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. 2019 Dec 1;17(4):e2470.
doi: 10.30498/IJB.2019.99581. eCollection 2019 Dec.

Physicochemical Characterization of Altebrel™, a Proposed Etanercept Biosimilar

Ramin Fazel  1 , Fereidoun Mahboudi  2 , Ehsan Seyedjafari  1 , Soroush Sardari  2 , Behrouz Vaziri  3
Affiliations

Physicochemical Characterization of Altebrel™, a Proposed Etanercept Biosimilar

Ramin Fazel et al. Iran J Biotechnol. .

Abstract

Background: Etanercept is prescribed for the rapid and effective treatment of chronic immune-mediated inflammatory disorders. Due to the expiration of etanercept patents and worldwide demand for comparable and more affordable substitutes, several biosimilars of etanercept have been approved in different countries and new ones are in the process of approval.

Objectives: In the present study, Altebrel™ as an etanercept proposed biosimilar was investigated in a side by side comparison using various orthogonal analytical methods.

Materials and methods: Three batches of the Altebrel™ and Enbrel® samples were used for the study. Several physicochemical properties of samples were compared according to international guidelines, incliding; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis sodium dodecyl sulfate (CE-SDS), size exclusion high performance liquid chromatography (SE-HPLC), hydrophobic interaction chromatography high performance liquid chromatography (HIC-HPLC) and its biological activity was evaluated using surface plasmon resonance affinity analysis and tumor necrosis factor alpha (TNFα) neutralization biological assay. Amino acid analysis was applied to check the primary sequence and far-UV circular dichroism (CD) spectroscopy investigated the secondary structure.

Results: The obtained results indicated a high degree of similarity between Altebrel™ and Enbrel®. Results of SDS-PAGE, CE-SDS, HIC-HPLC and SE-HPLC implied a comparable pattern of size variants for all samples. Based on the data achieved via in vitro bioactivity assays and SPR analysis, the functional property of Altebrel™ was proved comparable to that of the reference product. Moreover, amino acid analysis indicated similar primary structure and circular dichroism study implied a similar secondary structure for Altebrel™ and Enbrel®.

Conclusion: Overall, our data provide analytical evidence for structural and in vitro functional similarity between Altebrel™ and Enbrel®.

Keywords: Biosimilar; Comparability study; Etanercept; Physicochemical Characterization.

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Figures

Figure 1
Figure 1
A) Non-reduced and B) Reduced samples of Enbrel® and Altebrel™. C) SDS-PAGE densitometry of Enbrel G30909 and Altebrel 9202009 in percentage. Lanes 1 to 3 are Enbrel G30909, 6% (0.3 μg) and 8% (0.4 μg) of Enbrel G30909 respectively. Lanes 4 to 6 are Altebrel 9202009 (5 μg), 6% (0.3 μg) and 8% (0.4 μg) of Altebrel 9202009 respectively.
Figure 2
Figure 2
Non-reduced capillary gel electrophoresis analysis of etanercept samples. Etanercept electropherograms show the HMWC, main peak and LMWC.
Figure 3
Figure 3
SE-HPLC analysis of Altebrel™ and Enbrel® samples. The samples were separated into three major areas; Peak A is the main sample, Peak A’ is the degraded form and Peak B is the aggregated form.
Figure 4
Figure 4
HIC-HPLC analysis of Altebrel™ and Enbrel® samples. Peak 1: degraded and truncated species; Peak 2: Main product; and Peak 3: misfolded and aggregated species.
Figure 5
Figure 5
Sensorgrams from SPR analysis of A) Enbrel® and B) Altebrel™ as an example. Appropriate curvature of the sensorgram in the association and dissociation time would result in gaining the reliable results.
Figure 6
Figure 6
Biological assay of etanercept samples. In all of the samples, reference standard is pool of the three Enbrel® samples.
See this image and copyright information in PMC

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