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



This article corrects:

  1. Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines Volume 77, Issue 5, 419–428, Article first published online: 23 April 2013

Correspondence to: A. C. Vestrheim, T. E. Michaelsen, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, 0403 Oslo

Norway. E-mail: anne.cathrine.vestrheim@fhi.no;




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. 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.

Materials and methods

We have detected that the IgG3 amino acid sequence for the wild-type and mutant IgG3 antibodies, as originally published in Scand J Immunol. 2013 May;77(5):419-28. doi: 10.1111/sji.12046, was not EEQFNSTFR but rather EEQYNSTFR. Tables 2, 3 and 4 inserted below therefore replaces Tables 2, 3 and 4 from the original article.

Table 2. 20 IgG1 and IgG3 glycoforms were searched for and displayed below is the mass and charge state
Charge state 2Charge state 3Charge state 2Charge state 3
Table 3. The average percentage distribution for the different glycoforms found for IgG1 and IgG3 produced in transiently transfected HEK 293E cells from two harvest, harvest 1 (early) and harvest 2 (late). The last two IgG antibodies were also produced in HEK 293E cells, but these had only one harvest. Of these, IgG3 N297A had no glycoforms, as would be expected, as the oligosaccharides are situated at asparagine 297. Glycoforms that were not detected in any of the antibodies were not included in the table, and glycoforms detected in some, but not all, were marked nd, not detectable. Glycoforms present in <0,4% were excluded. H1 = harvest 1 and H2 = harvest 2
2 harvests
IgG1 wt H1 52 45 3 ndndndndndndndnd
IgG1 wt H2 87 13 ndndndndndndndndnd
IgG1 P329A H1 50 45 5 ndndndndndndndnd
IgG1 P329A H2 81 17 2 ndndnd <0,4 <0,4 <0,4 ndnd
IgG1 D270A H1 33 46 15 ndndnd 2 4 ndndnd
IgG1 D270A H2 66 27 2 1 ndnd 4 ndndndnd
IgG1 L234A H1 49 42 9 ndndndndndndndnd
IgG1 L235A H2 76 21 2 ndndnd <0,4 <0,4 1 ndnd
IgG1 L235A H1 50 45 5 ndndndndndndndnd
IgG1 L235A H2 78 20 1 ndndnd <0,4 <0,4 1 ndnd
IgG1 L234A/L235A H1 42 51 7 ndndndndndndndnd
IgG1 L234A/L235A H2 75 22 2 ndndnd <0,4 <0,4 1 ndnd
IgG3wt H1 45 27 5 ndnd 2 ndndndnd 21
IgG3wt H2 75 12 2 ndnd 2 1 <0,4 ndnd 8
IgG3 P329A H1 37 41 8 ndnd 1 ndndndnd 13
IgG3 P329A H2 70 16 2 nd 4 1 1 1 ndnd 5
IgG3 D270A H1 40 32 8 ndnd 2 ndndndnd 18
IgG3 D270A H2 73 12 2 ndnd 2 1 <0,4 ndnd 10
IgG3 m15 H1 45 45 10 ndndndndndndndnd
IgG3 m15 H2 75 20 4 ndnd 1 <0,4 ndndndnd
1 harvest
IgG1 H435A 56 32 9 ndndndnd 2 nd 1 nd
IgG3 N297A ndndndndndndndndndndnd
Table 4. The percentage distribution for the different glycoforms found for permanently transfected IgG1 and IgG3 from permanently transfected cell lines from one harvest only. Glycoforms that were not detected in any of the antibodies were not included in the table, and glycoforms detected in some, but not all, were marked nd, not detectable. Glycoforms present in <0,4% were excluded
Permanent cell lines
IgG1 chimer NS-0582611ndnd41ndnd
IgG1 chimer J558L31872ndnd<0,416nd
IgG3 wt NS-0154630ndndndndnd9
IgG3 m15 NS-0185328ndndndndnd1
IgG3 wt anti-NIO J558L274823ndndndndnd2
IgG3 m15 R435H NS-073360ndndndndndnd


Using transient transfection, IgG1 molecules contain more fucose than IgG3 molecules

IgG1 wild type and mutants all showed rather similar glycoforms and IgG3 wild type and mutants all showed rather similar glycoforms (see table 3), and G0F and G1F were the main glycoforms for both IgG1 and IgG3 antibodies. The glycoforms from IgG1 antibodies were generally fucosylated, while 3 of the transiently transfected IgG3 antibodies contained a portion of a non-fucosylated glycoform that was not observed in IgG1. The non-fucosylated glycoforms were mainly G2S, but also G1 and G2 to a minor degree (see Table 3).

IgG antibodies from transient transfection showed more complex glycoforms from the early harvest compared with the late harvest

IgG1 wild type and mutants and IgG3 wild type and mutants all showed rather similar profiles within the subclass in the early harvest. A few stood out, as IgG1 D270A displayed more G2F than any other IgG1, while the presence of G1 was slightly higher in IgG3wt and IgG3 D270A compared with the other two IgG3 antibodies from the early harvest (see Table 3).

The same trends were seen for the late harvest; the mutant IgG antibodies showed similar glycosylation patterns to their wild-type IgG subclass, although a decrease in the more complex glycoforms were seen here (See Table 3).

Choice of cell line for permanent transfection influence glycosylation pattern for IgG1, but not for IgG3

The four IgG3 antibodies from permanently transfected cell lines showed similar glycosylation profiles, with four glycoforms, G0F, G1F, G2F and G2S. Two of the antibodies from the NS-0 cell line, wt and m15, showed almost identical profiles to each other, while the antibody produced in J558L cells showed a relatively similar distribution, although not identical. IgG3 R435H from NS-0 cells showed a marked skewing towards G2F as the main glycoform. As this antibody was mutated, the difference from IgG3 m15 produced in NS-0 cells was likely caused by the mutation, not the cell line (see Tables 3 and 4).

Transient and permanent transfection affect glycosylation pattern of both IgG1 and IgG3

The permanently transfected IgG3 wild types (from NS-0 and J558L cells) and m15 (from NS-0 cells) showed similar profiles where 3 fucosylated glycoforms were observed; G0F, G1F and G2F and 1 non-fucosylated, G2S. When comparing these profiles with the transiently transfected antibodies, the permanently transfected IgG3 wild types had twice the amount of G1F as the transiently transfected IgG3 wild type from the early harvest, and 8 times the amount from the late harvest. On the other hand, the transiently transfected IgG3 wt showed a larger presence of G2S, a glycoform without fucose, particularly in the early harvest, compared with the permanently transfected IgG3 wt. The distribution of G0F and G2F between the permanently transfected IgG3 m15 and the transiently transfected IgG3 m15 from the early harvest was very different, while transiently transfected IgG3 m15 from the second harvest showed a completely different glycoform distribution (see Tables 3 and 4).

IgG3 glycoforms isolated from NS-0 and J558L are therefore rather similar, but differ from the ones produced by HEK 293E cells.


The percentage of IgG3 non-fucosylated glycoforms was noticeable, especially for the early harvest after transient transfection compared with the almost complete lack of non-fucosylated glycoforms in IgG1 antibodies. The non-fucosylated IgG3 glycoform G2S was not found in transiently transfected IgG3 m15, and only present as 1% on permanently transfected IgG3 m15. This could possibly indicate involvement of the long IgG3 hinge region, as IgG3 m15 has a shortened hinge region.