Biomedical Engineering Reference
In-Depth Information
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high degree of automation (including data analysis);
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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 poly-
meric 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 mi-
nor differences in hydrophobicity. In some instances a single amino acid substitution or the re-
moval of a single amino acid from the end of a polypeptide chain can be detected by RP-HPLC.
In most instances, modifi cations 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 fi nds extensive application in, for example, the analysis of insulin preparations. Modi-
fi ed 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
specifi c protein preparations should be undertaken only after extensive validation studies. HPLC
in general can have a denaturing infl uence on many proteins (especially larger, complex proteins).
Reverse-phase systems can be particularly harsh, as interaction with the highly hydrophobic sta-
tionary phase can induce irreversible protein denaturation. Denaturation would result in the gen-
eration 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 in-
clude silica-based supports and cross-linked agarose of defi ned pore size. Size-exclusion systems
are most often used to analyse product for the presence of dimers or higher molecular mass ag-
gregates 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 fi eld 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 pos-
sible 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
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