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 .
Claims Status
Claims 1-10 are pending in the application and are presently considered.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The present application and all claims are being examined with the earliest effective filing date of 12/25/2023.
Claim Objections
Claim 6 is objected to because of the following informalities: the claim recites “adding the fungal liquid with an equal volume of ethyl acetate followed by extracting to obtain an upper layer”. For clarity, this should be amended to recite “adding the fungal liquid to an equal volume of ethyl acetate followed by extracting to obtain an upper layer”. Appropriate correction is required.
Claim Interpretation
Per MPEP § 2111, During patent examination, the pending claims must be "given their broadest reasonable interpretation consistent with the specification." The Federal Circuit’s en banc decision in Phillips v. AWH Corp., 415 F.3d 1303, 1316, 75 USPQ2d 1321, 1329 (Fed. Cir. 2005) expressly recognized that the USPTO employs the "broadest reasonable interpretation" standard:
The Patent and Trademark Office ("PTO") determines the scope of claims in patent applications not solely on the basis of the claim language, but upon giving claims their broadest reasonable construction "in light of the specification as it would be interpreted by one of ordinary skill in the art." In re Am. Acad. of Sci. Tech. Ctr., 367 F.3d 1359, 1364[, 70 USPQ2d 1827, 1830] (Fed. Cir. 2004). Indeed, the rules of the PTO require that application claims must "conform to the invention as set forth in the remainder of the specification and the terms and phrases used in the claims must find clear support or antecedent basis in the description so that the meaning of the terms in the claims may be ascertainable by reference to the description." 37 CFR 1.75(d)(1).
Because applicant has the opportunity to amend the claims during prosecution, giving a claim its broadest reasonable interpretation will reduce the possibility that the claim, once issued, will be interpreted more broadly than is justified. In re Yamamoto, 740 F.2d 1569, 1571 (Fed. Cir. 1984).
Claim 1 recites an Aspergillus sp. L14-OE::laeA2 “capable of producing cyclic tripeptides”. Per MPEP 2111.02(II), statements in the preamble reciting the purpose or intended use of the claimed invention must be evaluated to determine whether or not the recited purpose or intended use results in a structural difference. Furthermore, a statement made in the preamble is not a limitation where the claim is directed to a product and the preamble merely recites an inherent property. See Rowe v. Dror, 112 F.3d 473, 478, 42 USPQ2d 1550, 1553 (Fed. Cir. 1997) ("where a patentee defines a structurally complete invention in the claim body and uses the preamble only to state a purpose or intended use for the invention, the preamble is not a claim limitation"); Kropa v. Robie, 187 F.2d at 152, 88 USPQ2d at 480-81 (preamble is not a limitation where claim is directed to a product and the preamble merely recites a property inherent in an old product defined by the remainder of the claim).
In the instant case, the limitation of “an Aspergillus sp. L14-OE::laeA2” describes a complete invention (an engineered fungal strain) which necessarily possesses the capability of producing cyclic tripeptides, as demonstrated in Applicant’s examples. Therefore, the limitation of “capable of producing cyclic tripeptides” is interpreted to be an inherent property of the claimed engineered Aspergillus strain and does not serve to further limit the claimed product.
Claim 6 recites the method step of:
…performing isocratic elution on the filtrate by an analytical column of high-performance liquid chromatography (HPLC) with a mobile phase of acetonitrile : water at a volume ratio of 23 : 65 to obtain effluents from 6th minute (min) to 7th min, 8th min to 9th min and 10.8th min to 11.2nd min respectively, and removing solvents from the effluents by rotary evaporation followed by drying at 25°C to obtain the compound (I), the compound (II) and the compound (III) respectively.
It is understood in view of Applicant’s disclosure, as well as the instant claims, that the three retention times recited above are directed to the elution of Compound I (aspochracin), Compound II (JBIR-15), and Compound III (sclerotiotide C) (see, e.g., instant specification at pg. 12, para. [0088]). It should be noted that the elution of each of these compounds naturally flows from performing the isocratic elution step. In other words, if one were to follow the same method as the inventors, as recited in the claims, wherein these particular compounds were present in the culture broth, these compounds would necessarily elute at the times observed by the inventors and recited in the claims. Therefore, the limitation “to obtain effluents from 6th minute (min) to 7th min, 8th min to 9th min and 10.8th min to 11.2nd min respectively” only requires obtaining an effluent when each compound elutes, and the recited retention times are directed to an inherent result of following the claimed method. It should be noted that this does not necessarily require an ordinary artisan to know beforehand when a particular compound will elute from the column when obtaining each effluent.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 2 and 8-10 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because they are directed to a method of use without reciting any active steps.
Claim 2 recites an “application method” comprising “applying the Aspergillus sp. L14-OE::laeA2 as claimed in claim 1 in producing the cyclic tripeptides”. The BRI of the claim is a method wherein the Aspergillus strain is “used” (i.e., applied) to produce cyclic tripeptides. However, the claim does not recite any active steps for a method to produce said cyclic tripeptides, and only recites “applying” the strain to produce these compounds. Therefore, the claimed “application method” fails to qualify as a process.
