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An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.

Gianoncelli A, Bonini SA, Bertuzzi M, Guarienti M, Vezzoli S, Kumar R, Delbarba A, Mastinu A, Sigala S, Spano P, Pani L, Pecorelli S, Memo M - BioDrugs (2015)

Bottom Line: The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa, 11, 25123, Brescia, Italy, alessandra.gianoncelli@unibs.it.

ABSTRACT

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Representative immunoblot of Eprex®, Binocrit®, and Retacrit® obtained using an anti-human rhEPO antibody. In the sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) precast gel, 10 µL of each recombinant human rhEPO solution was loaded, corresponding to 3.36 µg of protein content. The Western blot protein analysis revealed a single band in all three of the comparative drugs with an apparent molecular weight of 36 kDa. BIN Binocrit®, EPR Eprex®, MW molecular weight, RET Retacrit®
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Fig3: Representative immunoblot of Eprex®, Binocrit®, and Retacrit® obtained using an anti-human rhEPO antibody. In the sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) precast gel, 10 µL of each recombinant human rhEPO solution was loaded, corresponding to 3.36 µg of protein content. The Western blot protein analysis revealed a single band in all three of the comparative drugs with an apparent molecular weight of 36 kDa. BIN Binocrit®, EPR Eprex®, MW molecular weight, RET Retacrit®

Mentions: To evaluate the protein content present in the final product, an SDS-PAGE was also performed (Electronic Supplementary Material Fig. S4). Results showed the presence of a single diffuse band, due to the presence of glycoforms, with an apparent molecular weight of 36 kDa [32]. This result confirmed that, as already indicated by the HPLC analyses, there were no other protein impurities in the composition of each final product. Furthermore, Western blot analysis with a specific monoclonal anti-hEPO antibody confirmed that the band viewed by SDS-PAGE was effectively rhEPO (Fig. 3).Fig. 3


An Integrated Approach for a Structural and Functional Evaluation of Biosimilars: Implications for Erythropoietin.

Gianoncelli A, Bonini SA, Bertuzzi M, Guarienti M, Vezzoli S, Kumar R, Delbarba A, Mastinu A, Sigala S, Spano P, Pani L, Pecorelli S, Memo M - BioDrugs (2015)

Representative immunoblot of Eprex®, Binocrit®, and Retacrit® obtained using an anti-human rhEPO antibody. In the sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) precast gel, 10 µL of each recombinant human rhEPO solution was loaded, corresponding to 3.36 µg of protein content. The Western blot protein analysis revealed a single band in all three of the comparative drugs with an apparent molecular weight of 36 kDa. BIN Binocrit®, EPR Eprex®, MW molecular weight, RET Retacrit®
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4562010&req=5

Fig3: Representative immunoblot of Eprex®, Binocrit®, and Retacrit® obtained using an anti-human rhEPO antibody. In the sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) precast gel, 10 µL of each recombinant human rhEPO solution was loaded, corresponding to 3.36 µg of protein content. The Western blot protein analysis revealed a single band in all three of the comparative drugs with an apparent molecular weight of 36 kDa. BIN Binocrit®, EPR Eprex®, MW molecular weight, RET Retacrit®
Mentions: To evaluate the protein content present in the final product, an SDS-PAGE was also performed (Electronic Supplementary Material Fig. S4). Results showed the presence of a single diffuse band, due to the presence of glycoforms, with an apparent molecular weight of 36 kDa [32]. This result confirmed that, as already indicated by the HPLC analyses, there were no other protein impurities in the composition of each final product. Furthermore, Western blot analysis with a specific monoclonal anti-hEPO antibody confirmed that the band viewed by SDS-PAGE was effectively rhEPO (Fig. 3).Fig. 3

Bottom Line: The analogy between the originator and its biosimilar(s) is assessed through safety, purity, and potency analyses.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Translational Medicine, University of Brescia, Viale Europa, 11, 25123, Brescia, Italy, alessandra.gianoncelli@unibs.it.

ABSTRACT

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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus