HK1008937B - Production scale method of forming microparticles - Google Patents

Description

Large-scale production method of microparticles

Background of the invention

There is a need for maintaining a constant amount of drug in the body for a wide range of diseases to provide effective prophylactic, therapeutic and diagnostic results. In the past, drugs have been administered in a spaced manner, causing fluctuations in drug levels.

In order to control and stabilize the level of drug, numerous attempts have been made, including the use of biodegradable substances, such as polymeric or protein microspheres containing drug. The use of these microspheres improves controlled release of the drug because the biodegradability of the polymer itself improves the release of the drug, thereby providing a smoother, controlled level of drug release.

However, many of these methods have resulted in low yields of microspheres due to the method and equipment used. Further, some methods have not been scalable to the level of commercial production.

Therefore, there is a need to provide a method for producing microspheres in a commercial scale with low loss of bioactive agents and high yield.

Summary of the invention

The present invention relates to a method for producing microparticles from droplets of a solution, wherein the solution comprises a substance dissolved in a solvent. The method comprises the step of feeding the droplets into a freezing zone, wherein the freezing zone is surrounded by a liquefied gas, where the droplets are frozen. The frozen microdroplets are then mixed with a non-solvent liquid, causing the solvent to be extracted into the non-solvent portion, thereby producing microparticles.

The present invention has numerous advantages, for example, the method and apparatus of the present invention are high yielding, can enable commercial scale production of controlled release microparticles, can use a closed system to ensure sterility of the production process, can control the size of the microparticles, can be repeatable and reliable, etc.

Furthermore, the process of the invention can be carried out with a wide range of temperatures.

Brief description of the drawings

FIG. 1 is a sectional view showing the apparatus for producing fine particles of the present invention suitable for the method of the present invention, which can freeze droplets of a solution containing a substance in a solvent, the freezing zone being cooled by a circulating flow of a liquefied gas, and then extract the solvent from the frozen droplets with a non-solvent liquid;

FIG. 2 is a sectional view showing another embodiment of the apparatus for producing fine particles of the present invention according to the method of the present invention, which can freeze droplets of a solution containing a substance in a solvent, the freezing zone being cooled by a circulating flow of a liquefied gas, and then extract the solvent from the frozen droplets with a non-solvent liquid;

FIG. 3 is a sectional view showing still another embodiment of the apparatus for producing fine particles of the present invention according to the method of the present invention, which can freeze droplets of a solution containing a substance in a solvent, the freezing zone being cooled by a circulating flow of a liquefied gas, and then extract the solvent from the frozen droplets with a non-solvent liquid;

fig. 4 is a sectional view showing an alternative embodiment of the apparatus for producing microparticles of the present invention according to the method of the present invention, which can freeze droplets of a solution containing a substance in a solvent, the freezing region being cooled by a circulating flow of liquefied gas, and then extract the solvent from the frozen droplets with a non-solvent liquid.

Technical scheme of the invention

Technical features and other details of the apparatus and method of the present invention are described in more detail with reference to the accompanying drawings and pointed out in the claims. It should be understood that the specific embodiments of the present invention are provided for illustration and not for limitation. The essential features of the invention can be used in many embodiments without departing from the scope of the invention.

The present invention relates to a method and apparatus for preparing microparticles of a substance from a solution of the substance. Microparticles as described herein include particles of a substance having a diameter of less than about 1 millimeter. The microparticles may be spherical, non-spherical, or non-regular. Preferred microparticles are microspheres.

Materials suitable for producing microparticles of the present invention include, for example, polymers, peptides, polypeptides, proteins, small molecule drugs, and prodrugs.

The microparticles may additionally contain one or more substances, which may be dispersed within the microparticles. When the substance comprises a polymer, then a solution of the polymer contains at least one biologically active agent.

As used herein, a biologically active agent refers to an agent, or a metabolite of an agent, that has a therapeutic, prophylactic, or diagnostic function in vivo after administration, or after metabolism (e.g., a prodrug, such as hydrocortisone sodium succinate).

Figure 1 shows an apparatus according to the invention suitable for carrying out the process according to the invention. The apparatus includes a container 10, generally cylindrical, having a sidewall 12, a container top 14, a container bottom 16, and an inner wall 18. The side wall 12 and container bottom 16 are generally insulated using conventional insulation methods in order to minimize the amount of heat that can penetrate the container 10 from the external environment, thereby providing better control of the temperature within the container 10. Conventional insulation methods include, for example, covering the outer surfaces of the sidewall 12 and container bottom 16 with at least one layer of insulating material 17. Other insulation methods include, for example, vacuum sandwich type side walls 12, and radiation shielded container bottom 16. Suitable insulation materials include conventional insulation materials such as mineral fibers, polystyrene, polyurethane, foam rubber, balsa wood and cork.

In this embodiment, the container top 14 is generally not insulated, thereby allowing components of the device that are placed near the container top to be heated by heat that penetrates into the container 10 from the outside. In addition, the container top 14 may also be insulated with a suitable insulating material.

The material from which the container 10 is made may be any material that will withstand the conditions of steam sterilization performed inside the container 10 and the temperature and pressure conditions under which the microparticle formation of the present invention is performed. Suitable materials for container 10 include, for example, stainless steel, polypropylene, and glass.

