Patent Application
20140193456
This invention relates to methods of preserving, shredding, sampling, encapsulating, extracting and purifying bioactive substances from various biological origins. More specifically, it aligns to methods of preserving, extracting, manipulating and separating bioactive substances from various biological sources using deep freezing, fluid extraction and/or hydrocarbon solvent extract. The invention further relates to methods for manipulation and separation of bioactive substances contained in biological materials and extracts using instant freezing by direct contact to the biological material. The presented invention also relates to formulations, pharmaceutical preparations and dietary supplements that may be prepared with the original biological material and/or extracted bioactive substances and a use of such pharmaceutical preparations and dietary supplements to treat various human ailments and for cosmetic purposes. Most importantly, the invented process of biological material preservation and conservation is intended for commercial-scale manipulations and market value product preparations with no particular implementation for laboratory- and investigation practices and purposes alone.
Throughout history, humans have ingested and otherwise consumed a wide variety of plants, herbs, and extracts of such plants and herbs to help alleviate aches and pains, improve immunity to infection, treat various illnesses, or even to induce relaxation or stress reduction.
Although many types of herbs and fungi have been identified and used through the human civilization history for medicinal and cosmetic purposes, no simple and efficient method is available for both efficient preservation of all bioactive components within the biological material and for extraction of bio-components from the roots and other parts of the material, and separation of each individually extracted component. Traditionally, herbs are air-dried at an ambient temperature of choice and different extraction methods have been employed including boiling and steam distillation to obtain components suspected to have biologically important properties. In many cases plant slurry has been steam distilled and the first portion of distillate has been collected, filtered and lyophilized. Alternately, a liquid-solid extraction at room temperature has been reported wherein the above slurry has been intimately mixed in a blender and the mixture then has been filtered and lyophilized. In many recent cases, rather than lyophilization, the filtrate has been subjected to successive extractions with organic solvents such as chloroform and else. These purification operations removed impurities from the aqueous layer. The extraction yield for these methods varied depending on the solvent and methodology used. Importantly, most of the approaches employed for manipulating the biological material are known to have harmful effect on most of the components of the material leading to total loss or particular degradation of their biological activities resulting in quite limited pharmaceutical gains.
Much more practical approach is proposed in the methodology described in this invention for nearly-perfect preservation of the entire biological content obtained and for preparation of commercially viable products—preserving for future utilization all the biologically active components as good as they exist in vivo (alive) where particular separation of certain bio-chemicals is not especially desired. We employ rapid and instant freezing of the biological objects under conditions leading to residual water “glassification” (or vitrification—instant freezing without crystallization and volume increase) followed by water evaporation, other manipulations and encapsulation of the biological material as a commercial product without any treatments harmful for degrading the bio-chemical content nor cellular structure leading to supplements as natural as the alive original obtained initially.
The method can be traced back to prehistoric times and has been used by the Aztecs and Eskimo for preserving foodstuffs. The Andean civilizations had preserved potatoes using a freeze-drying process. They called this foodstuff Chuño. Toward the end of the 1880s the process has been used on animal material on a laboratory scale and the basic principles—well understood at that time. Practically, the method remained a laboratory technique until the 1930s when there was the need to process heat-labile antibiotics and blood products. At this time, refrigeration and vacuum technologies had advanced sufficiently to enable production freeze-dryers to be developed, and since then the process has been used industrially in both the food and pharmaceutical industries. Freeze-drying was actively developed during WWII. Serum being sent to Europe for medical treatment of the wounded required refrigeration, but because of the lack of simultaneous refrigeration and transport, many serum supplies were spoiling before reaching their intended recipients. Therefore, the freeze-drying process was developed as a commercial technique that enabled serum to be rendered chemically stable and viable without having to be refrigerated. Shortly thereafter, the freeze-dry process was applied to penicillin and bone, and lyophilization became recognized as an important technique for preservation of biologicals. Since that time, freeze-drying has been used as a preservation or processing technique for a wide variety of products. These applications include the following but are not limited to the processing of food, pharmaceuticals and diagnostic kits, and animal and human material; the restoration of water damaged documents; the preparation of river-bottom sludge for hydrocarbon analysis; the manufacturing of ceramics used in the semiconductor industry; the production of synthetic skin; the restoration of historic/reclaimed boat hulls.
The conversion of an aqueous system to an amorphous, noncrystalline solid solely by increase in viscosity is called “Vitrification”. The vitrification is defined by the viscosity of the solution reaching a sufficiently high value (˜1013 poises) to behave like a solid but without crystallization. In conventional cryopreservation, the concentration of solute in the remaining liquid increases during progressive freezing, and a temperature (Tg) is eventually reached with many systems where the residual liquid vitrifies in the presence of ice (
Freeze-drying or lyophilization describe precisely the same process. The term “lyophilization,” which means “to make solvent loving,” is less descriptive than the alternative definition “freeze-drying.” Several alternative definitions have been used to describe freeze-drying. Operationally we could define freeze-drying as a controllable method of dehydrating labile products by vacuum desiccation.
Earlier accounts of freeze-drying suggested that ice was only removed by sublimation and defined this step as primary drying. The cycle was then described as being extended by secondary drying or desorption. Although these definitions are applicable to ideal systems, they incompletely define the process for typical systems that form an amorphous matrix or glass when cooled.
Technically, freeze-drying may be defined as:
Freeze-drying has a number of advantages over alternative stabilizing methods. These may be summarized by the following criteria:
Freeze-dried products may be classified as:
However, freeze-drying is less appropriate for:
Freeze-drying is a complex process during which drying may proceed more or less rapidly within individual samples throughout the process batch, such that parts of the product will be frozen, whereas other areas are drying or will have dried depending on the nature of the sample and stage in the cycle. The precise freezing and drying behavior will be determined by the interrelationship between the sample and shelf temperature, system pressure, extent of product dryness, and variations in drying conditions throughout the cycle.
Often regarded as a gentle method of drying materials, freeze-drying is in reality a potentially damaging process where the individual process stages should be regarded as a series of interrelated stresses, each of which can damage sensitive bio-products. Damage sustained during one step in the process may be exacerbated at succeeding stages in the process chain and even apparently trivial changes in the process, such as a change in container, may be sufficient to transform a successful process to one which is unacceptable.
Freeze-drying will not reverse damage incurred prior to formulation and care must be exercised when selecting an appropriate cell type or technique used to culture or purify the cell or its extracts prior to freeze-drying. The essence of the formulation exercise should be to minimize freeze-drying damage, loss of viability, or activity. To ensure minimal losses of activity, the sample may require dilution in a medium containing protective additives, specifically selected for the product or application. Although frequently described as “protectants” these additives may not be effective at all stages of the process but may protect only during particular steps in the drying cycle. At other stages, the additive may fail to protect the active component and indeed may be incompatible with the process. It is also important to appreciate that individual stages in the process can result in damage, which initially remains undetected, becoming evident only when the dried sample is rehydrated. Particular attention must be applied to the selection and blending of the additive mixes in the formulation and the importance of formulation will be discussed at greater length later.
Freeze-dried products should be:
Products should be formulated to ensure batch product uniformity, whereas there may be particular requirements relating to product use. In this context, vaccines freeze-dried for oral or aerosol delivery may require the inclusion of excipients that minimize damage when the dried product is exposed to moist air.
