Optimization and Validation of the Cationization Method for the Fab' Fragment of Antibody Rituximab.

This study aimed to optimize and validate a cationization method for the Fab' fragment of antibody rituximab, following the guidelines set by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The optimization process involved fragmentation of antibody rituximab into an F(ab)(2) fragment using pepsin, followed by reduction into the Fab' fragment, and subsequent cationization. Various parameters, such as time intervals, enzyme ratios, reducing agent concentrations, pH levels, and reaction durations, were systematically investigated to achieve optimal cationization efficiency. The developed method was validated through spectrophotometry using the bromophenol blue (BPB) dye assay method. The validation process included assessment of linearity, robustness, and sensitivity. Results demonstrated the efficacy of the optimized cationization method, providing a reliable approach for analyzing rituximab antibody cationization of Fab'.