A method for freeze-drying biological matter

Abstract

The invention describes a method for freeze-drying biological matter (100), the method comprising the steps of introducing a fluid biological matter (100) into a flexible receptacle (200), closing the flexible receptacle (200), freezing the fluid biological matter (100), thereby obtaining a frozen biological matter (100), placing the flexible receptacle (200) in a freeze dryer, lowering the pressure in the freeze dryer to a pressure lower than the vapor pressure of water, wherein the flexible receptacle (200) remains sealed at least until it inflates, freeze drying the frozen biological matter (100) by providing heat to the frozen biological matter (100) such that a vapor pressure at the frozen biological matter (100) is larger than the vapor pressure at one or more cooling element(s) (260, 600) in gas communication with the frozen biological matter (100), thereby obtaining a freeze- dried biological matter (100) in the first portion (210) and frozen water at the one or more cooling element(s) (260, 600), and sealing the first portion (210) off from a second portion (220) of the flexible receptacle (200) not containing the freeze-dried biological matter (100).

Description

[0001] A method for freeze-drying biological matter

[0002] Field of invention

[0003] The invention relates to the field of freeze-drying of biological matter

[0004] Background

[0005]

[0001] Freeze-drying of various blood products for use in treatment of injured patients has been known since World War II. Freeze-drying of for example blood plasma for later use is thus generally a known process.

[0006]

[0002] A common way of freeze-drying blood plasma is to freeze dry the blood plasma directly in glass bottles, before sealing the glass bottle with a cap. When the blood plasma is needed, the blood plasma is commonly rehydrated in the bottle, transferred to a separate bag, and injected into for example a patient from the bag. The use of multiple receptacles for the blood plasma involves a risk of contamination of the blood plasma during the transfer process, and also a risk of loosing track of the origin of the blood plasma.

[0007]

[0003] Conventional methods for freeze-drying blood plasma involve open containers (US2009 / 0107001A1, US2441730), which necessitate post-drying handling and sealing, increasing the risk of contamination. Performing the entire process in cleanrooms or under inert gas (e.g., nitrogen or argon) adds complexity and cost, and introduces traceability challenges due to multiple handling steps.

[0008]

[0004] To address these issues, containers with gas-permeable membranes have been proposed (US6517526B1, JP2020054835A, US2008 / 0119818A1), allowing drying, storage, transport, rehydration, and use within a single sealed unit. In some variants, the dried product is transferred to a membrane-free compartment and sealed off. However, contamination of the membrane with unfrozen plasma has been shown to obstruct vapor transport. Solutions such as membrane elevation or spatial separation (US2019 / 0106254A1, US2020 / 0289728A1) have been proposed to mitigate this issue.

[0009]

[0005] A further challenge is that gas-permeable membranes remain permeable to moisture and oxygen after drying, necessitating temporary sealing measures such as clamps or barrier films. These membranes also require complex integration with the container and add to manufacturing costs.

[0006] Alternative approaches involve structurally reinforced containers capable of withstanding vacuum conditions, where water vapor exits through a vacuum channel during drying (US4973327, US5257983, US5309649). After drying, the reinforcement may be removed to allow the container to collapse for compact storage and transport.

[0010]

[0007] It is an aim of the present invention to address at least some of the issues with the prior art.

[0011] Summary of the invention

[0012]

[0008] In a first aspect, the present invention provides a method for freeze-drying biological matter, the method comprising the steps of introducing a fluid biological matter into a first portion of an otherwise closed flexible receptacle, closing the flexible receptacle, freezing the fluid biological matter, thereby obtaining a frozen biological matter, placing the flexible receptacle in a freeze dryer, lowering the pressure in the freeze dryer to a pressure lower than the vapor pressure of water, wherein the flexible receptacle remains sealed at least until it inflates, freeze drying the frozen biological matter by providing heat to the frozen biological matter such that a vapor pressure at the frozen biological matter is larger than the vapor pressure at one or more cooling element(s) in gas communication with the frozen biological matter, thereby obtaining a freeze- dried biological matter in the first portion and frozen water at the one or more cooling element(s), and sealing the first portion off from a second portion of the flexible receptacle not containing the freeze-dried biological matter.

[0013]

[0009] The flexible receptacle may further comprise an entry valve configured to be employed for introducing the fluid biological matter into the flexible receptacle.

[0014]

[0010] In an exemplary process of the first aspect, the method comprises an initial step of delimiting the first portion from the second portion. The restriction of the first portion from the second portion may preferably be performed by clamping the flexible receptacle. Such a restriction may restrict the fluid biological matter from entering the second portion before the step of freezing the fluid biological matter.

[0015] [Oil] During the step of freeze drying, the step of providing heat to the biological matter may increase the rate at which the frozen water or ice in the frozen biological matter is sublimated into water vapor at the first portion and subsequently desublimated into ice at the one or more cooling elements. For example, the heat provided at the first portion and the cooling provided at the one or more cooling elements increases the water vapor pressure at the first portion relative to the one or more cooling elements, leading to increased efficiency of the freeze drying of the biological matter. This pressure gradient allows for an increased flow of the water vapor towards the one or more cooling elements.

[0016]

[0012] In another exemplary process, after the step of freezing the fluid biological matter, the method further comprises a step of providing a fluid communication between the first portion and the second portion. For example, the restriction may preferably be removed by unclamping the flexible receptacle.

[0017]

[0013] Alternatively or in addition, the flexible receptacle may comprise one or more joints configured to prevent the fluid biological matter in the first portion from entering the second portion. The joints may furthermore, during the step of freeze drying the frozen biological matter, be configured to yield and separate by a force applied such that the first portion and the second portion are in fluid communication. For example, the joints may separate upon inflation of the flexible receptacle for enabling a controlled opening along predetermined joining regions. Such joints may include, but are not limited to temporary seals or at least one septum.

[0018]

[0014] In another exemplary process, the method further comprises the step of positioning a heating element at the first portion. Alternatively or in addition, the method may similarly comprise the step of cooling the second portion by positioning a cooling element at the second portion, thereby obtaining freeze dried biological matter in the first portion and frozen water in the second portion.

