THE GLYCOSYLATION PATTERN OF A HUMANIZED IGGI ANTIBODY (D1.3) EXPRESSED IN CHO CELLS

A humanized IgG antibody (D1.3) which retains murine complementarity d etermining regions specific for the antigen lysozyme has been expresse d 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 p urified from culture supernatants by Protein G chromatography. With th e recombinant D1.3 antibody as a model, this cell culture system was s hown to glycosylate the IgG Fc region in a similar manner to IgG isola ted from serum. The neutral, core fucosylated biantennary oligosacchar ides found are present in serum IgG and no novel carbohydrate sequence s were detected. The degree of terminal agalactosylation was also simi lar to normal serum, in contrast to the increased levels found in rheu matoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta 1,2) Ma n(alpha 1,6) Man(beta 1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combi nation of specific exoglycosidase digestions, gel permeation chromatog raphy of 5-aminobenzamide derivatives, high pH anion exchange chromato graphy and methylation analysis followed by gas-liquid chromatography- mass spectrometry.