Proposed Biosimilar Shows Similar Glycosylation, Primary Structure, In-Vitro Functionality to Enbrel

In a recent comparability analysis, a proposed biosimilar to etanercept (that the developers hope to sell as Altebrel) underwent direct comparison to the reference and was found to have a high similarity in terms of glycosylation, primary structure, and in-vitro functionality. However, the authors said additional investigations are needed in terms of quality, safety and efficacy.

Researchers used bottom-up mass spectrometric analysis for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation.

N/O-glycans were analyzed after permethylation derivatization. The effect of N-glycans on in-vitro functionality of etanercept was assayed.

Three enzyme peptide mapping resulted in complete identification of the primary structure, and new N/O-glycan structures were identified and all the N-glycans were quantified.

Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality.

The researchers said their findings were similiar to previous studies, 1 of which reported 100% sequence coverage of etanercept using trypsin, LysC and AspN. Another comparability study between the etanercept and a different biosimilar, Infinitam, showed 97.9% coverage of the primary sequence, (which the authors said was surprisingly high) using trypsin as the only protease.

In this report, the degree of methionine oxidation was investigated and statistical analysis (P = .05) showed no differences in oxidation rates between the reference drug and the proposed Altebrel. In addition, the potencies of the 2 drugs to induce necroptosis proved to be comparable.

The authors said their study describes a detailed strategy for the mass spectrometric characterization of etanercept primary sequence and glycosylation as a model and investigates the effect of N-glycosylation on its in-vitro functional properties. It also can help regulators set the requirements needed for biosimilarity or interchangeability demonstration by providing additional information about the glycosylation, primary structure and functionality of etanercept. Moreover, the same primary structure, comparable N-glycan profile, comparable rates of methionine oxidation and in-vitro functionality showed a high similarity of Altebrel to etanercept.

Reference

Fazel R, Guan Y, Vaziri B, et al. Structural and in vitro functional comparability analysis of Altebrel™, a proposed etanercept biosimilar: focus on primary sequence and glycosylation. Pharmaceuticals 2019; 12(1):14. doi:10.3390/ph12010014.