RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 63/153,002, filed on February 24, 2021. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Cannabinoids are a class of active compounds derived from the Cannabis sativa, Cannabis indica, or cannabis hybrid plants commonly known as marijuana. The most well- known cannabinoid is the phytocannabinoid tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis. Delta-9-tetrahydrocannabinol (A9-THC) and delta-8- tetrahydrocannabinol (A8-THC) mimic the actions of anandamide and 2- arachidonoylglycerol neurotransmitters produced naturally in the body. These cannabinoids produce the effects associated with cannabis by binding to the CB1 cannabinoid receptors in the brain. In addition to psychoactive effects, THC is therapeutically useful in decreasing nausea and vomiting in certain patients, such as in patients with chemotherapy-induced nausea and vomiting (CINV) and for AIDS patients. The cannabinoid, cannabidiol (CBD), does not produce the psychoactive effects of THC but has been described as useful for treating anxiety, insomnia, and chronic pain.

Typically, cannabinoids are isolated from plants. Various species of cannabis make complex mixtures of cannabinoids of varying composition. It can be difficult and costly to isolate specific cannabinoids. Chemical synthesis of cannabinoids has also been tried, which is also costly. Microbial fermentation can be more economical and have been described, for example, in W02019/071000 and W02020/208411 to Szamecz et ak, which are incorporated herein by reference in their entirety. In Szamecz’ s preferred processes, Saccharomyces cerevisiae has been recombinantly modified to express one or more polynucleotides encoding for i) acyl activating enzyme (AAE1); ii) a polyketide synthase (PKS), iii) an olivetolic acid cyclase (OAC), iv) a prenyltransferase, and/or (v) a THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS), or any combination thereof.

US Patent 10,894,952 to Mendez et al. and US Publication 20210024968 to Davidovich describe the use of bacteria for the biosynthetic production of a cannabinoid while US Publication 20200370073 to Leo describes the use of microalgae. US Publication 20160010126 to Poulos et al. also describes the use of yeast, specifically K. marxianis, to produce cannabinoids. These references are also incorporated herein by reference in their entirety.

There is a need in the art to improve purification methods of such processes.

SUMMARY OF THE INVENTION

The present invention is directed to a method of isolating a cannabinoid from fermentate, such as a fermentate from a recombinant microorganism engineered to express one or more enzymes of a cannabinoid synthesis pathway. The process results in a highly purified cannabinoid product which is substantially free of residual solvent, as described in more detail below.

The embodiments of the invention include, without limitation, the following numbered embodiments, also called “claims”:

1. A process of preparing substantially pure cannabinoid, such as cannabigerol (CBG), from a biosynthetic starting material, wherein the biosynthetic starting material is a cannabinoid fermentate, comprising the steps: a. Dehydrating the cannabinoid fermentate or broth; b. Adding a polar organic solvent, such as ethyl acetate, and collecting a cannabinoid extract, such as a CBGA extract; c. Optionally decarboxylating an anionic cannabinoid, such as CBGA, to form a decarboxylated cannabinoid extract, such as a CBG extract, wherein the decarboxylation step can be a dry heat decarboxylation, heating the CBGA in a solvent, such as heptane, and/or adding a base to the CBGA; d. Subjecting the optionally decarboxylated cannabinoid extract to (i) silica chromatography to yield a cannabinoid isolate, such as a CBG isolate; (ii) subjecting the optionally decarboxylated cannabinoid extract, such as CBG extract, to clarification to yield a clarified cannabinoid and/or (iii) a silica plug step comprising loading the optionally decarboxylated cannabinoid extract on silica and heptane elution via a filtration funnel; e. Crystallizing the product of step (d) to provide a substantially pure CBG.

2. The process of embodiment 1, wherein the cannabinoid is present in the cannabinoid fermentate in an amount less than about 5% by weight and/or at least about 2% by weight. 3. The process of embodiment 1, wherein the dehydration step (a) reduces the water content to less than about 50% by weight.

4. The process of embodiment 1, wherein the concentration of cannabinoid in the dehydrated fermentate is between about 8 to about 20% by weight.

5. The process of embodiment 4, wherein the concentration of cannabinoid in the dehydrated fermentate is between about 9 and about 17% by weight.

6. The process of embodiment 1, wherein the cannabinoid content in the cannabinoid extract is at least about 25% by weight.

7. The process of embodiment 1, wherein the yield of cannabinoid in the cannabinoid extract is greater than about 90% by weight.

8. The process of embodiment 1, wherein the decarboxylation comprises heating the cannabinoid extract, such as directly heating the cannabinoid extract, heating the extract in a solvent, such as heptane, and/or adding a base, such as NaOH.

9. The process of embodiment 8, wherein the decarboxylation is conducted under vacuum.

10. The process of embodiment 8, wherein the decarboxylation is conducted under inert atmosphere.

11. The process of embodiment 1 wherein decarboxylated cannabinoid concentration after decarboxylation is at least about 25% by weight.

12. The process of embodiment 1, wherein anionic cannabinoid concentration after decarboxylation is less than about 4% by weight.

13. The process of embodiment 12, wherein anionic cannabinoid concentration after decarboxylation is less than about 2% by weight.

14. The process of embodiment 1, wherein the cannabinoid extract is purified using silica chromatography, preferably eluting with heptane and acetone.

15. The process of embodiment 14, wherein the cannabinoid extract is eluted using heptane and acetone.

16. The process of embodiment 15, wherein the heptane and acetone is used at a 9:1 ratio.

17. The process of embodiment 16, wherein the concentration of cannabinoid in the cannabinoid isolate is greater than 80% by weight.

18. The process of embodiment 16, wherein the concentration of cannabinoid is greater than 85% by weight.

19. The process of embodiment 16, wherein the concentration of cannabinoid is greater than 88% by weight. 20. The process of embodiment 16, wherein the yield of cannabinoid in the cannabinoid isolate is at least about 80%.

