A Poly Acrylamide Gel Electrophoresis PAGE

Polyacrylamide gel electrophoresis (PAGE) is by far the most common mode of electrophoresis. In SDS-PAGE analysis, the protein of interest is denatured with a surfactant sodium dodecylsulfate (SDS) producing a protein-detergent complex whose net negative charge is proportional to its mass. When placed in an electric field, the protein molecules migrate towards the electrode at a rate that is proportional to their molecular weight. Following electrophoresis, the proteins in the slab gel are detected by means of direct staining techniques using either silver stains or Coomassie Brilliant Blue [30],

The identity of the protein may be confirmed by co-migration of the sample with a reference standard. However, as the extent of migration on an SDS-PAGE experiment is insufficient to establish the identity of a protein it must be used in conjunction with other methods for unambiguous identification. In the non-reducing mode, SDS-PAGE is an excellent technique for assessing the state of aggregation or oligomerization of a protein as these will appear as late migrating species in the gel provided these aggregates are stable to the denaturing conditions of SDS-PAGE. The method may also be run without detergent treatment in a "native gel" mode to evaluate the extent of aggregation although such separations tend to be of relatively poor resolution to yield reliable molecular weight information. An estimation of purity may be made by comparison of the sample with that of the reference standard to detect the presence of new impurities.

The amount of protein in the band may be determined either qualitatively using visual observation or quantitatively using laser densitometry. Although not readily amenable to laser densitometry due to high backgrounds, silver staining has much higher sensitivity allowing detection of protein bands at sub-ng levels [31], Detection in the range of 1 ng corresponding to a concentration detection limit of 200 ng/mL can routinely be achieved for a sample load of ~ 5 (xg of a typical sample. Estimation of the impurity levels requires running protein standards of increasing dilutions alongside the sample on the same gel until the intensity of the impurity band visually matches that of a known dilution of the standard. This quantitation technique is similar to that practiced for thin layer chromatography (TLC) and can be adequate for a limit test. While common proteins such bovine serum albumin (BSA) have been used as standards, accuracy may be improved by using a reference standard of the protein of interest to make such assessments. Still, the protein sample may contain several impurities below the detection limit, which may add up to a significant percentage of total impurity in the sample. Enzyme linked immuno-sorbent assays (ELISA), because of their increased sensitivity for the detection of antigenic protein impurities are recommended in combination with electrophoretic methods to achieve absolute quantitation of protein impurities in a given sample.

If a quantitative impurity test is warranted, the use of Coomassie Brilliant Blue staining with laser densitometric quantitation is preferable. In order to validate such a method for routine quantitative use, a number of variables need to be evaluated and controlled. The effectiveness of staining is highly dependent on sample loading, which can vary from protein to protein. This is a key factor in obtaining high quality gels suitable for quantitation and this parameter must be optimized for reliable analysis. Related protein impurities may not stain to the same degree as the protein of interest leading to potentially erroneous values of impurity content. In cases where the identity of the impurity is not known and/or unavailable for use as a standard, one has to assume that the staining of the impurity is similar to that of the protein of interest. In most cases, however, this is a relatively safe assumption. If Coomassie Brilliant Blue staining with laser densitometric detection does not have the desired limit of detection, then the use of other techniques such as capillary electrophoresis with laser induced fluorescence (LIF) detection may be considered.

There are several factors that impact the ruggedness of PAGE analysis. For the separation phase of the analysis, many of the factors that affect the extent of migration of the protein are not critical as the migration of the sample is usually compared with that of a reference standard of the protein of interest, usually run simultaneously on the same gel, as a confirmation of identity. Factors impacting resolution, on the other hand, are significant and must be controlled to assure adequate separation of protein impurities of similar molecular weight. Peak purities are difficult to ascertain in a slab gel experiment and could lead to an overestimation of protein purity because of co-migration of one or more related impurities with the parent peak.

An essential element of validation of SDS-PAGE analysis is evaluation of the gel under reducing conditions. Reduction of the intramolecular disulfide bonds of the protein using such reagents as p-mercaptoethanol or dithiothreitol unfolds the protein, which will then migrate at a slower rate consistent with a higher apparent molecular weight in the gel due to its increased hydrodynamic volume. Appearance of bands at lower apparent molecular weights may indicate the presence of multiple chains in the original protein, which are separated into their respective single chains under the reducing conditions of the experiment. Also, the presence of intramolecular cleavages known as "clips" can result in the same phenomenon. These new bands can be quantitated in much the same way as described earlier. Further treatment of the sample using iodoacetic acid or iodoacetamide results in alkylation of the free sulfhydryl groups leading to further structural changes detectable by SDS-PAGE. While these techniques are very effective in elucidation of secondary structure and identities of possible protein impurities, validation of the reproducibility of such procedures requires optimization and strict adherence to the experimental conditions used for the reduction and derivatization reactions. Otherwise, the gels may contain artifacts that could interfere with the separation precluding meaningful interpretation of the data. Finally, the quality of PAGE experiments are often operator dependent making validation of ruggedness difficult. For this reason, capillary electrophoresis is rapidly gaining popularity for the analysis of protein samples. A more detailed discussion follows.

b. Isoelectric Focusing

Isoelectric focusing (IEF) is a mode of electrophoresis in which proteins are separated by their migration in an electric field over a pH gradient. The pH gradient may be formed by casting a thin layer of gel containing a large series of carrier ampholytes. In an electric field, the carrier ampholytes are arranged in the order of increasing isoelectric point (pi) from the anode to the cathode thereby generating and maintaining a local pH corresponding to their pi. Thus a uniform pH gradient is created across the gel matrix. Samples are applied to the gel surface and upon application of an electric field, each protein component migrates to the region in the gradient where the pH corresponds to its pi. At that point the protein is electrically neutral and becomes stationary or focused in the gel. The protein bands may then be visualized by staining techniques similar to those described above for PAGE analysis. Alternatively, a pH gradient can be generated in the gel when the matrix is cast through addition of charge modified monomers such as immobilines to the gel solution prior to polymerization of the gel. Thus isoelectric focusing is complementary to PAGE analysis, a separation that is primarily based on molecular size. The combination of IEF and PAGE is very powerful in elucidating the nature of protein impurities or degradation products in biopharmaceuticals.

While IEF is a powerful tool used primarily for confirming the identity of a protein biopharmaceutical, upon suitable validation it can also be used as a stability-indicating method to monitor changes to the protein over time [32, 33], Isoelectric focusing is normally run in a native gel using wide pore polyacrylamide or agarose but the addition of nonionic detergents, nonionic chaotropic agents or reducing agents during sample preparation may serve to dissociate molecular complexes and aggregates resulting in enhanced resolution.

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