MX2008009175A - Lyophilization system and method - Google Patents

Abstract

System and method for lyophilization or freeze-drying is provided. During the freezing step, the material or solution to be frozen is initially brought to a temperature near or below its freezing temperature after which the pressure in the freeze-dryer chamber is reduced to induce nucleation of the material.

Description

SYSTEM AND METHOD OF LIOFILIZATION Field of the Invention The present invention relates to a lyophilization process, and more particularly, to a method for inducing nucleation of freezing in a material, wherein the material is initially cooled to a temperature below a transition temperature of phase and then depressurized so as to induce nucleation of freezing in the material. Background of the Invention Controlling the generally random process of nucleation in the freezing stage of a lyophilization or freeze drying process, for both it is necessary to decrease the processing time to complete freeze drying, and it would be very desirable in the art increase the uniformity of the container-to-container product in the final product. In a common pharmaceutical freeze drying process, multiple containers containing a common aqueous solution are placed on shelves that are cooled, generally at a controlled rate, at low temperatures. The aqueous solution in each vessel is cooled below the thermodynamic freezing temperature of the solution and remains in a liquid state subcooled with metastability until nucleation occurs. The range of nucleation temperatures across the vessels is randomly distributed between a temperature close to the thermodynamic freezing temperature and some value (eg, up to above 30 ° C) significantly lower than the thermodynamic freezing temperature. This distribution of nucleation temperatures causes container-to-container variation in the crystalline structure of ice and ultimately in the physical properties of the lyophilized product. In addition, the drying step of the freeze drying process must be excessively long to adjust the range of sizes and structures of the ice crystal produced by the phenomenon of natural stochastic nucleation. The additives have been used to increase the nucleation temperature of the subcooled solutions. These additives can take many forms. It is well known that some bacteria (for example, Pseudomonas syringae) synthesize proteins that help ice formation with nucleus in subcooled aqueous solutions. Bacteria or their isolated proteins can be added to the solutions to increase the nucleation temperature. Various inorganic additives also demonstrate a nucleation effect; the most common additive is silver iodide, Agí. Generally any additive or contaminant has the potential to serve as a nucleating agent. Freeze-drying vessels prepared in environments containing high levels of particles generally lead to nucleation and freeze to a lower degree of subcooling than containers prepared in environments with few particles. All of the nucleating agents described above are referred to as "additives," because they change the composition of the medium in which they undergo nucleation at a phase transition. These additives are not commonly acceptable by the FDA that regulates and approves pharmaceutical products I iof ilized. These additives also do not provide control of time and temperature when the containers are subjected to nucleation and are frozen. In fact, the additives operate only to increase the average nucleation temperature of the containers. The ice crystals by themselves can act as nucleating agents for the formation of ice in subcooled aqueous solutions. In the "ice vapor" method, a dehydrator based on wet freezing is filled with a cold gas to produce a vapor suspension of small ice particles. The ice particles are transported to the containers and the nucleation starts when they come into contact with the fluid interface. The "ice vapor" method does not control the nucleation of multiple vessels simultaneously at a controlled time and temperature. In other words, the nucleation event does not occur concurrently or instantaneously within all vessels during the introduction of cold vapor into the freeze-based dryer. The ice crystals will take some time to process the manner in which each of the vessels will initiate nucleation, and transport times are probably different for containers in different locations within the dryer based on freezing. For large-scale industrial freeze dryers, the implementation of the "ice vapor" method required system design changes since internal convection devices are required to help a more even distribution of "ice vapor" through of the dryer based on freezing. When the shelves of the freeze-based dryer are continuously cooled, the time difference between when the first container freezes and the last container freezes, will create a temperature difference between the containers, which will increase the container-to-container non-uniformity in freeze-dried products. The pre-treatment of the perforating, tearing, or scraping vessel has also been used to decrease the degree of sub-cooling required for nucleation. As with the other methods of the prior art, the pretreatment of the container also imparts no degree of control to the time and temperature when the individual containers are subjected to nucleation and freezing, instead in fact only increases the average nucleation temperature of the container. all the containers. The vibration has also been used to subject a nucleation to a phase transition in a material with metastability. Sufficient vibration to induce nucleation occurs at frequencies above 10 kHz and can be produced using a variety of equipment. Frequently vibrations in this frequency range are called "ultrasonic," although frequencies in the range of 10 kHz to 20 kHz are commonly within the audible range of humans. Ultrasonic vibration frequently causes cavitation, or the formation of small gas bubbles, in a subcooled solution. In the temporary cavitation or inertial regime, the gas bubbles grow and collapse rapidly, causing very high localized fluctuations of pressure and temperature. The ability of ultrasonic vibration to induce nucleation in a material with metastability is often attributed to alterations caused by temporary cavitation. The other cavitation regime, called stable or without inertia, is characterized by bubbles that exhibit stable oscillations of volume or shape without collapse. US Patent Application No. 20020031577 A1 discloses that ultrasonic vibration can induce nucleation even in the stable cavitation regime, but offers no explanation of the phenomenon. British Patent Application 2400901A also discloses that the probability of causing cavitation, and therefore nucleation, in a solution using vibrations with frequencies above 10 kHz can be increased by reducing the ambient pressure around the solution or dissolving a volatile liquid in the solution. An electric freezing method has also been used previously to induce nucleation in subcooled liquids. Electric freezing is generally achieved by supplying relatively high electric fields (~ 1 V / nm) in a continuous manner or by pulsations between closely spaced electrodes immersed in a liquid or subcooled solution. The disadvantages associated with an electric freezing process in common lyophilization applications include the relative complexity and cost to implement and maintain, particularly lyophilization applications using multiple containers or containers. Also, electric freezing can be applied directly to solutions containing ionic species (eg, NaCl). Recently, studies were made examining the concept of "vacuum induced surface freezing" (see for example, US Patent No. 6,684,524). In such "vacuum induced surface freezing", containers containing an aqueous solution are loaded in a temperature controlled rack in a freeze based dryer and initially maintained at about 10 ° C. The freeze drying chamber is then evacuated to near vacuum pressure (e.g., 1 mbar), which causes the surface freezing of the aqueous solutions at depths of a few millimeters. The subsequent release of vacuum and the decrease in shelf temperature below the freezing point of the solution allows the development of the ice crystals of the pre-frozen surface layer through the rest of the solution. A major disadvantage of executing this process of 'vacuum-induced surface freezing' in a common lyophilization application is the high risk of sudden boiling or degassing of the solution under indicated conditions. Improved control of the nucleation process can allow the freezing of all non-frozen pharmaceutical solution containers in a freeze-based dryer to occur within a narrower range of temperature and time, thereby providing a lyophilized product with greater container-to-container uniformity. The control of the minimum nucleation temperature can affect the crystal structure of the ice formed inside the container, and I allow a very accelerated freezing drying process. Therefore, there is a need to control the random nucleation process in various freezing systems including the freezing step of a freeze drying or lyophilization process to decrease the processing time to complete freeze drying and to improve uniformity of the container-to-container product in the final product. Therefore, it would be desirable to provide a process that possesses some, or preferably all, of the above features. Brief Description of the Invention The present invention can be characterized as a method for making a material, comprising the steps of: (i) cooling the material in a chamber at a preset cooling rate; (ii) decrease in pressure in the chamber dryer to induce nucleation of freezing in the material; (iii) additional cooling of the material without subjecting to nucleation or below a final temperature to freeze the material; and (iv) drying the material to produce a dried product that reduces moisture or solvent. The invention can also be characterized as a freezing-based drying system comprising: a chamber having a controlled gas atmosphere and one or more shelves adapted to hold one or more containers or containers of a material; means for controlling the temperature of the shelves inside the chamber in order to control the temperature of the material; a condenser coupled to the chamber and adapted to remove any solvent or moisture from the chamber; and means for controlling the pressure of the chamber so as to rapidly depressurize the chamber to subject nucleation to a phase change in the material during freezing, and to maintain a low pressure during drying.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other aspects, features, and advantages of the present invention will be more apparent from the following, more detailed description thereof, presented in combination with the following drawings, wherein: Figure 1 is a graph representing the temperature against the time diagram of a solution that undergoes a stochastic freezing process and also shows the nucleation temperature range of the solution; Figure 2 is a graph representing the temperature versus time diagram of a solution undergoing a freezing system equilibrated with the depressurized nucleation according to the present methods; Fig. 3 is a graph representing the temperature against the time diagram of a solution undergoing a dynamic freezing process with depressurized nucleation according to the present methods; and Figure 4 is a schematic representation of a lyophilization system according to the present invention. Detailed Description of the Invention Nucleation is the beginning of a phase transition in a small region of a material. For example, the phase transition may be the formation of a crystal of a liquid. The crystallization process (ie, formation of solid crystals of a solution) frequently associated with the freezing of a solution begins with a nucleation event followed by the development of crystals. In the crystallization process, nucleation is the stage where the selected molecules dispersed in the solution or other material begin to accumulate to create nanoscale groups when they become stable under current operating conditions. These stable groups constitute the nuclei. Groups need to reach a critical size to become stable nuclei. Such a critical size is specified by the operating conditions such as temperature, contaminants, degree of supersaturation, etc. and it can usually vary from one sample of solution to another. It is during the event of nucleation that the atoms in the solution organize in a definite and periodic way that defines the