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 .
Election/Restrictions
Applicant's election with traverse of Group II and Species II-A in the reply filed on 17 June 2026 is acknowledged. The traversal is on the ground(s) that the grouped invention contained claims that overlap and there was no mention burden to examine the grouped inventions. This is not found persuasive because:
The present application is a continuation of a PCT application filed as a U.S. continuation under 35 U.S.C. § 111(a). Accordingly, domestic restriction practice under 35 U.S.C. § 121 and 37 C.F.R. §§ 1.141–1.146 applies.
The restriction among Groups I–III remains proper because the claims are directed to distinct inventions and examination of all groups together would impose a serious search and examination burden. Group I is directed to cell-containing compositions, Group II to oil/composition products, and Group III to methods for producing the VLC-PUFAs.
Within elected Group II, the election of Species II-A, claims 16–31, is also maintained. Species II-A is directed to crude oil; Species II-B, claims 32–43, to quantitatively defined lipid compositions; and Species II-C, claims 44–61 and 76, to microbial oil. Although certain embodiments overlap, the species are not coextensive and require materially different searches.
Accordingly, the restriction requirement under 35 U.S.C. § 121 is maintained. Group II and Species II-A remain elected. Claims directed to Groups I and III and non-elected Species II-B and II-C remain withdrawn from consideration.
The requirement is still deemed proper and is therefore made FINAL.
Claim Rejections - 35 USC § 112
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 16, 22, 27, and 31 are rejected under 35 U.S.C. §112(a) as failing to comply with the enablement requirement because the specification, while enabling certain crude oils containing the disclosed and exemplified very-long-chain polyunsaturated fatty acids. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to reasonably provide enablement for crude oils containing a polyunsaturated fatty acid having any carbon-chain length of 30 carbons or greater, without an upper limit, and/or a derivative thereof at the concentration required by claim 16.
Claim 16 recites crude oil comprising a polyunsaturated fatty acid “having at least 30 carbons” and/or a derivative thereof at a content of at least 1.0 area%, with no upper limit on the carbon-chain length. Claims 22 and 27 ultimately depend from claim 16 and do not impose an upper carbon-number limit. Claim 31 depends through claims 29 and 28 from claim 16 and likewise does not cure this deficiency.
The specification defines VLC-PUFAs as PUFAs having at least 30 carbons and states that the upper limit is nominally not limited; however, the specification immediately identifies less than 40 carbons or less than 38 carbons from the standpoint of microbial production and particularly describes C30, C32, C34, and C36 species. (Goole Patents) The working examples similarly demonstrate production in the disclosed microbial systems principally within this finite chain-length region. For example, the AsELOVL4b examples demonstrate C30:5, C32:5 and C34:5 products, while the African sharptooth catfish ELOVL4a example demonstrates C32:5, C34:5 and C36:5.
Accordingly, the specification provides an enabling disclosure for at least particular C30-C36 VLC-PUFAs produced using the disclosed ELOVL4/host systems, but claim 16 is not limited to that scope. The claim also embraces C40, C42, C44, and progressively longer PUFAs and their derivatives at the claimed concentration, without providing a corresponding upper boundary.
Wands analysis
Breadth of the claims. The breadth weighs against enablement. Claim 16 is open-ended as to carbon-chain length. Once the minimum of C30 is met, no upper carbon number is specified. Thus, the claim is substantially broader than the particularly described C30-C36 species and the microbial-production range identified in the disclosure. The dependent limitations in claims 22, 27, and 31 do not restrict this open-ended feature.
Nature of the invention. The invention concerns biosynthetic production of VLC-PUFAs using PUFA-chain elongases in biological host systems. Production is dependent on enzyme activity, substrate utilization, host metabolism, and successive chain elongation. The specification itself recognizes that ELOVL4 enzymes capable of using PUFAs as substrates may nevertheless be incapable of producing VLC-PUFAs of at least C30. Thus, chain elongation cannot reasonably be treated as automatically extending to every progressively longer carbon-chain length merely because activity is shown for a shorter VLC-PUFA.
State of the prior art. The specification explains that conventional microbial production had resulted in total VLC-PUFA concentrations below 0.8% and individual VLC-PUFA concentrations below 0.5%, and attributes the disclosed higher production to particular recombinant cells and culture/induction conditions. The disclosed advance therefore involves achieving appreciable concentrations through specific biological production conditions rather than applying a universally established method for producing arbitrary VLC-PUFA chain lengths.
