抗体高产中国仓鼠卵巢细胞细胞株生理生化特征、糖代谢及单克隆抗体基因表达量的分析

摘要

Product specific productivity represents an overwhelming parameters for cell line, thus studies on cell lines with differed productivity provide deeper insights for understanding this parameter, and make suggestions for obtaining cell line with improved productivity. In this study, the productivity-enhanced cell variants from Chinese hamster ovary (CHO) cells producing monoclonal antibody (mAb) were artificially selected by limiting dilution method. Compared with the original cells, the specific productivity of the selected subclone NO.9 increases by 57.8%, thus its physiological and biochemical characteristics, gene expression and glucose metabolism were analysed in detail. In batch culture, the maximum viable cell density of NO.9 decreases by 68.6%. However, the specific productivity increases by 57.8%. During the batch culture, all the dry cell weight, cell diameter, intracellular protein and the ratio of intracellular protein to cell diameter of NO.9 increase in the logarithmic growing period, and the relative contents of gene and mRNA of heavy chain and light chain of NO.9 increase either. Besides, compared with the control, the specific consumption rate of glucose increases by 77.9%, the specific production rate of lactate increases by 68.0%;in logarithmic growing period, the relative mitochondrial synthetic activity increases by 172.5%, this will promote the product translation rate.The herein analyzed extensive features of the cell variant could help to understand the increased productivity during cell culture process, and it also offers advice for constructing and selecting the high-producer.%产物比生成速率是衡量细胞株表现的重要指标，研究产物比生成速率有差异的细胞株有助于深入理解此参数，进而为获得具有高产物比生成速率的细胞株提供借鉴。以表达单克隆抗体的中国仓鼠卵巢(CHO)细胞为研究对象，通过有限稀释法筛选获得了抗体比生产速率比对照提高57.8%的亚克隆。以此为基础，对其生理生化特征、葡萄糖代谢以及抗体轻重链基因表达量进行了详细的分析。此亚克隆在 Hyclone SFM4CHO 培养基批次培养中，最高活细胞密度比对照降低了68.6%，其在指数生长期细胞干重、细胞直径、胞内总蛋白含量及胞内总蛋白和细胞直径比与对照相比均有不同程度的增加，且其表达产物的轻重链基因和信使 RNA 相对含量均有所提高。代谢方面，此亚克隆的葡萄糖比消耗速率比对照增加了77.9%，指数生长期线粒体相对合成活性比对照增加了172.5%，有助于增加产物的翻译速率。通过研究对表达单克隆抗体的高产亚克隆生理生化特征、葡萄糖代谢以及抗体轻重链基因表达量的分析，可为深入理解细胞培养过程中产物比生成速率的提高提供借鉴，同时为高产细胞株的构建和筛选提供指导。