PROCESS FOR FREEZE-DRYING A CRYOGENICIZED CELLULAR COMPOSITION CONTAINING DISSOLVED GAS

Abstract

The invention relates to the field of freeze-dried biological materials. More particularly, the invention concerns a novel method for preparing a freeze-dried cell sample comprising a cryogenic step. In a preferred embodiment, this method is applied to lactic acid bacteria.

Description

Title of the invention: METHOD FOR FREEZE-DRYING A CELLULAR COMPOSITION CRYOGENIZED AND CONTAINING DISSOLVED GAS

[0001] The invention relates to the field of freeze-dried biological materials. More particularly, the invention concerns a new method for preparing a freeze-dried cell sample comprising a cryogenic step. In a preferred embodiment, this method is applied to lactic acid bacteria. State of the art

[0002] The preservation of cells in frozen form has been known for a long time. This method requires little equipment. The principle is simple: freezing induces a drop in temperature that slows down and then stops all the cell's biochemical reactions. These reactions can be reactivated after thawing. However, regardless of the method used, a significant loss of viability is observed upon thawing, caused in particular by the formation of intracellular crystals, and this loss increases with the freezing time.

[0003] Alternatively, the cells can be freeze-dried. Freeze-drying is a process of desiccating a previously frozen product by sublimation. This process yields a dry product that can be stored at 4°C or at room temperature. However, this process is lengthy, expensive, and very energy-intensive.

[0004] Conventionally, a cell lyophilization process comprises the steps of preparing a sample containing the cells, centrifuging the sample and transferring the cell pellet to a cryoprotective medium that allows dehydration, freezing the sample, and then lyophilizing it. The freezing step is generally carried out at temperatures between -20°C (slow freezing) and -80°C (rapid freezing); rapid freezing is less damaging to the cells but more difficult to implement on an industrial scale.

[0005] Attempts to freeze cells by cryogenics, alone or for the purpose of lyophilization, have been made, but the results have been inconclusive due to significant cell deterioration. This deterioration probably results from the fact that prior art processes do not allow control of the amounts of water and oxygen present in the product during the freezing and lyophilization steps. Water and oxygen are two factors that impair cell quality and long-term viability. Thus, none of the cryogenic solutions proposed to date have significantly improved the properties of the lyophilized products. In particular, most of the proposed solutions rely on a negative pressure applied during the freezing phase. In contrast, application WO2018 / 138461 describes a process for cooling biological material by cryogenics under high pressure, namely at a pressure between 10 and 1000 bar. This process improves the survival rate of a Lactobacillus bulgaricus strain by a factor of almost 400.

[0006] Among the many drawbacks associated with the processes available to date, it is regrettable that it is not possible to obtain preparations of lactic acid bacteria of probiotic interest that are stable in the long term at room temperature.

[0007] More generally, there is a need for a method of long-term preservation of cells of all types, including living cells. Description of the invention

[0008] The inventors have shown that the quality and long-term viability of lyophilized cells, in particular probiotic bacterial strains, can be significantly improved when the freezing step is carried out via pressurized cryogenics, allowing the dissolution of gas within the matrix to be frozen before lyophilization.

[0009] Thus, the invention relates to a freeze-drying process for a cell composition characterized in that the freezing step is coupled with a significant gas dissolution in the product, occurring just before said freezing. The dissolution is achieved either by freezing in a pressurized chamber or by subjecting the product to a very high density of gas molecules without necessarily resulting in overpressure. These two embodiments are equivalent in terms of the result, in that the matrix passes through a zone dense with gas molecules (gas dissolution step in the matrix) before being cryogenically frozen. Advantages of the invention

[0010] The inventors have shown the interest of cryogenics under pressure as a method of freezing biological materials, in particular bacteria such as lactic acid bacteria.

[0011] Indeed, this method allows better preservation of cells during the freezing and freeze-drying stages and therefore an improvement in the production yields of frozen cell preparations.

[0012] First of all, it is observed that cryogenics under pressure is advantageous in itself compared to other freezing methods for the preservation of the integrity and / or viability of cells.

