Abstract des Autors

Human erythropoietin (EPO) and recombinant human EPO (rEPO) are approximately 30-kDa glycosylated proteins comprising 165 amino acids. Darbepoetin alfa (NESP) is a glycosylated protein encompassing five changes in the amino acid sequence of human EPO, which contains two extra sugar chains. NESP is under patent protection in the USA until May 2024 and in Europe until July 2016, which suggests that the number of NESP biosimilars might substantially increase. The detailed characterisation of biosimilar products are required to ensure the identity and purity of the biosimilar products in terms of safety and efficacy for patients. In this study, a mass spectrometric characterisation of NESP biosimilar products is demonstrated. The study comprises a time-of-flight mass spectrometry characterisation for the asialo-NESPs after sialidase digestion and primary structure characterisation using bottom-up analysis after endoproteinase Glu-C digestion of the core protein. The study revealed that there was a wide range of glycoforms spaced by 365 Da intervals, namely, HexHexNAc units, which indicated that NESP biosimilars likely contained more N-acetyllactosamine units in their molecules. The bottom-up analysis also showed that the NESP biosimilars, as well as a rEPO biosimilar, contain not only the des-arginine product but also the C-terminal arginine product comprising 166 amino acids, whereas the innovator products contain des-arginine EPO comprising only 165 amino acids. The C-terminal arginine EPO would be used as a potential marker for doping with EPO bisimilaras. These findings also point to a need for the investigation of immunogenicity and comparability for the biosimilar products.