DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application no. EP22305320.8 filed March 18th, 2022, and PCT/EP2023/056850 filed March 17th, 2023, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is March 18th, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on March 3rd, 2025, was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
The listing of references on pages 39-40 of the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Status of Claims
Claims 1-20 filed September 17th, 2024 are pending and examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see for example, pg. 8, ln. 29 and pg. 10, ln. 24). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 10 objected to because of the following informalities: Line 2 of claim 10 appears to attempt to recite the acronym for optical density, “OD”, but recites “DO” as the acronym. As optical density is referred to as OD in the instant specification [pg. 6, ln. 18-19]. It is suggested to amend the acronym to be “OD”. Appropriate correction is required.
In claim 3, “MS”, “DKW”, “B5”, “WPM”, and “SH” are used as abbreviation. It is suggested to insert a definition for “MS”, “DKW”, “B5”, “WPM”, and “SH” without bringing in new matter, immediately before the first appearance of “MS”, “DKW”, “B5”, “WPM”, and “SH” in claim 3; and to enclose the appearance of “MS”, “DKW”, “B5”, “WPM”, and “SH” in parentheses (in claim 3 only).
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 8, 10, and 19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 8 recites the limitation "the abiotic stress condition used in step d)" in line 2. Claim 1, from which 8 depends, does not recite an abiotic stress condition. There is insufficient antecedent basis for this limitation in the claim. Claim 19 is also rejected insofar as it depends from claim 9, and do not overcome the stated rejection.
Claim 10 recites the method according to claim 1, wherein the optical density at 600 nm of the one or more bacterial and/or yeast strains in the liquid culture medium after inoculation in step c) is between 0.0001 and 0.1. OD has several challenges and limitations when used for tracking bacterial cell concentration in liquid culture1. OD depends on cell size and morphology, meaning that the specific bacterial and/or yeast strains in the liquid culture would vary in OD based on their specific properties. Stress, nutrient limitation, and approaching stationary phase all shift cell size, so OD can keep changing while the cell count is steady. OD reports an arbitrary number that is not convertible to cells/mL or any standard biological unit and the reading are instrument-dependent, requiring cross-calibration between instruments to get the same OD values. OD further does not distinguish cells by physiological state, meaning that there is no single OD reading at which cells stop counting toward the output.
The instant specification provides only one example of OD in practice, disclosing a “final OD” of 0.0057 for Roseomonas mucosa with Cannatonic cultivar. The claim simply states that the OD600 is measured after step c), the inoculation of the sterile liquid culture medium with one or more bacterial and/or yeast strains, but it is unclear if there is a specific timepoint at which the OD should be measured to get the claimed reading between 0.0001 and 0.1. This makes OD600 of 0.0001 and 0.1 a relative term because it cannot be determined in the exact parameters it was originally tested and may change depending on the context, comparison or reference point. Thus, it is not clear exactly what the Applicant is claiming and if the prior art can read on the claim.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Applicant describes:
Sterile apical meristems and nodal segments cultured in a temporary immersion bioreactor [Examples 1 & 2].
A method for producing plant tissue using plant material from Cannatonic cultivar, Jamaican Pearl cultivar, California indica cultivar, Amazing Haze cultivar, Michka cultivar, Sensi 741 cultivar, and Afghani cultivar.
Evaluation of decarboxylated cannabinoids THC, CBC and CBD [Examples 1 & 2].
Supplementation of sterile liquid culture MS and DKW medium with 0.5 mg/L BAP [Examples 1 & 2].
The identification of Curtobacterium spp. Staphylococcus epidermis, Micrococcus luteus, Microbacterium ginsengisoli, Cystobasidium minutum in Example 1, Roseomonas mucosa in Example 2, as well as Bacillus megaterium and Sphingomonas paucimobilis (data not shown; pg. 35, lns. 26-28).
The Applicant does not describe:
A method for producing plant tissues comprising inoculating a system with any sterile plant material.
A method for producing plant tissue containing decarboxylated cannabinoids from any Cannabis sativa cultivar.
A method for producing plant tissue containing any decarboxylated cannabinoids with inoculation of bacteria or yeast.
A method for producing plant tissue comprising a medium with any cytokinin and/or auxin at any concentration.
Inoculating the sterile liquid culture medium with any bacterial and/or yeast strains at any concentration.
The claims are broadly directed to a method for producing plant tissue containing one or more decarboxylated cannabinoids (claims 1-13 and 16-20), a method for producing one or more decarboxylated cannabinoids (claim 14), and a method for decarboxylating on or more cannabinoids in plant tissue (claim 15). The claims broadly require any plant tissue, any decarboxylated cannabinoid, and any bacterial and/or yeast strains, with some dependent claims being further narrowing. Applicant describes using sterile apical meristems and nodal segments for Cannabis sativa in culturing [pg. 31, lns. 29-30], but claims the broad genus of inoculating a temporary liquid immersion culture system with any sterile plant material. The success of propagation in a temporary liquid immersion culture system relies on the type of explant and the genotype of cannabis. Indeed, Monthony, A. et al. (2021, “The Past, Present and Future of Cannabis sativa Tissue Culture.” Plants. 10:185) teaches that micropropagation is highly dependent on the genotype, tissue type, and physiological state of the material [pg. 16, ¶2].
Monthony teaches that most protocols for micropropagation in cannabis rely on shoot multiplication from existing meristems found in the apical and axillary nodes, comprising regions of high cellular plasticity with cells early in their developmental state [pg. 13, ¶2]. Nodal cuttings can be grown In Vitro, much like vegetative greenhouse propagation. However, existing micropropagation protocols that rely on regeneration from non-meristematic tissues often report low levels of regeneration [pg. 15, ¶3]. The Applicant has not reduced to practice the broad genus of a method of producing plant tissue comprising cultivating any Cannabis sativa plant material as the genus of plant material can include highly variable tissue of any type or age that may not predictably achieve a successful result in the claimed method. Thus, it is not clear that the structure of plant material will necessarily perform the claimed function.
