Patent Description:
Biological materials are produced industrially in large batches that are stored for later use as needed, providing this way great management flexibility. In many cases, the biological materials are obtained as aqueous solutions, which are stored frozen with two main objectives: increase the shelf-life of the product and facilitate its transport. Usually, the produced batches are split in smaller amounts and place inside bottles, carboys and bags for storing, transporting, freezing and thawing. However, freezing, handling and transportation of containers at low temperature presents several risks, such as the degradation of the biological material and/or container rupture.

Currently, the freezing of biological materials involves placing a container (bottles and/or carboys) comprising the biological materials in a cabinet or chest freezer and allowing the biological materials to freeze. In other techniques, a moldable container (bags) enclosing biological materials is placed on a solid or wire-frame shelf in the cabinet or chest freezer. However, problems exist in such freezing techniques as currently configured.

At low temperatures, the physical properties of the plastics materials of the containers may change, leading to their fragility and consequently can reduce the containers' ability to absorb external forces, i.e., shocks without fracturing. Also, the volumetric expansion of the ice inside the containers can cause significant mechanical stress, leading to a container, tubing or connector break. Moreover, the heat transfer in the top of the containers, both by convection and radiation, can also lead to the formation of an ice-crust, consisting on an ice layer at the top of the liquid, at the air interface, in the head-space region of the containers, contributing to the cryoconcentration and increasing pressure in the containers and consequently resulting in their damage or rupture.

Rupture or damage to the integrity of the containers is undesirable, as it can compromise sterility or lead to contamination or leakage or loss of the biological material. The storing and transportation processes also present some hazard risks since one is dealing with fragile containers that were previously submitted to the freezing process, which can damage or induce mechanical failure. While it is well known that the containers and freezing technologies currently available do not adequately protect the frozen products, the pharmaceutical industry has not been adequately documented the incidence of containers damage during the freezing process.

Systems and methods for freezing, storage and transport of moldable containers containing biological materials, has been already disclosed in order to protect such containers from damage or mechanical failure. For example, the document <CIT> disclosed a system for freezing, thawing, transporting, and storing biopharmaceutical materials, which includes a container, a supporting structure, a temperature control unit, and a transportation cart. The supporting structure is configured to support a container of biopharmaceutical material and the transportation cart includes channels configured to receive supporting structures, such as frames. The frame is configured to receive and support bags in the vertical position. Also, the document <CIT> disclosed a system for cooling, freezing, preserving, processing and thawing biopharmaceutical materials. This system includes a moldable container configured to contain the biopharmaceutical materials and to be supported by a supporting and/or protective structure, such as a holder. The holder may have a pillow-shape and acts as a protector, supporting structure or frame for supporting a moldable container during filling, transport, storage, and/or freezing of biopharmaceutical materials. The document <CIT> also relates to a housing for a moldable container for transporting liquids, which is at least partially coated with an elastic foam. <CIT> discloses a system for maintaining temperature during transport of a moldable container such as a blood bag. The system comprises two complementary identical elements that can be joined to enclose the moldable container, wherein each element comprises a phase change material between two walls of a thermally insulating material. <CIT> discloses a container for holding temperature-sensitive items, e.g. in vials, wherein the container comprises a removable cover comprising a moderator material such as a phase change material.

Although there are already systems and methods that protect the moldable containers, mainly bags, during the freezing, transport, storage and thawing processes, these systems do not avoid the problem of heat transfer on the top of the containers that leads to the formation of an ice-crust, which leads to cryoconcentration and increased pressure in the containers, resulting in their damage or rupture. Moreover, a system capable of avoiding such problems in rigid containers, such as bottles and/or carboys comprising biological materials, does not yet exist. The present disclosure aims at solving the above-mentioned problems.

The invention is defined in the claims. In a first aspect, this disclosure discloses a device for freezing or thawing an aqueous biological solution, shaped to fit the top of a container, comprising:.

In an embodiment the internal and external walls are continuous, thus forming a single unit.

In an embodiment the phase change material is a pure liquid or liquid mixture, preferentially with a freezing temperature between -<NUM> and <NUM>, more preferentially between -<NUM> °Cand <NUM>.

