Abstract

Background: The majority of non-small cell lung cancer (NSCLC) creates advances in the knowledge of tumor biology and mechanisms of oncogenesis have granted the singling out of several molecular targets for NSCLC treatment. Bevacizumab is an anti-growth factor vascular endothelial growth factor (VEGF) monoclonal antibody, it is the antiangiogenic agent at the most advanced stage of development in the treatment of NSCLC.This drug was selected because of its inter individual differences in clinical response, showing its therapeutic importance in the treatment of NSCLC. The urgent need for proper analytical technique for determination of its concentration in plasma samples for the purpose of therapeutic drug monitoring (TDM) is highlighed.

Methods: This study describes the development and validation of a new enzyme-linked immunosorbent assay (ELISA) with high sensitivity and selectivity for bioanalysis of bevacizumab (BEV), a monoclonal antibody used for immunotherapy of different types of cancer. The assay was developed for the purpose of studying the pharmacokinetics (PK), pharmacodynamics (PD), and therapeutic drug monitoring (TDM) of BEV. The assay involved the non-competitive binding reaction of BEV to its specific antigen (human vascular endothelial growth factor protein; VEGF) followed by a chromogenic enzyme reaction for immune detection of the BEV-VEGF complex and color development. Technically, BEV (in the samples) was captured by VEGF antigen protein immobilized onto a 96-well assay plate. The BEV-VEGF complexes formed onto the plate wells were quantified using horseradish peroxidase labeled anti-human IgG (HRP-IgG) and 3,3`,5,5`-tetramethylbenzidine (TMB) as a chromogenic substrate for peroxidase enzyme. The optimum conditions for conducting the proposed ELISA were established and its analytical performance was evaluated as per the guidelines for the validation of immunoassays for bioanalysis.

Results: The optimum conditions for conducting the proposed ELISA were established and its analytical performance was validated according to the recommendations for the bioanalytical method validation of ELISA to support PK assessments of macromolecules. The assay limit of detection was 1.01 ng mL-1 and the effective working dynamic range was 1.56-100 ng mL-1 (r2 = 0.9999). The accuracy and precision of the assay were proved.

Conclusion: The present study described the development and validation of a highly sensitive and selective ELISA for determination of BEV in plasma samples. The developed ELISA method allowed quantitation limits in human plasma samples at 3.07 ng mL-1 which are 15-fold lower than the reported methods in the literature. The procedures of the proposed ELISA is very convenient to be carried out in a 96-well plate. An analyst can process a batch of ~ 200 samples, in triplicate, per day. This facilitates the processing of a large number of samples in a clinical setting. Because the assay produces color as the analytical signal, only a simple colorimetric plate reader is required. Unlike LC methods, the marked selectivity of the proposed ELISA eliminated the need for pretreatment of plasma samples by affinity chromatography or other sophisticated equipment. For these reasons, the proposed ELISA for BEV is expected to contribute in studying its PK, PD, TDM, and assessing the expected bioavalilability of biosimillars.

Keywords: Cancer immunotherapy, therapeutic monoclonal antibodies, bevacizumab, bioanalysis, ELISA, vascular endothelial growth factor (VEGF).

Graphical Abstract