In this embodiment, vessel 10 is a single unitary vessel divided into a freezing section 20 and an extraction zone 22. The freezing section 20 is located between and substantially enclosed by the side wall 12, the container top 14 and the inner wall 18. The extraction zone 22 is located between and substantially surrounded by the side wall 12, the vessel bottom 16 and the inner wall 18.

In another embodiment, the freezing section 20 and the extraction zone 22 comprise separate containers, wherein the freezing section container is generally placed above the extraction zone and the bottom of the freezing section container is in contact with the top or sides of the extraction zone container.

The vessel 10 includes means for introducing liquefied gas into the freezing section 20 and forming a liquefied gas flow 24. The liquefied gas flow 24 includes an injection of liquefied gas and/or at least one liquefied gas flow. The liquefied gas flow 24 begins in the freezing section 20 at or near the top 14 of the vessel and then flows generally downward toward the inner wall 18. Within the freezing section 20, at least a portion of the liquefied gas flow 24 flows in a direction substantially parallel to the side wall. The liquefied gas flow 24 is generally located at or near the sidewall 12. It is generally preferred to have the side wall 12 wetted by the liquefied gas stream 24. Further, the liquefied gas flow 24 substantially surrounds the freezing zone 26 and is at the central radial line of the freezing section 20. The spacing between the liquefied gas flow 24 as it surrounds the freezing zone 26 depends on the type and number of liquefied gas introduction devices used.

At or near the top 14 of the vessel, at least one liquefied gas conducting device is arranged, in particular radially from the center of the top 14 of the vessel. For radially placed liquefied gas guiding devices, their number is suitable as long as they do not significantly affect the formation of droplets 28, e.g. such that a portion of the solution is frozen within the droplet forming device 30, thereby partially blocking the droplet forming device 30. Likewise, the liquefied gas guiding device is disturbed if there are significant droplets 28 affecting it.

In the embodiment shown in FIG. 1, a suitable liquefied gas diversion device includes at least two nozzles, discharging in a linear or fan shape [ e.g., using a model 1/8-K-SS-1 nozzle from injection systems, Wheatstone, Ill.; (Flood Jet Atomizer Model 1/8-K-SS-1, Spray System Co., Wheaton, IL) ], which can inject a liquefied gas to form at least a portion of the liquefied gas stream 24. The nozzles 32 are positioned within the freezing section 20 of the container top 14 and are equally spaced in a circle centered about the center of the container top 14 or, when radial, centered on the droplet forming device 30. The number of nozzles 32 used will depend on the arc of injection of the nozzles used, the distance the liquefied gas flow 24 reaches from the nozzles 32 to the point of contact with the sidewall 12.

With two nozzles 32 equally spaced in the center of the top freeze zone 20, the circulating liquefied gas flow 24 will be two about 180 degrees apart, since the nozzles 30 are generally not capable of spraying in an arc greater than 180 degrees. In the preferred embodiment, at least 3 nozzles are installed in the freezing section 20 to form the liquefied gas flow 23 and to surround the freezing zone 24 without significant separation of the flow.

Typically, 3 nozzles 32 at equal distances will provide a 360 degree angle of liquefied gas flow 24. In a more preferred embodiment, 6 nozzles are positioned equidistantly around the center of the freezing section 20.

The liquefied gas guiding device receives liquefied gas from at least one liquefied gas inlet 34. The liquefied gas inlet 34 provides communication between a liquefied gas source 36 and a liquefied gas diversion device. It should be understood that other liquefied gas introduction means that can direct the liquefied gas into the liquefied gas flow guide can be used in place of, or in combination with, the liquefied gas inlet 34.

Fig. 2 shows another suitable embodiment of the liquefied gas guiding device of the apparatus according to the invention. The apparatus of fig. 2 bears many similarities to the apparatus of fig. 1 and is denoted by the same reference numerals. In this plant, suitable liquefied gas guiding means include a guiding dam 102 and a liquefied gas compartment 104. The deflector dam 102 is positioned within the freeze section 20 between the side wall 12 and the freeze zone 26. The deflector weir 102 extends upwardly from the inner wall 18 or from the side wall 12 toward the top 14 of the vessel. In one particular embodiment, the top of the deflector dam 102 is not in contact with the vessel top 14, thereby allowing liquefied gas to pass from the top of the deflector dam 102 to the freezing section 20. Additionally, when the deflector dam 102 contacts the vessel top 14, the deflector dam 102 is porous or slotted (not shown) at the top of the deflector dam 102, allowing liquefied gas to pass from the upper portion of the deflector dam 102 to the freezing section 20.

A liquefied gas compartment 104 is disposed within the freeze section 20 between the deflector dam 102 and the sidewall 12. The liquefied gas compartment receives liquefied gas from at least one liquefied gas inlet 34. The liquefied gas is then directed through the diversion dam 102 to the center of the freezing section 20.

