CAPILLARY ISOELECTRIC-FOCUSING AND SODIUM DODECYL-SULFATE CAPILLARY GEL-ELECTROPHORESIS OF RECOMBINANT HUMANIZED MONOCLONAL-ANTIBODY HER2

Capillary isoelectric focusing (cIEF) and IEF of recombinant humanized monoclonal antibody HER2 (rhuMAbHER2) show five charged isoforms with estimated pi values ranging from 8.6-9.1, The cIEF assay demonstrated good precision with relative standard deviations (R.S.D,) 0.7-3.7% an d 0.4-4.2% for intra and interassay analysis, respectively. The method was linear for the area of the main peak over the concentration range 2-250 mu g/ml with a Pearson correlation coefficient >0.99. The limit of detection for the main peak was determined to be 2 ppm. With both sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) and SDS -polyacrylamide gel electrophoresis, the nonreduced rhuMAbHER2 migrate d as a single major peak with minor peaks in the aggregate and clip re gions. After reduction, the electropherogram and the slab gel showed t he expected heavy chain and light chain fragments with minor peaks in the aggregate and clip regions. The SDS-CGE assay showed good precisio n with R.S.D, values of 0.1-7.8% and 0.1-8.1% for intra and interassay analysis, respectively, The Pearson correlation coefficient for the a rea of the main peak was >0.99 demonstrating linearity for the concent ration range 0.5-500 mu g/ml. The limit of detection for intact rhuMAb HER2 was determined To be 0.5 ppm. The data presented demonstrates the feasibility of replacing the slab gel techniques with capillary elect rophoresis in a quality control environment.