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

Abstract

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'.

Figure 1

Figure depicts the digestion process of rituximab analyzed through SDS-PAGE and Coomassie blue staining. In part (A), lane 2 exhibits the molecular weight standard, while lanes 3–9 present rituximab samples digested for varying durations (0 min, 30 min, 2, 4, 8, and 24 h). Lane 3 represents the intact antibody without treatment, at a 1:40 antibody to enzyme ratio. Part (B) mirrors this setup, with lanes 2–8 containing samples digested for different durations and a 1:20 antibody to enzyme ratio. Lane 9 also displays untreated rituximab under the experimental conditions. Lanes 1 and 10 remain vacant in both parts, offering a comparative assessment of the enzyme concentration’s influence on digestion.

Figure 2

Reduction process was conducted via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining. Lane 1 contained a molecular weight standard/ladder, while lanes 2–10 featured rituximab (F(ab)₂). Rituximab was reduced using β-mercaptoethanol (2-ME) across four different reducing buffer concentrations: 1 mM, 2 mM (A), 5 mM, and 10 mM (B), at five time intervals: 0 min, 1 h, 2 h, 4 h, and 8 h, with a 1:2000 antibody to 2-ME ratio. The optimized reduction process is depicted in (C), where a distinct band pattern at 5 and 10 mM confirmed the successful reduction.

Figure 3

Figure presents confirmation of rituximab Fab’ cationization through a concentration-dependent significant increase (p < 0.05, denoted by asterisk) in BPB dye absorbance observed across concentrations ranging from 0.6 to 1 mg/mL (A–C). Pearson correlation analysis (D) showcases the correlation of chimeric monoclonal antibody rituximab Fab’ cationization profiles across different time intervals and pH conditions. Linearity across concentration ranges is illustrated in panels (E–G), where concentrations of 0.1–2 mg/mL (E) show a notable increase in linearity (y = 0.073x + 0.0097, r² = 0.9624), 0.2–1 mg/mL (F) exhibit a slight increase in linearity (y = 0.0663x – 0.0111, r² = 0.9736), and 0.6–1 mg/mL (G) display a significant increase in linearity (y = 0.0529x + 0.0007, r² = 0.9961), demonstrating higher linearity compared to other concentration ranges.″.

Figure 4

In this figure, the robustness of rituximab’s Fab’ segment cationization is assessed through repeatability and reproducibility validations. Repeatability comparisons between Run1 and Run2 in (A–C), with (C) meeting the 10% acceptance criterion, while (A) exceeded it. Reproducibility was evaluated between R (averaging Run1 and Run2) and Run3 in (D–F), with (F) meeting the criterion and (D) exceeding it. Notably, parts (C) and (F) show significant decreases in %CV, ensuring adherence to acceptance criteria. Dotted lines represent the 10% acceptance criterion.