Returning to fig. 1, the container 10 further includes droplet-forming apparatus 30 located in the freezing section 20 at the top 14 of the container for forming suitable solutions into droplets 28. In this context, microdroplets refer to droplets of a solution that, after freezing and subsequent extraction of the solvent in the solution, will form microparticles. Examples of suitable droplet forming devices 30 include atomizers, nozzles, and various needle valves. Suitable atomizers include, for example, external air (or gas) atomizers [ e.g., SUE15A Model atomizer by injection Systems (Model SUE 15A; SpraySystems Co., Wheaton, IL) ], internal air atomizers (e.g., SU 12; SpraySystem Co., Ltd.), rotary atomizers [ (e.g., disc, bowl, cup, and wheel; (Niro, Inc., Columbia, MD) ], and ultrasonic atomizers [ e.g., atomizing probe 630; (sonic & Materials, Inc., Danbury, CT) ]. suitable nozzles include pressure atomizing nozzles [ e.g., vortex Spray drying nozzles; (sprayton Systems Co., Wheaton, IL) ]. needle valves typically used to form droplets 28 include 16 to 30 gauge needle valves.

In a preferred embodiment, droplet-forming device 30 is an air atomizer that can form microparticles ranging in diameter from about 1 micron or less to about 300 microns. Adjusting the pressure of the gas (e.g., nitrogen) supplied to the air atomizer can change the average size of the microparticles. The average diameter of the fine particles can be made small by increasing the pressure of the gas.

The droplet-forming apparatus 30 is made of a material that can withstand the low temperatures of the steam sterilization and freezing stages 20.

Droplet-forming device 30 receives solution from at least one solution inlet 38. The solution inlet 38 provides communication between a solution source 40 and the freezing section 20. It should be understood that other suitable solution introduction devices may be used, such as a spray gun, or other device capable of spraying a solution into a cryogenic environment, in place of or in combination with the solution inlet 38.

The vessel 10 also includes at least one three-phase valve 42 located in the inner wall 18 to provide communication between the freezing section 20 and the extraction zone 22. The three-phase valve 42 is sized to allow a mixed flow of frozen droplets 44, liquefied gas and volatilized gas to pass from the freezing section 20 to the extraction zone 22.

The extraction zone 22 includes means for separating the liquefied gas from the frozen droplets 44. In one embodiment, suitable separation means include means for heating the extraction zone 22 to volatilize the liquefied gas and thereby separate it from the frozen droplets 44, and to generally confine the frozen droplets 44 to a lower portion of the extraction zone 22. The heating device may also be used to heat the solvent within the frozen microdroplets 44. Suitable heating means may also include the infiltration of recent heat from the external environment through the side walls and container bottom 16. The heating means may or may not also include, for example, electrical means, such as heating coils, or circulating heat exchange tubes 46 to control the temperature inside the freezing section 22 by circulating a liquid to first volatilize the liquefied gas and then subsequently heat the solvent within the frozen droplets 44 to control the rate of solvent extraction.

Another type of separation device includes a filtered bottom tap 48 that opens out from the bottom of the extraction zone 22. The filtered bottom tap 48 includes a filter 50 having a filter pore size of less than the diameter of the microparticles 11, typically less than or equal to 1 micron, and adapted to filter liquids, such as liquefied gases, in the extraction zone 22 to retain the frozen droplets 44 and even the microparticles 11 within the extraction zone 22.

A gas outlet 52 is located in the inner wall 18 of the extraction zone 22 and is adapted to discharge volatilized liquefied gas out of the vessel 10. The gas outlet 52 may or may not include means for depressurizing the vessel 10, such as a Vacuum blower (e.g., cryogenic blower CP-21, Barber Nichols, Arvada, CO) or a Vacuum pump (e.g., E2M18 Vacuum pump, Edwards high Vacuum International, Crawley, West Sussex, England) suitable for venting gases. Furthermore, the gas outlet 52 typically also includes a filter 53 (e.g., a 0.2 micron sterile filter) in the path of the gas stream to support the sterilization process and to ensure that the resulting microparticles 11 meet sterility requirements.

The vessel 10 may or may not also include a gas outlet 52 located in the extraction zone 22 and/or in the freezing section 20 (not shown). It is preferable that no gas outlet is provided in the freezing section 20 because gas circulation is generated by discharging gas from the freezing section 20, resulting in a decrease in the yield of the fine particles 11.

In addition, the container 10 may or may not include at least one means for preventing over-pressurization (not shown) to protect the integrity of the substances from over-pressurization due to evaporation of the liquefied gas. Generally, the overpressure protection device includes, for example, a burst disk or a pressure relief valve.

The extraction zone 22 also includes at least one non-solvent inlet 54 located in the inner wall 18 and/or on the side wall 12. The extraction zone 22 receives a non-solvent liquid stream or spray from a non-solvent inlet 54. Preferably, the non-solvent forms an extraction bath 56 at least in the lower portion of the extraction zone 22. It should be understood that other means suitable for introducing liquid into the cryogenic vessel, such as a spray gun or other means suitable for introducing liquid at cryogenic conditions, may be used in place of, or in combination with, the non-solvent inlet 54.

In another embodiment, a suitable means 60 for mixing the frozen droplets 44 with a non-solvent is located within the extraction bath 56. The purpose of the mixing device 60 is to reduce the likelihood of an extraction gradient forming within the extraction bath 56, i.e., to prevent agglomeration of the frozen droplets 44 within the extraction zone 22. Examples of suitable mixing devices 60 include low shear devices such as swirlers (e.g., a P6X05E Lightning Sealmaster device with a310 blades and operating at 1-175 revolutions per minute), marine propellers, paddle mixers or external recirculation loops with low shear pumps.