[0019]

[0015] In another exemplary process, the method further comprises the step of providing an outlet opening at the second portion and wherein a filter and / or a gas permeable membrane is arranged within the interior of the flexible receptacle in a region between the outlet opening and the first portion such that any inert gas(es) inside the flexible receptacle may pass through the filter and / or the gas permeable membrane and escape the flexible receptacle, and wherein the filter and / or the gas permeable membrane and / or the outlet opening is(are) dimensioned to restrict the outflow of gas such that water vapor predominantly condense at the second portion within the flexible receptacle. The outlet opening may be arranged at an area of the second portion that is furthest away from the first portion.

[0016] In another exemplary process, the method further comprises the step of providing a small outlet opening at the second portion such that water vapor may escape the flexible receptacle and predominantly condense outside the flexible receptacle at one or more second cooling element(s).

[0020]

[0017] The heating element and the cooling element may have a size and shape that conforms to at least a part of the first portion and the second portion, respectively, for providing efficient heating at the first portion. This may increase the rate of water vapor formation, and subsequently cooling of water vapor and formation of ice at the one or more cooling elements, for example at the second portion, thereby increasing the rate of ice formation. Alternatively or in addition, at least one heating element may be positioned on a bottom surface and an upper surface at the first portion. Similarly, at least one cooling element may be positioned on a bottom surface and a top surface at the second portion. Such configuration may enable two thermally distinct zones comprising an efficient temperature gradient between the first portion and the second portion.

[0021]

[0018] The temperature at the first portion may be kept at a temperature sufficient to induce sublimation of ice from the frozen biological matter into water vapor. Furthermore, the temperature at the second portion may be kept at a temperature sufficient to promote desublimation of the water vapor from the first portion into ice at the second portion.

[0022]

[0019] Alternatively or in addition, the method may further comprise a step of simultaneously freeze drying a plurality of flexible receptacles. In this example, the flexible receptacles may be positioned on a carrier. Optionally, the heating element(s) and / or the cooling element(s) is an integrated part of the carrier.

[0023]

[0020] Alternatively or in addition, the flexible receptacles may be arranged in a stacked configuration, where a bottom surface of a first portion and a second portion of an upper flexible receptacle are configured to rest upon respective heating element and cooling element disposed on an upper surface of a corresponding first portion and second portion of a lower flexible receptacle.

[0024]

[0021] During or after the step of freeze drying the frozen biological matter, the step of sealing the first portion off from the second portion may be performed while the vapor pressure of water at the second portion is still lower than the vapor pressure at the first portion. Alternatively or in addition, the second portion may be removed entirely from the first portion.

[0022] In another exemplary process, the method further comprises, after the step of sealing the first portion from the second portion, a step of separating the first portion from the second portion. For example, the second portion may be completely separated from the first portion.

[0025]

[0023] In an exemplary process, the fluid biological matter is human blood plasma.

[0026]

[0024] In an exemplary process, the method further comprises a step of rehydrating the freeze-dried biological matter.

[0027]

[0025] In another exemplary process, the flexible receptacle comprises a drain valve for removing hydrated freeze-dried biological matter from the first portion, and an inlet valve for introducing liquid such as water into the first portion. The drain valve and the inlet valve are furthermore arranged at the first portion.

[0028]

[0026] The flexible receptacle may further be delimited between the flexible receptacle and the drain valve. Such a restriction may include, but is not limited to, a clamp, one or more joints, or a septum.

[0029]

[0027] In an exemplary process where the flexible receptacle comprises a drain valve and an inlet valve, the flexible receptacle further comprises a filter arranged at the drain valve. The filter is configured to block non-hydrated particles of freeze- dried biological matter from flowing through the drain valve.

[0030]

[0028] In an exemplary process, the flexible receptacle further comprises means for enabling the flexible receptacle to be hung onto a hook, wherein the drain valve is arranged such that the drain valve faces downwards when the flexible receptacle is hung onto the hook. Such means may include, but are not limited to, a through-going hole or a protrusion configured to receive the hook.

[0031]

[0029] In an exemplary process, the method further comprises the steps of placing the flexible receptacle comprising the freeze-dried biological matter inside an additional receptacle, and sealing the additional receptacle. Optionally, the additional receptacle comprises a metal foil.

[0032]

[0030] In an exemplary process where the method comprises the step of placing the flexible receptacle comprising the freeze-dried biological matter inside an additional receptacle, prior to the step of sealing the additional receptacle, the method further comprises any one or more of the steps of introducing an oxygen scrubber into the additional receptacle, evacuating the additional receptacle, and / or introducing a non-oxidizing gas into the additional receptacle.

[0033] Brief description of the drawings

[0034]

[0031] Figure 1 is a schematic illustration of a flexible receptacle that may be employed in the method according to the present invention,

[0035]

[0032] Figure 2 is a schematic illustration of an example of a flexible receptacle comprising a hole, where the hole may be employed to hang the flexible receptacle onto a hook,

[0036]

[0033] Figure 3 is a schematic illustration of a cross section of a flexible receptacle before freeze drying is employed and wherein the frozen biological matter is in a first portion of the flexible receptacle between two clamps,

[0037]

[0034] Figure 4 is a schematic illustration of a cross section of an inflated flexible receptacle during freeze drying where the first portion and a second portion are in fluid communication,

[0038]

[0035] Figure 5 is a schematic illustration of a cross section of a flexible receptacle during freeze drying where water vapor from the biological matter at the first portion is condensed into ice at the second portion,

[0039]

[0036] Figure 6 is a schematic illustration of a cross section of the second portion of the flexible receptacle comprising an opening with a filter and / or a gas permeable membrane

[0040]

[0037] Figure 7 is a schematic illustration of a flexible receptacle during freeze drying comprising an opening to allow water vapor to escape and condense at one or more second cooling elements,

[0041]

[0038] Figures 8A to 8C are schematic illustrations of a section of a flexible receptacle, showing various examples of a joint extending vertically along the outermost walls of the flexible receptacle, and which may yield and separate by a force applied such that, for example, the first portion and the second portion are in fluid communication, or such that a region may yield and create an opening at a distal end of the flexible receptacle,

[0042]

[0039] Figure 9 is a schematic illustration of a flexible receptacle during the final stages of freeze drying and where the first portion and the second portion are delimited from each other,

[0043]

[0040] Figures 10A to 10C are schematic illustrations of sections of a flexible receptacle and examples of arrangements at or near a valve of the flexible receptacle,

[0044]

[0041] Figure 11 is a schematic illustration of a flexible receptacle arranged within an additional receptacle,

[0042] Figure 12 is a schematic illustration of a method for freeze-drying biological matter.

