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

초록

Chinese hamster ovary (CHO) cell line is the mostly used mammalian cells for the production of therapeutic proteins. 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 initial feeding, exponential phase feeding and intermittent feeding. The highest titer was obtained when 0.5 mM of 4-PBA was added intermittently. When analyzing cell cycle distribution, it was observed that the G1 phase related to protein synthesis was found to be the largest proportion when 1 mM 4-PBA was added initially. 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. The degree of inducing apoptosis and endoplasmic reticulum stress was dependent on the concentration of 4-PBA and addition time. The content of sialic acid, which determines the circulatory time of glycoprotein, was found to be similar to control culture with intermittent feeding. 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.