Production of Protein-Loaded Polymeric Microcapsules by

biodegradable polymer particles is of great interest for the development of new ... been proposed so far, such as the rapid expansion from supercritic...
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Ind. Eng. Chem. Res. 2001, 40, 795-800

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MATERIALS AND INTERFACES Production of Protein-Loaded Polymeric Microcapsules by Compressed CO2 in a Mixed Solvent Nicola Elvassore* and Alberto Bertucco Istituto di Impianti Chimici, Universita` di Padova, via Marzolo 9, I-35131 Padova, Italy

Paolo Caliceti Dipartimento di Scienze Farmaceutiche, via Marzolo 5, I-35131 Padova, Italy

In view of developing new drug-delivery systems, microparticles of biocompatible polymer loaded with protein were produced using a supercritical antisolvent technique. As the polymer, poly(lactic acid) (PLA) of about 100 000 Da was used, whereas insulin was chosen as the protein model. To achieve microencapsulation, we started from a homogeneous solution of protein and polymer, to which the supercritical antisolvent is added; mixtures of dichloromethane and dimethyl sulfoxide were used to ensure the solubility of both the polymer and the protein. All experiments were performed in semicontinuous mode. A preliminary study with PLA alone was done in order to select the best operative condition and to control the particle dimension and their diameter distribution range, especially with mixtures of the two solvents. Then, the microspheres of PLA charged with insulin were produced; the average diameters ranged from 0.5 to 2 µm. The composition of these particles was determined analytically, and the protein bioactivity was checked. Introduction Microencapsulation of pharmaceutical compounds in biodegradable polymer particles is of great interest for the development of new controlled drug-delivery systems.1 Several medical methods, depending on the required particle morphology, can be used for this purpose: aerosol, inhalation, and systemic and subcutaneous injections. Typically, particles in the range of 5-100 µm are subcutaneously injectable, 1-5 µm particles are suitable for aerosol delivery and inhalation therapy to the lungs, and nanoparticles can be directly injected in the systemic circulation. Conventional pharmaceutical methods for the production of protein-loaded microparticles include emulsion and double-emulsion solvent extraction, liquid antisolvent, spray drying, and freeze drying. All of these techniques are associated with the use of organic solvents which lead to high residual contents of toxic solvent in the final product and low encapsulation efficiencies due to the partitioning of the pharmaceuticals between the two immiscible liquid phases. In this work, an alternative way, based on gas and supercritical CO2 antisolvent precipitation, was used to produce insulin-loaded microparticles of poly(L-lactic acid) (PLA). CO2 offers the advantage not only of being an environmentally benign solvent but also of reducing the residual solvent in the product to very low concentration, by a suitable tuning of the process variables.2 * To whom correspondence should be addressed. E-mail: [email protected]. Phone: [+39] (049) 8275472. Fax: [+39] (049) 8275461.

In the latest literature the production of pharmaceuticals-loaded biopolymer microparticles by supercritical techniques is widely considered3,4 even if proteins have been used as pharmaceuticals in only a few cases. All of these applications take advantage of the solvent or antisolvent power of CO2. Different techniques have been proposed so far, such as the rapid expansion from supercritical solution (RESS),5 the gas antisolvent (GAS)6 and supercritical antisolvent (SAS) precipitation,6 the precipitation with a compressed fluid antisolvent (PCA),7 and the aerosol solvent extraction system (ASES).8 However, because of the low solubility of polymers in supercritical CO2 (except in the case of perfluorinated polymers), major attention was paid to SAS, GAS, or PCA techniques.9-14 In a GAS (SAS) process, an organic solution of polymer and drug is atomized through a nozzle into a high-pressure vessel containing compressed CO2, so that liquid drops of solution are formed in a supercritical gas bulk phase. Because of the effect of CO2 diffusion in the organic solvent and of the organic solvent counterdiffusion in the gas phase, the liquid density of the drop is lowered until a critical value is reached, at which time the solute precipitation occurs. The supersaturation conditions allow fast and simultaneous precipitation of both polymer and drugs, so that, in most cases, the drugs can be trapped into the polymer matrix. The particularly high supersaturation condition, higher than that in the case of traditional liquid antisolvent methods, leads to a micro- or submicroparticle morphology. Among other types of drugs, proteins, peptides, enzymes, hormones, and genetic material have crucial

