Capillary electrophoresis of proteins

Abstract

Capillary electrophoresis (CE) is a separation technique that combines aspects of both gel electrophoresis and high performance liquid chromatography (HPLC). As is the case for gel electrophoresis, the separation in CE is based upon differential migration in an electrical field. Like HPLC, the detection of the migrating sample analytes may be monitored on-line or postcolumn/capillary for both quantitation and characterization, thereby eliminating the need for labor-intensive staining and destaining. The CE separation may take place within free solution or in the presence of a viscous gel medium. Since its initial description by Hjerten in 1967 (1), CE techniques analogous to most conventional electrophoretic methods have been demonstrated, including zone electrophoresis, displacement electrophoresis (isotachophoresis), isoelectric focusing, and molecular sieving separations. Data presentation and analysis are also similar to HPLC in that the detector output can be displayed as an " electropherogram" representing peaks on a baseline, and can therefore be integrated by area or height to provide quantitation. In contrast to conventional gel electrophoresis where multiple samples are run in parallel fashion on one gel, in CE (as in HPLC) a single sample is injected into the end of a capillary and a sample set is run in serial fashion. Electroosmotic flow (EOF), a phenomenon caused by a high-density charged surface such as that present in a uncoated fused silica capillary, is an additional force with which analytes can be transported down the capillary in the presence of an electrical field via plug-flow, resulting in very high efficiencies (i.e., narrow peaks). For the analysis of small molecules, the success of CE has been limited by poor reproducibility owing to capillary wall-Analyte interactions, variable EOF, and limited detection sensitivity. In the world of biopolymers, however, the story has been quite different as CE has achieved significant success for the enhancement of separation methods traditionally performed through the use of gels. The most notable example is the adaptation of CE for use in the multichannel DNA sequencers that were employed for the sequencing of the human genome in less than 2 yr. Lesser publicized but equally successful has been the application of CE as a replacement for gel electrophoresis analyses of protein therapeutics in the biopharmaceutical industry. Several major biopharmaceutical companies have developed and validated CE protein methods according to ICH and FDA guidelines for control and release of marketed products. This chapter will focus primarily upon three CE protein analysis techniques that have become routinely employed: (1) the use of CE for free solution analysis of glycoproteins (2) the use of CE as a replacement for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and (3) the use of CE as a replacement for gel isoelectric focusing. © 2008 Humana Press.