The increased number of approved therapeutic glycoproteins prompted rapid development of comprehensive and reproducible multilevel characterization methods for the biopharmaceutical industry and regulatory agencies. One of the largest groups of biotherapeutics has been monoclonal antibodies (mAbs) and recently new modalities such as multispecific Abs and fusion protein, possessing various post-translational modifications (PTMs) and potential degradation hotspots, which all should be analyzed during clone selection, manufacturing and lot release as potentially affecting efficacy and immunogenicity. The exceptional separation power of capillary electrophoresis (CE) in conjunction with laser induced fluorescence or high resolution mass spectrometry detection fulfills the multilevel characterization requirements of: Level 1) determination of accurate molecular mass and some degree of heterogeneity at the intact glycoprotein level; Level 2) measurement of exact molecular mass of the subunits (e.g.,

heavy and light chains of mAbs) as well as the degree of heterogeneity after reduction of the disulfide bonds with or without alkylation; Level 3) characterization of degradative hotspots such as aspargine-deamidation, methionine-oxidation, glutamic-acid-cyclization, C-terminal lysine heterogeneity and other posttranslational modifications at the peptide/glycopeptide level after proteolytic digestion of the reduced and alkylated glycoproteins; Level 4) characterization of released glycans. A comprehensive multilevel characterization example will be given for a representative therapeutic monoclonal antibody, trastuzumab, illustrating the benefits of the integration of capillary electrophoresis (CE) with laser induced fluorescence detection (CE-LIF) and with electrospray ionization (ESI) in a unified bioanalytical process (CESI) coupled with high resolution mass spectrometry. The low flow rate of the latter system (>20 nL/min) ensured maximized ionization efficiency and dramatically reduced ion suppression.