Table 1.
Mass spectrometry analysis of reduced monomeric forms of the purified IgG1-Fc proteins. The molecular weight column shows the masses of the reduced non-glycosylated IgG1-Fc obtained from MS analysis along with the masses measured for the most abundant glycoforms (Man8GlcNAc2) of the glycosylated Fc. The values for experimentally observed change in mass, and predicted change in mass are also shown. Based on these results, the modification expected due to enzymatic treatment and/or glycan variation of the IgG1-Fc proteins is then presented.
| IgG1-Fc proteins | Observed Molecular Weight (Da) | Observed Δ Mass (Da) | Reduced Fc fragment Modificationsa | ||
|---|---|---|---|---|---|
| Arm 1 | Arm 2 | ||||
| Non-glycosylated IgG1-Fc | Mutant (QQ) | 25,077 | +11 | N → Q (+14) | N → Q (+14) |
| Mono-glycosylated + PNGase F (DN) | 25,063b | −3 | Deglycosylated N → D (+1) | N (0) | |
| Di-glycosylated + PNGase F (DD) | 25,063b | −3 | Deglycosylated N → D (+1) | Deglycosylated N → D (+1) | |
| Glycosylated IgG1-Fc | Mono-glycosylated c | 26,766d / 25,062b | +1,700/−4 | +Man8GlcNAc2 (+1,704) | N (0) |
| Di-glycosylated c | 26,766d | +1,700 | +Man8GlcNAc2 (+1,704) | +Man8GlcNAc2 (+1,704) | |
D, aspartic acid; N, asparagine; Q, glutamine; GlcNAc, N-acetylglucosamine; Man, Mannose; Numbers in ( ) indicates predicted Δ mass change in Da.
The theoretical mass of the reduced, non-glycosylated IgG1-Fc protein is 25,066 Da.
Man8-12GlcNAc2 glycoforms were observed during MS analysis for the diglycosylated IgG1-Fc. Man8-9GlcNAc2 glycoforms were observed for the mono-glycosylated IgG1-Fc.
theoretical mass of reduced monomelic form of the purified IgG1-Fc protein with Man8GlcNAc2 glycan is 26,770 Da.