[0045] Detailed description of the invention

[0046]

[0043] In the following, general embodiments as well as particular exemplary embodiments of the invention will be described. References will be made to the accompanying drawings. It shall be noted, however, that the drawings are exemplary embodiments only, and that other features and embodiments may well be within the scope of the invention as claimed. Further, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality, and the term "disclosure" may herein be used interchangeably with the term "invention".

[0047]

[0044] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. Certain terms of art, notations, and other scientific terms or terminology may, however, be defined specifically as indicated below.

[0048]

[0045] The present invention provides, as schematically illustrated in figure 12, a method for freeze-drying biological matter 100. The biological matter 100 may here for example be fluid blood plasma or more generally a fluid blood product, for example fluid human blood plasma or a fluid human blood product. More specifically, the biological matter 100 may be a quantity of fluid blood plasma or more generally a quantity of a fluid blood product.

[0049]

[0046] The method according to the present invention generally relates to the employment of a flexible receptacle 200 for freeze drying biological matter 100. The flexible receptacle 200 may be used as a receptacle for the biological matter 100 prior to freeze drying, as a receptacle for the biological matter 100 during the process of freeze drying, and as a receptacle for the biological matter 100 once freeze dried. The method comprises in short the steps of introducing a fluid biological matter 100 into a flexible receptacle 200, closing the flexible receptacle 200, freezing the fluid biological matter 100, placing the flexible receptacle 200 in a freeze dryer, lowering the pressure in the freeze dryer to a pressure lower than the vapor pressure of water, wherein the flexible receptacle 200 remains sealed at least until it inflates, freeze drying the frozen biological matter 100 by providing heat to the frozen biological matter 100 such that a vapor pressure at the frozen biological matter 100 is larger than the vapor pressure at one or more cooling element(s) 260, 600 in gas communication with the frozen biological matter 100, thereby obtaining a freeze-dried biological matter 100 in the first portion 210 and frozen water at the one or more cooling element(s) 260, 600, and sealing the first portion 210 off from a second portion 220 of the flexible receptacle 200 not containing the freeze-dried biological matter 100.

[0050]

[0047] The biological matter 100 thus remains inside the closed flexible receptacle 200 from the step of introducing the fluid biological matter 100 into the flexible receptacle 200. The latter has been found to be beneficial for reducing contamination as compared to employing methods that are dependent on, for example, transferring the biological matter 100 between receptacles. The employment of one flexible receptacle 200 as described herein may further be beneficial for tracking the origin or the freeze-dried biological matter 100, as the flexible receptacle 200 may be marked when the fluid biological matter 100 is being introduced.

[0051]

[0048] The method may further comprise an initial step of delimiting or restricting a first portion 210 from a second portion 220 of the flexible receptacle 200. The step of introducing the fluid biological matter 100 into the flexible receptacle 200 may, as schematically illustrated in figure 1, be performed by introducing the fluid biological matter 100 into the first portion 210 of the flexible receptacle 200, for example through an entry valve 290 or may alternatively be introduced through an opening. As illustrated in figure 1, the entry valve 290 for introducing the fluid biological matter 100 into the flexible receptacle 200 may be positioned such that the fluid biological matter 100 may be introduced into the first portion 210 of the flexible receptacle 200. In other words, the fluid biological matter 100 may only contact a limited portion, i.e. the first portion 210, of the flexible receptacle 200. Introducing the fluid biological 100 matter into the first portion 210 of the flexible receptacle 200 is preferrable in order to obtain pre-defined shape of frozen biological matter 100, and later freeze-dried biological matter 100, but is particularly preferable to enable a subsequent flow of water vapor from the first portion 210 towards the second portion 220 during freeze drying. Furthermore, introducing the fluid biological matter 100 into the first portion 210 may avoid any contact between the fluid biological matter 100 and any one or more of of a single valve 276, a drain valve 270 and an inlet valve 275 of the flexible receptacle 200, and an area of the flexible receptacle 200 where a later sealing may be performed. Such contact may be preferrable to avoid, as a contact between the fluid biological matter 100 and a valve may affect the function of that valve, for example during a later rehydration of the biological matter 100 when the biological matter 100 has been freeze-dried. Introducing the fluid biological matter 100 into a delimited portion may further be preferable in order to enable a homogeneous and / or controlled heat transfer with the later frozen biological matter 100 during freeze drying.

[0052]

[0049] As schematically illustrated in figures 1 to 3 and figures 8A to 8C, a defined portion of the flexible receptacle 200 may be obtained by providing one or more temporary seals 230 in the flexible receptacle 200. Clamping the flexible receptacle 200 may here cause two sides of the flexible receptacle 200 to be forced against each other thereby providing a barrier, i.e., the temporary seal(s) 230 that the fluid biological matter 100 may not pass. The temporary seal 230 or seals may thus form two pockets, i.e., the first portion 210 and the second portion 220, inside the flexible receptacle 200. The clamping may here be performed by any number of suitable external clamps 230, for example flat clamps 230, optionally mounted with a rubber section for contacting the flexible receptacle 200. Figure 1 schematically illustrates an example of a defined portion of the flexible receptacle 200 delimited by providing a temporary seal 230 in the flexible receptacle 200. Other examples for restricting the first portion 210 from the second portion 220 may be, but is not limited to, joints 232 (see figures 8A to 8C), seals and / or a septum or other means for preventing the fluid biological matter 100, once introduced, from entering the second portion 220. The size of the portions may comprise a similar or approximately similar volume of the total volume of the flexible receptacle, and may also depend on the design and length of the flexible receptacle. Once the fluid biological matter 100 is frozen, the temporary seal or seals 230 may be broken.

[0053]

[0050] The step of closing the flexible receptacle 200 may involve closing the entry valve 290 or by sealing the opening, for example through heat sealing. As shown in figure 1, the closing may be performed along a stapled closing line 202. As will be appreciated by a person skilled in the art with knowledge of the present invention, the introduction of the fluid biological matter 100 into the flexible receptacle 200 may be performed by any suitable filling equipment of which there are several commercially available. Examples of suitable filling equipment include a sterile welding process, where the tube is cut, and the two ends - one from the flexible receptacle and one from the transfer system - are brought into abutment within a sterile tube welder, or equipment that comprises any one of a filling hose, a screw-on coupling for being connected to the entry valve 290, and a rubber plug comprising an insertion canal through which an insertion needle may be introduced. The entry valve 290 may alternatively comprise such a rubber plug, whereas the filling equipment may comprise an insertion needle for being introduced through the insertion canal.

