Quaternary conformational stability: the effect of reversible self-association on the fibrillation of two insulin analogs.

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far-UV CD) and second derivative Fourier transform infrared (2D-FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α-helical in native insulin to predominantly ß-sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self-associate. Composition gradient multi-angle light scattering (CG-MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant-induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first-order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers.

Aggregation of granulocyte colony stimulating factor under physiological conditions: characterization and thermodynamic inhibition.

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.