structure of the crystals. The development of crystals is the subsequent development of the nuclei that manage to reach the critical group size. Depending on the conditions, the nucleation or the development of crystals can predominate over the other, and therefore, crystals with different sizes and shapes are obtained. The control of the size and shape of the crystals constitutes one of the main challenges in industrial manufacturing, such as for pharmacists. The present method relates to a process to control the time and / or temperature, wherein a phase transition subjected to nucleation occurs in a material. In freezing applications, the probability that a material spontaneously submits to nucleation and begins to change the phase, is related to the degree of sub-cooling of the material and the absence or presence of contaminants, additives, structures, or alterations that provide a site or a surface for nucleation. The freezing or solidification step is particularly important in the freeze drying process, where the existing techniques give rise to differences in nucleation temperature through a multiplicity of containers or containers. The differences in nucleation temperature tend to produce a non-uniform product and a long drying time. The present methods, on the one hand, provide a higher degree of process control in the batch solidification processes (eg, freeze drying) and produce a product with a more uniform structure and characteristics. Contrary to some of the techniques of the prior art for inducing nucleation, the present methods minimal changes of equipment and operation for implementation. Primarily, the present methods can be applied to any material treatment step involving a phase transition subjected to nucleation. Examples of such processes include the freezing of a liquid, crystallization of ice from an aqueous solution, crystallization of polymers and metals from fusions, crystallization of inorganic materials from supersaturated solutions, crystallization of proteins, production of artificial snow , ice deposition from steam, food freezing, concentration by freezing, fractional crystallization, cryopreservation, or condensation of vapors to liquids. From a conceptual point of view, the present methods can also be applied to phase transitions such as melting and boiling. The presently described method represents an improvement to the present pharmaceutical lyophilization processes. For example, within a dryer based on large industrial freezing there may be more than 100,000 containers containing a pharmaceutical product that needs to be frozen and dried. The current practice in the industry is to cool the solution to a very high degree to ensure that the solution is frozen in all containers or containers in the freeze-based dryer. The contents of each container or container, however, freeze randomly at a temperature range below the freezing point, because the nucleation process is uncontrolled. Returning to the figures, and particularly to Figure 1, a temperature against the time diagram of six vessels of an aqueous solution undergoing a conventional stochastic nucleation process, which shows the common interval of nucleation temperatures of the solution within, is presented. of the containers (11, 12, 13, 14, 15, and 16). As considered therein, the content of the vessel has a thermodynamic freezing temperature of about 0 ° C, yet the solution within each vessel is naturally nucleated at a wide temperature range of about -7 ° C to -20 ° C. ° C or more, as indicated by area 18. Diagram 19 represents the temperature of the shelf inside the freeze-drying chamber. In contrast, Figure 2 and Figure 3 represent the temperature against the time diagrams of a solution which undergoes a freezing process with the depressurized nucleation according to the present methods. Particularly, Figure 2 shows the temperature against the time diagram of six vessels of an aqueous solution undergoing a balanced cooling process (see example 2) with nucleation induced via chamber decompression (21, 22, 23, 24, 25, and 26). The contents of the container have a thermodynamic freezing temperature of approximately 0 ° C, even the solution inside the container is subjected to nucleation at the same time as decompression and within a very narrow temperature range (ie, -4 ° C to -5 ° C) as seen in area 28. The diagram 29 represents the temperature of the shelf inside the freeze drying chamber and represents a balanced freezing process, wherein the temperature of the shelves is kept more or less constant before decompression. Similarly, Figure 3 shows the temperature against the time diagram of three vessels of an aqueous solution undergoing a dynamic cooling process (see Example 7) with nucleation induced via chamber decompression (31, 32, and 33). ). Again, the contents of the container have a thermodynamic freezing temperature of about 0 ° C, yet the solution within each container is nucleated at the same time during decompression at a temperature range of about -7 ° C to -10 ° C. ° C as considered in area 38. Diagram 39 represents the temperature of the shelf inside the freeze-drying chamber and generally represents a dynamic cooling process, where the temperature of the shelves is actively decreased during or before the decompression. As illustrated in the figures, the present methods provide improved control of the nucleation process by allowing the freezing of pharmaceutical solutions in a freezing-based dryer to occur within a narrower temperature range (e.g., above 0 ° C). at -10 ° C) and / or concurrently, thereby producing a freeze-dried product with greater container-to-container uniformity. Although not shown, it is obviously foreseeable that the range of induced nucleation temperatures may even be slightly extended over the phase transition temperature and may also extend to about 40 ° C of subcooling. Another benefit associated with the present methods is that by controlling the minimum nucleation temperature and / or the exact duration of nucleation, the ice crystal structure formed inside the frozen containers or containers can be altered. The ice crystal structure is a variable that affects the time it • takes for the ice to be sublimated. Thus, by controlling the ice crystal structure, it is possible to greatly accelerate the entire freeze drying process. Turning now to FIG. 4, the illustrated freezing dryer unit (200) has several major components plus additional auxiliary systems to perform the lyophilization cycle. Particularly, the dryer unit based on freezing (200) includes a freeze-drying chamber (202) containing shelves (204) adapted to hold the containers or containers of the solution to be lyophilized (not shown). The solution to be lyophilized is specially formulated and commonly contains the active ingredient, a system of solvents and various stabilizing agents or other pharmaceutically acceptable carriers or additives. The lyophilization of this formulation occurs in specialized containers located in hollow shelves. These packages may include containers with caps, ampoules, syringes, or, in the case of large-scale lyophilization, pans. The freeze-based dryer unit (200) illustrated also includes a condenser (206) which is adapted to remove the sublimed solvent and desorb it from the vapor phase by condensing or freezing it as ice to maintain the proper vacuum within the freeze-based dryer. The condenser (206) can be located internally in the lyophilization chamber (202) or as a separate external unit in communication with the lyophilization chamber (202) through the so-called isolation valve. The freeze-based dryer unit (200) also preferably includes a vacuum pump (208) operatively coupled to the condenser (206) and adapted to draw vacuum from the lyophilization chamber (202) and the condenser (206). The cryogenic refrigeration system (210) provides the temperature control means for the freeze-based dryer unit (200) by cooling a pre-set heat transfer liquid that is circulated to the shelves (204) inside the freeze-drying chamber ( 202) and the capacitor (206). As illustrated, the cryogenic refrigeration system (210) comprises a source of cryogen (218), for example liquid nitrogen, a cryogenic heat exchanger (220), and a heat transfer fluid circuit (222), vent duct (224). ), heater (226) and pumps (227.228). The cryogenic heat exchanger (220) is preferably a NCOOL ™ Non-Freezing Cryogenic Heat Exchange System available from Praxair, Inc. an important aspect of the cryogenic heat exchanger (220) is the vaporization of liquid nitrogen within or internally in the heat exchanger. heat still in a way that avoids direct contact of liquid nitrogen on the cooling surfaces exposed to the heat transfer fluid. Details of the structure and operation of such a heat exchanger can be found in U.S. Patent No. 5,937,656 (Cheng et al.), The disclosure of which is incorporated by reference herein. The preset heat transfer fluid circuit (222) is adapted to circulate a heat transfer fluid and is operatively coupled to the lyophilization chamber (202) as well as the condenser (206). More specifically, the heat transfer fluid circulates within the hollow shelves (204) within the freeze-drying chamber (202) to accurately communicate cooling or heating through the shelves (204) to the solution as needed. In addition, the pre-set heat transfer fluid also passes through the condenser (206) to provide the cooling means necessary to sublimate the ice and further desorb the solvent. The pump (227) and the heater (226) are located along the heat transfer fluid circuit (222) 'countercurrently from the lyophilization chamber (202) and downstream from the cryogenic heat exchanger (220). The pump (227) is sized to move the heat transfer fluid through the heat transfer circuit (222) to the required flow rates. The heater (226) is preferably an electric heater adapted to provide heat complementary to the heat transfer fluid and to the lyophilization chamber (202) as may be required during the drying processes. As observed in the embodiment of Figure 4, the condenser (206) is also cooled by the recirculation of a low temperature heat transfer fluid. Cooling of the heat transfer fluid through the condenser (206) is also provided by a cryogenic heat exchanger (220). The cryogenic heat exchanger (220) is capable of cooling the heat transfer fluid continuously without freezing. During the drying phases, the cryogenic heat exchanger (220) is set or adapted to reach the lowest temperature required by the condenser (206). As described above, the cryogenic heat exchanger (220) pre-evaporates the liquid nitrogen to a cold cryogenic gas for the transfer of heat to the heat transfer fluid. Through the pre-evaporation of liquid nitrogen, it is ensured that liquid nitrogen avoids boiling directly on a heat exchange surface where the heat transfer fluid is located on the other side. Such an arrangement prevents freezing of the cryogenic heat exchanger (220) since the liquid nitrogen boils at about -195 ° C at atmospheric pressure. The illustrated embodiment of Figure 4 also includes means for controlling the gas atmosphere of the lyophilization chamber (250), and particularly the composition and pressure of the gas within the chamber (202). The pressure control of the chamber (202) allows pressurization and rapid decompression of the chamber to induce nucleation of the solution. The described embodiment preferably utilizes one or more flow control valves (252) controllably adapted to facilitate the introduction of a pressurized gas atmosphere into the chamber (202) from a gas source (not shown), and to depressurize the chamber discharging the pressurized gas atmosphere out of the chamber (202) in a controlled and preferably rapid manner, thereby nucleating the solution in several containers or containers. Although not shown, the freeze-based dryer unit (200) also includes several software and control hardware systems adapted to control and coordinate various parts of the freeze drying equipment, and perform the preprogrammed freeze-drying cycle. Various software systems, and control hardware can also provide documentation, data logging, alarms, and also the system's security capabilities. In addition, the auxiliary systems of the freeze-based dryer unit (200) may include several sub-systems for cleaning and sterilizing the freeze-drying chamber (202), automatically charging and discharging the product in the freeze-drying chamber (202); and associated cryogenic system accessories such as cooling slides, liquid nitrogen tanks, pipes, valves, sensors, etc.

