Study and ICH validation of a reverse-phase liquid chromatographic method for the quantification of the intact monoclonal antibody cetuximab

Abstract

Cetuximab (CTX) is a potent chimeric mouse/human monoclonal antibody (mAb) approved worldwide for treatment of metastatic colorectal cancer. Among the various biological and physical analyses performed for full study on this biopharmaceutic, the determination of the concentration preparations throughout manufacturing and subsequent handling in hospital is particularly relevant. In the present work, the study and validation of a method for quantifying intact CTX by reverse-phase high-performance liquid chromatography with diode array detection ((RP)HPLC/DAD) is presented. With that end, we checked the performance of a chromatographic method for quantifying CTX and conducted a study to validate the method as stability-indicating in accordance with the International Conference on Harmonization guidelines (ICH) for biotechnological drugs; therefore, we evaluated linearity, accuracy, precision, detection and quantification limits, robustness and system suitability. The specificity of the method and the robustness of the mAb formulation against external stress factors were estimated by comprehensive chromatographic analysis by subjecting CTX to several informative stress conditions. As demonstrated, the method is rapid, accurate, and reproducible for CTX quantification. It was also successfully used to quantify CTX in a long-term stability study performed under hospital conditions.

Keywords: Biopharmaceuticals; Cetuximab; Diode detector; Reverse-phase high performance liquid chromatography; Stress study.

Graphical abstract

Fig. 1

General structure of IgG1.

Fig. 2

Standard solutions of 5000 mg/L (a), 2500 mg/L (b), and 1000 mg/L (c) of CTX standard samples analyzed by (RP)HPLC/DAD. UV absorption spectra recorded at the chromatographic peak and the graphic results for the spectral peak purity analysis using 99.3% as similarity factor. Chromatogram of a blank sample (d). UV absorption spectra recorded at the peak (e). Results from peak purity analysis (f).

Fig. 3

Stress study of medicine sample solutions of 2000 mg/L of CTX prepared in 0.9% NaCl. Stronger stress condition studied: (A) Temperature, 50 °C; (B) light stress; (C) weak basic media; (D) strong basic media; (E) weak ionic stress; and (F) strong oxidative stress.

Fig. 4

Graphical results of the long-term study of CTX sample solutions of 2000 mg/L prepared from dilution of Erbitux^® in 0.9% NaCl.