Level of ordinary skill. A person of ordinary skill would be expected to possess substantial knowledge of molecular biology, recombinant expression, microbial culture, fatty-acid metabolism, and chromatographic lipid analysis. This relatively high level of skill favors enablement to some extent. It does not, however, supply the missing teaching as to which elongases, substrates, hosts, or culture conditions would produce increasingly longer C40+ VLC-PUFAs at the concentration required by claim 16.
Predictability of the art. The predictability factor weighs against enablement. The disclosure itself distinguishes among ELOVL4 enzymes on the basis of their ability to generate C30+ VLC-PUFAs and reports different product distributions for different ELOVL4 enzymes. The record therefore does not establish that successful production of C30-C36 species reasonably predicts production of arbitrarily longer VLC-PUFAs at a concentration of at least 1.0 area%.
Amount of direction or guidance. The specification provides substantial guidance for the disclosed embodiments, including recombinant ELOVL4 expression, yeast hosts, inducible promoters, PUFA substrates, culture conditions, expression induction, oil extraction, and analytical procedures. This guidance favors enablement of the disclosed region. The specification does not, however, provide a general rule, additional elongase series, substrate-selection rule, or other technical roadmap explaining how to extend that methodology to progressively longer C40+ PUFAs while maintaining the minimum concentration required by claim 16.
Working examples. The working examples favor enablement only for a substantially narrower portion of the claimed scope. The examples establish C30, C32, C34, and C36 VLC-PUFAs under the disclosed conditions, including C36:5 using the catfish ELOVL4a system. They do not establish that the same methodology is generally operative throughout the unlimited C30+ range.
Quantity of experimentation. To practice the claim across its full scope, a skilled artisan outside the disclosed region would have to identify or engineer suitable elongases, select substrates and hosts, establish expression and culture conditions, and then analytically determine whether each candidate system actually generates the progressively longer VLC-PUFA at the required concentration. The disclosure does not provide a predictive principle that would eliminate this iterative selection and testing. This weighs toward undue experimentation when considered together with the open-ended scope and limited predictability.
The current MPEP requires the Wands factors to be considered as a whole and emphasizes that enablement must extend to the full claimed scope; a specification may require reasonable experimentation, but it may not leave a skilled artisan to undertake an undue research program to identify operative members throughout an overbroad class.
Considering the evidence as a whole, undue experimentation would be required to make crude oils containing the full range of VLC-PUFAs encompassed by the open-ended “at least 30 carbons” limitation at the claimed concentration. The disclosure therefore is not commensurate in scope with claims 16, 22, 27, and 31.
For purposes of advancing prosecution, an amendment limiting the VLC-PUFA carbon-chain length to a scope supported and enabled by the original disclosure, such as the particularly disclosed C30-C36 species or another adequately supported finite upper limit, would address this particular enablement issue, subject to consideration of all other statutory requirements.
Claim 31 is additionally rejected under 35 U.S.C. §112(a) as failing to comply with the enablement requirement because the specification, while enabling the specifically disclosed non-mammalian ELOVL4 polynucleotides and at least certain closely related functional sequences, does not reasonably enable the full scope of the genus recited in alternative (b).
Claim 31(b) encompasses a polynucleotide having at least 90% nucleotide-sequence identity to a polynucleotide encoding non-mammalian ELOVL4, provided that the encoded protein possesses PUFA-chain-elongation activity.
The specification states that the “specific ELOVL4” can be obtained from any species and identifies non-mammalian ELOVL4 broadly as including avian, fish, reptile, and amphibian enzymes. It particularly identifies black-seabream ELOVL4b and African-sharptooth-catfish ELOVL4a, with SEQ ID NOs. 12 and 14 as corresponding nucleotide sequences. The specification also generically states that polynucleotides over a range of sequence identities may be used when they encode a protein having PUFA-chain-elongation activity.
The presence of the functional requirement does not itself teach the skilled artisan which members of the structural sequence genus will satisfy that requirement. Rather, the functional limitation identifies successful members after their activity is known or experimentally determined.
