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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

Overlay of the three chromatograms obtained by qualitative analysis of Eprex® (blue), Binocrit® (magenta), and Retacrit® (black), showing the presence of the rhEPO peak (retention time 7.053, 7.098 and 7.038 min, respectively). BIN Binocrit®, EPR Eprex®, RET Retacrit®
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Fig1: Overlay of the three chromatograms obtained by qualitative analysis of Eprex® (blue), Binocrit® (magenta), and Retacrit® (black), showing the presence of the rhEPO peak (retention time 7.053, 7.098 and 7.038 min, respectively). BIN Binocrit®, EPR Eprex®, RET Retacrit®

Mentions: On this basis, EPR and its biosimilar products BIN and RET were analyzed using HPLC–UV. This experimental procedure allowed us to demonstrate that rhEPO in the originator EPR and in its biosimilar products BIN and RET could be identified by only one peak, with retention times of 7.053, 7.098, and 7.038 min, respectively (Fig. 1). Interestingly, as shown in Fig. 1, while chromatograms of EPR and BIN were characterized by a single peak, multiple peaks could be detected in the chromatogram of RET. Indeed, in addition to the major peak corresponding to the rhEPO, other peaks were eluted at 3.052 and 3.225 min, close to the solvent front. The extra peaks of RET were recovered by a fraction collector and analyzed in GC–MS (Electronic Supplementary Material Fig. S1). These peaks were attributed to amino acids used as excipients in RET pharmaceutical formulation and were not present in the other two drugs (Table 3).Fig. 1


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)

Overlay of the three chromatograms obtained by qualitative analysis of Eprex® (blue), Binocrit® (magenta), and Retacrit® (black), showing the presence of the rhEPO peak (retention time 7.053, 7.098 and 7.038 min, respectively). BIN Binocrit®, EPR Eprex®, RET Retacrit®
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Overlay of the three chromatograms obtained by qualitative analysis of Eprex® (blue), Binocrit® (magenta), and Retacrit® (black), showing the presence of the rhEPO peak (retention time 7.053, 7.098 and 7.038 min, respectively). BIN Binocrit®, EPR Eprex®, RET Retacrit®
Mentions: On this basis, EPR and its biosimilar products BIN and RET were analyzed using HPLC–UV. This experimental procedure allowed us to demonstrate that rhEPO in the originator EPR and in its biosimilar products BIN and RET could be identified by only one peak, with retention times of 7.053, 7.098, and 7.038 min, respectively (Fig. 1). Interestingly, as shown in Fig. 1, while chromatograms of EPR and BIN were characterized by a single peak, multiple peaks could be detected in the chromatogram of RET. Indeed, in addition to the major peak corresponding to the rhEPO, other peaks were eluted at 3.052 and 3.225 min, close to the solvent front. The extra peaks of RET were recovered by a fraction collector and analyzed in GC–MS (Electronic Supplementary Material Fig. S1). These peaks were attributed to amino acids used as excipients in RET pharmaceutical formulation and were not present in the other two drugs (Table 3).Fig. 1

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