Wolfgang Goetzinger

Examination of capillary zone electrophoresis, capillary isoelectric focusing and sodium dodecyl sulfate capillary electrophoresis for the analysis of recombinant tissue plasminogen activator

The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.

Use of acidic and basic pH and calcium ion addition in the capillary zone electrophoretic characterization of recombinant human deoxyribonuclease, a complex phosphoglycoprotein

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.