Abstract:
A process for forming small micron-sized (1-10 μm) protein particles is provided wherein a protein, a solvent system for the protein and an antisolvent for the protein solvent system are contacted under conditions to at least partially dissolve the protein solvent system in the antisolvent, thereby causing precipitation of the protein. The solvent system is made up of at least in part of a halogenated organic alcohol, most preferably 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Preferably, a solution of the protein in the solvent system is sprayed through a nozzle into a precipitation zone containing the antisolvent (preferably CO 2 ) under near- or supercritical conditions.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is broadly concerned with improved methods for the forming and precipitation of small protein or peptide particles making use of the precipitation using compressed antisolvents (PCA) process. More particularly, the invention is concerned with such a method and the resulting proteinaceous particles wherein the process is carried out using a halogenated organic alcohol as at least a part of the protein solvent; in particularly preferred forms, the solvent is 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and the process yields micron-sized particles suitable for pharmaceutical uses without substantially degrading the protein. 
     2. Description of the Prior Art 
     Micron-sized (1-10 μm) protein particles are often deemed necessary for drug delivery systems such as controlled release and direct aerosol delivery to the lungs. Consistent commercial production of small protein particles of this type can be difficult. For example, spray drying techniques often lead to thermal denaturation of the protein, while milling and similar processes yield unacceptably broad size distributions and/or denaturation. Lyophilization can give particles in the desired size range, but with a broad distribution and/or denaturation; moreover, not all proteins of interest can be lyophilized to stable products. 
     In an effort to overcome these problems, supercritical fluid precipitation processes have been employed. Two processes that use supercritical fluids for particle formation are: (1) Rapid Expansion of Supercritical Solutions (RESS) (Tom, J. W. Debenedetti, P. G., 1991,  The Formation of Bioerodible Polymeric Microspheres and Microparticles by Rapid Expansion of Supercritical Solutions . BioTechnol. Prog. 7:403-411), and (2) Gas Anti-Solvent (GAS) Recrystallization (Gallagher, P. M., Coffey, M. P., Krukonis, V. J., and Klasutis, N., 1989,  Gas Antisolvent Recrystallization: New Process to Recrystallize Compounds in Soluble and Supercritical Fluids . Am. Chem. Sypm. Ser., No. 406; U.S. Pat. No. 5,360,478 to Krukonis et al.; U.S. Pat. No. 5,389,263 to Gallagher et al.). See also, PCT Publication WO 95/01221 and U.S. Pat. No. 5,043,280 which describe additional SCF particle-forming techniques. 
     In the RESS process, a solute (from which the particles are formed) is first solubilized in supercritical CO 2  to form a solution. The solution is then sprayed through a nozzle into a lower pressure gaseous medium. Expansion of the solution across this nozzle at supersonic velocities causes rapid depressurization of the solution. This rapid expansion and reduction in CO 2  density and solvent power leads to supersaturation of the solution and subsequent recrystallization of virtually contaminant-free particles. The RESS process, however, is not suited for particle formation from polar compounds because such compounds, which include drugs, exhibit little solubility in supercritical CO 2 . Cosolvents (e.g., methanol) may be added to CO 2  to enhance solubility of polar compounds; this, however, affects product purity and the otherwise environmentally benign nature of the RESS process. The RESS process also suffers from operational and scale-up problems associated with nozzle plugging due to particle accumulation in the nozzle and to freezing of CO 2  caused by the Joule-Thompson effect accompanying the large pressure drop. 
     The relatively low solubilities of pharmaceutical compounds in unmodified carbon dioxide are exploited in the second process wherein the solute of interest (typically a drug, polymer or both) is dissolved in a conventional solvent to form a solution. The preferred ternary phase behavior is such that the solute is virtually insoluble in dense carbon dioxide while the solvent is completely miscible with dense carbon dioxide at the recrystallization temperature and pressure. The solute is recrystallized from solution in one of two ways. In the first method, a batch of the solution is expanded several-fold by mixing with dense carbon dioxide in a vessel. Because the carbon dioxide-expanded solvent has a lower solvent strength than the pure solvent, the mixture becomes supersaturated forcing the solute to precipitate or crystallize as microparticles. This process was termed Gas Antisolvent (GAS) recrystallization (Gallagher et al., 1989). 
