Evaluation of the Effects of Human Beta-Interferon Scaffold Attachment Region (IFN-SAR) on Expression of Vascular Endothelial Growth Factor- Fc (VEGF-Fc) Fusion Protein Expression in Chinese Hamster Ovary (CHO) Cells

Background: recombinant anti-vascular endothelial growth factor (vegf) monoclonal antibodies and fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. in this regard, vegfr-fc fusions, which act as strong vegf inhibitors, have been approved for the treatment of age-related macular degeneration (amd) and diabetic macular edema (dme). production of monoclonal antibodies and fc-fusion proteins relies on mammalian host systems such as chinese hamster ovary (cho) cells. application of genomic regulatory elements including scaffold/matrix attachment regions (sar/mars) can profoundly affect recombinant protein expression in cho cells. methods: to construct the vegfr-fc expression vectors, the enhanced green fluorescent protein (egfp) gene was replaced by the vegfr-fc coding sequence in pegfp-sar-puro and pegfp-puro vectors. recombinant plasmids were transfected to cho-k1 cells using turbofect transfection reagent. vegfr-fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (elisa). results: ifn-sar showed no significant effect on transient expression of vegfr-fc during 72 h of culture. however, a 2.2-fold enhancement in vegfr-fc fusion protein titer was observed in ifn-sar containing stable cell pools. further evaluation of the vegfr-fc expression level in single-cell clones also indicated that clones with the highest vegfr-fc expression belonged to the pools transfected with ifn-sar construct. conclusion: our results indicate that the incorporation of ifn-sar in expression vector can increase the expression of vegfr-fc in stable cell pools as well as single-cell clones. in contrast, transient expression of the fusion protein was not affected by ifn-sar. more studies are needed to investigate the mechanism underlying this effect, including the analysis of mrna expression and gene copy number in stable cell pools as well as clonal cells.