Biomedical Engineering Reference
In-Depth Information
techniques like reverse-phase high-performance liquid chromatography (RP-HPLC)
have proved to be a blessing in this case.
In a nutshell, with the methods of peptide synthesis it can be concluded that both
solid and solution phase methods have got their own advantages and disadvantages.
Solid phase peptide synthesis offers some salient advantages like high speed, auto-
mation, and absence of solubility related issues. Conversely, scale up becomes easy
in solution phase synthesis, with no involvement of expensive machineries, thereby
cutting down investment and inventory costs. Development in all these methods has
facilitated identification of the binding epitope of HIV virus with an antibody in
ELISA assay, providing deeper insight into the menace of AIDS.
8.7.1 Characterization of Proteins as Pharmaceuticals
The proteins developed as pharmaceuticals must be extensively studied for their size,
shape, conformational properties, toxicological properties, stability, safety, and effi-
cacy issues.
8.7.1.1 Size and Shape
A plethora of analytical methods is available to determine the size and shape of a protein
pharmaceutical. Sodium dodecyl sulfate polyacrylamide (SDS PAGE) electrophoresis is
one of the most popular techniques used. The underlying principle of this method is that
binding of SDS to proteins results in a nearly similar charge density on all molecules,
and mobility of proteins through gel is related to hydrodynamic volume and shape of the
protein. However, protein gets denatured in this process. Nondenaturing techniques, such
as gel permeation chromatography or ultracentrifugation, are used to assess the size of
proteins, particularly in the presence of formulation excipients.
8.7.1.2 Conformation
Although a protein has a higher degree of freedom in conformational flexibility, under
normal physiological conditions it exists as a single specific conformation. Besides, the
possibility of artifacts introduced by conformational impurities must be ruled out, as
they may pose a serious concern in the stability and performance of a formulation.
Ultraviolet-based spectroscopic methods and nuclear magnetic resonance (NMR),
Raman fluorescence, and circular dichroism are some of the well-documented tech-
niques that are used to determine the protein conformation.
8.7.1.3 Solubility and Charge
Conformational changes are also detected by a change in the solubility profile of a pro-
tein. The explanation for this can be put in this way: when the protein is denatured, more
hydrophobic groups are exposed to aqueous milieu from the interior of the protein. To
maintain negative changes in entropy, contact of hydrophobic groups with water is mini-
mized. Generally, conformation adjusts itself in such a way that avoids the thermodynam-
ically unfavorable interactions with the microenvironment. This leads to self-association
that may culminate in aggregation and, ultimately, precipitation of protein.
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