VARIATION IN N-LINKED CARBOHYDRATE CHAINS IN DIFFERENT BATCHES OF 2 CHIMERIC MONOCLONAL IGG1 ANTIBODIES PRODUCED BY DIFFERENT MURINE SP2 O TRANSFECTOMA CELL SUBCLONES/

Two chimeric human/murine monoclonal antibodies were constructed by su bstitution of the murine constant regions with human gamma 1 and kappa constant regions for heavy and light chains, respectively. The chimer ic human/murine molecules are anti-idiotypic antibodies, meaning that they were directed against the antigen binding site in the variable re gion of another antibody. Antibody batches were produced under identic al production conditions, using two selected SP2/0 myeloma cell subclo nes, which produce chimeric antibodies with different variable regions , but identical constant regions. Several samples were collected durin g the production of the antibodies in hollow-fibre reactors. The heavy chain, but not the light chain, of the two different chimeric IgG1 an tibodies is glycosylated. Structural analysis of the enzymically relea sed N-linked carbohydrate chains by H-1-NMR spectroscopy, as well as b y chromatographic profiling, demonstrated that the collection of N-gly cans comprises a small amount of monoantennary, and for the greater pa rt diantennary structures. The N-glycans are completely (alpha 1 --> 6 )-fucosylated at the innermost GlcNAc residue. The antennae of the neu tral diantennary N-glycans are built up from GlcNAc beta 1 --> 2, Gal beta 1 --> 4GlcNAc beta 1 --> 2 or Gal alpha 1 --> 3G alpha l beta 1 - -> 4GlcNAc beta 1 --> 2 elements, whereas the antennae of the neutral monoantennary carbohydrate chains have only (beta 1 --> 2)-linked GlcN Ac residues. Galactosylation of the GlcNAc beta 1 --> 2Man alpha 1 --> 6 branch occurs four times more frequently than that of the GlcNAc be ta 1 --> 2Man alpha 1 --> 3 branch, independently of the production ba tch. A small amount of the diantennary N-glycans are mono- or disialyl ated, carrying N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuramini c acid (Neu5Gc), exclusively (alpha 2 --> 6)-linked to beta Gal. Analy sis of the different production batches demonstrates that the structur es of the N-linked carbohydrate chains are identical in the two chimer ic antibodies, but that the relative amounts of the major oligosacchar ide components, the degree of sialylation and the molar ratio of Neu5A c to Neu5Gc varies with the SP2/0 cell subclone, and only slightly wit h cell age.