21. The process of embodiment 16, wherein the yield of cannabinoid is at least about 85%.

22. The process of embodiment 1, wherein the cannabinoid extract is subjected to clarification.

23. The process of claim 22 wherein the concentration of cannabinoid is at least about 60% by weight.

24. The process of claim 22 wherein the yield of cannabinoid is at least about 65% by weight.

25. The process of embodiment 22, wherein the clarification comprises dissolving the cannabinoid extract in a polar organic solvent, such as ethyl acetate, and precipitating with a cannabinoid nonsolvent, such as heptane and is, optionally, repeated.

26. The process of embodiment 1 wherein the cannabinoid isolate is subjected to a silica plug.

27. The process of embodiment 26, wherein the concentration of cannabinoid is at least about 60% by weight.

28. The process of embodiment 26, wherein the yield of cannabinoid is at least about 77%.

29. The process of embodiment 1, wherein the cannabinoid isolate or clarified cannabinoid was crystallized using a non-solvent comprising n-pentane or heptane.

30. The process of embodiment 1, wherein the concentration of cannabinoid in the substantially pure CBG is greater than 95% by weight as measured by HPLC.

31. The process of embodiment 1, wherein the concentration of cannabinoid in the substantially pure cannabinoid product is greater than 97% as measured by HPLC.

32. The process of embodiment 1, wherein the concentration of cannabinoid in the substantially pure cannabinoid product is greater than 98% as measured by HPLC.

33. The process of embodiment 1, wherein the concentration of cannabinoid in the substantially pure cannabinoid product is greater than 99% as measured by HPLC.

34. The process of embodiment 1, wherein the concentration of cannabinoid in the substantially pure cannabinoid product is about 100% as measured by HPLC.

35. The process of embodiment 1, wherein the substantially pure cannabinoid is a white or light yellow powder. 36. The process of embodiment 29, wherein the residual solvent content is less than about 20 ppm.

37. The process of embodiment 29, wherein the residual solvent content is about 10 ppm.

38. The process of embodiment 1, wherein the engineered fermentation broth is a yeast broth.

39. The process of any preceding embodiment where the cannabinoid is independently selected from CBGA and CBG.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1A and IB are schematics summarizing processes of the invention.

FIG. 2 is an HPLC analysis of the starting materials, intermediate product and pure CBG product by purification process method via chromatography and clarification, described below.

FIG. 3 is a Decarboxylation of Cannabigerolic acid (CBGA) crystal into Cannabigerol (CBG) crystal in vacuum. Crystallization can be conducted as the last process step using the following sequence: broth > CBGA extract > CBGA isolate > CBGA crystal. CBGA crystal can then be converted into its neutral form, CBG crystal, by incubation at 110°C in vacuum.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the purification of cannabinoids from biosynthetic starting materials, or fermentates. In one embodiment, the process comprises the steps of subjecting a cannabinoid fermentate to a process comprising an extraction step, chromatography step/silica plug step and a crystallization step. Alternatively, the process comprises the steps of subjecting a cannabinoid fermentate to a process comprising an extraction step, a clarification step and a crystallization step. It can be particularly desirable for the method to further comprise one or more chemical synthesis steps, such as a decarboxylation step where the cannabinoid fermentate comprises cannabigerolic acid (CBGA). The cannabinoid fermentate (or fermentate) can be a broth produced by fermenting a recombinant microorganism engineered to produce a cannabinoid. For example, the microorganism can be yeast (e.g., Saccharomyces. Cerevisiae or Kluyveromyces marxianis), bacterium (e.g., Escherichia cob), microalgae, fungi, insect, or animal cells (e.g., Chinese hamster ovary). Yeast is preferred. A preferred yeast is S. cerevisiae. Typically, the microorganism (or yeast) does not naturally produce CBGA or CBGVA.

The microorganism can be genetically engineered to express a cannabinoid synthesis pathway, such as the pathway found in cannabis. For example, the genetically modified microorganism comprises one or more heterologous nucleic acid encoding one or more of the enzymes including acyl activating enzyme (AAE1); polyketide synthase (PKS); olivetolic acid cyclase (OAC); prenyltransferase (PT); THCA synthase (THCAS); CBDA synthase (CBDAS), CBCA synthase (CBCAS); HMG-Co reductase (HMG1); famesyl pyrophosphate synthetase (ERG20); or a combination thereof.

In general, the microorganisms are fermented, or maintained, in an aqueous medium under conditions that convert a carbon source (such as a sugar, alcohol, and/or fatty acid) to CBGA or other cannabinoid (e.g., THC, CBD, CBC). Fermentation conditions that should be considered include media, temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition, as described by Szamecz et al. (supra).

The medium can include a carbon source, such as a sugar, alcohol, and/or fatty acid. For example, the sugar, alcohol or fatty acid can include without limitation hexanoic acid, glucose, fructose, xylose, sucrose, dextrins, starch, xylan, cellulose, hemicellulose, arabinose, glycerol, ethanol, butanol, methanol, or combinations thereof.

The medium used to ferment Saccharomyces, for example, can include a fatty acid, such as a C4-C8 alkanoic acid, including butyric acid, pentanoic acid, hexanoic acid, heptanoic acid and octanoic acid. For example, in some cases, the media can comprise a combination of hexanoic acid, yeast extract, peptone, and glucose. In certain cases, the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid. In some cases, hexanoic acid can be used in an amount of 1 mg/L to 1 g/L. In some cases, hexanoic acid can be used in an amount of 10 mg/ to 900 mg/L. In some cases, hexanoic acid can be used in an amount of 25 mg/ to 800 mg/L. In some cases, hexanoic acid can be used in an amount of 50 mg/ to 700 mg/L. In some cases, hexanoic acid can be used in an amount of 75 mg/ to 600 mg/L. In some cases, hexanoic acid can be used in an amount of 100 mg/ to 500 mg/L. In some cases, hexanoic acid can be used in an amount of 125 mg/ to 400 mg/L. In some cases, hexanoic acid can be used in an amount of 150 mg/ to 300 mg/L. In some cases, hexanoic acid can be used in an amount of 175 mg/ to 250 mg/L. In some cases, hexanoic acid can be used in an amount of 50 mg/ to 250 mg/L. In some cases, hexanoic acid can be used in an amount of 75 mg/ to 200 mg/L. In some cases, hexanoic acid can be used in an amount of 90 mg/ to 150 mg/L.

