Method of lyophilization of a sample of faecal microbiota

Abstract

Method for lyophilisation of a simple of fecal microbiota. The present invention relates to a method for lyophilisation of a sample of fecal microbiota from a donor subject, comprising the following steps: A) mixing of a sample of fecal microbiota from a donor subject with a diluent selected from polyols, disaccharides to pentasaccharides, maltodextrins and mixtures thereof, and B) freezing the mixture obtained in A) at a temperature of less than 50 C., preferably of between 70 C. and 100 C., followed by the lyophilisation thereof.

Claims

1. A method of preparing a lyophilizate of faecal microbiota from a donor subject, comprising the following steps: A) mixing a sample of faecal microbiota from a donor subject with a diluent which is a saline aqueous solution comprising (i) at least one cryoprotectant chosen from polyols, di- to pentasaccharides, or mixtures thereof, and (ii) a maltodextrins; and B) freezing the mixture obtained in A) at a temperature less than 50 C., and then lyophilizing the frozen mixture.

2. The method of preparing a lyophilizate according to claim 1, wherein the diluent is a saline aqueous solution comprising at least galactose-lactose or trehalose as cryoprotectant.

3. The method of preparing a lyophilizate according to claim 1, wherein the diluent is a saline aqueous solution comprising at least a mixture of maltodextrins.

4. The method of preparing a lyophilizate according to claim 1, wherein the total amount of cryoprotectant in the saline aqueous solution is between 3 and 30% by weight relative to the total volume of the solution.

5. The method according to claim 1, wherein step A) comprises the following steps: A1) optionally, preparing a continuous gradient of iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide formed by freeze-thaw; A2) mixing at least one faecal microbiota sample from a donor subject with a saline buffer optionally comprising iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide, under anaerobiosis; to form a mixture; A3) optionally, forming a deposit of the mixture obtained in step A2) of the present method under the gradient obtained in step A1) of the present method, A4) sequentially centrifuging at low acceleration under anaerobiosis, or ultracentrifuging the mixture obtained in step A2) or step A3) of the present method to obtain a bacterial ring and a supernatant; A5) recovering the bacterial ring or the supernatant formed from finishing step A4) of the present method, under anaerobiosis; and A6) mixing the bacterial ring or the supernatant recovered in step A5) of the present method with the diluent.

6. The method according to claim 5, wherein step A6) comprises the following steps under anaerobiosis: a) centrifuging the bacterial ring obtained in step A5) resuspended in a saline buffer or the supernatant obtained in step A5), at an acceleration between 3000 and 4000g for a time between 5 and 15 minutes, and at a temperature between 20 and 30 C.; to obtain a pellet; b) recovering the pellet obtained from finishing step a) of the present method, and resuspending in a saline buffer, then centrifuging at an acceleration between 200 and 500g for a time between 5 and 15 minutes, and at a temperature between 20 and 30 C.; to obtain a supernatant; c) recovering the supernatant obtained from finishing step b) of the present method, and resuspending in a saline buffer, then centrifuging at an acceleration between 3000 and 4000g for a time between 5 and 15 minutes, and at a temperature between 20 and 30 C. to obtain a pellet; d) recovering the pellet obtained from finishing step c) of the present method; and e) mixing the pellet recovered in d) of the present method with the diluent.

7. The method according to claim 1, wherein the lyophilization of step B) is carried out in the following conditions: B1) freezing the mixture obtained in A) at a temperature less than 50 C.; B2) loading the frozen mixture obtained in B1) into a freeze-drier lyophilizer with shelves to a temperature between 50 C. and 30 C., at atmospheric pressure; then B3) at least one step of primary drying of the mixture loaded at B2) comprising lowering the pressure to a value between 80 and 200 bar, then increasing the temperature of the shelves to a value between 20 C. and +25 C., while applying a heating rate between 0.2 and 0.5 C./min; then B4) secondary drying of the mixture obtained at B3) comprising lowering the pressure to a value less than or equal to 80 bar, and raising the temperature of the shelves to a value comprised between +25 C. and +35 C., at a heating rate comprised between 0.1 and 0.3 C./min, and maintaining the temperature for between 8 and 15 hours.