In the interest of compact prosecution, the claim is interpreted as being directed to the product of claim 1, and the limitation of “producing the cyclic tripeptides” is directed to an intended use which does not serve to limit the claim.
Claim 8 recites an “application method” comprising “applying the cyclic tripeptides produced by strain Aspergillus sp. L14-OE::laeA2 as claimed in claim 1 in preparing antioxidants drugs”. The BRI of the claim is a method wherein the Aspergillus strain is “used” (i.e., applied) to prepare antioxidant drugs. However, the claim does not recite any active steps for a method to prepare said drugs. Therefore, the claim fails to qualify as a process.
In the interest of compact prosecution, the claim is interpreted as being directed to the product of claim 1, and the limitation of “preparing antioxidants drugs” is directed to an intended use of the claimed product.
Claim 9 recites an “application method” comprising “applying the cyclic tripeptides produced by strain Aspergillus sp. L14-OE::laeA2 as claimed in claim 1 in preparing drugs to inhibit activity of tumor cells”. The BRI of the claim is a method wherein the Aspergillus strain is “used” (i.e., applied) to prepare drugs to inhibit activity of tumor cells. However, the claim does not recite any active steps for a method to prepare said drugs. Therefore, the claim fails to qualify as a process.
In the interest of compact prosecution, the claim is interpreted as being directed to the product of claim 1, and the limitation of “preparing drugs to inhibit activity of tumor cells” is directed to an intended use of the claimed product.
Claim 10 recites the “application method as claimed in claim 9, wherein the tumor cells comprise: a human brain glioma cell line (HEB) and a human hepatocellular carcinoma cell line (Hep-G2)”. However, the claim is rejected for the same reasons as claim 9 above, as it fails to recite any active steps for a method to prepare the recited drugs. Therefore, the claim fails to qualify as a process.
In the interest of compact prosecution, the claim is interpreted as being directed to the product of claim 1, and the limitation of “the tumor cells” is directed to an intended use of the claimed product.
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 2, 5 and 7-10 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.
As discussed under 35 U.S.C. 101, claims 2 and 8-10 are directed to an “application method” comprising “applying the cyclic tripeptides produced by strain Aspergillus sp. L14-OE::laeA2 as claimed in claim 1” without reciting any active method steps. Attempts to claim a process without setting forth any active, positive steps involved in said process raises an issue of indefiniteness, because it is unclear how this use is actually practiced. See MPEP 2173.05(q).
In the interest of compact prosecution, the claims are interpreted to be directed to the product of claim 1, as previously discussed.
The examiner notes that claim 3 recites the application method of claim 2 and further recites active method steps to delineate how the claimed process is carried out. Therefore, claim 3 and its dependents obviate this particular basis for rejection.
Claim 5 recites “wherein the Czapek medium comprises… with water as solvent and potential of hydrogen (pH) of natural”, which renders the claim indefinite, because it is unclear what a pH “of natural” includes or excludes. It is also unclear whether this is referring to the pH of the water or the medium as a whole. In view of the specification: “In an embodiment, the PDA medium includes… distilled water as a solvent and potential of hydrogen (pH) of natural (i.e., an unadjusted pH, which is about 7)” (see pg. 4, para. [0015]). This fails to clarify the limitation, because water or medium comprising “an unadjusted pH” does not necessarily result in a pH “of about 7”. It should also be noted that it is generally considered improper to read limitations from the specification into the claims.
For the sake of applying prior art, this limitation may be met by the water or medium having a neutral pH (i.e., about 7.0).
Claim 7 recites the method of claim 6, “wherein HPLC conditions comprise… an ultraviolet-visible (UV-Vis) liquid chromatography instrument, a detector, and a HPLC pump…. the analytical column is… a flow rate is… a column temperature is…” etc., which renders the claim indefinite because the language of the claim is inconsistent, recites broader and narrower limitations, and fails to provide clear antecedence for each of the claimed elements.
First, the plain meaning of the claim is that the “ultraviolet-visible (UV-Vis) liquid chromatography instrument” is a separate component from “the analytical column”, “a detector” and “a pump”. On one hand, a skilled artisan may reasonably interpret that the recited “liquid chromatography instrument” comprises the whole system, including each of the other components (e.g., the analytical column). However, each of these structures are recited as separate “conditions” without clearly delineating their relationship to one another.
It should also be noted that in order for the “HPLC conditions” of the claim to have proper antecedent basis, it must be clear that all of the recited limitations are specifically referring to the “elution” step of claim 6, which is where “HPLC” is first recited (i.e., “performing isocratic elution on the filtrate by an analytical column of high performance liquid chromatography (HPLC)”). For example, it should be clear that the “injection volume” is referring to an injection volume comprising “the filtrate” recited in claim 6, because there is otherwise no recitation of injecting said filtrate. While this may be reasonably implied (i.e., performing an elution on a filtrate necessarily requires injecting said filtrate), the present claim creates confusion by reciting multiple independent clauses (i.e., HPLC conditions comprise… the analytical column is… a flow rate is…) without any clear transitions, making the antecedence of these limitations difficult to interpret.