[0013] On the other hand, it is very advantageous to combine a cryogenic freezing step under pressure with freeze-drying for several reasons: - The production yield is better; a freeze-dried composition richer in cells is obtained at the end of the process. Consequently, the cell concentration of the freeze-dried product is improved; - The cells are less damaged, their membrane integrity is maintained; this is advantageous for preserving cell viability, but also in the case of damaged or dead cells whose membrane or cell wall is damaged. Indeed, it has been shown for several years that non-viable bacteria can retain certain probiotic activities (adhesion, stimulation of the immune system, etc.). - Cell viability is increased after the freeze-drying step compared with products obtained by conventional freeze-drying processes. - The stability of the cell lyophilized products is also observed over the long term, namely for several months, at 4°C as well as at room temperature.

[0014] The advantages associated with this process are observed when the starting cell composition contains cryoprotectants. Interestingly, they are also observed in the absence of cryoprotectants; by eliminating the use of cryoprotectants, the addition of increasingly criticized additives is limited.

[0015] On the other hand, this process allows the treatment of matrices whose size of the beads formed can reach 7 to 8 mm as well as aggregates; the size parameter does not affect the efficiency of the process, nor the quality of the lyophilized products obtained.

[0016] Following this process, it is proposed to package the lyophilized products under a protective atmosphere, particularly to prevent deterioration caused by oxidation. In order to limit contact with oxygen as much as possible, it is advantageous to perform a gassing of the lyophilized product with an inert gas before sealing the packaging, so as to further increase the stability of the lyophilized products over time. By proceeding in this manner, increased stability of up to one year is expected.

[0017] Thus, thanks to the incorporation of gas coupled with very rapid cooling allowing the gas to remain dissolved and the maintenance of anaerobiosis throughout the process, the quality of the lyophilized cells is greatly improved.

[0018] The process according to the invention makes it possible to control the three critical parameters related to the quality of a lyophilized cell preparation: cell concentration, oxidation, and the amount of free water (the presence of water being detrimental to the stability of lyophilized cells at room temperature and at 4°C), and thus allows the preparation of lyophilized probiotic bacteria whose properties are preserved long-term. Depending on the product, storage at 4°C is more or less suitable. In the case of preparations of probiotic bacteria, storage at room temperature makes them easier to use (transport, storage, application). It is therefore advantageous to have preparations of probiotic bacteria that can be stored at room temperature.

[0019] This process can be applied to many types of cells: probiotic and agri-food bacteria of interest (used for the manufacture of cheeses in particular), yeasts, plant cells, microalgae, microorganisms from the intestinal or fecal microbiota (for fecal microbiota transplantation in the treatment of Clostridium difficile infections), reproductive cells (oocytes and spermatozoa) for animal and human reproduction, blood cells and stem cells.

[0020] Without being bound by this theory, the inventors consider that the benefits of the process according to the invention for the preparation of frozen and / or freeze-dried cells lie in the fact that cryogenics under pressure makes it possible to obtain non-porous frozen products containing a large quantity of perfectly dissolved gas. Since this gas is not oxygen, oxidation reactions are avoided. Furthermore, the freeze-drying of such products makes it possible to eliminate most of the water contained in the product. As a result, the conditions implemented in this process are, on the whole, gentler, less aggressive, and less damaging to the cells.

[0021] From the perspective of the freeze-drying process itself and its industrialization, the pressurized cryogenics step also allows for significant improvements. First, the preparation time is much faster. Cryogenics is a near-instantaneous process, enabling the continuous production of initially fluid product pellets at high rates (several hundred kg per hour with standard equipment). The time savings are considerable compared to freezing in a cold room, even if these operate at very low temperatures (generally -40°C to -80°C). The cryogenically frozen pellets are extracted at temperatures generally between -80°C and -120°C, which allows freeze-drying to begin directly with products whose temperature is around -60°C, without a preliminary cooling step. The freeze-drying time itself is significantly reduced (by at least a factor of 2).Finally, this process allows for the treatment of larger quantities of raw materials while reducing processing time. This last advantage makes it possible to obtain quality products even in the absence of a cryoprotectant.

