Charge Heterogeneity of Monoclonal Antibodies by Multiplexed Imaged Capillary Isoelectric Focusing Immunoassay with Chemiluminescence Detection

Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional ...