US EPA

5408661 

Journal Article 

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

Perobelli, RF; Xavier, B; Silveira, ARD; Remuzzi, GL; Motta, LGJ; Dalmora, SL 

2018 

Yes 

International Journal of Biological Macromolecules
ISSN: 0141-8130
EISSN: 1879-0003 

119 

96-104 

English 

30048724 

10.1016/j.ijbiomac.2018.07.120 

WOS:000447682100012 

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.