Olivetolic acid can also be used to ferment CBGA or cannabinoids. For example, in some cases, the media can comprise a combination of olivetolic acid, yeast extract, peptone, and glucose. In certain cases, the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 40 mg/L hexanoic acid. In some cases, olivetolic acid can be used in an amount of 1 mg/ to 1 g/L. In some cases, olivetolic acid can be used in an amount of 5 mg/ to 900 mg/L. In some cases, olivetolic acid can be used in an amount of 10 mg/ to 800 mg/L. In some cases, olivetolic acid can be used in an amount of 15 mg/ to 700 mg/L. In some cases, olivetolic acid can be used in an amount of 20 mg/ to 600 mg/L. In some cases, olivetolic acid can be used in an amount of 25 mg/ to 500 mg/L. In some cases, olivetolic acid can be used in an amount of 30 mg/ to 400 mg/L. In some cases, olivetolic acid can be used in an amount of 35 mg/ to 300 mg/L. In some cases, olivetolic acid can be used in an amount of 40 mg/ to 200 mg/L. In some cases, olivetolic acid can be used in an amount of 50 mg/ to 150 mg/L. In some cases, olivetolic acid can be used in an amount of 10 mg/ to 100 mg/L. In some cases, olivetolic acid can be used in an amount of 20 mg/ to 75 mg/L. In some cases, olivetolic acid can be used in an amount of 30 mg/ to 50 mg/L.

The fermentate of the invention will include not only the cannabinoid product and, optionally, cannabinoid intermediates, but also components of the media (such as peptone and glucose), microbial extracts (e.g., yeast extract and microbial intracellular proteins and nucleic acids), and the like. The term “microbial” is used herein to mean that the extract, protein and/or nucleic acid is native to the microorganism host cell. The term is defined herein to specifically exclude extracts, proteins and nucleic acids native to cannabis. Typically, the fermentate is cell-free.