In the interest of compact prosecution the claim is interpreted as follows:
“The application method as claimed in claim 6, wherein the isocratic elution step is performed using an ultraviolet-visible (UV-Vis) liquid chromatography instrument comprising a C18 column of 4.6 x 250 millimeters (mm), a detector, and an HPLC pump; wherein the instrument is operated at a flow rate of 1.0 milliliters per minute (mL/min), a column temperature of 40°C, a detection wavelength of 210 nanometers (nm), and an injection volume of 10 microliters (µL)."
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 1-10 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The claims recite an Aspergillus sp. L14-OE::laeA2 strain deposited under preservation number CCTCC NO: M20232057 as being required to practice the claimed invention. As a required element it must be known and readily available to the public or obtainable by a repeatable method set forth in the specification. If it is not so obtainable or available, the enablement requirements of 35 U.S.C. § 112, first paragraph, may be satisfied by a deposit of Aspergillus sp. L14-OE::laeA2. See 37 CFR 1.802.
In the instant case, the specification does not provide a repeatable method for obtaining the same strain having the same deposit number of the claim, and the deposited strain does not appear to be readily available material. See 37 CFR 1.801 – 37 CFR 1.809.
If a deposit is made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants or someone associated with the patent owner who is in a position to make such assurances, or a statement by an attorney of record over his or her signature, stating that the deposit has been made under the terms of the Budapest Treaty and that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of a patent, would satisfy the deposit requirements. See 37 CFR 1.808.
If a deposit is not made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants or someone associated with the patent owner who is in a position to make such assurances, or a statement by an attorney of record over his or her signature, stating that the deposit has been made at an acceptable depository and that the following criteria have been met:
(a) during the pendency of this application, access to the invention will be afforded to one determined by the Commissioner to be entitled thereto;
(b) all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon granting of the patent;
(c) the deposit will be maintained for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposited material;
(d) a viability statement in accordance with the provisions of 37 CFR 1.807; and
(e) the deposit will be replaced should it become necessary due to inviability, contamination or loss of capability to function in the manner described in the specification.
In addition the identifying information set forth in 37 CFR 1.809(d) should be added to the specification. See 37 CFR 1.803 - 37 CFR 1.809 for additional explanation of these requirements.
Claim Rejections - 35 USC § 103
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-3 and 8-10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang, et al. (Genomic and AntiSMASH Analyses of Marine-Sponge-Derived Strain Aspergillus niger L14 Unveiling Its Vast Potential of Secondary Metabolites Biosynthesis. J Fungi (Basel). 2022 May 31;8(6):591; cited on Form 892), hereafter, “Wang 2022” and further in view of Wang, et al. (The chemical profile of activated secondary metabolites by overexpressing LaeA in Aspergillus niger. Microbiological Research. Volume 248. 2021; cited on From 892), hereafter, “Wang 2021”.
Regarding claim 1, Wang 2022 discloses the sequencing and assembly of the whole genome of the marine-sponge-derived Aspergillus niger strain L14 (see Abstract). Wang 2022 teaches that Aspergillus niger is one of the most important sources of secondary metabolites (SMs), with a wide array of pharmacological effects, including anti-inflammatory, antitumor, immunomodulatory and antioxidant effects (see Abstract). Wang 2022 teaches that marine-derived Aspergillus is appraised as a favorable source for discovering new bioactive secondary metabolites (SMs), and there have been reported about 200 SMs from this group, including cyclic peptides (see pg. 1, Introduction). Wang 2022 teaches that some of these metabolites have shown anti-inflammatory, anti-tumor, antibacterial, free radical scavenging and other biological activities, which may be promising resources for new therapeutic drugs (see pg. 1, Introduction).
Wang 2022 discloses that AntiSMASH analysis uncovered 69 secondary metabolite biosynthesis gene clusters (BGCs) in strain L14 and only 7 of them had high similarity with known gene clusters, suggesting most of these BGCs are unknown and yet to be unveiled. This is confirmed by a previous chemical investigation, since a great number of cryptic BGCs in strain L14 are silent or expressed at low levels under conventional conditions. These findings provide an important basis for further chemical study of strain L14 using the gene mining strategy to activate these cryptic BGCs for the production of novel bioactive SMs. See pg. 11, para. 2.
Wang 2021 teaches that the synthesis of many secondary metabolites (SMs) in Aspergilli could be activated by LaeA mutation (see Abstract). Wang 2021 teaches that genomic data has showed that the capacity of filamentous fungi in synthesizing SMs might be underrated, which was due to the silence of many SM gene clusters under general lab conditions (see pg. 1, col. 1, para. 1). For example, in A. niger, more than 70% of the SM gene clusters were repressed under normal lab cultivation conditions, making it valuable to investigate the regulation of synthesizing SMs in filamentous fungi (see pg. 1, para. 1, col. 1). By doing so, the production of many known SMs might be elevated and many silent gene clusters might be activated to produce novel SMs, which could enrich the diversity of fungal SMs and provide important lead compounds for drug exploration.