A wide range of containers can be used to freeze-dry vaccines, microorganisms, and others, including all glass ampoules, rubber stoppered vials, double chambered vials, and prefilled syringes that hold both dried vaccine and diluent, bifurcated needles, and so on. Alternatively, vaccines can be dried in bulk in stainless steel or plastic trays and the resultant powder tableted, capsulated, sachet filled, or dispensed into aerosol devices for lung or nasal delivery.
Freeze-drying is a complex process during which drying may proceed more or less rapidly within individual samples throughout the process batch, such that parts of the product will be frozen, whereas other areas are drying or will have dried depending on the nature of the sample and stage in the cycle. The precise freezing and drying behavior will be determined by the interrelationship between the sample and shelf temperature, system pressure, extent of product dryness, and variations in drying conditions throughout the cycle.
Often regarded as a gentle method of drying materials, freeze-drying is in reality a potentially damaging process where the individual process stages should be regarded as a series of interrelated stresses, each of which can damage sensitive bio-products. Damage sustained during one-step in the process may be exacerbated at succeeding stages in the process chain and even apparently, trivial changes in the process, such as a change in container, may be sufficient to transform a successful process to one that is unacceptable.
Freeze-drying will not reverse damage incurred prior to formulation and care must be exercised when selecting an appropriate cell type or technique used to culture or purify the cell or its extracts prior to freeze-drying. The essence of the formulation exercise should be to minimize freeze-drying damage, loss of viability, or activity. To ensure minimal losses of activity, the sample may require dilution in a medium containing protective additives, specifically selected for the product or application. Although frequently described as “protectants” these additives may not be effective at all stages of the process but may protect only during particular steps in the drying cycle. At other stages, the additive may fail to protect the active component and indeed may be incompatible with the process. It is also important to appreciate that individual stages in the process can result in damage, which initially remains undetected, becoming evident only when the dried sample is rehydrated. Particular attention must be applied to the selection and blending of the additive mixes in the formulation, and the importance of formulation will be discussed at greater length later.
Freezing refers to the abrupt phase change when water freezes as ice. Except for very complex biomolecules or cold sensitive cells, cooling in the absence of freezing (chilling) is generally not damaging to biomaterials.
When solutions or suspensions are frozen, they may cool appreciably below their measured freezing point prior to ice formation, a phenomenon defined as super-cooling (undercooling or sub-cooling). The extent of super-cooling depends on cooling rate, sample composition and cleanliness, dispensed fill volume, container type, method of sample cooling, and so on. Even when a simple solution is repeatedly cooled or warmed, the onset and extent of super-cooling will vary from cycle to cycle. In the super-cooled state, while the composition of the solution remains unchanged, the cooled liquid is thermodynamically unstable and sensitive to ice formation. As the solution is cooled to lower temperatures, the probability of ice crystallization will correspondingly increase. For optimized freeze-drying, the intention should be to induce super-cooling in the suspension to encourage uniform cooling and freezing throughout the sample contents.
Sample freezing may be defined as the abrupt conversion of the suspension into a mixture of ice and solute concentrate. Freezing is a two-step process during which water initially nucleates, followed by the growth of the ice crystals that pervade the solute phase resulting in a mixture of ice and solute concentrate. Under typical processing conditions, ice nucleates heterogeneously around microscopic particles within the suspension and is encouraged by reducing temperature and agitating the super-cooled suspension to increase the probability of contact between nucleating foci and water clusters. Nucleation depends on the number and physical nature of particulate impurities within the suspension or solution. Ice is a particularly effective nucleation focus and cryobiologists may deliberately seed samples with ice to induce nucleation. Other effective ice nucleators include glass shards and specifically formulated nucleation promoters. Whereas nucleation aids can be added to experimental systems, deliberate attempts to add ice inducers to pharmaceutical materials would be at variance with Good Pharmaceutical Manufacturing Practice.
In contrast to nucleation, ice growth (proliferation) is encouraged by raising the temperature, thereby decreasing the suspension viscosity. Ice nucleation and proliferation are inhibited at temperatures below the glass transition temperature (Tg), whereas above the melting temperature (Tm) the suspension or solution will melt. The consequences and measurements of these parameters are important elements in the formulation exercise.
To facilitate the sublimation of water vapor from the drying mass, the ice crystals should be large, wide, and contiguous, extending from the product base toward its surface, thereby providing an optimized structure for vapor migration. Crystal structures commonly observed during freeze-drying when solutions are frozen in trays or vials include dendritic structuring, where the ice crystal branches continuously from the nucleating focus and the spherulite form, and where sub-branching is discouraged because the solution viscosity is high, or fast rates of cooling are used.
Cooling or freezing rates are defined as slow (suboptimal), rapid (super-optimal), or optimal as assessed by criteria such as post-freezing cell survival or biopolymer activity, and is ambiguous unless conditions are more precisely defined. Cooling rates may be defined in terms of: 1) The rate at which the shelf temperature is cooled per unit time; 2) The rate at which the solution cools per unit time; 3) The depth of liquid within the vial (in mm) which cools per unit time.
The ice and solute crystal structure resulting from sample freeze has a major impact on subsequent freeze-drying behavior, encouraging the sample to dry efficiently or with defects such as melt or collapse depending on freezing rate used. The preferred ice structure comprising large contiguous ice crystals is induced by freezing the sample at a slow rate of 0.2-1.0° C./min. The slow cooling will also induce the crystallization of solutes reluctant to crystallize when faster rates of cooling are used. However, a slow rate of cooling may exacerbate the development of a surface skin, which inhibits sublimation efficiency. Slow cooling can also inactivate a bio-product by prolonging sample exposure to the solute concentrate biomolecules. However, a fast rate of cooling can result in the formation of numerous, small, randomly orientated ice crystals embedded in an amorphous solute matrix, which may be difficult to freeze-dry. Complicating the choice of freezing regimes is the fact that the optimal cooling rate cannot be sustained where the sample fill depth exceeds 10 mm. In short, defining cooling rates often requires a compromise in sample requirements.
The freeze-drying process may be divided into a number of discrete steps that may be summarized as:
For convenience, the freeze-drying process may be divided into a number of discrete steps that may be summarized as:
The preservation of biological material in a stable state is a fundamental requirement in biological/medical science, agriculture, and biotechnology. It has enabled standardization of experimental work over time, has secured lifesaving banks of cells and tissue ready for transplantation and transfusion at the time of need and has assured the survival of critical germplasm in support of programs for the conservation of species. Cryopreservation and freeze-drying are widely accepted as the preferred techniques for achieving long-term storage, and have been applied to an increasingly diverse range of biological materials. Although the basis for many methodologies is common, many laboratories lack expertise in applying correct preservation and storage procedures and many apply outdated or inappropriate protocols for storing samples or cultures. Importantly, the common lyophilization practices are employing techniques and conditions allowing for water-crystal formation within the biological material for stimulating the evaporation process with reducing the cost, and preventing a direct physical/chemical contact between the biological material and the cooling surfaces for preventing contamination—exactly in opposite to the concept utilized in this invention.
Samples may be frozen in a variety of ways depending on operational requirements:
There are four stages in the complete commercial drying process: pretreatment, freezing, primary drying, and secondary drying.
