MX2008009384A - Method of inducing nucleation of a material - Google Patents

Abstract

Methods of inducing nucleation of a material is provided. The disclosed methods comprise the steps of bringing the material to a temperature near or below a phase transition temperature and decreasing the pressure to induce nucleation of the material. The disclosed methods are useful in freeze-drying processes, particularly pharmaceutical freeze-drying processes.

Description

METHOD TO INDUCE NUCLEATION OF A MATERIAL Field of the Invention The present invention relates to a nucleation process, and more particularly, to a method for inducing the nucleation of a phase transition in a material wherein the material is initially brought to a temperature near or below a phase transition temperature and subsequently depressurized to induce nucleation of the material. Background of the Invention Controlling the generally random process of nucleation in the freezing stage of a lyophilization or freeze drying process at both reduction transformation times necessary to complete the dehydration by freezing and to increase the uniformity of the vial product. A-vial in the finished product would be highly desirable in the art. In a normal pharmaceutical freeze dehydration process, multiple bottles containing a common aqueous solution are placed in racks that are cooled, generally at a controlled rate, at low temperatures. The aqueous solution in each flask is cooled below the thermodynamic freezing temperature of the solution and remains in a subcooled metastable liquid state until nucleation occurs.

The temperature range of nucleation through the bottles is randomly distributed between a temperature close to the thermodynamic freezing temperature and a certain value perceptibly (for example, up to about 30 ° C) lower than the thermodynamic freezing temperature. This distribution of nucleation temperatures causes the variation of the bottle-to-bottle in the structure of the ice crystal and ultimately the physical properties of the product. In addition, the drying step of the freeze dehydration process must be excessively long to accommodate the range of sizes and structures of ice crystal produced by the natural stochastic nucleation phenomenon. The additives have been used to increase the nucleation temperature of subcooled solutions. These additives can take many forms. It is well known that certain bacteria (e.g., Pseudomonas syringae) synthesize proteins that aid in the formation of nucleated ice in subcooled aqueous solutions. Bacteria or their isolated proteins can be added to solutions to increase the nucleation temperature. Various inorganic additives also demonstrate a nucleation effect; the most common additive is silver iodide, Agí. In general, any additive or contaminant has the potential to serve as a nucleating agent. Freeze-drying bottles prepared in environments containing high levels of particles are generally nucleated and frozen to a lesser degree of subcooling where the bottles are prepared in low particle environments. All of the nucleation agents described above are labeled "additives", because they change the composition of the medium in which they nucleate a phase transition. These additives are not normally acceptable for FDA regulated and approved freeze-dried pharmaceuticals. These additives also do not provide control over time and temperature when the bottles are nucleated and cooled. Preferably, the additives operate only to increase the average nucleation temperature of the bottles. The ice crystals can by themselves act. as nucleating agents for the formation of ice in subcooled aqueous solutions. In the "ice fog" method, a wet lyophilizer is filled with a cold gas to produce a vapor suspension of small ice particles. The ice particles are transported in the jars and nucleation begins when they come into contact with the fluid interface. The "ice fog" method does not control the nucleation of multiple flasks simultaneously at a controlled time and temperature. That is to say, the nucleation event does not occur concurrently or instantaneously within all the flasks during the introduction of the cold vapor into the lyophilizer. Ice crystals will take some time to work their direction in each of the bottles to initiate nucleation, and transport times are likely to be different for the bottles in different locations within the freeze dryer. For large scale industrial freeze dryers, the implementation of the "ice fog" method will require system design changes as internal convection devices may be required to assist a more even distribution of "ice fog" through the freeze dryer. When the freeze dryer racks are cooled continuously, the time difference between when the first bottle freezes and the last one freezes will create a temperature difference between the bottles, which will increase the non-uniformity of "bottle-to-bottle" in products dried by The pre-treatment of the jar by scratching, scraping or roughing has also been used to lower the degree of sub-cooling required for nucleation.As with the other methods of the prior art, the pre-treatment of the flask also does not impart any degree of control over time and temperature when the individual flasks are nucleated and frozen, but instead increases only the average nucleation temperature of all the flasks.The vibration has also been used for the nuclear a phase transition in a metastable material Sufficient vibration to induce nucleation occurs at frequencies above 10 kHz and can be produced using a equipment variety. Often vibrations in this frequency range are termed "ultrasonic", although frequencies in the range of 10 kHz to 20 kHz are normally within the audible range of humans. Ultrasonic vibration often produces cavitation, or the formation of small gas bubbles, in a subcooled solution. In the transient or inertial cavitation regime, gas bubbles grow and collapse rapidly, causing very localized pressure and temperature fluctuations. The ability of ultrasonic vibration to induce nucleation in a metastable material is often attributed to disturbances caused by transient cavitation. The other cavitation regime, called stable or non-inertial, is characterized by bubbles that exhibit stable volume or shape oscillations without collapsing. US Patent Application 20020031577 A1 discloses that ultrasonic vibration can induce nucleation even in the stable cavitation regime. but no explanation of the phenomenon is offered. Patent application GB 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 by dissolving a volatile fluid in the solution. An electro-freezing method has also been used in the past to induce nucleation in sub-cooled liquids. Electro-freezing is generally achieved by supplying relatively high electric fields (~ 1 V / nm) in a continuous or pulsed manner between the closely spaced electrodes submerged in a liquid or subcooled solution. The disadvantages associated with an electrocooling process in normal refrigeration applications include the relative complexity and cost of implement and maintenance, particularly for refrigeration applications using bottles or multiple containers. Also, electro-freezing can not be applied directly to solutions containing the ionic species (eg, NaCl). Recently, these studies that examine the concept of 'vacuum-induced surface freezing' (See for example, US Patent No. 6684,524). In such 'vacuum-induced surface freezing', the flasks containing an aqueous solution are loaded onto a temperature controlled shelf in a and carried out initially at about 10 degrees centigrade. The freeze dehydration chamber is then evacuated to near vacuum pressure (eg, 1 mbar) which causes the surface freezing of the aqueous solutions - at depths of a few millimeters. The subsequent release of vacuum and decrease of shelf temperature below the freezing point of the solution allows the growth of ice crystals of the pre-frozen surface layer with the rest of the solution. An important disadvantage for implementing this process of 'freezing the vacuum induced surface' in a normal lyophilization application is the high risk of violent boiling or gassing of the solution under indicated conditions. Improved control of the nucleation process can allow the freezing of all nonfrozen pharmaceutical solution bottles in a freeze dryer to occur within a narrow temperature and time range, thereby producing a freeze-dried product with greater uniformity of bottle-a -jar. Controls the minimum nucleation temperature can affect the crystalline structure of the ice formed inside the bottle and allows a process of drying by freezing mostly accelerated. 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 necessary to complete freeze drying and improve uniformity of the product from the bottle-to-bottle in the finished product. Therefore it will be desirable to provide a process that has some, or preferably all, of the above features. Brief Description of the Invention The present invention can be characterized as a method for inducing the nucleation of a phase transition in a material, the method comprising the steps of bringing the material to a temperature close to or below a phase transition temperature of the material and to decrease the pressure to induce the nucleation of the phase transition in the material. The invention can also be characterized as a method for controlling the freezing process of a solution comprising the steps of: cooling the solution at a rate that cooled prescribed; rapidly decrease the pressure to induce the nucleation of the solution; and continuing the cooling of the nucleated solution to a prescribed final temperature to freeze the solution. Decompression is initiated when the solution achieves a desired nucleation temperature or at a desired time after the initiation of the cooling step.

