Aggregation of proteins has been one of the most significant obstacles in
the development for practical purposes. Aggregation is unacceptable in protein
pharmaceuticals because it can not only compromise biological activity but
also increases the chance of undesirable side effects, e.g., injection site
reactions and immunogenecity. Extensive studies have been carried out to understand
aggregation and to find practical ways to stabilize proteins against it.
Numerous physicochemical stresses can induce protein aggregation, including
heat, pressure, pH, agitation, freeze-thawing, dehydration, heavy metals,
phenolic compounds, and denaturants(1-4). Some proteins are more susceptible
to aggregation than others when exposed to these stresses. Extensive examples
of the role various additives can play in protecting proteins against stress-induced
aggregation can be found in the literature(5-7). However, with few exceptions,
little information is available that can be used to predict the susceptibility
of an individual protein to different stresses based upon its structure.
The unfolding of protein structure induced by heat or denaturants has been
very thoroughly examined in the literature(8-9). In fact, it has been the
primary focus of research into protein stability. Numerous biophysical techniques
including circular dichroism, fluorescence, FTIR, and calorimetry are available
to examine the changes in secondary structures associated with unfolding(10-11).
Many different additives have been shown to stabilize proteins against denaturation.
Sugars, amino acids, and salts are all preferentially excluded from proteins'
surfaces and thus protect them from unfolding(12-13). Due to great interest
and thorough research, an extensive database containing the thermodynamic
and kinetic results from the unfolding of proteins has been accumulated.
However, the application of results from unfolding studies to the problem
of protein aggregation has been limited because in practice most protein aggregates
are not a product of complete unfolding. For example, precipitation or aggregation
observed in the development of therapeutic proteins generally results from
minor stresses which are not sufficient to affect secondary structure. Proteins
precipitate at temperatures much below their thermal unfolding temperature.
Their activation energies, calculated crudely using kinetic rate constants,
are in the range of 20 - 30 kcal/mole, which is significantly smaller than
the 100 - 200 kcal/mole of thermal unfolding.
In addition, very little change in secondary structure is detected when
proteins aggregate during exposure to minor stresses like agitation and freeze-thaw.
Numerous studies have revealed that aggregated proteins still contain a majority
of their secondary structures(14-15). These lines of evidence all suggest
that a far more minor structural alteration than the unfolding of secondary
structure is sufficient to cause aggregation, and that this minor alteration
is the primary cause of the aggregation that we observe in practical conditions.
There are a number of reasons why the minor structural changes responsible
for undesirable aggregations have been difficult to examine. The degree of
structural change is probably too small for many analytical methods to resolve.
The change is also presumably a reversible process unless kinetically trapped
by aggregation and, therefore, the population of the changed species will
be relatively scarce when compared to that of the native molecules. In addition,
minor structural changes may be constantly occurring so that what is defined
as a "native" structure is actually an average of a continuous spectrum of
fluctuating molecules.
Since there is no defined species which can be analytically quantified,
examining the structural changes that lead to aggregation with routine spectroscopic
methods that are designed to determine protein secondary structure will be
difficult. For the same reason, obtaining thermodynamic parameters with equilibrium
constants will be practically impossible. The best approach to understanding
these undefined and transient structural species will be to obtain rate constants
by kinetically trapping and then characterizing the modified species. Kinetic
entrapment can be achieved by natural aggregation processes where aggregation
prevents the partially unfolded species from refolding to the native conformation.
Other kinetic traps can be achieved by chemical modifications of side chains
which occur specifically during the subtle structural changes. These include
such natural protein degradation mechanisms as oxidation, deamidation, and
disulfide scrambling as well as artificial modifications produced by the addition
of reagents which can react with specific side chains. Advantages of these
chemical modification techniques include the stability of modified species
during analysis and the freedom to choose specific targets. Solvent exchange
studies have been also useful to identify structurally flexible regions and
to compare their relative flexibilities by selectively labeling the solvent-exposed
surfaces(16-19). Both deuterium and tritium can be used along with analytical
methods such as FTIR, Raman, NMR, or mass spectrometry to evaluate the flexibility
of various structural components.
In order to inhibit aggregation or precipitation, it is important to restrict
molecular fluctuations. This can be achieved effectively with stabilizers
which are known to protect proteins against complete unfolding. A deuterium
exchange study and a kinetic analysis of side chain groups which are chemically
modified upon exposure to the surface have demonstrated that sucrose can keep
the native structure more compact and prevent the minor structural changes
which result in aggregation(20,21).
These results suggest that information obtained from complete unfolding
studies can be applied to the more subtle structural changes observed in practice.
Although the increase in surface area that occurs in subtle conformational
changes is quantitatively smaller than that of complete unfolding, it is qualitatively
same phenomenon and, therefore, it is ruled by the same underlying thermodynamic
principles.
Analytical methods for protein aggregates
• Non-denaturing methods
o HPLC (SEC-HPLC, Hydrophobic interaction chromatography,
ion-exchange chromatography, affinity chromarography, etc)
o Native gel electrophoresis
o Capillary electrophoresis
o Analytical ultracentrifuge
o Light scattering
• Denaturing methods
o SEC-HPLC with denaturant (SDS, Guanidium HCl, Organic
solvent) in the sample or in mobile phase
o SDS-PAGE
o RP-HPLC
o Disulfide reduction with reducing agent(s)
References
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Manning, and J.F. Carpenter. Proc. Natl. Sci. USA 94: 11917 (1997)
22. 1. Kim, Y.S., Jones, L.S., Dong, A., Kendrick, B.S., Chang, B.S., Randolph,
T.W., Manning, M.C., and Carpenter, J.F. Protein Sci. 12(6):1252-1261 (2003)
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