10.1021/ie0004904 CCC: $20.00 © 2001 American Chemical Society Published on Web 01/05/2001

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pharmaceutical importance in controlled drug-delivery systems. In fact, their delivery to the human body is affected by fast metabolization, decomposition, and degradation problems and a short half-time life. It is clear that techniques based on supercritical fluids offer an alternative way to classical antisolvent processes for low-temperature protein encapsulation. However, to apply these methods successfully, it is of fundamental importance that the biological activity of these labile compounds is maintained; in fact, the SAS process exposes proteins to organic and supercritical solvents, high pressure, and shearing strength. A first study on proteins processed by supercritical CO2 was proposed by Debenedetti and co-workers;15 they used a supercritical antisolvent precipitation to produce protein particles. Further studies16,17 evidenced a conformational change of the secondary structure of the proteins; surprisingly, despite the major conformation change induced during SAS precipitation, the precipitated proteins recovered their biological activity and native structure content upon redissolution in aqueous media. In particular, insulin and lyzozyme precipitated from dimethyl sulfoxide (DMSO) solutions fully recovered their biological activity upon redissolution, while higher molecular weight proteins were irreversibly denatured.18 To reduce the time of the possible labile compound denaturation, an alternative technique to SAS has been proposed by York and co-workers.19,20 This method, named solution-enhanced dispersion by supercritical fluids (SEDS), is able to precipitate proteins from an aqueous solution by extracting water with a supercritical CO2/ethanol mixture, and it has been successfully applied so far to lyzozyme and trypsin. A recent study by Young et al.21 deals with encapsulation of lyzozyme in biodegradable polymer microspheres. A 1-10 µm lyzozyme particle suspension in a polymer solution was sprayed into CO2 vapor phase through a capillary nozzle. The droplets solidified after falling into the liquid phase. By delay of the precipitation in the vapor phase, the larger microparticles obtained were able to encapsulate the suspended lyzozyme. The final capsules were in the range of 5-70 µm. This work is a nice attempt of protein encapsulation but led to products which are far away from the pharmaceutical requirements needed to develop microparticle drug-delivery systems. To improve the results obtained so far, i.e., to produce smaller particles with the drugs finely dispersed in the polymer matrix by a SAS process, a homogeneous solution of polymer and pharmaceuticals has to be used. Unfortunately, when (as usual) the drug cannot be solubilized in the same organic polymer solution, some expedients are necessary. Therefore, among various techniques recently proposed, such as the hydrophobic ion pairing (HIP)22 and the covalent conjugation of acyclic moieties, we observed that a suitable mixture of dichloromethane (DCM) and DMSO is capable of giving a homogeneous solution of PLA and insulin at sufficient amounts for the process to be carried out quantitatively. This easiest way of processing does not require addition of other chemicals to form the ion pairing or surfactants to form micelles. In the present work, microparticles of insulin and polymer were produced according to this technique, and the effect of process variables on particle morphology in the presence of a mixture of solvents was studied.

