Biomedical Engineering Reference
In-Depth Information
that are used in parenterals. It becomes highly complex in multicomponent systems
like proteins, where the presence of such excipients may be critical in determin-
ing the destiny of formulation. Even the cosolvents, buffers, and so forth, must be
screened. For example, the addition of cosolvents such as glycerol or polyethylene
glycol may stabilize protein structure by preferential hydration and by decreasing the
contact area of the protein surface with solvent.
8.7.3  Evaluation of Purity and Stability
Peptides form a substantial bulk of pharmacologically and therapeutically active
biomolecules. Although their whole potential in therapeutics is yet to be explored
completely, they have been widely acclaimed for cardiovascular, central nervous
disorders, and others. The peptide sequences have microscopic differences among
themselves, just differing by sequential arrangement of amino acids. Hence, it is very
difficult to develop a selective and analyte-specific analytical method for quantita-
tive determination of the same. The biological potency characteristic of these mol-
ecules, degradation products, impurities, and matrix components places tremendous
demands for selectivity on analytical methodology. The presence of intrinsic func-
tional groups further renders them reactive, adding to difficulties in analysis. During
analysis itself, the material tends to adhere to glass and similar surfaces due to
adsorption, thus leading to erroneous results.
The analytical methods for peptides may be classified into four major catego-
ries: (1) measurement of biological activity, (2) evaluation of purity and stability,
(3) quantitative determination, and (4) structural characterization.
8.7.3.1 Measurement of Biological Activity
Bioassays are performed to measure quantitatively the biological response of a given
species to a particular compound. Details and protocol guidelines are followed as per
United States pharmacopeia or as per those of the Food and Drug Administration.
For instance, activities of protein hormones like insulin and corticotrophin (ACTH)
are measured by this technique.
8.7.3.2 Radioreceptor Assays (RRAs)
Receptors present on the surface of biological cell membranes are major media-
tors for eliciting biological response. Thus, characterization of the peptide-receptor
interaction is studied to obtain an in depth idea about the quantification of biolo-
gical activity. In this method, mainly the principle of competitive assay is adopted.
The nonradioactive sample of interest is mixed with highly purified radiolabeled
peptide and incubated with a known concentration of cultured cells that possess the
peptide receptor with affinity to bind to the given peptide. The ability of the sample
to compete with the peptide trace compound for binding sites is evaluated from the
measurement or characterization of receptor-bound radioactivity. RRAs have been
utilized widely for a variety of peptides. However, this technique suffers from its
own limitations, especially that the technique is highly restricted to the evaluation of
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