Development of a stable freeze-dried formulation of recombinant human interleukin-1 receptor antagonist

Purpose. A formulation of recombinant human interleukin-l receptor antagonist (rhIL-1ra) was developed that provided both acute protection during lyophilization and storage stability in the dried solid. Methods. The formulation was optimized by monitoring the impact of excipients on protein degradation which was analyzed by turbidimetry and cation-exchange HPLC. Results. The most appropriate pH was 6.5. Sodium citrate buffer provided better stability than sodium phosphate buffer. Glycine was selected as a bulking agent because the greatest protein stability was noted when this bulking agent was used in combination with an amorphous protein stabilizer. Among the amorphous stabilizers tested, sucrose protected rhIL-1ra best in the presence of glycine. When the protein was freeze-dried in the presence of an inadequate mass ratio of sucrose/protein (< 0.3), the rate of degradation of rhIL-1ra increased. For a formulation containing 100 mg/ml of rhIL-1ra, increasing the sucrose/protein mass ratio to greater than or equal to 0.3 greatly increased storage stability. The moisture content of the dried solid affected the storage stability to a minor degree. Three different stoppers obtained from the WEST Company did not affect the stability of rhIL-1ra. Conclusions. An optimized formulation could be reconstituted without precipitation after 14 months at 30 or 50 degrees C. At 30 degrees C, there was no loss of native protein due to deamidation, and only a 4% loss at 50 degrees C. These results indicated that the optimized formulation could be stored at ambient temperatures for long periods, without damage to the protein.