Detection of protein-based product impurities

The use of silver-based stains increases the detection sensitivity up to 100 fold, with individual bands containing as little as lng of protein usually staining well. However, because silver binds to protein non-stoichiometrically, quantitative studies using densitometry cannot be undertaken. 

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. Two-dimensional electrophoretic analysis would overcome this eventuality in most instances. 

Two-dimensional 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 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 that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

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 

Isoelectric focusing 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 that alter protein charge characteristics. 

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A number of different techniques may be used to characterize protein-based biopharmaceutical products, and to detect any protein-based impurities that may be present in that product (Table 7.2). Analysis for non-protein-based contaminant is described in subsequent sections. 

Applications in Biotechnology. Biopharmaceutical products must be well characterized by means of analytical techniques. RP-HPLC with UV detection is suitable for the content assay and impurity profiling because of its high resolution. However, the bioactivity of protein-based pharmaceuticals not only relies on the primary structure but also on the higher order structure, i.e., the molecular confirmation, which is not revealed by UV absorbance. Circular dichroism and intrinsic fluorescence detection have been combined with UV detection in RP-HPLC to monitor the conformational properties of active compounds and impurities in biopharmaceutical products [57]. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

Having demonstrated production of designed protein-based polymers, the next issue is one of achieving purification adequate for the intended application. The most sensitive means with which to detect impurities of relevance to medical applications is the Western immunoblot technique. )Wth this technique, levels of purification are demonstrated in Figure 9.12 for the following model proteins ... 

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