The term “cannabinoid” and cannabinoid intermediates are defined to include biosynthetically produced tetrahydrocannabinol, (-)-(6aR, 9S,1 OS, 10aR)-9, 10- Dihydroxy hexahydrocannabinol-C 5 [(-)- Cannabiripsol] (CBR), (-)-(6aR,9S,10S,10aR)-9,10- Dihydroxyhexahydrocannabinolic acid-C5 [(-)- Cannabiripsolic acid] (CBRA), (-)-6a,7,10a- Trihydroxy-A9-tetrahydrocannabinol-C5 [(-)-Cannabitetrol] (CBTT), (-)-6a,7,10a- Trihydroxy-A9-tetrahydrocannabinobc acid-C5 ((-)-Cannabitetrol) (CBTTA), (-)-7- hydroxycannabichromane-C5, (-)-7-hydroxycannabichromanic acid-C5, (-)-Cannabidiol-C5 (CBD), (-)-Cannabidivarin-C3 (CBDV), (-)-trans-10-Ethoxy-9-hydroxy-A6a(10a)- tetrahydrocannabinol-C5 ((-)-trans-CBT-Oet), (-)-trans- 10-Etho\y-9-hydro\y-A6a( 10a)- tetrahydrocannabinobc acid-C5 ((-)-trans-CBTA-Oet), (-)-trans-10-Ethoxy-9-hydroxy- A6a(10a)-tetrahydrocannabivarin-C3 [(-)-trans-CBT-Oet], (-)-trans-10-Ethoxy-9-hydroxy- A6a(10a)-tetrahydrocannabivarinic acid-C3 ((-)-trans-CBT-Oet), (-)-trans-Cannabitriol-C5 ((- )-trans-CBT), (-)-trans-Cannabitriobc acid-C5 ((-)-trans-CBTA), (-)-trans-A8-THC, (-)-trans- A8-THCA-C4, (-)-trans-A8-THC-C4, (-)-A7-trans-(lR,3R,6R)-Isotetrahydrocannabinol-C5 (trans-iso- A7-THC), (-)-A7-trans-(lR,3R,6R)-Isotetrahydrocannabinobc acid-C5 (trans-iso- A7-THCA), (-)-A8-trans-(6aR,10aR)-Tetrahydrocannabinol-C5 (A8-THC), (-)-A8-trans- (6aR,10aR)-Tetrahydrocannabinobc acid-C5 (A8-THCA), (-)-A8-trans-(6aR,10aR)- Tetrahydrocannabiorcol-Cl (A8-THCO), (-)-A8-trans-(6aR,10aR)-Tetrahydrocannabiorcobc acid-Cl (A8-THCOA), (-)-A8-trans-THCA-C3 (A8-THCVA), (-)-A8-trans-THC-C3 (D8- THCV), (-)-A9- trans-Tetrahydrocannabiorcobc acid-Cl (A9-THCOA), (-)-A9-(6aS,10aR- cis)-Tetrahydrocannabinol-C5 ((-)-cis-A9-THC), (-)-A9-(6aS,10aR-cis)- Tetrahydrocannabinobc acid- C5 ((-)-cis-A9-THCA), (-)-A9-trans-Tetrahydrocannabinol-C4 (A9-THC-C4), (-)-A9-trans-Tetrahydrocannabinol-C5 (A9-THC), (-)-A9-trans- Tetrahydrocannabinobc acid-C4 (A9-THCA-C4), (-)-A9-trans-Tetrahydrocannabinobc acid- C5 (A9-THCA), (-)-A9-trans-Tetrahydrocannabiorcol-Cl (A9-THCO), (-)-A9-trans- Tetrahydrocannabivarin-C3 (A9-THCV), (-)-A9-trans-Tetrahydrocannabivarinic acid-C3 (A9-THCVA), (+)-trans-Cannabitriol-C5 ((+)-trans-CBT), (+)-trans-Cannabitriobc acid-C5 ((+)-trans-CBTA), (±)-(laS,3aR,8bR,8cR)-Cannabicyclol-C5 (CBL), (±)- (laS,3aR,8bR,8cR)-Cannabicyclobc acid-C5 (CBLA), (±)-(laS,3aR,8bR,8cR)- Cannabicyclovarin-C3 (CBLV), (±)-3”-hydroxy-A4”-cannabichromene-C5, (±)-3”- hydroxy-A4”-cannabichromenic acid-C5, (±)-4-acetoxycannabichromene-C5, (±)-4- acetoxycannabichromenic acid-C5, (±)-6,7-cis-epoxycannabigerol-C5, (±)-6,7-cis- epoxycannabigerobc acid-C5, (±)-6,7-trans-epoxycannabigerol-C5, (±)-6, 7-trans epoxy cannabigerobc acid-C5, (±)-Cannabichromene-C5 (CBC), (±)-Cannabichromenic acid- C5 (CBCA), (±)-Cannabichromevarin-C3 (CBCV), (±)-Cannabichromevarinic acid-C3 (CBCVA), (±)-cis-Cannabitriol-C5 ((±)-cis-CBT), (±)-cis-Cannabitriolic acid-C5 ((±)-cis- CBTA), (5aS,6S,9R,9aR)-Cannabielsoic acid-C5 (CBEA), (5aS,6S,9R,9aR)-Cannabielsoin- C5 (CBE), [(6aR,10aR)-3-[(lS,2R)-l,2-dimethylheptyl]-6a,7,10,10a-tetra hydro-6, 6,9- trimethyl-6H-dibenzo[b,d]pyran-l-ol], r,2',3',4',5'-pentanorcannabinol-3-carboxybc acid, 1'- hydroxycannabinol, 1 '-hydroxy -A9-THC (Isomer B), l'-oxocannabinol, 10- hydroxycannabidiol, 10-O\o-A6a( 10a)-Tetrahydrocannabinol-C5 (OTHC), 10-Oxo- A6a( l()a)-Tetrahydrocannabinolic acid-C5 (OTHCA), 10a-Hydroxy-A8- Tetrahydrocannabinol-C5 (10a-OH-A8-THC), 1 ()a-Hydroxy-A8-Tetrahydrocannabinolic acid-C5 ( 1 ()a-OH-A8-THC A). 10a-hydroxy-A9,ll-hexahydrocannabinol-C5, 10a-hydroxy- A9,ll-hexahydrocannabinobc acid-C5, 10 -Hydroxy-A8-Tetrahydrocannabinol-C5 (10b- OH-A8-THC), 10 -Hydroxy-A8-Tetrahydrocannabinobc acid-C5 ( 10b-OH-D8-TH€ A). 11- Acetoxy-A9-Tetrahydrocannabinoic acid-C5 (ll-Oac-A9-THCA), ll-Acetoxy-A9- Tetrahydrocannabinol-C5 (ll-Oac-A9-THC), 11 -hydroxy tetrahydrocannabinol, 11- hydroxycannabinol, ll-hydroxy-A8-THC, ll-hydroxy-A9-THC, ll-nor-9-carboxy- tetrahydrocannabinol, ll-nor-A8-THC-9-carboxylic acid, 2'-carboxy-3',4',5'-trinor-A9-THC, 2'-hydroxy-A9-THC, 2-acetoxy-6-geranyl-3-n-pentyl-l ,4-benzoquinone-C5, 2-acetoxy-6- geranyl-3-n-pentyl-l,4-benzoquinonic acid-C5, 2-geranyl-5-hydroxy-3-n-pentyl-l,4- benzoquinone-C5, 2-geranyl-5-hydroxy-3-n-pentyl-l,4-benzoquinonic acid-C5, 2-Methyl-2- (4-methyl-2-pentenyl)-7-propyl-2H-l-benzopyran-5-ol, 2-Methyl-2-(4-methyl-2-pentenyl)-7- propyl-2H-l-benzopyran-5-ol acid, 3'-hydroxy-A9-THC, 4'-hydroxy-A9-THC, 4-acetoxy-2- geranyl-5-hydroxy-3-n-pentylphenol-C5, 4-acetoxy-2-geranyl-5-hydroxy-3-n-pentylphenolic acid-C5, 4-acetoxy-2-geranyl-5-hydroxy-3-n-propylphenol-C5, 4-acetoxy-2-geranyl-5- hydroxy-3-n-propylphenolic acid-C5, 4-terpenyl cannabinolate-C5, 4-terpenyl-A9- tetrahydrocannabinolate-C5, 5'-azido-A8-tetrahydrocannabinol, 5'-carboxy-A9-THC, 5'- Dimethylamino-A8-THC, 5 '-hydroxy -A9-THC, 5'-methylamino-A8-THC, 5'-N-methyl-N-4- (7-nitrobenzofurazano)amino-A8-THC, 5'-Trimethylammonium-ll-hydroxy-A8-THC phenolate, 5'-trimethylammonium-A8-THC phenolate, 5-acetyl-4-hydroxycannabigerol-C5, 5-acetyl-4-hydroxycannabigerolic acid-C5, 8,9-Dihydroxy-A6a(10a)-tetrahydrocannabinol- C5 (8,9-Di-OH-CBT), 8,9-Dihydroxy-A6a(10a)-tetrahydrocannabinolic acid-C5 (8,9-Di-OH- CBT), 8-OH-CBNA-C5 (OH-CBNA), 8-OH-CBN-C5 (OH-CBN), 8a-ll-dihydroxy-A9- THC, 8a-Hydroxy-A9-Tetrahydrocannabinol-C5 (8a-OH-A9-THC), 8a-Hydroxy-A9- Tetrahydrocannabinolic acid-C5 (8a-OH-A9-THCA), 8a-hydroxy-A9-THC, 8b-11- Dihydroxy-A9-THC, 8b-Hydroxy-D9-Tetrahydrocannabinol-C5 (8b-OH-D9-TH€). 