The biological activity of proteins and the encapsulation yield were evaluated. Insulin was used as the model drug for two main reasons: its great therapeutic importance for human diabetic applications and the possibility of evaluating easily its biological activity by in vivo release profiles. Materials and Methods Chemicals and Solutions. Bovine insulin was obtained from Sigma (St. Louis, MO). Poly(L-lactic acid) (RESOMER L 206) of 102 000 Da molecular weight was purchased from Boehringer Ingelheim Pharma KG (Ingelheim, Germany). CO2 (99.95%) was purchased from Air Liquide (Padova, Italy). Organic solvents trifluoroacetic acid (TFA), DMSO, and DCM were purchased from Aldrich (Steinheim, Germany). These chemicals were used without further purification. Insulin/PLA solutions were prepared by dropping 20 mL of an insulin solution in DMSO (1 mg/mL) into 20 mL of DCM containing PLA at a concentration of 20 mg/mL. PLA and insulin concentrations in the DCMDMSO mixture were determined gravimetrically after lyophilization and colorimetrically by protein Biorad assay, respectively. SAS Experiments. SAS was applied in both discontinuous and semicontinuous modes; in the first case the thermodynamic behavior of the PLA solution was studied, whereas for the production of the loaded microparticles, a semicontinuous SAS was adopted. In the batch experiment, the precipitation unit was initially loaded with a given amount of a polymersolvent solution (or mixture of solvents) and then the supercritical antisolvent was added. The antisolvent was fed from the bottom of the cell to promote the CO2 dissolution in the liquid phase. The measure of volume expansion and the “unaided eye” precipitation pressure was recorded. This experiment provides information to characterize the precipitation of PLA from an organic mixture of two solvents. In the semicontinuous SAS experiment, the liquid solution and the supercritical antisolvent were continuously added to the precipitation unit in cocurrent or countercurrent mode and the particles formed were collected in the vessel. Then a washing step is carried out to reduce the contents of the residual organic solvents to the desired amount. The best operative condition to minimize and eliminate flocculation and agglomeration of drug-loaded microspheres was studied: in particular, we have examined the influence on the production of the microsphere of PLA of biopolymer concentration in the solution, of the ratio between antisolvent and solution flow rates, of the values of temperature and pressure, and of the hydrodynamics and geometry of the nozzle. We refer to Figure 1 (for the batch mode) and Figure 2 (for the semicontinuous mode). In the batch mode of operation, the CO2 is fed into a stationary bulk liquid phase from the bottom of the cell by a HPLC pump (Dosapro Milton Roy model 169-33), with a maximum flow rate of 250 cm3/h at 250 bar. A 70 cm3 highpressure windowed cell (Klinger model 100) was used in order to measure the volume expansion and the precipitation pressure. Compressed CO2 was added to increase the pressure at a rate of approximately 1 bar/ min; the CO2 flow rate was controlled by fine metering valves (Hoke model 1316G4y). At the end of the experiment, the CO2 was vented through an expansion valve

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from the microspheres: 10 mg of an insulin-loaded microsphere was dissolved in 400 µL of DCM, and 400 µL of a H2O-0.05% TFA mixture was added; the mixture was emulsified and then centrifuged for 15 min at 3500 rpm. The insulin content in the aqueous phase was estimated by Biorad DC1 colorimetric assay. Results and Discussion

Figure 1. Scheme of the apparatus for batch experiments: F, CO2 feed; P, piston pump; TG, thermostat; RH1, RH2, electric resistance; VM, metering valves; K, windowed precipitation cell; PI, pressure indicator; TI, temperature indicator; R, rotameter; W, vent.

and an expansion unit; both devices were heated by electric resistance to prevent freezing due to CO2 expansion. The temperature of the cell was controlled by electric resistance and the inlet CO2 temperature by a heat exchanger connected with an auxiliary thermostatic bath. The temperature was measured by Pt 100 Ω resistances and the pressure by a manometer. When operating in semicontinuous mode, the polymer solution was sprayed into a high-pressure vessel, as shown in Figure 2. In the first runs, a 70 cm3 windowed cell (Klinger model 100) was used in order to observe the precipitation visually, whereas all other experiments were performed in a 200 cm3 high-pressure cylinder. In both cases, CO2 was fed by a reciprocating pump (DOXE), from the top of the cell. In cocurrent mode the polymer solution was injected by a HPLC pump (Altex A1) through a 50 µm internal diameter fused silica capillary nozzle; to minimize the headloss, the capillary length was kept below 2 cm. The cell temperature control was achieved by either an electrical resistance or a heat exchanger connected to an auxiliary bath. The inlet CO2 temperature was controlled by a heat exchanger connected with another auxiliary thermostatic bath. The cell temperature was measured by two Pt 100 Ω resistances placed at the top and the bottom of the cell. The CO2 flow rate was controlled by fine metering valves (Hoke model 1316G4y). Typically, after steady conditions of P, T, and CO2 flow rate were reached, 20 mL of solution was injected into the precipitation unit at a constant flow rate. Compressed CO2 continuously fed from the top of the cell caused the precipitation. The temperature of the precipitation vessel was checked to remain constant in all experiments. Polymer microparticles were recovered at the bottom of the cell, where a glass wool and a 2 µm metallic filter were positioned. The CO2 was vented through an expansion valve and an expansion unit; both devices were heated by electric resistance to prevent freezing due to CO2 depressurization. The CO2 flow rate was measured with a rotameter (Rigas, Italy). In the final step, supercritical CO2 flowed into the vessel to wash and dry the precipitated products. There the dried materials were collected and analyzed. Morphological Studies. Morphology characterizations were performed by scanning electronic microscopy (SEM). All samples were gold sputtered under a high vacuum (0.05 mTorr), and photographs were taken at different magnifications from 1000 to 20 000. The mean particle size and the particle distribution were calculated by imaging analysis. Protein-Loading Determination. The yield of insulin loading was estimated after protein extraction

