Hypomyelination in the central nerves system (CNS) is one of the most obviously pathological features in Niemann-Pick Type C disease (NPC), which is a rare neurodegenerative disorder caused by mutations in the NPC intracellular cholesterol transporter 1 or 2 (Npc1 or Npc2). Npc1 plays key roles in both neurons and oligodendrocytes during myelination, however, the linkage between the disturbed cholesterol transport and inhibited myelination is unrevealed. In this study, mass spectrometry (MS)-based differential quantitative proteomics was applied to compare protein composition in the corpus callosum between wild type (WT) and NPC mice. In total, 3009 proteins from both samples were identified, including myelin structural proteins, neuronal proteins, and astrocyte-specific proteins. In line to hypomyelination, our data revealed downregulation of myelin structural and indispensable proteins in Npc1 mutant mice. Notably, the reduced ceramide synthase 2 (Cers2), UDP glycosyltransferase 8 (Ugt8), and glycolipid transfer protein (Gltp) indicate the altered sphingolipid metabolism in the disease and the involvement of Gltp in myelination. The identification of most reported myelin structural proteins and proteins from other cell types advocates the use of the corpus callosum to investigate proteins in different cell types that regulate myelination.

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel^® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. “Bottom-up” mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.

Background: The demand for reliable comparability studies of biosimilars grows with the increase of their market. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. Methods: In the current study, Enbrel® (the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. “Bottom-up” mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Results: Three enzyme peptide mapping resulted in an identification of the primary sequence completely. It was stated that total ion chromatograms should be considered as a highly valuable data for analyzing the biodrug primary sequence and variations. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, functional properties of N-deglycosylated and non-modified etanercept samples were investigated using surface plasmon resonance analysis and in-vitro bioassay. Conclusion: It was shown that N-glycosylation has no significant effect on in-vitro functionality of etanercept. Moreover, comparison of etanercept samples stated the high similarity between the originator and the proposed biosimilar.