     The second method involves spraying the solution through a nozzle into compressed carbon dioxide as fine droplets. In this process, a solute of interest (typically a drug, polymer or both) that is in solution or is dissolved in a conventional solvent to form a solution is sprayed, typically through conventional spray nozzles, such as an orifice or capillary tube(s), into supercritical CO 2  which diffuses into the spray droplets causing expansion of the solvent. Because the CO 2 -expanded solvent has a lower solubilizing capacity than pure solvent, the mixture can become highly supersaturated and the solute is forced to precipitate or crystallize. This process has been termed in general as Precipitation with a Compressed Fluid Antisolvent (PCA) (Dixon, D. J.; Johnston, K. P.; Bodmeier, R. A.  AIChE J.  1993, 39, 127-139.) and employs either liquid or supercritical carbon dioxide as the antisolvent. When using a supercritical antisolvent, the spray process has been termed Supercritical Antisolvent (SAS) Process (Yeo, S.-D.; Debenedetti, P. G.; Radosz, M.; Schmidt, H.-W.  Macromolecules  1993, 26, 6207-6210.) or Aerosol Spray Extraction System (ASES) Müller, B. W.; Fischer, W.; Verfahren zur Herstellung einer mindestens einen Wirkstoff und einen Träger umfassenden Zubereitung, German Patent Appl. No. DE 3744329 A1 1989). 
     U.S. Pat. No. 6,063,910 describes a specific process for the production of protein particles by supercritical fluid precipitation. In this process, a solution of protein is prepared using a variety of solvents such as ethanol, DMSO and glycols, whereupon the solution is sprayed through a nozzle into an antisolvent under supercritical conditions, thereby effecting precipitation of the protein as small micron-sized products. In the case of insulin, the process was found to create a substantial loss of α-helicity and a marked increased in β-sheet and β-reverse turn content. (Winters et al.,  J. Pharmaceutical Sciences,  85(6):586-594 (1996)). The solvents used in the process of the &#39;910 patent, and particularly DMSO, are not favored for pharmaceutical uses. For example, many such solvents leave a residuum in the precipitated particles, causing purity problems. 
     There is accordingly a real and unsatisfied need in the art for an improved protein precipitation process which avoids the solvent problems of many prior techniques while giving micron-sized particles of small size distribution and with little protein degradation. 
     SUMMARY OF THE INVENTION 
     The present invention overcomes the problems outlined above and provides an improved method for forming small protein particles of micron size, preferably from about 1-10 μm, and more preferably from about 1-5 μm. Broadly speaking, the process involves contacting a protein, a protein solvent system and an antisolvent for the protein solvent system under conditions to at least partially dissolve the protein solvent system in the antisolvent with consequent precipitation of the protein; the protein solvent system includes at least in part a halogenated organic alcohol, and preferably consists essentially of a single halogenated organic alcohol. Use of such solvents materially improves precipitation processes heretofore used and avoids many of the problems of the prior art. 
     A wide variety of solvent/antisolvent precipitation processes can be used in accordance with the invention. For example, the GAS and PCA processes can be employed. Preferably however, the solvents of the invention are used in the PCA process wherein the protein is first dissolved in the solvent system, and then droplets of the solution are sprayed into an antisolvent under conditions to precipitate protein particles. 
     The preferred halogenated organic alcohols are the halogenated alkyl alcohols, especially the C 1 -C 4  alcohols. Particular alcohol solvents are HFIP, trifluoroethanol, 2-chloroethanol and mixtures thereof. The single most preferred solvent is HFIP (CAS #920-66-1). This solvent has a boiling point of 59° C. and a density of 1.618 g/ml, and is very soluble in CO 2 . Normally, only a single halogenated organic alcohol will be used as a protein solvent. However, multiple-component solvent systems can also be employed, so long as such systems include a halogenated organic alcohol as at least a part thereof. 
     A variety of antisolvents can also be used in the invention, such as CO 2 , propane, butane, isobutane, nitrous oxide, sulfur hexafluoride, trifluoromethane, hydrogen and mixtures thereof. CO 2  is the most preferred antisolvent, owing to its low cost, ready availability and critical properties (T c =81.0° C. and P c =73.8 bar or 1070 psi). Furthermore, CO 2  is non-toxic, non-flammable, recyclable, and “generally regarded as safe” by the FDA and pharmaceutical industry. 
     During processing, the contact between the protein solution system and antisolvent is carried out at near or supercritical conditions for the antisolvent, e.g., from about 0.5-2 P c  and more preferably from about 0.9-1.5 P c ; when CO 2  is used as the antisolvent, pressure conditions are normally maintained at a level of from about 1000-2000 psig, and more preferably from about 1100-1600 psig. The temperature conditions during processing are generally relatively low in order to avoid heat denaturation of the protein. Generally, temperatures of up to about 60° C. and more preferably up to about 50° C. are used. When CO 2  is the antisolvent, such temperatures exceed the T c . 
