CHARACTERIZATION OF HUMANIZED ANTI-TAC, AN ANTIBODY-DIRECTED AGAINST THE INTERLEUKIN-2 RECEPTOR, USING ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY BY DIRECT INFUSION, LC MS, AND MS/MS/

Characterization of a humanized monoclonal antibody (Hu-anti-TAC) dire cted against a surface protein expressed On T-lymphocytes was performe d with an electrospray mais spectrometer. Capillary reversed-phase liq uid chromatography (LC)/mass spectrometry (MS) and direct infusion MS were utilized along with tandem MS/MS analysis tb confirm the sequence and to determine the sources of heterogeneity in Hu-anti-TAC. The MS analysis was performed on disulfide-reduced and trypsin-digested;sampl es of the antibody. Two forms of diantennary carbohydrate structures w ere identified and found to be consistent with those reported for the human IgG1 framework. The analysis demonstrated that the N-terminus wa s modified by conversion of a glutamine residue to pyroglutamic acid. Another source of heterogeneity was the partial removal of the C-termi nal lysine residue and was confirmed by mass calculations of tryptic p eptides followed by MS/MS sequencing. This study demonstrates that the high sensitivity of electrospray mass spectrometry when combined with capillary chromatography can allow detailed characterization of micro gram samples of high molecular weight proteins such as antibodies.