Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 181

a bioassay, immunoassays are rapid (undertaken in minutes to hours), inexpensive, and straight-
forward to undertake.
The obvious disadvantage of immunoassays is that immunological reactivity cannot be guaran-
teed to correlate directly to biological activity. Relatively minor modifi cations of the protein prod-
uct, although having a profound infl uence on its biological activity, may have little or no infl uence
on its ability to bind antibody.
For such reasons, although immunoassays may provide a convenient means of tracking product
during downstream processing, performing a bioassay on at the very least the fi nal product is usu-
ally necessary to prove that potency falls within specifi cation.

7.3.2 Determination of protein concentration

Quantifi cation of total protein in the fi nal product represents another standard analysis undertaken
by QC. A number of different protein assays may be potentially employed (Table 7.3).
Detection and quantifi cation of protein by measuring absorbency at 280 nm is perhaps the sim-
plest such method. This approach is based on the fact that the side chains of the amino acids tyro-
sine 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 content of tyro-
sine and tryptophan vary to any signifi cant extent. Hence, this method is rarely used to determine
the protein concentration of the fi nal product, but it is routinely used during downstream processing
to detect protein elution off chromatographic columns, and hence track the purifi cation process.

Table 7.3 Common assay methods used to quantitate proteins. The principle upon which each method is
based is also listed

Method Principle

Absorbance at 280 nm
( A 280 ; UV method)

The side chain of selected amino acids (particularly tyrosine and tryptophan)
absorbs UV at 280 nm

Absorbance at 205 nm
(far-UV method)

Peptide bonds absorb UV at 190-220 nm

Biuret method Binding of copper ions to peptide bond nitrogen under alkaline conditions generates

a purple colour

Lowry method 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 that displays an
absorbance maximum at 750 nm

Bradford method Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The

dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm

Bicinchonic acid method Copper-containing reagent that, when reduced by protein, reacts with bicinchonic

acid yielding a complex that displays an absorbance maximum at 562 nm

Peterson method Essentially involves initial precipitation of protein out of solution by addition of

trichloroacetic acid. The protein precipitate is redissolved in NaOH and the
Lowry method of protein determination is then performed

Silver-binding method Interaction of silver with protein - very sensitive method