The vessel 10 further includes a bottom spigot 62 extending outwardly from a lower portion of the extraction zone 22. The bottom tap 62 is adapted to remove the microparticles 11 and a liquid, such as a non-solvent liquid, from the vessel 10. Alternatively, dip tubes (not shown) may be used to remove the microparticles 11 and liquid from the vessel 10.

When it is desired to deliver a drug, the relevant interior of the device of the present invention is cleaned and sterilized to ensure sterility of the final product at each use.

In the method of the present invention, microparticles of a substance are prepared from a solution of the substance in a suitable solvent. Suitable materials for use in the method of the invention include those having a suitable solvent, and having a lower melting point than the solvent, and sufficient miscibility with the solvent to extract solids from the frozen microparticles and/or to melt the liquid solvent. Preferred substances in the present method are peptides, polypeptides, proteins, polymers, small molecule drugs and prodrugs.

Any suitable polymer may be used to make the microparticles. In a preferred embodiment, the polymers used in the present method are biologically compatible. Biologically compatible polymers are polymers or any degradation products thereof, such as by metabolism, that are non-toxic to humans and livestock and do not produce any significant side effects on the individual receiving the polymers, such as immunological reactions at the site of injection. The biologically compatible polymer may be a biodegradable polymer, a non-biodegradable polymer, or a mixture thereof.

Suitable biologically compatible, non-biodegradable polymers include, for example, polyacrylates, polymers of ethylene glycol diacetate, and other acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chlorides, polyvinyl fluorides, poly (vinylimidazoles), chlorosulfonated polyolefins, polyethylene oxides, and mixtures and copolymers thereof.

Suitable biocompatible, biodegradable polymers include, for example, poly (lactide), poly (glycolide), poly (lactide-co-glycolide), poly (lactic acid), poly (glycolic acid), polycarbonate, polyesteramide, polyanhydride, poly (amino acid), polyorthoester, polyacetal, polynitrile acrylate, polyetherester, polycaprolactam, poly (dioxane), poly (alkylene alkyl ester), polyurethane, and mixtures and copolymers thereof. Preferred polymers include poly (lactide), copolymers of lactide and glycolide, mixtures and blends thereof.

The polymers used in the present process may be blocked, unblocked, or a mixture of blocked and unblocked. Blocked polymers are defined as conventionally in the art as blocked, particularly carboxyl terminated. Unblocked polymers are as generally defined in the art, and in particular have free carboxyl terminal groups. Typically, the blocking group is derived from the starting material of the polymerization reaction and is typically an acyl group.

The molecular weight of the polymer useful in the present invention can be determined by those skilled in the art depending on factors such as desired polymer degradation rate, physical properties such as mechanical strength, dissolution rate of the polymer in a solvent, and the like. Generally, acceptable molecular weights range from about 2,000 daltons to about 2,000,000 daltons.

In a more preferred embodiment, the polymer is poly (lactide-co-glycolide) and the ratio of lactide to glycolide is about 1: 1 and the molecular weight is from about 5,000 daltons to about 70,000 daltons. In a more preferred embodiment, the poly (lactide-co-glycolide) used in the invention has a molecular weight of from about 5,000 daltons to about 42,000 daltons.

Typically, solutions of suitable polymers contain from about 1% (w/w) to about 30% (w/w) of a suitable biologically compatible polymer, which is typically dissolved in a suitable solvent. Preferably, the polymer solution contains from about 5% (w/w) to about 20% (w/w) polymer.

The method of forming the droplets may be a continuous freezing and extraction method or a batch method in which a batch of frozen droplets is formed in a first step and then in a second step the frozen batch of droplets is extracted to form microparticles.

In the present method, the freezing zone 26 includes a partial freezing section 20 that is substantially surrounded by the liquefied gas flow 24. A freezing zone 26 is formed within the freezing section 20 of the vessel 10 and is formed by the liquefied gas flow 24 being sprayed by at least two spray nozzles 32 in a substantially downward direction against the sidewall 12. Typically, the liquefied gas sprayed from the nozzles 32 is at an angle that causes the liquefied gas to reach the sidewall 12, creating a flow of liquefied gas 24 along the inner surface of the sidewall 12, thereby wetting the sidewall 12. In the preferred embodiment, the liquefied gas sprayed from each of the six nozzles 32 impinges on the sidewall 12 at an angle of less than about 30 degrees from the sidewall 12, thereby reducing the reflection and splashing of the liquefied gas by the sidewall 12.

In addition, the liquefied gas flow 24 is sprayed at an angle that is substantially parallel to the inner surface of the sidewall 12 but does not contact the sidewall 12, forming a separate wall of liquefied gas from the nozzle 32 to the inner wall 18.

Liquefied gas is provided to the nozzle 32 from a liquefied gas source 36 by a liquefied gas inlet 34.

Suitable liquefied gases for use in the present process include liquid argon (-185.6 deg.C), liquid nitrogen (-195.8 deg.C), liquid helium, and other liquefied gases having a temperature sufficiently low to enable droplets of the solution to be frozen while in the freezing zone 26 or in the liquefied gas stream 24. Liquid nitrogen is preferred.

