Characterization of the F(ab′) 2 fragment of a murine monoclonal antibody using capillary isoelectric focusing and electrospray ionization mass spectrometry

We characterized a F(ab′) 2 fragment obtained by pepsin cleavage from a murine monoclonal IgG3 by means of electrospray ionization (ESI) mass spectrometry (MS), capillary isoelectric focusing (cIEF), high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC–PAD) and LC–MS peptide mapping. Separation of the fragment by cIEF under nonreducing conditions resulted in a number of distinct peaks. Using reducing conditions the heavy chain and the light chain were separated into two peaks each. Analysis by ESI-MS revealed a high mass heterogeneity of the molecule. Digestion with neuraminidase simplified both the cIEF pattern and the mass spectrum. The cIEF of the reduced molecule showed that the sialic acids were located only on the heavy chain of the F(ab′) 2-fragment. By incubation with O-glycosidase a further reduction of the complexity of the mass spectrum was achieved showing 8 different isoforms. By LC–MS peptide mapping these isoforms could be attributed to the heterogeneity of the pepsin cleavage site in the hinge region of the antibody. The sugars of the O-linked carbohydrate chain were identified by HPAEC–PAD as galactosyl- N-acetyl-galactosamine (GalNAcGal) with terminal N-glycolylneuraminic acid. The glycosylation site was identified by peptide mapping and amino acid sequence analysis as Ser 222.