Capillary isoelectric focusing applications

FIGURE 12 Application of capillary isoelectric focusing (clEF) for the determination of apparent p/ values of rMAb samples. Capillary Bio-Rad Bio-CAP XL capillary (50 pm x 24 cm) ampholyte 80% clEF Bio-Lyte Ampholyte 3-10 (2% solution with 0.5% TEMED, 0.2% HPMC) anolyte 20 mM phosphoric acid catholyte 40 mM sodium hydroxide focusing l5kV (625V/cm) for 5 min mobilization 20 kV (833V/cm) for 25 min with zwitterions (cathodic mobilizer from Bio-Rad) capillary temperature 25°C. (Reprinted from reference 40, with permission.)... 

Among the electrophoretic methods of chiral resolution, various forms of capillary electrophoresis such as capillary zone electrophoresis (CZE), capillary isotachophoresis (CIF), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), affinity capillary electrophoresis (ACE), and separation on microchips have been used. However, in contrast to others, the CZE model has been used frequently for this purpose [44]. On the other hand, drawbacks associated with the electrophoretic technique due to lack of development of modem chiral phases have limited the application of these methods. Moreover, the electrophoretic techniques cannot be used at the preparative scale, which represents an urgent need of chiral separation science. 

B. Michalke, P. Schramel, Application of capillary zone electrophoresisDinductively coupled plasma mass spectrometry and capillary isoelectric focusing-inductively coupled plasma mass spectrometry for selenium speciation, J. Chromatogr. A, 807 (1998), 71D80. 

Capillary isoelectric focusing (CIEF) — An electrophoretic separation technique (- electrophoresis) for the separation of amphoteric analytes (-> ampholyte) according to their - isoelectric points by the application of an electric field along a pH gradient formed in a capillary. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

In spite of all these clear advantages, capillary isoelectric focusing is still not used as a routine method in bioanalysis, although more and more papers show important applications. Study of the proteome combined with microchip technology may, however, cause sudden progress in this innovative technique. In the past decade several reviews have appeared about CIEF separations [5-17]. This chapter summarizes the theory and the newest innovations, as well as applications of CIEF. 

Figure 4 Schematic representation of the sample application and the single-step capillary isoelectric focusing with electroosmotic zone displacement. A pressure application may standardize the migration of the focused zones toward the detection point.

Capillary isoelectric focusing has proven its separation potential and special advantages in proteome bioanalysis. Several applications have been reported using this unique methodology. Notwithstanding, further improvements are necessary to design fully automated chip-based devices and a more advanced coupling to various detection systems. 

The next four chapters discuss the basic principles underlying operation and method development of the most common electrodriven analytical techniques CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a comprehensive approach for method development in CE with an emphasis on small-molecule applications. This is followed by Kilar s chapter describing the principles of and method development in cIEF, as well as recent innovations... 

A range of electrophoretic techniques use the capillary prindple capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF) and micellar electrokinetic capillary chromatography (MECC). Some of these techniques, particularly CZE and CGE, have already established themselves as important analytical tools others, notably MECC, may open new approaches in analytical biochemistry. Table 4-11 summarises the areas of application of the various techniques in what follows we shall focus on CZE, CGE and MECC. 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Hempe JM, Vargas A, Graver RD. Clinical analysis of structural hemoglobin variants and Hb Ale by capillary isoelectric focusing. In Petersen JR, Mohammad AA, eds. Clinical and forensic applications of capillary electrophoresis. Totowa, NJ Humana Press, 2001 145-63. 

Kilar F (2003). Recent applications of capillary isoelectric focusing. Electrophoresis. 24 3908-3916. 

Over the last two decades, capillary electrophoresis (CE) has been developed as a powerful separation technique for complex mixtures. Its advantages include a high separation efficiency, short analysis time, small sample requirement, and applicability to a wide range of analytes. The basic modes of CE that are presently being exploited include capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), capillary isotachophoresis, capillary isoelectric focusing, and capillary gel electrophoresis. 

Capillary isoelectric focusing (CIEF) is designed to separate solutes based on their p/, the pH where they are electrically neutral. Because solutes do not migrate when they are neutral, the mobilization step distinguishes CIEF from other capillary electrophoretic techniques. This entry reviews the basis for CIEF including pH gradient formation, mobilization techniques, additives, and applications. 

Shih, M.L. Korte, W.D. Analysis of hemoglobin derivatives by capillary isoelectric focusing and its application in the antidotal research of cyanide poisoning. Anal. Biochem. 1996,238,137. 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

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