In another embodiment, as shown in FIG. 2, the freezing zone 24 is formed in the freezing section 20 by liquefied gas from a liquefied gas source 36 passing through a liquefied gas inlet 34 to a liquefied gas compartment 104 and then passing over the diversion dam 102 or from a tank (not shown) on the diversion dam to form the liquefied gas flow 24. The liquefied gas flow 24 then flows down the inner surface of the deflector dam 102.

Returning again to fig. 1, the solution droplets 28, preferably a polymer solution, flow in a substantially downward direction through the freezing zone 26 where the droplets 28 are frozen into frozen droplets 44. A portion of the droplets 28 may be frozen by contact with the liquefied gas flow 24. The droplets 28 are formed by previously directing the solution from the solution source through the solution inlet 38 to the appropriate droplet forming device 30. Typically, at least a portion of the liquefied gas will be volatilized in the freezing section 20 due to heat that permeates in and/or heat transferred from the droplets 28 to the liquefied gas.

The fluid, which is comprised of the volatilized gas, liquefied gas, and frozen droplets 44, then passes from the bottom of the freezing section 20 through the three-phase valve 42 to the extraction zone 22.

In one embodiment, at least a portion of the droplets 44 are encapsulated in the liquefied gas flow, which is then carried by the liquefied gas flow into the extraction zone 22. This phenomenon of entrapping frozen droplets in the liquefied gas stream 24, in accordance with the method of the present invention, is advantageous for the ultimate yield of microparticles produced, as it can transport frozen microparticles that would otherwise remain in the freezing section 20 by adhering to the side wall 12 and/or inner wall 18 to the extraction zone 22 and/or reduce the amount of frozen microparticles that are discharged into the atmosphere from the gas outlet 52 of the vessel 10.

The liquefied gas is then separated from the frozen droplets 44 using a suitable separation method, leaving the frozen droplets 44 in the lower portion of the extraction zone 22.

In one embodiment, the frozen droplets 44 are heated to a temperature below their melting point but above the boiling point of the liquefied gas, causing the liquefied gas to evaporate and separate from the frozen droplets 44.

Alternatively, the liquefied gas may be separated by evaporating the liquefied gas by creating a partial vacuum through the outlet 52 in the extraction zone 22 and heating the liquefied gas to a temperature below the boiling point of the liquefied gas but sufficient to raise the vapor pressure of the liquefied gas.

After heating, the liquefied gas is volatilized, thereby separating the liquefied gas and the frozen droplets 44. The liquefied gas can be heated by external heat from the infiltration of the side wall 12 and the container bottom 16. Extraction zone 22 is preferably heated by an electrical heating source or by a circulating heated liquid, such as nitrogen or a nitrogen/liquid nitrogen mixture through heat exchange tubes 46. In addition, the temperature within the extraction zone 22 can be controlled by circulating a liquid through a heat exchanger to first volatilize the liquefied gas in a controlled manner and then slowly heat the solvent in the frozen droplets 44 to cause the solvent to be extracted into the non-solvent liquid.

Alternatively, the liquefied gas and frozen droplets 44 can be separated by passing the liquefied gas through a filter 50 and then exiting the extraction zone 22 through a filtering bottom tap 48. Passing the liquefied gas through the filter 50 can remove the liquefied gas from the extraction zone 22 while retaining the frozen droplets 44 at the bottom of the extraction zone 22.

After heating to volatilize the liquefied gas, the volatilized liquefied gas can be discharged from the extraction zone 22 through the at least one gas outlet 52. The pressure within the vessel 10 is determined primarily by the amount of liquefied gas that is volatilized within the extraction zone 22 and by the rate at which the gas is discharged through the outlet 52. The vessel 10 may operate under atmospheric, low pressure and high pressure conditions. The upper pressure limit for carrying out the process of the present invention is determined by the pressure rating of the vessel 10.

Preferably, the method of the present invention is performed under partial vacuum conditions while forming the frozen droplets 44. For the method of creating a partial vacuum in extraction zone 22 and thus in vessel 10, methods well known to those skilled in the art are used, for example, providing a vacuum pump or blower at gas outlet 52 of extraction zone 22 to draw off gas.

After separating the frozen droplets 44 from the liquefied gas, the frozen droplets 44 are then contacted with a suitable cold non-solvent liquid, the non-solvent having a temperature below the melting point of the frozen droplets 44. In a preferred embodiment, the frozen non-solvent is maintained below the melting point of the frozen microdroplets 44 and is extracted from the solid state as a non-solvent liquid and forms porous microparticles 11 within 1 to 24 hours. Extraction of the solid solvent slows the extraction process, thereby providing better control of the extraction and formation of microparticles 11.

In another embodiment, the frozen non-solvent is warmed to the melting point of the frozen microdroplets 44 or higher. Thereby melting and extracting the solvent in the frozen microdroplets 44 into the non-solvent. The extraction of solvent as a solid and/or liquid is determined herein by a number of factors, such as the amount of solvent in the frozen droplets 44, the amount of solvent with which the frozen droplets 44 are exposed, the rate of temperature rise of the frozen droplets 44, and the like. The rate of temperature increase affects the porosity of the product, with lower rates of temperature increase producing microparticles that are porous, and with higher rates of temperature increase partial concentration of the particles occurs after rapid solvent extraction to produce microparticles 11 with significantly reduced porosity.