Solution Preparation. The simplest way to reach microencapsulation is spraying a homogeneous solution of protein and polymer into a supercritical antisolvent, obtaining, because of the supersaturated conditions, the simultaneous precipitation of both solutes. Unfortunately, in a few cases only, both the protein and the biopolymer are soluble in the same solvent; in addition, water, where proteins are naturally soluble, cannot be used directly for supercritical antisolvent precipitation. So, in this work, among various methods to enhance the solubility of hydrophilic pharmaceuticals in hydrophobic solvents for SAS process purposes, such as HIP,22 mixtures of solvents were used. It is well-known that PLA is highly soluble in DCM and an insulin-DMSO solution processed with a supercritical technique was demonstrated to maintain the biological activity of the protein.15 As a consequence, mixtures of DCM and DMSO were considered. A study of the mutual solubility of PLA and insulin in different concentrations of the DCM-DMSO mixtures evidences that a 50% (w/w) composition gives the best solubility compromise, as shown in Figure 3. In fact, this combination of solvents allows the stable solubilization of 1% of polymer and 5% of insulin with respect to the polymer. Thermodynamics. The presence of a solvent mixture complicated the theoretical understanding of the SAS process. The thermodynamic aspects were studied in the batch apparatus by measuring the liquid solvent expansions and the precipitation pressures. A direct observation through a window allows one to estimate that the volumetric expansion of the liquid solvent is strongly connected to the dissolution of the antisolvent in the liquid. Expansion curves, reported in Figure 4 for DCM, 50% (w/w) DMSO-DCM mixtures, and DMSO, move toward lower pressure when the DMSO concentration increases. Moreover, the mixtures seem to have properties closer to those of the low expandable fluid, i.e., DMSO; as a consequence, the polymer and protein precipitation should be mainly driven by the DMSO thermodynamic properties. Microsphere Production. In the first set of experiments polymer solutions were sprayed into a windowed cell containing high-pressure CO2 to get information to set up the best operative variables. The summary of relevant experimental runs are reported in Table 1. Particle formation was observed approximately 1 cm below the nozzle, and it appeared as a white dense cloud. The suspension moved slowly versus the bottom of the cell because of the CO2 convective flow. The liquid in the cell above the suspension formation layer was relatively clear, indicating poor retromixing. The polymer solution flow rate of 1 mL/min gave good jet atomization; at this condition, even if in the nozzle, there is a completely formed laminar flow (low Reynolds number); the high jet velocity of the solution (estimated to be around 10 m/s) and the high antisolvent density out of the nozzle favor the jet breakup. The pressure of the antisolvent and the solution flow rate should not

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Figure 2. Scheme of the semicontinuous SAS apparatus: F, feed; P, piston pump; S, heater; TG, thermostat; VM1, VM2, VM3, metering valves; VT, three-way valve; K, windowed cell (runs 1-3) and 200 cm3 cylindrical vessel (runs 5-18); RH, electric resistance; PI, pressure indicator; TI, temperature indicator; PL, pump injection device; S1, S2, S3, solution vessels; PE, expansion vessel; R, rotameter; W, vent.