Wang 2021 teaches that LaeA regulates the synthesis of many secondary metabolites, and it has been found to regulate the production of many medically important compounds in the Aspergillus genus (see pg. 1, para. 2), including in A. niger (see pg. 1, col. 2). Wang 2021 discloses an Aspergillus niger laeA overexpression mutant that was constructed by introducing a linearized plasmid comprising laeA under the control of a strong promoter into an A. niger strain (see pg. 2, col. 1, para. 4). Wang 2021’s study confirmed that LaeA acts as a non-local regulator in activating the production of many SMs and its overexpression is involved in the production of additional products (see pg. 6, col. 1, para. 2).
In view of the instant specification, the Aspergillus sp. L14-OE::laeA2 of the claim was obtained by transferring a recombinant plasmid containing an LaeA target gene fragment into Aspergillus niger L14 to obtain an LaeA overexpression mutant (see pg. 3, para. [0012]; pgs. 9-10, paras. [0071]-[0077]).
Per MPEP 2112.01, where the claimed and prior art products are identical or substantially
identical in structure or composition, or are produced by identical or substantially identical
processes, a prima facie case of either anticipation or obviousness has been established. In re
Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "When the PTO shows a sound
basis for believing that the products of the applicant and the prior art are the same, the applicant
has the burden of showing that they are not." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655,
1658 (Fed. Cir. 1990). Therefore, the prima facie case can be rebutted by evidence showing that
the prior art products do not necessarily possess the characteristics of the claimed product. In re
Best, 562 F.2d at 1255, 195 USPQ at 433.
In the instant case, Wang 2021 teaches the overexpression of the LaeA gene in an Aspergillus niger strain by the introduction of a plasmid comprising a copy of said gene to obtain the Aspergillus niger OElaeA strain, which is substantially the same process disclosed by Applicant to have been used to obtain the claimed Aspergillus sp. L14-OE::laeA2 strain.
Therefore, it would have been obvious at the time of filing for a person of ordinary skill to have arrived at the claimed invention by combining the teachings of Wang 2022 and Wang 2021, because Wang 2021 teaches the overexpression of LaeA activates the synthesis of many useful secondary metabolites (SMs) in Aspergillus niger. One would have recognized from Wang 2022 that the marine-sponge-derived Aspergillus niger strain L14 is capable of producing secondary metabolites which may be promising resources for new therapeutic drugs. Furthermore, Wang 2022 also discloses that many of the biosynthesis gene clusters in this strain are silent and suggests that their activation can lead to new drug discovery. Therefore, a person of skill would have immediately envisaged using the L14 strain for the method taught by Wang 2021, because Wang 2021 also suggests activating silent gene clusters to produce novel SMs for drug exploration, by the overexpression of LaeA. One would have also recognized that the structure and functions of this particular Aspergillus strain, as well as the LaeA gene, are well characterized in the art, and Wang 2021’s disclosure confirms the overexpression of LaeA in Aspergillus niger by use of recombinant plasmids comprising the LaeA gene. Therefore, there would have been a reasonable expectation of success when performing this transformation in A. niger L14 to increase the production of secondary metabolites. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 2, as discussed under 35 U.S.C. 101 and 35 U.S.C. 112(b), the claim is interpreted as being directed to the product of claim 1. Therefore, the claim is obvious for the same reasons as claim 1.
Regarding claim 3, Wang 2021 discloses that the A. niger OElaeA mutant was first cultivated for secondary metabolite collection, and the cultures were then extracted with ethyl acetate (see pg. 2, col. 1, para. 5). Wang 2021 discloses that a C18 solid-phase extraction (SPE) column was used to fractionate A. niger OElaeA crude extract, and the SPE stationary phase was conditioned by sequential washing, followed by purification by semi-prep HPLC (see pg. 2, col. 1, para. 6 to col. 2, para. 2), followed by a chemical workup of organic extracts of the culture broth (see pg. 6, col. 1, para. 2). Therefore, Wang 2021 teaches fermenting and culturing the Aspergillus niger OElaeA, to obtain a fermentation broth, and separating and purifying the fermentation broth to obtain secondary metabolites.
Regarding the limitation, “to obtain the cyclic tripeptide compounds; wherein the cyclic tripeptides comprise compound (I), compound (II) and compound (III)”, as discussed under Claim Interpretation, the capability of the Aspergillus sp. L14-OE::laeA2 to produce the cyclic tripeptides of the claims is presumed to be an inherent property of the claimed engineered Aspergillus, and these products would necessarily be present after separating and purifying the fermentation broth. A person of skill would have necessarily obtained these products when following the claimed method. Therefore, it would have been obvious in view of Wang 2021’s disclosure to have separated and purified the fermentation broth in order obtain the secondary metabolites produced by the strain, which would have necessarily included the claimed compounds.
Regarding claim 8, as discussed under 35 U.S.C. 101 and 35 U.S.C. 112(b), the claim is interpreted as being directed to the product of claim 1. Therefore, the claim is obvious for the same reasons as claim 1.
Regarding claim 9, as discussed under 35 U.S.C. 101 and 35 U.S.C. 112(b), the claim is interpreted as being directed to the product of claim 1. Therefore, the claim is obvious for the same reasons as claim 1.
