Isoelectric focusing standardization

P. Schmitt-Kopplin, Capillary Electrophoresis Methods and Protocols, Humana, 2007, ISBN 9781588295392 J.P. Landers, Handbook of Capillary Microchip Electrophoresis and Associated Microtechniques, CRC Press, Boca Raton, Florida, 2007, ISBN 9780849333293, C. Henry, Microchip Capillary Electrophoresis, Humana, 2006, ISBN 9781588292933.] The bands are eluted according to their isoelectric points. Isoelectric focusing standards are available which can be used in a preliminary run in order to calibrate the effluent from the column, or alternatively the pH of the effluent is recorded using a glass electrode designed for the purpose. Several efficient commercial equipment are available for separating proteins on a preparative and semi-preparative scale. 

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

The most characteristic abnormality in patients with multiple sclerosis is certainly the intrathecal synthesis of IgG. It can be demonstrated—with different sensitivity— by various methods, which can be divided into qualitative and quantitative methods. The gold standard for the demonstration of intrathecal synthesis of IgG is the detection of oligoclonal bands, which are not present in CSF, in the appropriately diluted serum (i.e., to the same concentration of IgG) by isoelectric focusing. This is a qualitative method and the description of its different modifications and interpretations goes beyond the scope of this chapter. This method is by far the most sensitive, and its sensitivity is reported between 90 and 100%. Here it is suitable to repeat that the detection of plasmocytic forms in cerebrospinal fluid may also be regarded as qualitative proof of intrathecal synthesis of immunoglobulins— although in this case the proof is obviously not specific for IgG from the theoretical point of view. 

Figure B3.1.3 An isoelectric focusing (IEF) gel, pH 3 to 10. Lane 1, 4 pg purified egg white cystatin. Lane M, broad-range pi standards trypsinogen (pi 9.3), lentil lectin-basic band (pi 8.65), lentil lectin-middle band (pi 8.45), lentil lectin-acidic band (pi 8.15), myoglobin-basic band (pi 7.35 visible as a broad band), myoglobin-acidic band (pi 6.85), human carbonic anhydrase B (pi 6.55), bovine carbonic anhydrase (pi 5.85), a-lactoglobulin A (pi 5.20), soybean trypsin inhibitor (pi 4.55), and amyloglucosidase (pi 3.50) in order shown from top of gel. The pi values of the two purified egg white cystatin isomers were determined to be 6.6 (upper band) and 5.8 (lower band). Adapted from Akpinar (1998) with permission from author.

The following table provides a list of proteins that may be used as internal standards, along with their isoelectric points, pi, in quantitative applications of polyacrylamide gel electrophoresis. These proteins may be used in isoelectric focusing or in SDS-PAGE. The isoelectric points are reported at 25°C.1... 

Isoelectric focusing of different samples of rhEPO (recombinant human erythropoietin).The different profiles of isoforms are observed in the different preparations. Lane 3 international standard. 

Kumaratilake, L. M. Thompson, R. C. A. (1979). A standardized technique for the comparison of tapeworm soluble proteins by thin-layer isoelectric focusing in polyacrylamide gels, with particular reference to Echinococcus granulosus. Science Tools, 26 21-4. 

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. 

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.

One of the most troublesome aspects of isoelectric focusing is how to modulate the slope of the pH gradient to increase resolution (equivalent to pH gradient engineering, as easily available in immobilized pH gradients). A simple solution is offered with addition, to the standard 2-pH-units interval, of separa-... 

While all of these devices used normal zone electrophoresis separation techniques, isoelectric focusing (IEF) methods on microchips have also been interfaced with ESI-MS for high-resolution separations of proteins [37], Figure 5 shows the electropherogram and corresponding mass spectra generated by this device. For on-chip sample preconcentration before separation, a polarityswitching technique was employed to achieve subnanomolar detection limits for many peptide standards [38],... 

Capillary isoelectric focusing is a rapid analysis technique with typical run times of 5-30 min, fully automated with on-line detection and real-time data acquisition, and minute sample consumption (a few microliters is enough for repetitive injections). A linear dynamic range over one order of magnitude is achievable, and a detectability down to 5-10 u.g/mL. A resolution of Ap/-0.01 is possible under optimized conditions. Reproducibility of pi determination is typically <0.5% (RSD) using internal standards. 

---