We have investigated the aggregation of recombinant human granulocyte colony stimulating factor (rhGCSF), a protein that rapidly aggregates and precipitates at pH 6.9 and 37 degrees C. We observed that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that this dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. We postulate that sucrose acts by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble. Thermodynamic stability data from unfolding curves and hydrogen-deuterium exchange experimental results support the above hypothesis. Thus, the strategy of stabilizing the native state of the protein under physiological conditions using thermodynamic stabilizers, especially ligands binding with high affinity to the native state, is expected to protect against protein aggregation occurring under such nonperturbing solution conditions.

The equilibrium dissociation of recombinant human IFN-␥ was monitored as a function of pressure and sucrose concentration. The partial molar volume change for dissociation was ؊209 ؎ 13 ml/mol of dimer. The specific molar surface area change for dissociation was 12.7 ؎ 1.6 nm 2 /molecule of dimer. The first-order aggregation rate of recombinant human IFN-␥ in 0.45 M guanidine hydrochloride was studied as a function of sucrose concentration and pressure. Aggregation proceeded through a transition-state species, N*. Sucrose reduced aggregation rate by shifting the equilibrium between native state (N) and N* toward the more compact N. Pressure increased aggregation rate through increased solvation of the protein, which exposes more surface area, thus shifting the equilibrium away from N toward N*. The changes in partial molar volume and specific molar surface area between the N* and N were ؊41 ؎ 9 ml/mol of dimer and 3.5 ؎ 0.2 nm 2 / molecule, respectively. Thus, the structural change required for the formation of the transition state for aggregation is small relative to the difference between N and the dissociated state. Changes in waters of hydration were estimated from both specific molar surface area and partial molar volume data. From partial molar volume data, estimates were 25 and 128 mol H2O/mol dimer for formation of the aggregation transition state and for dissociation, respectively. From surface area data, estimates were 27 and 98 mol H2O/mol dimer. Osmotic stress theory yielded values Ϸ4-fold larger for both transitions.A ggregation is of considerable concern to the medical, pharmaceutical, and biotechnology industries, as protein aggregation occurs in human diseases (1-4) and during the production, purification, and storage of protein products (5). Characterization of the conformational state(s) that lead(s) to aggregation is essential for a mechanistic understanding of the aggregation process.Protein aggregation was once viewed as a nonspecific hydrophobically driven process involving the fully unfolded random coil (6-8). However, recent research has shown protein aggregation to follow distinct pathways involving folding intermediates (6, 7, 9). Thus, the folding pathway and aggregation processes are critically linked (7,8). Characterization of the unfolding process, in conjunction with aggregation rate studies, should then provide insight into the mechanisms of protein aggregation and the role of folding intermediates therein. A major challenge in characterizing aggregation pathways is that under conditions that greatly favor the native state (e.g., physiological), the small populations of highly reactive aggregationcompetent species and the transition states leading to these species may be inaccessible to available spectroscopic techniques. To populate putative aggregation-competent species at levels sufficient for spectroscopic measurement, conditions that greatly perturb the native state are typically used (e.g., pH Ͻ4.0). Furthermore, even if the aggregation-competent species populated under nonn...