Figure 3. Fraction of concentration of PLA and insulin in the solution with respect to the initial amount dissolved (1.5 g/L of polymer and 75 mg/L of insulin) as a function of the concentrations of DCM-DMSO mixtures.

Figure 4. Volumetric expansion of DMSO, a 50/50 (w/w) mixture of DCM and DMSO, and DCM as a function of the pressure at a temperature of 25 °C.

be lowered below 80 bar and 0.5 mL/min, respectively, to ensure micrometric liquid drop formation. These values are fully consistent with the study, recently proposed by Eggers and Czerwonatis,23 on the disintegration of liquid jets in pressurized gases. Once the main criteria of the nozzle hydrodynamics were established, the influence of the process variables on the particle morphologies was studied in the case of DMSO-DCM mixtures. Microparticles were produced for different DMSODCM concentration mixtures (runs 3-5); it was observed that the higher DMSO concentration increased

the agglomeration and flocculation phenomena. As qualitatively shown in Figure 5, there are a large number of particles fused together; the presence of DMSO leads to adhesive particles. Based on preliminary runs, we focused on the optimization of the 50/50 DMSO-DCM mixtures, where both insulin and PLA are reasonably soluble. The pressure plays an important role not only for jet breakup of the organic solution but also for mass transport. Below 80 bar the density of the gas phase is too low to favor the atomization of the solution; as shown in Figure 6, microfibrils were produced. At higher values, pressure effects on the macroscopic morphology are not so relevant; however, smaller particles were produced at 130 bar. Temperature effects were more remarkable; microparticles produced at higher temperature evidence agglomeration and flocculation phenomena. It was evident that runs performed at 40 °C gave polymer plasticization on the vessel surface, whereas the runs at 20 °C led to soft particle powders; in fact, because of the high pressure and of the CO2 and organic solvent presence, the melting temperature of the polymer is likely to be considerably lowered. When the polymer concentration and the flow rate of the organic solution were varied, the flocculation was not accentuated appreciably; however, the best values were estimated to be 1% (w/w) and 1 mL/min, respectively. The high CO2 flow rate, as reported in the latest literature,24 decreased the residual solvent content but did not affect significantly the particle morphology; a value of 1500 NL/h was used for the experiments. The best operative conditions are given in Table 1, those corresponding to runs 15-18. In Figures 7 and 8 two SEM analysis examples of typical PLA and insulinloaded PLA microsphere production are presented. The diameter distribution profile (Figure 9) evidences a main fraction of microparticles of 0.5 µm diameter; furthermore, the particles produced from an insulin-PLA mixed-solvent solution in the best operative condition do not present agglomeration or flocculation. Insulin Yield Loading. Thanks to the high supersaturation conditions of the SAS process, full encapsulation of the protein is expected to occur. In fact, in all

Ind. Eng. Chem. Res., Vol. 40, No. 3, 2001 799 Table 1. Summary of Microparticle Production Performed by a Semicontinuous SAS Process

run

solvent

polym concn [g/L]

1a 2a 3a 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

DCM DCM DCM DCM DCM-DMSO (75/25) DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50% DCM-DMSO 50%

1.0 1.0 2.0 1.0 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

a

insulin concn [% w/w on polymer]

5.0 5.0

5.0

T [°C]

P [bar]

solution flow rate [mL/min]

CO2 flow rate [NL/h]

primary particle [µm]

28.5 28.7 37.0 34 33 34 38 37 37 39 32 38 39 23.5 22.5 22.5 25 19.8

95 115 90 100 100 100 105 102.5 85 105 105 130 130 130 130 130 130 130

1.0 1.2 1.0 1.0 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.3 0.6 1.0 1.0 1.0 1.0 1.0

290 700 575 720 900 960 1145 1800 1050 1100 1200 1211 1160 1239 1178 872 1580 1518

1-3 1-3 1-4b 1-3 1-4c 1-4c 1-5b 1-5b -d 1-5b 1-4c 1-4b 1-5c 0.5-2 0.5-2 0.5-2 0.5-2 0.5-2

Experiment performed with a windowed cell. b Agglomerated. c Partially agglomerated.

Figure 5. SEM micrographs of PLA microparticles produced at high temperature in mixed solvents (75/25 DCM-DMSO): run 5 of Table 1; 10 000×.

