Unambiguous characterization of N-glycans of monoclonal antibody cetuximab by integration of LC-MS/MS and ¹H NMR spectroscopy.
Monoclonal antibodies are most rapidly emerging as therapeutic drugs for the treatment of cancer and of various other diseases such as autoimmunity or inflammation. Recently, it was found that nonhuman glycosylation of recombinant antibodies can cause tremendous problems for some patients. Therefore, unambiguous assignment of the glycosylation pattern of therapeutic antibodies is of high importance for assessment of human compatibility. Here we present results from a broad and detailed N-glycan analysis of the therapeutic antibody cetuximab by LC-MS/MS analyses tightly integrated with (1)H NMR to obtain unambiguous structures. Thirty-seven N-glycan compositions were identified by LC-MS(/MS). Subsequently, ten abundant structures were structurally characterized by applying the recently introduced method called three-dimensional cross correlation (3DCC). It was possible to extract NMR spectra of pure N-glycans that were heavily overlapping in a chromatographic separation by mathematically dissecting the NMR spectra obtained from chromatographic fractions. Even mass isobaric structures that differ only in the branching position of one monosaccharide unit were distinguished and characterized. We also developed an improvement of the 3DCC method by introducing singular value decomposition (SVD) for processing of the data. The smallest amount of the N-glycan characterized by 3DCC was approximately 400 pmol (836 ng). Among the ten unambiguously identified glycans, six N-glycans, representing 24% of all detected glycans, possess the immunogenic α-1,3-Gal epitope and/or N-glycolylneuraminic acid. These results illustrate the importance of integrated use of LC-MS(/MS) and (1)H NMR for the glycome analysis of biopharmaceuticals in research, development, and quality control.
Organic Chemistry, Department of Chemistry, University of Hamburg , Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Alena
A
Wiegandt
Bernd
B
Meyer
D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D000970:Antineoplastic Agents / Q000737:chemistry*; D000068818:Cetuximab; D002851:Chromatography, High Pressure Liquid; D009682:Magnetic Resonance Spectroscopy; D011134:Polysaccharides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D011134:Polysaccharides; D000068818:Cetuximab