WO2022182844A1 - Cannabinoïdes purifiés isolés à partir d'un produit de fermentation - Google Patents

Cannabinoïdes purifiés isolés à partir d'un produit de fermentation Download PDF

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WO2022182844A1
WO2022182844A1 PCT/US2022/017659 US2022017659W WO2022182844A1 WO 2022182844 A1 WO2022182844 A1 WO 2022182844A1 US 2022017659 W US2022017659 W US 2022017659W WO 2022182844 A1 WO2022182844 A1 WO 2022182844A1
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cannabinoid
extract
weight
concentration
cbg
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PCT/US2022/017659
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Wenmin YUAN
Solange Valdes CURIQUEN
Karen Melissa MARCANTONIO
Bin Zhao
Oren Levy
Tuna Yucel
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Molecular Infusions Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/50—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by reactions decreasing the number of carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/82—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by solid-liquid treatment; by chemisorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/68—Purification; separation; Use of additives, e.g. for stabilisation
    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
    • C07C37/84—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment by crystallisation

Definitions

  • 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.
  • THC phytocannabinoid tetrahydrocannabinol
  • 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.
  • 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.
  • CINV chemotherapy-induced nausea and vomiting
  • CBD cannabidiol
  • 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.
  • 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.
  • AAE1 acyl activating enzyme
  • PES polyketide synthase
  • OAC olivetolic acid cyclase
  • THCAS THCA synthase
  • CBDA synthase CBDA synthase
  • CBCAS CBCA synthase
  • 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.
  • 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.
  • CBG cannabigerol
  • 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.
  • a solvent such as heptane
  • step (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.
  • 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.
  • 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.
  • CBD Cannabigerolic acid
  • the invention relates to the purification of cannabinoids from biosynthetic starting materials, or fermentates.
  • 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.
  • 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).
  • CBDA cannabigerolic acid
  • the cannabinoid fermentate can be a broth produced by fermenting a recombinant microorganism engineered to produce a cannabinoid.
  • 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 e.g., Saccharomyces. Cerevisiae or Kluyveromyces marxianis
  • bacterium e.g., Escherichia cob
  • microalgae fungi
  • insect e.g., Chinese hamster ovary
  • animal cells e.g., Chinese hamster ovary
  • Yeast is preferred.
  • a preferred yeast is S. cerevisiae.
  • the microorganism (or yeast) does not
  • the microorganism can be genetically engineered to express a cannabinoid synthesis pathway, such as the pathway found in cannabis.
  • 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.
  • AAE1 acyl activating enzyme
  • PES polyketide synthase
  • OAC olivetolic acid cyclase
  • PT prenyltransferase
  • THCAS THCA synthase
  • CBDAS CBDA synthase
  • 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).
  • a carbon source such as a sugar, alcohol, and/or fatty acid
  • 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.
  • a carbon source such as a sugar, alcohol, and/or fatty acid.
  • 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 can include a fatty acid, such as a C4-C8 alkanoic acid, including butyric acid, pentanoic acid, hexanoic acid, heptanoic acid and octanoic acid.
  • the media can comprise a combination of hexanoic acid, yeast extract, peptone, and glucose.
  • the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 100 mg/L hexanoic acid.
  • hexanoic acid can be used in an amount of 1 mg/L to 1 g/L.
  • 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.
  • 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.
  • the media can comprise a combination of olivetolic acid, yeast extract, peptone, and glucose.
  • the media can comprise 10 g/L of yeast extract, 20 g/L peptone, 20 g/L glucose and 40 mg/L hexanoic acid.
  • 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.
  • 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.
  • 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.
  • the fermentate is cell-free.
  • 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- hydroxycannabichroman
  • 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-benzoquino
  • 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.
  • CBD canabidibutol
  • CBDB canabidibutol
  • CBDB canabidibutol
  • CBDB cannabichromanone B-C5
  • cannabichromanone-C3 cannabichromanone-C5
  • cannabichromanonic acid D-C5 cannabichromanonic acid- C3, cannabichromanonic acid-C5
  • CBC cannabichromene
  • cannabichromene propyl analogue cannabichromevarin (CBCV), cannabicitran-C5 (CBR), cannabicitranic acid-C5 (CBRA), cannabicoumaronic acid-C5 (CBCONA), cannabicoumaronone-C5 (CBCON-C5), cannabicyclohexanol (CP-47,
  • 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.
  • an organic polar solvent is contacted with the optionally dehydrated fermentate crude for extraction.
  • 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.
  • Solvent e.g., ethyl acetate
  • cannabinoid fermentate in an amount sufficient to form a supersaturated solution of the cannabinoid (e.g., CBGA).
  • the ratio of fermentate (crude) to solvent can be about 1 : 1 (w/v).
  • 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
  • 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.
  • the cannabinoid is anionic, such as CBGA
  • 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.
  • 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.
  • the CBGA extract can be maintained under nitrogen gas.
  • 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.
  • the cannabinoid extract such as the CBGA or CBG extract
  • a silica gel e.g., a silica plug
  • charcoal e.g., a suitable solvent
  • 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
  • CBG extract can be additionally or alternatively subjected to clarification.
  • 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, Lac-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, Lactane, 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.
  • 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.
  • 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.
  • Ethyl acetate was selected for further study.
  • Example 2 Ethyl acetate extraction of CBGA from a fermentation broth
  • 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 5 Base-promoted decarboxylation of CBGA extract to CBG extract.
  • 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 7 Silica plug purification of CBG extract to CBG isolate
  • CBG Cannabigerol
  • Example 11 Alternative method 1 for CBGA/CBG purification:
  • 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.
  • CBGA crystal can then be converted into its neutral form, CBG crystal, by incubation at 110°C in vacuum.
  • 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.
  • 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.