Pretreatment includes any method of treating the product prior to freezing. This may include concentrating the product, formulation revision (i.e., addition of components to increase stability and/or improve processing), decreasing a high vapor pressure solvent or increasing the surface area. In many instances, the decision to pretreat a product is based on theoretical knowledge of freeze-drying and its requirements, or is demanded by cycle time or product quality considerations. Methods of pretreatment include Freeze Concentration, Solution-Phase Concentration, Formulation to Preserve Product Appearance, Formulation to Stabilize Reactive Products, Formulation to Increase the Surface Area, and Decreasing High Vapor Pressure Solvents.[1]
In a lab, freezing is often achieved by placing the material in a freeze-drying flask and rotating the flask in a bath, called a shell freezer, which is cooled by mechanical refrigeration, dry ice and methanol, or liquid nitrogen. On a larger scale, freezing is usually done using a freeze-drying machine. In this step, it is important to cool the material below its triple point, the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps. Larger crystals are easier to freeze-dry. To produce larger crystals, the product should be frozen slowly or can be cycled up and down in temperature. This cycling process is called annealing. However, in the case of food, or objects with formerly-living cells, large ice crystals will break the cell walls (a problem discovered, and addressed, by Clarence Birdseye), resulting in the destruction of more cells, which can result in increasingly poor texture and nutritive content. In this case, the freezing is done rapidly, in order to lower the material to below its eutectic point quickly, thus avoiding the formation of ice crystals. Usually, the freezing temperatures are between −50° C. and −80° C. The freezing phase is the most critical in the whole freeze-drying process, because the product can be spoiled if badly done.
Regarded as the first step in the process, the formulated product must be frozen before evacuating the chamber to induce sublimation. Freezing will:
Ideally freezing should minimize solute concentration effects and result in a sample where all the components are spatially arranged as in the dispensed solution. However, it may not be possible to achieve this ideal when typical solutions or suspensions are frozen. When addressing the freezing of aqueous solutions or suspensions, there is the need to consider both the solvent (water in the case of aqueous solutions) and solute(s) in the formulation. Frequently, the terms cooling and freezing are erroneously interchanged and confusion in understanding the process may occur and may be compounded by failing to distinguish between shelf or product cooling and freezing. Cooling refers to the reduction of temperature of the freeze-dryer shelves, the diathermy fluid circulating through the shelves, the vial and tray mass, interior of the freeze-dryer, and the dispensed solution or suspension. Cooling does not assume a change in state from liquid to solid and strictly should be used to describe reducing temperature during the initial stage of freeze-drying. Freezing refers to the abrupt phase change when water freezes as ice. Except for very complex biomolecules or cold sensitive cells, cooling in the absence of freezing (chilling) is generally not damaging to biomaterials. When solutions or suspensions are frozen, they may cool appreciably below their measured freezing point prior to ice formation, a phenomenon defined as super-cooling (undercooling or sub-cooling). The extent of super-cooling depends on cooling rate, sample composition and cleanliness, dispensed fill volume, container type, method of sample cooling, and so on. Even when a simple solution is repeatedly cooled or warmed, the onset and extent of super-cooling will vary from cycle to cycle. In the super-cooled state, while the composition of the solution remains unchanged, the cooled liquid is thermodynamically unstable and sensitive to ice formation. As the solution is cooled to lower temperatures, the probability of ice crystallization will correspondingly increase. For optimized freeze-drying, the intention should be to induce super-cooling in the suspension to encourage uniform cooling and freezing throughout the sample contents.
Sample freezing may be defined as the abrupt conversion of the suspension into a mixture of ice and solute concentrate. Freezing is a two-step process during which water initially nucleates, followed by the growth of the ice crystals that pervade the solute phase resulting in a mixture of ice and solute concentrate. Under typical processing conditions, ice nucleates heterogeneously around microscopic particles within the suspension and is encouraged by reducing temperature and agitating the super-cooled suspension to increase the probability of contact between nucleating foci and water clusters. Nucleation depends on the number and physical nature of particulate impurities within the suspension or solution. Ice is a particularly effective nucleation focus and cryobiologists may deliberately seed samples with ice to induce nucleation. Other effective ice nucleators include glass shards and specifically formulated nucleation promoters. Whereas nucleation aids can be added to experimental systems, deliberate attempts to add ice inducers to pharmaceutical materials would be at variance with Good Pharmaceutical Manufacturing Practice.
In contrast to nucleation, ice growth (proliferation) is encouraged by raising the temperature, thereby decreasing the suspension viscosity. Ice nucleation and proliferation are inhibited at temperatures below the glass transition temperature (Tg′), whereas above the melting temperature (Tm) the suspension or solution will melt. The consequences and measurements of these parameters are important elements in the formulation exercise.
To facilitate the sublimation of water vapor from the drying mass, the ice crystals should be large, wide, and contiguous, extending from the product base toward its surface, thereby providing an optimized structure for vapor migration. Crystal structures commonly observed during freeze-drying when solutions are frozen in trays or vials include dendritic structuring, where the ice crystal branches continuously from the nucleating focus and the spherulite form, and where sub-branching is discouraged because the solution viscosity is high, or fast rates of cooling are used.
Cooling or freezing rates are defined as slow (suboptimal), rapid (super-optimal), or optimal as assessed by criteria such as post-freezing cell survival or biopolymer activity, and is ambiguous unless conditions are more precisely defined. Cooling rates may be defined in terms of:
The shelf-cooling rate is the simplest parameter to control and programmed rates of cooling are standard options on research and production freeze-dryers. Because shelf temperature and product responses are not identical, defining shelf-cooling rate will not fully define product behavior. Although we are concerned with the cooling rate achievable within each vial, this parameter is less easy to monitor compared with shelf cooling, and freeze-drying cycles generally are controlled by programmed shelf cooling rather than feedback control from the sample. Cooling rates of the product/cell suspension will vary considerably from vial and throughout the sample within the vial and, consequently, measuring the temperature of vial contents at a fixed position will give only an approximation of the sample temperature variation.
Observing the freezing pattern of a number of vials arranged on a shelf will demonstrate that while the contents of some vials will freeze slowly from the vial base, neighboring vials may remain unfrozen and supercool appreciably before freezing instantly. This random freezing pattern will reflect differences in ice structure from vial to vial and translated into different drying geometries from sample-to-sample vials. In summary, freezing patterns will be related to:
The ice and solute crystal structure resulting from sample freeze has a major impact on subsequent freeze-drying behavior, encouraging the sample to dry efficiently or with defects such as melt or collapse depending on freezing rate used. The preferred ice structure comprising large contiguous ice crystals is induced by freezing the sample at a slow rate of c. 0.2-1.0° C./min. Slow cooling will also induce the crystallization of solutes reluctant to crystallize when faster rates of cooling are used. However, a slow rate of cooling may exacerbate the development of a surface skin, which inhibits sublimation efficiency. Slow cooling can also inactivate a bio-product by prolonging sample exposure to the solute concentrate biomolecules. However, a fast rate of cooling can result in the formation of numerous, small, randomly orientated ice crystals embedded in an amorphous solute matrix, which may be difficult to freeze-dry. Complicating the choice of freezing regimes is the fact that the optimal cooling rate cannot be sustained where the sample fill depth exceeds 10 mm. In short, defining cooling rates often requires a compromise in sample requirements.
