Dense gas techniques provide a suite of clean technology options for the processing of pharmaceuticals. Monodisperse, micron-sized particles can be produced at mild operating temperatures and with negligible solvent residue. In this study, protein was precipitated from organic solutions using dense carbon dioxide as antisolvent. The gas antisolvent precipitation process (GAS) was used to produce biologically active lysozyme, insulin, and myoglobin powders. The effects of operating temperature, solute concentration and the rate of antisolvent addition on the morphology, size, activity and residual solvent concentration of lysozyme and insulin precipitates have been examined. The powders produced consisted of uniformly sized non-aggregated spherical particles. Precipitate size was controlled between 0.05 mm and 2.0 mm by changes to the solvent and antisolvent compositions. In general the concentration of residual organic solvent was found to be dependent on the mass of antisolvent used during the washing cycle. Residual concentrations as low as 300 ppm were easily achievable in a single step.
Protein was precipitated from organic and aqueous solutions using carbon dioxide and ammonia as antisolvents. The gas antisolvent precipitation process (GAS) was used to produce lysozyme, insulin and myoglobin powders. Protein powders were produced with narrow size ranges, and particle size was controlled between 0.05 mm and 2.0 mm by changes to the solvent system. Typically the stronger the protein solvent the larger the precipitate size. The GAS process, although ideal for the micronisation of stable protein powders, was limited by the number of suitable protein solvents that were miscible with dense carbon dioxide and that did not irreversibly affect protein conformation. As a result, GAS precipitation from aqueous solutions was also assessed. Insulin was precipitated from aqueous solutions as discrete 0.2±0.3 mm spheres using ammonia as an antisolvent.
A novel protein fractionation technique using a volatile electrolyte has been developed. Carbon dioxide was used to isoelectrically precipitate 80% and 95% pure glycinin and beta-conglycinin fractions from soybean isolate. The protein fractions precipitated as primary particles 0.2-0.3 microm in diameter, which under optimum conditions may be recovered as aggregates up to 500 microm in diameter. The dependency of protein fractionation efficiency on aggregate settling rates has been demonstrated. The isoelectric points of the two main soybean fractions, glycinin and beta-conglycinin, were calculated to be pH 5.2 and 4.95, respectively. Solution pH was accurately controlled by pressure in the isoelectric pH range of the different soybean protein fractions, and a pH "overshoot" was eliminated. Volatile electrolyte technology was also applied to a continuous process in order to eliminate the particle recovery concerns associated with batch precipitation and to demonstrate the potential for scale-up. Glycinin was effectively recovered on-line (94% glycinin recovery) with a purity approaching that of the batch process (95%).
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