Development of iCIEF platform method for analysis of charge heterogeneity of monoclonal antibodies

Objective To develop a platform method using imaged capillary isoelectric focusing (iCIEF) for measuring charge heterogeneity of monoclonal antibody (mAb) and validate its suitability with different isoforms of mAbs (IgG1, IgG2, IgG4). Methods The volumes of carrier ampholyte and cathode stabilizer, focusing time, and urea concentration were optimized in iCIEF method development. The specificity, precision, linearity, accuracy, and robustness of this method were validated using 3 isoforms of mAbs (IgG1, IgG2, IgG4). Results Under following conditions: 70 μl 3 mol/L urea-0.5% methyl cellulose, 4 μl broad-range carrier ampholyte (pH3-10), 2 μl cathode stabilizer (500 mmol/L Arg), 2 μl each isoelectric point 6.14 and 9.99 Markers, 0.2 mg/ml mAb (sample) was prepared. The detection parameters were determined to be pre-focusing 1 min at 1 500 V and focusing 8 min at 3 000 V. The specificity of the platform method was good, and formulation buffer had no interference with detection. Repeated testing of 6 parallel samples and testing 12 samples by different persons at different time both showed that, the relative standard deviations of all compositions met requirements. The linear coefficients of determination (R2) were ≥0.99 for main and acidic compositions in mAb and ≥0.98 for basic composition, when the final concentrations of mAbs (samples) were 0.1-0.3 mg/ml. The accuracies of the platform method for detecting different composition contents in mAbs were 92%-105%. The robustness results of design of experiment showed that carrier ampholyte volume and capillary lot had significant impacts on this platform method. Conclusions The iCIEF platform method for analysis of charge heterogeneity of different isoforms of mAbs is developed, which has high separation resolution and good precision, accuracy, and robustness. This method can provide a more effective tool for charge heterogeneity characterization and quality control of mAbs. Key words: Electrophoresis, capillary; Isoelectric focusing; Antibodies, monoclonal; Laboratory techniques and procedures; Charge heterogeneity