Broadly speaking, currently described methods for inducing the nucleation of a phase transition within a material, comprise the steps of: (i) cooling the material to a temperature of about or below a phase transition temperature of the material; and (ii) rapidly decreasing the pressure to induce nucleation of a phase transition in the material. Each of these important stages will be discussed in more detail below. Step 1 - Material cooling The illustrative materials useful in the present method include substances, gases, suspensions, gels, liquids, solutions, mixtures, or pure components within a solution or mixture. Suitable materials for use in the present method may include, for example, pharmaceutical materials, biopharmaceutical materials, foodstuffs, chemical materials, and may include products such as wound care products, cosmetics, veterinary products and products related to diagnostics. in vivo / in vitro and the like. When the material is a liquid, it may be desirable to dissolve the gases in the liquid. Liquids in a controlled gas environment will generally have gases dissolved in them. Other illustrative materials useful in the present method include biological or biopharmaceutical material such as multicellular tissues, organs and structures. For certain biological and pharmaceutical applications, the material may be a solution or a mixture that includes: live or attenuated viruses; nucleic acids; monoclonal antibodies; polyclonal antibodies; biomolecules; non-peptide analogues; peptides, including polypeptides, peptide mimics and modified peptides; proteins, including fusion and modified proteins; RNA, DNA and subclasses thereof; oligonucleotides; viral particles; and the like such as materials or components thereof. Pharmaceutical or biopharmaceutical solutions contained in freeze-drying containers or containers would be a good example of a material that would benefit from the present method. The solutions are mainly water and are substantially incompressible. Such pharmaceutical or biopharmaceutical solutions are also highly pure and generally free of macroparticles that can form sites for nucleation. The uniform nucleation temperature is important to create a constant and uniform ice crystal structure from container-to-container or container-to-container. The converted ice crystal structure also greatly affects the time required for drying. In relation to a freeze drying process, the material is preferably placed in a chamber, such as a freeze drying chamber. Preferably, the chamber is configured to allow control of the temperature, pressure, and atmosphere of the gas within the chamber. The gas atmosphere may include, but is not limited to: argon, nitrogen, helium, air, water vapor, oxygen, carbon dioxide, carbon monoxide, nitrous oxide, nitric oxide, neon, xenon, krypton, methane, hydrogen, propane , butane, and the like, including permitted mixtures thereof. The preferred gas atmosphere comprises an inert gas, such as argon, at a pressure between about 7 to about 50 psig or more. The temperatures inside the freeze-based dryer chamber are frequently indicated by the freeze-drying process and are easily controlled via the use of a heat transfer fluid that cools or heats the shelves inside the chamber to control the temperature of the freezer. the containers or containers and the material inside each container or container. According to the present methods, the material is cooled to a temperature of approximately or below its phase transition temperature. In the case of a liquid-based solution that undergoes a freeze-drying process, the phase transition temperature is the thermodynamic freezing point of the solution. Where the solution reaches temperatures below the thermodynamic freezing point of the solution, it is commonly referred to as subcooled. When applied to a freezing process of a liquid-based solution, the present method is effective when the degree of sub-cooling ranges from about or below the phase transition temperature to about 40 ° C of sub-cooling, and preferably between approximately 3 ° C of sub-cooling and 10 ° C of sub-cooling. In some of the examples described below, the present method for inducing nucleation desirably operates even where the solution has only about 1 ° C of subcooling below its thermodynamic freezing point. Where the material is at a temperature below its phase transition temperature, it is frequently referred to as in a state with metastability. A state with metastability is an unstable and temporary, but relatively durable state of a chemical or biological system. A material with metastability exists in a phase or temporarily state that is not its phase or state of equilibrium. In the absence of any change in the material or its environment, a material with metastability will eventually experience the transition from its state of imbalance to its state of equilibrium. Materials with illustrative metastability include supersaturated solutions and subcooled liquids. A common example of a material with metastability would be liquid water at atmospheric pressure and at a temperature of -10 ° C. With a normal freezing point of 0 ° C, liquid water must not exist thermodynamically at this temperature and pressure, but it may exist in the absence of an event or nucleation structure to begin the process of ice crystallization. Extremely pure water can be cooled to very low temperatures (-30 ° C to -40 ° C) at atmospheric pressure and still remain in a liquid state. Such subcooled water is in a state with thermodynamically unbalanced metastability. It lacks only a nucleation event to cause the beginning of the phase transition, so it returns to equilibrium. As discussed above, the present methods for inducing the nucleation of a phase transition within a material, or for freezing a material can be used with various cooling profiles, including, for example, a balanced cooling environment or a cooling environment dynamic (see Figs 2 and 3). Step 2 - Quickly decrease the pressure When the material has reached the desired temperature of approximately or below the phase transition temperature, the chamber is depressurized either accelerated or rapidly. This decompression activates the nucleation and the phase transition of the solution inside the containers or containers. In the preferred embodiment, the depressurization of the chamber is achieved by partially opening or opening a large control valve that separates the high pressure chamber from the surrounding environment or a low pressure or ambient chamber. The high pressure is rapidly diminished by the total flow of the gas atmosphere of the chamber. Depressurization needs to be fast enough to induce nucleation. The depressurization must be completed in several seconds or less, preferably 40 seconds or less, preferably 20 seconds or less, and more preferably 10 seconds or less. Commonly freeze drying applications, the pressure difference between the initial chamber pressure and the final chamber pressure, after depressurization, should be greater than about 7 psi, although small decreases in pressure can induce nucleation in some situations. Most commercial freeze-based dryers can easily adapt the range of pressure decreases needed to control nucleation. Many freeze-based dryers are designed with pressure levels above 25 psig to withstand conventional sterilization procedures using saturated steam at 121 ° C. Such equipment levels provide a wide window to induce nucleation by following the protocols that depressurize from the initial pressures on the ambient pressure or pressure in the current surrounding environment. The elevated pressure and subsequent depressurization can be achieved by any known means (eg, pneumatic, hydraulic, or mechanical). In the preferred embodiments, the operating pressures for the present methods must remain below the supercritical pressure of any applied gas, and the subjection of the material at extreme low pressures (i.e., approximately 10 mTorr or less) should be avoided during the nucleation of the material. While not wishing to be limited to any particular mechanism, a possible mechanism explaining the controlled nucleation observed in the practice of the present method is where the gases in the solution in the material leave the solution during the depressurization and form bubbles which are subjected to nucleation to the material. An initial high pressure increases the concentration of dissolved gas in the solution. The rapid decrease in pressure after cooling reduces the gas solubility, and the subsequent release of the gas from the subcooled solution triggers the nucleation of the phase transition. Another possible mechanism is that where the decrease in the temperature of the next gas The material during depressurization causes a cold spot on the surface of the material that initiates nucleation. Another possible mechanism is that depressurization causes the evaporation of some liquid in the material and the cooling resulting from the endothermic evaporation process can initiate nucleation. Another possible mechanism is that the depressurized cold gas close to the material freezes some steam in equilibrium with the material before depressurization or release of the material by evaporation during depressurization; the resulting solid particles enter the material again and act as seeds or surfaces to initiate nucleation. One or more of these mechanisms can contribute to the initiation of nucleation of freezing or solidification to different degrees depending on the nature of the material, its environment and the phase transition that is subjected to nucleation. The process can be performed entirely at a pressure greater than ambient pressure or over a range of pressures that exceed the ambient pressure. For example, the initial pressure of the chamber may be above the ambient pressure and the final pressure of the chamber, after depressurization, may be above the ambient pressure but below the initial pressure of the chamber; the initial pressure of the chamber may be above ambient pressure and the final pressure of the chamber, after depressurization, may be above ambient pressure or slightly below ambient pressure. The index and magnitude of the pressure decrease is also believed to be an important aspect of the present methods. Have experiments shown that nucleation will be induced where the pressure drop (? P) is greater than about 7 psi. Alternatively, the magnitude of the pressure drop can be expressed as an absolute pressure ratio, - R = PJPf, where P, is the initial absolute pressure and Pf is the final absolute pressure. It is believed that nucleation can be induced during depressurization the absolute pressure ratio, R, is greater than about 1.2 in many practical applications of the present methods. The rate of pressure decrease also plays an important role in the present methods. A method to characterize the rate of pressure decrease is through the use of a parameter, A, where A =? P /? T. Again, it is believed that nucleation will be induced for A values greater than a preset value, such as approximately 0.2 psi / sec. Empirical data through experimentation should help to verify the decrease in pressure and the preferred rate of pressure decrease.

