Pairing of oligosaccharides in the Fc region of immunoglobulin G

Abstract

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter-chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.z 2000 Federation of European Biochemical Societies.

Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

“…The glycosylation profile featuring different number of hexoses was not resolved in this case. The measured mass agreed well with the calculated MW ϭ 147,353 Da for glycosylation structure without°any°terminal°hexose°residues° [13].°These°results have been confirmed by size exclusion chromatography and MALDI-TOF analyses in our laboratory (data are not shown). The control experiments confirmed that the degradations products of the IgG2 antibody were indeed caused by the storage conditions and not by the low pH, elevated temperature environment on the POROS column.…”

“…Da correlated with a loss of a hexose residue (Hex), which is a typical heterogeneity of glycosylation observed in IgG antibodies produced in mammalian cells [13].°When°the°same°ACN/ethanol°containing°mobile phase was used for IgG2 antibody, chromatographic resolution efficiency was significantly reduced (data are not shown). To improve eluotropic properties of the mobile phase and obtain better separation of the degradation products of IgG2 antibody, iso-propanol was added to Solvent B.…”

“…However, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) [7,8], ESI orthogonal-TOF [9,10] and ESI quadrupole [2,[11][12][13] mass spectrometers have been the instruments of choice for MAbs and macromolecules, largely due to the large m/z range of the TOF and high ion transmission efficiency of the quadrupole mass analyzers. Unfortunately, on-line RP-HPLC has not accompanied these previous studies of intact monoclonal antibodies.…”

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

“…The most common glycan structures for IgG possess zero, one, or two terminal galactose (G) residues with or without a fucose (F) and are defined as G0, G0F, G1F, and G2F [6,7].…”

“…More recently, several methods using mass spectrometry (MS) have been developed. These analyses are performed using HPLC coupled with either electrospray ionization single quadruple mass spectrometry (ESI-MS) [20,21], or ESI quadruple time-of-flight MS (ESI-Q-TOF-MS) [8,[22][23][24], or ESI orthogonal acceleration time-of-flight MS (ESI-oa-TOF-MS) [6,25,26]. Matrix assisted laser desorption/ionization-TOF-MS (MALDI-TOF-MS) is also a widely applied technique [14,22,[27][28][29].…”