The invention can be further characterized as a solidification process comprising the steps of: cooling a material at a temperature near or below a phase transition temperature; rapidly decrease the next pressure of the material to induce the nucleation of the material; and continuing the cooling of the nucleated material to a prescribed final temperature to facilitate the solidification of the material. Finally, the invention can be characterized as a method for controlling the condensation process of a gas comprising the steps of: cooling the gas at a temperature near or below a phase transition temperature; rapidly decrease the pressure to induce nucleation within the gas, and continue to cool the nucleated gas to a prescribed final temperature to condense the gas. Brief Description of the Drawings The foregoing and other aspects, features and advantages of the present invention will be more apparent from the following, the more detailed description thereof, presented together with the following drawings, in which: Fig. 1 is a graph representing the temperature against a time diagram of a solution that undergoes a stochastic nucleation process and also shows the nucleation temperature range of the solution; Fig. 2 is a graph representing the temperature versus time diagram of a solution undergoing a cooling process equilibrated with depressurized nucleation according to the present methods; and Fig. 3 is a graph representing the temperature against the time diagram of a solution undergoing a dynamic cooling process with depressurized nucleation according to the present methods. 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 can be the formation of a crystal from a liquid. The crystallization process (ie, solid crystal formation of a solution) often associated with freezing of a solution starts with a nucleation event followed by crystal growth. In the crystallization process, nucleation is the stage where the selected molecules dispersed in the solution or another start of material to collect to create clusters in the nanometer scale as soon as it becomes stable under current operating conditions. These stable groupings constitute the nuclei. Clusters need to reach a critical size to become stable cores. Such a critical size is normally dictated by the operating conditions such as temperature, contaminants, degree of supersaturation, etc. and it can vary from one sample of the solution to another. It is during the event of nucleation that the atoms in the solution are arranged in a definite and periodic way that defines the crystal structure. Crystal growth is the subsequent growth of nuclei that succeed in realizing the critical size of the cluster. Depending on the conditions, nucleation or crystal growth may predominate over the other, and as a result, crystals with different sizes and shapes are obtained.Control of the size and shape of the crystal constitutes one of the main challenges in industrial manufacturing, such as for pharmaceutical products The present method is related to a process to control the time and / or temperature at which a nucleated phase transition occurs in a material.In freezing applications, the probability that a material is spontaneously nucleated and begin The change in phase is related to the degree of sub-cooling of the material and the absence or presence of contaminants, additives, structures, or disturbances that provide a site or surface for nucleation.The freezing or solidification stage is particularly important in the freeze drying process where existing techniques give rise to difference s of nucleation temperature through a multiplicity of bottles or containers. The differences in nucleation temperature tend to produce a non-uniform product and an excessively long drying time. The current methods, on the other hand, they provide a greater degree of process control in batch solidification processes (eg, freeze drying) and produce a product with a structure and for example more uniform. Unlike some of the prior art techniques for inducing nucleation, the present methods require minimal equipment and operational changes for implementation. In principle, the present methods can be applied to any process step of material involving a nucleated phase transition. Examples of such processes include the freezing of a liquid, crystallization of the ice from an aqueous solution, crystallization of polymers and fusion metals, crystallization of inorganic materials from supersaturated solutions, crystallization of proteins, production of artificial snow, vapor deposition of ice , food freezing, frozen concentration, 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 for present pharmaceutical lyophilization processes. For example, inside a large industrial freeze dryer can contain over 100,000 bottles containing a pharmaceutical product that need to be frozen and dried. The current practice in the industry is to cool the solution to a very high degree so that the solution in all the bottles or containers in the freeze dryer freeze. The content of each bottle or container, however, is randomly frozen during a temperature range below the freezing point, because the nucleation process is uncontrolled. Returning now to the figures, and in particular FIG. 1, a temperature against the time diagram of six flasks of an aqueous solution undergoing a conventional stochastic nucleation process showing the normal range of nucleation temperatures of the solution is plotted against. inside the jars (11, 12, 13, 14, 15 and 16). As noted herein, the content of the flask has a thermodynamic freezing temperature of about 0 ° C however the solution within each flask naturally nucleated during the wide temperature range of about -7 ° C to -20 ° C or more, as highlighted by area 18. Diagram 19 represents the shelf temperature inside the freeze drying chamber. Conversely, Fig. 2 and Fig. 3 represent the temperature against the time diagrams of a solution undergoing a freezing process with depressurized nucleation according to the present methods. In particular, Fig. 2 shows the temperature. against the time diagram of six flasks of an aqueous solution undergoing a balanced cooling process (See Example 2) with induced nucleation via depressurization of the chamber (21, 22, 23, 24, 25 and 26). -The contents of the bottle have a thermodynamic freezing temperature of about 0 ° C, however the solution inside each bottle nucleated at the same time during depressurization and within a very narrow temperature range (ie, -4 ° C a -5 ° C) as seen in. the area 28. Diagram 29 represents the shelf temperature inside the freeze drying chamber and represents a balanced freezing process, one where the temperature of the racks is carried out more or less constantly before depressurization.

Similarly, Fig. 3 shows the temperature against the time diagram of three flasks of an aqueous solution undergoing a dynamic cooling process (See Example 7) with induced nucleation via depressurization of the chamber (31, 32 and 33). Again, the contents of the bottle have a thermodynamic freezing temperature of about 0 ° C however the solution within each bottle nucleated at the same time during depressurization in a temperature range of about -7 ° C to -10 ° C as it is seen in area 38. Diagram 39 represents the shelf temperature inside the freeze drying chamber and generally represents a dynamic cooling process, one where the. Shelf temperature is actively decreased during or before depressurization. As illustrated in the Figures, the present methods provide improved control of the nucleation process by allowing the freezing of pharmaceutical solutions in a freeze dryer to occur within a narrower temperature range (e.g., about 0 ° C to -10 ° C). C) and / or concurrently, thereby producing "a lyophilized product with greater uniformity from bottle to bottle." As long as it is not demonstrated, it is foreseeable that the range of induced nucleation temperatures may even extend slightly over the transition temperature. of phase and can also extend from about 40 ° C of subcooling Another benefit associated with the present methods is that which can control the lower nucleation temperature and / or the exact nucleation time, one can affect the crystalline structure of the ice formed inside frozen jars or containers The crystalline structure of ice is a variable that affects the time it takes for the ice to sublimate. Thus, by controlling the crystalline structure of the ice, it is possible to accelerate the drying process by total freezing. In a broad sense, currently described methods for inducing nucleation of a phase transition within the material comprise the steps of: (i) cooling the material to a temperature near or below a phase transition temperature of the material; and (ii) quickly decrease. the pressure to induce the nucleation of the material. Each of these important stages will be discussed in more detail below. STEP 1 - COOLING OF MATERIAL 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, edibles, chemical materials, and may include products such as wound care products, cosmetics, veterinary products and in products related to diagnosis in live / in vitro and similar. When the material is a liquid, it may be desirable to dissolve 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 tissues, organs and multicellular structures. For certain biological and pharmaceutical applications, the material may be a solution- or mixture that includes: a living or mitigating virus; nucleic acids; monoclonal antibodies; polyclonal antibodies; biomolecules; non-peptide analogues; peptides, including polypeptides, peptide mimetics 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. The pharmaceutical or biopharmaceutical solutions contained in bottles or containers for freeze drying will be a good example of a material that will benefit 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 particulates that can form sites for nucleation. The uniform nucleation temperature is important to create a constant and uniform crystalline ice structure from bottle to bottle or container to container. The crystalline structure of the developed ice also greatly affects the time required for dryness. As it applies 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 atmosphere of the gas 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 dryer chamber are often dictated by the freeze drying process and are easily controlled via the use of a heat transfer fluid that cools or warms the shelves inside the chamber to drive the temperature of the bottles or containers and the material inside each bottle or container. According to the present methods, the material is cooled to a temperature near or below its phase transition temperature. In the case of an aqueous 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, ie to sub-cool. When applied to a freezing process of an aqueous based solution, the present method is effective when the degree of subcooling goes from near or below the phase transition temperature to about 40 ° C of subcooling, and more preferably between about 3 ° C of sub-cooling and 10 ° C of sub-cooling. In some of the examples described below, the present method for inducing nucleation works desirably the same 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 often referred to as being in a metastable state. A metastable state is an unstable and transient, but relatively durable, state of a chemical or biological system. A metastable material temporarily exists in a phase or state other than its phase or equilibrium state. In the absence of any change in the material or its environment, a metastable material will eventually transition from its unbalanced state to its equilibrium state.