Wands analysis for claim 31
Breadth of the claim. Claim 31(b) is not referenced to one identified nucleotide sequence. The reference class itself encompasses polynucleotides encoding non-mammalian ELOVL4 across multiple taxonomic groups, and the claim then extends to sequences differing by as much as approximately 10% from members of that reference class, provided the encoded protein retains the recited activity. The resulting sequence space is substantially broader than the specifically exemplified genes.
Nature of the invention. The claimed subject matter concerns recombinant polynucleotides whose encoded membrane-associated elongase protein must retain PUFA-chain-elongation activity. Function therefore depends not merely on nucleotide similarity but on the resulting amino-acid sequence and preservation of enzymatic characteristics.
State of the prior art. Specific ELOVL4 proteins and genes were known, and the specification itself identifies particular fish ELOVL4 sequences. This favors some degree of routine cloning and expression. The present record, however, does not establish a general rule under which every nucleotide sequence meeting the 90% numerical threshold can be predicted to encode an operative PUFA-chain elongase.
Level of ordinary skill. The skilled artisan would be capable of sequence analysis, recombinant expression, microbial culture, and fatty-acid assays. This favors the ability to test individual candidates. The ability to test candidates does not by itself provide a predictive teaching identifying which candidates throughout the claimed genus possess the required activity.
Predictability. The disclosure indicates that even ELOVL4 proteins that use PUFAs as substrates may fail to produce C30+ VLC-PUFAs. Thus, membership in the general ELOVL4 class does not establish identical elongation behavior. A numerical nucleotide-identity threshold standing alone does not resolve this functional variability.
Direction and guidance. The specification provides SEQ ID NOs. 12 and 14 and identifies conserved protein motifs associated with chain elongases. Those teachings provide meaningful guidance. However, the specification does not identify which nucleotide or amino-acid substitutions throughout the claimed ≥90% genus can be made while retaining the required activity, nor does it provide a sequence/function rule that permits functional members across the genus to be selected without testing.
Working examples. The working examples demonstrate particular naturally derived ELOVL4 genes, including black-seabream ELOVL4b and African-sharptooth-catfish ELOVL4a, in yeast-expression systems. These examples establish operability of particular sequences, but do not demonstrate a representative series of altered ≥90%-identity polynucleotides distributed throughout the full claimed sequence genus.
Quantity of experimentation. Outside the specifically disclosed sequences, a skilled artisan would have to obtain or generate candidate polynucleotides, express the encoded proteins, expose the host system to appropriate PUFA substrates, recover and analyze the lipid products, and determine whether the encoded protein possesses the claimed PUFA-chain-elongation activity. Because the claim defines the genus in significant part by the result of this testing, practice throughout the claimed scope would require iterative screening rather than application of a disclosed predictive rule.
MPEP §2164 explains, following Wands and Amgen, that a broad functionally limited genus is not necessarily enabled merely because individual candidates can be made and screened; the pertinent question is whether the disclosure teaches the full claimed scope without undue experimentation.
Upon balancing all of the Wands factors, particularly the breadth of the sequence genus, biological variability, limited predictive guidance, finite examples, and necessity for functional screening of candidates, the specification does not enable the full scope of claim 31(b) without undue experimentation.
An amendment defining the variant genus relative to one or more particular disclosed reference sequences, together with additional structural limitations reasonably correlated with the recited activity, would more closely correspond to the enabling disclosure.
Claim 22 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.
Claim 22 recites crude oil comprising “an impurity having a chemical formula of C23H36O3.” Under the broadest reasonable interpretation, the claim is not limited to the particular chromatographic component identified in the examples because claim 22 does not require the disclosed APGC retention time, measured mass-to-charge value, or another characteristic tying the claimed impurity to that particular component.
The specification reports that fatty-acid analysis revealed an impurity having a composition formula of C23H36O3 and explains that it may be identified using GC/MS. In the working example, however, the component occurring at an APGC retention time of 18.034 minutes could not be structurally identified and was designated “impurity X”; the disclosure reports a presumed formula of C23H36O3 and measured [M+H]+ of 361.2710 for that particular analytical component.