Examiner notes that claim 11 overcomes this particular rejection with the recitation of the plant material described in the instant application, apical meristems and nodal segments, but remains rejected due to lack of written description of other claim limitations.
Additionally, claim 1 broadly claims producing plant tissues containing one or more decarboxylated cannabinoids comprising sterile plant material from any Cannabis sativa cultivar. Monthony teaches that the success of micropropagation is highly dependent on the genotype [pg. 16, ¶2]. The range of genetic variability in Cannabis can be seen by the physiological and chemical differences between hemp and drug-type cultivars. Hemp produces negligible (<0.3%) levels of THC and has been bred for a high fiber and oil content. In contrast, drug-type Cannabis can contain anywhere from 5% to over 20% THC. Monthony further teaches that, in an assessment of regeneration of young leaves, petioles, internodes, and axillary buds in five hemp cultivars, callusing and regeneration levels were cultivar- and tissue-dependent [pg. 17, ¶2].
Cannabis sativa displays extreme genetic and phenotypic variability. Aina, A. et al. (2025, “Genetic diversity, population structure, and cannabinoid variation in feral Cannabis sativa germplasm from the United States.” Sci Rep 15, 20423. https://doi.org/10.1038/s41598-025-07912-8) teaches that the collection of the USDA-ARS National Plant Germplasm System (NPGS), holds 617,467 accessions representing 17,482 species [pg. 2, ¶3]. The Cannabis sativa diversity also includes unclassified hemp populations that have escaped production and continued reproducing in the wild [pg. 2, ¶1].
The Applicant has not reduced to practice the broad genus of plant material from any Cannabis sativa cultivar given the large scope of Cannabis sativa and the variability of propagation success in cannabis genotypes.
Examiner notes that claim 12 overcomes this particular rejection with the recitation of the cultivars described in the instant application: Cannatonic cultivar, Jamaican Pearl cultivar, California indica cultivar, Amazing Haze cultivar, Michka cultivar, Sensi 741 cultivar, and Afghani cultivar, but remains rejected due to lack of written description of other claim limitations.
More than 100 cannabinoids have been identified in Cannabis plants, existing in two forms: the carboxylated form (acidic cannabinoids) and the decarboxylated form (neutral cannabinoids derived from the transformation of acidic cannabinoids) (Kanabus, J. et al. 2021. “Cannabinoids-Characteristics and Potential for Use in Food Production.” Molecules. 26(21):6723. doi: 10.3390/molecules26216723) [pg. 5, ¶1]. Neutral cannabinoids include Cannabigerol (CBG), Cannabidiol (CBD), Δ9-Tetrahydrocannabinol (Δ9-THC), Δ8-Tetrahydrocannabinol (Δ8-THC), Cannabichromene (CBC), Cannabinol (CBN), Cannabicyclol (CBL), Cannabivarin (CBV), Cannabidivarin (CBDV), Cannabielsoin (CBE), Cannabitriol (CBT), Cannabinodiol (CBDL), and Δ9-Tetrahydrocannabivarin (Δ9-THCV) [Table 1]. Currently, it is believed that cannabinoids are initially synthesized in an acidic form then converted to their neutral forms as a result of decarboxylation and/or appropriate enzymes [pg. 5, ¶2].
The Applicant describes only the evaluation of decarboxylated cannabinoids THC, CBD, and CBC in the above cultivars, stating that other cannabinoids were quantified but with no data provided [pg. 32, lns. 25-26]. As broadly claimed in claim 1, the method is for producing plant tissue containing one or more of any decarboxylated cannabinoids using any Cannabis sativa cultivar. Based on the variability of cannabis cultivars as detailed above and the small species of described in the instant disclosure (THC, CBD, CBC in Figs. 1-8), the structure of the method as claimed may not lead to the function of any plant tissue of any Cannabis sativa containing any one decarboxylated cannabinoid.
The Applicant further only describes the use of one cytokinin, BAP, at 0.5 mg/L used in the liquid culture medium. In cannabis cultivation, exposing cannabis cultures to excessive concentrations of either auxin or cytokinin can trigger lethal physiological abnormalities and developmental arrest. Burgel, L. et al. (2020. “Impact of Different Phytohormones on Morphology, Yield and Cannabinoid Content of Cannabis sativa L.” Plants (Basel). 9(6):725. doi: 10.3390/plants9060725) teaches that high concentrations of IAA are toxic and have been used to develop herbicides [pg. 13, ¶2], while Kurepa, J. et al. (2022. “Auxin/Cytokinin Antagonistic Control of the Shoot/Root Growth Ratio and Its Relevance for Adaptation to Drought and Nutrient Deficiency Stresses.” Int J Mol Sci. 23(4):1933. doi: 10.3390/ijms23041933) teaches that high cytokinin prevents nutrient accumulation to excessive and potentially toxic levels [pg. 7, ¶6]. As such, with the undefined concentrations in the medium as recited in the method of claim 1 and the small number of species described in the instant specification (0.5 mg/L BAP), the Applicant has not sufficiently described an adequate number of species to support the broad claim.
Examiner notes that claims 4-5 recite concentrations for auxins and cytokinins, respectively, but only in the alternative, and thus do not overcome the above rejection. Claim 15 does not require auxin or cytokinin and does not depend from claim 1. Thus, claim 15 overcomes the above rejection, but remains rejected under the other written description rejections.
Lastly, the Applicant claims the broad genus of a method for producing plant tissue containing one or more decarboxylated cannabinoid, comprising inoculating the sterile liquid medium with one or more bacterial and/or yeast strains. However, Applicant concedes that not every bacterial and/or yeast strain results in the production of any decarboxylated cannabinoid. Figures 3 and 4 show 0.00% ratio of decarboxylated cannabinoid content for CBC with solely contamination with either Micrococcus luteus or Microbacterium ginsengisoli. Further, the broad genus of bacterial and/or yeast strains would necessarily include phytopathogens that would result in plant diseases and physiological disfunction that may not allow for decarboxylation of cannabinoids. The Applicant describes only the identification of Curtobacterium spp. Staphylococcus epidermis, Micrococcus luteus, Microbacterium ginsengisoli, Cystobasidium minutum in Example 1, Roseomonas mucosa in Example 2, as well as Bacillus megaterium and Sphingomonas paucimobilis (data not shown; pg. 35, lns. 26-28), providing 7 bacterial species and one yeast strain identified in the contamination of the TIB.
