title: Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab.
abstract: The principal objective of this study was the evaluation of two-dimensional gel electrophoresis (2-DE) in combination with MALDI-TOF MS, after tryptic digest with regard to suitability for qualitative characterization and identification of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. Moreover, the impact of post-translational modifications of these glycoproteins on the electrophoresis behavior has been evaluated. 1-D SDS-PAGE, in reducing and non-reducing conditions, and 2-DE were used for the assessment of M(r) and the monitorization of deglycosylation efficiency. In addition, 2-DE was used for the determination of pIs. 2-DE gels revealed characteristic glycoprotein migration behavior, highly complex spot pattern, typical for recombinant monoclonal antibodies. N-linked oligosaccharides were released with PNGase F; enzymatic desialination was studied with sialidase and carboxypeptidase B was used for the study of lysine truncation. Peptide spots resolved in 2-DE gels were in gel tryptically digested, resulting peptides were subjected to MALDI-TOF MS analysis and peptide mass fingerprinting (PMF) has been used for the identity confirmation of both monoclonal antibodies.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Life Sciences, University of Vienna, Vienna, Austria.
authors.forename: Dashnor
authors.initials: D
authors.lastname: Nebija
authors.forename: Hannelore
authors.initials: H
authors.lastname: Kopelent-Frank
authors.forename: Ernst
authors.initials: E
authors.lastname: Urban
authors.forename: Christian R
authors.initials: CR
authors.lastname: Noe
authors.forename: Bodo
authors.initials: B
authors.lastname: Lachmann
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D058846:Antibodies, Monoclonal, Murine-Derived / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D000069283:Rituximab; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; D006023:Glycoproteins; D009844:Oligosaccharides; D010455:Peptides; D011994:Recombinant Proteins; D000069283:Rituximab; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000068878:Trastuzumab