C Capillary Electrophoresis

Capillary electrophoresis (CE) has recently emerged as the technique of choice in obtaining high resolution electrophoretic separations of proteins and peptides [34]. These separations are fast, relatively easy to perform, of very high resolution, amenable to automation and may be linked to a variety of detectors. Also, a variety of electrophoresis experiments such as conventional zone electrophoresis, IEF, isotachophoresis and micellar electrokinetic chromatography (MEKC) may be conveniently performed using the same instrument with relative ease. Capillaries may be also be packed with gels to operate in the capillary gel electrophoresis mode to mimic PAGE separations but with significantly higher resolution and ease of operation. Method development using CE is generally straightforward as a number of separation variables such as different buffers, pH, temperature, and additives may be evaluated in a short time by simply flushing the appropriate solutions through a single capillary. Capillaries may be coated with various agents to allow manipulation of selectivity using wide pH ranges at below and above the isoelectric points of the protein of interest.

Capillary electrophoresis is rapidly gaining popularity for assessing the purity of proteins in part due to its ability to separate charged structural variants such as deamidation products with very high resolution. For erythropoietin (EPO) whose carbohydrate structure makes up 40% of its weight, CE was demonstrated to resolve the various glycoforms by manipulation of pH and the incorporation of organic modifiers [35]. When linked to mass spectrometry, CE is a powerful technique for structural characterization of proteins, particularly for products of peptide mapping. IEF experiments may also be conducted in the CE mode by separation of the protein in a stable pH gradient. However, IEF by CE poses special challenges in achieving good accuracy and precision, such as minimization of endosmotic flow to facilitate on line detection, mobilization of the peaks to the detector upon completion of focusing without loss of electrophoretic resolution and maintenance of a stable pH gradient across the capillary column by maintaining a uniform ampholine distribution over the pH range of interest. The IEF experiment is further complicated by the high background absorbance of ampholines at 280 nm which are manifested as unstable baselines in the high sensitivity detections employed by CE. SDS-PAGE type separations to assess molecular size can be performed in the gel mode as a high resolution, high sensitivity alternative to slab gel electrophoresis. This is accomplished by covalently bonding polymerized polyacrylamide to the fused silica capillary by means of cross linking agents. However, CGE suffers from poor reproducibility due to difficulty in obtaining uniform gels. In addition, at the high voltages commonly used in CE, the carboxylate ions are formed as a degradation product of acrylamide leading to destruction of the gel matrix.

It is clear that CE is an indispensable technique for protein analysis as evidenced by its extensive use in various facets of drug development from in process control during fermentation to stability monitoring of protein drug products. Although CE is still primarily being used in a research mode, its utility in providing reliable analysis in a quality control environment is rapidly being realized. Capillary electrophoresis has been shown to be equivalent to RP-LC for the analysis of human growth hormone in terms of its linearity of response, precision and sensitivity. In addition, use of internal reference standards have been demonstrated to significantly improve reproducibility of migration times and detector response between injections. More recently, CE was shown to have adequate accuracy, precision, selectivity, linearity of response and limit of detection to assay the concentration of an oligonucleotide drug product for quality control release as well stability [62], As more laboratories explore the versatility of CE, method validation paradigms will evolve for the successful application of the various modes of this technique for routine pharmaceutical analysis.

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