The demand for reliable comparability studies of biosimilars grows with their increased market\nshare. These studies focus on physicochemical, structural, functional and clinical properties to ensure that\na biosimilar has no significant differences to the originator product and can be released into the market\nwithout extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrelâ?¢\n(the proposed biosimilar) underwent direct comparison. â??Bottom-upâ? mass spectrometric analysis\nwas used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of\nmethionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of\nN-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted\nin complete identification of the primary structure. It was confirmed that total ion chromatograms are\nvaluable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures\nwere identified and all the N-glycans were quantified. Finally, investigation of the functional properties\nof N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis\nand in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality.\nAnalysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary\nstructure and in-vitro functionality.
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