Figure 6. SEM micrographs of PLA microparticles produced at low pressure (85 bar): run 9 of Table 1; 500×.

runs a high protein incorporation yield was found, over 80%. This value is very high when considering the possibility of the low protein extraction process efficiency used for analytical determination. Only a small part of insulin, corresponding to around 5-10%, was recovered by washing the microspheres with DMSO (50%) and water-TFA (47/3) mixtures. These results

d

Fibrils.

Figure 7. SEM micrographs of PLA microparticles produced at the best operative condition: run 17 of Table 1; 2000×.

Figure 8. SEM micrographs of insulin-loaded PLA microparticles produced at the best operative condition: run 3 of Table 1; 8000×.

suggested that all of the insulin was loaded, and it was finely dispersed into the polymeric matrix. Moreover, insulin extracted from microspheres was found to fully maintain the hypoglycemic activity after subcutaneous injection to diabetic mice. The total amount of insulin loaded in the microspheres and its biological activity were unaffected by the production condition.

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Figure 9. Size distribution of microparticles produced at 130 bar and 20 °C (run 18 in Table 1).

Conclusions The problem of coprecipitation of PLA and insulin by a SAS technique was addressed. A study of the polymer precipitation in a solvent mixture, necessary to coprecipitate a protein and a polymer, was carried out. It was found that the use of mixed solvents gives a suitable solution for the application of the SAS process. The best operative condition for PLA particle production in a mixture of DCM and DMSO was determined. Submicron particles with no flocculation and agglomeration phenomena were obtained. Coprecipitation of the protein and biopolymer for a drug-delivery system was successfully achieved by a semicontinuous SAS process. The results reported show that the yield of the proposed process is very high compared to traditional microencapsulation techniques and that the protein still retains their biological activity. Furthermore, it is uniformly dispersed within the microspheres. Acknowledgment Partial support granted to this work by the Italian Ministry for University and Scientific Research (MURST 40%) is gratefully acknowledged. The authors thank Ms. Monica Daminiato and Mr. Lucio Gelmi for their assistance in performing experimental runs. Literature Cited (1) Okada, H.; Toguchi, H. Biodegradable Microspheres in Drug Delivery. Crit. Rev. Ther. Drug Carrier Syst. 1995, 12, 1. (2) Falk, R.; Randolph, T. Process variable implication for residual solvent removal and polymer morphology in the formation of gentamycin-loaded poly(L-lactide) microparticles. Pharm. Res. 1998, 15, 1233. (3) Subramaniam, B.; Rajewski, R. A.; Snavely, K. Pharmaceutical Processing with supercritical Carbon dioxide. J. Pharm. Sci. 1997, 86, 885. (4) Reverchon, E. Supercritical antisolvent precipitation of micro- and nanoparticles. J. Supercrit. Fluids 1999, 15, 1. (5) Matson, D. W.; Fulton, J. L.; Petersen, R. C.; Smith, R. D. Rapid Expansion of Supercritical Fluid Solution: Solute Formation of Powders, Thin Films and Fibers. Ind. Eng. Chem. Res. 1987, 26, 2298. (6) Gallagher, P. M.; Coffey, M. P.; Krukonis, V. J.; Klasutis, N. Supercritical Fluid Science and Technology; Johnston, K. P.; Penninger, J. M. L., Eds.; ACS Symposium Series 406; American Chemical Society: Washington, DC, 1988; p 334. (7) Dixon, D. J.; Johnston, K. P. Formation of Microporous Polymer Fibers and oriented Fibrils by Precipitation with a Compressed Fluid Antisolvent. J. Appl. Polym. Sci. 1993, 50, 1929.