Regarding claim 10, as discussed under 35 U.S.C. 101 and 35 U.S.C. 112(b), the claim is interpreted as being directed to the product of claim 1. Therefore, the claim is obvious for the same reasons as claim 1.
Claim(s) 4-5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang 2022 and Wang 2021 as applied to claims 1-3 and 8-10 above, and further in view of Zhang, et al. (Overexpression of global regulator LaeA increases secondary metabolite production in Monascus purpureus. Appl Microbiol Biotechnol. 2020 Apr;104(7):3049-3060; cited on Form 892), hereafter, “Zhang”, and Liu, et al. (Assignment of Absolute Configurations of Two Promising Anti-Helicobacter pylori Agents from the Marine Sponge-Derived Fungus Aspergillus niger L14. Molecules. 2021; 26(16):5061; cited of Form 892), hereafter, “Liu”.
Regarding claim 4, Wang 2022 discloses that the white mycelia of strain L14 grew from a potato dextrose agar (PDA) medium after incubation for 3 days at 28°C (see pg. 3, para. 5). Wang 2022 also discloses that one suspension of culture containing its mycelia in potato dextrose broth (PDB) was used for storing the strain (see pg. 3, para. 4).
Wang 2021 discloses that mediums used for culturing included PDA medium (see pg. 2, para. 3). However, the disclosure is not explicit as to when in the protocol this medium was used. Wang 2021 discloses that to collect secondary metabolites, the A. niger OElaeA mutant was first cultivated for 7 days at 25°C in the dark on 10 Petri dishes with WATM medium (See pg. 2, col. 1, para. 5).
Zhang discloses an LaeA-overexpressing filamentous fungus that produces several secondary metabolites (see Abstract). Zhang teaches the LaeA overexpressing strain was: (1) activated on a potato dextrose agar (PDA) medium for 2 generations; (2) inoculated into a seeding medium for 2 days at 30°C at about 200 rpm; (3) inoculated into fermentation medium and cultured for 2 days at 30°C at about 150 rpm; (4) and cultured further at 25°C at about 150 rpm for 13 days (see pg. 3050, col. 2, para. 2).
Therefore, Zhang teaches a protocol for obtaining secondary metabolites from an LaeA-overexpressing filamentous fungus, comprising inoculating the strain into a potato dextrose agar (PDA) medium to “activate” the strain and further culturing the strain for 4 days (2 days in seeding medium and 2 days in fermentation medium) at 30°C. This is similar to the method step “to obtain activated Aspergillus” recited in the instant claim, except Zhang teaches additional transfers of the culture (i.e., from PDA to seeding medium to fermentation medium). Zhang also teaches a further culturing step which is similar to method step “(2)” (“to obtain the fermentation broth”), except Zhang teaches lowering the temperature from 30°C to 25°C, a lower rpm of 150 (versus 180-200 rpm), and a shorter duration of 13 days (versus 15 days).
Liu discloses that the chemical investigation of the endozoic fungus Aspergillus niger L14 resulted in the isolation of two dimeric naphtho-g-pyrones (see Abstract). Liu discloses that strain L14 was cultured on potato dextrose agar (PDA) at 30°C for 5 days. Then, a balanced amount of fungal colony was transferred to the culture broth in a 500 mL Erlenmeyer flask containing 300 mL sterilized potato dextrose broth (PDB); it was then shaken at 180 rpm for 3 days under 30°C as seed broth. The seed broth was then inoculated to a solid rice medium in a 1000 mL Erlenmeyer flask with sterilized rice (160 g) and water (320 mL) and cultivated at 20°C for 40 days. See pg. 6, para. 2.
Therefore, Liu also teaches an initial culturing step using PDA at 30°C, but for 5 days instead of 3-4 days. Liu also teaches inoculating the activated Aspergillus into a potato dextrose broth (PDB) medium and then culturing the activated Aspergillus at 30°C and 180 revolutions per minute (rpm) for 3 days, which meets the limitations of the claimed method step “to obtain seed liquid”. However, Liu teaches a slower final culturing step (20°C for 40 days)
Regarding the limitation of a “Czapek medium”, Wang 2021 teaches that the WATM medium was composed of 30.0 g/l sucrose, 2.0 g/l sodium nitrate, 0.5 g/l magnesium sulfate, 0.2 g/l potassium chloride, 0.01 g/l ferrous sulfate, and 1.0 g/l dipotassium hydrogen phosphate with a pH of 7.0 (see pg. 2, col. 3, para. 1). This is highly similar to the Czapek medium of the claims comprising 30 g/L sucrose, 3 g/L sodium nitrate, 0.5 g/L magnesium sulfate heptahydrate, 0.5 g/L potassium chloride, 0.01 g/L ferrous sulfate and 1 g/L dipotassium hydrogen phosphate (see, e.g., dependent claim 5). As discussed under 35 U.S.C. 112(b), a pH “of natural” includes pH 7.0.