Further even strains described by the Applicant may have negative impacts for cannabis plants depending on the dosage. Comeau, D. et al. (2021, “Interactions Between Bacillus Spp., Pseudomonas Spp. and Cannabis sativa Promote Plant Growth.” Front. Microbiol. 12:715758. doi: 10.3389/fmicb.2021.715758) determined that, although Bacillus sp. inoculation increased plant yield, an increased dose negatively affected plant growth and vigor [pg. 4, col. 1, ¶1].
Thus, the Applicant has not reduced to practice a sufficient number of species at any concentration to describe the full genus of any bacterial and/or yeast strains at any concentration, based on the structure function relationship between the culturing process and the production of decarboxylated cannabinoids which would require a functional plant.
Examiner notes that although claims 9 and 10 limit claim 1 to specific bacterial and yeast strains and optical density in the liquid culture medium after inoculation, respectively, they do not specify both the specific strains and the concentrations as described in the instant disclosure as to limit the genus claim of method 1.
Scope of Enablement
Claims 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method for producing plant tissue containing one or more of select decarboxylated cannabinoids comprising contamination with select bacterial and yeast strains in combination with abiotic drought stress, does not reasonably provide enablement for producing any decarboxylated cannabinoid merely by contamination with any bacterial or yeast strain. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make or use the invention commensurate in scope with these claims.
In re Wands lists a number of factors for determining whether or not undue experimentation would be required by one skilled in the art to make and/or use the invention. These factors are: (1) the
quantity of experimentation necessary; (2) the amount of direction or guidance presented; (3) the presence or absence of working examples of the invention; (4) the nature of the invention; (5) the state of the prior art; (6) the relative skill of those in the art; (7) the predictability or unpredictability of the art; (8) the breadth of the claim. In re Wands, 858 F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988).
The Applicant teaches:
The identification of Curtobacterium spp. Staphylococcus epidermis, Micrococcus luteus, Microbacterium ginsengisoli, Cystobasidium minutum in Example 1, Roseomonas mucosa in Example 2, as well as Bacillus megaterium and Sphingomonas paucimobilis (data not shown; pg. 35, lns. 26-28).
That contamination and abiotic stress combined produce plant tissues consistently containing one or more decarboxylated cannabinoids as compared to the control.
The Applicant does not teach:
Inoculating the sterile liquid culture medium with any bacterial and/or yeast strains alone to produce plant tissues containing decarboxylated cannabinoids distinguishable from a control plant.
The claims are broadly directed to a method for producing plant tissue containing one or more decarboxylated cannabinoids (claims 1-13 and 16-20), a method for producing one or more decarboxylated cannabinoids (claim 14), and a method for decarboxylating on or more cannabinoids in plant tissue (claim 15), all comprising co-cultivating the plant material with any bacterial or yeast strain.
The Applicant provides two working examples. In the first example, some TIB in which Cannabis sativa cultivars were cultivated were contaminated, while some were not. Some of the contaminated TIB were subjected to abiotic stress (drought) and the decarboxylated cannabinoids were measured. Example 1, represented by Figs. 1-5, identified five different microoganisms, Curtobacterium spp. Staphylococcus epidermis, Micrococcus luteus, Microbacterium ginsengisoli, Cystobasidium minutum. Figs. 1-5 show that contamination and abiotic stress together produce a far larger ratio of decarboxylated cannabinoids than the control or the contamination alone, which were about equal in each figure, with the control having higher THC and CBC than the contaminated TIB in Fig. 2. Further, Figs. 3 and 4 show that no CBC was produced in the plant tissues of the control or the contaminated treatment, while the contaminated and abiotic stress condition combined did produce CBC.
Example 2 states that culture under abiotic stress improves decarboxylation but is not necessary to decarboxylation. While Example 2 shows that abiotic stress alone does not enhance decarboxylation, Fig. 7 shows that co-culture without abiotic stress for 30 days resulted in a lower decarboxylated cannabinoid ratio, indicating that the combination is the novel feature of the invention.
It is not clear that one of ordinary skill in the art would be able to know that they are practicing with the sterile liquid culture medium inoculated with the bacterial or yeast strain in place of the control treatment due to the high similarity in results. Undue experimentation would be required to ensure that the method actually has an impact over merely cultivating the plant material without bacterial or yeast strains or that one is using the invention as claimed. Additionally, given that the decarboxylated cannabinoid content for CBC was 0.0% in the contaminated treatment with Micrococcus luteus or Microbacterium ginsengisoli bacteria alone, while it was 60.72 and 26.7%, respectively, when combined with the abiotic drought stress, the use of the combination appears to be an essential limitation of the claimed invention. One of ordinary skill in the art would not be able to make and/or use the invention as claimed with reasonable expectation of success without the combination.
In the art, microbial contamination of liquid culture can lead to growth and development challenges for micropropagation. Klayraung, S. et al. (2017, “Diversity and control of bacterial contamination of plants propagated in temporary immersion bioreactor system.” Acta Hortic. 1155) teaches that bacterial contamination in plant tissue culture is a common problem which can slow down the development of all in vitro techniques [Abstract]. In rice micropropagation, Klayraung found that the contamination in 20 L TIB container was caused by Bacillus amyloliquifaciens, B. pumilus and B. subtilis, whereas in 700 mL TIB container was by Methylobacterium sp., Paenibacillus sp. and Sphingomonas sp. [Abstract]. The broad scope of bacterial and/or yeast strains as instantly claimed would necessarily include phytopathogens that would result in development dysfunction that may not allow for decarboxylation of cannabinoids.
