Abstract

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.

中文翻译：

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结合 PAT 和建模的重折叠动力学监测和控制进展

摘要

重组蛋白在大肠杆菌中的过度表达会导致细胞质中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 IB 的观念发生了变化：IB 不再被视为不需要的废品，而是在短时间内生产高产量、高纯度和稳定性产品的有效替代品。然而，为了获得正确折叠的活性产品，IB 的溶解和随后的重折叠是必要的。这种蛋白质重折叠过程是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。最先进的技术的缺点包括：大量的缓冲液和重折叠槽的容量、均匀混合的问题、低蛋白质浓度下的挑战性分析、不可使用的聚集体中的反应动力学以及普遍较低的重复性。折叠产量。没有可用的通用平台程序，也缺乏稳健的控制策略。质量源于设计 (QbD) 的引入是提供受控且可重复的重折叠环境的首选方法。然而，实时描述重折叠动力学的可靠在线监测技术很少。我们认为，只有监测和控制重折叠动力学才能确保重折叠过程的高效、可扩展和多功能平台技术。在本次综述中，我们筛选了当前文献，结合在线过程分析技术 (PAT) 和建模技术，以确保受控的重折叠过程。根据我们的研究，我们提出了一种集成方法，其理念是通过数字孪生来估计所有无法直接监控的方面，并实时用于过程控制。

关键点

• 监测和全面了解重折叠动力学对于基于模型的重折叠过程控制至关重要。

• 质量源于设计的引入结合了过程分析技术和建模，确保了包涵体重折叠的强大平台。