Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
Institute of Biomaterials and Biomolecular Systems, Department of Biobased Materials, University of Stuttgart, Pfaffenwaldring 57, 70569, Stuttgart, Germany.
Metab Eng. 2019 Jul;54:145-159. doi: 10.1016/j.ymben.2019.02.005. Epub 2019 Mar 28.
To fulfil the optimization needs of current biopharmaceutical processes the knowledge how to improve cell specific productivities is of outmost importance. This requires a detailed understanding of cellular metabolism on a subcellular level inside compartments such as cytosol and mitochondrion. Using IgG1 producing Chinese hamster ovary (CHO) cells, a pioneering protocol for compartment-specific metabolome analysis was applied. Various production-like growth conditions ranging from ample glucose and amino acid supply via moderate to severe nitrogen limitation were investigated in batch cultures. The combined application of quantitative metabolite pool analysis, C tracer studies and non-stationary flux calculations revealed that Pyr/H symport (MPC1/2) bore the bulk of the mitochondrial transport under ample nutrient supply. Glutamine limitation induced the concerted adaptation of the bidirectional Mal/aKG (OGC) and the Mal/HPO antiporter (DIC), even installing completely reversed shuttle fluxes. As a result, NADPH and ATP formation were adjusted to cellular needs unraveling the key role of cytosolic malic enzyme for NADPH production. Highest cell specific IgG1 productivities were closely correlated to a strong mitochondrial malate export according to the anabolic demands. The requirement to install proper NADPH supply for optimizing the production of monoclonal antibodies is clearly outlined. Interestingly, it was observed that mitochondrial citric acid cycle activity was always maintained enabling constant cytosolic adenylate energy charges at physiological levels, even under autophagy conditions.
为了满足当前生物制药工艺的优化需求，了解如何提高细胞特异性生产率至关重要。这需要在细胞质和线粒体等隔室中深入了解细胞代谢的亚细胞水平。使用 IgG1 产生的中国仓鼠卵巢（CHO）细胞，应用了一种开创性的区室特异性代谢组分析方法。在分批培养中，研究了各种类似于生产的生长条件，从充足的葡萄糖和氨基酸供应到适度至严重的氮限制。定量代谢物池分析、C 示踪剂研究和非稳态通量计算的综合应用表明，在充足的营养供应下， Pyr/H 协同转运（MPC1/2）承担了线粒体运输的大部分负担。谷氨酰胺限制诱导了双向 Mal/aKG（OGC）和 Mal/HPO 反向转运体（DIC）的协同适应，甚至安装了完全相反的穿梭通量。结果，NADPH 和 ATP 的形成根据细胞需求进行了调整，揭示了细胞质苹果酸酶在 NADPH 产生中的关键作用。最高的细胞特异性 IgG1 生产率与根据合成代谢需求强烈的线粒体苹果酸输出密切相关。为了优化单克隆抗体的生产而安装适当的 NADPH 供应的要求显然已经阐明。有趣的是，观察到线粒体柠檬酸循环活性始终保持不变，即使在自噬条件下，也能使细胞溶胶腺苷酸能量电荷保持在生理水平。