The Applicant describes only the identification of Curtobacterium spp. Staphylococcus epidermis, Micrococcus luteus, Microbacterium ginsengisoli, Cystobasidium minutum in Example 1, Roseomonas mucosa in Example 2, as well as Bacillus megaterium and Sphingomonas paucimobilis (data not shown; pg. 35, lns. 26-28), providing 7 bacterial species and one yeast strain identified in the contamination of the TIB, many of which did not produce a ratio of decarboxylated cannabinoids above that of the control without abiotic stress combined with contamination. Figures 3 and 4 show 0.00% ratio of decarboxylated cannabinoid content for CBC with solely contamination with either Micrococcus luteus or Microbacterium ginsengisoli.
The art teaches that even strains described by the Applicant may have negative impacts for cannabis plants depending on the dosage. For example, Comeau, D. et al. (2021, “Interactions Between Bacillus Spp., Pseudomonas Spp. and Cannabis sativa Promote Plant Growth.” Front. Microbiol. 12:715758. doi: 10.3389/fmicb.2021.715758) determined that, although Bacillus sp. inoculation increased cannabis plant yield, an increased dose negatively affected plant growth and vigor [pg. 4, col. 1, ¶1], demonstrating the unpredictability in the vast genus of any bacterial and/or yeast strain. Undue experimentation would be required to determine which bacterial and/or yeast strains may be useful in a method of producing plant tissues containing one or more decarboxylated cannabinoids without using the combination of contamination and abiotic drought stress. Further experimentation would be required to ensure that the treatment results in decarboxylated cannabinoids without negatively impacting the plant and is different than the control alone, allowing one of ordinary skill to use the invention as claimed.
Thus, the Applicant is not enabled to the full scope of a method for producing plant tissues containing one or more decarboxylated cannabinoids, the method comprising merely co-cultivation with any bacterial or yeast strain. Given the breadth of the claims, the lack of guidance and working examples, the unpredictability in the art, and the state of the art, undue experimentation would be required to make and use the claimed invention, and therefore, the invention is not enabled throughout the broad scope of the claims.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-7, 9, 11, 13-18 are rejected under 35 U.S.C. 103 as being unpatentable over Heinricher, J. et al. "Media for Rapid and Reliable Tissue Culturing of Plants." International Publication No. WO 2019/006470 A1, published 01/03/2019 (see IDS filed 03/03/2025), in view of Ahmed, B. et al. (2021). "Potential impacts of soil microbiota manipulation on secondary metabolites production in cannabis." J. Cannabis Res. 3:25 and Cantabella, D. et al. (January, 2022). "GreenTray® TIS bioreactor as an effective in vitro culture system for the micropropagation of Prunus spp. rootstocks and analysis of the plant-PGPMs interactions." Scientia Horticulturae, Volume 291,110622.
Claim 1 recites a method for producing plant tissues containing one or more decarboxylated cannabinoid(s), the method comprising: a) inoculating a temporary liquid immersion culture system containing a sterile liquid culture medium containing at least one cytokinin and/or at least one auxin with sterile plant material from a Cannabis sativa cultivar, b) cultivating the plant material in the temporary liquid immersion culture system under conditions suitable for growing plant biomass, c) inoculating the sterile liquid culture medium with one or more bacterial and/or yeast strain(s), d) cultivating the plant material in the temporary liquid immersion culture system inoculated by the one or more bacterial and/or yeast strain(s), and e) collecting plant tissues containing one or more decarboxylated cannabinoid(s).
Claim 2 recites the method of claim 1, wherein said temporary liquid immersion culture system is a temporary immersion bioreactor (TIB).
Claim 3 recites the method according to claim 1, wherein the sterile liquid culture medium is selected from MS medium, DKW medium, B5 medium, WPM medium, and SH medium.
Claim 4 recites the method according to claim 1, wherein: a) said cytokinin is a natural or artificial cytokinin belonging to the adenine-type or the phenylurea-type, and/or b) said sterile liquid culture medium comprises from 0.01 to 10 mg/L of said cytokinin.
Claim 5 recites the method according to claim 1, wherein: a) said auxin is selected from naturally occurring auxins; or synthetic auxin analogues, and/or b) the concentration of auxin in the sterile liquid culture medium is from 0.01 to 10 mg/L.
Claim 6 recites the method according to claim 1, wherein the duration and frequency of immersion of the plant material in the liquid culture medium in step b) varies from 30 seconds to 15 minutes every 30 minutes to 12 hours.
Claim 7 recites the method according to claim 1, wherein in step b): a photoperiod of 12 to 24 hours of light and 0 to 12 hours of darkness is used.
Claim 9 recites the method according to claim 1, wherein: a) the one or more bacterial strain(s) is(are) selected from i. the Actinomycetia class.
Claim 11 recites the method according to claim 1, wherein the plant material comprises apical meristems and nodal segments from surface-sterilized seeds germinated in vitro.
Claim 13 recites the method according to claim 1, wherein the one or more decarboxylated cannabinoid is tetrahydrocannabinol (THC).
Claim 14 recites a method for producing one or more decarboxylated cannabinoid(s), the method comprising: a) producing plant tissues containing one or more decarboxylated cannabinoid (s) using the method according to claim 1, and b) collecting or extracting the one or more decarboxylated cannabinoid(s) from the obtained plant tissues.
Claim 15 recites a method for decarboxylating one or more cannabinoid(s) in plant tissues of Cannabis sativa cultivars cultivated in a temporary liquid immersion culture system, wherein the method comprises a step of a co-cultivating the one or more bacterial and/or yeast strain(s) with the Cannabis sativa cultivars in the temporary liquid immersion culture system.
Claim 16 recites the method according to claim 2, wherein the volume of sterile liquid culture medium in the TIB is from 1 L to 10,000 L.
Claim 17 recites the method according to claim 4, wherein said cytokinin is selected from adenine and derivatives thereof having cytokinin activity.
Claim 18 recites the method according to claim 5, wherein: a1) the naturally occurring auxins are indole-3-acetic acid (IAA).
