Structural identification and absolute quantification of monoclonal antibodies in suspected counterfeits using capillary electrophoresis and liquid chromatography-tandem mass spectrometry.

ABSTRACT

Monoclonal antibodies (mAbs) represent a major category of biopharmaceutical products which due to their success as therapeutics have recently experienced the emergence of mAbs originating from different types of trafficking. We report the development of an analytical strategy which enables the structural identification of mAbs in addition to comprehensive characterization and quantification in samples in potentially counterfeit samples. The strategy is based on the concomitant use of capillary zone electrophoresis analysis (CZE-UV), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). This analytical strategy was applied to the investigation of different samples having unknown origins seized by the authorities, and potentially incorporating an IgG 4 or an IgG 1. The results achieved from the different techniques demonstrated to provide orthogonal and complementary information regarding the nature and the structure of the different mAbs. Therefore, they allowed to conclude unequivocally on the identification of the mAbs in the potentially counterfeit samples. Finally, a LC-MS/MS quantification method was developed which specificity was to incorporate a different mAbs labeled with stable isotopes as internal standard. The LC-MS/MS quantification method was validated and thus demonstrated the possibility to use common peptides with the considered IgG in order to achieve limit of quantification as low as 41.4 nM. The quantification method was used to estimate the concentration in the investigated samples using a single type of internal standard and experimental conditions, even in the case of mAbs with no stable isotope labeled homologues available.