Thus, the specification demonstrates possession of a particular analytically detected impurity X. Claim 22, as presently drafted, is broader because it identifies the impurity solely by molecular formula and does not incorporate the analytical characteristics by which the disclosed impurity X is distinguished. To the extent the formula-only limitation encompasses structurally different compounds satisfying C23H36O3, the specification does not describe representative species reflecting that structural variation or another structure/function or structure/property relationship showing possession of the full genus.
MPEP §2163 provides that a genus may be supported through representative species or other identifying structural, physical, chemical, or functional characteristics sufficient to show possession of the claimed genus; where substantial variation exists, the disclosure must reasonably reflect that variation.
Accordingly, the disclosure of one unresolved analytical component does not reasonably convey possession of every structurally distinct impurity encompassed by a formula-only construction of C23H36O3.
An amendment tying the limitation to the disclosed impurity X by appropriate originally disclosed analytical characteristics—such as the disclosed APGC retention time and/or measured m/z—would more closely correspond to the subject matter actually demonstrated in the specification, subject to compliance with 35 U.S.C. §132(a).
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 25, 27, 31 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.
Claim 25 — “8 to 22 hydrocarbons”
Claim 25 is rejected under 35 U.S.C. §112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter regarded as the invention.
Claim 25 recites that a fatty acid other than the specified PUFAs “is a fatty acid having from 8 to 22 hydrocarbons.”
It is unclear what physical or chemical parameter is defined by “8 to 22 hydrocarbons.” In particular, the claim does not specify whether the range is intended to refer to (1) the number of carbon atoms in the fatty acid, (2) a number of hydrocarbon groups or moieties associated with the fatty acid, or (3) some other property.
The specification does not provide a special definition that resolves this ambiguity. Rather, elsewhere the specification expressly defines PUFA carbon number in terms of the number of carbons in the constituent fatty acid and uses conventional C20, C22, C30, C32, C34, and C36 nomenclature. Although this context suggests that “8 to 22 carbon atoms” may have been intended, the Office may not rewrite the limitation in examination where the claim itself recites a materially different term.
Accordingly, the metes and bounds of claim 25 cannot be determined with reasonable certainty from the language presently recited.
Applicant is invited to consider amending “a fatty acid having from 8 to 22 hydrocarbons” to “a fatty acid having from 8 to 22 carbon atoms,” if that accurately reflects the intended scope and is supported by the application as originally filed.
Claim 27 — “non-productive PUFA externally added”
Claim 27 is rejected under 35 U.S.C. §112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter regarded as the invention.
Claim 27 recites that “the PUFA comprises a non-productive PUFA externally added.”
The phrase “non-productive PUFA” does not establish what characteristic makes the PUFA “non-productive.” It is unclear whether the language means a PUFA that is not synthesized by the host, a PUFA that is not the product of the ELOVL4 elongation reaction, a PUFA that remains unconverted after being supplied as a substrate, or some other category of PUFA.
The specification provides relevant context but does not eliminate the uncertainty in the claim language. In particular, the specification discusses non-PUFA-producing hosts, explains that yeast ordinarily does not produce PUFAs without artificial manipulation, and states that when such a host is employed the PUFAs in the culture are derived from added substrate. The specification further explains that substrate PUFAs may be present from the beginning of culture or added when expression is induced.
This disclosure defines a characteristic of the host and the origin of a substrate PUFA; claim 27 instead characterizes the PUFA itself as “non-productive.” The claim therefore does not clearly state whether “non-productive PUFA” is intended to mean a substrate PUFA not biosynthesized by the host.
The term “externally added” introduces a related uncertainty in this product claim because the claim does not state to what the PUFA is externally added. The specification contemplates adding PUFA to a culture medium at different stages, while claim 27 merely defines the resulting crude oil.
Accordingly, a person of ordinary skill is left to select among materially different interpretations of the recited limitation, and the scope of claim 27 is therefore indefinite.
If consistent with applicant's intended invention and the original disclosure, clarification that the PUFA is an externally supplied substrate added to the culture medium and not produced by the host would address the identified ambiguity.
Claim 31 — ≥90% “sequence identity”
Claim 31 is rejected under 35 U.S.C. §112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter regarded as the invention.
The deficiency concerns alternative (b), which recites a polynucleotide having at least 90% sequence identity to “a nucleotide sequence of the polynucleotide encoding non-mammalian ELOVL4.”