Regarding claim 1, Heinricher teaches media, kits, systems, and methods for achieving large scale cannabis micropropagations through tissue culture and the use of bioreactors [¶11-12]. Heinricher teaches a method for producing cannabis micropropagations comprising utilizing a temporary immersion bioreactor (TIB) comprising a growth vessel for incubating plant tissue in a sterile environment (i.e., a method for producing plant tissues, the method comprising a) inoculating a temporary liquid immersion culture system containing a sterile liquid culture) [claim 3]. Heinricher additionally teaches a medium for producing cannabis micropropagations comprising at least one cytokinin and at least one auxin (i.e., medium containing at least one cytokinin and/or at least one auxin) [claim 1]. Heinricher teaches Example 6, wherein initial explants of Cannabis sativa are surface sterilized and initiated in culture vessels before microshoots were moved into bioreactors (i.e., with sterile plant material from a Cannabis sativa cultivar) [¶577]. Heinricher teaches that the material is maintained in the bioreactors under standard environmental conditions (16/8 hour photoperiod and 25°C ± 2°C) (i.e., b) cultivating the plant material in the temporary liquid immersion culture system under conditions suitable for growing plant biomass) [¶577].
Heinricher teaches that most cannabinoids exist in two forms, as acids and in neutral decarboxylated forms, and that some decarboxylation occurs within the plant (i.e., plant tissues containing one or more decarboxylated cannabinoids) [¶111]. Heinricher teaches that references to cannabinoids in a plant include both the acidic and decarboxylated versions (CBDA vs. CBD). Modern cannabis production relies on asexual cuttings of single "mother" plants to produce uniform crops, but this faces several challenges such as time, space and resource constraints [¶119].
Heinricher teaches that cannabis is one of the world’s oldest and most useful cultivated genus of plants which has long been used for drug and industrial purposes [¶105]. Marijuana has historically consisted of the dried flowers of cannabis plants bred to produce high levels of THC and other psychoactive cannabinoids, in addition to various extracts such as hashish and hash oil produced from the plant. Heinricher teaches that microshoots are harvested at maturity (8 to 10 weeks) (i.e., e) collecting plant tissues containing one or more decarboxylated cannabinoids) [¶577]. Singulated shoots are moved to rooting media for rooting. Established plants are planted in soil or hydroponic applications.
Although Heinricher teaches that the media may also comprise nutritional elements for plant growth, including yeast extracts and other undefined media components [¶130], Heinricher does not explicitly teach inoculating the sterile liquid culture medium with one or more bacterial and/or yeast strains or the co-cultivation of the plant material with the one or more bacterial and/or yeast strains. However, Ahmed teaches that cannabis growing practices and indoor cultivation conditions have a great influence on the production of cannabinoids and that plant associated microbes may affect nutrient acquisition and thus cannabinoid production [Abstract]. Ahmed teaches that manipulation of cannabis-associated microbiome obtained through interventions of diversified microbial communities sourced from natural forest soil could potentially help producers of cannabis to improve yields of cannabinoids and enhance the balance of cannabidiol (CBD) and tetrahydrocannabinol (THC) proportions.
Ahmed teaches that biostimulant substances, such as beneficial microbes belonging to plant growth-promoting rhizobacteria (PGPR), have been proposed to aid in stabilizing or balancing proportions of THC and CBD in cannabis [pg. 3, col. 1, ¶1]. Plants and their associated plethora of microbes nurture multifactorial interactive relationships where specific microorganisms including bacteria and fungi can stimulate the biosynthetic and signaling pathways of the host plants for the production of pharmaceutically or agronomically important metabolic compounds [pg. 3, col. 1, ¶2]. PGPR has attracted scientists’ attention for its potential to increase the quality and quantity of production of desired cannabinoids; however, in most cases, scientists have focused on well-known PGPR genera, for example, Pseudomonas and Bacillus for cannabinoid yield and disease control effects demonstrated in other crops [pg. 3, col. 2, para 1].
The first report on the cannabis plant microbiome highlighted cultivar-specificity and soil determinants of the microbiome for five cannabis cultivars — Bookoo Kush, Burmese, Maui Wowie, White Widow, and Sour Diesel — and reported a core bacterial community composed of Pseudomonas, Cellvibrio, Oxalobacteraceae, Xanthomonadaceae, Actinomycetales, and Sphingobacteriales [pg. 4, col. 1, ¶2]. This study included the biochemical correlations with bacterial communities, highlighting that the concentration and composition of CBD were correlated with the structure of bacterial communities residing inside the root system, whereas THC concentrations were correlated with the soil’s edaphic factors. Another study on three Cannabis sativa L. reported bacterial genera of Pseudomonas, Pantoea, and Bacillus and three fungal genera of Aureobasidium, Alternaria, and Cochliobolus.
Ahmed teaches that many studies examine a variety of microbial communities that promote growth in an agricultural context or the microbial community in contaminated environments and that such studies could be adapted to apply to marijuana to decipher the underlying microbiota [pg. 5, col. 1, ¶1]. Ahmed teaches that cannabis plants recruit their specific core microbiota when they are inoculated with a microbial suspension prepared from naturally microbial rich environments [pg. 5, col. 2, ¶1]. Ahmed teaches microbial communities and their interactions with cannabis plants would be provide promising improvement of cannabis quality and cannabinoid production in sustainable agricultural practices [pg. 5, col. 2, ¶2].
While Ahmed teaches that bacterial stains are correlated with the concentration and composition of decarboxylated cannabinoids and Heinricher teaches a temporary liquid immersion culture system for propagating cannabis plants, Heinricher and Ahmed do not explicitly teach the use of bacterial strains in a temporary liquid immersion culture system. However, use of temporary immersion bioreactors for co-cultivation of bacterial strains and plant explants for micropropagation efforts has been demonstrated in the prior art. For example, Cantabella teaches the use of the GreenTray® TIS bioreactor for the in vitro analysis of the interaction between plantless and two Plant Growth-Promoting Microorganisms (PGPMs), (Pseudomonas oryzihabitans PGP01 and Cladosporium ramotenellum PGP02) [Abstract]. Cantabella teaches that 3-cm-long shoots were cultured in ½ MS medium supplemented with 10-μM indole-3-butyric acid (IBA) for one week in darkness for root induction [pg. 3, col. 1, ¶2]. The inoculation of GreenTray® bioreactors containing RP-20 explants took place by adding 3 mL of P. oryzihabitans PGP01 or C. ramotenellum PGP02 suspensions at 1 × 103 CFU mL-1 and 1 × 105 esp mL-1, respectively (i.e., inoculating the medium with one or more bacterial strains). The co-culture in the presence of the two PGPMs was maintained during 15 days (i.e., cultivating the plant material in the temporary liquid immersion culture system inoculated by the one or more bacterial strain). Bacterial cell concentration was estimated by measuring the absorbance at 420 nm, and the final concentration was set up with sterile distilled water at 1 × 103 colony forming unit per mL (CFU mL-1) in the first trial, and 1 × 106 CFU mL-1 in the second trial [pg. 3, col. 1, ¶3].