The non-solvent may be sprayed, a liquid stream, and/or an extraction bath 56. It is preferred that the frozen droplets 44 are submerged in the non-solvent extraction bath 56.

Suitable non-solvents are defined as being non-solvent for the material in solution, and the non-solvent is sufficiently miscible with the solvent of the solution to extract the solvent from the frozen microdroplets 44 as the solvent heats up, thereby forming microparticles 11. Furthermore, the non-solvent has a melting point lower than the melting point of the frozen microdroplets 44.

In another embodiment, a second non-solvent is added to the first non-solvent, for example, hexane is added to increase the rate of solvent extraction from the polymer, for example, poly (lactide-co-glycolide) or to increase the ratio of non-solvents.

In a preferred embodiment, at least a portion of the frozen droplets 44 are retained in the non-solvent, which improves the final yield of microparticles 11 of the method of the present invention, since this phenomenon transports the frozen droplets 44 to the extraction bath 56. If not, frozen droplets 44 may be lost by adhesion to the sidewall 12 and/or lost upon discharge of gas into the air from the gas outlet 52.

In another embodiment, the frozen droplets 44 are agitated by an agitation device 60 in the extraction bath 56, thereby reducing the solvent concentration gradient around each frozen droplet 44 or microparticle 11, thereby improving the efficiency of the extraction.

In another embodiment, the extraction process includes sequentially adding additional aliquots of non-solvent to the extraction zone 22 and removing the aliquots, thereby extracting solvent into each aliquot. Thus, the extraction is performed in a stepwise fashion. The rate of melting depends on the solvent and non-solvent selected, and the temperature of the non-solvent in the extraction zone 22. Table 1 gives the polymer/solvent/non-solvent systems that can be used in the process of the present invention, and their melting points.

TABLE 1

Suitable polymers, solvent and non-solvent systems, and melting points of solvents and non-solvents
			Polymer and method of making same	Solvent (. degree.C.)	Non-solvent (. degree.C.)
Poly (lactide)	Dichloromethane (-95.1) chloroform (-63.50)	Ethanol (-114.5) methanol (-97.5)
			Poly (lactide-co-glycolide)	Ethyl acetate (-83.6) acetone (-95.4) dichloromethane (-95.1)	Ethanol (-114.5) diethyl ether (-116.3) pentane (-130) isopentane (-160)
Poly (caprolactam)	Dichloromethane (-95.1)	Ethanol (-114.5)
			Poly (vinyl alcohol)	Water (0)	Acetone (-95.4)
Ethylene vinyl acetate	Dichloromethane (-95.1)	Ethanol (-114.5)

For proteins, it is preferred that the frozen microdroplets 44 be thawed slowly while the polymer solvent is being extracted to produce microparticles.

Microspheres can be produced in a wide range of sizes by varying the size of the droplets, for example, by varying the diameter of the nozzle or by varying the gas flow to the gas atomizer. If microparticles 11 of very large diameter are desired, the droplets can be added directly to the freezing zone 24 by means of a syringe. Increasing the inherent viscosity of the polymer solution may also increase the size of the microparticles. The microparticles 11 produced by the present method may range in size from above about 1000 microns in diameter to below about 1 micron, or even smaller. Generally, the microparticles are of a size suitable for injection into a human or animal. The preferred diameter of microparticles 11 is less than about 180 microns.

After extraction, the microparticles 11 are filtered and dried by conventional methods well known to those skilled in the art. For polymeric microparticles, it is preferred not to heat the microparticles above their glass transition temperature to reduce adhesion between the microparticles unless an active agent, such as mannitol, is added to reduce adhesion between the microparticles.

In another embodiment, the solution of a substance further comprises one or more other substances dispersed therein. The other materials may be dispersed in the solution by co-dissolving, suspending solid particles, such as freeze-dried particles, in the solution, or dissolving them in a second solvent that is miscible with the solution and mixing with the solution to form an emulsion. The solid particles suspended in the solution may be very large particles, which may be greater than 300 microns in diameter, or micronized particles, which may be as small as about 1 micron in diameter. In general, these other materials should not be soluble in non-solvents.

When the substance comprises a polymer, the polymer solution contains at least one biologically active agent. Suitable therapeutically and/or prophylactically biologically active agents include proteins, such as immunoglobulin-like proteins; an antibody; cytokines (e.g., lymphokines, monokines, and chemokines); an interleukin; an interferon; erythropoietin; hormones (e.g., growth hormone and corticotropin); a growth factor; a nuclease; tumor necrosis factor; a colony stimulating factor; insulin; an enzyme; antigens (e.g., bacterial and viral antigens); a tumor suppressor gene. Other examples of suitable therapeutically and/or prophylactically biologically active agents include nucleic acids, such as antisense molecules; small molecules such as antibiotics, steroids, decongestants, nerve activators, anesthetics, sedatives, cardiovascular drugs, antineoplastic agents, anticancer agents, antihistamines, hormones (e.g., thyroxine), and vitamins.

Suitable diagnostically and/or therapeutically active agents include radioactive isotopes and radiopacifiers.

