Comprehensive Physicochemical and Biological Characterization of the Proposed Biosimilar Darbepoetin Alfa, LBDE, and Its Originator Darbepoetin Alfa, NESP.

Background: For regulatory approval, the comparability of a biosimilar product to an originator product should be ensured through thorough physicochemical and biological characterization.

Objective: To evaluate the biosimilarity between LBDE, the proposed biosimilar darbepoetin alfa, and NESP, its originator, we performed a comprehensive physicochemical and biological characterization study.

Methods: Primary and higher-order protein structures were analyzed using Lys-C peptide mapping with liquid chromatography-mass spectrometry (LC-MS), disulfide bond identification, circular dichroism, and fluorescence spectroscopy. Glycosylation and isoform distribution were analyzed using MS, LC, and capillary zone electrophoresis. Size variants were evaluated with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Biological characterization included binding affinity for human erythropoietin receptor, in vitro cell proliferation, and in vivo potency. Pharmacokinetics (PK) were evaluated using rats through two injection routes.

Results: Non-reducing and reducing Lys-C peptide mapping showed a highly similar peak profile, confirming that LBDE and NESP have the same primary structure and disulfide bonds. Glycosylation and isoform analyses showed that the attached N-glycan and O-glycan structures were the same and their relative contents were similar. Spectroscopic analysis of LBDE showed indistinguishable spectra with NESP. For both LBDE and NESP, a very small amount of size variants was found in SEC-HPLC, and no minor bands were detected in SDS-PAGE. Furthermore, LBDE did not show any difference with NESP in the in vitro and in vivo functional analyses. PK parameters of LBDE were in good agreement with those of NESP.

Conclusion: LBDE shows high similarity to NESP with regard to structure and function.