In accordance with the invention the internal wall further comprises a moldable thermal insulating material, wherein the thermal insulating material of the internal wall is moldable to form an air-tight seal over the container opening.

In an embodiment the thermal insulating material of the internal and external walls are different.

In an embodiment the thermal insulating material of the internal and external walls comprises a low thermal conductivity material.

In an embodiment the thermal insulating material of the internal and external walls comprises a thermal conductivity of less than <NUM> W m-<NUM> K-<NUM>.

In an embodiment the thermal insulating material of the internal and external walls are plastic or polymer, such as poly-ethylene, polypropylene, polycarbonate, polylactic acid, or combinations thereof.

In an embodiment the volume of phase change material in the internal cavity is not more than <NUM>% of the volume of the aqueous biological solution, preferably wherein the volume of phase change material in the internal cavity is not more than <NUM>% of the volume of the aqueous biological solution.

In an embodiment the phase change material is water, a mixture of water and ethylene glycol, a mixture of water and sodium chloride, a mixture of water and ethanol, combinations thereof, among others solutions.

The phase change material may further comprise a nucleating agent, such as fine particles of silver iodide, lead iodide, or combinations thereof.

In an embodiment the moldable material is a resilient or a soft material, preferably extruded polystyrene foam, polyurethane foam, polychloroprene or acrylonitrile butadiene rubber, or combinations thereof.

In an embodiment the device is configured to cover the top of a container, and preferably <NUM>% of the height of aqueous biological solution,.

In an embodiment the device is configured to cover the top of a bottle, a vial, a tube, a bag or similar.

In another embodiment the invention discloses a kit as defined in claim <NUM>, comprising an ice-crust attenuator device and a holder.

The holder may be made of a plastic, polymer or other material having low thermal conductivity.

In a further embodiment, the holder comprises one or more surfaces made of a metal, alloy or a high thermal conductivity polymer, preferentially made of a material with a thermal conductivity higher than <NUM> W m-<NUM> K-<NUM>.

These and other objects, features and advantages of the disclosure will be evident from the following detailed description when read in conjunction with the accompanying drawings.

In this section, the fundamentals of the operation of the object of disclosure and of proposed embodiments will be described.

As presented above, many variables contribute to the rupture or damage of the containers during the freezing process, which can result in the degradation or loss of the biological material. The present disclosure describes devices for freezing, transporting, storing and thawing aqueous solutions of biological materials aiming to solve the above-mentioned problems.

It was observed that one of the main problems in the freezing process is the formation of an ice-crust at the top of the liquid, at the air interface, in the head-space region of the containers, due to the heat transfer, by convection and radiation, in the top of the containers (<FIG>). The ice-crust is defined as the thick layer of ice formed on the surface of the liquid and air interface, usually characterized by a "pyramidal" shape (<FIG>). This ice-crust leads to the increasing pressure in the containers, as shown in <FIG>, and consequently resulting in their damage or rupture and loss of the biological material.

We herein disclose that in order to freeze aqueous solutions of biological materials in a container avoiding such problems, it is necessary to have an insulator in the top of the container with heat resistance or with controlled heating to maintain the top part of the container under insulated conditions, avoiding the formation of a top ice-crust, as shown in <FIG>.

Therefore, the present disclosure discloses systems that allow the improvement of the freezing process of aqueous solutions of biological materials avoiding the ice-crust formation and the issue of increasing pressure inside the containers, while preventing cryoconcentration and the damage or rupture of the containers.

In an exemplary embodiment depicted in <FIG> and <FIG>, an ice-crust attenuator device <NUM> installed on a container of fixed shape <NUM> for freezing, transporting, storing and thawing aqueous solutions of biological materials is shown. The system includes the ice-crust attenuator device <NUM> configured to attach to the head-space <NUM> of a container of fixed shape <NUM> containing aqueous solutions of biological materials.

Biological materials may comprise protein, amino acid and peptide formulations, DNA, RNA and nucleic acid solutions, cell suspensions, tissue suspensions, cell aggregates suspensions, cell growth media, serum, biologicals, blood products, preservation solutions, fermentation broths, and cell culture fluids with and without cells, mixtures of the above and their fragments.