The following examples highlight various aspects and characteristics of the methods currently described for inducing nucleation in a material and should not be considered in a limiting sense. In fact, these examples are illustrative only and the scope of the invention should be determined only with respect to the claims appended hereto.

EXAMPLES All the examples described herein were carried out in a experimental scale-based VirTis 51-SRC freeze dryer having four shelves with approximately 1.0 m2 total shelf space and an internal condenser. This unit was adapted to "withstand positive pressures of up to approximately 15 psig.A 1.5" diameter circular opening was also added to the back wall of the freeze drying chamber with a 1.5"diameter stainless steel pipe that was extends from the hole through the insulation of the rear wall to emerge from the back of the dryer based on freezing.The full port of two 1.5", ball valves operated by air, were attached to this pipe via sanitary fittings. A ball valve allows a gas to flow into the freeze drying chamber and thereby provide positive pressures of up to 15 psig. The second ball valve allows gas to flow out of the freeze drying chamber and thereby reduce the chamber pressure to atmospheric conditions (0 psig). All refrigeration of the shelves and the freeze dryer condenser was performed via the circulation of the Dynalene MV heat transfer fluid cooled by liquid nitrogen using the Praxair NCool ™ -HX system.

All the solutions were prepared in a clean class 100. The dryer based on freezing was placed with the door, shelves, and controls all accessible from the clean space while. the other components (pumps, heaters, ...) were placed in a clean environment. All solutions were prepared with water of CLAR grade (Fisher Scientific, filtered through-membranes of 0.10 μm). The final solutions were filtered through 0.22 μm membranes before filling the lyophilization containers or containers. All gases were supplied via cylinders and filtered through 0.22 μm filters to remove the particulates. The glass containers (5 ml containers and 60 ml bottles) were obtained pre-cleaned for the Wheaton Science Products macroparticles. The pharmaceutically acceptable carriers were used when appropriate. The above steps were considered to ensure that the materials and methods met conventional pharmaceutical manufacturing standards for the particulates, which act as nucleating agents. As used herein, the "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, antioxidants, salts, coatings, surfactants, preservatives (e.g., methyl or propyl p-hydroxybenzoate, sorbic acid, antibacterial agents , anti-fungal agents), isotonic agents, solution retarding agents (e.g., paraffin), absorbents (e.g., kaolin clay, bentonite clay), drug stabilizers (e.g., sodium lauryl sulfate), gels , binders (e.g., syrup, acacia, gelatin, sorbitol, tragacanth, polyvinyl pyrrolidone, carboxymethyl cellulose alginates), excipients (e.g., lactose, milk sugar, polyethylene glycol), disintegrating agents (e.g., agar-agar, starch, lactose, calcium phosphate, calcium carbonate, alginic acid, sorbitol, glycine), wetting agents (eg, cetyl alcohol, glycerol monostearate), lubricants, absorption accelerators (for example, quaternary ammonium salts), edible oils (for example, almond oil, coconut oil, oily esters or propylene glycol), sweeteners, sweetening agents, flavoring agents, coloring agents, fillers, (eg, starch, lactose, sucrose, glucose, mannitol), tablet-forming lubricants (eg example, magnesium stearate, starch, glucose, lactose, rice flower, gypsum), carriers for inhalation (e.g., hydrocarbon propellants), buffering agents, or such materials and combinations thereof, as would be known to a skilled in the art. For the experimental conditions described herein and all the lyophilization formulations studied, it was observed that stochastic nucleation commonly occurred at container temperatures between about -8 ° C and -20 ° C and occasionally such hot as -5 ° C. The packages could generally be maintained at temperatures above -8 ° C for long periods of time without nucleation. The onset of nucleation and the subsequent development of crystals (i.e., freezing) was determined by the measurement of temperature as the point at which the package temperature increased rapidly in response to the latent exothermic heat of fusion. The initiation of freezing could also be determined visually through a transparent glass in the door of the dryer chamber based on freezing.

