Characterization of the conbercept gene localization in DHFR- amplified CHO cells using fluorescence in situ hybridization.

Chinese-hamster ovary (CHO) cells are most widely used for mammalian protein expression. After integration into the CHO genome, the exogenous gene may be lost in the process of large-scale protein production due to the removal of related selection pressures. Therefore, it is necessary to test its stability in the genome. Conbercept is a fusion protein that specifically binds to the various isoforms of vascular endothelial growth factor (VEGF)-A, VEGF-B, and placental growth factor (PlGF), thereby exerting anti-angiogenic activities. It has been approved for Phase Ⅲ clinical trials in the United States. In this study, fluorescence in situ hybridization (FISH) was used to localize the conbercept gene in dihydrofolatereductase (DHFR)-amplified CHO cell lines. Metaphase FISH showed that genomic integration of the conbercept gene was stable after 4 and 19 passages, and manifested three characteristics: first, the gene locates on one chromosome, rather than a number of chromosomes; second, the gene locates on the longer chromosomes; third, there are many copies located on the same chromosome. At the same time, the copy number of the conbercept gene in the CHO genome and the conbercept protein expression levels are also stable, as verified by qPCR and ELISA assays, respectively. These experiments demonstrated that the conbercept gene remained stable in the genome after 19 passages, and could be actively expressed, which strongly support the mass production and the quality control of conbercept.