Graham Ryan J, Mohammad Adil, Liang George, Fu Qiang, Kuang Bingyu, Polanco Ashli, Lee Yong Suk, Marcus R Kenneth, Yoon Seongkyu
Department of Chemical Engineering, University of Massachusetts Lowell, Lowell, Massachusetts, USA.
Division of Product Quality Research, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
Biotechnol Prog. 2021 Sep;37(5):e3181. doi: 10.1002/btpr.3181. Epub 2021 Jun 18.
Trace metals play a critical role in the development of culture media used for the production of therapeutic proteins. Iron has been shown to enhance the productivity of monoclonal antibodies during Chinese hamster ovary (CHO) cell culture. However, the redox activity and pro-oxidant behavior of iron may also contribute toward the production of reactive oxygen species (ROS). In this work, we aim to clarify the influence of trace iron by examining the relationship between iron supplementation to culture media, mAb productivity and glycosylation, and oxidative stress interplay within the cell. Specifically, we assessed the impacts of iron supplementation on (a) mAb production and glycosylation; (b) mitochondria-generated free hydroxyl radicals (ROS); (c) the cells ability to store energy during oxidative phosphorylation; and (d) mitochondrial iron concentration. Upon the increase of iron at inoculation, CHO cells maintained a capacity to rebound from iron-induced viability lapses during exponential growth phase and improved mAb productivity and increased mAb galactosylation. Fluorescent labeling of the mitochondrial hydroxyl radical showed enhanced environments of oxidative stress upon iron supplementation. Additional labeling of active mitochondria indicated that, despite the enhanced production of ROS in the mitochondria, mitochondrial membrane potential was minimally impacted. By replicating iron treatments during seed train passaging, the CHO cells were observed to adapt to the shock of iron supplementation prior to inoculation. Results from these experiments demonstrate that CHO cells have the capacity to adapt to enhanced environments of oxidative stress and improve mAb productivity and mAb galactosylation with minimal perturbations to cell culture.
痕量金属在用于生产治疗性蛋白质的培养基的开发中起着关键作用。铁已被证明可提高中国仓鼠卵巢（CHO）细胞培养过程中单克隆抗体的产量。然而，铁的氧化还原活性和促氧化行为也可能导致活性氧（ROS）的产生。在这项工作中，我们旨在通过研究培养基中铁补充、单克隆抗体产量和糖基化以及细胞内氧化应激相互作用之间的关系，来阐明痕量铁的影响。具体而言，我们评估了铁补充对以下方面的影响：（a）单克隆抗体的产生和糖基化；（b）线粒体产生的游离羟基自由基（ROS）；（c）细胞在氧化磷酸化过程中储存能量的能力；以及（d）线粒体铁浓度。接种时铁含量增加后，CHO细胞在指数生长期能够从铁诱导的活力下降中恢复，提高了单克隆抗体的产量并增加了单克隆抗体的半乳糖基化。线粒体羟基自由基的荧光标记显示，铁补充后氧化应激环境增强。活性线粒体的额外标记表明，尽管线粒体中ROS的产生增加，但线粒体膜电位受到的影响最小。通过在种子传代过程中重复铁处理，观察到CHO细胞在接种前适应了铁补充的冲击。这些实验结果表明，CHO细胞有能力适应增强的氧化应激环境，提高单克隆抗体的产量和单克隆抗体的半乳糖基化，同时对细胞培养的干扰最小。
