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. 2018 Jan 19;5(1):7.
doi: 10.3390/bioengineering5010007.

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment

Othman Montacir  1   2 , Houda Montacir  3 , Murat Eravci  4 , Andreas Springer  5 , Stephan Hinderlich  6 , Fereidoun Mahboudi  7 , Amirhossein Saadati  8 , Maria Kristina Parr  9
Affiliations

Bioengineering of rFVIIa Biopharmaceutical and Structure Characterization for Biosimilarity Assessment

Othman Montacir et al. Bioengineering (Basel). .

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.

Keywords: biopharmaceutical; biosimilar; coagulation assay; mass spectrometry; physicochemical characterization.

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Conflict of interest statement

Fereidoun Mahboudi and Amirhossein Saadati are employees of AryoGen Pharmed, the company which produces and sells AryoSeven™. All other authors declare no conflict of interest and disclose any financial and personal relationship with people or organizations that could inappropriately influence this scientifically oriented study.

Figures

Figure 1
Figure 1
(a) Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis of intact mass for NovoSeven®, and (b) for AryoSeven™.
Figure 2
Figure 2
(a) Mass spectra of γ-carboxylation detected using liquid chromatography accurate mass spectrometry (UHPLC-QTOF-MS)/MSE in NovoSeven®, and (b) AryoSeven™. Not assigned signals were artifacts.
Figure 3
Figure 3
(a) UHPLC-QTOF-MS/MSE spectra of N-glycopeptides representing N-glycosylation site Asn145 detected in NovoSeven®, and (b) in AryoSeven™; (c) site Asn322 in NovoSeven®, and (d) in AryoSeven™.
Figure 4
Figure 4
(a) Combined precursor ESI-QTOF low-energy scan mass spectra of doubly-charged O-glycopeptides from UHPLC-QTOF-MS/MSE peptide mapping analysis showing O-glycosylation sites Ser52 and Ser60 of NovoSeven®, and (b) AryoSeven™.
Figure 5
Figure 5
(a) MALDI-TOF-MS spectra of the most abundant permethylated N-glycans released from NovoSeven®, and (b) AryoSeven™. (c) Relative abundance of permethylated N-glycans from three batches of NovoSeven®, and (d) AryoSeven™ were calculated. Standard deviations were calculated from technical triplicates.
Figure 6
Figure 6
(a) The overlay of far-UV CD spectra obtained from innovator and AryoSeven™. (b) FTIR spectra indicating a high similarity of the amide I and II bands between both biologics (light blue; NovoSeven®, and red, AryoSeven™). (c,d) NMR spectral fingerprinting showing all protons in methyl and methine region (zero to three parts per million) as well as amide and aromatic region (six to eight parts per million) (green, NovoSeven®, and red, AryoSeven™).
Figure 7
Figure 7
(a) The drift time generated from traveling wave IMS after IM-MS analysis under denaturing conditions for NovoSeven®; and (b) AryoSeven™ displayed homogenous driftscope plots between 60 and 140 Bins.
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