Trap for MAbs: Characterization of intact monoclonal antibodies using reversed-phase HPLC on-line with ion-trap mass spectrometry

For the first time, the characterization of intact 150-kDa monoclonal antibodies (MAbs) using a commercially available three-dimensional ion-trap mass spectrometer (IT-MS) is reported. The IT-MS analysis was performed on-line with reversed-phase high performance liquid chromatography (RP-HPLC) on a POROS column using a nontraditional solvent system of acetonitrile, isopropanol, ethanol, and water in formic acid. The operating parameters of the IT-MS were optimized by extending the mass range to m/z 4000 and elevating the tube lens offset voltage value to around −100 V. Mass accuracy better than 300 ppm (±40 Da) has been routinely achieved for these macromolecules. Multiple peaks 162 Da apart due to the hexose variants of the monoclonal IgG antibodies were partially resolved in mass spectra. Several commercial and chimeric antibodies have been investigated in this study.