Illustrative metastable materials include supersaturated solutions and sub-cooled liquids. A normal example of a metastable material will be liquid water at atmospheric pressure and a temperature of -10 ° C. With a normal freezing point of 0 ° C, liquid water should not exist thermodynamically at this temperature and pressure, but it can exist in the absence of an event or nucleation structure at the beginning of the ice crystallization process. 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 thermodynamically unbalanced metastable state. Missing a single nucleation event causes the start of the phase transition to re-equilibrate. As discussed above, the present methods for inducing the nucleation of a phase transition within a material or freezing of a material can be used with various cooling profiles, including, for example, a balanced cooling environment or a dynamic cooling environment (See Figures 2 and 3). STAGE 2 - FAST PRESSURE DECREASE When the material has achieved the desired temperature near or below the phase transition temperature, the chamber is depressurized quickly or rapidly. This depressurization drives the nucleation and phase transition of the solution inside the bottles or containers. In the preferred embodiment, the depressurization of the chamber is achieved by opening or partially opening a large control valve that separates the high pressure chamber from the environment or a low pressure chamber or environment. The high pressure is rapidly decreased by the mass flow of the gas atmosphere of the chamber. Depressurization needs to be fast enough to induce nucleation. The depressurization should be completed in several seconds or less, preferably 40 seconds or less, more preferably 20 seconds or less, and more preferably 10 seconds or less. In normal freeze drying applications, the pressure difference between the initial chamber pressure and the final chamber pressure, after depressurization, must be greater than about 7 psi, although small pressure drops may induce nucleation In some situations. Most commercial freeze dryers can easily accommodate the range of pressure drops needed to control nucleation. Many freeze dryers are designed with pressure ratings in excess of 25 psig to withstand conventional sterilization procedures using saturated steam at 121 ° C. Such evaluations of the equipment provide a broad window to induce nucleation followed by protocols that depressurize the initial pressures on ambient pressure or pressure in the 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 should remain below the supercritical pressure of any applied gas, and subject the material to extreme low pressures (i.e., approximately 10 mTorr or less) should be avoided during nucleation of the material. While not wishing to join any particular mechanism, a possible mechanism for explaining the controlled nucleation observed in the practice of the present method is that the gases in solution in the material leave the solution during the depressurization and form bubbles that nucleate the material. An initial high pressure increases the concentration of dissolved gas in the solution. The rapid decrease in pressure after cooling reduces the solubility of the gas, and the subsequent release of the gas from the subcooled solution activates the nucleation of the phase transition. Other Possible mechanism is that the temperature decreases the gas close to the material during depressurization causing a cold spot on the surface of the material that initiates nucleation. Another possible mechanism is that the depressurization causes the evaporation of some liquid in the material and the cooling resulting from the endothermic evaporation process can initiate the nucleation. Another possible mechanism is that the cold gas depressurized next to the material freezes some steam in equilibrium with the material before depressurization or is released from the material by evaporation during depressurization; the resulting solid particles re-incorporate the material and act as seeds or surfaces to initiate nucleation. One or more of these mechanisms may contribute to the initiation of the nucleation of freezing or solidification to different degrees depending on the nature of the material, its environment and phase transition to nucleate. The process can be carried out completely at a pressure higher than the ambient pressure or during a range of pressures that reach the ambient pressure. For example, the pressure of the initial chamber may be above ambient pressure and the pressure of the final chamber, after depressurization, may be above the ambient pressure but less than the initial chamber pressure; the pressure of the initial chamber may be above ambient pressure and the pressure of the final chamber, after depressurization, may be above ambient pressure or slightly below ambient pressure. The speed and magnitude of the pressure drop are also believed to be an important aspect of the present methods. Experiments have shown that nucleation will be induced where the pressure drop (?) Is greater than about 7 psi. Alternatively, the magnitude of the pressure drop can be expressed as a ratio of the absolute pressure, R = Pj / Pf, where Pj is the initial absolute pressure and Pf is the final absolute pressure. It is believed that nucleation can be induced on depressurization in the absolute pressure ratio, R, is greater than about 1.2 in many practical applications of the present methods. The speed of the pressure drop also plays an important role in the present methods. A method to characterize the velocity of the pressure drop is through the use of a parameter, A, where A = ?? / ??. Again, it is assumed that nucleation will be induced for A values greater than a prescribed value, such as approximately 0.2 psi / sec. Empirical data through experimentation should help one to check the preferred pressure drop and velocity drop. The following examples highlight various aspects and characteristics of the methods currently described for inducing nucleation in a material and should not be taken in a limiting sense. Rather, 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 performed in a VirTis 51-SRC pilot scale freeze dryer having four shelves with approximately 1.0 m2 of total shelf space and an internal condenser. This unit is conditioned to maintain 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 extending from the hole through the insulation of the rear wall to emerge from the back of the freeze dryer. Two full-port 1.5"air activated ball valves were attached to this pipeline via sanitary fittings.A ball valve allows the gas to flow into the freeze drying chamber and therefore provides positive pressures of up to 15 psig. Second ball valve allows gas to flow out of the freeze drying chamber and consequently reduces the chamber pressure to atmospheric conditions (0 psig) All refrigeration of freeze dryer and condenser shelves is achieved via circulation of the Dynalene MV heat transfer fluid cooled by liquid nitrogen using the Praxair NCool ™ -HX system All solutions were prepared in a clean class 100 room. The freeze dryer was placed with the door, shelves, and all controls accessible from the clean room while the other components (pumps, heaters, etc.) were located in a room environment not clean. All solutions were prepared with water grade CALR (Fisher Scientific, filtered through a membrane of 0.10 and m). The final solutions were filtered through a membrane of 0.22 μ? T? before filling the bottles or lyophilization containers. All gases were supplied via cylinders and filtered through 0.22 and m filters to remove the particulates. The glass containers (5 ml bottles and 60 ml bottles) were obtained pre-cleaned for Wheaton Science Products particulates. The pharmaceutically acceptable carriers were used appropriately. The above steps were taken to ensure the materials and methods meet the conventional pharmaceutical manufacturing standards for particulates, which act as nucleating agents. . As used herein, "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 , antifungal agents), isotonic agents, solution retarding agents (e.g., paraffin), sorbents (e.g., kaolin clay, bentonite clay), drug stabilizers (e.g., sodium lauryl sulfate), gels, binders (e.g. example, syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, carboxymethylcellulose, alginates), excipients (e.g., lactose, milk sugar, polyethylene glycol), disintegrating agent (e.g., agar-agar, starch, lactose, calcium phosphate) , calcium carbonate, alginic acid, sorbitol, glycine), wetting agents (eg, cetyl alcohol, glycerol monostearate), lubricants, accelerate absorption agents (for example, quaternary ammonium salts), edible oils (for example, almond oil, coconut oil, oily esters or propylene glycol), sweetening agents, flavoring agents, coloring agents, fillers, (for example, starch, lactose, sucrose, glucose, mannitol), tableting lubricants (eg, magnesium stearate, starch, glucose, lactose, rice blossom, chalk), carriers for inhalation (eg, hydrocarbon propellants), buffering agents, or similar materials and combinations thereof, as known to one skilled in the art. For the experimental conditions described herein and all the lyophilization formulations studied, stochastic nucleation was normally observed by. occur at container temperatures between about -8 ° C and -20 ° C and occasionally as hot as -5 ° C. The packages are generally maintained at hot temperatures of -8 ° C for long periods of time without nucleation. The initiation of nucleation and subsequent crystalline growth (i.e., freezing) was determined by measuring the temperature as the point at which the temperature of the container rapidly increases in response to the exothermic latent heat of the melt. The initiation of freezing can also be visually determined through a viewing glass in the door of the freeze dryer chamber. Example 1 - Control of Nucleation Temperature Four separate bottles are filled with 2.5 ml of 5% by weight of mannitol solution. The predicted thermodynamic freezing point of 5% by weight of mannitol solution is approximately -05 ° C. The four flasks were placed on a shelf of the freeze dryer in close proximity to each other. The temperatures of the four flasks were monitored using the surface mounted thermocouples. The freeze dryer was pressurized with argon at 14 psig. The shelf of the freeze dryer was cooled to obtain the bottle temperatures of between about -1.3 ° C and about -2.3 ° C (+/- 1 ° C measuring accuracy of the thermocouples). The freeze 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 flasks. All four flasks were nucleated and started freezing immediately after depressurization. The results are summarized in Table 1 below. As seen in Table 1, the nucleation temperatures controlled in this example (ie, Initial Bottle Temperatures) are quite close to the predicted thermodynamic freezing point of the solution. Thus the present method allows control of nucleation to occur in solutions that have a very low degree of subcooling or at nucleation temperatures near or only slightly cooler than their freezing points.

