Limits...
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

Two-dimensional gel electrophoresis (2D-PAGE) of before EPR, BIN, and RET (a, c and e, respectively) and after (b, d and f, respectively) peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase F (PNGase F) digestion. EPR Eprex®, BIN Binocrit®, RET Retacrit®, MW molecular weight
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4562010&req=5

Fig6: Two-dimensional gel electrophoresis (2D-PAGE) of before EPR, BIN, and RET (a, c and e, respectively) and after (b, d and f, respectively) peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase F (PNGase F) digestion. EPR Eprex®, BIN Binocrit®, RET Retacrit®, MW molecular weight

Mentions: The 2D-PAGE technique was then applied to investigate the possible presence of isoforms in the three pharmaceutical preparations. In the literature, capillary electrophoresis was used to discover chemical differences between several isoform of these drugs [38]. Here, these isoforms were discovered and separated by 2D-PAGE (METHOD_A) [32, 39]. Results obtained with this approach clearly demonstrated that, although the peptide sequence was the same between the three drugs, differences could be observed (Fig. 6a, c, e). Comparing the three gels, the trend of BIN was different from RET and EPR; indeed, there were differences in the molecular weight of BIN that did not appear in the other two drugs. To better investigate if the variation in the molecular weight was due to the presence of different sugar chains in N-glycosylation, a 2D-PAGE (METHOD_B) was performed after PNGase F digestion of the three drugs. From this analysis (Fig. 6b, d, f), different isoforms but with the same molecular weight were obtained in each pharmaceutical preparation. Therefore, it could be concluded that differences in the molecular weight previously reported for BIN were due to the presence of different carbohydrate chains compared to EPR and RET.Fig. 6


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)

Two-dimensional gel electrophoresis (2D-PAGE) of before EPR, BIN, and RET (a, c and e, respectively) and after (b, d and f, respectively) peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase F (PNGase F) digestion. EPR Eprex®, BIN Binocrit®, RET Retacrit®, MW molecular weight
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Two-dimensional gel electrophoresis (2D-PAGE) of before EPR, BIN, and RET (a, c and e, respectively) and after (b, d and f, respectively) peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase F (PNGase F) digestion. EPR Eprex®, BIN Binocrit®, RET Retacrit®, MW molecular weight
Mentions: The 2D-PAGE technique was then applied to investigate the possible presence of isoforms in the three pharmaceutical preparations. In the literature, capillary electrophoresis was used to discover chemical differences between several isoform of these drugs [38]. Here, these isoforms were discovered and separated by 2D-PAGE (METHOD_A) [32, 39]. Results obtained with this approach clearly demonstrated that, although the peptide sequence was the same between the three drugs, differences could be observed (Fig. 6a, c, e). Comparing the three gels, the trend of BIN was different from RET and EPR; indeed, there were differences in the molecular weight of BIN that did not appear in the other two drugs. To better investigate if the variation in the molecular weight was due to the presence of different sugar chains in N-glycosylation, a 2D-PAGE (METHOD_B) was performed after PNGase F digestion of the three drugs. From this analysis (Fig. 6b, d, f), different isoforms but with the same molecular weight were obtained in each pharmaceutical preparation. Therefore, it could be concluded that differences in the molecular weight previously reported for BIN were due to the presence of different carbohydrate chains compared to EPR and RET.Fig. 6

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