Capillary Isoelectric Focusing (cIEF) Analysis of a Monoclonal Antibody under a Denatured and Reducing Condition (CE Pharm 2011)

Capillary isoelectric focusing (cIEF) is the best tool for protein charge heterogeneity characterization. Common sources of charge heterogeneity for monoclonal antibodies include heavy chain C-terminal lysine heterogeneity, deamidation, and sialyation associated with the glycosylation sites on the antibodies. Under the denatured and reduced condition, disulfide bonds in the antibodies can be reduced and the monoclonal antibodies are broken into heavy chains and light chains. The heavy chain and light chain are expected to have different pI values, thus, they can be separated from each other by IEF. In this way, the contributions of the heavy chain and light chain to the entire antibodies' charge heterogeneity can be observed by IEF analysis. In this presentation, monoclonal antibodies are analyzed by cIEF under a 8 M urea (denatured) and DTT condition. In the example shown in the presentation, the heavy chain and light chains are well separated and the charge heterogeneity of both is observed.