Plant responses in co-culture with inoculation of PGPMs, their ability to control endophytes growth in the culture media, and hormonal changes associated to plant growth were studied in the GreenTray® culture system, showing greater shoot length and fresh weight [Abstract]. Although the system was used for Prunus rootstock, Cantabella further teaches that the system was effective for plantlet and PGPM interactions [pg. 10, col. 1, ¶2].
Given that Heinricher teaches methods for cannabis propagation in a TIB system, including inoculating a culture system containing a sterile liquid culture medium containing at least one cytokinin and/or at least one auxin with sterile plant material from a Cannabis sativa cultivar, cultivating the plant material in the culture system under suitable conditions for growing plant biomass, teaching that the plant tissues contain one or more decarboxylated cannabinoids, and suggesting the addition of other nutritional elements for plant growth; given that Ahmed teaches that the use of beneficial microbes in cannabis propagation can enhance decarboxylated cannabinoids CBD and THC, providing several bacterial strains for inoculation; and given that Cantabella teaches the use of a temporary liquid immersion culture system for the production of plant tissues comprising inoculating the medium with one or more bacterial strains, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to combine the teachings of Heinricher, Ahmed, and Cantabella to enhance propagation efforts in cannabis.
As detailed above, Heinricher teaches that modern cannabis production relies on asexual cuttings of single "mother" plants to produce uniform crops, but this faces several challenges such as time, space and resource constraints. One would have been motivated to combine the teachings to improve modern cannabis propagation, especially given that Heinricher suggests the addition of other nutritional elements and Ahmed teaches that use of cannabis-associated microbiome could help producers of cannabis to improve yields of cannabinoids and enhance the balance of cannabidiol (CBD) and tetrahydrocannabinol (THC) proportions. One would have reasonable expectation of success due to Ahmed’s teachings of the bacterial communities improving decarboxylated cannabinoid content and Cantabella’s teachings of success with other plant species in co-cultivation with microbes in a temporary immersion culture system.
Regarding claim 2, Heinricher teaches that the bioreactor is a temporary bioreactor [¶15; claim 3].
Regarding claim 3, Heinricher teaches that at least one of the vitamins is provided by the Murashige and Skoog medium salts (Murashige and Skoog, 1962), Woody Plant (WPM) tissue culture salts, Driver Kuniyuki Walnut (DKW) tissue culture, and/or functional variations thereof (i.e., wherein the sterile liquid culture medium is selected from MS medium, DWK medium, B5 medium, WPM medium, and SH medium) [¶132].
Regarding claim 4, Heinricher teaches that the cytokinin is 2ip (i.e., wherein the cytokinin is a natural or artificial cytokinin beloning to the adenin-type) [claim 11], which is known in the art as an adenine-type cytokinin.
Regarding claim 5, Heinricher teaches the medium for producing cannabis micropropagates wherein the auxin is IAA (i.e., wherein said auxin is selected from naturally occurring auxins; or synthetic auxin analogues) [claim 13 and 14].
Regarding claim 6, Heinricher teaches that, in a cultivation cycle of the temporary immersion bioreactor, the chamber is filled with a predetermined amount of liquid or semi-liquid material and kept in the chamber for about 1-60 minutes before being drained [¶497]. Cantabella further teaches that the GreenTray® bioreactor was set at an immersion frequency of 2 minutes every 6 hours (i.e, wherein the duration and frequency of immersion of the plant material in the liquid culture medium in step b) varies from 30 seconds to 15 minutes every 30 minutes to 12 hours) [pg. 2, col. 2, ¶4].
Regarding claim 7, Heinricher teaches that bioreactors are kept under standard conditions (22- 24 °C and 16/8 hours day/night photoperiod) (i.e., a photoperiod of 12 to 24 hours of light and 0 to 12 hours darkness) [Example 3; ¶571].
Regarding claim 9, as detailed above, Ahmed teaches that the first report on the Cannabis plant microbiome highlighted a core bacterial community composed of Pseudomonas, Cellvibrio, Oxalobacteraceae, Xanthomonadaceae, Actinomycetales, and Sphingobacteriales (i.e., wherein: a) the one bacterial strain is selected from the Actinomycetia class) [pg. 4, col. 1, ¶2]. This study included the biochemical correlations with bacterial communities, highlighting that the concentration and composition of some dehydroxylated cannabinoids were correlated with the structure of bacterial communities residing inside the root system.
Regarding claim 11, Heinricher teaches that explants may be taken from seedlings which are aseptically grown from surface-sterilized seeds [¶274]. The explant can be any segment or collection of cells from apical meristems or immature nodal sections from stems, etc. and the micropropagated plants are grown in vitro in sterile media (i.e., wherein the plant material comprises apical meristems and nodal segments from surface-sterilized seeds germinated in vitro) [¶90 & 285].
Regarding claim 13, Heinricher teaches that references to cannabinoids in a plant include both the acidic and decarboxylated versions (CBDA vs. CBD) [¶111] and Ahmed teaches that diversified microbial communities sourced from natural forest soil could potentially help producers of cannabis to improve yields of cannabinoids and enhance the balance of cannabidiol (CBD) and tetrahydrocannabinol (THC) proportions [Abstract] (i.e., wherein the one or more decarboxylated cannabinoids are selected from THC and CBD).
