IgG glycosylation analysis.
A multitude of monoclonal IgG antibodies directed against a variety of therapeutic targets is currently being developed and produced by biotechnological companies. The biological activity of IgGs is modulated by the N-glycans attached to the fragment crystallizable (Fc) part. For example, lack of core-fucoses on these N-glycans may lead to a drastic enhancement of antibody-mediated cellular cytotoxicity. Moreover, sialylation of Fc N-glycans determines the immunosuppressive properties of polyclonal IgG from human blood, which stimulates research into Fc glycosylation of human plasma IgG in various disease settings. This review presents and evaluates the different approaches which are used for IgG glycosylation analysis: N-glycans may be enzymatically or chemically released from purified IgG, prior to chromatographic or mass spectrometric analysis. Moreover, IgGs may be treated with endoproteinases such as trypsin, followed by glycosylation analysis at the glycopeptide level, which is generally accomplished by HPLC with ESI-MS. Alternatively, intact IgGs or fragments thereof obtained by enzymatic cleavages in the hinge region and by reduction may be analyzed by a large number of analytical techniques, including MS and chromatography or CE.
Huhn
Carolin
C
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.
Selman
Maurice H J
MH
Ruhaak
L Renee
LR
Deelder
André M
AM
Wuhrer
Manfred
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; D010450:Endopeptidases