8. The method according to claim 1, wherein the saline buffer is an aqueous solution of HEPES comprising sodium chloride.

9. The method according to claim 1, which comprises the following steps: A1) preparing a continuous gradient of iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide formed by freeze-thaw, A2) mixing at least one faecal microbiota sample from a donor subject with a saline buffer added to an aqueous solution comprising iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide, to form a mixture under anaerobiosis, A3) forming a deposit of the mixture obtained in A2) under the gradient obtained in A1), A4) ultracentrifuging the mixture obtained in A3), for a time between 40 and 50 minutes, at a temperature between 2 C. and 6 C., and at a speed comprised between 13000 and 16000g to form a bacterial ring, A5) recovering the bacterial ring formed from finishing step A4), under anaerobiosis, A6) mixing the bacterial ring recovered in A5) with the diluent to form a mixture, and B) freezing the mixture obtained in A6) at a temperature less than 50 C., then lyophilizing the mixture.

10. The method according to claim 9, wherein the preparing of a continuous gradient of iodixanol formed by freeze-thaw in step A1) is carried out according to the following steps: A1.a) freezing a solution of iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide at a temperature between 70 C. and 100 C. for at least 12 hours; and then A1.b) thawing the solution obtained in A1.a) at an ambient temperature for 2 to 4 hours, in order to obtain a continuous gradient of iodixanol or 5-(N-2, 3-dihydroxypropylacetamido)-2, 4, 6-tri-iodo-N, N-bis (2, 3 dihydroxypropyl) isophthalamide.

11. The method according to claim 1, which comprises the following steps under anaerobiosis: A1) mixing at least one sample of faecal microbiota from a donor subject with saline buffer to obtain a mixture; A2) sequentially centrifuging at low acceleration the mixture obtained in A1) to obtain a supernatant; A3) recovering the supernatant formed from finishing step A2); A4) mixing the supernatant recovered in A3) with the diluent to obtain a mixture; and B) freezing the mixture obtained in A4) at a temperature less than 50 C., and then lyophilizing the mixture.

12. The method according to claim 11, wherein the sequential centrifugation at low acceleration of step A2) is carried out at an acceleration between 200 and 500g for a time between 5 and 15 minutes, and at a temperature between 20 and 30 C.

13. A lyophilizate of fecal microbiota from a donor subject obtainable by the method according to claim 1, for use in the transplantation of autologous or allogenic faecal microbiota, or for treating intestinal dysbioses.

14. A functional genomics, metaproteomics or immunology method, the improvement wherein a lyophilizate obtainable by the method according to claim 1 is used therein as a research tool.

15. The method of claim 1, wherein, in step B, freezing the mixture obtained in A) is performed at a temperature between 70 C. and 100 C.

16. The method according to claim 3, wherein the mixture of maltodextrins is in an amount between 4 and 20% relative to the total volume of the solution.

17. The method according to claim 4, wherein the total amount of cryoprotectant in the saline aqueous solution is between 4 and 20% by weight relative to the total volume of the solution.

18. The method according to claim 7, wherein, in step B1), freezing the mixture obtained in A) is performed at a temperature between 70 C. and 100 C.

19. The method according to claim 8, wherein the aqueous solution of HEPES comprising sodium chloride is at a concentration between 7 and 15 g/l.

20. The method according to claim 9, wherein, in step A2), said saline buffer is an aqueous solution of HEPES comprising sodium chloride.

21. The method according to claim 9, wherein, in step B), freezing the mixture obtained in A6) is performed at a temperature between 70 C. and 100 C.

22. The method according to claim 11, wherein, in step B), freezing the mixture obtained in A4) is performed at a temperature between 70 C. and 100 C.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a diagram which shows the Pearson correlations between lyophilizates and raw faeces.