     In order to maximize production rates, the preferred process is carried out in a pressurized precipitation chamber equipped with a nozzle. The protein solution is sprayed through the nozzle into a precipitation zone containing the antisolvent. The resultant protein particles are collected in a downstream recovery filter, and can easily be further processed for pharmaceutical uses. 
     In most instances, the starting protein is dissolved in a halogenated organic alcohol solvent or solvent system containing such an alcohol, thereby producing true solutions. However, the invention is not so limited. That is, it is possible that the protein may be only partially dissolved or dispersed within the solvent. Therefore, as used herein, “solution” should be understood to mean not only true solutions but also partial solutions and dispersions. Similarly, while complete proteins are often processed in accordance with the invention, protein fragments or peptides could also be treated. Accordingly, the term “protein” refers to all types of proteinaceous species. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic representation of preferred apparatus used in the precipitation of proteins in accordance with the invention; 
     FIG. 2 is a fragmentary schematic representation which, when viewed in connection with FIG. 1, depicts an alternative filtration apparatus; 
     FIG. 3A is a far-UV CD spectra comparing unprocessed insulin with insulin processed in accordance with the invention at a total pressure within the precipitation chamber of 1400 psig, as set forth in Example 1; 
     FIG. 3B is a far-UV CD spectra comparing unprocessed insulin with insulin processed in accordance with the invention at a total pressure within the precipitation chamber of 1200 psig as set forth in Example 1; 
     FIG. 4A is a near-UV CD spectra comparing unprocessed insulin with insulin processed in accordance with the invention at a total pressure within the precipitation chamber of 1400 psig as set forth in Example 1; 
     FIG. 4B is a near-UV CD spectra comparing unprocessed insulin with insulin processed in accordance with the invention at a total pressure within the precipitation chamber of 1200 psig as set forth in Example 1; 
     FIG. 5 is a CD spectra of unprocessed (U) and processed (P) albumin from Example 2; and 
     FIG. 6 is a CD spectra of unprocessed (U) and processed (P) albumin from Example 2. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The following examples set forth preferred techniques for the micronization of representative proteins, and the characterization of these proteins. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention. 
     EXAMPLE 1 
     In this example a series of biosynthetic insulin samples (Eli Lilly Lot No. 009LX9) dissolved in 20 mL HFIP were sprayed through an ultrasonic nozzle into supercritical CO 2  within a precipitation chamber using the techniques of the invention. The precipitated products were then tested to confirm that the final insulin products were not materially altered, as compared with the starting insulin samples. 
     The apparatus employed in this example is set forth in FIG.  1 . Broadly speaking, the apparatus  10  included a temperature-controlled water bath  12  including therein a pair of interconnected filters  14 ,  16  and a precipitation chamber  18  equipped with an ultrasonic nozzle (Misonix Sonimist 600-1)  20  having a solution input  80 . A protein-containing solution to be micronized is contained within a reservoir  22  and is directed through the nozzle  20  along with carbon dioxide from a supply  24 . Protein particles from the chamber  18  are recovered in a recovery system  16 . 
     In more detail, the heater for the water bath  12  is preferably a Fisher Scientific Allied Model 70 immersion heater (1000 W). A surge tank  28  (Whitey 304L-HDF4-2250CC) having a 2250 cm 3  capacity and a 1800 psig pressure rating is located within the bath  12 , along with a coil of {fraction (1/16)} inch stainless steel tubing  30 , the filters  14 ,  16 , and chamber  18 . A conduit  32  leads from the bottom of tank  28  to a three-way valve  34 . A first conduit  36  extends from an output of valve  34  to an inlet  21  of the nozzle  20 . A second conduit  38  extends from the other output of valve  34  to another three-way valve  40 . A first conduit  42  from the valve  40  is directed to three-way valve  44 , whereas a second conduit  46  leads to the outlet  48  of chamber  18 . 
