Stabilization of rasburicase and physico-chemical characterization of the resulting injectable formulation.

ABSTRACT

Rasburicase (Fasturtec/Elitek) is a new generation of recombinant urate oxidase administred therapeutically by intravenous infusion for the prevention or treatment of hyperuricemia during chemotherapy. To ensure a long storage period, a freeze-dried formulation was developed to guarantee the molecular integrity and enzyme activity. Screening of potential excipients was the first stage of the preformulation study. The selection was based on stability results (rasburicase solution with excipient) obtained with the isoelectric focusing profiles and residual enzyme activity. The different excipients were classified as stabilising, neutral or destabilising. A stability study was then carried out on different freeze-dried formulations containing the usual bulking agents for freeze-drying, excipients with a high glass transition temperature or competitive enzyme inhibitors having a stabilising effect. A mannitol/alanine mixture in phosphate buffer was selected from these preliminary results. Finally, the optimal content of mannitol and alanine in the freeze-dried powder was determined by an experimental design study. The water content and the appearance of the "cake", the osmolality, pH, clarity, and enzyme activity of the reconstituted solution were assessed. The formula with a mannitol/alanine ratio of 0.7 was found to be the best composition. Differential scanning calorimetry and ThermoStimulated Current technique experiments were carried out to study the amorphous phase. A glass transition temperature of about 45-500 degrees C was found. Glassy state is known to preserve stability, which was verified by the real stability data. X-ray diffraction studies have shown that alanine is in a crystallised state and that mannitol remains amorphous. Crystallised excipients participate in forming the structure of the powder and therefore help to prevent any collapse. Amorphous mannitol creates a surrounding medium favourable to the stability of the protein.