Etanercept is a soluble fusion protein of the tumor necrosis factor receptor 
(TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- 
and 13 O-glycosylation sites. Due to its complex structure, an analytical 
challenge is facing the development and approval of biosimilars. In the current 
study, physicochemical characterization using state-of-the-art analytics was 
performed to analyze intact and subunit masses, post-translational modifications 
(PTMs), higher order structure and potency of Etanercept originator Enbrel® and 
its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality 
attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a 
size of about 126 kDa for both biologicals. Similar glycoprotein species for the 
complete monomer and the Fc domain of originator and follow-on product were 
observed, however, small differences in lysine variants and oxidation were 
found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation 
sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and 
TNFR-specific highly sialylated glycans on N149 and N171 on both investigated 
products. Small quantitative variations in the N-glycan profile were detected, 
although the N-glycans were qualitatively similar. Four different 
O-glycopeptides bearing core 1-type glycans were detected. For both, N- and 
O-glycopeptide analysis, determination was achieved without prior cleavage of 
the sialic acid residues for the first time. In addition, ion mobility 
spectrometry data confirmed close similarity of higher-order structure of both 
biologics. Furthermore, a neutralization assay, investigating the impact of 
altered PTMs on potency, indicated that the differences within all batches are 
still in the acceptable range for biosimilarity.