Yamaguchi Keina, Ogawa Risa, Tsukahara Masayoshi, Kawakami Koichi
Laboratory of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka, 411-8540, Japan.
Bio Process Research and Development Laboratories, Kyowa Kirin Co., Ltd, 100-1 Hagiwara-machi, Takasaki, Gunma, 370-0013, Japan.
Sci Rep. 2025 May 27;15(1):18512. doi: 10.1038/s41598-025-03301-3.
The Chinese hamster ovary (CHO) cell line is a widely employed system for producing therapeutic proteins. In our previous study, we established an efficient method for generating stable cell lines by utilizing the Tol2 transposon system, combined with cycloheximide (CHX) resistance as a selection marker. This DNA-based transposon allows the integration of the gene of interest into various genomic loci within the host cell genome. To further develop this system, we performed gene transfer using single vectors carrying each subunit separately, rather than using dual vector linking subunits in tandem, to express monoclonal antibodies. The resulting cell lines exhibited stable protein production for an extended period of up to 12 weeks, as well as high productivity in fed-batch cultures. We found that the copy numbers of vector construct integrated in the genome varied for different mAbs, suggesting these cell lines maintained the vector constructs at copy numbers for effective gene expression. This study highlights the potential usefulness of the Tol2 transposon system in producing multi-subunit proteins, such as bispecific antibodies and Fc-fusion proteins, thereby promoting advancements in biopharmaceutical production.
中国仓鼠卵巢（CHO）细胞系是一种广泛用于生产治疗性蛋白质的系统。在我们之前的研究中，我们建立了一种利用Tol2转座子系统并结合环己酰亚胺（CHX）抗性作为选择标记来生成稳定细胞系的有效方法。这种基于DNA的转座子允许将感兴趣的基因整合到宿主细胞基因组内的各种基因组位点。为了进一步开发该系统，我们使用分别携带每个亚基的单个载体而不是使用串联连接亚基的双载体进行基因转移来表达单克隆抗体。所得细胞系在长达12周的时间内表现出稳定的蛋白质生产，以及在补料分批培养中的高生产力。我们发现整合到基因组中的载体构建体的拷贝数因不同的单克隆抗体而异，这表明这些细胞系以有效基因表达的拷贝数维持载体构建体。这项研究突出了Tol2转座子系统在生产多亚基蛋白质（如双特异性抗体和Fc融合蛋白）方面的潜在有用性，从而推动生物制药生产的进步。
