Glycoproteomic Characterization of FUT8 Knock-Out CHO Cells Reveals Roles of FUT8 in the Glycosylation

Abstract

The α1,6-fucosyltransferase (encoded by FUT8 gene) is the key enzyme transferring fucose to the innermost GlcNAc residue on an N-glycan through an α-1,6 linkage in the mammalian cells. The presence of core fucose on antibody Fc region can inhibit antibody-dependent cellular cytotoxicity (ADCC) and reduce antibody therapeutic efficiency in vivo. Chinese hamster ovary (CHO) cells are the predominant production platform in biopharmaceutical manufacturing. Therefore, the generation of FUT8 knock-out (FUT8KO) CHO cell line is favorable and can be applied to produce completely non-fucosylated antibodies. The characterization of monoclonal antibodies as well as host cell glycoprotein impurities are required for quality control purposes under regulation rules. To understand the role of FUT8 in the glycosylation of CHO cells, we generated a FUT8 knock-out CHO cell line and performed a large-scale glycoproteomics to characterize the FUT8KO and wild-type (WT) CHO cells. The glycopeptides were enriched by hydrophilic chromatography and fractionated 25 fractions by bRPLC followed by analysis using high-resolution liquid chromatography mass spectrometry (LC-MS). A total of 7,127 unique N-linked glycosite-containing intact glycopeptides (IGPs), 928 glycosites, and 442 glycoproteins were identified from FUT8KO and WT CHO cells. Moreover, 28.62% in 442 identified glycoproteins and 26.69% in 928 identified glycosites were significantly changed in the FUT8KO CHO compared to wild-type CHO cells. The relative abundance of all the three N-glycan types (high-mannose, hybrid, and complex) was determined in FUT8KO comparing to wild-type CHO cells. Furthermore, a decrease in fucosylation content was observed in FUT8KO cells, in which core-fucosylated glycans almost disappeared as an effect of FUT8 gene knockout. Meantime, a total of 51 glycosylation-related enzymes were also quantified in these two cell types and 16 of them were significantly altered in the FUT8KO cells, in which sialyltransferases and glucosyltransferases were sharply decreased. These glycoproteomic results revealed that the knock-out of FUT8 not only influenced the core-fucosylation of proteins but also altered other glycosylation synthesis processes and changed the relative abundance of protein glycosylation.