EXAMPLE 1: PURIFICATION OF A SAMPLE OF FAECAL MICROBIOTA FROM A DONOR SUBJECT BY CONTINUOUS GRADIENT OF IODIXANOL (OPTIPREP) ACCORDING TO THE INVENTION

(2) Principle:

(3) Separation of the total bacterial fraction, by buoyancy within a continuous gradient of OptiPrep auto-formed by freeze-thaw. The heavy faecal dilution in OptiPrep is deposited under a continuous gradient of OptiPrep, pre-formed by freeze-thaw. During centrifuging, the bacteria rise within the gradient up to their buoyant density (1.110-1.190), while the food and endogenous debris sink to the bottom of the gradient. All of the method is under anaerobiosis.

(4) Materials & Methods:

(5) Hepes-NaCl Buffers

(6) TABLE-US-00001 NaCl Hepes QS MilliQ For diluent for faecal suspensions 0.9 g 953.2 mg QS <100 mL 40 mM Hepes-9 g/L NaCl then adjust pH to 7.0 The diluent for faecal suspensions then QS 100 mL finally comprises, for 100 ml: 25 mL of 40 mM Hepes-9 g/L NaCl and 75 mL of OptiPrep-60 (commercial solution at 60% iodixanol in water) For gradients 0.9 g 357.5 mg Qs <100 mL 15 mM Hepes-9 g/L NaCl then adjust pH to 7.0 The diluent for gradients then QS 100 mL (OptiPrep-20) is finally a dilution of 3 times OptiPrep-60 in 15 mM Hepes-9 g/L NaCl For bacterial phase washings 18.0 g 4.766 g QS <2 L 10 mM Hepes-9 g/L NaCl then adjust pH to 7.0 then QS 2 L

(7) Preparation of the Continuous Gradients by Freeze-Thaw (Step A1)): pipette 16 mL of degassed OptiPrep-20 solution, and transfer into tube while avoiding aerating the solution freeze the tubes overnight at 80 C. thaw the undisturbed, immobile tubes at ambient temperature for 2-3 hours before use; a continuous density gradient of 1.03-1.22 forms automatically.

(8) Preparation of the Faecal Dilutions in an Anaerobic Chamber (Step A2)): Put the sample of faeces in the anaerobic chamber Weigh on the balance, on which a double filtered sterile Seward bag is placed, the desired weight of faeces (maximum 3.5 g for 1 gradient, possibly less; in this case make up to 3.5 g with 10 mM Hepes9 NaCl buffer) Add 24 mL of diluent for faecal suspensions in the filter of the Seward bag, Homogenize the mixture Transfer the filtered homogenized faecal suspension into a 50 mL Falcon type tube From this tube, fill a 20 mL syringe equipped with a needle with the faecal dilution

(9) Producing the Gradient Outside of the Anaerobic Chamber (Steps A3) and A4)): Plunge the needle of the syringe obtained in A2) to the bottom of the gradient pre-formed by freeze-thaw obtained in A1) Gently load the 20 mL of faecal dilution under the pre-formed gradient Recommence for the other gradients Weigh all the tubes and adjust the tubes in pairs to exactly the same weight with 10 mM Hepes9 g/L NaCl buffer Carefully insert the tubes into the cold centrifuging buckets for the oscillating rotor Centrifuge for 45 min at 4 C. 14,567g

(10) Recovering and Washing the Bacterial Cells in an Anaerobic Chamber (Steps A5), A6) and a) to d)): Insert the centrifuging buckets into the anaerobic chamber without opening them Open a bucket Using a pipette, remove the upper phase Pipette the intermediate cellular phase and distribute it into two 50 mL Falcon tubes Add washing buffer (10 mM Hepes9 g/L NaCl) up to graduation 50 of the two Falcon tubes Remove the bacterial phases of all the gradients in the same way Centrifuge for 10 min 4000g 22 C. in the oscillating rotor Remove the supernatant by aspiration Add the washing buffer up to graduation 25 mL of the Falcon tubes Carefully resuspend the bacteria by using a pipette; make up to 50 mL Centrifuge for 5 min 300g 22 C. in the oscillating rotor in order to remove the residual debris Transfer the supernatant (containing the bacteria without residue) with a pipette into two new 50 mL Falcons; discard the pellet of debris Centrifuge for 10 min 3500g 22 C. in the oscillating rotor Remove the supernatants

(11) The bacterial pellet without residue obtained can be resuspended in the chosen excipient.