Example 1: Control of nucleation temperature Four separate containers were filled with 2.5 ml of % by weight of mannitol solution. The expected thermodynamic freezing point of 5% by weight of mannitol solution is about -0.5 ° C. The four containers were placed on a dryer rack based on freezing in close proximity to each other. The temperatures of the four vessels were monitored using the surface mounted thermocouples. The freeze-based dryer was pressurized with argon at 14 psig.

The freezer-based dryer rack was cooled to obtain container temperatures between about -1.3 ° C and about -2.3 ° C (+/- 1 ° C measuring accuracy of the thermocouples). The freeze-based dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the containers. The four vessels were subjected to nucleation and began to freeze immediately after depressurization. The results are presented briefly in the following table 1.

As observed in Table 1, the nucleation temperatures controlled in this example (i.e. initial temperatures of the container) are very close to the expected thermodynamic freezing point of the solution. Thus the present method allows nucleation control to occur in solutions that have a very low degree of subcooling or at nucleation temperatures of approximately or only slightly cooler than their freezing points.

Table 1. Control of nucleation temperature.

Example 2 - Control of nucleation temperature In this example, ninety-five containers were filled with 2.5 ml of 5% mannitol solution. The thermodynamic freezing point of 5% mannitol solution is approximately -0.5 ° C. The ninety-five containers were placed on a dryer rack based on freezing in close proximity to each other. The temperature of six vessels placed in different locations on the rack of the dryer based on freezing, was continuously monitored using the surface mounted thermocouples. The freeze-based dryer was pressurized in an argon atmosphere at approximately 14 psig. The dryer rack based on freezing was then cooled to obtain container temperatures of almost -5 ° C. The freeze-based dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the containers. It was observed visually that the ninety, and five vessels were subjected to nucleation and began to freeze immediately after depressurization. The thermocouple data for the six monitored vessels confirmed the visual observation. The results are briefly presented in Table 2. As considered in the table, the nucleation temperatures controlled in this example (i.e. initial temperatures of the vessel) are partially below the expected thermodynamic freezing point of the solution. Thus, the present method allows nucleation control to occur in solvents that have a moderate degree of subcooling. This example also demonstrates the expandability of the present method in a multiple container application.

Table 2. Control of nucleation temperature Example 3 - Control of the depressurization amount In this example, the multiple containers were filled with 2.5 ml of 5% mannitol solution. Again, the predicted thermodynamic freezing point of 5% mannitol solution was about -0.5 ° C. For each test run, the containers were placed on a rack of the dryer based on freezing in close proximity to each other. As with the examples described above, the temperatures of the containers were monitored using the surface mounted thermocouples. The argon atmosphere in the freeze-based dryer was pressurized at different pressures and the freeze-based dryer rack was cooled to obtain container temperatures of about -5 ° C. In each test run, the freeze-based dryer is then rapidly depressurized (i.e., in less than five seconds) from the selected pressure at atmospheric pressure in an effort to induce nucleation of the solution within the containers. The results are presented briefly in Table 3. As observed in Table 3, controlled nucleation occurred where the pressure decreased approximately 7 psi or more and the nucleation temperature (ie, initial temperature of the container) was between approximately -4.7 ° C and -5.8 ° C. Table 3. Effect of the magnitude of depressurization Example 4 - Control of depressurization rates For this example, the multiple containers were filled with approximately 2.5 ml of 5% mannitol solution having a predicted thermodynamic freezing point of about -0.5 ° C. For each execution of varying times of depressurization, the containers were placed in a rack of the dryer based on freezing in close proximity to each other. As in the examples described above, container temperatures were monitored using the surface mounted thermocouples as the examples described above, the argon atmosphere in the freeze-based dryer was pressurized to approximately 14 psig and the rack was cooled to obtain the container temperatures of about -5 ° C. In each test run, the freeze-based dryer was then depressurized at different depressurization rates of 14 psig at atmospheric pressure in an effort to induce nucleation of the solution within the containers. To study the effect of the depressurization rate or depressurization time, a restriction ball valve was placed at the outlet of the depressurization control valve on the back of the dryer based on freezing. When the restriction valve is fully open, depressurization of approximately 14 psig to approximately 0 psig is achieved in approximately 2.5 seconds. By only partially closing the restriction valve, it is possible to vary the depressurization time of the chamber. Using the restriction ball valve, several test runs were performed with the dryer chamber based on depressurized freezing at different indices to check or determine the effect of depressurization rate on nucleation. The results are presented briefly in table 4. Table 4. Effect of depressurization time As observed in Table 4, nucleation only occurred where the depressurization time was less than 42 seconds, the pressure decrease was about 14 psi or more and the nucleation temperature. (ie, the initial temperature of the container) was between about -4.6 ° C and about -5.8 ° C. These results indicate that depressurization needs to be achieved relatively quickly in order for the method to be effective.

Example 5 - Control of the gas atmosphere Again, each of the multiple containers was filled with approximately 2.5 ml of 5% mannitol solution and placed in a rack of the freezing-based dryer in close proximity to each other. As with the examples described above, the temperature of the test vessels was monitored using the surface mounted thermocouples. For the different test runs, the gas atmosphere in the dryer based on freezing was varied maintaining always a positive pressure of approximately 14 psig. In this example, the shelf of the freeze-based dryer was cooled to obtain container temperatures of about -5 ° C to -7 ° C. At each test run, the freeze-based dryer was then rapidly depressurized from about 14 psig at atmospheric pressure in an effort to induce nucleation of the solution within the vessels. The results are presented briefly in table 5.

As observed in the table, controlled nucleation occurred in all gas atmospheres, except in the helium gas atmosphere where it was approximately 14 psi and the nucleation temperature (ie, the initial container temperature) was between approximately - 4.7 ° C and approximately -7.4 ° C. Although not shown in the examples, it is believed that the alternative conditions will likely allow controlled nucleation in a helium atmosphere.

Table 5. Effect of the composition of the gas atmosphere Example 6 - Large volume solutions In this example, six lyophilization bottles (60 ml capacity) were filled with about 30 ml of 5% by weight of mannitol solution having a predicted thermodynamic freezing point of about -0.5 ° C. . The six lyophilization bottles were placed in a rack of the dryer based on freezing in close proximity to each other. The temperature of the six bottles placed in different locations on the rack of the dryer based on freezing, was monitored using the surface mounted thermocouples. The freeze-based dryer was pressurized in an argon atmosphere at approximately 14 psig. The rack of the dryer based on freezing was then cooled to obtain the temperatures of the bottle of almost -5 ° C. The freeze-based dryer was then depressurized from 14 psig under approximately atmospheric pressure in less than five seconds to induce nucleation of the solution within the bottles. The results are presented briefly in table 6.