First, the claim does not identify a particular reference nucleotide sequence against which the 90% identity requirement is to be calculated. The specification states that ELOVL4 may be from any species, describes non-mammalian ELOVL4 as including avian, fish, reptile, and amphibian ELOVL4, and specifically discloses at least the black-seabream and African-sharptooth-catfish polynucleotides corresponding to SEQ ID NOs. 12 and 14. Thus, the phrase “the polynucleotide encoding non-mammalian ELOVL4” does not point to a single reference sequence.
Second, the application does not identify how the percentage sequence identity is to be determined. No definition specifying a global or local comparison, alignment methodology, treatment of insertions or deletions, or gap parameters is provided in the published disclosure. Searches of the disclosure do not identify a BLAST, alignment, or gap-penalty methodology.
Accordingly, for a candidate sequence near the 90% boundary, the claim does not provide an objective intrinsic standard establishing (1) the reference sequence to be used and (2) the comparison methodology governing whether the threshold is satisfied. The additional requirement that the encoded protein possess PUFA-chain-elongation activity does not resolve the uncertainty regarding whether the nucleotide sequence satisfies the separate ≥90%-identity limitation.
MPEP §§2173 and 2175 require an indefiniteness rejection to identify the particular language that prevents the skilled artisan from determining the claim boundaries and to explain the resulting uncertainty; mere breadth is insufficient. The deficiency here is not merely that claim 31 covers many sequences, but that the numerical boundary itself lacks a specified reference and measurement convention.
Applicant may address this issue by identifying one or more particular reference sequences—for example, an appropriate disclosed SEQ ID NO.—and by providing an objective sequence-identity calculation standard, provided the amendment is supported by the application as originally filed.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 16–21, 26–29 and 31 are rejected under AIA 35 U.S.C. §103 as being unpatentable over Anderson et al. (US 2009/0203787 A1) in view of Oboh et al., and further in view of US 2013/0046106 A1.
Rejection I — Claims 16–21, 26–29 and 31
Claim 16
Anderson teaches production of C28–C38 VLC-PUFAs by expression of ELOVL4 in a host cell. Anderson teaches that ELOVL4 host cells produce mixtures of C28–C38 VLC-PUFAs and expressly identifies yeast, including Saccharomyces, as a suitable host. Anderson ¶¶[0021], [0025]–[0027].
Anderson further teaches recovery of the resulting lipid by rupturing harvested cells and extracting the cellular lipid with, inter alia, hexane, and distinguishes the initially recovered oil from subsequent optional refining or further purification. Anderson ¶¶[0044]–[0047]. Thus, Anderson teaches the pertinent crude microbial-oil state rather than requiring use of a separately purified VLC-PUFA concentrate.
Anderson teaches host strains capable of producing total VLC-PUFA contents of at least about 5–20 dry wt.% and expressly teaches an oil recovered by extraction from the host organism containing at least about 20 dry wt.% of at least one VLC-PUFA, including oil derived from yeast. Anderson ¶¶[0063]–[0064]. Anderson further expressly contemplates extracted oils containing C30, C32, C34 and C36 PUFAs. Anderson ¶¶[0068]–[0069].
Oboh confirms operability of the relevant genus in yeast. Oboh expresses non-mammalian ELOVL4 proteins in S. cerevisiae and demonstrates production of C30-, C32-, C34- and C36-PUFAs, including C30:5, C32:5, C34:5 and C36:5 from EPA. Oboh, Table 3.
To the extent Anderson expresses VLC-PUFA concentration on a weight basis rather than the claimed area basis, Liang supplies the analytical teaching. Liang determines the percentage of each fatty acid by dividing its FAME GC peak area by total FAME peak area and multiplying by 100, and states that the resulting relative-area calculation approximates the weight-percent fatty-acid profile to approximately ±0.1 wt.%. Liang ¶¶[0119]–[0120], [0136]–[0137].
It would have been obvious to a person of ordinary skill to characterize Anderson's extracted ELOVL4 microbial oil by the conventional FAME-GC relative-area technique taught by Liang. Anderson is expressly directed to producing substantial concentrations of particular VLC-PUFAs, and Liang establishes a conventional analytical means for measuring the fatty-acid composition of recombinant yeast oil. Anderson's expressly contemplated amounts, including an extracted oil containing approximately 20 wt.% of a VLC-PUFA, are far above the claimed lower threshold of 1.0 area%, even accounting for the approximately ±0.1% analytical approximation disclosed by Liang.