Bottle Atomos Solution Temperature Drop Result of Bottle # Pressure Initial Depressurization [° C] [psi] 1 2.5 ml of 5% by weight Argon -2.3 14 Nucleation of mannitol 2 2.5 ml of 5% by weight Argon | 1.3.1 Nucleation of mannitol 3 2.5 ml of 5% by weight Argon -2.1 14 Nucleation of mannitol 4 2.5 ml of 5% by weight Argon -1.7 14 Nucleation of mannitol Table 1. Controlling the Nucleation Temperature Example 2 - Controlling the nucleation temperature In this example, ninety-five flasks were filled with 2.5 ml of 5% by weight of mannitol solution. The thermodynamic freezing point of 5% by weight of mannitol solution is approximately -0.5 ° C. The ninety-five flasks were placed on a rack of the freeze dryer in close proximity to each other. The temperatures of six flasks placed in different locations on the freeze dryer shelf were continuously monitored using the surface mounted thermocouples. The freeze dryer was pressurized in an argon atmosphere at about 14 psig. The rack of the freeze dryer was then cooled to obtain the bottle temperatures near -5 ° C. The freeze dryer was then depressurized from about 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution in the flasks. All ninety-five flasks were visually observed nucleated and freezing was initiated immediately after depressurization. The thermocouple data for the six monitored flasks confirmed the visual observation. The results are summarized in Table 2.

As seen herein, the nucleation temperatures controlled in this example (i.e., Initial Flask Temperatures) are rather below the predicted thermodynamic freezing point of the solution. Thus the present method allows nucleation control to occur in solutions that have a moderate degree of subcooling. This example also demonstrates the scalability of the present method for an application of the multiple bottle.

Flask # Solution Atoms Temperature Drop Result of the Initial Depressurization Bottle [° C] Pressure [psi] 1 2.5 ml of 5% in Argon -4.2 14 Nucleation weight of mannitol 2 2.5 mi of 5% in Argon -4.4 14 Nucleation weight of mannitol 3 2.5 mi of 5% in Argon -4.6 14 Nucleation weight of mannitol 4 2.5 mi of 5% in Argon -4.4 14 Nucleation weight of mannitol 2.5 ml of 5% in Argon -4.6 14 Nucleation weight of mannitol 6 2.5 mi of 5% in Argon -5.1 14 Nucleation weight of mannitol Table 2. Controlling the Nucleation Temperature Example 3 - Control the magnitude of the depressurization In this example, the multiple bottles were filled with 2.5-ml of 5% by weight of mannitol solution. Again, the predicted thermodynamic freezing point of 5% by weight of mannitol solution is about -0.5 ° C. For each test series, the bottles were placed on a shelf of the freeze dryer in close proximity to each other. As with the examples described above, the jar temperatures were monitored using surface mounted thermocouples. The argon atmosphere in the freeze dryer was pressurized at different pressures and the freeze dryer shelf was cooled to obtain the bottle temperatures of about -5 ° C. In each test series, the freeze dryer was then depressurized rapidly (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 flasks. The results are summarized in Table 3. As seen in Table 3, controlled nucleation occurred where the pressure drop was about 7 psi or greater and the nucleation temperature (ie, temperature of the initial flask) was between approximately - 4.7 ° C and -5.8 ° C.

Bottle Atomos Solution Temperature Drop Result of Bottle # Pressure Initial Depressurization [° C] [psi] 1 2.5 ml of 5% by weight Argon -4.7 7 Mannitol nucleation 2 2.5 ml of 5 wt% Argon -5.1 7 Mannitol nucleation 3 2.5 ml of 5 wt% Argon -5.3 7 Mannitol nucleation 4 2.5 ml of 5 % by weight Argon -5.6 7 No Mannitol nucleus 5 2.5 ml 5% by weight Argon -5.6 7 Mannitol nucleation 6 2.5 ml 5% by weight Argon -5.8 7 Mannitol nucleation 7 2.5 ml 5% by weight Argon -5.4 6 Without Mannitol nucleation 8 2.5 ml of 5 wt.% Argon -5.7 6 No Mannitol nucleation 9 2.5 ml of 5 wt.% Argon -5.8 6 No Mannitol nucleation 10 2.5 ml of 5 wt.% Argon -5.1 5 Without Mannitol Nucleation Flask Solution Atoms Temperature Result Drop of # of Bottle Pressure Initial Depressurization [° C] [psi] 11 2.5 ml of 5% by weight Argon -5.4 Without Nuclear of mannitol 12 2.5 ml of 5% by weight Argon -5.5 Without Mannitol Nucleation 13 2. § 5% wt Argon -4.7 No Mannitol Nucleation 14 2.5 mL 5% by Weight Argon -5.1 No Nucleation of mannitol 15 2.5 ml of 5% by weight Argon -5.3 Without nucleation of mannitol Table 3. Effect of the Magnitude of Depressurization Example 4 - Controlling Depressurization Speeds For this example, the multiple bottles were filled with approximately 2.5 ml of 5% by weight of mannitol solution having a predicted thermodynamic freezing point of about -0.5 ° C. For each test series of the variant depressurization time, the bottles were placed on a shelf of the freeze dryer in close proximity to each other. As with the examples described above, the flask temperatures were monitored using surface mounted thermocouples. As the examples described above, the argon atmosphere in the freeze dryer was pressurized to approximately 14 psig and the rack was cooled to obtain the bottle temperatures of about -5 ° C. In each test series, the freeze 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 flasks. 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 at the rear of the freeze dryer. When the restriction valve is fully open, depressurization of approximately 14 psig to approximately 0 psig was achieved in approximately 2.5 seconds. By only partially closing the restriction valve, it is possible to variably increase the depressurization time of the chamber. Using the restriction ball valve, several test series were performed with the depressurized freeze dryer chamber at differentiated rates to confirm or determine the effect of the depressurization rate on nucleation. The results are summarized in Table 4. Bottle Solution Atoms Temperature Drop Time Result of Bottle # of [sec] Initial Depressurization [° C] Pressure [psi] 1 2.5 ml of 5% Argon -4.6 14 300 Without Nucleation by weight manitol Flask Solution Atomos Temperature Drop of Time Result of # of Bottle Pressure [sec] Initial Depressurization [° C] [psi] 2.5 mi of 5% Argon -5.4 14 300 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.8 14 300 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -4.6 14 200 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.4 14 200 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.4 14 200 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -4.6 14 100 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.2 14 100 Without Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.2 14 100 Without Nucleation by weight of mannitol 10 2.5 ml of 5% Argon -4.7 14 60 Without Nucleation by weight of mannitol F scratch Solution Atoms Temperature Drop of Time Result of # of Bottle Pressure [sec] Initial Depressurization [° C] [psi] 11 2.5 ml of 5% Argon -5.1 14 60 Without Nucleation by weight of mannitol 12 2.5 ml of 5% Argon -5.1 14 60 Without Nucleation by weight of mannitol 13 2.5 ml of 5% Argon -5. · 1 14 50 No Nucleation by weight of mannitol 14 2.5 ml of 5% Argon -5.3 14 50 Without Nucleation by weight of mannitol 15 2.5 ml of 5% Argon -4.9 14 50 Without Nucleation by weight of mannitol 16 2.5 ml of 5% Argon -5.4 14 42 Without Nucleation by weight of mannitol 17 2.5 ml of 5% Argon -5.5 14 42 Without Nucleation by weight of mannitol 18 2.5 ml of 5% Argon -5.0 14 42 Without Nucleation by weight of mannitol 19 2.5 mi of 5% Argon 14 32 Nucleation by weight of mannitol Flask Solution Atomos Temperature Drop of Time Result of # of Bottle Pressure [sec] Initial Depressurization [° C] [psi] 2.5 ml of 5% Argon -5.7 14 32 Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.6 14 32 Nucleation by weight of mannitol 2.5 ml of 5 % Argon -4.7 14 13 Nucleation by weight of mannitol 2.5 ml of 5% Argon -5.3 14 13 Nucleation in weight of mannitol 2.5 ml of 5% Argon -5.5 14 13 Nucleation in weight of mannitol Table 4. Effect of Depressurization Time As seen in Table 4, nucleation only occurred where the depressurization time was less than 42 seconds, the pressure drop was about 14 psi or greater, and the nucleation temperature (ie, temperature of the initial flask) was between approximately -4.6 ° C and approximately -5.8 ° C. These results indicate that depressurization needs to be achieved relatively quickly for the method to be effective.

