Probing the conformational behavior of a monoclonal antibody with surfactant affinity capillary electrophoresis (SurfACE).

Multiple peaks are observed for a monoclonal antibody (mAb) when analyzed by "protein MEKC" (micellar electrokinetic capillary chromatography) using SDS-containing run buffers. We present our efforts to understand the mechanism of peak formation and the factors that affect the distribution of the mAb between these peaks. We used "intrinsic" charge ladders of the mAb to determine that peak-to-peak differences in the amount of bound surfactant are comparable to the aggregation numbers of protein-bound micelles.

Intrinsic charge ladders of a monoclonal antibody in hydroxypropylcellulose-coated capillaries.

Capillary zone electrophoresis (CZE) has been used to resolve the charge heterogeneity of an intact ( approximately 150 kDa) monoclonal IgG antibody (mAb). Although this microheterogeneity can also be observed by isoelectric focusing, CZE allows the net charge of each variant to be measured as a function of pH and other solution conditions. Separation was achieved in both borate and Tris run buffers using capillaries that had been statically coated with hydroxypropylcellulose (HPC).