Regarding claim 14, as detailed above, Heinricher, Ahmed, and Cantabella render obvious the method of claim 1, producing plant tissues containing one or more decarboxylated cannabinoids with co-cultivation of the plant material with one or more bacterial and/or yeast strains. Heinricher further teaches that cannabis is one of the world’s oldest and most useful cultivated genus of plants which has long been used for drug and industrial purposes [¶105]. Marijuana has historically consisted of the dried flowers of cannabis plants bred to produce high levels of THC and other psychoactive cannabinoids, in addition to various extracts such as hashish and hash oil produced from the plant (i.e., extracting the one or more decarboxylated cannabinoids from the obtained plant tissues). Thus, Heinricher teaches that decarboxylated cannabinoids are produced and extracted from plant tissue, such as the cannabis plant tissue from the taught method, for extracts such as hashish, hash oil and other products.
Regarding claim 15, as detailed above, Heinricher, Ahmed, and Cantabella combined render obvious the method of producing plant tissues containing one or more decarboxylated cannabinoids with co-cultivation of the plant material with one or more bacterial and/or yeast strains. As Heinricher teaches that decarboxylation occurs within the Cannabis sativa plant, Ahmed teaches increased decarboxylated cannabinoids in Cannabis sativa plants inoculated with bacterial strains, and Cantabella teaches co-cultivation of plant material with bacterial strains, the combined teachings further read on a method for decarboxylating one or more cannabinoids in plant tissues of Cannabis sativa cultivars cultivated in a temporary liquid immersion culture system, wherein the method comprises a step of co-cultivating the one or more bacterial strains with the Cannabis sativa cultivar in the temporary liquid immersion culture system.
Regarding claim 16, Heinricher teaches that the size of bioreactor vanes from 0.1 to 20 L depending on production requirements (i.e., wherein the volume of sterile liquid culture medium in the TIB is from 1 L to 10,000 L) [¶571]. As the size can vary between 0.1 L to 20 L, the volume of sterile liquid culture medium in the TIB could fall within the claim limitation of 1 L to 10,000 L depending on the production requirements, as taught by Heinricher.
Regarding claim 17, Heinricher teaches that the cytokinin is 2ip (i.e., wherein the cytokinin selected from adenine and derivatives thereof having cytokinin activity) [claim 11; ¶133].
Regarding claim 18, Heinricher teaches that the at least one auxin is IAA (i.e., wherein the naturally occurring auxins are selected from IAA) [claim 13].
Claims 8 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Heinricher, Ahmed, and Cantabella, as applied to claims 1-7, 9, 11, 13-18 above, and further in view of Caplan, D. et al. (2019). "Increasing Inflorescence Dry Weight and Cannabinoid Content in Medical Cannabis Using Controlled Drought Stress." HortScience, 54(5), 964–969. https://doi.org/10.21273/HORTSCI13510-18.
Claim 8 recites the method according to claim 1, wherein the abiotic stress condition used in step d) is drought.
Claim 19 recites the method according to claim 8, wherein the abiotic stress condition used in step d) is drought caused by decreasing the duration and/or frequency of immersion of the plant material in the liquid culture medium or by stopping the immersion of the plant material in the liquid culture medium in step d).
As detailed above, Heinricher teaches methods for cannabis propagation in a TIB system, including inoculating a culture system containing a sterile liquid culture medium containing at least one cytokinin and/or at least one auxin with sterile plant material from a Cannabis sativa cultivar, cultivating the plant material in the culture system under suitable conditions for growing plant biomass, teaching that the plant tissues contain one or more decarboxylated cannabinoids, and suggesting the addition of other nutritional elements for plant growth. Ahmed teaches that the use of beneficial microbes in cannabis propagation can enhance decarboxylated cannabinoids CBD and THC, providing several bacterial strains for inoculation. Lastly, Cantabella teaches the use of a temporary liquid immersion culture system for the production of plant tissues comprising inoculating the medium with one or more bacterial strains, thus collectively rendering obvious the method of claim 1 (see 103 rejection above).
Ahmed further teaches that cultivation methods and different abiotic and biotic factors are important considerations for cannabinoid biosynthesis [pg. 2, col. 1, ¶2]. Ahmed teaches that poor soil and inadequate moisture was found to increase THC production in hemp plants [pg. 2, col. 2, ¶1]. Although Ahmed merely suggests that lower moisture was found to increase THC production in hemp plants, additional research supports the finding that controlled drought stress can increase cannabinoid content in cannabis. Indeed, Caplan teaches that controlled drought stress may be an effective horticultural management technique to maximize both inflorescence dry weight and cannabinoid yield in cannabis.
Caplan teaches that drought-stressed plants had increased concentrations of major cannabinoids tetrahydrocannabinol acid (THCA) and cannabidiol acid (CBDA) by 12% and 13%, respectively, compared with the control. Further, yield per unit growing area of THCA was 43% higher than the control, CBDA yield was 47% higher, ∆9-tetrahydrocannabinol (THC) yield was 50% higher, and cannabidiol (CBD) yield was 67% higher. Caplan teaches drought stress induced by withholding fertigation from drought treatment plants until the water pressure reached between -1.4 and -1.5 MPa, as compared to the control, which was automatically fertigated when the substrate moisture content of an individual plant reached 20% [pg. 965, col. 3, ¶1].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to cultivate the plant material in the temporary liquid immersion culture system inoculated by the one or more bacterial strains under abiotic stress to further increased the decarboxylated cannabinoid content in the plant tissue for greater cannabinoid extraction. As Heinricher teaches that plant growth and development can be controlled by manipulating the frequency and duration of immersion in liquid medium [¶386], one would have reasonable expectation of success in causing drought by decreasing the duration and/or frequency of immersion of the plant material in the liquid culture medium. One would have been motivated to decreased the frequency or duration of the immersion as Caplan teaches that both the THC and the CBD yields were 50% and 67% higher, respectively.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Heinricher, Ahmed, and Cantabella, as applied to claims 1-7, 9, 11, 13-18 above, and further in view of Rocha, E. et al. (2020). "Qualitative terpene profiling of Cannabis varieties cultivated for medical purposes." Rodriguésia 71: e01192019.
Claim 12 recites the method according to claim 1, wherein the Cannabis sativa cultivar is selected from Cannatonic cultivar.
