The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells.
A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta1,2) Man(alpha1,6) Man(beta1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas-liquid chromatography-mass spectrometry.
Routier
F H
FH
Department of Biochemistry and Molecular Biology, University College London, UK.
Davies
M J
MJ
Bergemann
K
K
Hounsell
E F
EF
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D016466:CHO Cells / Q000276:immunology* / Q000378:metabolism; D002240:Carbohydrate Sequence; D002849:Chromatography, Gas / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics / Q000276:immunology; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000737:chemistry
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; C498150:IgG D1.3; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; D062367:ortho-Aminobenzoates; D006026:Glycoside Hydrolases; D009113:Muramidase; C000219:anthranilamide