[0054]

[0051] Once inside the first portion 210, the fluid biological matter 100 will according to the method of the present invention undergo a step of freezing the fluid biological matter 100, thereby resulting in a solid frozen biological matter 100. As schematically illustrated in figure 3 showing a cross section of the flexible receptacle once the biological material has been frozen, the frozen biological matter 100 is contained between two clamps 230 arranged at each side of the first portion 210. The freezing of the fluid biological matter 100 may here be performed in a separate freezing unit, and the freezing process may for example be conventional freezing or flash freezing. As a way of example, the fluid biological matter 100 may be frozen by lowering the closed flexible receptacle 200 into a dry-ice ethanol bath or a dry-ice antifreeze bath, or by introducing the flexible receptacle 200 into a blast freezer or mechanical freezer. A person skilled in the art with knowledge of the present invention will appreciate that alternative methods for freezing may be employed.

[0055]

[0052] The time between the initial step of introducing the fluid biological matter 100 into a first portion 210 of a flexible receptacle 200 and the step of freezing the fluid biological matter 100 may generally be kept as short as possible. Particularly when the fluid biological matter 100 is blood plasma it is preferable to perform the step of freezing the blood plasma within 8 hours of the time when the blood plasma was separated from the blood. The latter implies that the time between the initial step of introducing the fluid biological matter 100 into a flexible receptacle 200 and the step of freezing the fluid biological matter 100 may preferably be less than 8 hours, preferably less than 6 hours, as some time should be allocated to perform the step of introducing a fluid biological matter 100 into a flexible receptacle 200 and the step of closing the flexible receptacle 200.

[0056]

[0053] Once frozen, the then solid frozen biological matter 100 inside the first portion 210 of the flexible receptacle 200 may preferably be kept at a temperature of maximum - 18 °C, preferably maximum - 30 °C. A temperature of - 18 °C has been found to be particularly preferable when the solid frozen biological matter 100 is frozen blood plasma, as a storage temperature of maximum - 18 °C has been found to preserve coagulation factors in the blood plasma. A temperature of - 30 °C is generally employed by most blood storage facilities.

[0057]

[0054] As illustrated in figure 4, the method may, after the freezing step, further comprise a step of providing a fluid communication between the first portion 210 and the second portion 220 where the temporary seal(s) 230 is(are) broken. In an example where the flexible receptacle 200 has been restricted by clamping between the first portion 210 and the second portion 220, the fluid communication may be performed by unclamping the flexible receptacle 200, at least between the first portion 210 and the second portion 220.

[0058]

[0055] Alternatively or in addition, the first portion 210 and the second portion 220 may undergo fluid communication at a later step, i.e. during the step of freeze drying the frozen biological matter 100. In the latter example, this may be achieved by providing one or more joints 232 as illustrated in figures 8A to 8C. The joints 232 are configured to delimit the fluid biological matter 100 at the first portion 210 from entering the second portion 220, and configured to yield and separate caused by an external mechanical force and / or upon inflation of the first and second portions 210, 220 of the flexible receptacle 200. This may increase the effectiveness of the procedure by reducing the need for additional handling of the flexible receptacle, for example before introducing biological matter into the first portion 210 and after freezing the biological matter 100. In figure 8A, the joint 232 is illustrated as substantially linear and extending across an inner side of an upper surface and a lower surface of the flexible receptacle. The joints may be arranged along the stapled portion line 204 (see e.g. figures 1 and 2) and / or along an area at a distal region 206 of the second portion 220 as indicated in figure 1. Figure 8B illustrates an alternative example of a joint 232 comprising a spike 233. The force applied to the flexible receptacle may be concentrated at or near this spike, enabling an easier yield of the temporary sealing. Other examples may include, but are not limited to, less flexible materials such as fibre integrated in the flexible material of the flexible receptacle 200 along, for example, the indicated stapled portion line 204 (see figures 1 and 2). Figure 8C illustrates an example where the flexible receptacle 200 expands at the first portion 210 but to a lesser degree or to no extent at the second portion 220. In this example, the flexible receptacle 200 comprises an outlet opening 240 at the distal end of the second portion 220 (see figures 6 and 7). The applied force may here eventually cause the joint(s) 232 to yield, thereby allowing water vapor and / or inert gas(es) to escape the flexible receptacle 200 through the outlet opening 240.

[0059]

[0056] The flexible receptacle 200 containing the frozen biological matter 100 may, after the freezing step, be freeze dried by providing heat to the frozen biological matter 100 while cooling the second portion 220 of the flexible receptacle 200 not containing the frozen biological matter 100. As illustrated in figure 4, this may be achieved by providing a heating element 250 and / or a cooling element 260 arranged at the first portion 210 and the second portion 220, respectively. The heating element(s) 250 may supply heat to the first portion 210 during freeze drying, without the temperature at any point in the biological matter 100 becoming high enough to cause melting. The cooling element(s) 260 may cool the second portion 220, allowing the water vapor to condense at the second portion 220. Alternatively or in addition, additional flexible receptacles 200 comprising frozen biological matter 100 may be stacked atop the uppermost heating and / or cooling elements 250, 260 shown in figure 4, with corresponding heating and cooling elements 250, 260 disposed above each subsequent flexible receptacle 200. This arrangement enables simultaneous freeze-drying of multiple flexible receptacles 200 in a stacked configuration.

[0060]

[0057] In an alternative example of the invention, as will be explained in more detail below, the condensation of water vapor may be effected outside the flexible receptacle 200 (see figure 7). In this specific example, the cooling elements 260 in figure 4 may be interpreted solely as mechanical constraints limiting the expansion of the flexible receptacle 200, without performing any cooling function. In the absence of such constraining elements, the flexible receptacle 200 may be permitted to fully expand within the available volume. This external condensation configuration also permits the stacking and simultaneous freeze drying of multiple flexible receptacles 200 in a manner similar to the example with condensation within the flexible receptacle 200. Furthermore, it is envisaged that water vapor from multiple flexible receptacles 200 may be condensed concurrently using a common condensation arrangement.

[0061]

[0058] As illustrated in figure 5 showing a cross section of the flexible receptacle 200 during freeze drying, water vapor from the biological matter 100 at the first portion 210 is desublimated into ice 110 at the second portion 220.

