Compositional monosaccharide quantification of IgG1-Fc
| IgG1-Fc . | Monosaccharide molar ratios determined by HPAEC-PAD analysis* . | |||
|---|---|---|---|---|
| Fuc . | GlcN . | Gal . | Man† . | |
| Biantennary GP‡ | na | 3.86 | 2.08 | 3.00 |
| HuIgG1 | 0.95 | 3.50 | 0.80 | 3.00 |
| GG | 0.56 | 3.60 | 1.40 | 3.00 |
| GA | na | na | na | na |
| CTM | 1.10 | 5.20 | 1.90 | 3.00 |
| IgG1-Fc . | Monosaccharide molar ratios determined by HPAEC-PAD analysis* . | |||
|---|---|---|---|---|
| Fuc . | GlcN . | Gal . | Man† . | |
| Biantennary GP‡ | na | 3.86 | 2.08 | 3.00 |
| HuIgG1 | 0.95 | 3.50 | 0.80 | 3.00 |
| GG | 0.56 | 3.60 | 1.40 | 3.00 |
| GA | na | na | na | na |
| CTM | 1.10 | 5.20 | 1.90 | 3.00 |
PAD indicates pulsed amperometric detection; Fuc, fucose; GlcN, glucosamine; Gal, galactose; Man, mannose; HuIgG1, polyclonal human IgG1; GG, glycosylated chimeric anti-CD137 mAb; GA, aglycosylated chimeric anti-CD137 mAb; CTM, cetuximab; and na, not applicable.
Fc fragments were hydrolyzed in 2 M trifluoroacetic acid (TFA) at 100°C for 4 hours. The hydrolysates were lyophilized and subject to Dionex HPAEC-PAD analysis.
For quantification, the peaks corresponding to the different monosaccharides were integrated and peak areas were normalized to molar quantity by comparison with biantennary GP. The molar ratios were normalized by setting mannose as 3.
Biantennary GP represents a standard glycopeptide isolated from hen's egg yolk that contains a full-size biantennary complex–type N-glycan with the following molar ratios of monosaccharides: Man/Gal/GlcN = 3:2:4.