A major limitation in the successful development of multidose protein formulations is protein aggregation induced by antimicrobial preservatives such as benzyl alcohol, which are included to maintain product sterility. Studies were conducted to evaluate the strategy of developing lyophilized formulations of a therapeutic protein, recombinant human interlukin-1 receptor antagonist (rhIL-1ra), to be reconstituted with a bacteriostatic amount (0.9% w/v) of benzyl alcohol in water. The strategy was based on the following hypotheses. The first was that benzyl alcohol would foster aggregation during reconstitution of the lyophilized sample. The second hypothesis was that the extent of benzyl alcoholinduced protein aggregation would correlate directly with the degree of structural perturbation of rhIL-1ra in the dried solid after lyophilization. Differential structural retention of rhIL-1ra in the dried solid was obtained by using a combination of formulation variables important for lyophilization and included: protein concentration, type of stabilizer, and presence or absence of NaCl. Infrared spectroscopic analysis of the lyophilized samples indicated that high initial solution protein concentration and the stabilizer sucrose minimized structural perturbation of rhIL-1ra during lyophilization. In contrast, NaCl was destabilizing. Reconstitution of the dried solid with 0.9% (w/v) benzyl alcohol caused a greater degree of protein aggregation than reconstitution with water, confirming our first hypothesis. In support of our second hypothesis, the extent of aggregation induced by benzyl alcohol during reconstitution was strongly modulated by the degree of retention of native rhIL-1ra secondary structure during lyophilization. During storage of the reconstituted lyophilized samples at room temperature, benzyl alcohol did not accelerate aggregation of rhIL-1ra. This study demonstrated that for development a multidose lyophilized protein formulation involving reconstitution with a solution of benzyl alcohol, protein structural perturbations during freeze-drying should be minimized with a stabilizing excipient and appropriate choice of protein concentration and tonicity modifier. Furthermore, postreconstitution storage at reduced temperature (e.g., room temperature or 48C) could minimize the risk of preservative-induced protein aggregation.