期刊
MABS
卷 7, 期 3, 页码 571-583出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/19420862.2015.1029215
关键词
ECL, Erythrina Cristagalli lectin; MAL-II, Maackia Amurensis lectin II; alpha 2,3SA, alpha 2,3-linked sialic acid; alpha 2,6SA, alpha 2,6-linked sialic acid; SNA, Sambucus Nigra agglutinin; LC-ESI-MS, liquid chromatography coupled to electrospray ionization mass spectrometry; cIEF, capillary zone electrophoresis isoelectric focusing; HILIC, hydrophilic interaction liquid chromatography; mAbs, monoclonal antibodies; TZM, trastuzumab (Herceptin (R)); PEI, polyethylenimine; GT, beta 1,4-galactosyltransferase 1; ST6, alpha 2,6-sialyltransferase 1
资金
- NSERC Strategic Network for the Production of Single-Type Glycoform Monoclonal Antibodies (MabNet)
The presence of alpha 2,6-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 alpha 2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan alpha 2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human alpha 2,6-sialyltransferase 1 (ST6) and beta 1,4-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 alpha 2,6-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 alpha 2,6-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.
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