Example 5 - Controlling the Gas Atmosphere Again, the multiple bottles were each filled with approximately 2.5 ml of 5% by weight of mannitol solution and placed on a shelf of the freeze dryer in close proximity to each other. As with the examples described above, the temperature of the test bottles was monitored using the surface mounted thermocouples. For the different test series, the gas atmosphere in the freeze dryer was varied to always maintain a positive pressure of approximately 14 psig. In this example, the shelf of the freeze dryer was cooled to obtain bottle temperatures of about -5 ° C to -7 ° C. In each test series, the freeze dryer was then rapidly depressurized from about 14 psig to atmospheric pressure in an effort to induce nucleation of the solution within the flasks. The results are summarized in Table 5. As seen in the present, controlled nucleation occurred in all gas atmospheres except for the helium gas atmosphere where the pressure drop was approximately 14 psi and the nucleation temperature (is say, initial bottle temperature) was between about -4.7 ° C and about -7.4 ° C. Although not shown in the Examples, it is believed that alternating conditions will likely allow controlled nucleation in a helium atmosphere.

Bottle Solution Atoms Temperature Drop Result of # of Bottle of Initial Depressurization [° C] Pressure [psi] 1 2.5 ml of 5% by weight Argon -4.9 14 Nucleation of mannitol 2 2.5 ml of 5% by weight Argon -5.2 14 Nucleation of mannitol 3 2.5 ml of 5 wt.% Nitrogen -4.7 14 Mannitol nucleation 4 2.5 ml of 5 wt.% Nitrogen -5.1 14 Mannitol nucleation 5 2.5 ml of 5 wt.% Xenon -4.8 14 Mannitol nucleation 6 2.5 ml of 5% by weight Xenon -5.0 14 Nucleation of mannitol 7 2.5 ml of 5% by weight Air -7.4 14 Nucleation of mannitol 8 2.5 ml of 5% by weight Air -7.2 14 Nucleation of mannitol 9 2.5 ml of 5% by weight Helium -5.8 14 Without Mannitol Nucleation 10 2.5 ml of 5% by weight Helium -5.5 14 Without Mannitol Nucleation Table 5. Gas Atmosphere Composition Effect Example 6 - Large Volume Solutions In this example, six lyophilization bottles (60 ml capacity) were filled with approximately 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 on a rack of the freeze dryer in close proximity to each other. The temperature of the six bottles placed in different locations on the shelf of the freeze dryer was monitored using the surface mounted thermocouples. The freeze dryer was pressurized in an argon atmosphere at about 14 psig. The shelf of the freeze dryer was then cooled to obtain bottle temperatures near -5 ° C. The freeze dryer was then depressurized from 14 psig to atmospheric pressure of approximately less than five seconds to induce nucleation of the solution within the bottles. The results are summarized in Table 6. In a separate experiment, a thick freeze drying tray of plastic (Gore LYOGUARD, capacity 1800 ml) was filled with approximately 1000 ml of 5% by weight of mannitol solution. The tray was obtained pre-cleaned to meet the particulate requirements of USP. The tray was placed on a rack of the freeze dryer, and the temperature of the tray was monitored by a thermocouple mounted on the outer surface of the tray near the center of one side. The shelf of the freeze dryer was then cooled to obtain a near-tray temperature of -7 ° C. The freeze dryer was then depressurized from 14 psig to about atmospheric pressure in less than five seconds to induce nucleation of the solution within the tray. The results are also summarized in Table 6. As the examples described above, all the packages were nucleated and the freezing started immediately after depressurization. Also as the examples described above, the nucleation temperatures (ie, Packaging Temperatures) in this example were very much controllable being a little close to about the thermodynamic freezing temperature of the solution. More importantly, this example illustrates that the present method allows nucleation control to occur in larger volume solutions and several pack formats. It should be noted that one will expect the effectiveness of the depressurization method to improve while the volume of the formulation increases, because the nucleation event is more likely to occur when more molecules are present in the aggregate and form critical nuclei.

Bottle Solution Atoms Dropout Temperature of Initial Flask Pressure Depressurization [° C] [psi] bottle 30 ml of 5% in Argon -5.3 14 Nucleation # 1 weight of mannitol bottle 30 ml of 5% in Argon -5.1 14 Nucleation # 2 weight of mannitol Packaging Solution Atoms Dropout Temperature of Initial Flask Pressure Depressurization [° C] [psi] bottle 30 ml of 5% in Argon -5.3 14 Nucleation # 1 weight of mannitol bottle 30 ml of 5% in Argon -5.1 14 Nucleation # 2 weight of mannitol bottle 30 ml of 5% in Argon -5.9 14 Nucleacion # 3 weight of mannitol bottle 30 ml of 5% in Argon -5.2 14 Nucleation # 4 weight of mannitol bottle 30 ml of 5% in Argon -5.9 14 Nucleation # 5 weight of mannitol bottle 30 ml of 5% in Argon -6.1 14 Nucleation # 6 weight of mannitol Table 6. Effect of Volume of Solution and Type of Container Example 7 - Dynamic Cooling versus Balanced Cooling The present methods for controlling nucleation can be used in several ways. Examples 1-6, described above, each demonstrate the aspect for controlling the nucleation temperature of a lyophilization solution which is essentially equilibrated at a temperature below its thermodynamic freezing point (i.e., temperature changing very slowly) . 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 is undergoing rapid changes in temperature).

In this example, bottles 1 to 6 represent the samples described above with reference to Example 2. In addition, three separate bottles (Bottles 7-9) were also filled with 2.5 ml of 5% by weight of mannitol solution. In a separate test series, the three additional bottles were placed on a shelf? of the freeze dryer in close proximity to each other. The shelf of the freeze dryer was rapidly cooled to a final shelf temperature of -45 ° C. When one of the bottles reached a temperature of about -5 ° C, as measured by the surface-mounted thermocouples, the freeze dryer rapidly depressurized from about 14 psig to 0 psig in an effort to induce nucleation. All three flasks were nucleated and started freezing immediately after depressurization. Bottle 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 summarized in Table 7 below.

