Recovery, decarboxylation, and purification of cannabinoids from engineered cell cultures

Patent metadata

Field	Value
Publication number	US-12043859-B2
Application number	US-202017426973-A
Country	US
Kind code	B2
Filing date	Jan 30, 2020
Priority date	Jan 30, 2019
Publication date	Jul 23, 2024
Grant date	Jul 23, 2024

Abstract

Methods of recovering cannabinoids from cell cultures include methods comprising steps of separating the cell culture at a temperature above the melting point of the cannabinoid to separate a light phase comprising liquid state cannabinoid from a heavy phase; and methods comprising treating the cell culture at a temperature below the melting point of the cannabinoid to separate a light phase from a heavy phase comprising solid state cannabinoid. Other methods include contacting the culture with a water-miscible solvent to form a water-miscible phase and an aqueous phase, separating the two phases and recovering the cannabinoid. Other methods include contacting the culture with a water-immiscible solvent to form a water-immiscible phase and an aqueous phase, separating the two phases, and recovering the cannabinoid. Other methods include washing the inner surface of a fermentation vessel with alkaline solution to recover cannabinoid attached to the vessel surface. Various methods make use of aqueous solvent systems comprising no organic solvent, aqueous solvent systems comprising added water-miscible organic solvent, and dual-phase aqueous/water-immiscible solvent systems.

First claim

We claim:

1. A method of recovering a cannabinoid from a cell culture, the method comprising:
a) providing a cell culture comprising cells engineered to produce a cannabinoid in a culture medium;
b) treating the culture at a temperature below the melting point of the cannabinoid to generate a first pellet comprising cells, insoluble cellular material, and the cannabinoid, and a first supernatant comprising culture medium;
c) removing the first supernatant from the first pellet;
d) adding a water-immiscible organic solvent to the first pellet to generate a solvent-extracted pellet mixture;
e) separating, the solvent-extracted pellet mixture to generate a heavy phase and a light phase, wherein the light phase comprises the water-immiscible organic solvent and the cannabinoid; and
f) recovering the light phase comprising the cannabinoid.

2. The method of claim 1 wherein step b) treating comprises separating using g-force, separating by centrifugation, filtrating, by filtration under dead end microfiltration mode, evaporating, or flocculating; wherein step e) separating comprises g-force separating or both b) and e).

3. The method according to claim 1 additionally comprising one or more of the steps:
g) filtering the light phase;
h) adding water to the filtered light phase, wherein added water removes water soluble impurities;
i) separating the aqueous phase from the light phase by separation using g-force, by centrifugation.

4. The method according to claim 3 , wherein filtering uses a membrane having a molecular weight cutoff in the range of 100 to 5,000 kilodaltons.

5. A method of recovering a cannabinoid from a cell culture, the method comprising:
a) providing a cell culture comprising cells engineered to produce a cannabinoid in a culture medium;
b) contacting the culture with a water-immiscible solvent, wherein the contacting forms a water-immiscible phase and an aqueous phase from the culture medium;
c) separating the water-immiscible phase from the aqueous phase, wherein the water-immiscible phase comprises the cannabinoid;
d) separating the aqueous phase from light phase by separation using g-force by centrifugation.

6. The method of claim 5 comprising one or more of the following steps:
e) filtering the light phase;
f) adding water to the filtered light phase, wherein added water removes water soluble impurities; and
g) separating the aqueous phase from the light phase by separation using g-force, by centrifugation.

7. The method of claim 6 , wherein step c), step g), or both steps c) and g) separating comprises separating using g-force, by centrifugation.

8. The method of claim 6 which i) performs step e) before steps f) and g); or ii) which performs step e) after steps f) and g).

9. The method of claim 6 , further comprising heating the water-immiscible phase.

10. The method of claim 6 , further comprising removing the water-immiscible solvent from the water-immiscible phase of step (c) and resuspending the cannabinoid in a second solvent, wherein the second solvent is optionally ethanol.

