Rapid level-3 characterization of therapeutic antibodies by capillary electrophoresis electrospray ionization mass spectrometry.

With the increase of the number of approved protein therapeutics in the market, comprehensive and reproducible characterization of these new generation drugs is crucial for the biopharmaceutical industry and regulatory agencies. One of the largest groups of biotherapeutics is monoclonal antibodies (mABs) possessing various posttranslational modifications and potential degradation hotspots during the manufacturing process that may affect efficacy and immunogenicity. The exceptionally high separation power of capillary electrophoresis (CE) in conjunction with mass spectrometry fulfills Level-3 characterization requirements necessary to reveal such modifications and degradations. In this paper, a comprehensive characterization example will be given for a representative mAB Trastuzumab (Herceptin), illustrating the benefits of the integration of CE and electrospray ionization in a unified bioanalytical process coupled with high-resolution mass spectrometry. Peptides separated in a wide size range (3-65 amino acids) were identified with 100% sequence coverage and quantified, including degradative hotspots such as glutamic acid cyclization, methionine oxidation, aspargine deamidation and C-terminal lysine heterogeneity using only 100 fmol of a single protease digest sample. The low flow rate of the system (>20 nL/min) ensured maximized ionization efficiency and dramatically reduced ion suppression.