Per MPEP 2144.05(I), in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985). See also Warner-Jenkinson Co., Inc. v. Hilton Davis Chemical Co., 520 U.S. 17, 41 USPQ2d 1865 (1997) (under the doctrine of equivalents, a purification process using a pH of 5.0 could infringe a patented purification process requiring a pH of 6.0-9.0); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%).
In view of the instant specification, there is no discussion regarding the criticality of the cultivation steps selected by the inventors for the fermentation and culturing of the claimed Aspergillus strain. There is also no comparison to any other culturing methods in Applicant’s Examples to demonstrate that any of the specific conditions of the claimed culture method achieved some particular (unexpected) result.
Per MPEP 2144.05, Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.").
In the instant case, the prior art references each disclose a similar method of cultivating a filamentous fungi in order to obtain secondary metabolites from said fungi, each further disclosing method steps which are analogous to the steps of the claims, and the concentrations (i.e., g/L), temperatures (i.e., °C), conditions (i.e., rpm) and timeframes (i.e., days) taught collectively by these references either lie within or near the claimed ranges or values. The similarities and differences amongst the methods taught by each of the prior art references is also evidence to support that the limitations of the claimed method lie entirely within prior art conditions, and a person of skill would have had a sufficient starting point for arriving at the claimed invention through no more than a routine amount of effort.
Therefore, it would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of the aforementioned references, because each of these references provide a sufficient amount of guidance for obtaining useful products from the claimed engineered strain of Aspergillus. One would have recognized that each of the claimed method steps were known in the art using known media components and similar concentrations and conditions. One would have also recognized that each of the applied references are in the same field of endeavor and pertain to solving similar problems. For example, Wang 2022, Wang 2021 and Liu each relate to cultivating Aspergillus niger strains to obtain secondary metabolites, with Wang 2021 and Liu specifically relating to the L14 strain. Further, both Wang 2021 and Zhang each relate to cultivating filamentous fungi overexpressing the LaeA gene in order to obtain secondary metabolites. Therefore, a person of skill would have recognized that the results of combining these teachings would have been predictable, and there would have been a reasonable expectation of success given the ordinary skill in the art at the time of filing. Therefore, it was well within the ordinary skill in the art to have developed a method to cultivate the engineered Aspergillus strain to obtain useful compounds, as evidenced by the prior art references, and it would have required no more than routine optimization to have arrived at the claimed method.
Regarding claim 5, Wang 2021 teaches that the WATM medium was composed of 30.0 g/l sucrose, 2.0 g/l sodium nitrate, 0.5 g/l magnesium sulfate, 0.2 g/l potassium chloride, 0.01 g/l ferrous sulfate, and 1.0 g/l dipotassium hydrogen phosphate with a pH of 7.0 (see pg. 2, col. 3, para. 1). This is highly similar to the Czapek medium of the claims comprising 30 g/L sucrose, 3 g/L sodium nitrate, 0.5 g/L magnesium sulfate heptahydrate, 0.5 g/L potassium chloride, 0.01 g/L ferrous sulfate and 1 g/L dipotassium hydrogen phosphate (see dependent claim 5). As discussed under 35 U.S.C. 112(b), a pH “of natural” includes pH 7.0.
As previously discussed, a person of skill could have arrived at the claimed Czapek medium through no more than routine optimization. Therefore, the claim is obvious for the same reasons discussed regarding claim 4.
Claim(s) 6-7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang 2022, Wang 2021, Zhang and Liu as applied above, and further in view of Song, et al. (Discovery, Identification, and Insecticidal Activity of an Aspergillus flavus Strain Isolated from a Saline-Alkali Soil Sample. Microorganisms. 2023 Nov 16;11(11):2788; cited on Form 892), hereafter, “Song” and Altiero, P. (Getting Off to a Good Start: Isocratic method development. Agilent Technologies. February 13, 2020; cited on Form 892), hereafter, “Altiero”.
Regarding claim 6, Wang 2021 discloses that cultures were extracted with ethyl acetate plus 1% formic acid for 24 h, and the crude extract was filtered and dried on a rotary evaporator at 38°C (see pg. 2, col. 1, para. 5). Wang 2021 discloses that a C18 solid-phase extraction (SPE) column was used to fractionate A. niger OElaeA crude extract. The SPE stationary phase was conditioned by sequential washing with 60 mL 100% methanol (MeOH) and 240 mL 100% deionized water. A. niger OElaeA crude extract (1.86 g) was chromatographed with four subfractions (480 ml each) generated stepwise from deionized water to methanol: 25% methanol, 50% methanol, 100% methanol, and 100% methanol plus 0.1% trifluoroacetic acid (TFA). Subfraction 2 (50% methanol, 60 mg) and 3 (100% methanol, 780 mg) were further purified by semi-preparative HPLC (semi-prep HPLC). See pg. 2 col. 1, para. 6 to col. 2 para. 1. Wang 2021 discloses that Semi-prep HPLC was used to purify subfractions obtained by SPE separation using a semi-prep HPLC column comprising a Solvent A consisting of 95 % H2O, 5% methanol, and 0.05 % TFA and a Solvent B comprising 100% acetonitrile (see pg. 2, col. 2, para. 2). Wang also discloses that separations were performed at room temperature (20-25°C) (see pg. 2, col. 2, para. 2).