[0022] This process opens up new perspectives for the preservation of cells in the form of lyophilized material whose properties are preserved during the lyophilization process and restored after re-solution, both at the level of active molecules and at the level of the viability of whole cells. DETAILED DESCRIPTION OF THE INVENTION

[0023] A first object of the invention relates to a freeze-drying process for a cellular composition characterized in that the freezing step is carried out by cryogenics and which comprises the steps of:

[0024] a) have a cellular composition comprising cells in an aqueous medium;

[0025] b) dissolve a gas in said composition by passing through a zone dense in gas molecules, such density being obtained (i) either by means of the gas flow generated by the evaporation of a cryogenic fluid, (ii) or by an increase in pressure;

[0026] c) cryogenically freeze said gas-rich matrix obtained in step b) at a pressure enabling said gas to remain dissolved in said matrix to obtain frozen granules, particles or beads;

[0027] d) lyophilization of said granules, particles or frozen beads to obtain a lyophilized cell preparation.

[0028] This process is therefore characterized by freezing the matrix, composed of a cellular material, by cryogenics in contact with a gas so as to dissolve the gas in the matrix. Dissolution is achieved by passing the matrix through a zone dense with gas molecules.

[0029] The zone rich in gas molecules within the enclosure containing the cryogenic gas can be obtained in two ways: (i) either through the gas flow generated by the evaporation of a cryogenic fluid, (ii) or by an increase in pressure.

[0030] When the molecularly dense zone is obtained by means of a gas flow generated by the evaporation of a cryogenic fluid, the quantity of gas dissolved in the matrix is ​​typically equivalent, when no pressure is applied, to that which would be obtained by the application of relative pressures between 0.001 bars and 2 bars.

[0031] When the molecularly dense zone is obtained by raising the pressure, this pressure is greater than atmospheric pressure, and may in particular be greater than 0.1 bar, 0.5 bar, 1 bar, 2 bars, 5 bars, 10 bars, 15 bars, 20 bars, 25 bars, 30 bars, 50 bars or 100 bars.

[0032] The term "under pressure" in the context of the invention refers to conditions that allow the dissolution of a gas in a matrix and / or the retention of the dissolved gas in said matrix during freezing. Pressurization can be achieved either by increasing the pressure or by bringing the matrix into contact with a cryogenic fluid, the evaporation of this gas creating a gas molecule density equivalent to pressurization, such that the gas molecules dissolve in the matrix. Furthermore, "pressurization" corresponds to the application of relative pressures, i.e., atmospheric pressure is considered as a pressure of 0 bar. All pressures expressed in this document are relative pressures. In a preferred embodiment of the invention, the process is not carried out under partial vacuum.

[0033] Thus, step b) is carried out at a relative pressure sufficient to allow the dissolution of gas in the matrix, and an equivalent pressure is maintained in step c) to retain the dissolved gas within the matrix during cryogenics. In a particular embodiment of the invention, the pressure applied in step c) is greater than or equal to atmospheric pressure.

[0034] The gas may be an inert gas. The gas may be nitrogen, nitrous oxide, carbon dioxide, a noble gas such as argon or a mixture of these gases.

[0035] In a preferred embodiment, the gas used is nitrogen, and the cryogenic fluid is liquid nitrogen.

[0036] Regarding the implementation of the process as a whole, it is possible to link the process steps sequentially, and in particular to carry out the freeze-drying step immediately after the cryogenics step. Furthermore, the process can be carried out continuously. It is also possible to store the product in frozen form after step c) and to perform the freeze-drying later, after a period of storage at sub-zero temperatures to maintain the products in a solid state (for example, at -40°C). In both cases, the advantages of the process are retained.

[0037] The process conditions can be adapted according to the product to be dehydrated, in particular the pressure during the cryogenic stage and the freeze-drying parameters. Those skilled in the art will be able to make such adaptations.

[0038] For the purposes of this invention, "cell" means a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include lactic acid bacteria, probiotic bacteria, and bacteria that constitute the microbiota (intestinal, fecal, etc.). Examples of eukaryotic cells include yeasts, reproductive cells, blood cells and stem cells, plant cells, and microalgae. The cells treated according to the processes described in this document may be living or non-living. In particular, they may be tyndallized bacteria, that is, bacteria that have been previously killed by heat. The cells may be isolated from one another or organized into tissues.