8b- Hydroxy-A9-Tetrahydrocannabinolic acid-C5 (8b-OH-D9-TH€A). 8b-hydroxy-D9-THC, 9- carboxy-ll-nor-(2 or 4)-chloro-A8-THC, 9-carboxy-ll-norcannabinol, 9-carboxy-ll-nor-A8- THC, 9-carboxy-ll-nor-A9-THC, 9b,10b-Epoxyhexahydrocannabinol-C5, 9b,10b- Epoxyhexahydrocannabinolic acid-C5, AM411, AM708, AM836, AM855, AM919, AM926, AM938, AMG-1, AMG-3, bisnor-cannabielsoin-Cl (CBEO), bisnor-cannabielsoinic acid-Cl (CBEOA), bornyl-A9-tetrahydrocannabinolate-C5. canabidibutol (CBDB), canabidibutol (CBDB), cannabichromanone B-C5, cannabichromanone C-C5, cannabichromanone D-C5, cannabichromanone-C3, cannabichromanone-C5, cannabichromanonic acid B-C5, cannabichromanonic acid C-C5, cannabichromanonic acid D-C5, cannabichromanonic acid- C3, cannabichromanonic acid-C5, cannabichromene (CBC), cannabichromene propyl analogue, cannabichromevarin (CBCV), cannabicitran-C5 (CBR), cannabicitranic acid-C5 (CBRA), cannabicoumaronic acid-C5 (CBCONA), cannabicoumaronone-C5 (CBCON-C5), cannabicyclohexanol (CP-47,497 C8 homolog), cannabicyclol (CBL), cannabicyclolyc acid- C3 (CBLVA), cannabidiol (CBD), cannabidiol monomethyl ether-C5 (CBDM), cannabidiol propyl analogue (CBDV), cannabidiol-C4 (CBD-C4), cannabidiolic acid (CBD A), cannabidiolic acid-C5 (CBDA), cannabidiorcol-Cl (CBDO), cannabidiorcolic acid-Cl (CBDOA), cannabidiphorol (CBDP), cannabidiphorol (CBDP), cannabidivarin (CBDV), cannabidivarinic acid-C3 (CBDV A), cannabielsoic acid-C3 (CBEVA), cannabielsoin (CBE), cannabielsoin-C3 (CBEV), cannabifuran-C5 (CBF), cannabifuranic acid-C5 (CBFA), cannabigerol (CBG), cannabigerol monomethyl ether (CBGM), cannabigerol monomethyl ether-C5 (CBGM), cannabigerol-C5 (CBG), cannabigerolic acid (CBGA), cannabigerolic acid monomethyl ether-C5 (CBGMA), cannabigerolic acid-C5 (CBGA), cannabigerovarin (CBGV), cannabigerovarin-C3 (CBGV), cannabigerovarinic acid (CBGVA), cannabigerovarinic acid (CBGVA), cannabigerovarinic acid-C3 (CBGVA), cannabiglendol- C3 (OH-iso-HHCV-C3), cannabiglendolic acid-C3 (OH-iso-HHCVA-C3), cannabimovone- C5, cannabimovonic acid-C5, cannabinodiol (CBDL), cannabinodiol-C5 (CBND), cannabinodiolic acid-C5 (CBND A), cannabinodivarin-C3 (CBNDV), cannabinol (CBN), cannabinol methyl ether-C5 (CBNM), cannabinol-C2 (CBN-C2), cannabinol-C4 (CBN-C4), cannabinol-C5 (CBN), cannabinolic acid (CBNA), cannabinolic acid-C4 (CBNA-C4), cannabinolic acid-C5 (CBNA), cannabiorchromene-C 1 (CBCO), cannabiorchromenic acid- Cl (CBCOA), cannabiorcol-Cl (CBNO), cannabiorcolic acid-cl (CBNOA), cannabiorcyclol Cl (CBLO), cannabiorcyclolic acid Cl (CBLOA), cannabioxepane-C5 (CBX), cannabioxepanic acid-C5 (CBXA), cannabitriol (CBTL), cannabitriol-Cl (CBTO), cannabitriol-C3 (CBTV), cannabitriolic acid-Cl (CBTOA), cannabivarin (CBV), cannabivarin-C3 (CBNV), carmagerol-C5, carmagerolic acid-C5, CBCA-C2, CBCA-C4, CBC-C2, CBC-C4, CBDA-C2, CBDA-C4, CBD-C2, CBDMA-C5 (CBDMA), CBEA-C2, CBEA-C4, CBE-C2, CBE-C4, CBGA-C1 (CBGOA), CBGA-C2, CBGA-C4, CBG-C1 (CBGO), CBG-C2, CBG-C4, CBLA-C2, CBLA-C4, CBL-C2, CBL-C4, CBNA-C2, CBNA- C3 (CBNV A), CBNDA-C 1 (CBNDOA), CBNDA-C2, CBNDA-C3 (CBNDV A), CBND A- C4, CBND-C1 (CBNDO)*, CBND-C2, CBND-C4, CBNDMA-C5 (CBNDMA), CBNDM- C5, CBNMA-C5 (CBNMA), CBTA-C2, CBTA-C3 (CBTVA), CBTA-C4, CBT-C2, CBT- C4, CP 47497, CP 50556, CP 55940, CP 55244, CT-3 or IP-751 (ajulemic acid), dehydrocannabifuran-C5 (DCBF), dehydrocannabifuranic acid-C5 (DCBFA), desacetyl-L- nantradol, dexanabinol, dimethylheptyl HHC, epi-bomyl-A9-tetrahydrocannabinolate-C5, HU-210, HU-211, HU-308, JWH-051, JWH-133, L-5 759633, levonantradol, nabilone, O- 1184, OH-CBDA-C5 (OH-CBDA), OH-CBD-C5 (OH-CBD), OH-CBNDA-C5 (OH- CBNDA), OH-CBND-C5 (OH-CBND), obvetobc acid, sesquicannabigerol-C5 (SesquiCBG), sesquicannabigerobc acid-C5 (SesquiCBGA), tetrahydrocannabinobc acid (THCA), tetrahydrocannabiphorol (THCP), tetrahydrocannabivarin (THCV), tetrahydrocannabutol (THCB), WIN 55212-2, a-fenchyl-A9-tetrahydrocannabinolate-C5, a-terpenyl-A9- tetrahydrocannabinolate-C5, -fenchyl-A9-tetrahydrocannabinolate-C5, D7- tetrahydrocannabivarin-C3 (A7-THCV), A7-tetrahydrocannabivarinic acid-C3 (A7-THCVA), A8-tetrahydrocannabinol (A8-THC), A8-tetrahydrocannabinol-DMH, A8-THCA-C2, D8- THC-C2, A9-tetrahydrocannabinol (A9-THC), A9-tetrahydrocannabinol propyl analogue (THCV), A9-tetrahydrocannabiphorol (THCP), A9-tetrahydrocannabutol (THCB), D9- THCA-C2, A9-THC-C2, A9-THCMA-C5 (A9-THCMA), A9-THCM-C5.