Therefore, Wang 2021 teaches the separating and purifying of the fermentation broth, comprising: adding the fungal liquid to ethyl acetate followed by extracting (i.e., extracted with ethyl acetate), except Wang 2021 does not state “in an equal amount”; concentrating the upper layer to dryness by rotary evaporation to obtain a crude fermentation extract (i.e., dried on a rotary evaporator at 38°C), except Wang 2021 does not state “under reduced pressure”; dissolving the crude fermentation extract with methanol alcohol followed by diluting with the methanol alcohol to obtain a diluted solution (i.e., conditioned by sequential washing with methanol and deionized water to create subfractions that were 480 ml each, which reasonably meets the limitations of “dissolving” and “diluting”); performing elution on the filtrate by a HPLC analytical column with a mobile phase of acetonitrile and water (i.e., solvent comprising water and a solvent comprising acetonitrile), except Wang 2021 does not appear to use an isocratic elution method using the claimed volume ratio of acetonitrile to water. Wang 2021 does not explicitly disclose removing solvents by rotary evaporation. However, Wang 2021 does teach the separations were performed at 20-25°C which includes the temperature of 25°C recited in the drying step of the claim, and there is no disclosure that this temperature was changed following purification. It should be noted that Wang also discloses rotary evaporation was used in a previous step to dry the crude extract, and a skilled artisan would recognize the same could be performed to remove solvents.
Liu discloses that at the end of fermentation, all fermented material was collected and extracted with the same volume of ethyl acetate 3 times. The organic layer was concentrated under vacuum at 38°C to obtain crude extract (see pg. 6, para. 2), and the ethyl acetate extract was separated into six fractions, A-F, under a gradient condition of CH3CN (acetonitrile) and H2O on a preparative HPLC column. Fraction D was further subjected to HPLC fractionation to generate fonsecinone A and isoaurasperone A using a semi-preparative C18 column under a gradient condition of mobile phase (CH3CN and H2O) with a flow rate of 3.0 mL/min. See pg. 6, para. 3.
Therefore, Liu teaches that the extraction step used an equal volume of ethyl acetate (i.e., “same volume”), the crude extract was concentrated under reduced pressure (i.e. vacuum), and the crude extract was separated into fractions for elution on an HPLC column. Liu also teaches that the mobile phase comprised acetonitrile (CH3CN) and water, but does not disclose a volume ratio. Liu also appears to teach a step-gradient elution method, not an isocratic elution method.
However, it should be noted that in view of Applicant’s Examples, a step-gradient elution was performed by the inventors (see pg. 11, para. [0087]) prior to the isocratic elution step (see pg. 12, para. [0088]).
The examiner notes that Zhang also teaches the use of HPLC to detect fermentation products in the culture broth (see pg. 3051, col. 2, para. 2), but does not disclose any purification steps.
Therefore, the aforementioned references do not explicitly teach filtering the broth through eight-layer gauze, filtering the diluted solution with a .22 micrometer organic filter to obtain a filtrate, or performing isocratic elution on the filtrate at a volume ratio of 35:65.
Song teaches a protocol for obtaining fermentation products from Aspergillus flavus (see Abstract). Song discloses that after fermenting the Aspergillus strain, the fermented broth was filtered with eight layers of gauze and eluted three times (see pg. 3, para. 2). Song teaches that after filtration with 8 layers of gauze, the fermentation solution was centrifuged for 5 min and the supernatant was filtered using a 0.22 micrometer microporous filtration membrane (see pg. 3, para. 4).
Altiero teaches that the benefits of an isocratic separation (i.e., one where the composition of the solvent remains constant throughout separation) include simple adjustability, no baseline drift, no re-equilibration time, and no impacts due to delay volume (i.e., easily transferable) (see pg. 4). Altiero teaches the selection of this method and its parameters depend on the goals of the separation, the desired speed of the separation, and accuracy and precision requirements (see pg. 5). Altiero teaches that when exploring organic modifiers, acetonitrile (ACN) and methanol (MeOH) are readily available and work on any bonded phase (i.e., optimize separation no matter the column choice (see pg. 29). Aliero recommends using a scouting gradient run of 5-95% acetonitrile (see pg. 27) to estimate isocratic conditions, including organic solvent concentration (see pg. 28), and exemplifies the use of water and acetonitrile as solvents (see pg. 13). Therefore, Altiero teaches the advantages of using isocratic elution methods and teaches that the ideal concentration of acetonitrile for a given application can be readily determined by a scouting gradient test run.
Therefore, a person of ordinary skill could have easily determined an effective volume ratio of acetonitrile and water using a gradient test run of 5-95% acetonitrile.
In view of the instant specification, there is no discussion regarding the criticality of the separating and purifying steps selected by the inventors for the separating and purifying of the claimed compounds. There is also no comparison to any other separating and purifying methods in Applicant’s Examples to demonstrate that any of the specific parameters of the claimed culture method achieved some particular (unexpected) result.
Per MPEP 2144.05, "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.").
