Profiling of oligosaccharide-mediated microheterogeneity of a monoclonal antibody by capillary electrophoresis.
Based on complex formation of borate with carbohydrates in alkaline solutions, the oligosaccharide microheterogeneity of a monoclonal antibody was studied using capillary zone electrophoresis. In borate buffers characteristic separation patterns were found that could be attributed to the same antibody by their UV spectra, while in a phosphate buffer, under otherwise the same conditions, only a single peak was observed. N- and O-glycans were chemically hydrolyzed by trifluoromethane sulfonic acid, resulting in a completely deglycosylated protein; alternatively, N-glycans were enzymatically cleaved by incubation with peptide N-glycosidase F (PNGase F). In both approaches a changed antibody pattern was detected, indicating that the separation is due to carbohydrate microheterogeneity of the protein. Deglycosylation of the antibody by treatment with PNGase F was investigated by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A shift to lower molecular masses of approximately 1500 Da for the enzymatically treated protein, compared with the intact glycoprotein, was found. The separation method was validated for linearity and reproducibility of migration time and peak area and optimized in terms of buffer pH, capillary temperature and borate concentration. This technique is sensitive to analyze batch-to-batch consistency in production and to test the stability of galenical formulations. After antibody storage in glass vials for 3 months at 37 degrees C, the separation profile changed distinctly due to degradation at the carbohydrate or sialic acid moiety of the antibody, as indicated by MALDI-TOF-MS.
Institut für Angewandte Forschung, FH Reutlingen, Germany.
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Hoffstetter-Kuhn
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Alt
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Kuhn
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002241:Carbohydrates; D019075:Electrophoresis, Capillary / Q000379:methods*; D009844:Oligosaccharides / Q000032:analysis*
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D009844:Oligosaccharides