Risk-Based Approach and Design of Experiments in the development of a Capillary Isoelectric Focusing Analytical Procedure Platform for the charge heterogeneity characterization of monoclonal antibodies

The research provides a regulatory-compliant conventional electrokinetic methodology for biopharmaceutical quality control. A Capillary IsoElectric Focusing (cIEF) Analytical Procedure Platform for the charge heterogeneity characterization of monoclonal antibodies (mAbs) was developed. The Analytical Quality by Design approach was implemented to define a robust Method Operable Design Region (MODR). The Analytical Target Profile consisted in an Analytical Procedure Platform able to measure the mAbs isoforms pI values with a bias lower than 4%. Risk Management tools were used to evaluate and control the risk of Analytical Target Profile failure. Risk Analysis identified potential Critical Procedure Parameters, including urea and methyl cellulose concentrations, cathodic/anodic stabilizer levels, carrier ampholytes composition. The carrier ampholytes composition was optimized utilizing a mixture design. A Scheffé mixture model allowed the selection of experiments, and the Partial Least Squares regression provided high predictive power models for the three defined responses, i.e. the calibration biases at the bracketing markers of Infliximab (pI 9.5 and pI 7.0) and the overall fit of the pI vs. migration time relationship. Within the MODR, the optimized conditions were achieved using as the specific carrier ampholyte a blend constituted of 1.5% at pH 3-10, 1.5% at pH 5-8, and 2.0% at pH 8-10.5. The cIEF method was validated and applied to successfully profile charge heterogeneity of mAbs for monitoring the stability, supporting hospital pharmacy practices and contributing to the sustainable management of biopharmaceuticals.