As detailed above, Heinricher teaches methods for cannabis propagation in a TIB system, including inoculating a culture system containing a sterile liquid culture medium containing at least one cytokinin and/or at least one auxin with sterile plant material from a Cannabis sativa cultivar, cultivating the plant material in the culture system under suitable conditions for growing plant biomass, teaching that the plant tissues contain one or more decarboxylated cannabinoids, and suggesting the addition of other nutritional elements for plant growth. Ahmed teaches that the use of beneficial microbes in cannabis propagation can enhance decarboxylated cannabinoids CBD and THC, providing several bacterial strains for inoculation. Lastly, Cantabella teaches the use of a temporary liquid immersion culture system for the production of plant tissues comprising inoculating the medium with one or more bacterial strains, thus collectively rendering obvious the method of claim 1 (see 103 rejection above).
Heinricher, Ahmed, and Cantabella do not explicitly teach the method of claim 1 wherein the Cannabis sativa cultivar is the Cannatonic cultivar, however, Rocha teaches that Cannatonic cultivar is a known marijuana variety, with a THC/CBD ratio of around 9.8 and higher concentration of more volatile monoterpenes [Table 1; pg. 5, col. 2, ¶2]. As this is a known cultivar that has already been the subject of cannabis studies related to terpene content and cannabinoid content, it would have been prima facie obvious to one of ordinary skill to choose Cannatonic from a finite number of cultivars previously researched and with an established commercial presence. It would have been an obvious variant to try as Rocha is primarily teaching terpene profiling of cannabis varieties cultivated for medical purposes and there are a finite number of cultivars available to meet the problem of improving cannabis propagation, as taught by Heinricher and Ahmed. One would have reasonable expectation of success as it is a Cannabis sativa cultivar and is already in production, demonstrating that it is able to be successfully propagated commercially.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Heinricher, Ahmed, and Cantabella, as applied to claims 1-7, 9, 11, 13-18 above, and further in view of Carlin, D. et al. "Biosynthesis of Cannabinoids and Cannabinoid Precursors." International Publication No. WO 2022/081615 A1. Effectively filed 12/10/2021.
Claim 20 recites the method for producing one or more decarboxylated cannabinoid(s) according to claim 14, further comprising the steps of: c) purifying the collected or extracted one or more decarboxylated cannabinoid(s), and d) adding an acceptable diluent, excipient or carrier to the one or more decarboxylated cannabinoid(s).
As detailed above, Heinricher teaches methods for cannabis propagation in a TIB system, including inoculating a culture system containing a sterile liquid culture medium containing at least one cytokinin and/or at least one auxin with sterile plant material from a Cannabis sativa cultivar, cultivating the plant material in the culture system under suitable conditions for growing plant biomass, teaching that the plant tissues contain one or more decarboxylated cannabinoids, and suggesting the addition of other nutritional elements for plant growth. Ahmed teaches that the use of beneficial microbes in cannabis propagation can enhance decarboxylated cannabinoids CBD and THC, providing several bacterial strains for inoculation. Lastly, Cantabella teaches the use of a temporary liquid immersion culture system for the production of plant tissues comprising inoculating the medium with one or more bacterial strains, thus collectively rendering obvious the method of claim 1 (see 103 rejection above). Further, the combined teachings render obvious claim 14, as Heinricher teaches that cannabis plants produce decarboxylated cannabinoids and would thus produce decarboxylated cannabinoids according to the method of claim 1. Additionally, Heinricher teaches that cannabinoids can be extracted for hashish, hash oil, and other products for plant tissue [¶105].
Heinricher, Ahmed, and Cantabella do not explicitly teach purifying the collected or extracted decarboxylated cannabinoids and adding an acceptable diluent, excipient, or carrier to the one or more decarboxylated cannabinoids. However, Carlin teaches biosynthesis of cannabinoids and cannabinoid precursors in vitro [Abstract], including methods for producing and extracting cannabinoids, and a bioreactor for producing a cannabinoid compound [¶36]. Carlin teaches that cannabinoids or cannabinoid precursors produced by any of the recombinant cells disclosed in this application or any of the in vitro methods described in this application may be identified and extracted using any method known in the art. Mass spectrometry (e.g., LC-MS, GC-MS) is an example of a method for identification and may be used to extract a compound of interest [¶466].
Carlin teaches that any of the methods may include isolation and purification of the cannabinoids and/or cannabinoid precursors produced in a bioreactor [¶465], and teaches adding a pharmaceutically acceptable excipient, including an inert diluent [¶473]. Carlin teaches that cannabinoids comprise both the acidic and decarboxylated acid forms of the naturally occurring plant-derived cannabinoids [¶115].
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the extraction and purification methodologies as taught by Carlin with the plant tissue and decarboxylated cannabinoids produced by the instant claim 14, as well as to include an appropriate excipient. As excipients can improve bioavailability, stability and handling of the final cannabis product, one would be motivated to add this to the method of producing one or more decarboxylated cannabinoids. Carlin additionally provides a number of acceptable diluents, excipients and carriers to improve the final product. Further, purification of the extracted compound can provide a higher purity of the compound of interest and a higher quality cannabis product [¶247]. One would have reasonable expectation of success as Carlin provides several examples of successful isolation or purification including one or more of cell lysis, centrifugation, extraction, column chromatography, distillation, crystallization, and lyophilization [¶465].
Subject Matter Free of Art
Claim 10 appears to be free of prior art in light of the lack of clarity and demonstrated unpredictability of OD600 between 0.0001 and 0.1 as a measure of the bacterial and/or yeast inoculation. As detailed in the 112(b) indefiniteness rejection above, in light of the lack of guidance on the time the measurement was taken, the specificity of bacterial and/or yeast strains that may be inoculated in the medium, and the fact that OD cannot be easily converted to other bacterial measurement standards, such as Colony Forming Units (CFU), claim 10 cannot be appropriately compared to the prior art.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Skands, G. 2026, “Principle and Considerations: Understanding OD600.” https://sbtinstruments.com/knowledge/understanding-od600.
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