Rapid Level-3 Characterization of Therapeutic Antibodies by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Abstract

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.

Investigating capillary electrophoresis‐mass spectrometry for the analysis of common post‐translational modifications

“…Due to significant differences in the pKa values of aspartate and isoaspartate and of glutamate and isoglutamate, respectively, these isomerized forms could be assigned based on a shift in migration time. In a similar manner peptide and protein deamidation has been investigated in the course of peptide drug analysis , the analysis of a protein standard mixture , and in the course of monoclonal antibody studies . Even site‐specific effects can influence the overall charge of peptides thus causing a CE separation of phosphopeptides positional isomers .…”

Recent advances in CE‐MS coupling: Instrumentation, methodology, and applications

“…Besides a conductive liquid, the electrical connection can be provided also by a conductive component eliminating the sample dilution. This instrumentally various group of sheathless interfacing is currently dominated by the design proposed by Moini in 2007, commercially available under the trademark CESI 8000 (AB SCIEX, USA) routinely used for complex studies . This construction is based on 30 μm id emitter constructed from porous glass membrane surrounded by a conductive liquid, providing electrical connection without any noticeable sample dilution.…”

“…The construction was used by Lew et al. for detailed characterization of trastuzumab with 100% primary sequence coverage and identification of modified amino acids . Within the same separation, structural identification of glycopeptides was done identifying 14 various glycoforms.…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…155 A characterization of therapeutic antibodies by CE-ESI-MS allows for rapid sequencing and degradation hotspot analysis, and this work achieved enhanced sensitivity for poorly ionizing molecular species like glycoproteins. 156 …”