The presence of α2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized α2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan α2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human α2,6-sialyltransferase 1 (ST6) and β1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by α2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The α2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl- and sialyl-transferases substrate specificities.
Keywords: B4GALT1; CHO cells; ECL, Erythrina Cristagalli lectin; GT, β1,4-galactosyltransferase 1; HILIC, hydrophilic interaction liquid chromatography; IgG1; LC-ESI-MS, liquid chromatography coupled to electrospray ionization mass spectrometry; MAL-II, Maackia Amurensis lectin II; N-glycosylation; PEI, polyethylenimine; SIAT1; SNA, Sambucus Nigra agglutinin; ST6, α2,6-sialyltransferase 1; TZM, trastuzumab (Herceptin®); cIEF, capillary zone electrophoresis isoelectric focusing; mAbs, monoclonal antibodies; sialylation; transfection; α2,3SA, α2,3-linked sialic acid; α2,6SA, α2,6-linked sialic acid.