Accordingly, the combined teachings would have rendered obvious a crude oil containing at least 1.0 area% of a C30-or-higher PUFA as required by claim 16.
Claim 17 — Species II-A
Claim 17 further specifies C30, C32, C34 or C36 PUFA. For Species II-A, the elected species is the C30 PUFA.
Anderson expressly identifies C30:4n3 through C30:8n3 and corresponding n-6 C30 species among the VLC-PUFAs produced by the ELOVL4 system, Anderson ¶¶[0022]–[0024], and further teaches an extracted VLC-PUFA oil containing 0.1–99.9% C30 PUFA, ¶¶[0068]–[0069].
Oboh directly demonstrates that a non-mammalian ELOVL4 expressed in yeast produces C30 PUFA. For EPA, Table 3 reports C30:5, followed by C32:5, C34:5 and C36:5 elongation products.
Thus, selection of the C30 member from Anderson's expressly identified VLC-PUFA species would have been a selection of a known species having its known function, and Oboh supplied a reasonable expectation that C30 PUFA would actually be produced in the contemplated yeast host.
Claim 18
Claim 18 requires at least one selected C30/C32/C34/C36 PUFA to be present at ≥0.5 area%.
Anderson expressly teaches an extracted oil containing 0.1–99.9% C30 PUFA, with the claimed 0.5% amount lying within Anderson's disclosed range. Anderson ¶[0068]. Anderson also teaches substantially higher VLC-PUFA production targets, including ≥20 dry wt.% of at least one VLC-PUFA in extracted oil, ¶[0064].
Liang teaches determining fatty-acid content from relative FAME GC peak areas, ¶¶[0119]–[0120], and establishes that the area-derived percentage approximates weight percentage to within about ±0.1 wt.%, ¶¶[0136]–[0137].
Thus, one of ordinary skill implementing Anderson's express C30 embodiment and measuring the resultant oil by the conventional GC/FAME technique would have been led to compositions containing at least 0.5 area% C30 PUFA. The claimed threshold is within Anderson's expressly contemplated C30 composition range and far below Anderson's preferred VLC-PUFA production levels.
Claim 19
Claim 19 requires a total content of the selected C30/C32/C34/C36 PUFAs of at least 1.0, 1.5, 2.0 or 2.5 area%.
Anderson teaches mixtures containing C30, C32, C34 and C36 PUFAs, ¶¶[0068]–[0069], and an extracted oil containing at least approximately 20 dry wt.% of at least one VLC-PUFA, ¶[0064]. Oboh independently demonstrates simultaneous generation of the C30:5, C32:5, C34:5 and C36:5 series in ELOVL4-expressing yeast.
Liang provides the conventional GC/FAME relative-area measurement, ¶¶[0119]–[0120].
Accordingly, the claimed total content of at least 1.0 area% would have been encompassed by and substantially below the VLC-PUFA production levels toward which Anderson expressly directs the skilled artisan.
Claim 20
Claim 20 requires the ratio of stearic acid content to total C30/C32/C34/C36 PUFA content to be less than 6.0, alternatively less than 5.0, 2.5 or 2.0.
Anderson teaches that production of particular VLC-PUFAs can be enhanced by selecting an appropriate host having particular fatty-acid-synthesizing genes and by selection of PUFA precursor substrate. Anderson ¶[0021]. Anderson identifies EPA as a suitable precursor for C28–C38 VLC-PUFA production, ¶[0027], and Oboh directly establishes that EPA is converted by ELOVL4 in yeast through C30:5, C32:5, C34:5 and C36:5.
Liang identifies a particularly suitable recombinant yeast lipid background. Y. lipolytica Y8672 contains 61.8% EPA while containing only 2.0% stearic acid, Liang ¶¶[0157]–[0159]. Those values are analytically tied to relative FAME peak area because Liang determines its fatty-acid profile from the individual FAME GC peak area divided by total FAME peak area, ¶¶[0119]–[0120].
