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. 2018 Apr;37(2):164-179.
doi: 10.1007/s10930-018-9757-y.

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept

Othman Montacir  1   2 , Houda Montacir  1 , Andreas Springer  3 , Stephan Hinderlich  2 , Fereidoun Mahboudi  4 , Amirhossein Saadati  4 , Maria Kristina Parr  5
Affiliations

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept

Othman Montacir et al. Protein J. 2018 Apr.

Abstract

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.

Keywords: Biopharmaceutical; Glycosylation; Mass spectrometry; Physicochemical characterization; Potency assay.

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References

    1. J Chromatogr B Analyt Technol Biomed Life Sci. 2007 May 1;850(1-2):285-94 - PubMed
    1. Mol Cell Proteomics. 2010 Aug;9(8):1716-28 - PubMed
    1. Mass Spectrom Rev. 2008 May-Jun;27(3):207-36 - PubMed
    1. J Immunol. 1999 Nov 15;163(10):5427-34 - PubMed
    1. Adv Biochem Eng Biotechnol. 2012;127:187-219 - PubMed

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