenbergerstraße 69, 4040, Linz, Austria.
Appl Microbiol Biotechnol. 2021 Mar;105(6):2243-2260. doi: 10.1007/s00253-021-11151-y. Epub 2021 Feb 17.
Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In recent years, a change in the mindset regarding IBs could be observed: IBs are no longer considered an unwanted waste product, but a valid alternative to produce a product with high yield, purity, and stability in short process times. However, solubilization of IBs and subsequent refolding is necessary to obtain a correctly folded and active product. This protein refolding process is a crucial downstream unit operation-commonly done as a dilution in batch or fed-batch mode. Drawbacks of the state-of-the-art include the following: the large volume of buffers and capacities of refolding tanks, issues with uniform mixing, challenging analytics at low protein concentrations, reaction kinetics in non-usable aggregates, and generally low re-folding yields. There is no generic platform procedure available and a lack of robust control strategies. The introduction of Quality by Design (QbD) is the method-of-choice to provide a controlled and reproducible refolding environment. However, reliable online monitoring techniques to describe the refolding kinetics in real-time are scarce. In our view, only monitoring and control of re-folding kinetics can ensure a productive, scalable, and versatile platform technology for re-folding processes. For this review, we screened the current literature for a combination of online process analytical technology (PAT) and modeling techniques to ensure a controlled refolding process. Based on our research, we propose an integrated approach based on the idea that all aspects that cannot be monitored directly are estimated via digital twins and used in real-time for process control. KEY POINTS: • Monitoring and a thorough understanding of refolding kinetics are essential for model-based control of refolding processes. • The introduction of Quality by Design combining Process Analytical Technology and modeling ensures a robust platform for inclusion body refolding.
在大肠杆菌中过量表达重组蛋白会导致细胞质中出现错误折叠和无活性的蛋白聚集体，即所谓的包涵体（IB）。近年来，人们对包涵体的看法发生了变化：包涵体不再被视为一种不需要的废物产物，而是一种有效的替代方法，可以在短时间内以高产率、高纯度和高稳定性生产产品。然而，要获得正确折叠和有活性的产物，需要对包涵体进行溶解和复性。该蛋白复性过程是一个关键的下游单元操作，通常以分批或补料分批模式进行稀释。现有技术的缺点包括以下几个方面：缓冲液体积大，复性罐的容量大；混合均匀性问题；在低蛋白浓度下进行分析具有挑战性；非有用聚集体中的反应动力学问题；以及一般较低的复性产率。目前还没有通用的平台程序，也缺乏稳健的控制策略。引入质量源于设计（QbD）是提供可控和可重复的复性环境的首选方法。然而，实时描述复性动力学的可靠在线监测技术却很少见。在我们看来，只有监测和控制复性动力学才能确保复性过程具有生产性、可扩展性和多功能性的平台技术。在本综述中，我们筛选了当前文献中的在线过程分析技术（PAT）和建模技术的组合，以确保复性过程的可控性。基于我们的研究，我们提出了一种基于以下理念的集成方法，即所有不能直接监测的方面都通过数字孪生体进行估计，并实时用于过程控制。关键点：
监测和深入了解复性动力学对于基于模型的复性过程控制至关重要。
将质量源于设计与过程分析技术和建模相结合，可确保包涵体复性的稳健平台。
