Sialic Acids Content Analysis of the Innovator and Biosimilar Darbepoetin Alfa by Fluorometric HPLC Assay

Background: Erythropoietin is a highly glycosylated protein hormone, used medically for the treatment of anemia associated with chronic renal disease. Due to patent expirations, we expect biosimilar erythropoietins to play an increasing role in healthcare in coming years. Objective: Here, we have developed a chromatograpy method for determination of Neu5AC and Neu5Gc in darbepoetin alfa. Methods: Sialic acids (Sias) analysis provides the contents of the two most abundant sialic acids Neu5Ac and Neu5Gc. It has often been utilized for the evaluation of sialylated glycoprotein to ensure the biopharmtherapeutic safety and efficacy. In this work, the application of the DMB derivatization of Sias from darbepoetin alfa followed by the separation of the resulting adducts based on HPLC-FLD was presented. Results: Our work demonstrated that the method is characteristics of simple operation, and high reproducibility and sensitivity, and easy to set up for compliance analysis in a routine laboratory. Conclusion: By using a water:acetonitrile gradient, high resolution of Sias was obtained within a 1 min analysis time. The detection limits of NeuAc and NeuGc were 2.9 μg/L and 1.5 μg/L, respectively. The RSDs of the intraday and interday precisions in terms of retention time and peak areas of the analytes are below 1%, and from 3.8% to 4.2% respectively. Therefore, this developed method can be widely applied for simple determination of Neu5Ac, Neu5Gc, and O-acetylated Sias in the derivatized samples. Especially, high levels of O-acetylation were detected on darbepoetin alfa N-glycans, which are critical to establishing the bioequivalence of darbepoetin alfa biosimilars.