Effect of cryoprotectants on the stability and aerosol performance of nebulized aviscumine, a 57-kDa protein

Nebulization of aqueous drug solutions is a suitable delivery system for pulmonary application of proteins because it can easily produce droplets small enough to reach the alveolar region. However, proteins are sensitive to nebulization. Therefore, stabilizers need to be added which on the other hand influence the aerosol performance, such as average droplet size or mass output. This research presents the effect of various cryoprotectants such as Na-polyphosphate, CaCl(2) x 6H(2)O and MgSO(4) x 7H(2)O on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization using three different nebulizers. Formulations containing Tris-buffer, polysorbate 80, Na(2)-EDTA and HES450 were lyophilized and reconstituted with a buffered isotonic solution containing 100 mmol/l Tricine-buffer pH 8, 0.03% (w/v) octanoyl-N-methylglucamide, 150 mmol/l NaCl and a cryoprotectant. The aviscumine activity was determined by a binding assay. The addition of 0.2% Na-polyphosphate to the reconstitution medium led to retention of approx. 73% of the aviscumine activity after 20 min nebulization with the Systam ultrasonic nebulizer. It has been observed that 84 and 72% of the activity were retained by the addition of 10 mmol/l CaCl(2) x 6H(2)O using PariBoy air-jet and Multisonic ultrasonic nebulizer, respectively. In addition, a decrease in the mean droplet size with increasing the cryoprotectant concentration has been observed. A relationship between the average droplet size, surface tension and viscosity depending on the used cryoprotectant type and concentration could be established.