11. The method of claim 6 , wherein the water-immiscible solvent is selected from the group consisting of: C4-C10 acyl ester, Anisole, Butylacetate, tert-Butylmethyl ether, Chlorobenzene, Chloroform, Cumene, Cyclohexane, 1,2-Dichloroethene, Dichloromethane, 1,2-Dimethoxyethane, 1,4-Dioxane, 2-, Ethylacetate, Ethylether, Ethylformate, Heptane, Hexane, Isobutyl acetate, Isopropylacetate, Methylacetate, 3-Methyl-1-butanol, Methylbutylketone, Methylcyclohexane, Methylethylketone, Methylisobutyl ketone, 2-Methyl-1-propanol, N-Methylpyrrolidone, Pentane, 1-Pentanol, Propylacetate, Tetralin, Toluene, 1,1,2-Trichloroethene, Triethylamine, and Xylene.

12. A method of recovering a cannabinoid from a cell culture, the method comprising:
a) providing a cell culture comprising cells engineered to produce a cannabinoid in a culture medium;
b) allowing the flow of at least liquid components of the cell culture through a filtration membrane wherein a retentate comprising cells and cannabinoid is collected on a portion of the filtration membrane;
c) contacting the retentate with a solvent, wherein the solvent extracts the cannabinoid from the retentate, optionally comprising a step of separating the retentate and solvent to form a liquid phase comprising the solvent and cannabinoid and a heavy phase comprising semi-solids, solids, or a mixture thereof; and
d) recovering the cannabinoid from the solvent.

13. The method of claim 12 wherein prior to, or during step b) a filter aid selected from the group consisting of silica, alumina, zeolites, diatomaceous earth, sand, cellulosic material, lignocellulosic material, pumice, and perlite, is added to the cell culture in an amount up to about 5% (wt).

14. The method of claim 12 wherein the filtration membrane has a normal pore size in the range of 0.2 micrometer to 20 micrometer.

15. The method of any of claim 12 wherein the retentate is contacted with an amount of solvent to provide a weight ratio of retentate:solvent in the range of 2:1 to 1:2.

16. A method of recovering a cannabinoid from a cell culture, the method comprising:
a) removing a cell culture from a fermentation vessel, the cell culture comprising cells engineered to produce a cannabinoid in a culture medium, wherein the fermentation vessel comprises a surface that contacts the cannabinoid in the culture medium;
b) contacting the surface of the fermentation vessel with an alkaline solution, wherein contacting removes cannabinoid from the surface.

17. The method of claim 16 wherein the alkaline solution comprises an alkali metal hydroxide, an alkaline earth metal hydroxide, or a combination thereof, wherein the alkali metal hydroxide, alkaline earth metal hydroxide, or combination thereof are optionally present at a concentration of about 0.1N or greater.

18. The method of claim 16 further comprising a step of neutralizing the alkaline solution comprising the cannabinoid, wherein neutralizing optionally comprises adding hydrochloric acid, sulfuric acid, acetic acid, nitric acid, or a combination thereof, to the alkaline solution comprising the cannabinoid, optionally comprising a step of contacting neutralized solution comprising cannabinoid with a water immiscible solvent, wherein contacting extracts the cannabinoid from the solution to the solvent.

19. The method according to claim 1 wherein the cannabinoid is selected from the group consisting of cannabichromene (CBC) type, cannabigerol (CBG) type, cannabidiol (CBD) type, Δ9-trans-tetrahydrocannabinol (Δ9-THC) type (e.g. Δ9-tetrahydrocannabinolic acid), Δ8-trans-tetrahydrocannabinol (Δ8-THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, cannabigerolic acid (CBGA), cannabigerolic acid, monomethylether (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C1), Δ9-tetrahydrocannabinolic acid A (THCA-A), Δ9-tetrahydrocannabinolic acid B (THCA-B), Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinolic acid-C4 (THCA-C4), Δ9-tetrahydrocannabinol-C4 (THC-C4), Δ9-tetrahydrocannabivarinic acid (THCVA), Δ9-tetrahydrocannabivarin (THCV),

Assignees

• Genomatica Inc

Inventors

• Suominen Lauri H
• Lievense Jefferson
• Liu Aiguo
• Martinez-Gonzalez Jorge Jesus
• Ko Myong Kon
• Nan Wei

Classifications

• C12M33/10

  by centrifugation (centrifuges in general B04B); Cyclones (cyclones in general B04C) · CPC title

• B01D11/0292

  Treatment of the solvent · CPC title

• B01D11/0288

  Applications, solvents · CPC title

• C07D307/91

  Dibenzofurans; Hydrogenated dibenzofurans · CPC title

• C07D311/58

  other than with oxygen or sulphur atoms in position 2 or 4 · CPC title