Effects of ubiquitous chromatin opening element (UCOE) on recombinant anti-TNFα antibody production and expression stability in CHO-DG44 cells
Effects of ubiquitous chromatin opening element (UCOE) on recombinant anti-TNFα antibody production and expression stability in CHO-DG44 cells
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DOI：
10.1007/s10616-021-00503-1
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发表时间：
2021-10-23
期刊：
影响因子：
2.2
通讯作者：
Nghia Son Hoang
中科院分区：
文献类型：
--
作者：
Chinh Chung Doan;Thanh Long Le;Nghia Son Hoang
到目前为止，抗体的产生通常面临转基因表达减少和不稳定的风险，特别是在长期培养之后。有报道称，在CHO细胞系中，将普遍存在的染色质开放元件(UCOE)整合到表达载体中可以提高蛋白质产量和维持转基因表达的稳定性。因此，我们研究了UCOE对CHO-DG44细胞中重组抗肿瘤坏死因子α单抗(mAbTNFα)产生和表达稳定性的影响。在我们的研究中，我们构建了非UCOE和基于UCOE的mAbTNFα编码载体，并将其导入CHO-DG44细胞。用G418筛选转染细胞，用甲氨蝶呤(MTX)扩增，有限稀释分离，获得细胞库和单细胞克隆。通过mAb产量与基因表达水平变化、基因拷贝数变化的相关性，分析UCOE对mAb产量和转基因稳定表达的影响。与非UCOE池相比，UCOE池表现出更高的mAb产量。在MTX介导的基因扩增后，UCOE与较高的转基因转录活性有关，导致mAb产量的提高。UCOE产生的细胞的掺入允许分离出更多的高表达的阳性克隆。尽管单抗产量略有下降，但UCOE克隆在没有选择压力的情况下仍然保持稳定的长期表达，这是由于转基因拷贝的丢失而不是转录活性的下降。此外，纯化的mAb具有与adalimumab相似的主要化学和生物学特性。结果表明，在载体中掺入UCOE在获得高产克隆、提高单抗产量和提高基因表达稳定性方面具有显著优势。
To date, the production of antibodies (mAbs) usually faces the risks of transgene expression reduction and instability, especially after long-time culture. The inclusion of ubiquitous chromatin opening element (UCOE) into expression vectors was reported to enhance protein production and maintain transgene expression stability in CHO cell lines. Thus, we investigate the effects of UCOE on recombinant monoclonal anti-TNF alpha antibody (mAbTNF alpha) production and expression stability in CHO-DG44 cells. In our study, non-UCOE and UCOE-based vectors encoding mAbTNF alpha were constructed and introduced into the CHO-DG44 cells. Cell pools and single-cell clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate (MTX), and isolating them by limiting dilution. The effects of UCOE on mAb production and stable transgene expression in transfected cells were analyzed via the correlation between mAb yields and mRNA expression level variations, and gene copy number changes. The UCOE pool exhibited higher mAb yield compared to non-UCOE pool. The UCOE was associated with higher transgene transcriptional activity, leading to improvement of mAb production after MTX-mediated gene amplification. The incorporation of UCOE generated cells allowed isolation of greater numbers of positive clones with higher expression. Despite the slightly decreased mAb yield, UCOE clones still retain stable long-term expression in the absence of selective pressure, which was explained by the loss of transgene copies rather than due to the decline of transcriptional activity. In addition, the purified mAb had primary chemical and biological characteristics similar to those of adalimumab. The results showed that the incorporation of UCOE within vectors provides significant advantages in the generation of high-producing clones, enhancement of mAb production, and improvement of gene expression stability.