Detection of proteinbased product impurities

SDS polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 7.1). This technique is well established and easy to perform. It provides high-resolution separation of polypeptides on the basis of their molecular mass. Bands containing as little as 100 ng of protein can be visualized by staining the gel with dyes such as Coomassie blue. Subsequent gel analysis by scanning laser densitometry allows quantitative determination of the protein content of each band (thus allowing quantification of protein impurities in the product).

The use of silver-based stains increases the detection sensitivity up to 100 fold, with individual bands containing as little as 1ng of 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 P-mercaptoethanol or dithiothreitol (DTT) disrupts interchain (and intrachain) disulfide linkages. Individual polypeptides held together via disulfide 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 fragment, 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.

Well 1:

Well 2: Purified protein

Well 3:

Molecular mass standards

Crude protein mix

Well 2: Purified protein

Crude protein mix

Mobility through gel (mm)

Log molecular mass

Polyacrylamide slab gell n.

Mobility through gel (mm)

Log molecular mass


Figure 7.1 Separation of proteins by SDS-PAGE. Protein samples are incubated with SDS (as well as reducing agents, which disrupt disulfide 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

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.

Isoelectric focusing 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). Isoelectric focusing 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 such instances, isoelectric focusing analysis seeks to establish batch-to-batch consistency in terms of the banding pattern observed.

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.

7.4.1 Capillary electrophoresis

Capillary electrophoresis systems are also likely to play an increasingly prominent analytical role in the QC laboratory (Figure 7.2). As with other forms of electrophoresis, separation is based upon different rates of protein migration upon application of an electric field.

As its name suggests, in the case of capillary electrophoresis, this separation occurs within a capillary tube. Typically, the capillary will have a diameter of 20-50 ^m and be up to a 1 m long (it is normally coiled to facilitate ease of use and storage). The dimensions of this system yield greatly increased surface area:volume ratio (compared with slab gels), hence greatly increasing the efficiency of heat dissipation from the system. This, in turn, allows operation at a higher current density, thus speeding up the rate of migration through the capillary. Sample analysis can be undertaken in 15-30 min, and on-line detection at the end of the column allows automatic detection and quantification of eluting bands.

The speed, sensitivity, high degree of automation and ability to quantitate protein bands directly render this system ideal for biopharmaceutical analysis.

Figure 7.2 Photograph of a capillary electrophoresis system (the HP-3D capillary electrophoresis system manufactured by Hewlett Packard). Refer to text for details. Photograph courtesy of Hewlett Packard GmbH, Germany

7.4.2 High-performance liquid chromatography

HPLC occupies a central analytical role in assessing the purity of low molecular mass pharmaceutical substances (Figure 7.3). It also plays an increasingly important role in analysis of mac-romolecules, such as proteins. Most of the chromatographic strategies used to separate proteins under 'low pressure' (e.g. gel filtration, ion exchange, etc.) can be adapted to operate under high pressure. Reverse-phase-, size-exclusion- and, to a lesser extent, ion-exchange-based HPLC chro-matography systems are now used in the analysis of a range of biopharmaceutical preparations. On-line detectors (usually a UV monitor set at 220 or 280 nm) allows automated detection and quantification of eluting bands.

HPLC is characterized by a number of features that render it an attractive analytical tool. These include:

• excellent fractionation speeds (often just minutes per sample);

• superior peak resolution;

Figure 7.3 Photograph of a typical HPLC system (the Hewlett Packard HP1100 system). Photograph courtesy of Hewlett Packard GmbH, Germany

• high degree of automation (including data analysis);

• ready commercial availability of various sophisticated systems.

Reverse-phase HPLC (RP-HPLC) separates proteins on the basis of differences in their surface hydophobicity. The stationary phase in the HPLC column normally consists of silica or a polymeric support to which hydrophobic arms (usually alkyl chains, such as butyl, octyl or octadecyl groups) have been attached. Reverse-phase systems have proven themselves to be a particularly powerful analytical technique, capable of separating very similar molecules displaying only minor differences in hydrophobicity. In some instances a single amino acid substitution or the removal of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC. In most instances, modifications such as deamidation will also cause peak shifts. Such systems, therefore, may be used to detect impurities, be they related or unrelated to the protein product. RP-HPLC finds extensive application in, for example, the analysis of insulin preparations. Modified forms, or insulin polymers, are easily distinguishable from native insulin on reverse-phase columns.

Although RP-HPLC has proven its analytical usefulness, its routine application to analysis of specific protein preparations should be undertaken only after extensive validation studies. HPLC in general can have a denaturing influence on many proteins (especially larger, complex proteins). Reverse-phase systems can be particularly harsh, as interaction with the highly hydrophobic stationary phase can induce irreversible protein denaturation. Denaturation would result in the generation of artifactual peaks on the chromatogram.

Size-exclusion HPLC (SE-HPLC) separates proteins on the basis of size and shape. As most soluble proteins are globular (i.e. roughly spherical in shape), separation is essentially achieved on the basis of molecular mass in most instances. Commonly used SE-HPLC stationary phases include silica-based supports and cross-linked agarose of defined pore size. Size-exclusion systems are most often used to analyse product for the presence of dimers or higher molecular mass aggregates of itself, as well as proteolysed product variants.

Calibration with standards allows accurate determination of the molecular mass of the product itself, as well as any impurities. Batch-to-batch variation can also be assessed by comparison of chromatograms from different product runs.

Ion-exchange chromatography (both cation and anion) can also be undertaken in HPLC format. Though not as extensively employed as reverse-phase or size-exclusion systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated forms.

7.4.3 Mass spectrometry

Recent advances in the field of mass spectrometry now extend the applicability of this method to the analysis of macromolecules such as proteins. Using electrospray mass spectrometry, it is now possible to determine the molecular mass of many proteins to within an accuracy of ± 0.01 per cent. A protein variant missing a single amino acid residue can easily be distinguished from the native protein in many instances. Although this is a very powerful technique, analysis of the results obtained can sometimes be less than straightforward. Glycoproteins, for example, yield extremely complex spectra (due to their natural heterogeneity), making the significance of the findings hard to interpret.

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