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.