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Open AccessArticle

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment

1
Institute of Pharmacy, Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin, Germany
2
Labor für Biochemie, Department of Life Sciences & Technology, Beuth Hochschule für Technik Berlin, Seestraße 64, 13347 Berlin, Germany
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Institut für Chemie and Biochemie, Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
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Core Facility BioSupraMol, Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany
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AryoGen Pharmed, No. 140, Cross Tajbakhsh Street, 24th Kilometer Makhsous Road, Tehran, Iran
6
Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Iran
*
Author to whom correspondence should be addressed.
Bioengineering 2018, 5(1), 7; https://doi.org/10.3390/bioengineering5010007
Received: 29 November 2017 / Revised: 11 January 2018 / Accepted: 12 January 2018 / Published: 19 January 2018
Eptacog alfa (NovoSeven®) is a vitamin K-dependent recombinant Factor VIIa produced by genetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues. After activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino acids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten glutamic acids of the N-terminal moiety are γ-carboxylated, Asn145 and Asn322 are N-glycosylated, and Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted for the originator and the first biosimilar AryoSeven™ to evaluate comparable bioengineering. Physicochemical properties were analyzed based on mass spectrometry, including intact mass, PTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their N-glycan profiles. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed N-glycosylation sites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and Ser52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry (TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of both biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating comparable bioactivity. Consequently, careful process optimization led to a stable production process of the biopharmaceuticals. View Full-Text
Keywords: biopharmaceutical; biosimilar; mass spectrometry; physicochemical characterization; coagulation assay biopharmaceutical; biosimilar; mass spectrometry; physicochemical characterization; coagulation assay
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Montacir, O.; Montacir, H.; Eravci, M.; Springer, A.; Hinderlich, S.; Mahboudi, F.; Saadati, A.; Parr, M.K. Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment. Bioengineering 2018, 5, 7.

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