Estudos de métodos cromatográficos e biológico para análise do anticorpo monoclonal ramucirumabe

Abstract

Ramucirumab (RAM) is a human anti–VEGFR–2 monoclonal antibody that develops its anti–angiogenic function by decreasing vascularization to reduce tumor growth. . It is clinically used for the treatment of adult patients with gastric or advanced gastroesophageal junction cancer, lung cancer, colorectal cancer and liver cancer. Structurally it is an IgG class immunoglobulin produced by recombinant DNA technology in murine myeloma (NS0) cells. In this work, reversed-phase liquid chromatography (RP–LC) and size exclusion liquid chromatography (SE–LC) methods were developed and validated for the evaluation of RAM in biopharmaceuticals. In addition, in vitro A549 cell culture bioassay was developed to evaluate the potency of the biotechnological product. In the RP–LC method the ZORBAX 300SB–C18 column was maintained at 80 °C. The mobile phase consisted of 0.1% v / v trifluoracetic acid (TFA) in water and 0.1% v / v TFA in acetonitrile (ACN), with gradient elution, flow rate1 mL / min, and diode array detection (DAD) at 214 nm. For the SE–LC method, a BioSepSECS 2000 column was maintained at 35 °C, mobile phase composed of monobasic potassium phosphate (140 mM), dibasic potassium phosphate (60 mM) and potassium chloride (250 mM), pH 7.0 with isocratic elution of 0.8 mL / min and DAD detection at 214 nm. The RAM was eluted with retention times at 9.7 and 8.7 min, being linear in the concentration range of 0.125 – 10 mg / mL (r2 = 0.9998) and 0.250 – 10 mg / mL (r2 = 0. 9994), respectively, for the RP–LC and SE–LC methods. The specificity of the methods was verified and confirmed by studies of forced degradation, interference of formulation excipients and peak purity. The limits of detection and quantification were 0.03 and 0.08 mg / mL for the RP–LC method and 0.04 and 0.13 mg / mL for SE–LC. The mean values for accuracy were 99.72 and 100.08 with bias of 0.57 and 0.60%, respectively. The antiproliferative A549 cells in vitro bioassay was developed and applied to evaluate the biology activity of RAM. Correlation between the RP–LC and SE–LC analytical methods with the cell culture bioassay was calculated by Pearson's correlation coefficient (r), which gave the values of r = 0.9053 and r = 0.9353, respectively, demonstrating the significance of the data. Thus, it is suggested that the methods developed and validated by RP–LC and SE–LC could be applied in conjunction with the bioassay to improve quality control, contributing to ensure the safety and therapeutic efficacy of the biotechnological product.