[0039] In a preferred embodiment, the cellular composition consists of bacteria of probiotic interest to humans and animals.

[0040] In another preferred embodiment, the cellular composition consists of a sample of fecal microbiota.

[0041] By "aqueous medium" is meant water, a culture medium, or an aqueous plant extract. This aqueous medium may or may not contain cryoprotectants.

[0042] Thus, in a particular embodiment, the cellular composition does not contain a cryoprotectant.

[0043] A second object of the invention relates to a method for preparing a cryogenically frozen cell composition under pressure, comprising the steps:

[0044] a) have a cellular composition comprising cells in an aqueous medium in the form of a matrix;

[0045] b) dissolve a gas in said matrix by passing through a zone dense in gas molecules, such density being obtained (i) either by means of the gas flow generated by the evaporation of a cryogenic fluid, (ii) or by raising the pressure up to 10 bars;

[0046] c) cryogenically freeze said gas-rich matrix obtained in step b) at a pressure enabling said gas to remain dissolved in said matrix to obtain frozen granules, particles or beads.

[0047] This process makes it possible to obtain frozen cells whose survival is increased compared to the freezing processes described previously.

[0048] The pressure applied in step b) is less than 10 bars, but in some embodiments may be less than 5 bars, or even 2 bars.

[0049] In a preferred embodiment, the pressure for maintaining said gas dissolved in said matrix is ​​greater than or equal to atmospheric pressure.

[0050] A third object of the invention relates to a lyophilized cellular composition obtained by the lyophilization process as defined above.

[0051] In a particular embodiment, the composition comprises a lyophilized powder and superdried maltodextrins.

[0052] In a particular embodiment, the lyophilized cell composition comprises lyophilized and stabilized probiotic bacteria of interest obtained by the process as defined above and superdried maltodextrins. In a preferred embodiment, such a composition comprises 20-25% lyophilized powder and 75-80% superdried maltodextrins.

[0053] The present invention will be better understood by reading the following examples, which are provided by way of illustration and should in no way be considered as limiting the scope of the present invention. DESCRIPTION OF THE FIGURES

[0054] [Fig.1] [Fig.1]: Viability of an L. plantarum SD5209 strain after cryogenic freezing at different pressures.

[0055] [Fig.2] [Fig.2]: Concentrations in CFU and IC of the lyophilized products of a Lactobacillus plantarum strain.

[0056] [Fig.3] [Fig.3] : Loss of viability under accelerated aging conditions of lyophilized products of a Bifidobacterium animalis ssp lactis strain.

[0057] [Fig.4] [Fig.4]: Total cell yield after 3 months of storage under accelerated aging conditions of lyophilized Bifidobacterium animalis ssp lactis strain. EXAMPLES

[0058] EXAMPLE 1: Impact of the freezing method on the properties of a frozen lactic acid bacteria composition

[0059] A - Preparation of frozen lactic acid bacteria compositions

[0060] A bacterial sample is cultured for 24 h in 500 mL of MRS Vegetal type culture medium (Ref BK176HA from Biokar Diagnostics). The culture medium is then centrifuged for 10 min at a speed of 3400 g. The pellet, containing the microorganisms studied, is resuspended in fresh culture medium so that the concentration of intact cells is adjusted to 5 x 10⁹ cells / mL.

[0061] The preparation is then separated into 4 fractions allowing the 4 different freezing methods to be tested:

[0062] - The "standard process control" samples are prepared by aliquoting the preparation in 30mL bottles and placing these in an enclosure at -80°C for at least 48 hours to ensure that the entire product is frozen and has reached a temperature of -80°C;

[0063] - The "nitrogen bead control" samples are made by manually producing a drip, using a syringe, over an open container, such as a bowl or salad bowl, containing liquid nitrogen, at atmospheric pressure and ambient temperature;

[0064] - The "5 bar cryogenic" samples are obtained using equipment enabling the process to be implemented as described in patent EP07858410, under a relative pressure of 5 bars in the cryogenic chamber; similarly the "cryogenic 10 bar", "cryogenic 15 bar" samples as well as the samples prepared by applying other pressures during the cryogenic stage are obtained by applying the same process and modifying the pressure inside the chamber.