A period of consolidation (defined as the hold time), is necessary at the end of sample cooling to ensure that all the vial contents in the sample batch have frozen adequately, although excessive hold times will increase the time of sample freeze and impact on the overall cycle time. It is a fallacy to assume that the ice structure induced remains unchanged during this consolidation period and an ice structure comprising a large number of small ice crystals, induced by rapid cooling, is thermodynamically less stable than an ice structure comprising fewer, larger crystals. The thermodynamic equilibrium can be maintained by recrystallization of ice from small-to-large crystals, a process termed grain growth. Although ice structure changes take place randomly from vial to vial, the hold period is a major factor in ice recrystallization resulting in significant variation in crystal structure and subsequent sublimation efficiency from sample to sample the longer the hold period is employed.
As an alternative to increasing the length of the hold time to encourage ice recrystallization, a more controlled and time-efficient method of inducing recrystallization is to heat anneal the frozen sample. Essentially heat annealing is achieved by:
Heat annealing (also defined as tempering) is particularly useful to:
Although heat annealing will increase the length of the freezing stage of the cycle, overall freeze-drying cycle times may be significantly reduced because of improvements in drying efficiency resulting from heat annealing. Care should be exercised when selecting temperatures and hold times for heat annealing, particularly when defining the upper temperature for sample warming. Subjecting a labile product, such as a vaccine, to temperatures above the eutectic temperature will expose the sample to hypertonic solution concentrates as the sample partially melts, which can damage sensitive biomolecules.
Amorphous materials do not have a eutectic point, but they do have a critical point, below which the product must be maintained to prevent melt-back or collapse during primary and secondary drying.
Regardless of the precise freezing pattern, the formation of ice will concentrate the remaining solution within the container. As the proportion of ice increases within the mixture, solute concentration will correspondingly increase. In the case of an aqueous 1% (w/v) saline solution, this concentration effect will be considerable, increasing to approximately 30% (w/v) just prior to freezing, and damage to biomolecules results as a consequence of solute concentration exposure rather than direct damage by ice crystals. The behavior of the solute(s) within the solute concentrate depends on the nature, concentration, cooling rate, and interactions between individual solutes present in the medium and forms the basis for experimental review during a formulation development exercise.
Overall, four patterns of solute response are observed during freeze-drying:
For a crystallizing solute, the eutectic point is the lowest temperature in a system in which a residual liquid phase and solid phase are in equilibrium. Above the eutectic point, ice and solute concentrate persist, whereas below the eutectic point, a mixture of ice and solute crystals is formed. Eutectic temperatures for aqueous solutions containing crystallizing salts are characteristic for each solute and are significantly below the freezing point of water (for example, eutectic temperature for sodium chloride =−21.4° C.). Exposing cells or proteins for prolonged periods to a eutectic solution comprising hypertonic salt concentrations can cause damage by plasmolysis or precipitation by “salting out”. The eutectic zone is the range of temperatures encompassing all the eutectic temperatures within the system. For a two-part water/solute system, the eutectic temperature is a discrete, quantifiable temperature in contrast to multi-solute systems where a eutectic zone may be observed that represents a range of temperatures where the minimum eutectic temperature is lower than that of any individual eutectic temperatures in the medium.
Typical freeze-dried vaccine formulations fail to crystallize completely when cooled, and a proportion of the solutes in the sample persist as an amorphous, non-crystalline, glass-like. When exposed to temperatures above their glass transition (Tg') or collapse temperature (Tc), these samples may warm during sublimation causing the amorphous mass to soften so that freeze-drying progresses with sample collapse to form a sticky, structure-less residue within the vial. Less severe collapse will result in the formation of a shrunken, distorted, or split cake. Collapsed cakes are not only cosmetically unacceptable but may be poorly soluble, exhibit reduced activity, or compromised shelf stability. Collapse may be exacerbated by the formation of a surface skin, which impedes vapor migration from the drying structure. To avoid sample collapse, it is necessary to maintain the sublimation interface below Tg′ or Tc throughout primary drying and to include excipients in the formulation, which reduce the severity of collapse. It is therefore essential to characterize formulations experimentally during the process development program. Although collapse may cause operational difficulties during freeze-drying, the induction and maintenance of the amorphous state may be essential for protecting labile biomolecules during freezing, drying, and storage.
For clarity, it is usual to separate the drying cycle into primary drying (the sublimation stage) and secondary drying or desorption.
The first step in the drying cycle is defined as primary drying and represents the stage where ice, which constitutes between 70 and 90% of the sample moisture, is converted into water vapor. Sublimation is a relatively efficient process although the precise length of primary drying will vary depending on the sample formulation, cake depth, and so on. During primary drying, the sample dries as a discrete boundary (the sublimation interface), which recedes through the sample from surface to base as drying progresses.
During the primary drying phase, the pressure is lowered (to the range of a few millibars), and enough heat is supplied to the material for the water to sublime. The amount of necessary heat can be calculated using the sublimating molecules' latent heat of sublimation. In this initial drying phase, up to 95% of the water in the material is sublimated. This phase may be slow (can be several days in the industry), because, if too much heat is added, the material's structure could be altered.
In this phase, pressure is controlled through the application of partial vacuum. The vacuum speeds up the sublimation, making it useful as a deliberate drying process. Furthermore, a cold condenser chamber and/or condenser plates provide a surface(s) for the water vapor to re-solidify onto. This condenser plays no role in keeping the material frozen; rather, it prevents water vapor from reaching the vacuum pump, which could degrade the pump's performance. Condenser temperatures are typically below −50° C. (−60° F.).
It is important to note that, in this range of pressure, the heat is brought mainly by conduction or radiation; the convection effect is negligible, due to the low air density.
Variously described as the drying front, freeze-drying front, and so on, macroscopically the sublimation interface can be observed as a discrete boundary that moves through the frozen sample to form an increasingly deeper layer of dried sample above the frozen sample. Heat is conducted from the shelf through the vial base and the frozen sample layer to the sublimation front where ice is converted into water vapor. Several consequences result from this progressive recession of the sublimation front through the dry layer, which include:
Although the sublimation interface is defined as a discrete boundary, this is true only for ideal eutectic formulations, where ice crystals are large, open, and contiguous with each other. For typical amorphous formulations, such as vaccines, the sublimation front is much broader and comprises individual ice crystals imbedded in the amorphous phase. Under these conditions, although ice sublimes within the isolated crystals, the water vapor must diffuse through the amorphous phase (which is itself progressively drying) until it can migrate freely from the drying sample matrix. Under these conditions, sublimation rates are much lower than those anticipated from data derived using eutectic model systems. Complicating a precise prediction of sublimation rate is the fact that fractures in the dry cake between the ice crystals can improve drying efficiency. All of these factors, including system impedances caused by the development of a surface skin on the sample, have to be considered during sample formulation and cycle development programs.
Notwithstanding these complications in precisely defining primary drying, sublimation is nevertheless a relatively efficient process and conditions used for primary drying include the use of shelf temperatures high enough to accelerate sublimation without comprising sample quality by inducing collapse or melt, combined with high system pressures designed to optimize heat conduction from shelf into product. Removing the product when sublimation has been judged as complete will provide a vaccine which appears dry but which displays a high-moisture content that is invariably too high (7-10%) to provide long-term storage stability, and the drying cycle is extended to remove additional moisture by desorption or secondary drying.
