Establishment of a glycoengineered CHO cell line for enhancing antennary structure and sialylation of CTLA4-Ig

Abstract

Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we identified and over expressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and alpha 2,6-sialyltransferase (alpha 2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and alpha 2,6-ST), and co-overexpression (GnT-IV and alpha 2,6-ST, and GnT-V and alpha 2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnTIV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (-10.9% and-18.6%, respectively), while bi-and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV.ST (cells co-overexpressing GnT-IV and alpha 2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV.ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and alpha 2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.