Elshazly Mohamed, Leeb Benedikt, Brichtova Eva Prada, Gisperg Florian, Klausser Robert, Vijayakumar Shilpa, Lendl Bernhard, Voigtmann Martin, Berkemeyer Matthias, Spadiut Oliver, Kopp Julian
Research Division Integrated Bioprocess Development, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Gumpendorferstraße 1A, Vienna 1060, Austria; Christian Doppler Laboratory for Inclusion Body Processing 4.0, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Gumpendorferstraße 1A, Vienna 1060, Austria.
Research Division of Environmental Analytics, Process Analytics and Sensors, Institute of Chemical Technologies and Analytics, Technische Universität Wien, Getreidemarkt 9, Vienna 1060, Austria.
J Biotechnol. 2025 Sep;405:182-190. doi: 10.1016/j.jbiotec.2025.05.014. Epub 2025 May 20.
Ever since the potential of inclusion bodies (IBs) has been recognized, substantial advances have been made towards understanding IB processes and enabling efficient and controlled development strategies. Still, the influence of the chosen upstream processing (USP) strategy on the properties of inclusion bodies (IBs) and their refolding performance remains poorly understood. This work aims to target this challenge by investigating the influence of two chosen USP parameters, namely the specific substrate uptake rate and the temperature during induction, on IB titer, IB properties, namely IB purity, size and secondary protein structure of the IBs, as well as refolding yield of single-chain variable fragment M (scFvM) IBs. Contrary to findings in the literature, USP conditions neither had a statistically significant effect on the aforementioned IB properties nor on the refolding yield, but could clearly alter the IB titer. Our results provide detailed analytical insights on the independence of IB properties from USP conditions for this protein, while increasing the volumetric IB productivity proved feasible through variations in USP parameters. Therefore, titer maximization appears to be the sole optimization strategy for scFvM IBs and these findings may also apply to other target proteins with similar structural properties.
自从包涵体（IBs）的潜力被认识以来，在理解包涵体形成过程以及制定高效且可控的开发策略方面已经取得了重大进展。然而，人们对所选上游加工（USP）策略对包涵体（IBs）性质及其重折叠性能的影响仍知之甚少。这项工作旨在通过研究两个选定的USP参数，即比底物摄取速率和诱导期间的温度，对IB滴度、IB性质（即IB纯度、大小和IBs的二级蛋白质结构）以及单链可变片段M（scFvM）IBs的重折叠产率的影响，来应对这一挑战。与文献中的研究结果相反，USP条件对上述IB性质和重折叠产率均无统计学上的显著影响，但能明显改变IB滴度。我们的结果提供了关于该蛋白质的IB性质与USP条件无关的详细分析见解，同时通过改变USP参数提高体积IB生产力被证明是可行的。因此，滴度最大化似乎是scFvM IBs的唯一优化策略，并且这些发现可能也适用于其他具有相似结构性质的目标蛋白质。