In contrast to primary drying, which is a dynamic process associated with high vapor flow rates, secondary drying is much less efficient with secondary drying times representing 30-40% of the total process time but only removing 5-10% of the total sample moisture. Under secondary drying conditions, the sample approaches steady-state conditions where moisture is desorbed or absorbed from or into the sample in response to relative humidity and shelf temperatures. Desorption is favored by increasing shelf temperature, using high-vacuum conditions in the chamber, thereby reducing the system vapor pressure or relative humidity. Conversely, when the shelf temperature is reduced and the vapor pressure in the system increased by warming the condenser, dried samples will reabsorb moisture and exhibit an increase in moisture content. Although sample collapse during secondary drying is generally less likely than collapse during primary drying, it is possible to induce collapse in the dried matrix by exposing the sample to temperature above its glass transition temperature (Tg).
The secondary drying phase aims to remove unfrozen water molecules, since the ice was removed in the primary drying phase. This part of the freeze-drying process is governed by the material's adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0° C., to break any physico-chemical interactions that have formed between the water molecules and the frozen material. Usually the pressure is also lowered in this stage to encourage desorption (typically in the range of microbars, or fractions of a pascal). However, there are products that benefit from increased pressure as well.
After the freeze-drying process is complete, the vacuum is usually broken with an inert gas, such as nitrogen, before the material is sealed.
At the end of the operation, the final residual water content in the product is extremely low, around 1% to 4%.
Damage to a freeze-dried product may occur:
Lowering the temperature of biological material reduces the rate of metabolism until all internal water is frozen and no further biochemical reactions occur [7]. This is quite often lethal and at the very least causes cellular injury. Although fungi are quite often resistant to ice-induced damage, cooling must be controlled to achieve optimum survival. The avoidance of intracellular ice and the reduction of solution, or concentration, effects are necessary [8]. Little metabolic activity takes place less than −70° C. However, recrystallization of ice can occur at temperatures greater than −139° C. [9] and this can cause structural damage during storage. Consequently, cooling protocols have to be carefully designed for cells in order to inflict the least damage possible. The cryopreservation of microfungi at the ultra-low temperature of −196° C. in liquid nitrogen or the vapor above is currently regarded as the best method of preservation [20,10,11]. It can be widely applied to sporulating and non-sporulating cultures. Initial work with fungi was undertaken by Hwang [12] who employed a method designed for freezing avian spermatozoa [13]. Similar methods have been used successfully [11,14,15]. Provided adequate care is taken during freezing and thawing, the culture will not undergo any change either phenotypically or genotypically. Optimization of the technique for individual strains has enabled the preservation of organisms that have previously been recalcitrant to successful freezing [14]. The choice of cryoprotectant is a matter of experience and varies according to the organism. Glycerol gives very satisfactory results but requires time to penetrate the organism; some fungi are damaged by this prolonged incubation step. Dimethyl sulfoxide penetrates rapidly and is often more satisfactory [16,17], but is often toxic to sensitive organisms. Sugars and large molecular substances such as polyvinylpyrrolidone [18,19] (
The cryopreservation of dedifferentiated cells, grown in suspension culture, is one of the portfolio of techniques employed for the long-term conservation of higher plant germplasm. Suspension cultures are also important in biotechnology, particularly in transformation studies and for the production of specific metabolites, and, here, there is also a pressing need for genetically stable, long-term storage of cell lines. Cryopreservation of suspension cell cultures can be exploited by either slow, or rapid, cooling techniques. During slow cooling the extracellular solutions are nucleated and the cells cryodehydrate during controlled cooling as a consequence of extracellular ice, to the point where their intracellular fluids will vitrify on subsequent transfer to liquid nitrogen. In the rapid cooling protocols, the cells are prepared by extreme osmotic dehydration, with cryoprotection, before plunging the samples directly into liquid nitrogen to achieve vitrification. Extensive success has been achieved with both techniques but rapid cooling is, currently, widely favored because of its simplicity.
The consequences and advantages of cryoconservation and its application to plant suspension cultures are well documented [27-31]. The first generation of techniques, still current and widely employed, were based on a protocol reliant on relatively slow-cooling rates, at or near 1° C./min [27-32] and a cryoprotectant mixture containing dimethyl sulfoxide (DMSO), glycerol, and sucrose. For many culture types, acceptable levels of success with this basic method depended on empirical optimization of:
Typically, suspensions are concentrated from the growth medium at a point in the culture cycle where the proportion of small, more densely cytoplasmic cells is at a maximum, with these being viewed as the likely survivors of the freezing protocol. Dependent on the particular genotype, the suspensions may benefit from pre-growth with enhanced concentrations of an osmotic such as mannitol or sorbitol and proline and DMSO have also been used in pretreatment [26,28,33,34]. This pre-growth takes advantage of any natural, adaptive responses that result from osmotic stress and/or activation of the synthesis of natural protectants [35]. Pre-growth at low temperature may also be beneficial [36,37]. Thereafter, the cells are exposed to a cryoprotectant solution containing, in the original protocol, DMSO, glycerol, and sucrose at final concentrations of 0.5, 0.5, and 1 M, respectively [28,38]. The optimum concentrations will vary with circumstance, and empirical optimization is likely to be needed. Other protectant compounds used include proline and polyethylene glycol. Once pretreated, volumes of cell suspension of less than 2 mL are cooled at a rate typically between 0.5 and 2.0° C./min to −40° C., before plunging into liquid nitrogen. At these slow-cooling rates, the unfrozen suspension cells become embedded in ice following nucleation of the extracellular solution, resulting in ongoing cryodehydration [30,31]. As temperature decreases, the dehydration continues and intracellular solutions become increasingly concentrated and viscous. A point is reached where the residual intracellular solutions will vitrify when the cell sample is plunged directly into liquid nitrogen. A holding period at an appropriate subzero temperature may be required to achieve a sufficient level of dehydration. This pattern of cooling, followed by the plunge to ultra-low temperature, provides the classic “two-step” freezing protocol. The expectation is that, with rapid rewarming to avoid intracellular ice crystallization, a proportion of the cells in suspension will retain organization and viability following thawing [28,38,39].
A second major step in the cryopreservation of plant cell suspensions is seen in the more recent proliferation of vitrification techniques, whereby cells can be cooled rapidly to ultra-low temperatures avoiding the phase transition from liquid water to crystalline ice and, instead, producing an amorphous glass [39]. Two mechanisms are at work in the successful vitrification protocol, the first being extensive cytoplasmic dehydration resulting from high concentrations of essentially non-penetrating protectants, e.g., glycerol at 30% (w/v) in the extracellular medium [39]. The second is the increase in viscosity of the cytoplasmic solution that follows from the penetration of high concentrations of protectant, such as DMSO, included in the added Vitrification medium into the cells.
Following treatment with vitrification medium, both the extra- and intracellular solutions are radically modified as a consequence of osmotic dehydration and loading with high concentrations of cryoprotectants. The cells that survive are those that become sufficiently dehydrated so that their intracellular solutions form a stable glass when, with rapid cooling by direct immersion of the sample in liquid nitrogen, they pass the appropriate glass transition point. Typically, the cooling rates employed are in excess of 200° C./min. The extracellular solutions must undergo a similar transition. For the widely used plant vitrification solution PVS2 containing high levels of glycerol, ethylene glycol, and DMSO, this occurs in the region of −115° C. [39]. The presence of a glass provides stability in the system to be preserved, but successful recovery depends on a thawing procedure that transforms the aqueous glass phase directly to a liquid without the intervention of crystalline material, which can only be achieved by rapid rewarming. Precise rates are not usually quoted for this part of published protocols, perhaps because of the difficulties in making appropriately cited, accurate measurements. However, immersion of 2-mL cryo-vials, foil packets, or straws in water at 40° C. for 1-2 min is widely reported [37,39,40]. On occasion, the vitrification protocol can be modified to include an encapsulation stage, whereby the cells are embedded in alginate beads prior to the vitrification procedures [41].
