Details

Autor(en) / Beteiligte

• Reinert, Tessa
• Houzé, Pascal
• Francois, Yannis-Nicolas
• Gahoual, Rabah

Titel

Enhancing affinity purification of monoclonal antibodies from human serum for subsequent CZE-MS analysis

Ist Teil von

• Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2024-02, Vol.1234, p.123974-123974, Article 123974

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2024

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• •CE-MS/MS was used to characterize mAbs post-translational modification in serum.•The sample preparation was optimized to be appropriate with CE separation.•Final approach allows quantification simultaneously to characterization of mAb. Due to the separation technique employed, capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis performances are significantly influenced by the chemical composition and the complexity of the sample. In various applications, 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 purification, 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.