In the present disclosure the container of fixed shape <NUM> configured to contain aqueous solutions of biological materials can take several shapes and structural characteristics, such as bottles or carboys. Such container of fixed shape <NUM> should maintain its shape when empty and do not significantly deform when filled with product. Said container of fixed shape <NUM> can be made of a rigid and biocompatible material to promote compatibility with biological materials. The rigid materials can be, for instance, glass, polyethylene terephthalates, polycarbonate, polytetrafluoroethylene, polyethylene, polyesters, polyamides, polypropylenes, ethylene-vinyl alcohol copolymer, polyvinylidenefluoride, polyvinylchlorides, and copolymers, mixtures or laminates that comprise the above. Said container of fixed shape <NUM> may vary in size and volumetric capacity. In a preferred embodiment, container of fixed shape <NUM> has a volumetric capacity in a range from approximately <NUM> to approximately <NUM>, preferably in a range from approximately <NUM> to approximately <NUM> and more preferably in a range from approximately <NUM> to approximately <NUM>. Said container of fixed shape <NUM> configured to contain aqueous solutions of biological materials can comprise a head-space region <NUM> and one cap <NUM>. Said cap <NUM> may take several forms, with at least one port with tubing <NUM> for aseptic filling and venting operations.

The embodiment depicted in <FIG> comprises an ice-crust attenuator device <NUM> with heat resistance or with controlled heating configured to attach to the head-space <NUM> of the container of fixed shape <NUM>. The main purpose of the ice-crust attenuator device <NUM> is to prevent the formation of the ice-crust that leads to the increasing pressure inside the containers and consequently resulting in their damage. Thus, the ice-crust attenuator device <NUM> has two main functions that allow the desired effect to be achieved (do not form the ice-crust avoiding the damage of the containers): a) eliminate the loss of heat at the interface of the liquid by radiation and b) do not let the air in the head-space <NUM> of the container cool during the freezing period through an external insulation and a specific volume of phase change material (PCM). The ice-crust attenuator device <NUM> is configured to attach to the head-space <NUM> region of a container of fixed shape <NUM> with defined volumetric capacity, in order to cover the head-space <NUM> region and preferentially <NUM>% of the total height of aqueous solution of biological materials.

In the embodiment depicted in the <FIG>, the ice-crust attenuator device <NUM> has an external wall made of an insulating material <NUM>, such as plastic, polymer or other material having low thermal conductivity. Preferentially, the thermal insulating material <NUM> can be any material with a thermal conductivity less than <NUM> W m-<NUM> K-<NUM>, such as poly-ethylene, polypropylene, polycarbonate, polylactic acid. To assure that the air in the head-space <NUM> of the container does not cool during the freezing period, the ice-crust attenuator device <NUM> has an internal cavity <NUM> arranged to be filled with a phase change material (PMC) to improve the thermal insulation.

In an embodiment, the Phase Change Material (PCM), preferably, is a pure liquid or liquid mixture with a freezing temperature identical to the one of the biological material solution, which lies typically between -<NUM> and <NUM>. The PCM can be, for instance, a mixture of water and ethylene glycol, a mixture of water and sodium chloride, or a mixture of water and ethanol, provided that the phase change material has the same osmolality of the aqueous solution of biological materials. Moreover, the PCM may further comprise a nucleating agent, such as fine particles of silver iodide or lead iodide, to ensure that the phase change material will not supercool during the freezing process. The internal cavity <NUM> can be filled with the PCM through a port <NUM>, which is subsequently closed with a plug. The ice-crust attenuator device <NUM> should be configured with a determined design to assure that the quantity of PCM is not higher than <NUM>% of the volume of the aqueous solutions of biological materials, preferentially not higher than <NUM>% of the volume of the aqueous solutions of biological materials. The quantity of PCM can be calculated based on the PCM used, on the thickness and type of insulating material <NUM>, on the total area to insulate, and external heat transfer coefficient. For example, the ice-crust attenuator device <NUM> depicted in <FIG> was designed to be used in a <NUM> square bottle. The insulating material <NUM> chosen was polylactic acid (PLA) with a wall thickness of <NUM>. Therefore, to freeze an aqueous solution during <NUM>, the minimal amount of PCM should be approximately <NUM>.

