Production of IgGs with a human-like sialylation in CHO cells

Abstract

IgGs that possess α2,6-sialylated Fc-glycans are thought to be responsible for the anti-inflammatory properties of intravenous immunoglobulins (IVIGs) [1], through a mechanism which has not been elucidated yet. The impact of this sialylation on the classic IgG's effector functions also remains unclear [2, 3]. The understanding of these mechanisms has been impeded by the heterogeneity of the sialylated glycan species together with the relative rarity of α2,6-sialylated IgGs.

N-glycan sialylation is a post-translational modification where a sialic acid (SA) residue is added to the terminal galactose residue (Gal) of a growing glycan chain. The SA type (NANA or NGNA), the nature of the linkage between the SA and the Gal (α2,3 or α2,6), or the number of glycan antennae being sialylated, may vary according to the IgG subtype, the host cell in which it is expressed and the cell culture environment. A closer attention must thus be paid to the precise sialylation profiles leading to the reduced ADCC activity and to the gain of anti-inflammatory properties.

In humans, 10 to 15% of the circulating IgG1s are sialylated, carrying mostly complex di-antennary glycans with two Gal and one α2,6-linked SA residue (G2FS(6)1, where G stands for galactose, F for fucose and S(6) for α2,6SA). Most of the therapeutic monoclonal antibodies (mabs) are produced in Chinese hamster ovary (CHO) cells, which have a glycosylation machinery close to that of humans, but possess only α2,3-sialyltransferases (ST3) whereas humans have both α2,3- and α2,6-sialyltransferases. The Fc domain of mabs produced in CHO typically possesses N-glycans with low galactosylation and very low sialylation (0-2% of α2,3-sialylated glycans).

In this study, we show that the α2,6-sialylation of IgG1's Fc domain can be efficiently achieved by the transient coexpression of the human β1,4-galactosyltransferase 1 (GT) and α2,6-sialyltransferase 1 (ST6) in CHO cells, whereas the expression of one or the other glycosyltransferase alone does not significantly improve sialylation [4]. The process allows for the production of milligrams of human-like sialylated mabs within two weeks. We present a panel of four orthogonal assays for the fine characterization of the mabs' glycoprofile that are in very good agreement with each other.