Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells
Assessment of UCOE on Recombinant EPO Production and Expression Stability in Amplified Chinese Hamster Ovary Cells
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DOI：
10.1007/s12033-015-9877-y
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发表时间：
2015-09-01
影响因子：
2.6
通讯作者：
Dickson, Alan J.
中科院分区：
文献类型：
--
作者：
Betts, Zeynep;Dickson, Alan J.
CHO细胞是商业化生产治疗性蛋白质最常用的宿主，DHFR介导的基因扩增被广泛应用于产生蛋白质产量增加的细胞系。然而，在扩大规模所需的时间内，蛋白质生产率的下降是不可预测的，并对产量、时间、财务和监管审批产生影响。在这项研究中，我们研究了普遍存在的染色质开放元件 (UCOE) 和 DHFR 相关扩增之间与细胞表达稳定性相关的相互作用。总之，包含 UCOE 元件产生的细胞 (1) 实现了更高的细胞密度，并表现出每个细胞的重组 mRNA 产量和蛋白质产量增加，(2) 允许分离更多数量的高产克隆，(3) 导致每个重组基因拷贝的 mRNA 回收率更高，(4) 如果甲氨蝶呤 (MTX) 存在，则扩增后保留稳定的 mRNA 和蛋白质表达（但在观察到不稳定时不存在 MTX 的情况下），并且 (5) 赋予连接的拷贝数依赖性表达转基因，表明它们对位置基因沉默效应具有抵抗力。结论是，在表达构建体中包含 UCOE 对细胞系生成的确定性（以及回收的克隆数量以进行更详细的表征/优化）提供了显着的优势，并且 UCOE 与 DHFR 扩增方案兼容。数据表明，可以通过选择标记（例如 DHFR）的转录增强来增强细胞系恢复。
CHO cells are the most frequently used host for commercial production of therapeutic proteins, and DHFR-mediated gene amplification is extensively applied to generate cell lines with increased protein production. However, decreased protein productivity is observed unpredictably during the time required for scale-up with consequences for yield, time, finance and regulatory approval. In this study, we have examined the interaction between Ubiquitous Chromatin Opening Elements (UCOE) and DHFR-linked amplification in relation to cell expression stability. In summary, the inclusion of UCOE elements generated cells that (1) achieved higher cell densities and exhibited increased production of recombinant mRNA per cell and protein yield, (2) allowed isolation of greater numbers of high-producing clones, (3) resulted in greater mRNA recovery per recombinant gene copy, (4) retained stable mRNA and protein expression after amplification provided Methotrexate (MTX) was present (but not in the absence of MTX when instability was observed) and (5) conferred copy number-dependent expression to linked transgene, suggesting that they are resistant to positional gene-silencing effects. It was concluded that the inclusion of UCOEs within expression constructs offers significant advantages for certainty of cell line generation (and the number of recovered clones for more detailed characterisation/optimisation) and that UCOEs are compatible with DHFR amplification protocols. The data suggested that enhanced cell line recovery by transcriptional enhancement of selection markers, such as DHFR, could be achieved.