A separate experiment, a plastic bulk freeze drying tray (Gore LYOGUARD, 1800 ml capacity), was filled with about 1000 ml of 5% by weight of mannitol solution. The tray was obtained previously cleaned to meet the requirements of low USP particle content. The tray was placed on a rack of the dryer based on freezing, and the temperature of the tray was monitored by a thermocouple mounted on the outer surface of the tray almost in the middle of one side. The rack of the dryer based on freezing was then cooled to obtain a tray temperature of about -7 ° C. The freeze-based dryer was then depressurized from 14 psig to approximately atmospheric pressure in less than five seconds to induce nucleation of the solution within the tray. The results are also presented briefly in table 6.

As with the examples described above, all packages were subjected to nucleation and began to freeze immediately after depressurization. Like the examples described above, the nucleation temperatures (ie, container temperatures) in this example were much controllable than those that were approximately the thermodynamic freezing temperature of the solution. Importantly, this example illustrates that the present method allows nucleation control to occur in larger volume solutions and various pack formats. It should be noted that the effectiveness of the depressurization method would be expected to improve while the formulation volume increases, because the nucleation event is more likely to occur when more molecules are present to aggregate and form critical nuclei.

Table 6. Effect of solution volume and type of container Example 7 - Dynamic cooling against balanced cooling The present methods for controlling nucleation can be used in several ways. Examples 1-6, described above, each demonstrate the control aspect of the nucleation temperature of a lyophilization solution that essentially equilibrates at a temperature below its thermodynamic freezing point (i.e., very slowly changing temperature) . This example demonstrates that nucleation can also occur at a temperature below the thermodynamic freezing point in a dynamic cooling environment (i.e., the solution undergoes rapid temperature changes). In this example, containers 1 to 6 represent the samples described above with reference to example 2. In addition, three separate containers (containers 7-9) were also filled with 2.5 ml of 5% mannitol solution. In a separate test run, the three additional containers were placed in a dryer rack based on freezing in close proximity to each other. The freezer-based dryer rack was rapidly cooled to a final shelf temperature of -45 ° C. When one of the containers reached a temperature of about -5 ° C, as measured by the surface mounted thermocouplings, the freeze-based dryer was rapidly depressurized from about 14 psig to 0 psig in an effort to induce nucleation. The three vessels were subjected to nucleation and began to freeze immediately after depressurization. The container temperatures decreased significantly between -6.8 ° C and -9.9 ° C before nucleation as a result of the dynamic cooling environment. The comparative results are presented briefly in the following table 7. Table 7. Effect of dynamic cooling on nucleation The effectiveness of the present methods for controlling nucleation in freeze-dried solutions in a specified temperature range or lyophilization solutions that are dynamically cooled, it provides the end user with two potential modes of application with different advantages and disadvantages. By allowing the lyophilization solutions to equilibrate, the nucleation temperature range will be narrow or minimized to the operating limits of the dryer based on freezing by itself. The equilibrium stage may require additional time to relatively reach conventional or dynamic freezing protocols where the chamber and container temperatures are lowered to less than about -40 ° C in one stage. However, the use of the equilibrium step must produce a much improved nucleation uniformity through all the containers or containers as well as the realization of other benefits associated exactly with the control of the nucleation temperature of the material. Alternatively, if the equilibrium of the temperatures of the material solution or lyophilization is undesirable, the depressurization step can simply be implemented at an appropriate time during the normal freezing or dynamic cooling protocol. Depressurization during dynamic cooling will result in a broader extension in nucleation temperatures for the material within the lyophilization packages, but it will add very little time to the freezing protocol and still allow the problems of extreme sub-cooling to be alleviated. Example 8 - Effect of different excipients The present method for controlling or inducing nucleation in a material can be used to control the nucleation temperature of subcooled solutions containing different lyophilization excipients. This example demonstrates the use of the present methods with the following excipients: mannitol; hydroxyethyl starch (HES); polyethylene glycol (PEG); polyvinylpyrrolidone (PVP); dextran; glycine; sorbitol; sucrose; and trehalose. For each excipient, two containers were filled with 2.5 ml of a solution containing 5% by weight of excipient. The containers were placed in a rack of the dryer based on freezing in close proximity to each other. The freeze-based dryer was pressurized in an argon atmosphere at approximately 14 psig. The dryer rack based on freezing was cooled to obtain container temperatures of about -3 ° C and then rapidly depressurized to induce nucleation. The results are presented briefly in table 8.

Table 8. Effect of different lyophilization excipients Example 9 - Nucleation control of protein solutions The present methods and systems described herein can be used to control the nucleation temperature of subcooled protein solutions without negative or deleterious effects on protein solubility or enzymatic activity . Two proteins were used in this example, bovine serum albumin (BSA) and lactate dehydrogenase (LDH). BSA was dissolved in 5% by weight of mannitol at a concentration of 10 mg / ml. Three lyophilization vessels were filled with 2.5 ml of BSA-mannitol solution and placed on a rack of freezing dryer in close proximity to each other. The dryer based on freezing was pressurized in an argon a. approximately 14 psig. The freezer-based dryer rack was cooled to obtain container temperatures of about -5 ° C. The freeze-based dryer was rapidly depressurized to induce nucleation. All BSA solution containers were subjected to nucleation and began to freeze immediately after depressurization. No precipitation of the protein was observed during thawing. The LDH proteins were obtained from two different suppliers and for clarity are designated as LDH-1 or LDH-2 to distinguish the two different batches. LDH-1 was dissolved in 5% by weight of mannitol at a concentration of 1 mg / ml. Six freeze-drying vessels were filled with 2.5 ml of LDH-1 / mannitol solution and placed in a rack of the freezing-based dryer in close proximity to each other. The freeze-based dryer was pressurized in an argon atmosphere at approximately 14 psig. The freeze-based dryer rack was cooled from room temperature to obtain container temperatures of about -4 ° C. The freeze-based dryer was then rapidly depressurized to induce nucleation. All vessels were nucleated and began to freeze immediately after depressurization. The containers were kept in this state for approximately 15 minutes. The freeze-based dryer rack was then cooled to an index of about 1 ° C / min to obtain container temperatures of about -45 ° C and held for an additional 15 minutes to ensure completion of the freezing process. After the freezing step, the rack of the dryer based on freezing was then heated to an index of about 1 ° C / min to raise the container temperatures to about 5 ° C. No precipitation of the protein was observed during thawing. The content of the container was tested to determine enzyme activity, and the results were compared with a control sample of non-frozen solution of LDH-1 / mannitol.