In certain embodiments, the cannabinoid is CBGA or a compound derived from CBGA, such as CBG, A9-THC, THCA, THCV, A9-THCV, A9-THCVA, CBD, CBDA, CBDV, CBDL, CBC, CBCA, CBCV, CBCN, CBV, CBGV, CBN, CBL, and CBE. Preferred cannabinoids are anionic cannabinoids.

A preferred cannabinoid fermentate is an aqueous composition comprising CBGA, yeast extracts and one or more cannabinoid intermediates.

The concentration of cannabinoid in the cannabinoid fermentate is typically less than about 20% by wt, including less than 10% by wt, or possibly less than 5% by wt. and/or at least about 2% by wt.

The cannabinoid fermentate, isolated from the reactor can optionally be dehydrated. The dehydration step can desirably reduce the water content by at least about 30% by weight, such as at least about 40% by wt., more preferably at least about 50% by wt. The concentration of cannabinoid in the dehydrated fermentate crude is between about 8 to about 20% by weight, such as between about 9 and about 17% by weight.

The optionally dehydrated cannabinoid fermentate crude is then subjected to an extraction step. Preferably, an organic polar solvent is contacted with the optionally dehydrated fermentate crude for extraction. Examples of polar solvents include ethyl acetate, acetonitrile, acetone, dimethyl sulfoxide, dimethyl formamide, ethylene glycol, tetrahydrofuran, chloroform, dichloromethane, and ethanol, for example. Preferred solvents are considered safe per the FDA or the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Preferred solvents are a Class 3 or better per ICH as of February 8, 2021 at its website at ICH.org. A preferred solvent is ethyl acetate.

Several solvents were assessed. The results of the assessment are set forth below:

Ethyl acetate was shown to be superior in CBGA yields.

Solvent (e.g., ethyl acetate) is added to the cannabinoid fermentate in an amount sufficient to form a supersaturated solution of the cannabinoid (e.g., CBGA). For example, the ratio of fermentate (crude) to solvent can be about 1 : 1 (w/v). Alternatively, the solvent can be added in excess. The ratio of solvent: fermentate crude can be between 1 : 1 and 20: 1, such as between 1:1 and 10:1, preferably between about 5:1 to 8:1, w/w in each case.

The mixture can be maintained to allow for the cannabinoid (e.g., CBGA) to transfer from the fermentate crude to the organic phase. In the experiments described below, 90 minutes was deemed to be sufficient. It will be generally desirable to conduct the extraction in a closed vessel to minimize solvent evaporation. The organic phase containing the cannabinoid (CBGA), (i.e., the extraction supernatant) can then be collected. The insoluble precipitate can then be recycled, if desired. Solvent can then be removed from the supernatant. Evaporation, for example, such as under a vacuum or reduced pressure (e.g., 1 atm to about 10 torr) is desirable. The cannabinoid content in the dried extract can be at least about 25 wt.%, such as at least about 30% by wt. and yields can be at least about 90%, such as at least about 95% by wt. The product of the extract step is referred to herein as the cannabinoid extract.

Where the cannabinoid is anionic, such as CBGA, it is preferred to decarboxylate the compound after extraction, i.e., the CBGA extract. Decarboxylation can be achieved with heat treatment, optionally under vacuum and/or under an inert atmosphere. Decarboxylation of CBGA can occur, for example, by maintaining a CBGA extract at a temperature of at least about 80 °C or 90°C, at least about 100°C, such as between about 100°C to about 150°C, or at about 110°C. The decarboxylation step can be maintained at a temperature approaching the reflux temperature of the mixture, for example. Solvents that can be used include ethyl acetate, isopropyl acetate, heptane and/or toluene, for example. Heptane is a preferred solvent for decarboxylation. In embodiments, a base, such as NaOH can be added.

The decarboxylation can be conducted under conditions of a vacuum, such as in a vacuum oven at a temperature of at least about 100°C for at least about 10 hours, such as at least about 12 hours, such as at least about 15 hours. The decarboxylation conditions can optionally be conducted under inert atmosphere, such as nitrogen or a noble gas, such as argon. For example, the CBGA extract can be maintained under nitrogen gas. In some embodiments, decarboxylation can be conducted under a nitrogen gas blanket at a temperature of at least about 110°C for at least about 15 hours, such as at least about 16 hours, such as at least about 18 hours. Decarboxylation is preferably maintained until at least about 75%, preferably at least about 80%, such as at least about 90%, more preferably at least about 95%, and most preferably at least about 99% of the anionic cannabinoid, such as CBGA, is decarboxylated. The concentration of the decarboxylated cannabinoid is preferably at least about 25% wt. The concentration of the remaining anionic cannabinoid is preferably less than about 4% by wt., such as less than 2% by wt. CBG is the preferred product of CBGA decarboxylation. The product produced by the decarboxylation step of CBGA is referred to herein as the CBG extract. The concentration of decarboxylated cannabinoid extract can be at least about 25% by weight.

The cannabinoid extract, including the CBGA extract and/or the CBG extract, can then be subjected to chromatography, such as flash chromatography, or a silica plug. A preferred chromatographic method utilizes a silica column and an isocratic mobile phase of heptane and acetone and monitored at an absorbance of at least about 210 nm, preferably about 220 nm. Other alkane solvents can also be used, such as pentane or heptane. Other polar organic solvents, such as ethyl acetate, can be used as well. The ratio of alkane (e.g., heptane) to polar solvent (e.g., acetone) can be 9: 1 by volume. The CBG fraction isolated from a CBG extract can have a purity of at least about 65%, such as at least about 80%, preferably at least about 85%, more preferably at least about 88% by wt. Yields of cannabinoid can be at least about 80%, such as at least about 85% by wt.

