Stability Evaluation of Epidermal Growth-Factor Receptor (EGFR) Inhibiting Monoclonal Antibody, Cetuximab

Abstract

Introduction: Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor and approved for treatment of colorectal cancer and squamous cell carcinoma of head and neck. Objectives: The aim of this research was to study the stability of cetuximab under various stress conditions including mechanical, thermal, light stress, and various freeze-thaw cycles. Methods: To determine the effects of environmental stresses on the physicochemical properties and bioactivity of cetuximab, a combination of physicochemical and cell-based biological methods including size exclusion chromatography(SEC), cation exchange chromatography(CEX), flow cytometry-based binding assay, and MTS cell viability/proliferation assay was used. SEC and CEX methods were developed and validated according the International Conference on Harmonization guidelines. Results: The results of validation studies showed that the developed SEC and CEX methods were linear (r > 0.99) in the investigated concentration range of cetuximab. The precision, accuracy and robustness of the developed methods meets the demands of pharmaceutical analysis and both methods were stability-indicating. The results obtained by the SEC and CEX methods revealed that incubation of cetuximab at 25 and 30°C, shaking, and various freeze-thaw cycles caused no physicochemical instability. However, functional analysis of the samples exposed to the above mentioned conditions revealed a significant decrease in the bioactivity of cetuximab indicated by a significant reduction in the cell binding and growth inhibitory effects of cetuximab in EGFR overexpressing cancer cell line(A431). Incubation of cetuximab at 40 and 50°C led to polymerization and fragmentation of the mAb and caused a significant decrease in the bioactivity of the mAb. Our findings show that the light exposure had the most destructive effects on physicochemical and biological characteristics of cetuximab. Conclusion: we found that all mentioned stress conditions significantly affect the bioactivity of cetuximab. Our findings highlight the importance of bioactivity evaluation of biopharmaceuticals in their quality control assessment.