Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment.

Othman Montacir,Andreas Springer,Murat Eravci,Maria Parr,Fereidoun Mahboudi,Stephan Hinderlich,Houda Montacir,Amirhossein Saadati

doi:10.3390/bioengineering5010007

Othman Montacir, Andreas Springer + Show 6 more

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https://doi.org/10.3390/bioengineering5010007

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Abstract

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.

Highlights

One of the major classes of therapeutical biopharmaceuticals are blood factors
Bioengineering was successfully performed for the FVIIa follow-on biopharmaceutical
Small batch-to-batch variations were observed between three different batches of AryoSevenTM and for the originator NovoSeven®, which was analyzed in parallel

Summary

Introduction

One of the major classes of therapeutical biopharmaceuticals are blood factors. They represent six percent of the approved biotherapeuticals up to 2014 [1]. Bioengineering 2018, 5, 7 a hereditary (chromosome X) blood-coagulation disorder, due to a deficiency of the clotting factors. Activated factor VII (FVIIa) initiates the extrinsic coagulation pathway and thereby activates factor IX and factor X to bind to tissue factor on the surface of cells that were exposed to circulating blood due to an injury. The application of pharmacologic doses of FVIIa results in the binding of sufficient amounts of FVIIa to activated platelets and subsequent activation of factor X, further passing of the tenase complex and an inducing of thrombin burst [3].

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