EXAMPLE 2: PURIFICATION OF A SAMPLE OF FAECAL MICROBIOTA FROM A DONOR SUBJECT BY SEQUENTIALLY CENTRIFUGING AT LOW ACCELERATION ACCORDING TO THE INVENTION

(12) Principle:

(13) Separation of the total bacterial fraction, by sequentially centrifuging at low acceleration.

(14) Materials & Methods:

(15) Hepes-NaCl Buffer

(16) TABLE-US-00002 NaCl Hepes QS MilliQ for faecal suspensions and 18.0 g 4.766 g QS <2 L washings of bacterial phases then adjust pH to 7.0 10 mM Hepes-9 g/L NaCl then QS 2 L

(17) Preparation of the Faecal Dilutions in an Anaerobic Chamber: Put the sample of faeces in the anaerobic chamber Transfer the desired weight of faeces into the filter of the Seward bag Make up QS 350 g with 10 mM Hepes9 g/L NaCl for 14 g of faeces (i.e. a 1:25 dilution) and homogenize (step b)) Transfer 50 mL of filtered homogenized faecal suspension into six 50 mL Falcon type tubes Centrifuge for 10 min 300g 22 C. in the oscillating rotor in order to remove the debris (step d)) Distribute the supernatants (they contain the bacteria) into twelve new 50 mL tubes (25 mL/tube) Resuspend the 6 pellets in 50 mL 10 mM Hepes9 g/L NaCl and centrifuge for 10 min 300g 22 C. in the oscillating rotor (step d)) Distribute the 6 new supernatants into the waiting 12 tubes already containing the first supernatants; QS 50 mL for the 12 tubes with 10 mM Hepes9 g/L NaCl Centrifuge the 12 tubes for 10 min 3500g 22 C. in oscillating rotor in order to pellet the bacteria Using a pipette, carefully remove the supernatant

(18) Finally, the bacterial pellets without residue obtained are resuspended in the chosen excipient.

EXAMPLE 3: OBTAINING LYOPHILIZATES OF FAECAL MICROBIOTA OBTAINED IN EXAMPLES 1 AND 2

(19) The fractions obtained at the end of the methods of Examples 1 and 2 were mixed with the following diluents: NaCl: 9 g/L Maltodextrins: trehalose 15/5 or 5/15 in 9 g/mL NaCl Iodixanol: commercial aqueous solution at 60% diluted 3 times in 40 mM Hepes9 g/L NaCl buffer

(20) Then they were frozen at 80 C. or 100 C.

(21) Then they were subjected to the following lyophilization cycle:

(22) TABLE-US-00003 Temperature of Gradient in Pressure Stages shelves min bar Step in min Pre-cooling 40 C. 60 atmo Until loading Primary drying 10 C. 360 150 1080 Primary drying +25 C. 120 150 720

(23) Secondary drying is carried out at +25 C., at a pressure of 80 bar for approximately 900 minutes.

(24) The lyophilizer was pre-cooled to 40 C. before loading. From the moment of receiving the samples, the flasks stored in dry ice were loaded into the device and the lyophilizer was placed under vacuum from the end of loading. Two PT100 temperature probes were placed in two flasks. As the products were frozen before loading, the probes are above the product and not in the product.

(25) The duration of the cycle is 45 h. At the end of the cycle, the flasks are stoppered under vacuum in the lyophilizer then capped after unloading.