Recent advances in shoot-tip cryopreservation have been significant, this is largely because of: (1) development of vitrification-based cryoprotection protocols, (2) refinements in tissue culture practices, (3) identification of critical points in cryopreservation technology transfer, and (4) the wider uptake and validation of cryostorage technologies in international germplasm repositories. There still remain some genotypes intractable to cryogenic storage, and fundamental research is progressively facilitating the identification of decisive factors in recalcitrance, with a view to aiding storage protocol development. With these issues in mind, this chapter will report the routine storage and investigative methodologies currently applied to shoot cryopreservation. Generic cryopreservation protocols will be described and Ribes (currants) are used as an exemplar, as this genus has been studied in detail with regard to critical point thermal analysis, protocol validation, and technology transfer to germplasm repositories.
There are two main approaches to cryopreservation based on cryoprotective modality. The first is termed traditional, controlled rate, or two-step freezing and involves the application of single or combined mixtures of colligative cryoprotectants, sometimes in conjunction with osmotic additives. Controlled cooling of tissues to an intermediate freezing temperature causes a water vapor deficit to be created between the inside and outside of the cell initiated by the preferential formation (termed “seeding” or ice nucleation) of extracellular ice. Thus, intracellular water moves across the plasmalemma and cellular dehydration results; this process is called equilibrium freezing. Under optimum freezing rates this is a cryoprotective process, as the amount of water available for intracellular ice formation is reduced. However, excessive dehydration can lead to colligative damage owing to the harmful concentration of solutes. Critical cryogenic factors in controlled-rate freezing are:
The second approach to plant cryopreservation is vitrification which usually comprises different permutations of chemical additive as well as vitrification, and encapsulation, osmotic-, evaporative-, and chemical (silica gel)-dehydration. The cryoprotective basis is the concentration of solutes to such an extent that cell viscosity becomes so high that on exposure to cryogenic temperatures, a glass, rather than ice, is formed. The process, termed vitrification, involves the creation of a metastable amorphous glassy state, characterized by the glass transition temperature, Tg, the thermal point at which a glass is formed. However, glasses can be thermally unstable and it is possible that de-vitrification occurs on cooling and rewarming (as this risks the formation of ice it is critical that glasses are stabilized). This can be achieved by the manipulation of dehydration and cryoprotective additives, particularly the inclusion of sugars and careful control of rewarming. Vitrification is a useful alternative to controlled-rate cooling and freezing, as it has the major advantage that tissues are directly immersed into liquid nitrogen, circumventing the need for expensive programmable freezing equipment. In addition to cryogenic factors, the development of successful cryopreservation protocols also depends on the physiological status of the tissues, and pre- and post-treatments are applied to maximize the ability of shoot tips to survive cryopreservation. Detailed information as to the theory and applications of plant cryopreservation can be found in a fundamental theory of cryopreservation by Mazur [42]; cryo-physics and instrumentation reviewed by Benson et al. [43]; historical perspectives and wider areas of in vitro plant conservation are highlighted by Benson [44,45]; and a comprehensive overview of the contemporary aspects of fundamental and applied cryobiology is detailed by Fuller et al. [46].
Freeze-dried vaccines should be formulated to minimize storage decay and should tolerate storage at ambient temperatures for distribution purposes. However, it is a fallacy to suppose that a freeze-dried product remains immune to damage during storage and factors which damage freeze-dried products include:
The interrelationship between sample formulation, dried cake moisture cake, storage conditions, and glass transition temperature (Tg) are complex. In general terms, any physical distortion of the dry cake during storage will often result in a much more rapid loss of sample activity than predicted using the Arrhenius equation for reviewing similar samples.
If a freeze-dried substance is sealed to prevent the reabsorption of moisture, the substance may be stored at room temperature without refrigeration, and be protected against spoilage for many years. Preservation is possible because the greatly reduced water content inhibits the action of microorganisms and enzymes that would normally spoil or degrade the substance.
Freeze-drying also causes less damage to the substance than other dehydration methods using higher temperatures. Freeze-drying does not usually cause shrinkage or toughening of the material being dried. In addition, flavors, smells and nutritional content generally remain unchanged, making the process popular for preserving food. However, water is not the only chemical capable of sublimation, and the loss of other volatile compounds such as acetic acid (vinegar) and alcohols can yield undesirable results.
Freeze-dried products can be rehydrated (reconstituted) much more quickly and easily because the process leaves microscopic pores. The ice crystals that sublimate, leaving gaps or pores in their place, create the pores. This is especially important when it comes to pharmaceutical uses. Freeze-drying can also be used to increase the shelf life of some pharmaceuticals for many years.
Main goal in herbal-, pharmaceutical-, and food-oriented industries is the initial preservation of biological material immediately at or after the vitality-state is altered or destroyed. The basic reason for this is to preserve all biologically active ingredients as-is in their native state and function with almost no alterations. Moreover, the methodology maintaining the state of preservation must be both effective on all bio-material manipulation stages and commercially viable—handling large amounts of biological material and maintaining perfect preservation over extended periods of time up to years after the vitality is interrupted or destroyed.
In opposite to the common commercial lyophilization practices where water crystallization is well sought approach and result in order to facilitate the fast evaporation, in our approach for optimal bio-chemical and structural preservation we employ rapid and instant deep freezing of the biological objects under conditions leading to residual water “glassification” (or so called vitrification—instant freezing without crystallization and volume increase) entirely outsourcing the crystallization followed by water evaporation, manipulations and encapsulation of the biological material as a commercial product without any treatments harmful for degrading the bio-chemical content nor cellular structure—leading to preparation of supplements as natural as the alive biological original obtained initially.
In an optimized setting, the method for preservation of biological substances is based upon the construction and utilization of volume-enclosed container-type vessels comprising deep-freezing fluids, solids and/or gases capable of both engulfing, and instantly freezing any biological material added into vessel, and maintaining deep-freeze temperatures (optimally—below minus−25 degree Celsius) during the bio-material collection, transportation and initial pre-manipulation storage process. Best instant freezing is achieved by collecting the biological material from the field and submersing it immediately into a vessel containing liquefied gas or mixture of gases including but not limited to nitrogen in amounts ensuring the instant engulfing of the entire biological material within the frozen gas molecules leading to immediate deep-freezing. It is essential for the initial freezing to employ frozen liquids and/or liquefied gases at lowest possible temperatures (preferably from minus−180 and minus−80 to minus−30 degree Celsius) in order to best manage achieving water glassification (vitrification) state for best bio-material preservation. Further temperature increase up to minus−20 degree Celsius can be tolerated since it can increase water sublimation with no interference on the glassification (vitrification) state—i.e. no water crystals will be formed and no cells rupture will occur due to volume expansion once a rapid deep-freezing is achieved at temperatures preferably below −35° C. and the temperature after-on is maintained below −20° C.