     One conduit  50  of the valve  44  leads to the input of filter  14 , whereas the second conduit  52  leads to the input of filter  16 . The output conduits  54 ,  56  from the filters  14 ,  16  are connected to a three-way valve  58 . The third conduit  60  from the valve  58  is equipped with a two-way valve  62  and leads to a heated micrometering valve  64  (Autoclave Engineers 30VRMM 4812) equipped with a thermocouple  65 . As shown, the bath  12  is also provided with three thermocouples  66 ,  68  and  70 , with the latter extending into chamber  18 , as well as a pressure transducer  71 . 
     The solution reservoir  22  is coupled with a syringe pump  72  (Isco 260D) having inlet and outlet valves  72   a ,  72   b , with the latter having an output conduit  74  leading into bath  12  and particularly to the inlet side of coil  30 . The outlet of coil  30  is connected to a conduit  76  coupled with transducer  71  and leading to a three-way valve  78 ; one output leg  80  from the valve  78  leads to the solution inlet of nozzle  20 . The other output leg  82  is equipped with a two-way valve  84  and leads to the atmosphere. 
     The CO 2  supply  24  includes a pair of CO 2  tanks  86 ,  88  with valved outputs  90 ,  92  leading to a common outlet conduit  94  equipped with a pressure gauge  96 . The conduit  94  is connected to a valve  98 , the output conduit  100  of which passes through a 7μ filter  102  (Swagelok SS-4FW-7) and leads to a gas booster  104  (Haskell AGD-7, C8 single stage, double acting). The output conduit  106  from booster  104  includes a valve  108 , pressure gauge  110 , proportional pressure relief valve  112  (Nupro SS-4R3A-E, 2250-3000 psig), flow meter  114  and valves  115 ,  115   a . As shown, the conduit  106  passes into bath  12  and is coupled to the input of surge tank  28 . 
     The solvent recovery system  26  includes, in addition to micrometering valve  64 , a heated solvent separation cylinder  116 . As shown, a heated output line  118  from the valve  64  includes a thermocouple  119  and leads to the input of cylinder  116 , whereas an output line  120 , equipped with thermocouple  122  and valve  124 , allows recovery of solvent. A gas line  126  extends from the top of cylinder  116  and leads to ¼ inch coiled copper tubing  128 . The output  130  from the latter has a thermocouple  132  and leads to a rotameter  134  (Gilmont Accucal GF-4540-1250, 0-126 SLM CO 2 ). 
     In order to provide further process control, a transducer  136  and pressure gauge  138  are connected via line  140  to a port  141  of chamber  18 . Similarly, a transducer  142  and pressure gauge  144  are connected by way of line  146  to conduit  36  as shown. Finally, an observation light  148  is situated exteriorly of the chamber  18  to allow observation of the micronization process through one of the observation ports  150 ,  151  of the chamber  18 . 
     The operating, control and monitoring components of the apparatus  10  are conventionally connected with a personal computer (not shown). This computer has a known control/data logging program loaded thereon. 
     This set of experiments was conducted as a 2 3  factorial design with a center point replicate. The eight experiments were run in random order, followed by the three replicates. The three variables (along with their low and high values) were CO 2  pressure (1200 and 1400 psig), solution concentration (15 and 30 mg/mL), and solution flow rate to the nozzle  20  (2 and 4 mL/min). The rationale behind the selected variable ranges is as follows. The low value of the CO 2  pressure is above the critical pressure of CO 2  (˜74 bar), whereas the high value was limited by the output of the gas booster used to pressurize the CO 2 . This output is constrained by the house air pressure (85 psig) used to drive the booster. The design limitation of the booster is 2500 psig, for an air supply pressure of 150 psig. The selected range of concentrations takes advantage of the high solubility of insulin in HFIP (˜40 mg/mL). The solution flow rates are within the design specifications of the nozzle. 
     The remaining parameters were maintained constant throughout each experiment. Temperature was maintained by the bath 12 at 37° C., and above the critical temperature of CO 2  (˜31° C.). CO 2  mass flow rate was 75 SLM (137 g/min). 
     The procedure used in all of the separate runs is set forth below. 
     1. In order to ensure adequate CO 2  was present in the cylinders  86 ,  88  the pressure on gauge  96  upstream of the gas booster  104  was noted. For these dip tube cylinders  86 ,  88 , the pressure remained constant (˜900 psig) while liquid CO 2  is being withdrawn, then the pressure began to drop. A minimum pressure is required to achieve adequate outlet pressure from the gas booster—a higher outlet pressure (e.g. 1400 psig) requires a higher inlet pressure. 
     2. The data acquisition and control program was placed in RUN mode. A new file for data logging was opened. 
     3. The amount of insulin to dissolve in 20 mL HFIP was weighed out, and placed in a 25 mL Erlenmeyer flask with ground glass stopper. A stir bar was added to the flask and 20 mL HFIP directly from the solvent bottle was pipetted into the flask. The stopper was replaced and the joint was sealed with Parafilm. The mixture was stirred at medium setting (4) for at least two hours. 
