An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.
BACKGROUND
Authorization to market a biosimilar product by the appropriate institutions is expected based on biosimilarity with its originator product. The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses.


OBJECTIVE
In this study, we proposed a useful quality control system for rapid and economic primary screening of potential biosimilar drugs. For this purpose, chemical and functional characterization of the originator rhEPO alfa and two of its biosimilars was discussed.


METHODS
Qualitative and quantitative analyses of the originator rhEPO alfa and its biosimilars were performed using reversed-phase high-performance liquid chromatography (RP-HPLC). The identification of proteins and the separation of isoforms were studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and two-dimensional gel electrophoresis (2D-PAGE), respectively. Furthermore, the biological activity of these drugs was measured both in vitro, evaluating the TF-1 cell proliferation rate, and in vivo, using the innovative experimental animal model of the zebrafish embryos.


RESULTS
Chemical analyses showed that the quantitative concentrations of rhEPO alfa were in agreement with the labeled claims by the corresponding manufacturers. The qualitative analyses performed demonstrated that the three drugs were pure and that they had the same amino acid sequence. Chemical differences were found only at the level of isoforms containing N-glycosylation; however, functional in vitro and in vivo studies did not show any significant differences from a biosimilar point of view.


CONCLUSIONS
These rapid and economic structural and functional analyses were effective in the evaluation of the biosimilarity between the originator rhEPO alfa and the biosimilars analyzed.
Department of Molecular and Translational Medicine, University of Brescia, Viale Europa, 11, 25123, Brescia, Italy, alessandra.gianoncelli@unibs.it.
Alessandra
A
Gianoncelli
Sara A
SA
Bonini
Michela
M
Bertuzzi
Michela
M
Guarienti
Sara
S
Vezzoli
Rajesh
R
Kumar
Andrea
A
Delbarba
Andrea
A
Mastinu
Sandra
S
Sigala
Pierfranco
P
Spano
Luca
L
Pani
Sergio
S
Pecorelli
Maurizio
M
Memo
D000595:Amino Acid Sequence; D000818:Animals; D059451:Biosimilar Pharmaceuticals / Q000737:chemistry* / Q000494:pharmacology / Q000592:standards; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D017277:Drug Approval; D000068817:Epoetin Alfa / Q000737:chemistry* / Q000494:pharmacology / Q000592:standards; D006397:Hematinics / Q000737:chemistry* / Q000494:pharmacology / Q000592:standards; D006801:Humans; D011786:Quality Control; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D015027:Zebrafish / Q000196:embryology
D059451:Biosimilar Pharmaceuticals; D006397:Hematinics; D011994:Recombinant Proteins; D000068817:Epoetin Alfa