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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

Sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) purification of Eprex®, Binocrit®, and Retacrit® before (+) and after (−) PNGase F digestion. BIN Binocrit®, EPR Eprex®, MW molecule weight, RET Retacrit®
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Fig4: Sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) purification of Eprex®, Binocrit®, and Retacrit® before (+) and after (−) PNGase F digestion. BIN Binocrit®, EPR Eprex®, MW molecule weight, RET Retacrit®

Mentions: To better characterize the chemical structure of rhEPO, the originator and biosimilar drugs were also studied with a proteomic approach. Samples were separated by SDS-PAGE (see Electronic Supplementary Material Fig. S4), digested by trypsin, and analyzed by MALDI-TOF/TOF mass spectrometry. The results are reported in Table 4, which shows that 15 identical peptides were present in each drug. The data were then compared to the peptides obtained from in silico digestion of EPO alfa, based on peptide mass [33] (Table 4—MH+ calculated column). The small peptides consisting of only one or two amino acids were lost during sample purification or suppressed due to interference by matrix ions in the low m/z range. Fifteen peptides were found to be identical to the peptides obtained from in silico digestion except for two peptides containing N-glycosylation (Table 4—peptides 48–72 and 104–124), which have not been determined after tryptic digestion. This could be due to the fact that, as reported in the literature, the MALDI ionization of peptides cannot occur in the presence of the N-linked carbohydrates [31, 34–36]. In order to clarify this point and to complete the analysis starting from whole proteins, the carbohydrate chains were cleaved from the peptides by treatment with PNGase F [37]. Before purifying the sample by SDS-PAGE (Fig. 4), an aliquot (1 µL) of the reaction mixture was analyzed by MALDI-TOF–MS in linear mode (Fig. 5). Figure 5 shows that the principal peak at an m/z value of approximately 18,900 represented the single charge of rhEPO without N-glycosylation. The m/z value of about 9500 represented the double charge of relative rhEPO and the m/z value of about 37,800 represented the single charge of relative rhEPO dimer.Table 4


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)

Sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) purification of Eprex®, Binocrit®, and Retacrit® before (+) and after (−) PNGase F digestion. BIN Binocrit®, EPR Eprex®, MW molecule weight, RET Retacrit®
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS-PAGE) purification of Eprex®, Binocrit®, and Retacrit® before (+) and after (−) PNGase F digestion. BIN Binocrit®, EPR Eprex®, MW molecule weight, RET Retacrit®
Mentions: To better characterize the chemical structure of rhEPO, the originator and biosimilar drugs were also studied with a proteomic approach. Samples were separated by SDS-PAGE (see Electronic Supplementary Material Fig. S4), digested by trypsin, and analyzed by MALDI-TOF/TOF mass spectrometry. The results are reported in Table 4, which shows that 15 identical peptides were present in each drug. The data were then compared to the peptides obtained from in silico digestion of EPO alfa, based on peptide mass [33] (Table 4—MH+ calculated column). The small peptides consisting of only one or two amino acids were lost during sample purification or suppressed due to interference by matrix ions in the low m/z range. Fifteen peptides were found to be identical to the peptides obtained from in silico digestion except for two peptides containing N-glycosylation (Table 4—peptides 48–72 and 104–124), which have not been determined after tryptic digestion. This could be due to the fact that, as reported in the literature, the MALDI ionization of peptides cannot occur in the presence of the N-linked carbohydrates [31, 34–36]. In order to clarify this point and to complete the analysis starting from whole proteins, the carbohydrate chains were cleaved from the peptides by treatment with PNGase F [37]. Before purifying the sample by SDS-PAGE (Fig. 4), an aliquot (1 µL) of the reaction mixture was analyzed by MALDI-TOF–MS in linear mode (Fig. 5). Figure 5 shows that the principal peak at an m/z value of approximately 18,900 represented the single charge of rhEPO without N-glycosylation. The m/z value of about 9500 represented the double charge of relative rhEPO and the m/z value of about 37,800 represented the single charge of relative rhEPO dimer.Table 4

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