Studies for improving COMP-Angiopoietin-1 production in chinese hamster ovary cells

Abstract

Angiopoietins are a family of vascular growth factors that collaborate with members of the vascular endothelial growth factor family to regulate vascular and lymphatic vessel growth, acing via the endothelial receptor tyrosine kinase, Tie2. Among of vascular endothelial growth factors, angiopoietin-1 (Ang1) has potential therapeutic applications in enhancing endothelial cell survival and preventing vascular leakage. However, high-level expression of Ang1 is hindered by the aggregation and insolubility of the protein due to its unique structure of Ang1. To avoid problems of aggregation and insolubility of Ang1, COMP-Ang1 was developed by replacing an N-terminal portion of Ang1 with a short coiled-coil domain of cartilage oligomeric matrix protein (COMP). The COMP-Ang1 is found to be more potent than native Ang1 for therapeutic angiogenesis in vitro and in vivo. However, its aggregation was still detected at a later phase of batch culture of recombinant CHO (rCHO) cells expressing COMP-Ang1 although aggregation was less significant than that of Ang1. Protein aggregates usually exhibit reduced or no biological activity, and often lead to immunogenicity. Therefore, aggregation of COMP-Ang1 during rCHO cell culture is undesirable and should be minimized. For the first step of this research, we studied the effect of culture temperature and pH on cell growth and flag tagged COMP-Ang1 (FCA1) production. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37℃). Lowering culture temperature suppressed cell growth, while maintaining high cell viability for a longer culture period. The specific FCA1 productivity (qFCA1) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33℃, which was approximately 2.6-fold higher than that at pH 7.2 and 37℃. However, aggregates and a monomeric form of FCA1, which are undesirable because of their reduced biological act...