Using enhanced development tools offered by analytical Quality by Design to support switching of an analytical quality control method

Quality by Design (QbD) principles play an increasingly important role in pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis method under reducing conditions (rCE-SDS), with the aim of replacing SDS-PAGE as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile (ATP), followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments (DoE) studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a correlation factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of AQbD principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to critical quality attributes and the definition of method performance requirements were found to be most relevant for de-risking the analytical method switch, regarding impact on the control strategy

Restoring the biological activity of crizanlizumab at physiological conditions through a pH-dependent aspartic acid isomerization reaction

In this study, we report the isomerization of an aspartic acid residue in the CDR of crizanlizumab as a major degradation pathway. The succinimide intermediate and iso-aspartic acid degradation products were successfully isolated by ion exchange chromatography for characterization. The isomerization site was identified at a DG motif in the CDR by peptide mapping and the biological characterization of the isolated variants showed that the succinimide variant exhibited a loss in target binding and biological activity compared to the aspartic acid and iso-aspartic acid variants of the molecule. The pH influence of this isomerization reaction was investigated using capillary zone electrophoresis (CZE). Below pH 6.3, the succinimide formation was predominant, whereas at pH values above 6.3, iso-aspartic acid was formed and the initial amounts of succinimide dropped to levels even lower than those observed in the starting material. Importantly, while the succinimide accumulated at long term storage conditions of 2 to 8ºC at pH values below 6.3, complete hydrolysis of succinimide was observed at physiological conditions (pH 7.4, 37°C) resulting in full recovery of the biological activity. In this study, we demonstrate that the critical quality attribute succinimide with reduced potency has little or no impact on the efficacy of crizanlizumab due to the full recovery of the biological activity within a few hours under physiological conditions