Analytical Comparison of the Originator Granulocyte-colony Stimulating Factor Filgrastim and its Biosimilars

Purpose: Filgrastim, a recombinant human granulocyte colony-stimulating factor (rhG-CSF) produced in Escherichia coli, is indicated for treatment of neutropenia-related conditions in cancer patients. It has been marketed as Neupogen since 1991. In 2006, biosimilar rhG-CSF products have been approved in the European Union (EU). The aim of this study was to compare quality attributes of the originator filgrastim with its three biosimilars which came from the EU market in 2014 to verify whether their similarity is maintained since their market approval. Materials/Methods: Spectrophotometric analysis was used to determine protein content in analyzed products. Chromatographic and electrophoretic analyses were applied to verify the presence of high and low-molecular weight impurities. Secondary and tertiary structure of the drugs were investigated with circular dichroism and intrinsic fluorescence. Finally, biological activity of the drugs was assessed using cell proliferation assay. Results: All products displayed protein content close to the label concentration with a +/- 6% variation. Two oxidized forms and a deamidated form were present at <0.5%. Levels of dimers and other high molecular-weight impurities were similar except for one product, which contained higher amount of the dimer. Profiles and levels of process-related impurities were comparable. The three-dimensional conformation of the molecules with respect to exposed tryptophan residues was similar. The relative potencies of the products were comparable to the reference standard with a +/- 2% variation. Conclusion: This study shows that a high level of similarity is maintained among originator and three biosimilar filgrastims up to 5 years from their first registration in the EU.