In the instant case, the prior art references disclose similar methods of separating and purifying culture broths in order to obtain secondary metabolites from fungi, while further disclosing method steps which are analogous to the steps of the claims and reasonably appear to serve the same purpose. The similarities and differences amongst the methods taught by each of the prior art references is further evidence that the limitations of the claimed method lie entirely within prior art conditions, and a person of skill would have had a sufficient starting point for arriving at the claimed invention through no more than a routine amount of effort.
Regarding the limitation of “to obtain effluents from 6th minute (min) to 7th min, 8th min to 9th min and 10.8th min to 11.2nd min respectively”, as discussed under Claim Interpretation, this limitation only requires obtaining an effluent when each compound elutes, and the recited retention times are directed to an inherent result of following the claimed method. When these compounds are present, their observed retention time naturally flows from performing the isocratic elution step. Therefore, a person of skill would have necessarily obtained each of these effluents by following the method as claimed, whether or not they had been informed beforehand of what compounds would be present or at what retention times they would elute. Furthermore, one motivated to obtain fermentation products from this process would have reasonably known to collect each effluent and remove any remaining solvents in order to isolate whatever compound was present for further characterization and use.
Therefore, it would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of the aforementioned references, because each of these references provide a sufficient amount of guidance for obtaining useful fermentation products from the claimed engineered strain of Aspergillus. One would have recognized that each of the claimed method steps were known in the art, utilizing known materials and conditions that have been evidenced to be within prior art conditions. One would have also recognized that each of the applied references are in the same field of endeavor and pertain to solving similar problems. For example, Wang 2021, Liu, Zhang and Song each relate to obtaining products produced by the fermentation of filamentous fungi, while Wang 2022, Wang 2021, Liu and Song each specifically relate to the production of useful products from Aspergillus. Furthermore, Wang 2021 and Liu teach the purification of such compounds using HPLC elution methods, and one would have been motivated to combine these teachings with Altiero to arrive at the most efficient method for purifying these products (i.e., isocratic elution). Furthermore, Altiero specifically teaches result-effective variables and guidance for routinely optimizing such methods for their desired application. Therefore, a person of skill would have recognized that the results of combining these teachings would have been predictable, and there would have been a reasonable expectation of success given the ordinary skill in the art at the time of filing. Therefore, it was well within the ordinary skill in the art to have developed a method to separate and purify useful compounds from the fermentation of the engineered Aspergillus strain, and it would have required no more than routine optimization to have arrived at the claimed method.
Regarding claim 7, Wang 2021 discloses that for the purification of the metabolites, a semi-prep HPLC (i.e., high performance liquid chromatography) column ACE C18-HL was connected to an Agilent 1200 series binary pump and monitored by an Agilent photodiode array detector at 230, 254, 280 and 410 nm (see pg. 2, col. 2, para. 2), which are all within the ultraviolet range (i.e., UV-Vis).
Zhang discloses that the content of fermentation products in the culture broth was detected by HPLC using a flow rate of 1 mL/min with an ultraviolet detector, a detection wavelength of 237 nm, a temperature of 30°C, and an injection volume of 10 microliters (µL).
Song discloses that the UPLC conditions were set to 40°C (see Supplementary Material cited on Form 892 at pg. 2, para. 4.3).
Altiero teaches that HPLC columns for isocratic method development include C18 columns of 4.6 x 250 mm (see pg. 19), and the optimal flow rate for columns with a 4.6 mm inner diameter (4.6 mm i.d.) is 1.0 mL/min (see pg. 13).
Therefore, the prior art combination discloses every limitation of the claim, except Wang 2021 teaches a detection wavelength of 230 nm, which is close but not the same as the claimed 210 nanometers (nm). Similarly, Zhang teaches a detection wavelength of 237 nm, as discussed above.
Per MPEP 2144.05(I), in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985) (Emphasis added). See also Warner-Jenkinson Co., Inc. v. Hilton Davis Chemical Co., 520 U.S. 17, 41 USPQ2d 1865 (1997) (under the doctrine of equivalents, a purification process using a pH of 5.0 could infringe a patented purification process requiring a pH of 6.0-9.0); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%).
In view of the instant specification, there is no discussion regarding the criticality of the HPLC conditions selected by the inventors. There is also no comparison to any other HPLC conditions in Applicant’s Examples to demonstrate that any of the specific parameters of the claimed HPLC conditions achieved some particular (unexpected) result.
Per MPEP 2144.05, "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Emphasis added); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 ("The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.").
In the instant case, the prior art references disclose similar HPLC conditions which reasonably appear to serve the same purpose as those of the claim. Therefore, once a desirable fermentation product is successfully identified and/or purified using the methods explicitly described in the prior art, it would be the normal desire of an ordinary artisan to routinely optimize the HPLC conditions in order to repeat this success in a more efficient and/or cost-effective manner. The similarities and differences amongst the methods taught by each of the prior art references is further evidence that the limitations of the claimed method lie entirely within prior art conditions, and a person of skill would have had a sufficient starting point for arriving at the claimed invention through no more than routine optimization.
Conclusion
No claims are allowed.
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/DENNIS IGNATIUS ARMATO JR/Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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