(8) Bleich, J.; Kleinebudde, P.; Mueller, B. W. Influence of Gas density and pressure on Microparticles Produced with the ASES Process. Int. J. Pharm. 1994, 106, 77. (9) Randolph, T. W.; Randolph, A. D.; Mebes, M.; Yeung, S. SubMicrometer-Sized Biodegradable Particles of Poly(L-Lactic Acid) via the Gas Antisolvent Spray Precipitation Process. Biotechnol. Prog. 1993, 9, 429. (10) Bodmeier, R.; Wang, H.; Dixon, D. J.; Mawson, S.; Johnston, K. P. Polymeric Microspheres Prepared by Spraying into Compressed Carbon Dioxide. Pharm. Res. 1995, 12, 1211. (11) Bleich, J.; Mueller, B. W. Production of Drug Loaded Microparticles by the Use of Supercritical Gases with the Aerosol Solvent Extraction System (ASES) Process. J. Microencapsulation 1996, 13, 131. (12) Falk, R.; Randolph, T.; Meyer, J. D.; Kelly, R. M.; Manning, M. C. Controlled Release of Ionic Pharmaceuticals from Poly(Llactide) Microspheres Produced by Precipitation with a Compressed Antisolvent. J. Controlled Release 1997, 44, 77. (13) Mawson, S.; Kanakia, S.; Johnston, K. P. Coaxial Nozzle for Control of Particle Morphhology in Precipitation with a Compressed Fluid Antisolvent. J. Appl. Polym. Sci. 1997, 64, 2105. (14) Thies, J.; Mu¨ller, B. D. Size controlled production of biodegradable microparticles with supercritical gases. Eur. J. Pharm. Biopharm. 1998, 45, 67. (15) Yeo, S. D.; Lim, G. B.; Debenedetti, P. G.; Bernstein, H. Formation of microparticulate protein powders using a supercritical fluid antisolvent. Biotechnol. Bioeng. 1993, 41, 341. (16) Yeo, S.; Debenedetti, P. G.; Sugunakar, Y. P.; Przybycien, T. M. Secondary Structure Characterization of Microparticulate Insulin Powders. J. Pharm. Sci. 1994, 83, 1651. (17) Winters, M. A.; Knuston, B. L.; Debenedetti, P. G.; Sparks, H. G.; Przybycien, T. M.; Stevenson, C. L.; Prestrelski, S. J. Precipitation of protein in supercritical Carbon Dioxide. J. Pharm. Sci. 1996, 85, 586. (18) Winters, M. A.; Frankel, D. Z.; Debenedetti, P. G.; Carey, J.; Devaney, M.; Przybycien, T. M. Protein Purification with VaporPhase Carbon Dioxide. Biotechnol. Bioeng. 1999, 62, 247. (19) Sloan, R. M.; Hollowood, E.; Kibria, I.; Humphreys, G. O.; Ashraf, W.; York, P. Supercritical Fluid Processing: Preparation of Stable Protein Particle. 5th Meeting of Supercritical Fluids, Nice, France, Mar 23-25, 1998. (20) Forbes, R. T.; Sloan, R.; Kibria, I.; Hollowood, M. E.; Humphreys, G. O.; York, P. Production of Stable Protein Particles: a Comparison of Freeze, Spray and Supercritical Drying. 3rd World Congress on Particle Technology, Brighton, U.K., July 7-9, 1998. (21) Young, T. J.; Johnston, K. M.; Tanaka, H. Encapsulation of lysozyme in a biodegradable polymer by precipitation with a vapor-over-Liquid Antisolvent. J. Pharm. Sci. 1999, 88, 640. (22) Powers, M. E.; Matsuura, J.; Manning, M. C.; Shefter, E. Enhenced solubility of proteins and peptides in non polar solvents through hydrophobic ion paiting. Biopolymers 1990, 33, 927. (23) Eggers, R.; Czerwonatis, N. Disintegration of Liquid Jets and Drop Drag Coefficients in Pressurized Gases. 16th Annual Conference on Liquid Atomization and Spray Systems, Darmstadt, Germany, Sept 11-13, 2000. (24) Ruchatz, F.; Kleinebudde, P.; Mu¨ller, B. W. Residual solvents in biodegradable micro-particles. Influence of process parameters on the residual solvent in micro-particles produced by the aerosol solvent extraction system (ASES) process. J. Pharm. Sci. 1997, 86, 101.

Received for review May 15, 2000 Revised manuscript received September 18, 2000 Accepted September 22, 2000 IE0004904