Effects of 4-phenylbutyrate addition time on the productivity and glycan structure of hCTLA4-Ig in CHO cell culture

초록

Most of the therapeutic proteins are produced from Chinese hamster ovary (CHO) cell line. Cell cycle inhibitors have been applied for enhancing protein productivity [1]. Among them, 4-phenylbutyrate (4-PBA) is known to improve productivity in mammalian cell cultures, but it can also inhibit cell growth and induce cellular apoptosis [2]. In this study, we controlled the addition time of 4-PBA to determine the effects on protein productivity and glycan structure. Three concentrations (0.5, 1 and 2 mM) of 4-PBA were added with 5 culture conditions (day 0, day 1, day 2, day 3 addition and repeated addition). The highest titer was obtained when 0.5 mM 4-PBA was added 4 times repeatedly to make final concentration at 2 mM and it was 1.57-fold increase. When analyzing cell cycle distribution, it was observed that G1 phase related to protein synthesis was 10% higher than that of control when 1 mM 4-PBA was added on day 0. Moreover, each addition time showed different pattern of cell cycle arrest, the proportion of cell cycle could be controlled by regulating the addition time of 4-PBA. 4-PBA also acts as a chemical chaperone and reduces endoplasmic reticulum stress. When 0.5 mM 4-PBA was added repeatedly, the percentage of spliced X-box binding protein 1 gene levels was 35% lower than that of control. The content of sialic acid, which determines the circulatory time of glycoprotein, was found to be similar to that of control culture with repeated addition. It was proved that controlling addition time of cell cycle inhibitors can improve the production level and maintain the quality of hCLTA4-Ig. In conclusion, controlling the addition time of 4-PBA can effectively increase the production of hCTLA4-Ig without affecting the quality.