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THE DRUG MANUFACTURING PROCESS 163
Table 3.27. Common assay methods used to quantify proteins. The principle upon which each method is based is also listed
Absorbance at 280 nm (A280; UV method)
Absorbance at 205 nm (far UV method) Biuret method
Bicinchonic acid method
The side chain of selected amino acids (particularly tyrosine and tryptophan) absorbs UV at 280 nm Peptide bonds absorb UV at 190-220 nm Binding of copper ions to peptide bond nitrogen under alkaline conditions generates a purple colour Lowry method uses a combination of the Biuret copper-based reagent and the ‘Folin-Ciocalteau’ reagent, which contains phosphomolybdic-phosphotungstic acid. Reagents react with protein, yielding a blue colour which displays an absorbance maximum at 750 nm Bradford reagent contains the dye, Coomassie blue G-250, in an acidic solution. The dye binds to protein, yielding a blue colour which absorbs maximally at 595 nm Copper-containing reagent which, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid (TCA). The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein — very sensitive method
For such reasons, while immunoassays may provide a convenient means of tracking product during downstream processing, performance of a bioassay on at very least the final product is often considered necessary to prove that potency falls within specification.
Determination of protein concentration
Quantification of total protein in the final product represents another standard analysis undertaken by QC. A number of different protein assays may be potentially employed (Table 3.27).
Detection and quantification of protein by measuring absorbency at 280 nm is perhaps the simplest such method. This approach is based on the fact that the side-chains of tyrosine and tryptophan absorb at this wavelength. The method is popular, as it is fast, easy to perform and is non-destructive to the sample. However, it is a relatively insensitive technique, and identical concentrations of different proteins will yield different absorbance values if their contents of tyrosine and tryptophan vary to any significant extent. For these reasons, this method is rarely used to determine the protein concentration of the final product, but it is routinely used during downstream processing to detect protein elution off chromatographic columns, and hence track the purification process.
Measuring protein absorbance at lower wavelengths (205 nm) increases the sensitivity of the assay considerably. Also, as it is the peptide bonds that are absorbing at this wavelength, the assay is subject to much less variation due to the amino acid composition of the protein. The
most common methods used to determine protein concentration are the dye-binding procedure using Coomassie brilliant blue, and the bicinchonic acid-based procedure. Various dyes are known to bind quantitatively to proteins, resulting in an alteration of the characteristic absorption spectrum of the dye. Coomassie brilliant blue G-250, for example, becomes protonated when dissolved in phosphoric acid, and has an absorbance maximum at 450 nm. Binding of the dye to a protein (via ionic interactions) results in a shift in the dye’s absorbance spectrum, with a new major peak (at 595 nm) being observed. Quantification of proteins in this case can thus be undertaken by measuring absorbance at 595 nm. The method is sensitive, easy and rapid to undertake. Also it exhibits little quantitative variation between different proteins.
Protein determination procedures using bicinchonic acid were developed by Pierce chemicals, who hold a patent on the product. The procedure entails the use of a copper-based reagent containing bicinchonic acid. Upon incubation with a protein sample, the copper is reduced. In the reduced state it reacts with bicinchonic acid, yielding a purple colour which absorbs maximally at 562 nm.
Silver also binds to proteins, an observation which forms the basis of an extremely sensitive method of protein detection. This technique is used extensively to detect proteins in electrophoretic gels, as discussed in the next section.
Detection of protein-based product impurities
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) represents the most commonly used analytical technique in the assessment of final product purity (Figure 3.31). 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 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).