Generation of micro-particles of proteins for aerosol delivery using high pressure modified carbon dioxide

Purpose. To investigate the feasibility of using the Aerosol Solvent Extrac tion System (ASES) to generate microparticles of proteins suitable for aero sol delivery from aqueous-based solutions. Methods. The ASES technique using high- pressure carbon dioxide modified wi th ethanol was utilised for the generation of microparticles of proteins (l ysozyme, albumin, insulin and recombinant human deoxyribonuclease (rhDNase) ) from aqueous solutions. Particle size, morphology, size distributions and powder aerosol performance were examined. The biochemical integrity of the processed proteins was assessed by testing the level of molecular aggregat ion using size exclusion chromatography and by bioassay technique for lysoz yme. Results. Proteins were precipitated as spherical particles ranging in size from 100 to 500 nm. The primary nano-sized particles agglomerated to form m icron-sized particles during the precipitation process. The median size of the particles was a function of the operating conditions. In-vitro aerosol performance tests showed that the percent fine particle mass (< 5<mu>m) was approximately 65%, 40% and 20% for lysozyme, albumin and insulin, respecti vely. Negligible loss in the monomer content or biological activity was obs erved for lysozyme. Insulin exhibited slight aggregation and 93% of the mon omer was retained after processing. Albumin was affected by processing and only 50-75% of the monomer was retained compared with 86% in the original m aterial. However, rhDNase was substantially denatured during processing as shown by the significantly reduced monomer content. Conclusions. Micron-sized particles of lysozyme, albumin and insulin with s atisfactory inhalation performance were successfully generated from aqueous solutions using the modified ASES technique. The biochemical integrity of the processed proteins was a function of the operating conditions and the n ature of the individual protein.