Physicochemical and biologic comparability of a biosimilar granulocyte colony-stimulating factor with its reference product.

Background: Development of biosimilars requires physicochemical and biologic characterization to show comparability with a reference product. Zarzio (filgrastim) is a biosimilar recombinant human granulocyte colony-stimulating factor (G-CSF) that has been approved in the EU using Neupogen as its reference product.

Objective: The aim of this study was to compare the drug identity, purity, and bioactivity of Zarzio (300 and 480 μg/0.5 mL solution) with Neupogen, using a broad range of standard and advanced analytical methods.

Methods: Peptide mapping with UV detection and mass determination, circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and liquid chromatography electrospray ionization (LC-ESI) mass spectrometry were among the analyses used to compare primary and higher-order protein structure. Cation-exchange chromatography (CEX) and reversed-phase high-performance liquid chromatography (RP-HPLC) were used to compare polarity and charge. Biologic characterization included comparison of G-CSF receptor binding affinity by surface plasmon resonance spectroscopy, an in vitro cell proliferation assay, and Western blot immunologic binding.

Results: The primary structures of Zarzio and Neupogen were shown to be identical by peptide mapping and other tests. CD and NMR spectroscopy demonstrated that the two products have comparable secondary and tertiary structures. RP-HPLC and other methods showed that the products have similar purity profiles. Comparable affinity with the G-CSF receptor GCSFR/CD114 was obtained using surface plasmon resonance spectroscopy, and comparable in vitro bioactivity was shown in a cell proliferation assay.

Conclusion: These results show the physicochemical and biologic comparability of Zarzio and its reference product, Neupogen.