Additionally or alternatively, the cannabinoid extract, such as the CBGA or CBG extract, can be mixed with a silica gel (e.g., a silica plug) or charcoal in a suitable solvent, such as ethylacetate, isopropylacetate or heptane. The mixture can then be concentrated to a solid, which can be filtered and washed to elute the cannabinoid.

The cannabinoid extract, e.g., CBG extract, can be additionally or alternatively subjected to clarification. For example, a CBG extract can be dissolved in an organic solvent, such as ethyl acetate. A cannabinoid non-solvent can be added to the cannabinoid extract or isolate. Cannabinoid non-solvents include olive oil, sesame oil, castor oil, coton-seed oil, soybean oil, butane, pentane, hexane, heptane, octane and toluene. Heptane is preferred for CBG extract. The cannabinoid concentration of the clarified cannabinoid can be at least 40% by wt, such as at least about 60% by wt., such as at least about 70% by wt. The cannabinoid yield in the clarified cannabinoid is preferably at least about 60%, such as at least about 75% by wt., such as at least about 77% by wt.

The cannabinoid isolate and/or clarified cannabinoid can be further crystallized with a cannabinoid non-solvent. Cannabinoid non-solvents include olive oil, sesame oil, castor oil, coton-seed oil, soybean oil, butane, pentane, hexane, heptane, octane and toluene. Pentane or heptane is preferred for CBG. A substantially pure cannabinoid is thereby obtained. A substantially pure cannabinoid is characterized by a purity of at least about 95%, such as at least about 97%, or 98% or 99% or more purity as measured by HPLC. A substantially pure cannabinoid has very low levels of microbial impurities, as described above, and/or residual solvent/nonsolvent. In embodiments, microbial impurities are present at minimally detectable amounts. The residual solvent/nonsolvent (e.g., pentane, hexane or heptane) level can be less than about 500 ppm, such as less than about 100 ppm, for example, or less than about 20 ppm.

Solvent/non-solvent can be removed, for example, by evaporation, rotary evaporation, vacuum distillation, tangential flow filtration (TFF), ultracentrifugation, and/or freeze drying.

The cannabinoids (e.g., CBG or CBGA) isolated in accordance with the invention can be optionally further chemically modified to form other cannabinoid products (e.g., CBD) of high purity. Cannabinoids made in accordance with the invention are pharmaceutical-grade. Accordingly, they can be readily encapsulated, for example, by a taste-neutral cationic polymer, and further comprising a non-ionic surfactant and methods for the preparation thereof. A nanoprecipitates of the cannabinoids of the invention can be prepared by nanoprecipitation (also referred to as solvent displacement or interfacial deposition). Methods of nanoprecipitation have been described, for example, in U.S. Pat. No. 5,118,528, the contents of which are expressly incorporated by reference herein. The nanoprecipitate is generally of a size less than 1000 nm. In certain aspects, the nanoprecipitate has a diameter less than about 500 nm. In certain aspects, the nanoprecipitate has a z-average particle size is between about 20 to about 400 nm, about 25 to about 300 nm, about 30 to about 200 nm, about 40 to about 150 nm, about 50 to about 130 nm, or about 70 to about 300 nm.

The invention is illustrated by the following examples which are not meant to be limiting in any way.

EXEMPLIFICATION Example 1: CBGA solubility analysis:

The solubility of CBGA fermentate crude in a number of organic solvents was assessed by creating a supersaturated solution of crude: solvent. The summary of data is below:

Ethyl acetate was selected for further study. Example 2: Ethyl acetate extraction of CBGA from a fermentation broth

Fermentation broth was centrifuged and the supernatant was discarded to reduce the water content to <50%. CBGA content of the semi dried fermentate crude was approx. 9- 17wt. %. 149g of the semi dried fermentate crude (containing approx. 60-70g water) and l,188g ethyl acetate were added into a 2L glass beaker. The mixture was mixed at 600rpm and room temperature for 90min using a KA Eurostar 40 digital mixer. The beaker was covered with foil to minimize solvent evaporation. After stirring, the mixture was allowed to settle at room temperature for another 30min. The supernatant was transferred to a flask and dried using a rotary evaporator, from 1500 to lOtorr. Typical CBGA content and percent yield values of the extract were 35-43wt.% and >95%, respectively.

Example 3: Decarboxylation of CBGA extract

(a) in vacuo : 13g dried CBGA extract was incubated at 110°C for 17h in a vacuum oven. Reaction yielded 12g of extract containing 38.8% CBG and 3.8% CBGA.

(b) under inert atmosphere: 60.6g dried CBGA extract was incubated at 110°C with 400 rpm stirring for 18h under inert atmosphere (nitrogen gas). The yield was 54.6g of extract containing 25 to 39.4% CBG and 0-1.1% CBGA.

Example 4: Thermal Decarboxylation of CBGA extract to CBG extract

104 g of extract containing 42 wt% CBGA was warmed in a 50°C water bath and transferred to a 500 mL 3 -neck round bottom flask with overhead stirring at 200 rpm under nitrogen. 100 mL heptane as added and the mixture was heated, dissolving at 45°C, to 96- 99°C for 40 hours. HPLC assay indicated 100% conversion to CBG with non-detectable (<0.03A%) CBGA.

Example 5: Base-promoted decarboxylation of CBGA extract to CBG extract.

133 g of biosynthetic extract containing 28 wt% CBGA was added to a 1-L 3-neck round bottom flask with overhead stirring at 300 rpm. 310 mL aqueous sodium hydroxide (2N, 6 equiv, 2.3 vol) was added and the mixture was heated to 84 °C. After 40 min a sample was quenched into IN HC1 and ACN for HPLC, indicating complete conversion to CBG.

The solution was cooled to 35 °C and diluted with 300 mL isopropyl acetate. The mixture was stirred for 5 minutes to dissolve all solids and transferred to a separatory funnel. After settling for 10 minutes the dark brown bottom layer was removed. The top greenish-brown layer was washed with 100 mL aqueous sodium hydroxide (IN) and the layers were separated. The organic layer was transferred back to the reaction flask and stirred vigorously with 100 mL aqueous hydrochloric acid for 10 minutes until the color lightened to orange. The aqueous was removed. The organic layer washed with 100 mL 10% aqueous sodium chloride and dried over sodium sulfate. The solution was filtered and concentrated to 93.22 g orange oil contained 31 wt% CBG with 88 % yield.

