Evaluation of Analytical Quality by Design Approaches on the Development and Optimization of Size Exclusion...

The development of analytical procedures, following the International Council of Harmonisation (ICH) regulatory requirements, is crucial for managing the lifecycle of methodologies. This study applies analytical quality by design (AQbD) principles to optimize size exclusion chromatography (SEC) determination for bovine serum albumin (BSA), chosen as a model drug. Key method variables, including mobile phase buffer concentration, flow rate, and column type, were systematically evaluated using risk assessment and design of experiments (DoE). A consistent and rigorous workflow was applied to prove and ensure the robustness of this methodology. The SEC method was established through a framed analytical target profile (ATP), complying with chromatography parameters like tailing, resolution, capacity, and sensitivity. Those were achieved with a Waters XBridge BEH200A SEC column (200 Å, 3.5 µm, 7.8 mm × 300 mm) at 20°C, using a phosphate buffer saline (PBS) solution at pH 7.4 in isocratic elution with a flow of 0.3 mL/min and UV detection of 280 nm. The method was validated according to ICH Q2 (R²) and Q14 guidelines. The linear response was demonstrated over concentration ranges of 100 µg/mL and 10 mg/mL (R² = 0.9998), and desirable standards of accuracy and precision were met. Although this work highlights the benefits of AQbD in developing trustworthy analytical methods, SEC constitutes a challenge for applying these principles considering the short lifetime of the column, reproducibility, and sensitivity.