Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
2000-10-31
2003-05-13
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S419000, C530S422000, C530S424000, C530S426000, C530S427000, C424S044000, C424S045000, C424S489000, C210S651000, C210S666000, C210S710000, C210S711000
Reexamination Certificate
active
06562952
ABSTRACT:
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 &mgr;m) 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 &agr;-helicity and a marked increased in &bgr;-sheet and &bgr;-reverse turn content. (Winters et al.,
J. Pharmaceutical Sciences,
85(6):586-594 (1996)). The solvents used in the process of the '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 &mgr;m, and more preferably from about 1-5 &mgr;m. 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 alcoh
Niu Fenghui
Rajewski Roger A.
Snavely William K.
Subramaniam Bala
Hovey & Williams, LLP
Low Christopher S. F.
Mohamed Abdel A.
The University of Kansas
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