Phrase Solution Mode Temperature Drop Result o # Nucleation Pressure Depressurization 2. 5 ml of 5% in Balanced Nucleation weight of mannitol 2.5 ml of 5% in Balanced -5.1 14 Nucleation weight of mannitol 2.5 ml of 5% in Balanced -5.9 14 Nucleation weight of mannitol 2.5 ml of 5% in Balanced -5.2 14 Nucleation mannitol weight 2.5 ml of 5% in Balanced -5.9 14 Nucleation weight of mannitol 2. 5 ml of 5% in Balanced -6.1 14 Nucleation weight of mannitol 2.5 ml of 5% in Dynamic -6.8 14 Nucleation weight of mannitol 2.5 ml of 5% in Dynamic -7.2 14 Nucleation weight of mannitol 2.5 ml of 5% in Dynamic - 9.9 14 Nucleation weight of mannitol Table 7. Test Results - Effect of Dynamic Cooling in Nucleation The effectiveness of the present methods for controlling nucleation in freeze-dried solutions in a given temperature range or lyophilization solutions was cooled dynamically, providing the end user with two potential application mobilities with different advantages and offsets. By allowing the lyophilized solutions to equilibrate, the nucleation temperature range is narrowed or minimized to the limits of the freeze dryer performance by itself. The equilibrium stage may require additional time to be performed in relation to conventional or dynamic freezing protocols where chamber and bottle temperatures drop to less than about -40 ° C in one stage. Nevertheless, using the equilibrium step should provide much improved nucleation uniformity throughout all the bottles or packages as well as the realization of the other advantages associated with the precise control of the nucleation temperature of the material. Alternatively, if compensation of the temperatures of the material or solution of lyophilization is undesirable, one can simply place the depressurization step at an appropriate time during the normal freezing or dynamic cooling protocol. The depressurization during a dynamic cooling down will produce a broader extension in the nucleation temperatures for the material within the lyophilization containers, but will add minimal time to the freezing protocol and still allow one to mitigate the problems of extreme sub-cooling. 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 for its acronym in English); polyvinyl pyrrolidone (PVP); dextran; glycine; sorbitol; sucrose; and trealosa. For example for each excipient, two bottles were filled with 2.5 ml of a solution containing 5% by weight of the excipient. The bottles were placed on a shelf of the freeze dryer in close proximity to each other. The freeze dryer was pressurized in an argon atmosphere at about 14 psig. The shelf of the freeze dryer was cooled to obtain the bottle temperatures near -3 ° C and then rapidly depressurized to induce nucleation. The results are summarized in Table 8.

Bottle Solution Atoms Temperature Drop Result of # of Bottle of Initial Depressurization [° C] Pressure [psi] 2.5 ml of 5% by weight Argon 14 Nucleation of mannitol 2.5 ml of 5% by weight Argon 14 Nucleation of mannitol 2.5 ml of 5% by weight Nitrogen 14 Nucleation of HES Flask Solution Atoms Temperature Drop Result of # of Bottle of Initial Depressurization [° C] Pressure [psi] 2.5 ml of 5% by weight Argon -3.7 14 Nucleation of HES 2.5 ml of 5% by weight Argon -3.8 14 Nucleation of PEG 2.5 ml of 5% by weight Argon -3.4 14 Nucleation of PEG 2.5 ml of 5% by weight Argon -3.5 14 Nucleation of PVP 2.5 ml of 5% by weight Argon -3.3 14 Nucleation of PVP 2.5 ml 5% by weight Argon -4.0 14 Dextran nucleus 10 2.5 ml of 5 wt.% Argon -3.1 14 Dextran nucleation 11 2.5 ml of 5 wt.% Argon -3.8 14 Nuclein of glycine 12 2.5 ml of 5 wt.% Argon -6.9 14 Glycine nucleation 13 2.5 ml of 5% by weight Argon -3.6 14 Nucleation of sorbitol 14 2.5 ml of 5 wt% Argon -3.4 14 Nucleation of sorbitol 15 2.5 ml of 5 wt.% Argon -3.3 14 Nucleation of sucrose 16 2.5 ml of 5 wt.% Argon -3.4 14 Nucleation of sucrose Flask Solution Atomos Temperature Drop Result of # of Initial Depressurization Bottle [° C] Pressure [psi] 17 2.5 ml of 5% by weight Argon -3.7 14 Nucleation of trehalose 18 2.5 ml of 5% by weight Argon. -3.1 14 Nucleation of trehalose Table 8. Effect of Different Lyophilization Excipients Example 9 - Nucleation Control of Protein Solutions The methods described herein can be used to control the nucleation temperature of subcooled protein solutions without negative or deleterious effects on protein solubility or enzyme activity. Two proteins, bovine serum albumin (BSA) and lactate dehydrogenase (LDH) were used in this example. BSA was dissolved in 5% by weight of mannitol at a concentration of 10 mg / ml. Three lyophilization flasks were filled with 2.5 ml of the BSA-mannitol solution and placed on a shelf of the freeze dryer in close proximity to each other. The freeze dryer was pressurized in an argon atmosphere at about 14 psig. The shelf of the freeze dryer was cooled to obtain flask temperatures near -5 ° C. The freeze dryer was rapidly depressurized to induce nucleation. All flasks of BSA solution were nucleated and freezing initiated immediately after depressurization. No precipitation of the protein was observed during thawing. The LDH proteins were obtained from two different suppliers and for clarity purposes were 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 lyophilization flasks were filled with 2.5 ml of the LDH-1 / mannitol solution and placed on a shelf of the freeze dryer in close proximity to each other. The freeze dryer was pressurized in an argon atmosphere at about 14 psig. The shelf of the freeze dryer was cooled by starting at room temperature to obtain bottle temperatures of about -4 ° C. The freeze dryer was then rapidly depressurized to induce nucleation. All the bottles were nucleated and started freezing immediately after depressurization. The bottles were kept in this state for approximately 15 minutes. The freeze dryer shelf was then cooled at an index rate of about 1 ° C / min to obtain flask temperatures close to -45 ° C and maintained for an additional 15 minutes to ensure completion of the freezing process. After the freezing step, the shelf of the freeze dryer was then heated at a rate of about 1 ° C / min to raise the bottle temperatures to about 5 ° C. No precipitation of the protein was observed during thawing. The content of the bottle was tested for enzymatic activity, and the results were compared for a control sample of the unfreened LDH-1 / mannitol solution. As part of Example 9, the depressurized nucleated samples of the LDH-1 / mannitol solution were compared to the stochastic nucleated samples. In the stochastic nucleated samples 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 lyophilization flasks were filled with 2.5 ml of the LDH-1 / mannitol solution and placed on a shelf of the freeze dryer in close proximity to each other. The rack of the freeze dryer was cooled starting from room temperature at a rate of approximately 1 ° C / min to obtain the flask temperatures close to -45 ° C and were maintained for 15 minutes to ensure the completion of the process. freezing. After the freezing step, the shelf of the freeze dryer was heated at a rate of about 1 ° C / min to raise the bottle temperatures to about 5 ° C. No precipitation of the protein was observed during thawing. The contents of the vial were tested for enzymatic activity, and the results were compared with the same control sample of the 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 nucleation temperature close to -3 ° C for LDH-2 rather close to -4 ° C for LDH-. As seen in Table 9, nucleation and controlled freezing process were achieved via depressurization clearly not decrease the enzymatic activity in relation to a comparable stochastic nucleation and freezing protocol. In fact, the controlled nucleation process was achieved via depressurization that appears to improve 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. Bottle Solution Atoms Temperature Drop Loss Result of # of Bottle of Initial Depressurization [° C] Pressure activity [psi] enzymatic [%] 1 2.5 mi of Argon -4.9 14 - Nucleation solution of BSA 2 2.5 ml of Argon -4.3 14 - Nucleation BSA solution Flask Solution Atoms Temperature Drop Loss Result of # of Bottle of Initial Depressurization [° C] Pressure activity [psi] enzymatic [%] 2.5 ml of Argon -5.3 14 Nucleation BSA solution 2.5 ml of Argon -3.8 14 9.0 Nucleation solution of LDH-1 2.5 ml of Argon -4.0 14 16.2 Nucleation solution of LDH-1 2.5 ml of Argon -3.7 14 18.4 Nucleation of LDH-1 solution 2.5 ml of Argon -4.0 14 23.4 Nucleation solution of LDH-1 2.5 mi de Argon ° -3.9 14 18.5 Nucleation solution of LDH-1 2.5 ml of Argon -4.0 14 21.2 Nucleation solution of LDH-1 10 2.5 ml of Air -10.4 35.7 Nucleation solution of LDH-1 11 2.5 ml of Air -16.5 35.4 Nucleation solution Bottle Solution Atoms Temperature Drop Loss Result of # of Bottle of Initial Depressurization [° C] Pressure activity [psi] enzymatic [%] 12 2.5 mi of Air -15.5 0 36.1. Nucleation solution of LDH-1 14 2.5 ml of Air -9.8 24.9 Nucleation solution of LDH-1 15 2.5 ml of Air -11.0 39.2 Nucleation solution of LDH-1 16 2.5 ml of Argon -3.1 14 29.9 Nucleation solution of LDH-2 17 2.5 m! of Argon -2.9 14 18.9 Nucleation of LDH-2 solution 18 2.5 ml of Argon -3.1 14 23.3 Nucleation of LDH-2 solution 19 2.5 ml of Argon -2.7 14 19.6 Nucleation of LDH-2 solution 20 2.5 ml of Argon -3.1 14 32.1 Nucleation solution of LDH-2 21 2.5 ml of Argon -2.6 14 35.2 Nucleation solution of Flask Solution Atomos Temperature Drop Loss Result of # of Bottle of Initial Depressurization [° C] Pressure activity [psi] enzymatic 22 2.5 mi of Air -5.0 38.3 Nucleation solution of LDH-2 23 2.5 mi of Air -5.5. 40.0 Nucleation solution of LDH-2 24 2.5 ml of Air -2.3 36.5 Nucleation solution of LDH-2 25 2.5 ml of Air -3.8 42.0 Nucleation solution of LDH-2 26 2.5 ml of Air -5.1 50.2 Nucleation solution of LDH-2 27 2.5 ml of Air -5.9 40.6 Nucleation solution of LDH-2 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 some 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, however the improvements in retention of the enzymatic activity obtained via the controlled nucleation for LDH-1 and LDH-2 are similar in 18.1% and 14.8%, respectively. This result suggests that improvements in retention of enzymatic activity can be partially attributed to the characteristics of the self-controlled nucleation process, it does not justify the prescribed hot nucleation temperatures obtained via depressurization. Example 10 - Reducing the primary drying time 5% by weight of mannitol solution was prepared by mixing approximately 10.01 grams of mannitol with approximately 190.07 grams of water. The flasks were filled with 2.5 ml of 5% by weight of mannitol solution. The bottles were weighed empty and with the solution to determine the mass of the water added to the bottles. The twenty flasks were placed in a rack on a shelf of the freeze dryer in close proximity to one another. The temperatures of six flasks were monitored using surface mounted thermocouples; all the supervised bottles were surrounded by other bottles to improve the uniformity of the bottle's behavior. The freeze dryer was pressurized to approximately 14 psig in a controlled gas atmosphere of the argon gas. The shelf of the freeze dryer was cooled from room temperature to about -6 ° C to obtain the bottle temperatures between about -1 ° C and -2 ° C. The freeze 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 flasks. All flasks visually observed or monitored via thermocouples nucleated and initiated freezing immediately after depressurization. The shelf temperature was then quickly lowered to approximately -45 ° C to complete the freezing process. Once all bottle temperatures were above -40 ° C or less, the freeze-dried chamber was evacuated and the primary drying process (ie, sublimation) was initiated. During this drying process, the shelf of the freeze dryer was heated to about -14 ° C via a one hour ramp and kept at this temperature for 16 hours. The condenser was maintained at approximately -60 ° C through the drying process. The primary drying was stopped by turning the vacuum pump and filling the chamber with argon at atmospheric pressure. The bottles were promptly removed from the freeze dryer and weighed to determine how much water was lost during the primary drying process. In a separate experiment as part of Example 10, other bottles were filled with 2.5 ml of the same 5% by weight of mannitol solution. The jars were weighed empty and with the solution to determine the mass of water added to the jars. The bottles were loaded into the freeze dryer in the same manner described above, and the temperatures of six bottles were again monitored using the surface mounted thermocouples. The shelf of the freeze dryer was rapidly cooled from room temperature to about -45 ° C to freeze the bottles. Nucleation occurred stochastically between about -15 ° C and about -18 ° C during the cooling step. Once all the flask temperatures were above -40 ° C or less, the bottles were dried in a manner identical to the method described above. Upon completion of the primary drying, samples were promptly removed from the freeze dryer and weighed to determine how much water was lost during the primary drying process.

