Ocrelizumab is a second generation recombinant humanized IgG1 monoclonal antibody used for the treatment of multiple sclerosis that selectively target B cells expressing the CD20 antigen. This study aimed to develop and validate a UPLC-MS/MS method for quantification of ocrelizumab in human serum, which can be used in clinical applications for therapeutic drug monitoring. The analysis of ocrelizumab was performed using a bottom-up approach on a liquid chromatography coupled to tandem mass spectrometry detection. The method involved immunoglobulin precipitation with cold methanol followed by peptide digestion with trypsin. The resulting specific peptides were separated on an Acquity UPLC BEH C18 column at 55 °C using gradient elution. The method was validated according to European Medicines Agency (EMEA) guidelines and demonstrated intra- and inter-assay precision with coefficients of variation ranging from 1.6 % to 6.1 % and accuracies between 90.2 % and 107.2 %. Stability testing, including autosampler, long-term and freeze-thaw stability, showed no more than 15 % variation. The method was successfully applied to 169 patient samples, revealing ocrelizumab concentrations ranging from 0.5 to 21.8 mg/L in patients on 6-month dosing regimen and 20.5-65.0 mg/L in 16 patients receiving an initial two-week dose of 300 mg. The newly developed UPLC-MS/MS method met all criteria for accuracy, precision and stability, confirming its suitability for clinical use in monitoring ocrelizumab levels in multiple sclerosis patients.

Department of Clinical Pharmacology Faculty of Medicine University of Ostrava Syllabova 19 703 00 Ostrava Czech Republic; Department of Clinical Pharmacology Institute of Laboratory Medicine University Hospital Ostrava 17 listopadu 1790 5 708 52 Ostrava Czech Republic

Department of Neurology University Hospital Ostrava 17 listopadu 1790 5 Ostrava 70852 Czech Republic; Department of Clinical Neurosciences Faculty of Medicine University of Ostrava Syllabova 19 Ostrava 703 00 Czech Republic

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