As part of Example 9, the samples subjected to depressurized nucleation of LDH-1 / mannitol solution were compared to the samples subjected to nucleation in a stochastic manner. In the samples subjected to stochastic nucleation of LDH-1, the freezing procedure was repeated without pressurization and depressurization and without argon atmosphere. Specifically, LDH-1 was dissolved in 5% by weight of mannitol at a concentration of 1 mg / ml. Six freeze-drying vessels were filled with 2.5 ml of LDH-1 / mannitol solution and placed in a rack of the freezing-based dryer in close proximity to each other. The freeze-based dryer rack was cooled from room temperature to an index of about 1 ° C / min to obtain container temperatures of about -45 ° C and maintained for 15 minutes to ensure completion of the freezing process. After the freezing stage, the shelf of the dryer based on freezing was heated at an index of about 1 ° C / min to raise the container temperatures to about 5 ° C. No precipitation of the protein was observed during thawing. The content of the container was tested to determine the enzymatic activity, and the results were compared with the same control sample of non-frozen LDH-1 / mannitol solution. Also as part of Example 9, the experiments described above for LDH-1 were repeated using LDH-2. The only difference was a controlled nucleation of approximately -3 ° C for LDH-2 instead of -4 ° C for LDH-1. Table 9. Control of nucleation temperature of subcooled protein solutions As observed in Table 9, the controlled nucleation and freezing process achieved via depressurization, do not clearly diminish the enzymatic activity related to a comparable stochastic nucleation and a freezing protocol. In fact, the controlled nucleation process achieved via depressurization appears to preserve enzyme activity with an average activity loss of only 17.8% for LDH-1 and 26.5% for LDH-2 compared to the average activity loss of 35.9% for LDH-1 and 41.3% for LDH-2 after stochastic nucleation. It should be noted that the stochastic nucleation temperatures observed for LDH-2 were substantially warmer than the stochastic nucleation temperatures for LDH-1. This difference may be due to a little contaminant that acts as a nucleating agent in LDH-2. The stochastic nucleation temperatures are much closer to the controlled nucleation temperatures for LDH-2 compared to LDH-1, even with the improvements in the retention of enzymatic activity obtained via controlled nucleation for LDH-1 and LDH-2, are similar in 18.1% and 14.8%, respectively. This result suggests that improvements in the retention of enzymatic activity can be partially attributed to the characteristics of the controlled nucleation process by itself, not only at the preestablished heater nucleation temperatures obtained via depressurization. Example 10 - Reduction of primary drying time A solution of 5% by weight mannitol was prepared by mixing approximately 10.01 grams of mannitol with approximately 190.07 grams of water. The containers were filled with 2.5 ml of 5% mannitol solution. The containers were weighed empty and with the solution to determine the mass of the water added to the containers. The twenty containers were placed in a rack on a dryer rack based on freezing in close proximity to each other. The temperatures of the six vessels were monitored using the surface mounted thermocouplings; all the monitored containers were surrounded by other containers to improve the uniformity of the container's behavior. The freeze-based dryer was pressurized to approximately 14 psig in a controlled gas atmosphere of argon gas. The freezer-based dryer rack was cooled from room temperature to about -6 ° C to obtain container temperatures between about -1 ° C and -2 ° C. The freeze-based dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the containers. All vessels observed or monitored visually via the thermocouples were nucleated and began to freeze immediately after depressurization. The shelf temperature was then rapidly decreased to approximately -45 ° C to complete the freezing process. Once all container temperatures were about -40 ° C or less, the freeze drying chamber was evacuated and the primary drying process (i.e., sublimation) was initiated. During this drying process, the rack of the dryer based on freezing was heated to about -14 ° C via a ramp for one hour and kept at that temperature for 16 hours. The condenser was maintained at approximately -60 ° C through the drying process. The primary drying was stopped by turning off the vacuum pump and filling the chamber with argon "at atmospheric pressure." The containers were quickly removed from the freeze-based dryer and weighed to determine how much water was lost during the primary drying process. separated as part of example 10, other containers were filled with 2.5 ml of the same solution % by weight mannitol. The containers were weighed empty and with the solution to determine the mass of the water added to the containers. The containers were loaded in the freeze-based dryer in the same manner as described above, and the temperatures of the six vessels were again monitored using the surface mounted thermocouples. The dryer shelf based on freezing was rapidly cooled from room temperature to about -45 ° C to freeze the containers. The nucleation occurred stochastically between about -15 ° C and about -18 ° C during the cooling step. Once all container temperatures were approximately -40 ° C or less, the containers were dried in an identical manner to the method described above. During the conclusion of the primary drying, the samples were quickly removed from the dryer based on freezing and weighed to determine how much water was lost during the drying. process-primary drying. Table 10. Increasing the nucleation temperature improves the primary drying The results of the freeze-drying process with controlled nucleation and stochastic nucleation1 are presented briefly in the following table 10. It should be noted that these two experiments differ only in the addition of controlled nucleation via the depressurization step to an experiment. As considered in Table 10, the controlled nucleation process achieved via depressurization, in this example allows nucleation at very low degrees of subcooling, between about -1.1 ° C and -2.3 ° C. The much warmer nucleation temperatures for the case of controlled nucleation compared to the stochastic nucleation case produce an ice structure and a resulting lyophilized cake with dramatically improved drying properties. For the same amount of drying time, the containers subjected to nucleation using the described depressurization methods between approximately -1.1 ° C and -2.3 ° C lost an average of 86.1% of their water while the containers subjected to stochastic nucleation between I approximately -14.5 ° C and -17.9 ° C lost only an average of 65.3%. Therefore, containers subjected to stochastic nucleation would require much more primary drying time to reach the same degree of water loss as the containers subjected to nucleation in a controlled manner according to the methods currently described. The improvement in drying time is probably attributed to the formation of larger ice crystals at warmer nucleation temperatures. These larger ice crystals produce larger pores during sublimation, and the larger pores offer less resistance to the flow of water vapor during further sublimation.

Industrial Applicability The present method provides an improved method for controlling the temperature and / or time where subcooled materials, ie liquids or solutions, are subjected to nucleation and then frozen. Although this application is partially focused on freeze drying, a similar problem occurs for any material treatment step that involves a phase transition under nucleation. Examples of such processes include the crystallization of polymers and metals from melting, crystallization of supersaturated solution materials, protein crystallization, artificial snow production, food freezing, freeze concentration, fractional crystallization, cryogenic preservation, or condensation of vapors to the liquids. The most immediate advantage of controlling the nucleation temperature of a liquid or solution is the ability to control the number and size of solid domains produced by the phase transition. In freezing water, for example, the nucleation temperature directly controls the size and number of ice crystals formed. Speaking in general terms, ice crystals are few in number and large when the nucleation temperature is hotter. The ability to control the number and size of l? Solid domains produced by a phase transition can provide additional advantages. In a freeze drying process, for example, the number and size of the ice crystals strongly affect the drying properties of the lyophilized cake. Larger ice crystals produced by warmer nucleation temperatures leave larger pores during sublimation, and larger pores offer less resistance to the flow of water vapor during subsequent sublimation. Therefore, the process and methods described provide the means to increase the primary drying rates (i.e., sublimation) in freeze drying processes by increasing the nucleation temperature. Another possible advantage can be realized in the applications where the sensitive materials are conserved via freezing processes (that is, they are conserved in a cryogenic way). For example, a biological material that includes but is not limited to, mammalian tissue samples (e.g., bone marrow blood, tissue biopsy, stem cells and sperm, etc.), cell lines (e.g., mammalian, yeast, prokaryotic, fungal, etc.) and biological molecules (eg, proteins, DNA, RNA and subclasses thereof), frozen in an aqueous solution may experience several problems during the freezing process, which may impair the function or activity of the material. The formation of ice can physically damage the material or create severe changes in the interfacial junction, osmotic forces, solute concentrations, etc. Experienced by the material. Since nucleation controls the structure and kinetics of ice formation, it can significantly alter these problems. The current process and methods therefore provide unique means to diminish the problems associated with cryogenic preservation processes and to improve the recovery of the function or activity of conservative materials in a cryogenic manner. This represents an improvement in conventional nucleation control methods (e.g., seeding or contacting cold surfaces) used to initiate extracellular ice formation in two-stage cryogenic conservation algorithms designed for living cells. i: The present methods can also be applied to complex solutions or mixtures containing various components in cryogenic preservation and lyophilization applications. These formulations are often solutions with an aqueous, organic, organic, or mixed aqueous organic solvent containing a pharmaceutically active ingredient (eg, a synthetic chemical, protein, peptide, or vaccine) and optionally, one or more reducing components, including Volume agents that help prevent the physical loss of active ingredient during drying (eg, dextrose, glucose, glycine, lactose, maltose, mannitol, polyvinylpyrrolidone, sodium chloride, and sorbitol), the buffering agents or toxicity modifiers that they help maintain the pH or environmental toxicity appropriate for the active component (eg, acetic acid, benzoic acid, citric acid, hydrochloric acid, lactic acid, maleic acid, phosphoric acid, tartaric acid, and sodium salts of the aforementioned acids ); stabilizing agents that help to preserve the structure and function of the active component during the process or in its final liquid or dried form (for example, alanine, dimethylsulfoxide, glycerol, glycine, human serum albumin, polyethylene glycol, lysine, polysorbate, sorbitol, sucrose , and trehalose); agents that modify the vitreous transition behavior of the formulation (eg, polyethylene glycol and sugars), and antioxidants that protect the active component against degradation (eg, ascorbate, sodium bisulfite, sodium formaldehyde, sodium metabisulfite, sulfite sodium, sulfoxylate, and thioglycerol). Since nucleation is commonly a random process, a plurality of the same material subjected to identical process conditions can be subjected to nucleation at different temperatures. Therefore, the properties of the materials that depend on the nucleation behavior will probably be different despite the identical process conditions. The process and methods described provide the means to control the nucleation temperatures of a plurality of materials simultaneously and thereby offer a way to increase the uniformity of those product properties that depend on the nucleation behavior. In a common freeze drying process, for example, the same solution in separate containers may be subjected to stochastic nucleation at a wide temperature range, and therefore, the final lyophilized products may possess significant variability in the critical properties. as the residual humidity, activity and time of reconstitution. By controlling the nucleation temperature via the presently described process, the container-to-container uniformity of the product properties of a freeze drying process can be greatly improved. The ability to control the nucleation behavior of a material can also provide the substantial advantage in reducing the time needed to develop an industrial process that relates to a normally uncontrolled nucleation event. For example, it often takes many months to develop a successful freeze drying cycle that can be achieved in a reasonable amount of time, that produces desired product properties within the specified uniformity, and that retains sufficient activity of the pharmaceutically active ingredient (API). . By providing means to control nucleation and thereby potentially improve drying time, product uniformity, and API primary activity, the time needed to develop successful freeze drying protocols must be dramatically reduced.