Bottle Atomos Solution Temperature 'Drop Loss Result of Water bottle # Depressurization Initial [° C] Pressure 2. 5 ml Argon -1.3 89.9 Nucleation 5% by weight of mannitol 2.5 ml of Argon -1.9 14 85.2 Nucleation 5 wt.% Of mannitol 2.5 ml of Argon -1.3 14 87.1 Nucleation 5 wt.% Of mannitol Bottle Solution Atomos Temperature Drop of Loss Result of Bottle # Initial Depressurization Pressure [° C] [psi] water [%] 2.5 ml of 5% Argon -2.3 14 88.8 Nucleation by weight of mannitol 2.5 ml of 5% Argon -2.1 14 85.0 Nucleation by weight mannitol 2.5 ml of 5% Argon -1.1 14 80.7 Nucleation by weight of mannitol 2.5 ml of 5% Air -15.7 65.7 by weight of mannitol 2.5 ml of 5% Air -16.7 66.9 by weight of mannitol 2.5 ml of 5% Air -14.5 64.6 in weight of mannitol 10 2.5 ml of 5% Air -15.6 64.7 in weight of mannitol 11 2.5 ml of 5% Air -16.5 64.1 in weight of mannitol 12 2.5 ml of 5% Air -17.9 65.7 in weight of mannitol Table 10. Increase the Nucleation Temperature Improving the Primary Drying The results of the freeze drying process with Nuclea Controlled nucleation and stochastic nucleation are summarized in 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 seen in Table 10, the controlled nucleation process was achieved via depressurization allowing nucleation at very low degrees of subcooling, between about -1.1 ° C and -2.3 ° C in this example. The much warmer nucleation temperatures for the case of controlled nucleation compared to the case of stochastic nucleation produce an ice structure and a resulting lyophilized cake with dramatically improved drying properties. For the same amount of drying time, the nucleated flasks used described depressurization methods between approximately -1.1 ° C and -2.3 ° C lost an average of 86.1% of their water while the bottles stochastically nucleated between approximately -14.5 ° C and -17.9 ° C only lost an average of 65.3%. Therefore, stochastically nucleated flasks would require a much longer primary drying time to achieve the same degree of water loss as the nucleated flasks 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 go behind the 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 in such subcooled materials, that is liquids or solutions, nucleated and then frozen. Although this application focuses in part on freeze drying, a similar problem occurs for any stage of material processing involving a nucleated phase transition. Examples of such processes include the crystallization of polymers and fusion metals, crystallization of supersaturated solution materials, protein crystallization, production of artificial snow, food freezing, frozen concentration, fractional crystallization, cryo-preservation, or condensation. vapors to 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 the solid domains produced by the transition phase. In frozen water, for example, the nucleation temperature directly controls the size and number of ice crystals formed. Generally speaking, ice crystals are smaller in number and larger in size when the nucleation temperature is hotter. The ability to control the number and size of solid domains produced by a phase transition may provide additional advantages. In a freeze drying process, for example, the number and size of the ice crystals strongly influence the drying properties of the lyophilized cake. The larger ice crystals produced by warmer nucleation temperatures go behind the larger pores during sublimation, and the larger pores offer less resistance to the flow of water vapor during subsequent sublimation. Therefore, the present methods provide a means to increase the primary drying rates (i.e., sublimation) in the freeze drying processes by increasing the nucleation temperature. Another possible advantage can be realized in applications where sensitive materials are preserved via freezing processes (ie, cryopreserved). For example, a biological material that includes but is not limited to, samples of mammalian tissue (e.g., cord blood, tissue biopsy, egg and sperm cells, etc.), cell lines (e.g., mammalian, yeast, prokaryotic, fungicides, etc.) and biological molecules (for example, proteins, DNA, RNA and subclasses thereof) frozen in an aqueous solution can undergo several stresses during the freezing process that can deteriorate the function or activity of the body. The formation of ice can physically disrupt the material or create severe changes in the interfacial junction, osmotic forces, dissolved concentrations, etc. experienced by the material.Since nucleation controls the structure and kinetics of icing, it can influence These tensions are significant, and the currently described methods therefore provide a single means to mitigate the stresses associated with processes of cryopreservation and improve the recovery of the function or activity of the cryopreserved materials. The present methods also represent improvements over conventional nucleation control methods (e.g., seeding or contacting cold surfaces) used to initiate extracellular ice formation in two-stage cryopreservation algorithms designed for living cells. The present methods can also be applied to solutions or complex mixtures containing various constituents in cryopreservation and lyophilization applications. These formulations are often solutions with an aqueous, organic, or mixed aqueous-organic solvent containing a pharmaceutically active ingredient (e.g., a chemical, protein, peptide, or synthetic vaccine) and optionally, one or more mitigating constituents, including bulking agents that help prevent physical loss of the active ingredient during drying (eg, dextrose, glucose, glycine, lactose, maltose, mannitol, polyvinyl pyrrolidone, sodium chloride, and sorbitol); damping agents or toxicity modifiers that help maintain the appropriate environmental pH or toxicity for active constituent (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 constituent 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 trealosa); agents that modify the transition behavior of the formulation glass (eg, polyethylene glycol and sugars), and antioxidant that protect the active constituent from degradation (eg, ascorbate, sodium bisulfite, sodium formaldehyde, sodium metabisulfite, sodium sulfite, sulfoxylate and thioglycerol). Since nucleation is normally a random process, a plurality of the same material subjected to identical processing conditions can be nucleated at different temperatures. As a result, the properties of these materials that depend on nucleation behavior will probably differ in spite of the identical processing conditions. The described methods provide a 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 normal freeze drying process, for example, the same solution in separate bottles can be stochastically nucleated over a wide range of temperatures, and as a result, the final freeze-dried products may possess significant property variability-it is critical as the residual humidity, activity and time of reconstitution. By controlling the nucleation temperature via the process currently described, the jar-to-jar uniformity of the properties of the freeze-dried product can be processed to be dramatically 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 is based on a normally uncontrolled nucleation event. For example, often taking many months to develop a successful freeze drying cycle that can be achieved in a reasonable amount of time, yields the desired product properties within the specified uniformity, and preserves the sufficient activity of the pharmaceutically active ingredient. (API). By providing the means to control nucleation and thereby potentially improving drying time, product uniformity, and API activity, the present methods should dramatically reduce the time needed to develop successful freeze drying protocols. In particular, the potential advantages of the present nucleation process provide increased flexibility in specifying the composition of the formulation to be lyophilized. Since controlled nucleation can better preserve the API during the freezing stage, users should be able to minimize the addition of mitigation constituents (eg, stabilizing agents) to the formulation or choose simpler combinations of formulation constituents to achieve stability combined and processing goals. Synergistic advantages can occur in cases where controlled nucleation minimizes the use of stabilizing agents or other mitigating constituents that inherently lengthen the primary drying times (e.g., lowering glass transition temperatures of 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 easily adjust or adapt for manufacturing a wide range of products. The process provided for the nucleation of materials uses a process where all the manipulations can be carried out in a single chamber (for example, a freeze dryer) and where the process does not require the use of vacuum, use of additives, vibration, electro-freezing or the like to induce nucleation. In contrast to the prior art, the present method does not add anything to the lyophilized product. It only requires that the materials, (for example, liquids in the flasks), 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 bottles during the freeze drying cycle. The jars or their contents will not be contacted or touched with anything except gas. The simple manipulation of the environmental pressure and gas environment is sufficient in its own goal to achieve. Relying solely on the change of environmental pressure to induce nucleation, the present method described herein uniformly and simultaneously affects all flasks inside a freeze dryer. The present embodiment is also less expensive and easier to implement and maintain than the prior art methods for influencing nucleation in materials in freeze drying applications. The present method allows a significantly faster primary drying in lyophilization processes, thereby reducing processing costs for freeze dried pharmaceuticals. The present method produces much more uniform lyophilized products than the methods of the previous technique, thereby reducing product loss and creating entry barriers for processors unable to meet narrow uniformity specifications. This method achieves these advantages without contaminating the lyophilized product. The greater process control should lead to an improved product and shortened process times. From the foregoing, it should be appreciated that the present invention thus provides a method for inducing nucleation in a material and / or method for freezing the material. Various modifications, changes, and variations of the present methods will be apparent to one skilled in the art and it should be understood that such modifications, changes, and variations should be included within the scope of this application and spirit and scope of the claims.

