Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated From Transiently as Well as Permanently Transfected Cell Lines

Abstract

AbstractThe effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc portion. In this report, glycosylation profiles of recombinant wild‐type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC‐ESI‐Orbitrap. Differences were detected between IgG1 and IgG3, where IgG1 generally contained fucosylated glycoforms, while IgG3 showed a noticeable percentage of non‐fucosylated glycoforms. When using NS‐0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, while IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgG's harvested late showed simpler and less developed glycosylation structure compared with those harvested early. IgG's harvested early were slightly more effective in complement activation than those harvested late, while the antibody‐dependent cell‐mediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild‐type IgG's. The difference in glycosylation pattern of IgG1 compared with IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q‐binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.