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Abstract

L'invention concerne des procédés de purification de cannabinoïdes à partir de produits de fermentation, tels qu'un produit de fermentation de levure.
PCT/US2022/017659 2021-02-24 2022-02-24 Cannabinoïdes purifiés isolés à partir d'un produit de fermentation WO2022182844A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100168448A1 (en) * 2002-09-23 2010-07-01 Gw Pharma Limited Methods of Preparing Cannabinoids From Plant Material
US20160214920A1 (en) * 2015-01-22 2016-07-28 Phytoplant Research S.L. Methods of Purifying Cannabinoids, Compositions and Kits Thereof
WO2017139496A1 (fr) * 2016-02-09 2017-08-17 Cevolva Biotech, Inc. Génie microbien pour la production de cannabinoïdes et de précurseurs de cannabinoïdes
WO2020028198A1 (fr) * 2018-08-03 2020-02-06 Biomass Oil Separation Solutions, Llc Procédés et appareil d'extraction de substances et d'extraits enrichis à partir d'une matière végétale
US20200399194A1 (en) * 2017-07-07 2020-12-24 Orochem Technologies Inc. Process for separating a constituent/cannabinoid using a chromatographic resin
US20210189444A1 (en) * 2019-12-20 2021-06-24 Demetrix, Inc. Method of purifying cannabinoids from yeast fermentation broth

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100168448A1 (en) * 2002-09-23 2010-07-01 Gw Pharma Limited Methods of Preparing Cannabinoids From Plant Material
US20160214920A1 (en) * 2015-01-22 2016-07-28 Phytoplant Research S.L. Methods of Purifying Cannabinoids, Compositions and Kits Thereof
WO2017139496A1 (fr) * 2016-02-09 2017-08-17 Cevolva Biotech, Inc. Génie microbien pour la production de cannabinoïdes et de précurseurs de cannabinoïdes
US20200399194A1 (en) * 2017-07-07 2020-12-24 Orochem Technologies Inc. Process for separating a constituent/cannabinoid using a chromatographic resin
WO2020028198A1 (fr) * 2018-08-03 2020-02-06 Biomass Oil Separation Solutions, Llc Procédés et appareil d'extraction de substances et d'extraits enrichis à partir d'une matière végétale
US20210189444A1 (en) * 2019-12-20 2021-06-24 Demetrix, Inc. Method of purifying cannabinoids from yeast fermentation broth

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