     4. The 0.2 μm PTFE filter was weighed and installed in the filter  16 . 
     5. The precipitation chamber  18 , surge tank  28  connected to valves  34 ,  40  and the parallel filters  14 ,  16  were placed in the bath  12 , and the outlet of the chamber  18  was connected to conduit  46  leading to valve  40 . Valves  34 ,  40  were turned such that valve  34  directed CO 2  flow from the surge tank  28  to the inlet of the chamber  18 , and valve  40  directed CO 2  flow from the outlet of the chamber  18  to the parallel filter system. The two three-way valves  44 ,  58  of the parallel filter system were turned to direct flow through the 0.2 μm filter  16 . 
     6. The rest of the tubing and thermocouple connections for the complete setup were then made as described previously and illustrated in FIG.  1 . Valve  78  was then turned to isolate the chamber  18  from the syringe pump  72 , and the two-way valve  84  that connected valve  78  to the atmosphere was opened. This prevented high pressure CO 2  from entering the solution line and syringe pump. 
     7. The bath  12  was filled with water to a level covering the filters and outlets of the chamber  18  and surge tank  28 . 
     8. Valve  62  was closed and the system was pressurized with CO 2  by operating the air drive of gas booster  104 . CO 2  flowed from the cylinders  86 ,  88  simultaneously; this maintained CO 2  cylinder pressure for a longer period of time, thereby reducing the frequency of adjustments necessary to maintain gas booster outlet CO 2  pressure (and thereby chamber  18  pressure) during the runs. 
     9. If no leaks were present, pressurization was continued with CO 2  to the experimental pressure, and the bath  12  was filled with water until the fittings on top of the chamber  18  were covered. The temperature of the water during filling was monitored and controlled, to minimize the time required for the immersion heater to achieve and maintain 37° C. 
     10. The immersion heater was started to heat the water to the desired temperature. The temperature was checked with an ASTM 38C thermometer. The temperatures of the water bath and CO 2  in the chamber  18  were allowed to reach 37° C. 
     11. The insulin solution was filtered through a 0.2 μm PTFE syringe filter into a 25 mL graduated cylinder. This cylinder was sealed with Parafilm to create the reservoir  22 . 
     12. Valve  62  was opened and micrometering valve  64  was adjusted to achieve a 75-76 SLM CO 2  at ±4° C. 
     13. The program was then used to turn on the heaters: associated with micrometering valve  64 , cylinder  116 , and transfer line  118 . 
     14. The air drive pressure on the gas booster  104  was adjusted to obtain the downstream, or chamber  18 , pressure, as read off the downstream pressure gauge  138 . Typically, the gas booster outlet pressure is 40 psi greater than the downstream pressure. 
     15. The micrometering valve  64  was then adjusted as necessary to obtain a 60 on the scale of the rotameter. 
     16. The downstream temperature and pressure recorded by thermocouple  70  and transducer  136  were allowed to stabilize, as indicated by graphs displayed on the monitor output. 
     17. The syringe pump  72  was then filled with 3 mL solvent, and this solvent was pumped into the conduit  74 . The initial flow of solvent through the nozzle  21  was designed to prevent plugging of the capillary. 
     18. The observation light  148  was then turned on. 
     19. The two-way valve  84  connected to valve  78  was closed, along with the syringe pump outlet valve  72   b . The syringe pump  72  was filled with solution, at a flow rate of 20 mL/min. The syringe pump inlet valve  72   a  was closed and the syringe pump outlet valve  72   b  was opened. The contents of the syringe pump  72  were pressurized at the desired flow rate (e.g. 2 mL/min) until the syringe pump pressure (as indicated on the pump&#39;s display) was greater than the chamber  18  pressure; at this point, the valve  78  was turned to permit flow of the solvent/solution to the nozzle  20 . 
     20. Data logging on the control program was enabled and timing was begun with the stopwatch. This constituted the beginning of a test run. 
     21. While the solution was flowing through the nozzle  20 , the spray and/or particle formation was observed through the window  151 . 
     22. Solution was continually pumped at the desired flow rate until the syringe pump  72  was emptied; at this point the outlet valve  72   b  was quickly closed. The syringe pump was depressurized, then the syringe pump inlet valve  72   a  was opened to rapidly fill the syringe pump (20 mL/min) with ˜7 mL solvent. The syringe pump inlet valve  72   a  was closed to pressurize the syringe pump at the experimental flow rate until the syringe pump pressure was greater than the chamber  18  pressure, whereupon the syringe pump outlet valve  72   b  was opened. This step was designed to flush the remaining solution from the line and from the ˜1 mL dead volume in the nozzle  20 . 
