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#527 SCF Application Precipitation ofProteinPowders into a CompressedAntisolvent Introduction The pharmaceutical and health care industries areseeking new methods of administering proteinpowders. Controlled release systems are typicallyimplantable devices or injectable microparticulatesystems that can dispense appropriate amounts ofpeptides or proteins into the biologicalenvironment. Such systems are becomingincreasingly popular because they can reduceinjection frequency， decrease the total administeredprotein dose， and can increase patient compliancefor chronic indications. Since controlled release systems are made up ofprotein or peptide particles dispersed within apolymeric matrix， it is important to regulate theparticle size. Ideally， protein particles should be inthe 1- to 5-um size range. Small particles allow ahigher percentage of solids to be incorporated， andthus distributed more uniformly within thepolymeric matrix. Conventional methods for reducing the particlesize of proteins and peptides include spray drying，milling， fluid energy grinding， lyophilization， andusing miscible organic antisolvents. Unfortunately， these processes can also inactivateor denature proteins， produce small final yields，lead to electrostatically charged powders， produceparticles with a broad distribution， or rely on largevolumes of organic solvent. The precipitation by compressed antisolvent (PCA)process is an alternative technique that producesdry， biologically active microparticulate powdersof proteins， enzymes， and peptides. In the PCAmethod， a solid (such as a protein) is dissolved in aorganic solvent. This liquid is then continuously sprayed in smallamounts into a vessel filled with supercritical CO2.As the liquid dissolves in the SC-CO2 nano sizedparticles of proteins are produced. This application will describe a method forreducing the particle size of insulin usingsupercritical CO2. Equipment Applied Separations’Helix SupercriticalSystem √ Modifier Pump Materials Insulin (bovine， Zn， low endotoxin) CO2-bone dry grade (99.8%) DMSO (Dimethylsulfoxide) DMFA (N，N，-dimethylformamide) Method Prepare a 15 mg/mL DMSO/insulin solution and a5 mg/mL DMFA/insulin solution. Add a fewdrops of 1 N HCL to the DMFA/insulin solution inorder to fully solubilize the protein. Next， fillcrystallization vessel with SC-CO2. Spray organicsolvent containing solute into supercritical fluid.The organic solvent will dissolve into the SC-CO2，causing supersaturation and protein precipitation.The supercritical fluid containing the dissolvedsolvent is carried to a separator vessel. As thevessel is depressurized， the solvent becomes aliquid and SC-CO2 can be vented. 930 Hamilton Street Allentown，PA 18101 610.770.0900 (TEL) 610.740.5520 (FAX) SCF Application Stop the organic solvent/protein solution pump and continue the supercritical CO2 flow to insure protein is solvent free. PCA Conditions Crystallizer Vessel： Pressure： 86 BAR Temperature： 35C Solvent Flow Rate： 0.3 mL/min (liquid) Solvent Nozzle： 30 micron diameter. .24mm thickness CO2 Flow Rate： 8.0 L/min Time： Crystallization： 40 minutes SC-CO2 Drying： 120 minutes Separator Vessel： Pressure： 35 BAR Temperature： 35°C Analysis Examine the size of the protein particles byscanning electron microscopy (SEM). Conclusion PCA processing with supercritical antisolventproduces biologically active， dry， and fine powdersof proteins， enzymes， and peptides quickly anduniformly. When applied to insulin， PCAproduced particles between the 2- to 3-um rage，outperforming conventional methods. References Yeo， S.； Lim， G.； Debenedetti， P.； Bernstein，H."Formation of Microparticulate Protein PowdersUsing a Supercritical Fluid Antisolvent.”Biotechnology and Bioengineering. 41 (1993)‘341-346. 930 Hamilton Street Allentown，PA 18101 610.770.0900 (TEL) 610.740.5520 (FAX) www.appliedseparations.com