In the embodiment depicted in the <FIG>, the ice-crust attenuator device <NUM> has an internal wall made of a low thermal conductivity material. In accordance with the invention, the internal wall can be made of a moldable material <NUM> configured to attain a good thermal contact between the ice-crust attenuator device <NUM> and the outer surface of the head-space <NUM> of the container of fixed shape <NUM> to ensure that there is no air within the two surfaces. The better the thermal contact between the ice-crust attenuator device <NUM> and the outer surface of the head-space <NUM> of the container of fixed shape <NUM>, the better the insulation. Accordingly, pressing the head-space <NUM> of the container of fixed shape <NUM> against the moldable material <NUM> improves the quality and repeatability of thermal contact, enhancing the thermal insulation. Said moldable material <NUM> may be made of any resilient or soft material, preferentially, with low thermal conductivity, such as extruded polystyrene foam, polyurethane foam, polychloroprene or acrylonitrile butadiene rubber. Said moldable material <NUM> may be attached to the ice-crust attenuator device <NUM> by means of compatible adhesive materials, by mechanical means or by magnetic contact using magnetic materials for that purpose.

In another embodiment depicted in <FIG>, the ice-crust attenuator device <NUM> can be split in two bodies to be easily connected and/or removed from the container of fixed shape <NUM>. This feature associated with a suitable and effective locking system <NUM>, can also be used to compress the ice-crust attenuator device <NUM> against the container of fixed shape <NUM>. This embodiment promotes the compression to obtain satisfactory thermal contact and air tightness. Therefore, it is important that both bodies are closely connected and locked to assure the desired functions of the ice-crust attenuator device <NUM>. The two parts of the ice-crust attenuator device are connected and locked by means of locking system <NUM>. This locking system <NUM> can be standard methods, such as pins, springs, hinges, pivots, or other means to lock.

The ice-crust attenuator device <NUM> previously described was tested to freeze a volume of <NUM> of a <NUM>% (m/V) sucrose aqueous solution in a Polyethylene terephthalate (PET) bottle of <NUM> (h) x <NUM> (w) x <NUM> (d) mm of dimensions. The test was performed with and without the ice-crust attenuator device <NUM> described above. The bottle was frozen inside a chamber with a vertical (unidirectional) flow of gas at <NUM>/s and -<NUM>, during <NUM>. <FIG> illustrates the common freezing process without the ice-crust attenuator device <NUM>, showing the formation of the ice-crust <NUM> with the typical "pyramidal" shape on the head-space <NUM> region of the bottle. After <NUM> of freezing it was observed the formation of the ice-crust, and after <NUM> the ice-crust <NUM> was completely formed, while in the center of the container the solution is still liquid. Moreover, the cryoconcentration in the center of the container was observed by using a dye. In turn, <FIG> illustrates the freezing process with the ice-crust attenuator device <NUM>. The ice-crust attenuator device <NUM> herein used has an internal cavity filled with a phase change material. <FIG> shows that the ice-crust attenuator device <NUM> promotes a controlled ice front formation, avoiding the formation of the ice-crust, characterized typically by a "pyramidal" shape, as it undergoes freezing until the total freezing of the solution. Test results have demonstrated that the device described previously can avoid the formation of the ice-crust and consequently decreasing the pressure inside the container. It was also evaluated the pressure inside a <NUM> PET bottle, with <NUM> (h) x <NUM> (w) x <NUM> (d) mm of dimensions, during freezing of a volume of <NUM> of <NUM>% (m/V) sucrose aqueous solution. The bottle was frozen inside a chamber with a vertical (unidirectional) flow of gas at -<NUM>, during <NUM>. <FIG> shows the increasing pressure inside the bottle during the freezing process without the ice-crust attenuator device <NUM>.