     23. Solvent was pumped at the desired flow rate until the syringe pump  72  was emptied, whereupon the outlet valve  72   b  was closed and the valve  78  was turned to isolate the chamber  18 . 
     24. CO 2  was passed continuously through the chamber  18  for a given length of time (e.g., 1.5 h), at least until powder could no longer be seen floating in the chamber  18 . Chamber pressure was monitored on the downstream pressure gauge  138  and the control program display, and the inlet pressure (gas booster outlet pressure) was adjusted via the air drive to maintain the chamber pressure, if necessary. The micrometering valve was adjusted to maintain constant pressure. 
     25. The valves  38 ,  40  were then turned to direct flow of CO 2  from the surge tank  28  directly through the 0.2 μm filter  16 , isolating the chamber  18 . The 0.2 μm filter was flushed with CO 2  for 30 minutes. 
     26. The outlet from the gas booster  104  was shut off to allow the surge tank  28  to depressurize through the 0.2 μm filter  16 , at constant pressure. The immersion heaters were turned off toward the end of depressurization, when the temperatures began to rise. 
     27. The micrometering valve  64  was closed and the valves  44 ,  58  were turned to direct flow through the 0.5 μm filter  14 , whereupon the valve  40  was turned to direct flow from the chamber  18  outlet  48  to the filter  14 . 
     28. The heaters (except the condenser heater) were turned on and the micrometering valve  64  was opened to in order to depressurize the contents of the chamber  18 . 
     29. The heaters, including the immersion heater, were turned off and data logging was disabled. This is the end of the run. 
     30. Water was then siphoned from the bath  12  and the tubing and thermocouples were disconnected. 
     31. The tubing from the outlet  48  of the chamber  18  was disconnected, and the surge tank/valves/parallel filter assembly was removed along with the chamber  18 . 
     32. The lid of the chamber  18  was unscrewed and the lid was carried, with the nozzle attached, to the syringe pump  72 . 
     33. The outlet line  74  from the syringe pump was removed and the pump was filled with 20 mL DMSO. The pump was allowed to sit, giving time for the DMSO to solubilize any insulin remaining in the pump. 
     34. Helium was blown through the outlet line  74  and attached lid/nozzle, to remove the DMSO. 
     35. The nozzle from the lid was removed and the nozzle was sonicated in a beaker full of sufficient DMSO to cover the annular resonator cavity and tip of the capillary. The nozzle was rinsed with water and acetone, and dried with helium. The capillary inlet was connected to a helium cylinder to flush the remaining liquid from the capillary. 
     36. A weigh tray was tared, and powder was collected from the windows using a scoopula, with the powder being placed in the tray. Powder was also collected from the walls of the chamber  18 . The collected powder was then weighed and placed in a labeled glass vial under helium. The vial was stored at −20° C. 
     37. The 0.2 μm filter holder was disassembled and the filter was carefully dislodged and weighed. Using the weight of the PTFE filter, the amount of precipitate collected on the filter was calculated. The powder plus the filter was placed in a labeled glass vial under helium. This vial was also stored at −20° C. 
     38. The 0.5 μm filter was disassembled and if any powder was collected therein the powder was optionally weighed and stored. The purpose of the 0.5 μm filter was to trap any powder that leaves the chamber during depressurization, rather than to collect significant amounts of product. 
     A series of tests was performed to characterize the micronized insulin products, both physically and chemically. HPLC and CD were used to characterize the insulin in solution; IR and Raman spectroscopy were used to characterize the insulin in the solid state. Aerosizer and SEM provided particle size distributions and particle morphologies. Thermogravimetric analysis (TGA) determined the level of volatiles in the processed powder. 
     Three HPLC methods (Potency, Purity and Polymer) were run on the processed insulin powder reconstituted in aqueous solution. The methods gave an indication of the potency of the insulin, the purity and the polymer content. The processed insulin is referred to as PCA (precipitation with compressed antisolvents) insulin. 
     Tables 1 and 2 summarize the HPLC results. HMWP refers to high-molecular weight polymer. When reconstituted in water, the PCA insulin was as potent as unprocessed insulin, with some slight pressure and concentration factor effects. PCA insulin was also slightly degraded, containing more polymer and insulin related substances. Over the range of variables studied, the experimental factors (pressure, concentration and flow rate) had no significant effect on purity or polymer content of the processed insulin. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 HPLC Results - Effect of PCA on Reconstituted Insulin 
               
