Effects of high passage cultivation on CHO cells: a global analysis
Effects of high passage cultivation on CHO cells: a global analysis
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DOI：
10.1007/s00253-011-3806-1
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发表时间：
2012-05-01
影响因子：
5
通讯作者：
Noll, T.
中科院分区：
文献类型：
--
作者：
Beckmann, T. F.;Kraemer, O.;Noll, T.
用于工业药物蛋白质生产过程的细胞系需要坚固、生长快速且产量高。为了找到这样的细胞，我们在摇瓶中对产生单克隆抗体的中国仓鼠卵巢（CHO）细胞进行了420多天的高传代培养。对细胞生长、生产力、细胞内蛋白质和代谢物特征以及产物转录物和基因组整合水平的检查揭示了不同时间点长期培养的亚群之间存在显着差异。在高传代培养期间检测到的生长性能以及细胞内腺苷酸能量电荷增加。此外，蛋白质组分析表明糖酵解的利用增加，传代次数更高，并且基于抗应激蛋白的稳健性增强。有趣的是，尽管施加了选择压力，但产物形成在最初增加，但在后来的传代过程中急剧下降。利用流式细胞术和定量实时聚合酶链式反应，我们进一步检查了观察到的表型的翻译、转录和基因组基础。检测到的抗体表达减少，特别是重链的减少，归因于抗体转录物的减少，这是由基因拷贝数的损失以及可能是双顺反子mRNA的剪接机制故障引起的。据我们所知，这是第一个使用过程分析和靶向组学技术来阐明长期培养表达治疗性蛋白的 CHO 细胞的影响的系统方法。
Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.