Claims (15)

1. A method for inducing the nucleation of a phase transition in a material comprising the steps of: bringing the material to a temperature near or below a phase transition temperature; and decreasing the close pressure of the material to induce nucleation of the phase transition in the material.

The method according to claim 1, further comprising the step of continuing the cooling of the nucleated material after depressurization or below a final temperature ensuring the complete phase transition of the material.

3. The method according to claim 1, wherein the material is selected from the group consisting of gases, liquids, solutions, suspensions, mixtures, or constituents within a suspension, solution or mixture.

4. The method according to claim 1, wherein the material is a solution and the phase transition temperature is the thermodynamic freezing point of the solution.

5. The method according to claim 1, wherein the material is a solution with one or more dissolved substances and the phase transition temperature is a saturation temperature at which a dissolved substance will precipitate or crystallize out of the solution.

6. The method according to claim 1, further comprising the step of pressurizing the surrounding atmosphere of the material.

The method according to claim 1, wherein the material is cooled to a temperature ranging from the phase transition temperature to about 5 ° C below the phase transition temperature before depressurizing.

The method according to claim 1, wherein the pressure is decreased by an amount greater than about 7 psi 9.

The method according to claim 1, wherein the pressure is decreased such that an absolute pressure ratio, P¡ / Pf, is approximately 1.2 or greater.

The method according to claim 1, wherein the pressure is decreased by one drop of pressure velocity,? / ??, greater than about 0.2 psi per second.

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

12. The method according to claim 6, wherein the material contains a constituent comprising living or mitigating viruses; nucleic acid; monoclonal or polyclonal antibodies; biomolecules; non-peptide analogs, peptides; and proteins.

13. A method for controlling the freezing process of a material comprising the steps of: cooling the material at a prescribed cooling rate; quickly decrease the pressure to nuclear the material; and continuing the cooling of the nucleated material to a prescribed final temperature to completely freeze the material. The method according to claim 1 or 13, wherein the depressurization is initiated when the material achieves a desired nucleation temperature. The method according to claim 1 or 13, wherein the depressurization is initiated at a desired time after the initiation of the cooling step and when the temperature of the material is below the phase transition temperature.