We studied the non-native aggregation of recombinant human granulocyte stimulating factor (rhGCSF) in solution conditions where native rhGCSF is both conformationally stable compared to its unfolded state and at concentrations well below its solubility limit. Aggregation of rhGCSF first involves the perturbation of its native structure to form a structurally expanded transition state, followed by assembly process to form an irreversible aggregate. The energy barriers of the two steps are reflected in the experimentally measured values of free energy of unfolding (⌬G unf ) and osmotic second virial coefficient (B 22 ), respectively. Under solution conditions where rhGCSF conformational stability dominates (i.e., large ⌬G unf and negative B 22 ), the first step is rate-limiting, and increasing ⌬G unf (e.g., by the addition of sucrose) decreases aggregation. In solutions where colloidal stability is high (i.e., large and positive B 22 values) the second step is ratelimiting, and solution conditions (e.g., low pH and low ionic strength) that increase repulsive interactions between protein molecules are effective at reducing aggregation. rhGCSF aggregation is thus controlled by both conformational stability and colloidal stability, and depending on the solution conditions, either could be rate-limiting.
In this study, we found that the denaturation of proteins during freezing is closely related to surface-induced denaturation. Several proteins with varying sensitivities to freezing were tested, and the results were compared with susceptibilities to surface denaturation in unfrozen aqueous solution. Also, the influence of the surfactant Tween 80 on the denaturation of each of the proteins was examined during freeze-thawing, as were the effects of Tween 80 and several other surfactants on the stability of lactate dehydrogenase. Proteins formed insoluble precipitates when they were subjected to a quench cooling by dipping in liquid nitrogen, although freezing followed by supercooling caused less precipitation. A strong correlation (r = 0.99) was observed between the tendency of a protein to freeze denature and its tendency to surface denature. Also, the addition of small amounts of surface-active agents protected proteins from both freeze- and surface-induced denaturation. Freeze-induced denaturation of IL-1ra at the ice-water interface during freeze-drying was effectively prevented by adding a small amount of Tween 80. These results suggest that the denaturation of proteins during freeze-thawing can be ascribed primarily to the increase in the area of the ice-water interface during freezing.
Understanding the mechanism for sucroseinduced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucroseinduced restriction of rhIL-1ra conformational f luctuations, which were studied by hydrogen-deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein's interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.
Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-␥ (rhIFN-␥) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-␥ aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-␥ and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-␥ native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-␥ against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.Formation of biologically inactive proteins by aggregation is a problem of considerable importance in many disciplines (1-3). For example, protein aggregates can be formed in vivo and in vitro during folding of nascent polypeptide chains, eliminating or reducing the protein's biological function (4, 5). Misfolded protein aggregates often can be observed as inclusion bodies (6, 7) and are implicated in amyloid deposition in vivo (8). In the biotechnology industry, protein aggregation is encountered routinely during purification, refolding, sterilization, shipping, and storage processes because of the presence of chemical, physical, and thermal stresses (9).In addition to significant losses in protein activity, clinical dangers result from parenteral administration of aggregated material (10). Even aggregation levels as low as 1% over a 2-year shelf life can render a product clinically unacceptable. Thus, proteins must be protected against even relatively mild stresses by the addition of proper excipients. Rational choice of these excipients requires insight into the mechanism of aggregation.The detailed mechanism of protein aggregation is still unclear. Usually, the aggregation pathway is modeled as shown in Scheme 1 by using the Lumry-Erying framework (11,12). The model involves a first-order reversible unfolding of the protein and subsequent aggregation of nonnative species in a higher order process (2, 11-13):In this scheme, N refers to native protein, and A refers to an intermediate conformational state preceding aggregation. A m refers to an aggregated form composed of m pro...
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