             
          
           
               
                   
                   
                   
                 Unprocessed 
               
               
                 HPLC Method 
                 Measurement 
                 PCA Average 
                 Insulin 
               
               
                   
               
             
          
           
               
                 Potency 
                 “as is” Potency (U/mg) 
                 26.8 
                 25.9 
               
               
                 Purity 
                 Main Peak Insulin % 
                 97.9% 
                 99.1% 
               
               
                 Polymer 
                 HMWP % 
                 0.65% 
                 0.10% 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 HPLC Results - Significant Factor Effects (5% Level) 
               
             
          
           
               
                 Measurement 
                 Main Effect 
                 Interaction 
               
               
                   
               
               
                 “as is” Potency (U/mg) 
                 Pressure (+) 
                 Concentration-Flow rate (−) 
               
               
                   
                 Concentration (+) 
               
               
                 A21 Desamido 
                 None 
                 None 
               
               
                 Insulin (%) 
               
               
                 Other Insulin Related 
                 None 
                 Pressure-Flow rate (+) 
               
               
                 Substances (%) 
                   
                 Concentration-Flow rate (+) 
               
               
                 HMWP % 
                 None 
                 None 
               
               
                   
               
             
          
         
       
     
     CD was also performed on the processed insulin, reconstituted in water. FIGS. 3A,  3 B,  4 A and  4 B show the far-UV and near-UV CD spectra of the processed insulin and unprocessed insulin (UPI), respectively. The three numbers for each spectrum of processed insulin (e.g. 1200, 15, 2) represent the factor levels for pressure (1200 psig), concentration (15 mg/mL) and flow rate (2 mL/min). Other than the 1200,15,2 datum, the CD spectra are quite similar, meaning the processed and unprocessed insulins have similar secondary to quaternary structure when reconstituted in water. The anomalous scan of the 1200,15,2 sample in the lower graphs of FIGS. 3B and 4B was due to inaccurate concentration of insulin for this sample. The y-axis scale (mean residue ellipticity, or [Θ]) is obtained by multiplying the angle obtained from the raw CD data by a factor that incorporates the concentration of the sample. This concentration is obtained from a UV absorbance measurement at 280 nm. In the case of the 1200,15,2 sample, some additional component in the sample was absorbing at this frequency, such that the calculated concentration of insulin was greater than the actual concentration in the sample. 
     The secondary structure (α-helix mainly) of the unprocessed insulin is similar to that of the processed insulin, based on the similarity of the far-UV CD spectra (180-260 nm) in FIGS. 3A and 3B. Electronic transitions of the amide chromophore occur in this region. The amide forms the peptide bond in the backbone of the protein, and its CD absorbance is influenced by secondary structure. 
     The tertiary/quaternary structure of the unprocessed insulin is similar to that of the processed insulin. based on the similarity of the near-UV CD spectra (250-400 nm) in FIGS. 4A and 4B. Electronic transition of the tryosine chromophore occurs in this region. There are four tyrosine amino acid residues in the insulin molecule (monomer). The folding of the monomer (tertiary structure) and association with other monomers (quaternary structure) influence the CD absorbance of these residues. The unprocessed insulin contains zinc, and exists as a hexamer (non-covalent aggregate of six monomers) in solution at neutral pH. The similar near-UV CD spectra suggest the processed insulin contains hexameric material as well. 
     In summary, CD demonstrated that the PCA process does not significantly affect the structure of insulin when reconstituted in aqueous solution. In addition, qualitatively there is little difference among the experimental treatments, over the range of pressure, concentration and flow rate studied. 
     CD was also performed on unprocessed insulin dissolved in both water and HFIP. The far-UV spectra indicate some secondary structural changes in HFIP. The near-UV spectra point to unfolding and dissociation of the insulin hexamer into monomers in HFIP. Hence, dissolution of insulin in HFIP appears to change the structure of insulin; however, these changes are reversible. 
     IR and Raman spectroscopy were used to determine the solid-state structure of the processed insulin powder, collected from both the filter  16  and the precipitation chamber. IR was conducted using a Nicolet Nic-Plan IR™ microscope connected to a Nicolet Magna-IR 850 Spectrometer Series II. 
     In each IR spectroscopy case, based on a qualitative comparison of the spectra with that of native insulin, little difference was observed among treatments and the Fourier Self-Deconvoluted (FSD) spectra were similar to that of native insulin. However, the data suggested a higher sheet content and some denaturation for PCA insulin. Each FSD spectra was integrated and factorial analyses were run on both the helix and sheet content, for filter and chamber product. In all cases, there were no significant factors or interactions. 
     For Raman spectroscopy, a Nicolet Raman 950 spectrometer was used, along with OMNIC 4.1a software. Samples were pelletized by compression in a hydraulic press and the cylindrical pellets were placed in a sample holder for scanning. Laser power was limited to 250 mW, to avoid burning the samples. For each sample, 6000 scans were taken. 
     These analyses allowed some conclusions to be drawn about the solid-state structure of the PCA insulin. Qualitatively, both IR and Raman indicate that the PCA product contains less α-helix than native insulin, but the amount of degradation is not large. A comparison of spectra shows that Raman spectroscopy is a more sensitive technique than IR for detecting structural differences in insulin. Note the spike in the Raman spectrum for insulin fibrils, corresponding to β-sheet. This discrepancy may be the inaccurate method of quantifying relative structural content. 
     Particle size distributions (PSD) and morphology were determined using Aerosizer and SEM. For the Aerosizer, the true density of insulin crystals from Lilly Lot No. 002LX9 (density=1.30) was used as input. This density should be the same for Lot No. 009LX9 assuming the two lots have the same crystal form. The Aerosizer results are summarized in Table 3. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PSD Results from Aerosizer for PCA Insulin 
               
             
          
           
               
                   
                   
                 Mean 
                   
                 10% 
                 50% 
                 90% 
               
               
                 Collection 
                 Distribution 
                 Dia. 
                 S.D. 
                 Under 
                 Under 
                 Under 
               
               
                   
               
             
          
           
               
                 Filter 
                 Number 
                 1.87 
                 1.69 
                 1.05 
                 1.71 
                 3.85 
               
               
                   
                 Volume 
                 6.90 
                 1.83 
                 2.88 
                 7.82 
                 13.79 
               
               
                   
                 d V /d N   
                 3.68 
               
               
                 Chamber 
                 Number 
                 0.97 
                 1.67 
                 0.55 
                 0.92 
                 2.35 
               
               
                   
                 Volume 
                 4.22 
                 1.99 
                 2.26 
                 4.18 
                 9.03 
               
