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

Abstract

The 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. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst 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. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cellmediated 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 IgGs. The striking difference in glycosylation pattern of IgG1 compared to 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.

Transient pentameric IgM fulfill biological function—Effect of expression host and transfection on IgM properties

“…This might be due to the oligomannosidic carbohydrate structure at GS4 (Asn402) which is important for the C1q binding and complementdependent cytolysis [7,35]. In contrast to previous observations [7,33,35], we did not observe differences in effective C1q binding due to glycosylation variations. However, sensitivity of the assay may not allow determining minor differences of activation, as an active fraction of pentamers may be sufficient to provoke a high signal and therefore complement activation did not reveal expression host dependency.…”

“…These differences could result from different expression levels of enzymes participating in glycosylation in the two host cell lines [31,32]. In accordance with Vestrheim et al, 2013, a lower sialic acid content was observed in HEK-derived immunoglobulins [33]. This is highly relevant when it comes to therapeutic usage, as sialic acid-rich proteins exhibit an extended half-life compared to non-sialylated proteins [34].…”

A pilot study showing differences in glycosylation patterns of IgG subclasses induced by pneumococcal, meningococcal, and two types of influenza vaccines

“…Whether this was due to individual differences or the actual vaccine formulation is uncertain, but the difference was not observed when the same volunteers received other vaccines. We have previously studied recombinant chimeric mouse‐human IgG3, which we found to contain more non‐fucosylated glycoforms compared to IgG1 . Interestingly, IgG3 antibodies in general appear superior to IgG1 regarding capacity for both ADCC and opsonophagocytic activity .…”

Impact of host cell line choice on glycan profile

“…Terminal sialylation was detected at low amounts of under 5% on IgG derived from human plasma, CHO-K1, J558L and HEK293 cells [105,106,[108][109][110]112,113]. Due to low abundance, sialylated glycoforms of CHO-derived IgG was not clearly distinguishable through HPLC chromatograms and was thus not depicted during the two studies that utilized only liquid chromatography for glycan profiling [108,110].…”

“…Figure 2. Proposed structures of major N-linked glycans on IgG produced using mammalian cells [105][106][107][108][109][110][111][112][113][114]. Relative abundance of glycostructures, when determined by studies, is shown as percentage values.…”