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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 mm and be up to 1m long (it is normally coiled to facilitate ease of use and storage). The dimensions of this system yield greatly increased surface area:volume ratio (when compared to 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 DRUG MANUFACTURING PROCESS 167
Figure 3.33. Photograph of a typical HPLC system (the Hewlett-Packard HP1100 system). Photo courtesy of Hewlett-Packard GmbH, Germany
The speed, sensitivity, high degree of automation and ability to directly quantify protein bands renders this system ideal for biopharmaceutical analysis.
High-pressure liquid chromatography (HPLC)
HPLC occupies a central analytical role in assessing the purity of low molecular mass pharmaceutical substances (Figure 3.33). It also plays an increasingly important role in analysis of macromolecules 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 chromatography systems are now used in the analysis of a range of biopharmaceutical preparations. On-line detectors (usually a UV monitor set at 220 nm or 280 nm) allows automated detection and quantification of eluting bands.
HPLC is characterized by a number of features which render it an attractive analytical tool. These include:
• excellent fractionation speeds (often just minutes per sample);
• superior peak resolution;
• 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 hydrophobicity. 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 proved 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 analysis of insulin preparations. Modified forms or insulin polymers are easily distinguishable from native insulin on reverse-phase columns.
While RP-HPLC has proved 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), in most instances separation is essentially achieved on the basis of molecular mass. 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. Although not as extensively employed as RP or SE systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated forms.
Recent advances in the field of mass spectrometry now extends 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%. 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.