Example 6: Chromatographic isolation of CBG

2% of CBG extract solution in EtOAC containing 25wt.% CBG and 2.5wt.% CBGA was loaded into a pre-rinsed and equilibrated silica column (Sorbtech). Elution was achieved using an isocratic mobile phase of heptane: acetone (9:1) and monitored at UV220nm. The purity and yield of collected CBG isolate fraction were 89wt.% and 85%, respectively.

Example 7: Silica plug purification of CBG extract to CBG isolate

104 g of CBG extract and 104 g silica (60-230 micron) were slurried in 200 mL heptane in a 500 mL round bottom flask. The mixture was concentrated by rotary evaporation to a dry solid. The solid was transferred to a 600 mL sintered glass funnel, wetted with a minimal amount of heptane, and covered with filter paper. The silica cake was eluted with 3 L heptane, and the solution was concentrated to an oil which solidified. The 50.1 g wet solid assayed for 60 wt% CBG in 77 % recovery.

Example 8: Clarification for CBG

888 mg of Cannabigerol (CBG) extract (containing 34.1wt.% CBG and l.lwt.% CBGA) was dissolved in 4mL EtOAC in a lOOmL round flask. The solution was mixed with magnetic stirring at 250 rpm/min and room temperature. 40mL n-heptane was slowly added at a rate of lOmL/min. The mixture was allowed to settle for 5 min to facilitate precipitation. The resulting milky supernatant was separated. The precipitate was re-suspended in 4mL EtOAC, and n-heptane precipitation was repeated. The supernatant from 2 precipitation steps were combined. 6g of silica chromatography gel (Millipore) was added, and the mixture was stirred at 800rpm and room temperature for 5 min. The mixture was filtered using a Buchner funnel coupled with filter paper. The retentate was rinsed twice with lOmL mixture of heptane: EtOAC (10:1). The rinsing solution was filtered using a Buchner funnel and combined with the filtrate. The combined solution was dried using rotary evaporation. The dried material was dissolved in MeOH at 30mg/mL. Upon gentle shaking for 2min, an oily droplet formed, which was removed using a glass pipette. The solution was dried using rotary evaporation to obtain clarified CBG. The purity and yield values of clarified CBG were 64.5wt.% and 77.7%, respectively.

Example 9: Crystallization CBG isolate to final CBG product

20g of CBG isolate was dissolved in n-pentane at 90mg/mL in a flask by bath sonication at 32°C for lOmin. The mother liquor was transferred into a 4°C water bath, while stirring using a stainless steel spatula for 5min, until the formation of crystals. The crystals were vacuum filtered and the retentate was rinsed twice with pre-cooled n-pentane. The filtrate was crystallized, and vacuum filtered. The (2nd) retentate was rinsed twice with pre cooled n-pentane. The yield of both crystallization steps were combined and dried in a vacuum oven for 16h to obtain CBG crystal. The CBG crystal was white powder. The CBG purity and yield values of the product were 100 ± lwt.% (N=3) and 76% as measured using HPLC, respectively. The residual n-pentane content measured using GC-FID was approx. 10 ppm.

Example 10: Isolation CBG isolate to final product CBG crystal

50.1 g CBG isolate (60 wt% CBG) was slurried in 160 mL heptane in a 500 mL 3 neck round bottom flask with overhead stirring. The slurry was heated to dissolution at 40 °C and the heat was removed. When the solution reached 25 °C it was seeded with 250 mg CBG crystal. The resulting slurry thickened for 30 min and was cooled to 0 °C to -10 °C for 2h. The cooled slurry was filtered over a sintered glass funnel. 2 x 30 mL heptane was used to transfer residual solid from the flask. The filter cake was then washed with 100 mL heptane and dried on the funnel for lh. The isolated 27.78 g solid had a purity of 98.7 wt% in 91% yield of CBG from crude and 70 % yield from CBGA extract.

Example 11: Alternative method 1 for CBGA/CBG purification:

Alternatively, CBGA broth can first be processed into CBGA isolate (broth > CBGA extract > CBGA isolate) before crystallization and decarboxylation. CBGA isolate can further be purified to pure CBGA product via crystallization. Furthermore, CBGA crystal can then be converted into its neutral form, CBG crystal, by incubation at 110°C in vacuum.

Example 12: Alternative method 2 for CBGA/CBG purification

CBGA extract also could be clarified prior to decarboxylation. CBGA extract was dissolved in EtOAC at a concentration of 10wt.% CBGA, and 4ml extract solution was transferred into a lOOmL round bottle. The solution was mixed with magnetic stirring at 500rpm and room temperature, followed by addition of 40mL heptane at a rate of lOml/min. The mixture was stirred at 500rpm for another 10 min. The stirring was stopped, and the mixture was allowed to settle for 10 min to facilitate precipitation of impurities. Following precipitation, the milky supernatant was collected by pouring. The remaining precipitate was re-dissolved in 4mL EtOAC, and heptane precipitation was repeated. The supernatant from 2 precipitation steps were combined, to which 5.2g of silica chromatography gel (Millipore) was added, and the mixture was magnetically stirred at 500 rpm and room temperature for 5 min. The solution was filtered using a Buchner funnel coupled with a filter paper. The retentate was rinsed twice using 7mL of heptane: EtOAC (10: 1) mixture. The rinsing solution was filtered using a Buchner funnel and combined with the filtrate. The combined solution was dried using rotary evaporation. The dried filtrate was dissolved in MeOH at 20 mg/mL. Upon gentle shaking for 2min, an oily droplet formed, which was removed using a glass pipette. The solution was dried using rotary evaporation to obtain clarified CBGA. The CBGA purity and yield values of clarified CBGA were 64.8 wt.%, and 58.4%, respectively.

Clarified CBGA can further be purified to pure CBGA product via crystallization, or via a combination of chromatography and crystallization. CBGA crystal can then be converted into its neutral form, CBG crystal, by incubation at 110°C in vacuum.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.