Rüdt Matthias, Bussien Aymerick, Cortada-Garcia Joan, Morschett Holger, Holm Hauke, Mazzucchelli Chiara, Flicker Karlheinz
Institute of Life Sciences, School of Engineering, HES-SO University of Applied Sciences and Arts Western, Delémont, Switzerland.
Lonza AG, Visp, Switzerland.
Biotechnol Bioeng. 2025 Nov;122(11):3098-3113. doi: 10.1002/bit.70033. Epub 2025 Aug 5.
Inclusion bodies (IBs) frequently form during the expression of heterologous proteins in Escherichia coli and are therefore important in pharmaceutical production. While it is accepted that cultivation conditions affect cultivation performance, the link to refolding performance remains unexplored. This study proposes that this interplay relies, at least partially, on the inherited biophysical properties of the IBs. Using a design of experiments approach, this study systematically explored how cultivation conditions-postinduction temperature, pH, and feed rate-affect the production of IBs containing anti-desipramine single-chain variable fragment antibodies as a model therapeutic protein. Various biophysical properties of the IBs-including hydrophobicity, secondary structure, and particle size-were characterized, and their relationship to cultivation parameters and refolding performance was analyzed. Key findings revealed that higher feed rates and temperatures increased the product titer and IB size. Larger IBs facilitated refolding, while a higher content of amyloid structures, occurring locally without a strong link to the cultivation parameters, hampered protein solubilization and refolding efficiency. Higher protein content in the IBs adversely affected the refolding yield due to a hidden coupling between cultivation and refolding. This study establishes IB biophysical properties as critical factors for linking upstream and refolding process performance, offering actionable insights to enhance bioprocess robustness and efficiency.
包涵体（IBs）在大肠杆菌中表达异源蛋白时经常形成，因此在药物生产中很重要。虽然人们普遍认为培养条件会影响培养性能，但与复性性能的联系仍未得到探索。本研究提出，这种相互作用至少部分依赖于包涵体的固有生物物理特性。本研究采用实验设计方法，系统地探讨了诱导后温度、pH值和进料速率等培养条件如何影响含有抗地昔帕明单链可变片段抗体作为模型治疗蛋白的包涵体的产生。对包涵体的各种生物物理特性，包括疏水性、二级结构和粒径进行了表征，并分析了它们与培养参数和复性性能的关系。关键研究结果表明，较高的进料速率和温度提高了产物滴度和包涵体大小。较大的包涵体有助于复性，而淀粉样结构含量较高，在局部出现且与培养参数没有紧密联系，会阻碍蛋白质的溶解和复性效率。由于培养和复性之间存在隐藏的耦合，包涵体中较高的蛋白质含量对复性产率产生了不利影响。本研究将包涵体生物物理特性确立为连接上游和复性过程性能的关键因素，为提高生物过程的稳健性和效率提供了可行的见解。
