Use of acidic and basic pH and calcium ion addition in the capillary zone electrophoretic characterization of...

This paper describes the analysis of recombinant human deoxyribonuclease (rhDNAse), an acidic and complex phosphoglycoprotein, by capillary zone electrophoresis (CZE). Separation performance was found to be dramatically improved by the addition of calcium ions to the CZE running buffer, due to the influence of calcium binding on the charge and the electrophoretic behavior of rhDNAse. The pH dependent calcium binding effects on the electrophoretic separation were demonstrated at both acidic and basic pH, resulting in a two-dimensional (pH 4.8 and 8.0) calcium aided analysis that achieved multipeak resolution of the complex, glycosylation based, charge microheterogeneity of rhDNAse. Two-dimensional investigation of neuraminidase- and alkaline phosphatase-digested protein further demonstrated that the acidic pH resolved acidic charge heterogeneity and that the basic pH discriminated neutral heterogeneity. This work demonstrates the resolving power of CZE for the analysis of a complex microheterogeneous glycoprotein, and emphasizes the importance of employing multiple separation conditions in accordance with known structural characteristics of the protein.