In an exemplary embodiment depicted in <FIG>, another ice-crust attenuator device <NUM> for freezing, transporting, storing and thawing aqueous solutions of biological materials is shown. This ice-crust attenuator device <NUM> should be used preferentially, when freezing, transporting, storing and thawing aqueous solutions of biological materials in moldable containers <NUM>. Said moldable container <NUM> configured to contain aqueous solutions of biological materials can take several forms of configuration, such as bags, and comprises at least tubing <NUM> at one end for aseptic filling and venting operations. The moldable container <NUM> can deform when filled with product and can be made of a biocompatible polymeric material to promote compatibility with biological materials. The biocompatible polymeric materials can be, for instance, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, polytetrafluoroethylene, polyethylene, polyesters, polyamides, polypropylenes, polyvinylidene fluoride, polyurethanes, polyvinylchlorides, and copolymers, mixtures or laminates that comprise the above. An advantage of the moldable container <NUM> relies on the intrinsic characteristic of conforming to the shape of a holder <NUM> (described below). This is important for promoting a good thermal contact and repeatability between the moldable container <NUM> and the ice-crust attenuator device <NUM>. The moldable container <NUM> may vary in size and volumetric capacity. In a preferred embodiment, moldable container has a volumetric capacity in a range from approximately <NUM> to approximately <NUM>, preferably in a range from approximately <NUM> to approximately <NUM> and more preferably in a range from approximately <NUM> to approximately <NUM>.

The ice-crust attenuator device <NUM>, depicted in <FIG>, has particularly relevance in a common freezing process, when a moldable container (bag) is placed directly in a cavity of a refrigerated chamber. Therefore, by having an ice-crust attenuator device <NUM> configured to be placed in the cavity and in the top of the container in contact with its upper surface, the upper face of the container is kept under insulated conditions avoiding the formation of a top ice-crust and consequently avoiding the damage of the container, as described previously. The ice-crust attenuator device <NUM> should have the same technical characteristics of the previously described ice-crust attenuator device <NUM>. The ice-crust attenuator device <NUM> can be made of an insulating material <NUM>, such as plastic, polymer or other material having low thermal conductivity. Preferentially, the thermal insulating material <NUM> can be any material with a thermal conductivity less than <NUM> W m-<NUM> K-<NUM>, such as poly-ethylene, polypropylene, polycarbonate, polylactic acid. In addition, the ice-crust attenuator device <NUM> has an internal cavity <NUM> arranged to be filled with a phase change material (PMC) to improve the thermal insulation.

The ice-crust attenuator device <NUM> may also comprise a moldable material <NUM>, as described previously. Said moldable material <NUM>, may be made, preferentially, of any resilient or soft material with low thermal conductivity, such as extruded polystyrene foam, polyurethane foam, polychloroprene or acrylonitrile butadiene rubber. The moldable material <NUM> is configured to be pressed against the upper surface of the moldable container <NUM>, promoting a good thermal contact between the ice-crust attenuator device <NUM> and the outer surface of the moldable container <NUM>, ensuring no air between the two surfaces. Said moldable material <NUM> can be attached to the ice-crust attenuator device <NUM> by means of compatible adhesive materials, by mechanical means or by magnetic contact using magnetic materials for that purpose.

In another embodiment depicted in <FIG>, the ice-crust attenuator device <NUM> may be connected to a holder <NUM> to accommodate the moldable container <NUM>. The advantage of having the holder <NUM> is to protect the moldable container <NUM> during freezing, transporting, storing and thawing aqueous solutions of biological materials, avoiding the damage of moldable container <NUM>. Said holder <NUM> can be made of a plastic, polymer or other material having low thermal conductivity.