[0059] The temperature at the first portion 210 may further be maintained at a temperature sufficiently low to ensure the frozen water in the frozen biological matter 100 remains in the solid phase but sufficiently high to allow water vapor pressure for sublimation to occur. Similarly, the temperature at the second portion 220 may be maintained at a lower temperature than the temperature at the first portion 210 to allow subsequent desublimation of the water vapor flowing from the first portion 210 to the second portion 220. In figure 4, the heating and cooling elements 250, 260 are configured to maintain the respective predetermined temperature zones sufficient to induce sublimation and desublimation of water in its solid and gaseous phase, respectively, inside the flexible receptacle 200.

[0062]

[0060] As illustrated in figure 5, the provision of two thermally distinct zones, i.e. where the first portion 210 is maintained at a higher temperature relative to the temperature at the second portion 220, may facilitate sublimation of water molecules from the frozen biological matter 100 into water vapor at the first portion 210 and subsequently desublimation or deposition of the water vapor into ice 110 at the second portion 220. The higher temperature at the first portion 210 results in a higher water vapor pressure over the frozen biological matter 100 in contrast to the lower temperature and hence a lower water vapor pressure at the second portion 220. Furthermore, this temperature-induced water vapor pressure gradient along the inside of the flexible receptacle 200 from the first portion 210 towards the second portion 220, creates a driving force for the flow of water vapor from the first portion 210 to the second portion 220. The resulting pressure differential thus facilitates directional water vapor transport and phase transition within the closed environment of the flexible receptacle 200.

[0063]

[0061] The heating and / or cooling elements 250, 260 may be planar, curved and / or have a size and shape that conforms to at least a part of the first portion 210 and second portion 220, respectively. The heating and / or cooling elements 250, 260 are preferably dimensioned to provide uniform temperature distribution across a surface area covering at least a part of the first portion 210 and the second portion 220, respectively. The heating element 250(s) may furthermore include, but is not limited to, resistive heating coils, thermoelectric modules or circulating, fluid-based thermal jackets. The cooling element 260(s) may comprise thermoelectric coolers, or cryogenic coils. The heating and cooling elements 250, 260 may furthermore be controlled via a feedback-regulated temperature control system.

[0062] In a preferred example of the method according to the invention, the freeze drying of the frozen biological matter 100 may be performed by way of exposing the outside of the flexible receptacle 200 to a pressure P. The pressure P may be lower than the atmospheric pressure, preferably lower than the water vapor pressure. The step of lowering the pressure P may further be performed by introducing the flexible receptacle 200 into a pressure chamber before lowering the pressure P. The chamber may furthermore be configured to receive the flexible receptacle 200 and to exert a pressure P inside the chamber.

[0064]

[0063] As will be appreciated by a person skilled in the art with knowledge of the present invention, the chamber may form part of an apparatus for a freeze- drying system, wherein the freeze dryer may further comprise any one or more of a process condenser, cooling system, vacuum system, and a control unit. Optionally, the chamber may form part of an apparatus configured solely to exert an internal pressure in the chamber, without incorporating additional functionalities typically associated with freeze-drying equipment.

[0065]

[0064] The flexible receptacle 200 containing the frozen biological matter 100 may further according to the method of the present invention, once positioned inside the chamber and at a pressure P lower than the vapor pressure of water, undergo a step of freeze drying until a freeze-dried biological matter 100 with a predetermined humidity is obtained. A predetermined humidity may here for example be obtained by performing the step of freeze drying for a predetermined period of time under a set of predetermined conditions, e.g., pressure, temperature and time. As will be appreciated by a person skilled in the art, the predetermined humidity may be obtained by routine optimization for the operating conditions of the apparatus employed, and the properties and amount of the frozen biological matter 100 to be freeze dried.

[0066]

[0065] During the step of freeze drying the frozen biological matter 100, water molecules will sublimate from the frozen biological matter 100 at the first portion 210 and then desublimate into ice 110 at the second portion 220, as previously described. The provision of a pressure P outside the flexible receptacle lower than the internal pressure of the flexible receptacle, may cause the flexible receptacle 200 to expand.

[0067]

[0066] The step of lowering the pressure P in the chamber to a pressure lower than the vapor pressure of water may herein comprise lowering the pressure P in the chamber to a pressure lower than the water pressure caused by the presence of the biological matter 100. The pressure P in the chamber may in other words be lowered to a pressure that is lower than the pressure inside the flexible receptacle 200, more specifically, the pressure P in the chamber may be lowered to a pressure that is lower than the vapor pressure of water at the surface of the frozen biological matter 100.

[0068]

[0067] The lowered pressure on the inside of the flexible receptacle 200 combined with two thermally distinct zones where the temperature at the first portion 210 is higher relative to the temperature at the second portion 220, and conversely a reduced water vapor pressure at the second portion 220 compared to the first portion 210, enables an efficient freeze drying of the frozen biological matter 100 where water molecules sublimate from the frozen biological matter 100 at the first portion 210 and desublimate into ice 110 at the second portion 220 (see figure 5). More specifically, the increased volume and the decreased pressure P inside the flexible receptacle 200 together with the temperature-induced water vapor pressure gradient, allows for an efficient driving force of the flow of water vapor form the first portion 210 towards the second portion 220.

[0069]

[0068] Once a pressure P outside the flexible receptacle 200 is lower than the internal pressure of the flexible receptacle 200, the flexible receptacle 200 containing the frozen biological matter 100 may be opened, thereby providing an outlet opening 240. As schematically illustrated in figure 6 showing a cross section of the second portion 220, the outlet opening 240 is positioned in a distal region 206 (see also figure 1) of the flexible receptacle 200, located furthest away from the frozen biological matter 100. The outlet opening 240 is here provided with a filter and / or a gas permeable membrane 242 arranged on the inside of the interior of the flexible receptacle 200 in a region between the outlet opening 242 and the frozen biological matter 100. The filter 242 is configured to allow any inert gas(es) inside the flexible receptacle 200 to pass through, whereas the water vapor predominantly condense at the second portion 220 within the flexible receptacle 200. The outlet opening 240 may be opened before or during freeze drying by way of cutting. Cutting may be made in straight cut line or generally in any type of shape. Once opened, the frozen biological matter will then according to the method of the present invention undergo a step of freeze drying in the freeze dryer until a freeze-dried biological matter with a predetermined humidity is obtained. Alternatively, as illustrated in figures 8A to 8C when the flexible receptacle 200 comprises one or more joints 232, the outlet opening 240 may be provided by a force such that the one or more joints 232 along the area 232 eventually yield, thereby providing the outlet opening 240.

