Proteomics - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 391

albumin stored at 37°C at pH 6.8 to 7.0 using 0.15 and 0.3 M NaCl solutions have
been found to be stable up to 7 days, while lipoproteins are considered to be most
susceptible to all sorts of chemical degradation. Certain enzymes are extremely ther-
molabile and exhibit loss of activity when exposed to thermal stresses. For example,
alpha 1-antitrypsin loses its activity due to thermolability [10] and oxidation of methi-
onine fragment [11] . Falcoff et al. [12] reported that stability of proteins varies from
protein to protein. For example, crude human gamma interferon is resistant to inacti-
vation when exposed to 56 °C for 10 min. However, most proteins are affected by pH,
temperature, ionic strength, presence of surfactants, presence of proteolytic enzymes,
and other factors. Paradoxically, addition of certain organic solvents and substances
tends to accelerate the stabilization process of proteins. The exact mechanism is
unknown. However, they might act by stabilizing the conformation of proteins or
by promoting self-association of proteins, thereby reducing unfavorable interactions
with the solvent domain [13] . Sugars, amino acids, and salts act as protein stabiliz-
ers by increasing surface or interfacial tension. However, interestingly, some poly-
hydric alcohols and betaine have been found to improve stability of proteins despite
their intrinsic property of lowering surface tension. The underlying mechanism
for this may be hydration of proteins or alteration in protein alignment [14] .
8.2.1.3 Manifestations of Protein Instability
�.2.1.�.1 Aggregation
Proteins tend to undergo hydrophobic interactions in the bulk phase of solvents in
which they are dissolved and form self-associating structures resulting in crystal-
lization of proteins in severe cases and agglomeration leading to stability issues.
Lougheed et al. [15] concluded on the basis of their findings that self-association of
insulin is a critical issue in the stability of insulin infusion pumps designed for pre-
determined delivery of insulin to diabetic patients. Presence of certain trace metallic
ions is a major contributor to such aggregation. Addition of Zn 2 to insulin results in
aggregation of insulin by the formation of hexamers [16] . A common factor affecting
the conformational stability of proteins is the application of shear to proteins. It has
been observed that agitation leads to altered flow pathways in proteins [17] .
Aggregation of insulin is prevented by the addition of substances like urea
[18] and dicarboxylic amino acids such as aspartic acid and glutamic acid [19] .
An extensive study of 60 additives and 1125 formulations by Massey and Sheliga
[20] revealed that nonionic surfactants such as Pluronic F68 (poloxamer 188), a
polyoxyethylene-polyoxypropylene glycol surfactant, were promising stabilizers.
Human insulin appeared to aggregate more readily than porcine or bovine insulin.
The self-association structures may not always lead to loss of pharmacological activ-
ity of a protein. There are some examples to support this observation: dimeric forms
of insulin obtained by recombinant deoxyribonucleic acid (DNA) technology using
E. coli and natural gamma interferon are physiologically active in spite of forming
self-associating dimers. In fact, at acidic pH, a number of proteins dissociate into
monomers due to high electrostatic repulsion, leading to denaturation of the protein
and thereby loss of activity [21] .