Estudo de métodos cromatográficos e ensaio biológico para avaliação do anticorpo monoclonal denosumabe

Abstract

Denosumab (DmAb) is a fully human monoclonal antibody that inhibits osteoclast formation, function and survival, reducing bone turnover and resorption. Structurally, it is a fully human immunoglobulin G2, yielding a molecular mass of approximately 147 kDa, that was expressed using recombinant DNA technology. DmAb has been approved for the treatment of postmenopausal osteoporosis at increased risk of fracture, to avoid bone loss in hormone-ablation therapy during cancer treatment and for prevention of skeletal related events in patients with bone metastases from solid tumors. In this study, it was developed and validated size-exclusion liquid chromatography (SE−LC) and reversed-phase liquid chromatography (RP−LC) methods to quantitate DmAb in biopharmaceutical formulations. Equally, an in vitro bioassay using cultures of RAW 264.7 macrophages, that served as osteoclast precursors, was developed for the quantitation of DmAb. The analysis for the SE−LC method were performed on a TSKGel G2000SWXL column (300 mm × 7,8 mm i.d.) maintained at 25 ºC. The mobile phase consisted of 1 mM monobasic potassium phosphate, 8 mM dibasic potassium phosphate and 200 mM sodium chloride, pH 7.4, run isocratically at a flow rate of 1.0 mL min-1 and detection by a photodiode array detector (PDA) set at 214 nm. The gradient RP-LC method was carried out on a Vydac 214TP C4 column (250 mm × 4.6 mm i.d.) maintained at 60 ºC. The mobile phase consisted of 0.1% v/v trifluoracetic acid (TFA) in water and 0.1% v/v TFA in acetonitrile, at a flow rate of 1 mL min-1, and detection by a PDA at 214 nm. The chromatographic separation was obtained with retention times of 7.6 and 18.2 min, and the calibration curves were linear over the concentration range of 5 – 200 μg mL-1 (r2 = 0.9993) and 5 – 300 μg mL-1 (r2 = 0.9997), respectively, for SE and RP−LC methods. The limits of detection and quantitation were 1.83 and 5.54 μg mL-1, respectively, for the SE−LC and 1.94 and 5.87 μg mL-1, for the RP−LC. The specificity of the methods was confirmed by degradation studies, interference of the excipients and peaks purity. The accuracy was 100.53 and 100.80%, with bias lower than 0.84 and 1.25%, respectively, for SE and RP−LC. The in vitro bioassay was performed based on the ability of DmAb to inhibit the effect of human RANKL to stimulate the formation of osteoclasts derived from RAW 264.7 cells, by the addition of human RANKL and M-CSF. The LC methods were applied for the determination of DmAb and degraded forms in biotechnology-derived products, and the results were correlated to those of the osteoclastogenesis antiproliferative bioassay, showing significant correlation (p < 0.05). Thus, it is suggested the application of the methods developed and validated by SE and RP−LC to improve quality control of DmAb biotechnology-derived products, thereby contributing to ensure its safety and therapeutic efficacy.