[0070]

[0069] Figure 6 also illustrates how a filter and / or gas permeable membrane 242 may be positioned within a sealed internal barrier 244 disposed between the frozen biological matter 100 and the outlet opening 240. The barrier 244 is secured to the inner surface of the flexible receptacle 200 such that any gas originating from the region containing the biological matter 100 must pass through the filter and / or membrane 242 prior to exiting through the outlet opening 240. The barrier 244 may be configured in various alternative geometries, provided it establishes a substantially gas-tight separation that ensures directed gas flow through the filter or membrane 242. The combination of the outlet opening 240 and the membrane 242, in conjunction with condensation occurring within the flexible receptacle 200, serves to enable the controlled release of inert gases. This prevents the accumulation of such gases from impeding the drying process. Accordingly, only a relatively small outlet opening 240 and / or a compact membrane 242 is required to facilitate the effective removal of inert gases from the flexible receptacle 200.

[0071]

[0070] As schematically illustrated in figure 7, the flexible receptacle may alternatively be opened, once a pressure P outside the flexible receptacle 200 is lower than the internal pressure of the flexible receptacle 200, to allow water vapor to escape the flexible receptacle 200 during freeze drying. In this example, water molecules will sublimate from the frozen biological matter 100 and then flow out of the flexible receptacle 200 through the outlet opening 240, before optionally being evacuated out of the freeze dryer or condensed inside the freeze dryer using one or more second cooling elements 600, for example a condenser. Sine the outlet opening 240 is sufficiently large to allow water vapor from the frozen biological matter 100 to escape, the internal pressure within the flexible receptacle 200 does not exceed the level required to expand the flexible receptacle 200, overcome frictional resistance to vapor flow, and counteract the dynamic forces generated by the water vapor. As a result, the flexible receptacle 200 does not become fully inflated, but expands only to the extent necessary to permit a continuous flow of water vapor from the flexible receptacle 200.

[0072]

[0071] As illustrated in figure 9, once a freeze-dried biological matter 100 with a predetermined humidity is obtained, the flexible receptacle 200 is according to the present invention sealed by a sealing unit 234 between the first portion 210 and the second portion 220 while the flexible receptacle 200 is maintained inside the chamber. As schematically illustrated in figure 11, the seal can be made between the first portion 210 and the second portion 220 along the stapled portion line 204. The flexible receptacle 200 may here be sealed at the same pressure P in the chamber as was used during the step of freeze drying, or more generally with a pressure P in the chamber that is lower than the pressure inside the flexible receptacle 200. In the alternative example of the invention where the flexible receptacle 200 is opened during freeze drying and as illustrated in figures 6 and 7, the flexible receptacle 200 is sealed using sealing unit(s) 234 in the same manner as illustrated in figure 9 and figure 11. During this sealing process, water vapor continues to flow through the gap between the sealing unit(s) 234 until the opening is fully closed. After sealing, no further vapor from the freeze-dried biological matter 100 can escape, causing the first portion 210 to inflate while the remainder of the flexible receptacle 200 collapses. When air is reintroduced into the chamber, the first portion 210 collapses, enclosing the freeze-dried biological matter 100 in a sealed flexible receptacle 200, with no functional difference between whether the drying occurred with condensation inside or outside the flexible receptacle 200.

[0073]

[0072] As will be appreciated by a person skilled in the art, the step of sealing the flexible receptacle 200 may here be understood as a process of closing the opening of the flexible receptacle 200 securely. More specifically, the step of sealing the flexible receptacle 200 may be understood as a process where a fluid communication between the freeze-dried biological sample at the first portion 210 and the second portion 220 is removed so that free flow of water vapor or other gases no longer may occur through the internal opening. The step of sealing the flexible receptacle 200 may for example be performed by clamping or heat sealing or similar, and may be performed by a clamp 230, a sealing unit 234, such as a heat-sealing unit, arranged inside the chamber.

[0074]

[0073] As schematically illustrated in figures 1 to 6, the flexible receptacle 200 may comprise at least one valve. The flexible receptacle 200 may for example comprise an entry valve 290 configured to being employed for introducing the fluid biological matter 100 into the flexible receptacle 200. The entry valve 290 may here be configured to be connected to a suitable filling equipment, for example equipment configured to transfer blood plasma between receptacles. The flexible receptacle 200 may additionally or alternatively comprise a single valve 276 configured to being employed for introducing a hydrating fluid and configured for being employed for extracting hydrated freeze-dried biological matter 100 from the flexible receptacle 200. Alternatively or in addition, the flexible receptacle may comprise an inlet valve 275 configured to being employed for introducing a hydrating fluid, such as water, into the flexible receptacle 200. The inlet valve 275 may thus be employed to hydrate the freeze- dried biological matter 100 subsequent to having performed the method according to the present invention. In this latter example, the flexible receptacle 200 may additionally comprise a drain valve 270 configured to being employed for extracting hydrated freeze-dried biological matter 100 from the flexible receptacle 200 subsequent to having performed the method according to the present invention.

[0075]

[0074] The flexible receptacle 200 may, as schematically illustrated in figure 1 comprise both an inlet valve 275 and a drain valve 270, which may enable hydration of the freeze-dried biological matter 100 to occur simultaneously with extraction of hydrated freeze-dried biological matter 100 from the flexible receptacle 200. The presence of an inlet valve 275 and a drain valve 270 is particularly preferable when the freeze-dried biological matter 100 is freeze-dried blood plasma, and it is desirable to inject said freeze-dried blood plasma into a severely injured human or animal. Water may here be introduced through the inlet valve 275 simultaneously with extracting a water-diluted blood plasma from the drain valve 270. As it will inevitably take some time to dissolve the freeze-dried blood plasma, injection of diluted blood plasma into the severely injured human or animal may start immediately, where the concentration of the diluted blood plasma increases over time until the freeze-dried blood plasma is fully hydrated. The method according to the present invention may thus comprise a step of rehydrating the freeze-dried biological matter 100, for example using water or more specifically, a saline solution.