The microspheres produced by the present method may be homogeneous or heterogeneous blends of polymers and active agents. Homogeneous mixtures are prepared by dissolving the active agent and the polymer in a solvent, as is the case with certain hydrophilic drugs, particularly steroids. Heterogeneous two-phase systems have discrete domains of polymer and active agent formed by the active agent being insoluble in the polymer/solvent and are added to the polymer/solvent as a suspension or emulsion, for example, a hydrophilic material such as is the case for proteins in methylene chloride.

The amount of biologically active agent, i.e., contained in a particular batch of microparticles, is a diagnostically, prophylactically or therapeutically effective dose, which one of skill in the art can achieve, taking into account body weight, condition, type of polymer used, rate of release from the microparticles, and the like.

In one embodiment, a controlled release polymeric microparticle contains about 0.01% (w/w) to about 50% (w/w) of a biologically active agent. The particular amount of agent employed will depend on the potency of the agent desired, the level of release planned, the time of release of the drug, etc. A preferred loading range is from about 0.1% (w/w) to about 30% (w/w) active agent.

Other substances may also be added to the microparticles with the biologically active agent, as desired. These materials include salts, metals, sugars, surfactants, and the like. Additives, such as surfactants, may also be added to the non-solvent during solvent extraction to reduce agglomeration of the microparticles.

The biologically active agent may also be mixed with additional excipients, for example, stabilizers, solubilizers, or fillers. Stabilizers are added in order to maintain the efficacy of the drug during its release. Suitable stabilizers include, for example, carbohydrates, amino acids, fatty acids, surfactants, and others skilled in the artSpecies well known to the person. The amount of stabilizer used depends on the weight ratio between it and the reagent. For amino acids, fatty acids, and carbohydrates such as sucrose, lactose, mannitol, dextran, and heparin, the molar ratio of carbohydrate to agent is about 1: 10 to 20: 1. For surfactants, e.g. the surfactant Tween^TMAnd Pluronic^TMThe molar ratio is from about 1: 1000 to about 1: 20.

In another example, the biologically active agent may be lyophilized with a metal cation component to stabilize the agent and control the release of the biologically active agent from the microparticles, as described and taught in U.S. patent co-pending application No. 08/279,784, filed on 25/7/1994, which is incorporated herein by reference in its entirety.

The solubilizing agent is added in order to change the solubility of the reagent. Suitable solubilizing agents include complexing agents such as albumin and protamine, which can be used to control the rate of release of the agent by the polymer or protein matrix. The weight ratio of lytic agent to biologically active agent is generally from about 1: 99 to about 20: 1.

The filler generally comprises an inert substance. Suitable fillers are well known to those skilled in the art.

Further, the polymeric matrix may contain dispersed metal cation components to modulate the rate of release of the biologically active agent from the polymeric matrix, as described in U.S. patent co-pending application No. 08/237,057 filed on 5/3.1994, and International patent co-pending application No. PCT/US95/05511 filed on 5/3.1995, both of which are incorporated herein by reference in their entirety

In another embodiment, the microparticles further comprise at least a pore former, such as a water soluble salt, sugar, or amino acid, to adjust the microstructure of the microparticles. The porogen is added to the polymerization solution at a ratio of about 1% (w/w) to about 30% (w/w). Preferably, at least one porogen is included in the non-biodegradable polymeric matrix of the present invention.

Figure 3 shows another embodiment of the apparatus of the invention suitable for carrying out the process of the invention. The apparatus of figure 3 has many of the same parts as the apparatus of figure 1 and is indicated by the same numerals. In this apparatus, the freezing section 20 is located in a freezing container 202 and is substantially enclosed by the side wall 12, the container top 14 and the bottom 204 of the freezing container. The extraction zone 22 is similarly located in the extraction vessel 206 and is surrounded by the side wall 12(a), the extraction vessel top 206 and the vessel bottom 16. The freezing vessel 202 is generally located above the extraction vessel 206. A conduit 210 is located between the freezing vessel 202 and the extraction vessel 206. The conduit 210 includes a conduit inlet 212 at or near the bottom of the freezing vessel 204 and a conduit outlet 214 at or near the top 208 of the extraction vessel. Conduit 210 provides three-phase transport, i.e., transport of solids, liquids, and gases, between freezing section 20 and extraction zone 22.

In addition, conduit 210 may or may not include a three-phase mixing device for mixing the three-phase substances in a three-phase fluid such that at least a portion of the frozen droplets 44 contained in the gas phase are captured by the liquid phase, thereby increasing product yield and reducing the loss of frozen droplets 44 caused by the discharge of gas from gas outlet 52. Suitable three-phase mixing devices 216 include cascaded baffles, or preferably one or more electrostatic mixers (e.g., Model # KMR-SAN; Chemineer, Inc.). The preferred three-phase mixing device 216 provides a tortuous flow. More preferably, the three-phase mixing device 216 includes a number of electrostatic mixing elements in series and is sufficient to generate the swirling flow, typically 4 elements being used.

In another embodiment, the solution source 40 includes a mixing tank 218 having a second mixing device (not shown) and a dispersion loop 222. The second mixing device may be any solution, suspension or emulsion mixing device.