EXAMPLE 4: VIABILITY OF THE BACTERIA PRESENT IN THE LYOPHILIZATES OF FAECAL MICROBIOTA OBTAINED IN EXAMPLE 3

(26) Protocol:

(27) The viability of the bacteria present in the lyophilizates of faecal microbiota obtained in Example 3 was measured according to the following protocol: successive decimal dilutions under an anaerobic atmosphere, in order to achieve a final dilution of around 10.sup.6 bacteria/mL in the sample intended for staining; this operation must be immediately consecutive to resuspending the bacterial pellets without residue in the chosen excipient; the staining of the last dilution must also be immediate, Less than 30 min must elapse between the resuspending and the staining, as the living populations proliferate rapidly if the excipient contains a nutritive substrate: over a 4 hour wait at ambient temperature, under anaerobiosis and in the presence of a single nutritive substrate, the population increases by a half-log and the percentage of bacteria increases by 10%, staining the bacteria, using the LIVE/DEAD BacLight Bacterial Viability Kit according to the manufacturer's instructions, quantification of the living and dead bacteria by flow cytometry: the time between the staining and the quantification must not exceed 20 minutes, whether the stained samples (always shaded from the light) wait at ambient temperature or on crushed ice.

(28) Results:

(29) The results are shown in the table below:

(30) TABLE-US-00004 Viability 1 Viability 1 Viability 3 week after Standard month after Standard months after Standard Sample lyophilization deviation lyophilization deviation lyophilization deviation Lyophilizate with NaCl 8.50% 2.50% 5.70% 3.00% 12.10% 3.10% Lyophilizate with maltodextrins 44.80% 8.00% 48.10% 5.80% 50.20% 2.20% Lyophilizate with trehalose 31.10% 6.80% 37.60% 6.90% 40.30% 9.80% Lyophilizate with iodixanol 45.30% 6.60% 32.50% 15.40% 22.20% 9.70%

(31) Thus, the results show that the viability of the bacteria present in the purified samples according to the invention is very good when the bacteria are lyophilized with maltodextrins.

EXAMPLE 5: OBTAINING AND VIABILITY OF BACTERIA PRESENT IN NON-PURIFIED LYOPHILIZATES OF FAECAL MICROBIOTA

(32) A microbiota was collected and suspended in the following solutions: NaCl: 9 g/L Maltodextrins: trehalose 15/5 or 5/15 in 9 g/L NaCl

(33) Then the suspensions were frozen at 80 C. or 100 C.

(34) Then they were subjected to the following lyophilization cycle:

(35) TABLE-US-00005 Temperature of Gradient in Pressure Stages shelves min bar Step in min Pre-cooling 45 C. 60 atmo Until loading Primary drying 10 C. 360 150 1080 Primary drying +25 C. 60 150 480 Secondary drying +25 C. 1 80 960

(36) The lyophilizer was pre-cooled to 45 C. before loading. On receiving the samples, the stored products in dry ice were loaded into the device and the lyophilizer was placed under vacuum from the end of loading.

(37) The duration of the cycle is 48 h. At the end of the cycle, the flasks are stoppered under vacuum in the lyophilizer then capped after unloading.

(38) The quality of the bacterial populations was assessed in terms of diversity via an extraction of DNA and its analysis by sequencing the rDNA 16S gene. A phylogenetic analysis was then carried out in order to establish the profiles of the different samples in order to compare them. The results are shown in FIG. 1.

(39) Thus, the results show that the correlation levels between the taxonomic profiles are very high for the 3 formulations, demonstrating that the method effectively preserves approximately 90% of the bacterial populations.

(40) The viability was assessed in the same way as that presented in Example 4 and the results after storage for 10 months are presented below:

(41) TABLE-US-00006 Subject A Subject B Lyophilizate with trehalose 24.90% 31.20% Lyophilizate with NaCl 11.30% 20.20% Lyophilizate with 33.80% 30.70% Maltodextrins

(42) After 10 months, the suspensions produced with NaCl have viabilities clearly lower than the other 2.

(43) Thus, the microbiotas obtained according to the method make it possible at the same time to preserve the bacterial populations, since the correlations of the phylogenetic profiles are very high, and in addition they make it possible to store the bacteria alive for longer than the NaCl formulation.