When any partitioning of the biological materials is desired (such as separating roots, stems, leafs and/or flowers from an intact plant or else) it is preferable that such manipulations are performed immediately upon the collection of the biological material before the deep-freezing is employed since it is highly inconvenient and costly to perform separation manipulations further on while maintaining a frozen conditions. For the purpose of such a practical setup, the deep-freezing collection container is separated on sections allowing for separate collection and freezing of each part of the biological material unless entirely separate deep-freezing collection vessels are employed when needed. In cases when a cross-contamination is anticipated and is essentially not desired, employing separate deep-freezing vessels (containers) is a preferred choice.
After the initial rapid deep-freezing the biological material is transported and stored in freezing containers maintaining temperatures below −20° C. The construction type and the type of the system maintaining the freezing temperatures is not of importance, however the presence of inert gaseous atmosphere (such as Nitrogen, Argonne, Neon, Xenon) is highly desirable since it prevents from oxidation of the biological components—especially if the material has been mechanically disrupted to a higher degree prior or after the deep-freezing.
Commercial manipulation of the biological material starts with reducing the volume of the material for optimizing the drying process. This involves procedures for bio-material disruption such as chopping, cutting, grinding, mulching, etc.—all performed in frozen conditions not increasing −20° C. and resulting in downsizing the biological material to a particles of a preferred size. This step is necessary for two main reasons: first, for down-sizing the entire volume of the total bio-material amount since the following drying step requires minimizing the volume of the drying vessel for economical reasons and switching from submersible freezing (the bio-material is mixed and/or in molecular contact) with the freezing substance) to an external freezing (the bio-material is in contact only with the vessel walls and the gases inside it, and the coolant is outside the vessel walls); second, the size-reduction of the biomaterial particles increases the total contact-surface of the particles, which by itself promotes an increased efficiency of the drying and water evaporation from the bio-material.
The drying of the biomaterial is performed preferably by vacuumization of the storage vessel of a limited total volume while maintaining temperature below −20° C. Were convenient, the drying chamber may be equipped with a mixing device to promote equal surface access for all bio-material particles subjected to drying. The drying chamber is usually constructed with glass- and/or metal walls and parts for best freeze conductivity and mechanical strength. Were reasonable—optical and electro-chemical sensors may be employed for monitoring the processes of bio-material disruption, drying and other manipulations. A water sublimation and removal from the bio-material up to 85-95% completes the initial step of drying.
The secondary drying of the bio-material is usually employed for maximum preservation and increased shelf-life of the final commercial products. The secondary drying is performed after the removal of 90% of the water-content from the bio-material—by gradually increasing the temperature within the drying chamber. The preferable time of the gradual increase and the temperature of the secondary drying vary depending upon the total amount and size of the bio-material particles, the type—and size of the drying chamber, and the construction type of the freezing device. In general, the higher is the water-content of the bio-material—the lower must be the drying temperature with or without vacuumization of the drying chamber a vacuumization; when the residual water becomes below 5% (w/w)—the chamber temperature may be increased to 0° C. and above, preferably to up to 40° C. in a gradual manner usually (but not—obligatory) by a step of −5° C. per hour per kilogram of bio-material. Once the water-content within the bio-material falls below 1-2%, the secondary drying is accomplished. For certain highly-hygroscopic bio-materials, the temperature of the secondary drying may be increased up to 60-75° C. with certain considerations that some enzymes and other bio-components (such as aromatic oils and chemicals) are not stable and/or evaporate and increased temperatures. The main principle during the bio-material drying is to maintain the drying at lowest possible temperature while achieving 97-99.5% of water removal.
After the bio-material is dried it must be stored at low temperatures and humidity that prevent from water condensation. A preferred temperature range is 5-15° C. Large stock-volumes of bio-material may be stored in large buckets of a sizes 5-20-50 gallons filled with inert-gas atmosphere to prevent bio-components' oxidation. The commercial-size containers and packages should be stored at the same conditions.
The dried bio-material can be processed for mixing, combining, aliquoting, encapsulating, etc. after primary drying (at approximately 85-95% of water removal) and/or after secondary drying (at approximately 95-99.5% of water removal). Often manipulating the bio-material at low-temperatures after primary drying is more preferable option since maintaining extremely low humidity levels (below 5%) required after the secondary drying is very challenging during bio-material's manipulation. Therefore only the encapsulation procedures and vial-filling have to be performed after the secondary drying and at humidity level preferable below 1-2%.
All additional manipulations and storage of the bio-material are performed preferably at inert atmosphere, relatively low ambient temperature (not increasing 15-20° C.) and minimal humidity. For best preservation of the final product, a final-step vacuumization of the storage vials may be performed and introduction of inert atmosphere (Nitrogen, Argon, etc.) may be desired before sealing hermetically the packing vials.
The present invention will be described in further detail herein below.
Preservation of plans and fungi by drying has evolved along with the evolution of humanity. During the last century, however, the commercialization and the development of food and supplements industries had let to dramatic progress in implementing varieties of methods and technologies advancing the lyophilization method for preservation and drying of biological material and substances because of its efficiency and commercial viability. While some methods employing liquid nitrogen had been developed for laboratory isolation of biological substances such as RNA, DNA and proteins from limited amounts (of a gram-scale) of raw biological material and for storage with the addition of antifreeze additives, the commercial approaches and technologies have been employing entirely opposite principle of contact-freezing and lyophilization promoting the formation of ice crystals and cell disruption followed by material heating for water evaporation as a way to facilitate more economical preparation of frozen and dry nutrition goods.
The exploitation of instant deep-freezing with water glassification (vitrification) followed by cold lyophilization, however, is much more preferable choice for the preparation of herbal and pharmaceutical remedies. The availability of liquefied and solidified gases (such as nitrogen and carbon dioxide) at low prices and the price increase of herbal and supplement remedies had made the submersion-lyophilization commercially viable approach—especially because for the herbal industry there is no need to lyophilize as extremely large amounts of bio-material as it is needed in the food industry. The quality of preservation of biological material becomes of much higher importance in herbal and pharmaceutical industries making the submersion lyophilization a preferred choice.
The method of this invention is dedicated for commercial preparation and preservation of plants, funguses (fungi), lichens and/or algae, entire plant-, fungus-, lichen- and/or algae material, parts of entire plant-, fungus-, lichen- and/or algae species and/or any desired combinations of different biological species or parts of them, including but not limited to those serving herbal, medicinal and/or supplemental properties and purposes. It is not intended for implementation on biological material from animal-, bacterial- and viral origin nor purposes other than commercial ones thereby excluding direct laboratory- and research usage prior to complete bio-material drying and final product preparation. However, since the dried remedies consist of near-perfectly preserved bio-compounds, the final commercial products and dried substances are highly recommended for any nutrient-, culinary-, herbal-, medical-, laboratory- and research purposes.
