文献类型：期刊文献

英文题名：Understanding the effect of temperature downshift on CHO cell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state

作者：Zhu, Ziyu[1];Chen, Xiaoqian[2];Li, Wenhao[1];Zhuang, Yingping[1,3];Zhao, Yuzheng[2,4];Wang, Guan[1,3,5]

机构：[1]East China Univ Sci & Technol ECUST, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]East China Univ Sci & Technol ECUST, Optogenet & Synthet Biol Interdisciplinary Res Ctr, Shanghai Frontiers Sci Ctr Optogenet Tech Cell Met, Sch Pharm,State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[3]East China Univ Sci & Technol, Qingdao Innovat Inst, Shanghai, Peoples R China;[4]Chinese Acad Med Sci, Res Unit New Tech L cell Metab Imaging, Beijing, Peoples R China;[5]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2023

卷号：39

期号：4

外文期刊名：BIOTECHNOLOGY PROGRESS

收录：;EI(收录号：20231914067485);WOS:【SCI-EXPANDED(收录号:WOS:000981349700001)】；

基金：ACKNOWLEDGMENTS This research was funded by the National Key R&D Program of China (Grant no. 2021YFC2101100), Shanghai Rising-Star Program (Grant no. 21QA1402400), Scientific Research Think Tank of Biological Manufacturing Industry in Qingdao (QDSWZK202004), and the Fundamental Research Funds for the Central Universities (JKF01231708).

语种：英文

外文关键词：antibody; CHO cell culture; intracellular sensor; metabolomics; NAD(+)/NADH ratio; NADPH; redox state; temperature downshift

摘要：The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (qp) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, anti -body expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37 degrees C) and temperature downshift (37 degrees C -> 33 degrees C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra-and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA) cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD(+)/NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD(+)/NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.