High-level expression of biologically active human follicle stimulating hormone in the Chinese hamster ovary cell line by a pair of tricistronic and monocistronic vectors

Recombinant human follicle stimulating hormone (FSH), produced in Chinese hamster ovary (CHO) cells, is widely used for treatment of fertility disorders and is subject to biosimilars development. Cell lines with high specific productivities may simplify the FSH production process. Here, we used our previously established expression system based on vector p1.1 to create new cell lines secreting heterodimeric FSH protein. To this end, we linked open reading frames of both FSH subunits by the wild-type internal ribosome entry site from the encephalomyocarditis virus (EMCV IRES). Intact and double-negative for the dihydrofolate reductase CHO cells were stably transfected by the FSH-coding plasmids. Stably transfected intact cells showed higher level of the FSH secretion and were utilized for subsequent methotrexate-driven transgene amplification, which doubled their productivity. The excess of the free α-subunit was corrected by transfecting the cells by the additional p1.1-based plasmid encoding the β-subunit of the FSH. Clonal cell lines obtained secreted mostly the heterodimeric FSH and possessed specific productivities up to 12.3±1.7 pg/cell/day. Candidate clonal cell line C-P1.3-FSH-G4 maintained a constant specific productivity for at least 2 months of culturing without the section pressure. The resulting FSH protein conformed to the international pharmaceutical quality criteria as evidenced by the receptor binding kinetics, distribution pattern of hormone isoforms and biological activity. In conclusion, our expression system offers a simple and cost-effective approach to production of FSH.

由中国仓鼠卵巢（Chinese hamster ovary, CHO）细胞制备的重组人促卵泡激素（recombinant human follicle stimulating hormone, FSH）广泛用于不孕症的治疗，且其生物类似药研发工作亦在推进中。具备高比生产力的细胞系可简化FSH的生产流程。本研究基于前期构建的以载体p1.1为基础的表达系统，成功构建了可分泌异二聚体FSH蛋白的新型细胞系。为此，本研究利用脑心肌炎病毒（encephalomyocarditis virus, EMCV）的野生型内部核糖体进入位点（internal ribosome entry site, IRES），连接了FSH两个亚基的开放阅读框。将携带FSH编码序列的质粒稳定转染至野生型及二氢叶酸还原酶（dihydrofolate reductase, DHFR）双阴性的CHO细胞中。经稳定转染的野生型CHO细胞表现出更高的FSH分泌水平，随后被用于甲氨蝶呤介导的转基因扩增，使其生产力提升一倍。通过额外转染携带FSH β亚基编码序列的p1.1载体质粒，纠正了游离α亚基过量表达的问题。所获得的单克隆细胞系主要分泌异二聚体FSH，其比生产力可达12.3±1.7皮克/细胞/天。候选单克隆细胞系C-P1.3-FSH-G4在无筛选压力的条件下，至少连续培养2个月仍可维持稳定的比生产力。经受体结合动力学、激素异构体分布模式及生物学活性验证，最终获得的FSH蛋白符合国际药品质量标准。综上，本研究构建的表达系统为FSH的生产提供了一种简便且经济高效的方案。