Our approach and methodology involves the utilization of collection containers filled with chemically inert gases and/or liquids cooled to temperatures from −30° C. to −180° C. that allow for instant and immediate cooling of any biological material collected and submersed within the enclosed volume of these containers—without addition of any antifreeze chemicals. A preferred choice of deep-freezing inert gases are nitrogen, carbon dioxide, neon, argon, xenon and a preferred choice of deep-freezing liquids are light-molecular weight hydrocarbons such as ethanol, methanol, ether, pentane, hexane, octane, decane, acetone etc. or mixes of the mentioned. While the liquefied nitrogen and solidified carbon dioxide are the least expensive, the hydrocarbon alcohols are more hydrophilic, they mix with the water and precipitate the biological content within the tissue on one hand while promoting the water evaporation during the drying phase—on the other hand. Overall, the liquid nitrogen remains the preferred choice because it offers the lowest possible freezing temperature at the range of −80° C. coupled with perfect chemical inactivity, which makes it an ideal carrier for biological substances and materials. However if extraction of aromatic oils, lipids phenols and other bio-substances is desired, the raw bio-material may preferably be submersed in deep-freezing alcohols or other organic solvents such as hydrocarbons.
The optimal setup for bio-material collection is its immediate preliminary processing at the growing field followed by submersion in the deep-freezing containers dedicated for preliminary storage employing submersion liquids (such as liquid nitrogen). The preliminary processing is performed preferably within 10-20 minutes after cutting-off the vital nutritions from the bio-matrial and includes but is not limited to separating and sorting desired parts and tissues (i.e. roots, stems, leafs, flowers, etc.), and/or reducing the material in size by cutting, chopping, mulching, shedding or else. In worse-case scenario, the collected biomaterial is cooled down to temperatures 5-20° C. for reducing the bio-degradation damage and transported to a closest processing facility where the preliminary processing is performed and the bio-material is immediately deep-frozen. The freezing at close to −180° C. (liquid nitrogen) leads to instant water glassification (vitrification) and near-perfect preservation of the entire biological content for as long as the liquid nitrogen is present in a liquid state within the thermostat container. For extended storage, dry carbon dioxide can be added to gradually replace the liquid nitrogen increasing the storage temperature to approximately −80° C. (that is still an excellent storage temperature to maintain) offering longer storage time since the solid carbon dioxide sublimates slower than the liquid nitrogen, it is easier to handle and cheaper to produce. Any addition of organic solvents must be done at temperatures near- or above the temperature of the dry carbon dioxide, since they will solidify immediately at so low temperatures (within the range of −176° C. to −79° C.).
Best technical setup for down-sizing the bio-material is by mulching and grinding it to a sand- or powder-size particles in metal grinders with double-wall chambers that allow a liquid nitrogen to be present between the double-walls for maintaining deep-frozen temperatures (at −170° C. range) while processing the bio-material.
The preparation of desired herbal remedies including combinations of different parts of plants, algae, lichens, fungi and/or molls, or combinations of entire species is performed immediately after the collection of the bio-material at the preliminary processing level (before the deep-freezing) and/or after the primary or secondary drying. Preferably, the preparation of combinatorial remedies is performed after the first-stage drying (at 5-10% of water content remaining within the material and low temperatures of 5-10° C.) since maintaining complete de-humidification is much more challenging and economically unreasonable at higher levels of bio-material drying (above 95% of water removal). However, the re-introduction of certain volatile compounds removed on earlier stages of cold bio-material processing (such as extracted aromatic oils, lipids, phenols, etc.) is preferably performed after the final drying of the bio-material and/or after the final composition of the commercial remedies—just before the encapsulation.
The preservation of all or any particular volatile (light-molecular-weight) bio-compound (such as, but not limited to aromatic light-molecular-weight oils, pheromones, lipids, phenols, etc.) are extracted directly from the biomaterial at a cold stage (−40 to −10° C.) with organic solvents (hydrocarbons) and/or extracted from the collected evaporated water after melting it at temperatures of 5° C. to 30° C. (but below the evaporation temperature of a particular light-weight bio-compound. The latest approach is employed for bio-compounds that remain together with the water vapor removed from the biological material due to the technical impossibility to be separated from the water vapor; these bio-substances are sub-sequentially removed from the water evaporate via all appropriate physical and chemical means, they are biophysically isolated and/or condensated, and re-introduced into the volume containing the dry biological material—where such an re-introduction is optional and/or desired for the final product preparation in cases of particular applications, but may not always be an obligatory option.
Further on, a first-stage drying of the grinded bio-material can be performed by transferring the frozen material into a vacuumization chamber or using the grinder's chamber as a vacuumization chamber where possible by design. As an optimal design, the vacuum-evaporation chamber must have double-walls maintaining the presence of liquid nitrogen or dry carbon dioxide for maintaining deep-freeze temperatures until more than 85% of the residual water is evaporated from the bio-material. The vacuum-dryer device must have a water condensation section to facilitate the removal of the evaporated water in order to maintain low humidity promoting the efficient drying of the bio-material. The vacuum dryer and the condenser might be electrically operated; however, water-absorbing materials may be implemented if desired upon appropriate design. Were convenient, the drying chamber may be equipped with a mixing device to promote equal surface access for all bio-material particles subjected to drying. Were reasonable—optical and electro-chemical sensors may be employed for monitoring the processes of bio-material disruption, drying and other manipulations. The initial step of drying is to achieve up to 85-95% of water sublimation and removal from the bio-material.
The secondary drying of the bio-material is employed for ensuring maximum preservation and increased shelf-life of the commercial products prepared. The secondary drying is performed after the removal of approximately 90% of the water-content from the bio-material. During the secondary drying the temperature within the drying chamber is gradually increased to promote efficient water removal. Depending upon the optimal technical design, the drying chamber for the secondary drying may have much large surface area for depositing the bio-material in order to promote the enhanced drying since it does not require to maintain deep-freezing temperatures. The presence of cold temperatures and vacuum are preferable but not obligatory and a gradual temperature increase from −50° C. up to +30° C. are desired. Increasing the temperature up to 0° C. are preferably performed by a gradual increase of the temperature with a rate of 5° C. per every 30-60 minutes if vacuum is applied to quickly remove the water vapor and with a rate of 5° C. per hour per each 1 kilogram of bio-material within the temperature range above 0° C. until reaching 15° C. Increasing the temperature above 15° C. up to as high as 40° C. must be performed in accordance with a humidity measurement, water-content of the sample and the specificity of the biological material processed. For perfect preservation of the bio-components the temperature of drying may not increase above 15° C.
For best preservation, all subsequent steps of transferring, sampling, mixing, stock-storage, encapsulation and vial-filling should preferably be performed at ambient temperature not above 15-18° C. and nitrogen- or other inert-gas atmosphere; ambient air is less desired due to its oxidation properties affecting many biological components. The dry biological material obtained by the described method is stored and processed under dry conditions into enclosed volumes (vessels, containers, bags, capsules etc.) ensuring a dry storage and/or (preferably) an absence of chemically harmful gases such as but not limited to oxygen, volatile chemical elements, molecules and vapors; thereby the storage volumes and conditions should maintain chemically inert atmosphere (preferably but not limited to vacuum- or nitrogen-), or any transitional atmospheric conditions between the last mentioned (i.e.—vacuum, nitrogen and/or other chemically inert gases or combinations of- and transitions between them) while handling the material of biological origin.
All bio-material manipulation stages require the implementation of design-appropriate equipment for volumes, cameras, chambers, ventilation, cooling, vapor entrapment and removal, absorption, extraction, lightening, sterilization, sensor and/or optical monitoring, and encapsulation and sampling.
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This application is claiming the priority benefit of the provisional patent application U.S.-61/750,518 “Method for Drying-Conservation of Natural Substances”.
| Number | Date | Country | |
|---|---|---|---|
| 61750518 | Jan 2013 | US |