               
                   
                 d V /d N   
                 4.37 
               
               
                   
               
             
          
         
       
     
     As seen in Table 3, both number and volume distributions are narrow, and the mean diameter of the number distribution falls within the 1-5 micron range suitable for pulmonary delivery. 
     SEM was run on the three of the PCA insulin samples. The samples were prepared under different conditions, and were examined for particle morphology, size uniformity, and the occurrence of aggregation. Examination of the powder by SEM revealed that the particles have a fibrous matrix structure. 
     The PCA samples were also analyzed by TGA (25-195° C.), giving a PCA powder volatile content of from 3-6% probably due to moisture absorbed from the atmosphere. 
     EXAMPLE 2 
     In this example albumin samples dissolved in HFIP were recrystallized using the invention. The resultant particulate albumin was characterized by Aerosizer and SEM. 
     The apparatus used in this example is identical to that described in Example 1 and depicted in FIG. 1, except that a filter (55-6TF-7, 0.5 μm) was used in lieu of the parallel filter assembly  14 ,  16  of FIG. 1. A similar procedure recited in Example 1 was used in these experiments. 
     The albumin samples (Sigma Chemical Co., Lot 29H0684) were dissolved in HFIP at concentrations ranging from 25-30 mg/mL. The nozzle spray rate was 2 mL/min. The CO 2  flow rate was 75 sL/min. (0.161 kg/min.). In each experiment about 24-35 mL of albumin solution was sprayed into the chamber  18 . The recrystallized samples were characterized by CD to determine any alteration in the conformation of the precipitated samples as compared with the starting albumin, by Aerosizer particle size analyzer (Amherst Process Instruments, Inc.) to determine particle size and size distribution, and by SEM (HITACHI, S-570) to determine the particle size and morphology. 
     The following Table 4 shows the experimental results (harvested particle amounts and recovery yields) and Aerosizer analysis results for the albumin samples. Micron-size particles were obtained with reproducible yield and particle size distribution. All the samples contained very few particles below 0.7 μm (about 10% or less). A large number of particles were found to be very near to 2 μm. Most experiments produced particles which had a single (unimodal) population distribution. The recovery yield was about 57%, with the majority of particles harvested from the external filter. 
     
       
         
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
             
             
               
                   
                   
               
               
                   
                 Particle size (μm) by Aerosizer analysis 
               
             
          
           
               
                   
                 Flow Rate 
                   
                 Number distribution 
                 Volume distribution 
               
             
          
           
               
                 Run 
                 SL/min 
                 Other conditions 
                 Mean 
                 10% 
                 95% 
                 Mean 
                 10% 
                 95% 
               
               
                   
               
             
          
           
               
                 1 
                 75 
                 30.0 mg/mL, 35 mL, 2.0 mL/min, ultrasonic nozzle 
                 1.30 
                 0.55 
                 5.91 
                 9.23 
                 4.40 
                 16.1 
               
               
                 2 
                 75 
                 25.0 mg/mL, 35 mL, 2.0 mL/min, ultrasonic nozzle 
                 1.48 
                 0.73 
                 5.91 
                 11.1 
                 5.13 
                 19.3 
               
             
          
           
               
                   
                 Not Analyzed 
               
             
          
           
               
                 3 
                 75 
                 25.0 mg/mL, 23.9 mL, 2.0 mL/min, ultrasonic nozzle 
                 1.42 
                 0.66 
                 5.31 
                 11.4 
                 4.97 
                 19.7 
               
               
                   
                   
                   
                 1.37 
                 0.64 
                 5.37 
                 9.39 
                 4.49 
                 15.7 
               
               
                 4 
                 75 
                 25.0 mg/mL, 27 mL, 2.0 mL/min, ultrasonic nozzle 
                 1.96 
                 0.56 
                 10.4 
                 12.8 
                 6.24 
                 21.0 
               
               
                   
                   
                   
                 2.88 
                 1.11 
                 11.8 
                 12.7 
                 6.12 
                 20.5 
               
               
                   
               
               
                 • V indicates precipitation vessel or chamber 18; F indicates filter  
               
               
                 • The data of the first row in particle size for every run are from vessel sample.  
               
               
                 • The data of the second row in particle size for every run are from filter sample.  
               
             
          
         
       
     
     The CD scans of both unprocessed and processed albumin samples were very similar, indicating that there was no significant change in the protein conformation and the precipitated particles could attain native protein conformation. FIGS. 5 and 6 are representative spectra of processed (P) and unprocessed (U) albumin obtained from the precipitation chamber and the filter, respectively. Differences in intensity result from differences in protein concentrations. 
     SEM analysis of the samples of unprocessed and processed albumin under different magnifications (20 and 100μ) demonstrates that the particle size of processed albumin samples is much smaller than unprocessed samples.