A high propensity to aggregate into intractable deposits is a common problem limiting the production and use of many peptides and proteins in a wide range of biotechnological and pharmaceutical applications. Many therapeutic polypeptides are frequently abandoned at an early stage in their development because of problems with stability and aggregation. It has been shown recently that parameters describing the physicochemical properties of polypeptides can be used as predictors of protein aggregation. Here we demonstrate that these and similar tools can be applied to the rational redesign of bioactive molecules with a significantly reduced aggregation propensity without loss of physiological activity. This strategy has been exemplified by designing variants of the hormone calcitonin that show a significantly reduced aggregation propensity, yet maintain, or even increase, their potency when compared to the current therapeutic forms. The results suggest that this approach could be used successfully to enhance the solubility and efficacy of a wide range of other peptide and protein therapeutics.protein aggregation ͉ protein design ͉ biopharmaceuticals ͉ amyloid ͉ misfolding T he number of pharmacologically active peptides and proteins under development for the prevention and treatment of human disorders is increasing, and as a result, so is the pressure to overcome problems associated with aggregation and stability. Up to 96% of all drug candidates in trials are abandoned during preclinical or clinical development, often because of low solubility or aggregation problems (1). Many peptide-based drugs with great therapeutic potential are rendered ineffective simply because of an intrinsic propensity to aggregate irreversibly (2). Aggregation is one of the most significant obstacles to the development of protein-based drugs because it cannot only compromise their bioavailability and therapeutic activity but it may also increase the risk of immunogenic reactions (3, 4).A good example of a bioactive peptide with limited pharmaceutical potential due to a high tendency to aggregate is human calcitonin (hCT), a 32-residue polypeptide hormone synthesized and secreted by the C cells of the thyroid, and involved in calcium regulation and bone dynamics. In vivo, calcitonin (CT) causes a rapid, but short-lived, decline in calcium and phosphate levels in the blood by promoting the incorporation of these ions into bone (5). This activity has lead to the use of CT in the treatment of conditions such as osteoporosis and Paget's disease, as well as malignancy-caused hypercalcemia and musculoskeletal pain (5-7). However, hCT shows an extremely high tendency to selfassociate in the form of amyloid fibrils. As a result hCT amyloid deposits can occur in vivo in patients with medullar carcinoma of the thyroid (8, 9) and in vitro in preparations designed for patient administration (10). Not only does aggregation constitute a serious problem during the production, storage, and administration of hCT, but it can also lead to a significant decr...