In another embodiment, the holder <NUM> may also comprise one or more surfaces made of a metal, alloy or a high thermal conductivity polymer. Preferentially, is made of a material with a thermal conductivity higher than <NUM> W m-<NUM> K-<NUM>. Preferentially, the holder may comprise only a bottom surface that is made of a metal, alloy or a high thermal conductivity polymer, configured to attain a good thermal contact between the bottom of the holder and the bottom surface of the moldable container <NUM>, maximizing the heat transfer. An advantage of this embodiment is that, by keeping the ice-crust attenuator device <NUM> in the top of the holder and a heat transfer surface in the bottom, the aqueous solution of biological materials will freeze under unidirectional conditions from the bottom upwards. In the present disclosure unidirectional freezing, specifically unidirectional bottom-up freezing, means the creation of a unidirectional temperature gradient along the vertical axis that causes the ice-front to develop and progress from bottom to up of the container. The unidirectional bottom-up freezing allows the improvement of the freezing process of aqueous solutions of biological materials, preventing cryoconcentration and the damage or rupture of the containers.

In another embodiment depicted in <FIG>, it may be useful to freeze, store and thaw an aqueous solution of biological materials in a small-volume moldable container <NUM> at vertical position. However, freezing small-volumes using moldable containers, such as bags, can lead to the problems above mentioned (formation of the ice-crust and deformation of the container), and problems associated to quality and reproducibility. Therefore, as depicted in <FIG>, to avoid such problems, it may be useful freezing aqueous solution of biological materials in moldable container <NUM>, using a holder <NUM> comprising a heat transfer bottom <NUM> design to accommodate the moldable container <NUM> in a cavity <NUM>. The holder <NUM> has the heat transfer bottom <NUM> to considerably accelerating the heat transfer in the bottom of the moldable container <NUM>, increasing the reproducibility and scalability of freezing and nucleation of the aqueous solution of biological materials. The heat transfer bottom <NUM> can be made of a metal, alloy or a high thermal conductivity polymer. The heat transfer bottom <NUM> can hold a contacting fluid to enhance the thermal contact between the heat transfer bottom <NUM> and the bottom of the moldable container <NUM>, thus enhancing the reproducibility of the controlled nucleation between several containers and also decreasing the nucleation time.

In the embodiment depicted in <FIG>, the holder <NUM> will insulate the lateral walls of the moldable container <NUM> and acts as support to allow the unidirectional bottom-up freezing and to maintain the shape of the moldable container <NUM> in response to an expansion of biological material held due to freezing. The holder <NUM> can be made of a plastic, polymer or other material having low thermal conductivity. It is important to promote thermal contact between the moldable container <NUM> and the holder <NUM>.

In another embodiment, the holder <NUM> can have multiple cavities <NUM>, each one adjacent to each other, to receive multiple moldable container <NUM>. With this strategy it is possible to increase the number of moldable containers <NUM> per holder <NUM> assuring that multiple moldable containers <NUM> will experience similar time-temperature profiles and thus increase the freezing reproducibility. Besides having multiple cavities <NUM>, all the remaining features are identical to the ones previously described.

In accordance with the invention, to avoid the ice-crust formation in the top of the moldable container <NUM>, it is useful to freeze the aqueous solution of biological materials using the holder <NUM> placed in an isothermal temperature chamber or compartment with an ice-crust attenuator device <NUM> at the top. The ice-crust attenuator device <NUM> in the top of the chamber will eliminate the loss of heat at the top interface of the liquid by radiation and do not let the air in the head-space of the container cool during the freezing period.

Other embodiments, not forming part of the present invention, can be obtained through the assembling of controlled heating, by means of internal flow of a temperature-controlled fluid, by an electrical resistance, or by a thermoelectric element (Peltier) whose temperature is controlled by electric current.

Claim 1:
Device (<NUM>) for being used when freezing or thawing an aqueous biological solution and for reducing and/or preventing ice-crust formation on the liquid and air interface, the device being shaped to fit the top of a container (<NUM>) of fixed shape, comprising:
an external wall and an internal wall each comprising a thermal insulating material (<NUM>);
an internal cavity (<NUM>) comprising a phase change material;
wherein the cavity is between the internal and the external wall;
a recess configured for receiving a container;
wherein the freezing-temperature of the phase change material is substantially close to the freezing point of the aqueous biological solution, reducing and/or preventing ice-crust formation on the liquid and air interface;
wherein the internal wall further comprises a moldable thermal insulating material (<NUM>);
wherein the thermal insulating material of the internal wall is moldable to form an air-tight seal over the container top.