Determination of Charge Heterogeneity and Level of Unconjugated Antibody by Imaged cIEF

Imaged capillary isoelectric focusing (icIEF) is capable of monitoring the charge heterogeneity profile of conjugated antibodies. The electropherogram from icIEF can be integrated to quantitate the amount of unconjugated antibody present in a conjugate sample. This chapter describes an icIEF method where a conjugate sample was first prepared by mixing with appropriate ampholytes, pI markers, and additives. Then, the sample was focused in a fluorocarbon-coated fused silica capillary, where absorbance images were taken. Quantitation of the unconjugated antibody was achieved by using a calibration curve.