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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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The use of silver-based stains increases the detection sensitivity up to 100-fold, with individual bands containing as little as 1 ng protein usually staining well. However, because silver binds to protein non-stoichiometrically, quantitative studies using densitometry cannot be undertaken.
SDS-PAGE is normally run under reducing conditions. Addition of a reducing agent such as b-marceptoethanol or dithiothreitol (DTT) disrupts inter-chain (and intra-chain) disulphide linkages. Individual polypeptides held together via disulphide linkages in oligomeric proteins will thus separate from each other on the basis of their molecular mass.
The presence of bands additional to those equating to the protein product generally represent protein contaminants. Such contaminants may be unrelated to the product, or may be variants of the product itself (e.g. differentially glycosylated variants, proteolytic fragments, etc.). Further characterization may include Western blot analysis. This involves eluting the protein bands from the electrophoretic gel onto a nitrocellulose filter. The filter can then be probed using antibodies raised against the product. Binding of the antibody to the ‘contaminant’ bands suggests that they are variants of the product.
One concern relating to SDS-PAGE-based purity analysis is that contaminants of the same molecular mass as the product will go undetected, as they will co-migrate with it. 2-dimensional (2-D) electrophoretic analysis would overcome this eventuality in most instances.
2-D electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with
Well 2: ,
Purified Wdl
Weill: protein Molecular mass
Figure 3.31. Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulphide linkages). The electric field is applied across the gel after the protein samples to be analysed are loaded into the gel wells. The rate of protein migration towards the anode is dependent upon protein size. After electrophoresis is complete, individual protein bands may be visualized by staining with a protein-binding dye (a). If one well is loaded with a mixture of proteins, each of known molecular mass, a standard curve relating distance migrated to molecular mass can be constructed (b). This allows estimation of the molecular mass of the purified protein. Reproduced by permission of John Wiley & Sons Inc. from Walsh (2002)
separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment which renders 2-D electrophoretic separation routine is freely available. Application of biopharmaceutical finished products to such systems allows rigorous analysis of purity.
Isoelectric focusing (IEF) entails setting up a pH gradient along the length of an electrophoretic gel. Applied proteins will migrate under the influence of an electric field, until they reach a point in the gel at which the pH equals the protein’s isoelectric point (pI; the pH at which the protein exhibits no overall net charge — only species with a net charge will move under the influence of an electric field). IEF thus separates proteins on the basis of charge characteristics.
This technique is also utilized in the biopharmaceutical industry to determine product homogeneity. Homogeneity is best indicated by the appearance in the gel of a single protein band, exhibiting the predicted pI value. Interpretation of the meaning of multiple bands, however, is less straightforward, particularly if the protein is glycosylated (the bands can also be stained for the presence of carbohydrates). Glycoproteins varying slightly in their carbohydrate content will vary in their sialic acid content, and hence exhibit slightly different pi values. In
Figure 3.32. Photograph of a capillary electrophoresis system (the HP-3D capillary electrophoresis system manufactured by Hewlett-Packard). Refer to text for details. Photo courtesy of Hewlett Packard GmbH, Germany
such instances IEF analysis seeks to establish batch-to-batch consistency in terms of the banding pattern observed.
IEF also finds application in analysing the stability of biopharmaceuticals over the course of their shelf-life. Repeat analysis of samples over time will detect deamidation or other degradative processes which alter protein charge characteristics.
Capillary electrophoresis
Capillary electrophoresis systems are also likely to play an increasingly prominent analytical role in the QC laboratory (Figure 3.32). As with other forms of electrophoresis, separation is based upon different rates of protein migration upon application of an electric field.
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