Eptacog alfa (NovoSevenÃ?®) is a vitamin K-dependent recombinant Factor VIIa produced by\ngenetic engineering from baby hamster kidney (BHK) cells as a single peptide chain of 406 residues.\nAfter activation, it consists of a light chain (LC) of 152 amino and a heavy chain (HC) of 254 amino\nacids. Recombinant FVIIa undergoes many post-translational modifications (PTMs). The first ten\nglutamic acids of the N-terminal moiety are Ã?³-carboxylated, Asn145 and Asn322 are N-glycosylated,\nand Ser52 and Ser60 are O-glycosylated. A head-to-head biosimilarity study was conducted\nfor the originator and the first biosimilar AryoSevenââ??¢ to evaluate comparable bioengineering.\nPhysicochemical properties were analyzed based on mass spectrometry, including intact mass,\nPTMs and higher-order structure. Both biotherapeutics exhibit a batch-to-batch variability in their\nN-glycan profiles. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed N-glycosylation\nsites as well as two different O-glycopeptide sites. Ser60 was found to be O-fucosylated and\nSer52 had O-glucose or O-glucose-(xylose)1,2 motifs as glycan variants. Ion mobility spectrometry\n(TWIMS) and NMR spectroscopy data affirm close similarity of the higher-order structure of\nboth biologicals. Potency of the biodrugs was analyzed by a coagulation assay demonstrating\ncomparable bioactivity. Consequently, careful process optimization led to a stable production process\nof the biopharmaceuticals.
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