Analysis of Denosumab by a Validated CZE Method and Determination of Sialic Acids by the RP–HPLC Method

Abstract

A capillary zone electrophoresis (CZE) method was developed and validated to quantitate the monoclonal antibody denosumab (DmAb) and its charge variants in pharmaceutical products, demonstrating excellent precision, linearity and accuracy. Separations were obtained with migration times of 11.3 min for DmAb and the calibration curve was linear in the range of 0.95–20 mg/mL. The analytical comparability of seven batches of Prolia® showed mean differences of the estimated content/potencies of 1.87% lower, and 0.84 and 1.21% higher compared with the size-exclusion and reversed-phase liquid chromatography (SE–HPLC and RP–HPLC) methods and the osteoclast antiproliferative bioassay, respectively, with non-significant differences (P > 0.05). An RP–HPLC method with fluorescence detection (RP–HPLC–F), performed on a Kinetex® EVO C18 column (5 μm, 100 Å, 250 mm × 4.6 mm), was optimized to determine the levels of sialic acids of DmAb biomolecules, giving mean concentrations of 0.16 and 0.17 μg N-acetylneuraminic acid/mg DmAb for Prolia® and Xgeva® pharmaceutical products, respectively. The results demonstrated the capability of each one of the methods, and their use in combination constitutes a strategy to monitor instability, thereby assuring the quality and the batch-to-batch consistency of the biotechnology-derived medicine.