Optimization of microchip-based electrophoresis for monoclonal antibody product quality analysis revealed needs for...

Microchip-based electrophoresis has gained increasing popularity in biopharmaceutical development and testing laboratories because of its automation and rapid analysis capabilities. One application of microchip-based electrophoresis is the assessment of size-based variants for product purity analysis. However, monoclonal antibodies analyzed by this technique sometimes exhibited different electrophoretic behaviors. In this study, when three IgG1 and five IgG4 were analyzed using microchip-based electrophoresis under reducing conditions, one of the IgG1s, denoted as mAb1, exhibited an atypical profile attributed to its specific heterogeneity resulting in separation of its heavy chain into two main species. During investigation of the atypical profile, several parameters that were critical to optimal resolution were evaluated, and the data pointed toward incomplete denaturation of mAb1 due to lack of sufficient surfactant in the vendor provided sample buffer (0.7% surfactant). Denaturation studies demonstrated that, although typical antibody profiles could be achieved at 0.7% surfactant for most antibodies analyzed, five out of eight antibodies were not fully denatured until the surfactant concentration reached 0.9% or higher, and mAb1 required a surfactant concentration of 1.3% for complete denaturation. Molecular modeling analysis revealed features in surface charge, hydrophobicity, and structure from mAb1 that led to its unique surfactant concentration-dependent electrophoretic behaviors observed. The optimized method was further evaluated for specificity, linearity, precision, and limit of quantitation for mAb1, and compared with that of conventional CE-SDS.