In particular, the potential advantages of the controlled nucleation process will provide increased flexibility by specifying the composition of the formulation to be lyophilized. Since controlled nucleation can best conserve the API during the freezing stage, users should be able to minimize the addition of attenuation components (eg, stabilizing agents) to the formulation, or choose simpler combinations of formulation components to achieve the combined stability and process objectives. The synergistic advantages can occur in the event that controlled nucleation minimizes the use of stabilizing agents or other attenuation components that inherently lengthen the primary drying times (e.g., decreasing the glass transition temperatures of the aqueous solutions). The methods described are particularly well suited for large-scale production or manufacturing operations, since they can be conducted using the same equipment and process parameters that can be easily expanded or adapted to manufacture a wide range of products. The process is provided for the nucleation of materials using a process where all manipulations can be performed in a single chamber (for example, a dryer based on freezing) and where the process does not require the use of vacuum, the use of additives, vibration , electric freezing or the like to induce nucleation.

In contrast to the prior art, the present method adds nothing to the lyophilized product. It only requires that the materials, (for example, liquids in the containers), be initially maintained at a specified pressure under a gas environment and that the pressure be reduced rapidly to a lower pressure. Any applied gas will be removed from the containers during the lyophilization cycle. The containers or their contents do not come into contact with or touch anything other than gas. The simple manipulation of the ambient pressure and the gas environment is sufficient to achieve the objective. Based Relying on the change of ambient pressure to induce nucleation, the present method described herein uniformly and simultaneously affects all containers within a freeze-based dryer. The present embodiment is also less expensive and easier to execute and maintain than the prior art methods for altering nucleation in materials in lyophilization applications. The present method allows a significantly faster primary drying in lyophilization processes, thereby reducing the processing costs of lyophilized pharmaceutical products. The present method produces lyophilized products much more uniform than the methods of the prior art, thereby reducing product losses, and creating barriers for entry into the processors avoids meeting the more stringent specifications of uniformity. This method achieves these advantages without contamination of the lyophilized product. Most control processes should lead to an improved product and shorter process time. From the foregoing, it should be appreciated that the present invention therefore provides a lyophilization process and method. Various modifications, changes, and variations of the present methods will be apparent to the person skilled in the art. For example, the means for controlling the temperature may be cooling systems based on alternate cryogenic or conventional or advanced mechanical cooling processes. Also, the means for controlling the gas pressure and atmosphere in the chamber are specifically contemplated to include the known pressurization and depressurization techniques. It should be understood that any configuration, modification, change, and alternate variation must be included within the domain of this application and the spirit and scope of the claims.

Claims (20)

1. A method for cleaning a material, comprising the steps of: cooling the material in a chamber to a pre-set cooling rate; decrease in chamber pressure to induce nucleation of freezing in the material; additional cooling of the material subjected to nucleation at or below a final temperature to freeze the material; drying the frozen material to produce a dried product that has reduced moisture or solvent.

The lyophilization method according to claim 1, wherein the step of cooling the material further comprises cooling the material to a state with metastability.

The lyophilization method according to claim 1, wherein the additional cooling step of the material subjected to nucleation additionally comprises cooling a material subjected to nucleation at or below a final temperature ensuring that the freezing of the material is complete .

4. The lyophilization method according to claim 1, wherein the step of decreasing the pressure is initiated when the material reaches a desired nucleation temperature.

The lyophilization method according to claim 1, wherein the step of decreasing the pressure starts upon reaching the desired time after the start of the cooling step where the material is at or below a transition temperature of phase.

The lyophilization method according to claim 1, wherein the material additionally comprises a biopharmaceutical material, pharmaceutical material, chemical material, biological material, food product or combinations thereof.

7. The lyophilization method according to claim 1, wherein the step of decreasing the pressure occurs in a pressurized gas atmosphere within the chamber. . I

8. The lyophilization method according to claim 7, wherein the gas atmosphere within the chamber comprises argon, nitrogen, helium, air, water vapor, oxygen, carbon dioxide, neon, xenon, krypton, hydrogen, or mixtures thereof.

9. The lyophilization method according to claim 7, wherein the gas atmosphere is pressurized between ambient pressure and 25 psi above the ambient pressure.

10. The lyophilization method according to claim 1, wherein the material is initially cooled to a temperature ranging from the phase transition temperature to 20 ° C below the phase transition temperature before depressurization. .

The lyophilization method according to claim 1, wherein the pressure is decreased to approximately 7 psi or more.

The lyophilization method according to claim 1, wherein the pressure is decreased such that an absolute pressure ratio, PJPf, is about 1.2 or greater.

The lyophilization method according to claim 1, wherein the pressure is decreased to a pressure index decrease,? P /? T, greater than about 0.2 psi per second.

The lyophilization method according to claim 1, wherein the pressure is decreased by 40 seconds or less.

The method of claim 6, wherein the material additionally comprises one or more components comprising a live or attenuated virus; nucleic acid; monoclonal antibody; polyclonal antibody; protein; peptide; or polypeptide.

16. The method of claim 15, wherein the components of reconstituted material exhibit an improved function or activity greater than the function or activity associated with the reconstituted material components of a stochastic material subjected to nucleation.

17. The lyophilization method according to claim 1, wherein the material is held in a plurality of containers or containers and the dried product is obtained from the plurality of containers or relatively relatively exhibits a relatively uniform reconstitution time.

18. The lyophilization method according to claim 1, wherein the material is held in a plurality of containers or containers and the dried product obtained from the plurality of containers or containers exhibits residual moisture or relatively uniform solvent levels.

The method of claim 1, wherein the time required to dry the frozen material is less than the time required to dry the frozen material that is nucleated in a stochastic manner.

20. A lyophilization system comprising: a chamber having a controlled gas atmosphere and one or more shelves adapted to hold one or more containers or containers of a material; means for controlling the temperature of the shelves inside the chamber to control the temperature of the material; a condenser coupled to the chamber and adapted to remove any solvent or moisture from the chamber; and means for controlling the pressure of the chamber to rapidly depressurize the chamber to nucleate the material during freezing and maintain a low pressure during drying.