Development of a methodology for molecular weight determination of Biosimilars

Introduction: Antineoplastic monoclonal antibodies (mAbs), such as pembrolizumab and nivolumab, have shown efficacy in the treatment of various types of cancer.However, in the context of the Brazilian public health system, the domestic production of biological medicines becomes even more crucial to ensure access to innovative therapies for the entire population.Demonstrating that the product has comparable characteristics to the reference biological product is a fundamental requirement for regulatory approval.One of the main approaches to achieve this comparison is the determination of the molecular weight of the different components of the drug.This can be achieved through high-performance liquid chromatography (HPLC) coupled with a multi-angle light scattering (MALS) detector, a Refractive Index (RI), and a Photo Diode Array (PDA).This technology allows the separation and analysis of the monomers, dimers and aggregates present in the sample.From this analysis, it is possible to determine the molecular weight of each group of molecules, as well as to calculate the percentage of each component type.This approach provides essential data for evaluating the comparability between the new biological product and the reference product.Objectives: Development of a methodology for molecular weight determination of Biosimilars produced by Bio-Manguinhos.Methodology: Different batches of the commercially available drugs Keytruda (pembrolizumab) and Opdivo (nivolumab), as well as their biosimilars, were tested using HPLC equipment coupled with MALS, RI, and PDA detectors.The system suitability preparation included an injection of Bovine Serum Albumin (BSA).The analysis was performed with a mobile phase of 0.1 M phosphate buffer, pH 7.0, containing 0.01% (w/v) azide, and a TSK Gel G3000 SWxL column.The samples were diluted to a target concentration of 2.0 mg/mL.Data were processed using Empower 3 software and Astra 8.1.2. Results:The biosimilar samples showed a molecular size similar to the reference samples.Dimers and aggregates were identified only in the biosimilar samples of Pembrolizumab using the PDA detector, but it was not possible to identify them using MALS.Dimers were identified in the reference samples of Nivolumab using the PDA detector, but it was also possible to identify dimers and aggregates in the biosimilar samples using both detectors and quantify them using MALS. Conclusion:The proposed method allows comparison between biosimilars with reference biologics and can be used in the development and quality control of biosimilar mAbs produced by Bio-Manguinhos.