Analytical Quality by Design-driven development of a robust CIEF platform for the charge heterogeneity profiling of moderately basic monoclonal antibodies

Monoclonal antibodies (mAbs) are complex glycoproteins characterized by significant structural heterogeneity. Post-translational modifications, lead to various charge isoforms; these variants can impact both safety and efficacy, thus their reliable monitoring is a Critical Quality Attribute (CQA) in biopharmaceutical development. The present communication presents the development of a Capillary IsoElectric Focusing (cIEF) platform for the characterization of moderately basic mAbs. Following Analytical Quality by Design (AQbD) principles, we established a robust Method Operable Design Region (MODR) using Infliximab as a model analyte. Our Analytical Target Profile (ATP) focused on achieving a robust separation allowing the estimation of the apparent isoelectric point (pI) of the mAb with bias below 4%. Owing to the complex composition of the mixture subjected to the analysis, comprising the mAb and the components of the electrokinetic running buffer, a comprehensive Risk Analysis was undertaken identifying urea, methyl cellulose, and carrier ampholytes (CAs) composition as Critical Procedure Parameters. To manage their complex interactions, a Scheffé mixture design was employed. Partial Least Squares (PLS) regression provided high-predictivity models for three key responses: calibration biases at the two markers bracketing the mAb and the overall linearity of the pI vs. migration time relationship. The optimized MODR conditions utilized a CA blend consisting of 1.5% pH 3-10, 1.5% pH 5-8, and 2.0% pH 8-10.5. The resulting platform was validated showing pI measurement biases < 0.3%. The method demonstrated exceptional linearity, trueness, and precision (RSD < 0.52% for the main peak area) for Infliximab charge variants. The versatility of this horizontal platform was successfully demonstrated through the distinctive profiling of other therapeutic mAbs, including Bevacizumab and Daratumumab, under identical experimental conditions. This work offers a refined, regulatory-aligned electrophoretic methodology suitable for routine biopharmaceutical quality control.