Background The active substance of MabThera is rituximab. It is a genetically engineered chimeric mouse/human monoclonal antibody which binds specifically to the transmembrane antigen CD20. It is currently indicated for the treatment of several follicular lymphomas. MabThera is available in single-use vials containing 100 mg/10 ml and 500 mg/50 ml concentrate for solution for infusion.

Purpose The purpose of this study was to investigate the suitability of a proteomic approach using MALDI-TOF mass spectrometry to test the molecular integrity of rituximab in its pharmaceutical presentation form and diluted in typical hospital conditions.

Materials and methods Rituximab (100.0 mg/10 ml) was diluted with SSF (to 4.0 mg/ml and 1.0 mg/ml). 10 μL of three types of samples containing the antibody were reduced with DTT and alkylated by iodoacetamide in darkness for 30 min then digested by trypsin at pH 8.5 for 4 h at 37°C. The digest was loaded onto the MALDI target plate using 5 mg/ml α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile as the matrix. Each digest was analysed five times by MALDI-TOF mass spectrometry using a Voyager DE-PRO (Applied Biosystems) in positive reflector mode.

Results The peptide fingerprint map (PFM) of rituximab was obtained for the three types of samples (100.0 mg/10 ml and the dilutions) immediately after their preparation. In this way, the molecular integrity of rituximab could be described and characterised.

Conclusions This proteomic approach to the analysis of rituximab is suitable for use in a long stability study of the antibody diluted with SSF and stored refrigerated (4°C) and frozen (-20 °C) since possible changes in the antibody structure may be detected by changes in the corresponding PFM. This stability study is currently being performed by our research group.