A turbidimetric assay was used to determine the extent of thermally-induced aggregation in low molecular weight urokinase (LMW-UK). Previous work has shown that, under 60°C heat treatment, LMW-UK denatures and the unfolded protein proceeds to form soluble aggregates. The effects of excipients on the extent of aggregation were examined. Both salts (ammonium sulfate and magnesium chloride) and sugars (sucrose, glucose, trehalose, raffinose) were found to be effective, concentration-dependent inhibitors of aggregation, although excessive salt concentrations did lead to salting out of the protein. Addition of Tween 80, a nonionic detergent, was ineffective. Overall, the effect of these additives on the stability of thermally-stressed LMW-UK can be understood in terms of preferential exclusion of the solute from the surface of the protein. These interactions affect the extent of denaturation, or unfolding, of LMW-UK at 60°C, thereby controlling the degree of aggregation. Purification and incubation experiments indicate that a thermally-unstable subpopulation of LMW-UK exists and is responsible for the majority of the aggregation observed.

Stabilization of proteins through proper formulation is an important challenge for the pharmaceutical industry. Two approaches for stabilization of proteins in solution are discussed. First, work describing the effect of additives on the thermally induced denaturation and aggregation of low molecular weight urokinase is presented. The effects of these additives can be explained by preferential exclusion of the solute from the protein, leading to increased thermal stability with respect to denaturation. Diminished denaturation leads to reduced levels of aggregation. The second approach involves stoichiometric replacement of polar counter ions (e.g., chloride, acetate, etc.) with anionic detergents, in a process termed hydrophobic ion pairing (HIP). The HIP complexes of proteins have increased solubility in organic solvents. In these organic solvents, where the water content is limited, the thermal denautration temperatures greatly exceed those observed in aqueous solution. In addition, it is possible to use HIP to selectively precipitate basic proteins from formulations that contain large amounts of stabilizers, such as human serum albumin (HSA), with a selectivity greater than 2000-fold. This has been demonstrated for various mixtures of HSA and interleukin-4. (c) 1995 John Wiley & Sons, Inc.

Turbidimetric or light scattering assays can be used to determine the extent of aggregation in protein formulations. Using low molecular weight urokinase (LMW-UK) as a model protein, the effect of polymeric additives on heat-induced aggregation was evaluated. Previous work has shown that under 60°C heat treatment, LMW-UK initially denatures and the unfolded protein associates to form soluble aggregates. Eventually, these aggregates associate to form a precipitate. The effects of polymers on the initial aggregation phase was examined. Hydroxyethyl (heta) starch, polyethylene glycol 4000, and gelatin were found to be effective, concentration-dependent inhibitors of aggregation, whereas polyvinylpyrrolidone (PVP) and polyethylene glycol 300 were ineffective. Overall, the effect of polymeric additives on the stability of thermally-stressed LMW-UK can be accounted for by preferential exclusion of the solute from the surface of the protein.