Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis
Amélioration de la purification par affinité d'anticorps monoclonaux à partir de sérum humain pour l'analyse CZE-MS
Abstract
Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis per-formances are significantly influenced by the chemical composition and the complexity of the sample. In various applica-tions, that impact has prevented the use of CE-MS for the characterization and quantification of proteins in biological samples. Here we present the development and evaluation and a sample preparation procedure, based on affinity purifica-tion, for the specific extraction of the monoclonal antibody (mAbs) infliximab from human serum in order to perform subsequent proteolytic digestion and CE-MS/MS analysis. Three distinctive sample preparation strategies were envisaged. In each case, the different steps composing the protocol were thoroughly optimized and evaluated in order to provide a sample preparation addressing the important complexity of serums samples while providing an optimal compatibility with CE-MS/ MS analysis. The different sample preparation strategies were assessed concerning the possibility to achieve an appropriate absolute quantification of the mAbs using CE-MS/MS for samples mimicking patient serum samples. Also, the possibility to perform the characterization of several types of post-translational modifications (PTMs) was evaluated. The sample preparation protocols allowed the quantification of the mAbs in serums samples for concentration as low as 0.2 μg⋅mL$^{-1}$ (2.03 nM) using CE-MS/MS analysis, also the possibility to characterize and estimate the modification level of PTMs hotspots in a consistent manner. Results allowed to attribute the effect on the electrophoretic separation of the different steps composing sample preparation. Finally, they demonstrated that sample preparation for CE-MS/MS analysis could benefit greatly for the extended applicability of this type of analysis for complex biological matrices.
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