[0065] - The "cryozero" samples are obtained using equipment that allows implement a cryogenic step under a relative pressure of 0 bars, such that the saturation in dissolved nitrogen obtained is approximately 3 times greater than that which would be obtained at this same pressure with a standard process (condition "nitrogen beads control").

[0066] After the freezing step, all samples are kept in an enclosure at -80°C before being analyzed.

[0067] B - Analysis of frozen samples

[0068] The results obtained are presented in [Fig.1].

[0069] The results of [Fig.1] were obtained from a commercial strain of L. plantarum SD5209 (ATCC).

[0070] The bacteria were solubilized in a sugar-free, plant-based MRS medium containing 1% sucrose and 5% maltodextrin (cryoprotectants). The -80°C control corresponds to freezing 40 mL at -80°C for a minimum of 48 hours. The -20°C control corresponds to freezing 40 mL at -20°C for a minimum of 48 hours. The samples were then thawed (Ih at 37°C) and viability was measured. Different pressures were tested for the cryogenically frozen strains.

[0071] It is observed that the loss of viability of the control frozen at -20°C is much greater than for the various samples cryogenically frozen, particularly at 8 bar. This result is highly significant and demonstrates a real benefit of cryogenics. This result is all the more interesting given that the tested strains were not fermented before freezing, but simply solubilized from a lyophilized sample. The initial lyophilization of the bacteria has already eliminated the least resistant strains, and those contained in the sample have therefore already been "pre-selected" with regard to their resistance to dehydration under industrial conditions. In this context, we can expect a greater improvement when cryogenically freezing freshly fermented strains.

[0072] EXAMPLE 2: Impact of the freezing method on the properties of a lyophilized bacterial composition

[0073] The samples are prepared under the same conditions as those described in Example 1(a). The samples frozen according to the four methods are further freeze-dried in a Drywinner CT60 freeze dryer (Heto Holten) for 48 to 72 hours. The samples are carefully ground into a powder using a mortar and pestle. This powder is then stored in a sterile container at -20°C before analysis. For the analyses, the powdered samples are first dispersed in buffered peptone water to which 1% (w / v) Tween 80 has been added and homogenized using a Stomacher. The quantities of CFU and IC are obtained by counting on MRS agar and by flow cytometry analysis (protocol B of ISO 19344 IDF 232:2015).

[0074] The results obtained are presented in [Fig.2].

[0075] This graph shows the results obtained according to 2 parameters:

[0076] - the concentration of live cells in the lyophilized powder, in CFU (viability test) consisting of testing the ability of bacteria to form colonies on an agar medium) and

[0077] - the membrane integrity in intact cells (IC, measured by flow cytometry) of cells of the lyophilized powder.

[0078] The results show that using a higher cooling rate (cryogenics: Nitrogen bead control) doubles and triples the CFU and IC concentrations of the lyophilized powder compared to the -80°C control. The "Cryozero" condition further increases these concentrations, reaching three and four times the CFU and IC concentrations of the standard process control (-80°C). Finally, the "5 bar cryogenics" condition achieves the highest CFU and IC concentrations, four and six times higher than those of the standard process.

[0079] EXAMPLE 3: Impact of the freeze-drying process on the cellular integrity of a freeze-dried bacterial composition (dry powder)

[0080] The samples are prepared under the same conditions as those described in Example 2. The powdered samples are stored for 3 months, after dilution in superdried maize maltodextrin, in three-layer paper / aluminum / PE bags and placed under different temperature and humidity conditions (4°C / uncontrolled RH, 20°C / uncontrolled RH, 25°C / 60% RH, and 30°C / 65% RH) before analysis. For the analyses, the powdered samples are first dispersed as indicated in Example 2. The CFU counts are obtained by counting on MRS agar at t=0 immediately after sample production, and then after 1.5 months and 3 months of storage, respectively. The loss of viability is calculated as the difference between the CFU count at time t and that corresponding to t=0.

