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.

中文翻译：

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质谱法的开发和验证，该方法用于基于利妥昔单抗的微异质性分析确定其身份。

在过去的40年中，分析方法被认为是生物治疗剂开发，表征和批量释放的“眼睛”。用于生物治疗分析的最强大的分析平台之一是质谱联用了液相色谱（LC-MS）。由于其广泛的灵活性和仪器配置，LC-MS可以确定蛋白质的不同物理化学属性，例如分子量，一级序列和翻译后修饰。治疗性蛋白质的完整分子质量分析对于确认其身份至关重要。在批准过程中，必须验证分析方法以支持药物质量信息。尽管有国际准则提供了有关分析方法验证的一般信息，但有关设计的实际示例，鉴定LC-MS方法的验证属性和接受标准的选择很少。在这里，根据Q2R1 ICH指南的建议，我们展示了通过确定利妥昔单抗的完整和去糖基化的分子质量图谱来鉴定利妥昔单抗的LC-MS-TOF方法的验证。拟议的方法从去糖基化的利妥昔单抗和赋形剂空白（特异性）中特异性鉴定了利妥昔单抗的m / z分布图和去卷积质量分布图（特异性），最大误差为76.63 ppm（准确度），最大相对标准偏差（RSD）为0.00315％（精确度） ）。此外，基于质量校准器的预期质量值的系统适用性测试证实了分析结果的可靠性。总之，