[0076]

[0075] The flexible receptacle 200 may generally according to the present invention be a bag, more specifically a blood bag, and may additionally or alternatively be made of a polymer, for example polyethylene, polypropylene, polyvinyl chloride. However, as will be appreciated by a person skilled in the art with knowledge of the present invention the flexible receptacle 200 may be made from other suitable materials. The flexible receptacle 200 is preferably made from a material or a plurality of materials that enable the flexible receptacle 200 to expand, be heat sealed and / or transparent. The flexible receptacle 200 may be made from a laminate, optionally with at least one layer being made from a metal. The flexible receptacle 200 may be cleaned and / or sterilized prior to use or may undergo a cleaning and / or sterilization procedure as a part of its production process and may optionally be stored in a sterile environment prior to use.

[0077]

[0076] As schematically illustrated in figure 2, the flexible receptacle 200 may comprise means for enabling the flexible receptacle 200 to be hung onto a hook, and a drain valve 270 for removing hydrated freeze-dried biological matter 100 from the flexible receptacle 200. Here the drain valve 270 is preferably arranged such that the drain valve 270 faces downwards when the flexible receptacle 200 is hung onto a hook, which may enable the hydrated freeze-dried biological matter 100 to be extracted from the flexible receptacle 200 by means of gravity. The hydrated freeze-dried biological matter 100 may thus be removed without the need for an external pump or the like, which may be preferable in situations where such equipment is not available, or power is not available. The means for enabling the flexible receptacle 200 to be hung onto a hook may for example be a sealed off hole 280 in the flexible receptacle 200, an externally mounted loop, or an externally mounted hook.

[0078]

[0077] Figures 10A to 10C schematically illustrates sections of a flexible receptacle and examples of arrangements at or near a valve 276. Figure 10A schematically illustrates an arrangement 300 such as a protective cover that protects the valve 276 from being contaminated. The protective cover may comprise a tab 301 configured to be easily grasped by a user, thereby facilitating the removal or opening of the protective cover when the valve is to be activated. The protective cover may further comprise a tear guide 302 configured to serve the same function as the tab 301. Furthermore, the protective cover 300 may comprise a filter or a gas-permeable membrane 310 configured to allow release of any trapped gas that may expand and potentially rupture the protective cover 300 during freeze drying. The membrane 310 may be of a type that prevents the ingress of harmful contaminants, thereby maintaining the sterility and integrity of the internal environment. Alternatively or in addition, the protective cover 300 may be vacuum-sealed to ensure that it contains insufficient gas to cause rupture during freeze drying.

[0079]

[0078] In figure 10B, the flexible receptacle 200 may comprise a single valve 276 for introducing liquid into the flexible receptacle 200 and for removing hydrated freeze-dried biological matter 100 from the flexible receptacle 200 and a filter 272 arranged adjacent to the valve 276. Alternatively, although not illustrated, the filter 272 may alternatively or in addition be arranged adjacent to the drain valve 270. The filter 272 is here configured to block non-hydrated particles of freeze-dried biological matter 100 from flowing through the valve 276, 270. The employment of such a filter 272 is preferable in situations where the freeze-dried biological matter 100 is blood plasma, and the freeze-dried blood plasma is needed in a time of emergency to treat a severely injured human or animal. Saline water may here for example be used to hydrate the freeze-dried blood plasma, and the severely injured human or animal may thus initial be injected with mainly saline water before more and more of the freeze-dried blood plasma is hydrated and thereafter injected. The filter 272 may in the latter case hinder non-hydrated particles of freeze-dried blood plasma to be injected into the severely injured human or animal.

[0080]

[0079] Figure 10C schematically illustrates the single valve 276 operatively connected to a dual-function device 500 enabling both the introduction of rehydration fluid and the extraction of rehydrated biological matter 100. The device comprises a coupling unit 510 configured to establish fluid communication with the interior of the flexible receptacle 200 and to open access to the freeze-dried biological matter 100. A conduit 520 extends from the coupling unit and bifurcates at a junction 530 into a first branch 522 connected to a reservoir containing rehydration fluid, and a second branch 524 leading to a point of administration. A first and second control element 550, 560, such as clamps or valves, are disposed on each branch to regulate fluid flow. In this example, rehydration may be initiated by opening the first flow control element 550, allowing fluid to enter the flexible receptacle 200. Upon completion, the first branch is closed, optionally via a one-way valve to prevent backflow. The second flow control element 560 is then opened to permit withdrawal of rehydrated biological matter 100. Optionally, the device 500 may be replaced by a three-way valve connected to the coupling unit 510. Although figures 10A to 10C only illustrate a single valve 276, a drain valve 270 and an inlet valve 275 may, alternatively or in addition, also be employed.

[0081]

[0080] Residual water and oxygen are generally considered factors that limit the shelf life of the freeze-dried biological matter 100, particularly freeze-dried blood plasma. Oxygen may react with the freeze-dried biological matter 100 and may cause the freeze-dried biological matter 100 to deteriorate. In order to reduce the oxidation of the freeze-dried biological matter 100, some examples include limiting any oxygen diffusion through the flexible receptacle 200 and limiting the availability of oxygen that may diffuse through the flexible receptacle 200. The latter may be particularly preferable if the flexible receptacle 200 is made from a polymer or plastic, as some oxygen diffusion through these materials may occur. The method according to the present invention may for example comprise the additional steps of placing the flexible receptacle 200 comprising the freeze-dried biological matter 100 inside a additional receptacle 400 and sealing the additional receptacle 400. The additional receptacle 400 may here be flexible, and may optionally comprise a metal foil, e.g., comprise a laminate comprising a metal foil. The use of a metal foil may here be preferable, as a metal foil may have a low diffusivity of oxygen. Optionally or alternatively, the additional receptacle 400 may be made from a specific material or comprise a foil made from a specific material, where said specific material has a lower diffusivity to oxygen than the flexible receptacle 200.

[0082]

[0081] The employment of a additional receptacle 400 may, prior to the step of sealing the additional receptacle 400, further be combined with any one or more of the steps of introducing an oxygen scrubber 410 into the additional receptacle 400, evacuating the additional receptacle 400, and / or introducing a non-oxidizing gas into the additional receptacle 400. By reducing the amount of oxygen inside the sealed additional receptacle 400, any diffusion of oxygen through the flexible receptacle 200 may be reduced, which consequently further may reduce the oxidation of the freeze-dried biological matter 100. Figure 11 schematically illustrates a flexible receptacle 200 arranged inside an additional receptacle 400 where an oxygen scrubber 410 is arranged inside the additional receptacle 400.