[0081] The experiments were carried out using B. lactis spp animalis bacteria.

[0082] The results obtained are presented in [Fig.3].

[0083] It is observed that the loss of viability is very significant under standard conditions, as this loss is close to half of the CFUs initially present. Conversely, the loss observed for the sample obtained through cryogenics at 5 bar is very low and close to 0. The results obtained for the nitrogen bead control (N2 Control) and for the cryozero conditions are comparable and intermediate, which also demonstrates the positive impact of cryogenics according to the proposed process for the preservation of strains in lyophilized form.

[0084] EXAMPLE 4: Impact of the freeze-drying process on the cell lysis of a freeze-dried bacterial composition (dry powder)

[0085] The samples are prepared under the same conditions as those described in Example 2. The resulting powder samples are stored for 3 months in three-layer paper / aluminum / PE bags and placed under different temperature and humidity conditions (4°C / uncontrolled RH, 20°C / uncontrolled RH, 25°C / 60% RH and 30°C / 65% RH) before being analyzed. For the analyses, the powder samples are first dispersed as indicated in the example. 2. Total cell counts (TC) are obtained by flow cytometry analysis. Yields are obtained by relating the values ​​obtained to the number of TC counted in the preparation before the freezing step.

[0086] The experiments were carried out using Bifidobacterium animalis ssp lactis bacteria.

[0087] The results obtained are presented in [Fig.4].

[0088] It is observed that the percentage of lysed cells is higher with the standard process, while the number of preserved cells increases with increasing amounts of gas dissolved in the preparation during the freezing step in liquid nitrogen. The use of cryogenics at 5 bar pressure thus increases the cell yield by approximately 12% compared to the standard process, even though the strain used already exhibits high resistance to freezing and lyophilization processes.

[0089] The proposed process is therefore also of interest for the treatment of strains that are not very sensitive to current constraints, by making it possible to increase production yields.

[0090] Conclusion: The freeze-drying process of a cell composition comprising a freezing step carried out by cryogenics under pressure allows for both qualitative and quantitative improvement in the production of cell freeze-dried products.

Claims

Claims

1. A method of freeze-drying a cellular composition characterized in that the freezing step is carried out by cryogenics under pressure, said method comprising the steps of: a) providing a cellular composition comprising cells in an aqueous medium; b) dissolving a gas in said composition by passing through a zone dense in gas molecules, such density being obtained (i) either by the flow of gas generated by the evaporation of a cryogenic fluid, (ii) or by an increase in pressure; c) cryogenizing the matrix consisting of said gas-rich cellular composition obtained in step b) at a pressure making it possible to maintain said gas dissolved in said matrix to obtain frozen granules, particles or beads; d) freeze-drying said frozen granules, particles or beads to obtain a freeze-dried cellular preparation.

2. A method for preparing a composition of frozen cells by cryogenics under pressure comprising the steps of: a) providing a cellular composition comprising cells in an aqueous medium in the form of a matrix; b) dissolving a gas in said matrix by passing through a zone dense in gas molecules, such density being obtained by an increase in pressure of less than 10 bars; c) cryogenizing said gas-rich matrix obtained in step b) at a pressure making it possible to maintain said gas dissolved in said matrix to obtain frozen granules, particles or beads.

3. A method according to any preceding claim wherein said gas is nitrogen.

4. Method according to one of the preceding claims in which said cellular composition comprises lactic acid bacteria and probiotic bacteria, bacteria forming the microbiota, yeasts, plant cells, micro-algae, reproductive cells, blood cells, stem cells.

5. Method according to claim 4 wherein said cellular composition comprises bacteria of probiotic interest for humans and animals.

6.

7.

8.

9.

10. Method according to one of claims 1 to 5 in which the cellular composition does not contain cryoprotectant. The method of claim 4 wherein said cellular composition is a sample of fecal microbiota. Freeze-dried cellular composition obtained by the process as defined in one of claims 1, 3 to 7. A composition according to claim 8 further comprising superdried maltodextrins. Composition according to one of claims 8 or 9 in which the cellular composition comprises bacteria of probiotic interest.

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