School of Chemical and Bioprocess Engineering, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Biotechnol Bioeng. 2011 Apr;108(4):880-92. doi: 10.1002/bit.22994. Epub 2010 Nov 30.
Heterogeneity in gene expression and phenotypic traits is an inherent feature within the "clonal" cell lines used for biopharmaceutical production. This feature can allow the selection of cell lines with improved phenotypes and adaptation to growth under preferential conditions to improve productivity or provide a platform to study the molecular basis of improved characteristics. A repeated process of extended batch culture of a recombinant antibody producing GS-NS0 myeloma cell line generated a stable variant cell line displaying increased resistance to both environmental stresses and chemical apoptosis inducers, and resulted in extended culture viability and increased antibody production. An interesting feature of the variant cell line was an altered metabolic state with consumption of lactate as the culture progressed. The variant cell line also showed altered expression of proteins associated with autophagy suggesting a role for this process in extending cell survival in culture. Targeted transcriptomic analysis was carried out on the parental and variant cell lines using a qRT-PCR array containing a panel of apoptosis-associated genes to elucidate both the predominant apoptotic pathways in the NS0 cell line with batch culture progression, and the altered gene expression contributing to increased survival in the variant line. Results indicated a balance between pro- and anti-apoptotic signaling is triggered with the onset of cell death in the NS0 cell line. Pro-survival pathways such as NFκB signaling and the unfolded protein response were implicated along with death receptor, endoplasmic reticulum stress and p53 mediated apoptotic pathways. The identification of altered gene expression in the variant cell line also provides several potential targets for cell engineering strategies to create improved cell lines for production.
在用于生物制药生产的“克隆”细胞系中，基因表达和表型特征的异质性是固有特征。该特征可以允许选择具有改进表型的细胞系，并适应在有利条件下的生长，以提高生产力或提供一个研究改进特征分子基础的平台。对表达重组抗体的 GS-NS0 骨髓瘤细胞系进行延长批次培养的反复过程，产生了一种稳定的变体细胞系，该细胞系对环境压力和化学凋亡诱导剂的抗性增强，延长了培养活力并增加了抗体产量。变体细胞系的一个有趣特征是代谢状态发生改变，随着培养的进行，乳酸被消耗。该变体细胞系还显示出与自噬相关的蛋白质表达改变，表明该过程在延长细胞在培养中的存活中起作用。使用包含凋亡相关基因的 qRT-PCR 阵列对亲本和变体细胞系进行了靶向转录组分析，以阐明 NS0 细胞系在批培养过程中的主要凋亡途径，以及导致变体系中存活增加的改变基因表达。结果表明，在 NS0 细胞系中，细胞死亡开始时触发了促凋亡和抗凋亡信号之间的平衡。涉及 NFκB 信号传导和未折叠蛋白反应等存活途径，以及死亡受体、内质网应激和 p53 介导的凋亡途径。变体细胞系中改变的基因表达的鉴定也为创建用于生产的改良细胞系的细胞工程策略提供了几个潜在的靶点。
