Quantification of Monoclonal Antibodies in Pharmaceutical Matrices by Middle-Up Capillary Zone Electrophoresis-Mass Spectrometry.

Monoclonal antibodies are biopharmaceuticals used to treat various diseases, including autoimmune disorders and cancer. Due to their structural complexity and inherent heterogeneity, robust analytical methods are required for their accurate quantification and quality control. This study presents a novel middle-up (MU) capillary zone electrophoresis-mass spectrometry (CZE-MS) method for the quantitative analysis of infliximab (IFX), a widely used therapeutic mAb. The method relies on disulfide bond reduction to separate heavy and light chains, followed by CZE separation using a 2 M acetic acid background electrolyte and MS detection in single ion monitoring mode. Adalimumab was used as an internal standard, providing a suitable alternative to isotopically labeled monoclonal antibody analogues. Evaluation of the analytical method performance followed ICH Q2(R1) guidelines and demonstrated an acceptable linearity range (4-35 µg/mL), precision, accuracy, and a limit of quantification of 4 µg/mL. The method was successfully applied to quantify IFX in a commercial pharmaceutical (Remicade). Moreover, the method was preliminarily tested for the quality control of bevacizumab in Avastin and rituximab in MabThera pharmaceutical formulations. To our knowledge, this is the first report of quantitative MU CZE-MS analysis of therapeutic monoclonal antibodies directly from pharmaceutical matrices, highlighting its potential for quality drug control applications.