[0083]

[0082] The handling and transport of the flexible receptacle 200 may generally throughout the method according to the present invention be manual, or automatic. The flexible receptacle 200 may for example be aligned according to a suitable cutting tool and aligned according to a filling unit for introducing a fluid biological matter 100 into the flexible receptable 100. The flexible receptable 100 may thus be positioned with a certain arrangement by suitable handling means, such as a robotic handling and transport system, such that further manual interaction with the flexible receptacle 200 may be avoided before the method is completed. The latter may have the advantage of reducing the risk of contamination of the biological matter 100. The method may further comprise an initial step of positioning the flexible receptacle 200 in a predetermined position on a carrier. The carrier may be handled manually or automatically, i.e., be moved in and out of the chamber etc. and may further facilitate for the handling of multiple flexible receptacles 200 at once. List of reference numerals:

[0084] 100 Biological matter 200 Flexible receptacle 202 Stapled closing line 204 Stapled portion line 206 Distal region of the second portion 210 First portion 220 Second portion 230 Clamp / Temporary seal 232 Joint(s) / temporary seal(s) / septum 233 Spike

[0085] 234 Sealing unit / heat-sealing unit 240 Outlet opening / cut opening 242 Filter / gas permeable membrane 244 Barrier / Internal barrier 250 Heating element 260 Cooling element 270 Drain valve 272 Filter 275 Inlet valve 276 Single valve 280 Means / sealed off hole 290 Entry valve 300 Arrangement / Protective cover 301 Tab

[0086] 302 Tear guide 310 Gas-permeable membrane / membrane / filter 400 Additional receptacle 410 Oxygen scrubber 500 Dual-function device 510 coupling unit 520 Conduit 522 First branch 524 Second branch 530 Junction 550 First flow control element 560 Second flow control element 600 Second cooling element(s)

Claims

Claims1. A method for freeze-drying biological matter (100), the method comprising the steps of introducing a fluid biological matter (100) into a first portion (210) of a flexible receptacle (200), closing the flexible receptacle (200), freezing the fluid biological matter (100), thereby obtaining a frozen biological matter (100), placing the flexible receptacle (200) in a freeze dryer, lowering the pressure in the freeze dryer to a pressure lower than the vapor pressure of water, wherein the flexible receptacle (200) remains sealed at least until it inflates, freeze drying the frozen biological matter (100) by providing heat to the frozen biological matter (100) such that a vapor pressure at the frozen biological matter (100) is larger than the vapor pressure at one or more cooling element(s) (260, 600) in gas communication with the frozen biological matter (100), thereby obtaining a freeze-dried biological matter (100) in the first portion (210) and frozen water at the one or more cooling element(s) (260, 600), and sealing the first portion (210) off from a second portion (220) of the flexible receptacle (200) not containing the freeze-dried biological matter (100).

2. The method according to claim 1, wherein the method comprises an initial step of delimiting the first portion (210) from the second portion (220), preferably by clamping the flexible receptacle (200).

3. The method according to claim 2, wherein, after the step of freezing the fluid biological matter (100), the method further comprises a step of providing a fluid communication between the first portion (210) and the second portion (220), preferably by unclamping the flexible receptacle (200).

4. The method according to any of the preceding claims, wherein, during the step of providing heat to the frozen biological matter (100), the method further comprises the step of positioning a heating element (250) at the first portion (210).

5. The method according to any of the preceding claims, wherein, during the step of providing heat to the frozen biological matter (100), the method further comprises the step of cooling the second portion (220) by positioning a cooling element (260) at the second portion (220), thereby obtaining freeze dried biological matter (100) in the first portion (210) and frozen water in the second portion (220).

6. The method according to any of the preceding claims, wherein the method further comprises the step of providing an outlet opening (240) at the second portion (220) and wherein a filter and / or a gas permeable membrane (242) is arranged within the interior of the flexible receptacle (200) in a region between the outlet opening (240) and the first portion (210) such that any inert gas(es) inside the flexible receptacle (200) may pass through the filter and / or the gas permeable membrane (242), and wherein the filter and / or the gas permeable membrane (242) and / or the outlet opening (240) is(are) configured to restrict the outflow of gas such that water vapor predominantly condense at the second portion (220) within the flexible receptacle (200).

7. The method according to any of the claims 1 to 5, wherein the method further comprises the step of providing an outlet opening (240) at the second portion (220) such that water vapor may escape the flexible receptacle (200) and predominantly condense outside the flexible receptacle (200) at one or more second cooling element(s) (600).

8. The method according to any of the preceding claims, wherein the method further comprises, after the step of sealing the first portion (210) off from the second portion (220), a step of separating the first portion (210) from the second portion (220).

9. The method according to any one of the preceding claims, wherein the flexible receptacle (200) comprises a drain valve (270) for removing hydrated freeze-dried biological matter (100) from the first portion (210), and an inlet valve (275) for introducing liquid into the first portion (210) wherein the drain valve (270) and the inlet valve (275) are arranged at the first portion (210).

10. The method according to claim 9, wherein the flexible receptacle (200) further comprisesa filter (272) arranged at the drain valve (270), wherein the filter (272) is configured to block non-hydrated particles of freeze- dried biological matter (100) from flowing through the drain valve (270).

11. The method according to any one of the preceding claims, wherein the flexible receptacle (200) comprises means for enabling the flexible receptacle (200) to be hung onto a hook, wherein the drain valve (270) is arranged such that the drain valve (270) faces downwards when the flexible receptacle (200) is hung onto the hook.

12. The method according to any one of the preceding claims, wherein the method further comprises the steps of placing the flexible receptacle (200) comprising the freeze-dried biological matter (100) inside an additional receptacle (400), and- sealing the additional receptacle (400), wherein the additional receptacle (400) optionally comprises a metal foil.

13. The method according to claim 12, further comprising any one or more of the steps of introducing an oxygen scrubber (410) into the additional receptacle (400), evacuating the additional receptacle (400), and / or introducing a non-oxidizing gas into the additional receptacle (400), prior to the step of sealing the additional receptacle (400).

14. The method according to any one of the preceding claims, further comprising the step of rehydrating the freeze-dried biological matter (100).

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