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

Eprex®, Binocrit®, and Retacrit® injection effects on 48 and 72 hpf zebrafish embryos. Negative controls were injected with 0.05% phenol red solution. Positive controls were E. coli JM109 bacteria. Hemoglobin (HB) content was quantified by a O-dianisidine staining and b modified Drabkin protocol. Macrophages and neutrophils quantification was performed by whole-mount in situ hybridization with cl-plastin and dpu1 probes. Data are the mean ± standard deviation of 3 experiments. Epr Eprex ®, Bin Binocrit®, Ret Retacrit®, ctrl control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4562010&req=5

Fig8: Eprex®, Binocrit®, and Retacrit® injection effects on 48 and 72 hpf zebrafish embryos. Negative controls were injected with 0.05% phenol red solution. Positive controls were E. coli JM109 bacteria. Hemoglobin (HB) content was quantified by a O-dianisidine staining and b modified Drabkin protocol. Macrophages and neutrophils quantification was performed by whole-mount in situ hybridization with cl-plastin and dpu1 probes. Data are the mean ± standard deviation of 3 experiments. Epr Eprex ®, Bin Binocrit®, Ret Retacrit®, ctrl control

Mentions: EPR, BIN, and RET, at a 24 IU/mL concentration, were injected into the common cardinal vein of healthy zebrafish embryos at 48 hpf. Groups of 20–25 embryos coming from the same batch of fertilized eggs were used for each experimental point. Embryos were incubated at 28 °C for 2–4 h after injection to let the drugs act. Preliminary experiments have been conducted, injecting different rhEPO doses (2–48 IU/mL), to establish the most efficient treatment (data not shown). o-Dianisidine staining was performed to detect red blood cells. Figure 8 shows the percentage of o-dianisidine-positive area, proportional to the amount of red blood cells, measured in the trunk and in the tail of each embryo. Embryos treated with all the three compounds showed a statistically significant increase of o-dianisidine-positive area compared with the negative controls. Indeed, embryos injected with EPR, BIN, and RET showed a 1.69-, 1.72-, and 1.58-fold increase of erythrocytes content, respectively, when compared with the negative controls.Fig. 8


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)

Eprex®, Binocrit®, and Retacrit® injection effects on 48 and 72 hpf zebrafish embryos. Negative controls were injected with 0.05% phenol red solution. Positive controls were E. coli JM109 bacteria. Hemoglobin (HB) content was quantified by a O-dianisidine staining and b modified Drabkin protocol. Macrophages and neutrophils quantification was performed by whole-mount in situ hybridization with cl-plastin and dpu1 probes. Data are the mean ± standard deviation of 3 experiments. Epr Eprex ®, Bin Binocrit®, Ret Retacrit®, ctrl control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Eprex®, Binocrit®, and Retacrit® injection effects on 48 and 72 hpf zebrafish embryos. Negative controls were injected with 0.05% phenol red solution. Positive controls were E. coli JM109 bacteria. Hemoglobin (HB) content was quantified by a O-dianisidine staining and b modified Drabkin protocol. Macrophages and neutrophils quantification was performed by whole-mount in situ hybridization with cl-plastin and dpu1 probes. Data are the mean ± standard deviation of 3 experiments. Epr Eprex ®, Bin Binocrit®, Ret Retacrit®, ctrl control
Mentions: EPR, BIN, and RET, at a 24 IU/mL concentration, were injected into the common cardinal vein of healthy zebrafish embryos at 48 hpf. Groups of 20–25 embryos coming from the same batch of fertilized eggs were used for each experimental point. Embryos were incubated at 28 °C for 2–4 h after injection to let the drugs act. Preliminary experiments have been conducted, injecting different rhEPO doses (2–48 IU/mL), to establish the most efficient treatment (data not shown). o-Dianisidine staining was performed to detect red blood cells. Figure 8 shows the percentage of o-dianisidine-positive area, proportional to the amount of red blood cells, measured in the trunk and in the tail of each embryo. Embryos treated with all the three compounds showed a statistically significant increase of o-dianisidine-positive area compared with the negative controls. Indeed, embryos injected with EPR, BIN, and RET showed a 1.69-, 1.72-, and 1.58-fold increase of erythrocytes content, respectively, when compared with the negative controls.Fig. 8

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