Objective: The aim of the study was to demonstrate the analytical similarity of the bevacizumab biosimilar MB02 (Alymsys®) to its reference product (RP) Avastin® sourced from both the United States and the European Union. 
Method: The primary and higher order structure, post-translational modifications, purity and biological activity of up to 15 MB02, 19 US-Avastin® and 17 EU-Avastin® lots were evaluated using independent, orthogonal and state-of-the-art physicochemical and biological assays, according to international regulatory guidelines recommendations. 

Results: The analysis of the primary structure by LC-MS techniques at intact and peptide level revealed similar intact and reduced de-N-glycosylated mass profiles and confirmed same amino acid sequence of MB02 and Avastin® by peptide mapping. 
MB02 showed similar protein conformation and conformational stability to Avastin®, analyzed by non-reduced peptide mapping, circular dichroism, fluorescence and µDSC. 
Charge variants and glycosylation profile were studied as part of post-translational modifications assessment. Similar levels of total charge variants were reported by CEx HPLC and cIEF and similar pI was found for MB02 and Avastin®. Some differences were observed regarding glycoprofile (afucosylation, galactosylation and high mannose) however they are not clinically meaningful in regard to bevacizumab mechanism of action.
The purity of MB02 and Avastin® products was evaluated by SE HPLC and reduced/non-reduced CE-SDS, showing overall comparable purity profiles.
Specific focus was put on the characterization of Fab-related activities which reflect bevacizumab mechanism of action. Similar binding of MB02 and Avastin® to VEGF A was demonstrated, including VEGF165, the main VEGF variant (ELISA and SPR) and other splice variants VEGF 121, 189 and 206 (ELISA). Similar potency was also demonstrated using three different bioassays: an antiproliferation assay using HUVECs, a gene reporter neutralization assay and a VEGF receptor dimerization assay. 
Regarding Fc related activities, similar binding to FcRn, indicative of similar PK was shown. 
Comparative stability studies under forced degradation conditions (e.g. thermal degradation) showed similar behavior and degradation pathways for MB02 and Avastin®. Likewise, MB02 and Avastin® showed comparable process related impurities in terms of residual protein A, residual insulin HCPs and host cell DNA with values below the limits of methods quantification.  

Conclusion: Based on the exhaustive in vitro analytical similarity assessment carried out, MB02 was demonstrated to be highly similar to US and EU Avastin®. Although some minor differences have been found mainly regarding N-glycan profile, this is not relevant to bevacizumab mechanism of action and therefore not clinically meaningful. All methods used to assess biological activity supported similarity between MB02 and Avastin®. In addition to analytical similarity assessment, clinical data have been generated to complete the biosimilarity assessment and demonstrate the totality-of-the-evidence, confirming the similarity of MB02 to Avastin®. MB02 (Alymsys®) recently obtained a positive opinion from the CHMP as a biosimilar product.

Analytical Similarity Assessment of Bevacizumab Biosimilar MB02 Using Multiple State-of-the-art Assays

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