Development and validation of a mass spectrometric method to determine the identity of rituximab based on its microheterogeneity profile.

Analytical methods have been considered the "eyes" for development, characterization and batch releasing of biotherapeutics over the past 40 years. One of the most powerful analytical platform for biotherapeutic analysis is mass spectrometry coupled to liquid chromatography (LC-MS). Due to its wide flexibility and instrumental configurations, LC-MS can determine different physicochemical attributes of proteins, e.g. molecular mass, primary sequence, and posttranslational modifications. Intact molecular mass analysis of therapeutic proteins is essential to confirm their identity. Analytical methods must be validated to support drug quality information during its approval process. Although there are international guidelines that provide general information on validation of analytical methods, practical examples about the design, selection of validation attributes and acceptance criteria of identity LC-MS methods are scarce. Here, according to the recommendations of Q2R1 ICH guideline, we showcase the validation of an LC-MS-TOF method to identity rituximab by determining its intact and deglycosylated molecular mass profiles. The proposed method specifically identified the m/z profile and deconvoluted mass profile of rituximab from deglycosylated rituximab and from excipient blank (specificity) with a maximum error of 76.63 ppm (accuracy) and a maximum Relative Standard Deviation (RSD) of 0.00315% (precision). Besides, the system suitability test, which was based on the expected mass value of the mass calibrator, confirmed the reliability of the analytical results. In summary, validation showed that the proposed method is suitable for identifying rituximab based on its glycosylated (intact) and deglycosylated mass profile.