The Protein Purification Facility
The Alexander Silberman Institute of Life Sciences
The Hebrew University of Jerusalem
Dr. Mario Lebendiker  Tel: 972-2-6586920  

Protein Aggregation
According to Integrity Biosolution

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    RP-HPLC
o    Disulfide reduction with reducing agent(s)

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Dr. Mario Lebendiker The Protein Purification Facility
The Alexander Silberman Institute of Life Sciences,    The Hebrew University of Jerusalem  Tel: 972-2-6586920  FAX: 972-2-6758963

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