Quantitation of the monoclonal antibody Denosumab by bioassay and validated LC methods

Abstract

The monoclonal antibody Denosumab (DmAb) is clinically used to treat osteoporosis and bone loss. We developed a bioassay based on the ability of DmAb to inhibit the effect of human receptor activator of nuclear factor-κB ligand (RANKL) to stimulate the formation of osteoclasts derived from RAW 264.7 cells. This bioassay was applied in conjunction with size exclusion high-performance liquid chromatography (SE–HPLC) and reversed-phase high-performance liquid chromatography (RP–HPLC) methods, with diode array detection (DAD), validated for the quantitation of this biotechnology-derived medicine. The SE–HPLC(DAD) method was carried out on a TSKGel G2000SWXL column and the mobile phase consisted of potassium phosphate buffer with sodium chloride, pH 7.4. The gradient RP–HPLC(DAD) method was carried out on a Vydac 214TP C4 column at 60 °C. The mobile phases consisted of 0.1% v/v trifluoroacetic acid (TFA) in water and 0.1% v/v TFA in acetonitrile. Calibration curves were linear over the concentration ranges 6–200 μg mL−1 and 6–300 μg mL−1 for the SE–HPLC(DAD) and RP–HPLC(DAD) methods respectively. The bioassay results correlated with the LC methods results, indicating the capabilities of these methods to quantitate DmAb, which will contribute to ensure the batch-to-batch consistency and efficacy of this biotherapeutic.