The dispersion loop 222 includes a dispersion inlet 224 at or near the bottom of the dispersion tank 218 and a dispersion outlet 226 at the dispersion tank 218 generally above the dispersion inlet 224. The dispersion loop 222 further comprises a dispersion device 228, located between the dispersion inlet 224 and the dispersion outlet 226, which reduces the size or miniaturizes the particles suspended in the material solution; then a more refined, more thoroughly mixed emulsion of immiscible liquids is formed. Suitable dispersing means 228 include means capable of dispersing solids to a diameter of about 1 micron or less to a diameter of about 10 microns. Examples of suitable dispersing devices 228 include rotor/stator homogenizers, colloid mills, ball mills, sand mills, media mills, high pressure homogenizers, and the like.

In another embodiment, dispersion occurs within mixing tank 218 using agitation energy, such as with an acoustic wave, high shear mixer, or homogenizer.

When proteins or other heat sensitive materials are involved, the temperature of the dispersion tank 218 and/or the dispersion loop 222 is controlled by methods well known in the art to prevent protein denaturation.

In the method shown in fig. 3, the volatilized gas, liquefied gas, and frozen droplets 44 are all directed from the freezing section 20 through a conduit 210, the conduit 210 including a three-phase mixing device 216, preferably 4 or more electrostatic mixers, to swirl the three-phase material and wash the frozen droplets 44 in the gas phase into the liquefied gas, thereby improving yield.

In another embodiment, the solution contains additional material, either as a solid or in emulsion with the solvent, and flows through a dispersion device 228, such as a homogenizer, to micronize the solid particles into particles preferably about 1-10 microns in diameter, or to further mix the emulsion to form smaller emulsion droplets.

Where there are no suspended particles in the solution, or where larger suspended particles are desired, a disperser is not necessarily required.

Alternatively, the second mixing device may be used as a dispersing device, for example when the second mixing device used is a high speed/high shear mixer.

Figure 4 shows another embodiment of the apparatus of the invention suitable for carrying out the process of the invention. The device in fig. 4 bears many similarities to the devices of fig. 1 and 3 and is denoted by the same numerals. The apparatus includes a plurality of freezing containers 202, each containing a respective freezing section 20. The apparatus also includes an extraction vessel 206 having an extraction zone 22. Each freezing section 20 and extraction zone 22 have separate conduits 210 providing three-phase communication. Each conduit 210 includes a separate three-phase mixing device 216.

In the method shown in fig. 4, the frozen droplets 44 form frozen droplets 44 in each freezing section and are then transferred to the common extraction zone 22.

The compositions prepared according to the methods of the present invention may be administered to humans and animals by oral, suppository, injection, subcutaneous implant, intramuscular injection, intraperitoneal, intracranial, intradermal injection, mucosal administration, such as intranasal administration or by suppository administration, or locally (e.g., enema or aerosol spray) administration to treat a desired dose of the biologically active agent for a variety of conditions according to known parameters.

Equivalents of

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention. Such equivalents are intended to be encompassed by the present claims.

Claims (10)

1. A method of preparing microparticles of a substance from microdroplets of a solution of the substance in a solvent, the method comprising the steps of:

(a) directing said droplets into a freezing zone containing liquefied gas to freeze said droplets; and

(b) contacting the frozen microdroplets in an extraction zone with a non-solvent liquid to extract the solvent into the non-solvent to form said microparticles; wherein the freezing zone and the extraction zone are separate.

2. A method of preparing microparticles of a substance from microdroplets of a solution of the substance in a solvent, the method comprising the steps of:

(a) directing said droplets into a freezing vessel containing liquefied gas to freeze said droplets; and

(b) contacting the frozen microdroplets with a non-solvent liquid in an extraction vessel to extract the solvent into the non-solvent to form said microparticles; wherein the freezing vessel and the extraction vessel are separate.

3. The method of claim 1 or 2, wherein the substance comprises a biologically active agent or a stabilized biologically active agent, such as a protein, peptide, drug or prodrug.

4. The method of claim 3, wherein the biologically active agent is selected from the group consisting of an immunoglobulin protein, an interleukin, an interferon, an erythropoietin, an antibody, a cytokine, a hormone, an antigen, a growth factor, a nuclease, a tumor necrosis factor, a colony stimulating factor, an insulin, an enzyme, a tumor suppressor gene, an antisense molecule, an antibiotic, a steroid, a decongestant, a neuroactive agent, an anesthetic, a sedative, a cardiovascular drug, an antineoplastic agent, an anti-cancer agent, an antihistamine, and a vitamin.

5. The method of claim 3, wherein the substance further comprises a polymer.

6. The method of claim 5, wherein the polymer is selected from the group consisting of poly (lactide), poly (glycolide), poly (lactide-co-glycolide), poly (lactic acid), poly (glycolic acid), polycarbonate, polyesteramide, polyanhydride, poly (amino acid), polyorthoester, polyacetal, polynitrile acrylate, polyetherester, polycaprolactam, poly (dioxane), poly (alkylene alkyl ester), polyurethane, and mixtures and copolymers thereof.

7. The method of claim 1 or 2, wherein the temperature of step (a) is lower than the temperature of step (b).

8. A method according to claim 1 or 2, wherein the liquefied gas is injected into a freezing zone or a freezing container.

9. A method according to claim 1 or claim 2 wherein the droplets are formed by atomising a solution of the substance into a freezing zone or freezing chamber.

10. The method of claim 1 or 2 wherein the frozen microdroplets are collected in the bottom of a freezing zone or freezing vessel and directed to an extraction zone or extraction vessel.