It would have been obvious to select a known high-EPA, low-stearic-acid oleaginous yeast background such as that taught by Liang for implementation of Anderson's ELOVL4 process. Anderson expressly identifies precursor availability and host fatty-acid biosynthetic capacity as variables for enhancing production of a selected VLC-PUFA, and Liang's Y8672 supplies both an exceptionally abundant EPA precursor pool and a low C18:0 background. Oboh establishes that ELOVL4 uses that very EPA substrate to produce the claimed C30–C36 series. Thus, the reason for selecting Y8672 is derived from the prior art itself—maximizing availability of Anderson's identified precursor—not from the present specification.
The claimed ratio is not relied upon as an unrecognized inherent property. Rather, the prior art provides two known compositional characteristics to be combined deliberately: a yeast oil having approximately 2.0% stearic acid and an ELOVL4 system expressly directed to substantial production of C30–C36 VLC-PUFAs. Where Anderson's system is operated to provide only 1.0 area% total C30–C36 PUFA, the 2.0% C18:0 starting profile corresponds to a ratio of approximately 2; at higher C30–C36 contents, the ratio decreases. At a minimum, this provides a reasonable expectation of obtaining the broader claimed alternatives of less than 5.0 and less than 6.0 while practicing the prior-art combination.
Absolute predictability is not required; a reasonable expectation of achieving the claimed combination is sufficient. MPEP §2143.02. Accordingly, claim 20 would have been obvious, at least as to the recited alternatives <6.0 and <5.0.
Claim 21 — Species II-A
For Species II-A, claim 21 requires a ratio of stearic acid to C30 PUFA of less than 50, alternatively less than 30, 20 or 10.
Anderson expressly teaches extracted oil containing 0.1–99.9% C30 PUFA, ¶[0068]. Liang teaches a recombinant yeast lipid profile containing only 2.0% C18:0 together with 61.8% EPA, ¶¶[0157]–[0159]. Oboh establishes conversion of EPA to C30:5 and longer products by ELOVL4 in yeast.
The combination has a direct technical rationale. Anderson teaches choosing host fatty-acid biosynthetic characteristics and substrate availability to enhance a desired VLC-PUFA; Liang provides a known yeast having abundant EPA precursor and low C18:0; and Oboh demonstrates that ELOVL4 converts EPA to C30 PUFA in yeast.
Even at Anderson's expressly disclosed 0.1% C30 amount, a 2.0% stearic-acid background provides a numerical C18:0/C30 ratio of 20. At C30 contents above 0.1%, the ratio decreases. Thus, the combination reasonably directs the skilled artisan to compositions falling well within at least the claimed <50 and <30 alternatives without requiring any allegation that the ratio is inherently produced.
Claim 26
Claim 26 requires at least 0.5 area% PUFA. Claim 16 already requires at least 1.0 area% of a C30-or-higher polyunsaturated fatty acid. Thus, the composition rendered obvious for claim 16 necessarily satisfies the broader ≥0.5 area% PUFA limitation of claim 26 by express claim logic rather than an inherency allegation concerning the prior art.
Claim 27
Claim 27 requires that the PUFA include a non-produced PUFA externally added to the system.
Anderson expressly teaches that C26 substrate or other PUFA substrate of less than C26 carbons may be exogenously supplied to the ELOVL4 host. Anderson ¶[0027]. Anderson further describes fermentation in which additional substances such as PUFA precursors are supplied to permit or enhance VLC-PUFA production, ¶[0060].
Oboh supplies direct analytical evidence that the externally added material remains in the recovered yeast lipid rather than requiring an inherency inference. Oboh grows ELOVL4-transformed S. cerevisiae in the presence of one externally added PUFA substrate, including EPA, ARA, DPA and DHA. After growth, the cells are harvested, washed twice and freeze-dried before fatty-acid analysis.
Oboh calculates conversion according to:
> elongated product areas / (elongated product areas + substrate area)
and Table 3 reports, for example, only 20.4% and 6.3% conversion of externally supplied EPA at the first elongation step for Elovl4a and Elovl4b, respectively. The calculation expressly includes the chromatographic substrate area, demonstrating analytically that unconverted externally supplied PUFA remains detectable in the harvested yeast lipid sample.
It would have been obvious to supply Anderson's ELOVL4 yeast with EPA in accordance with Oboh because Anderson expressly recommends exogenous PUFA precursor supply and Oboh demonstrates the effectiveness of that exact technique for production of C30–C36 VLC-PUFAs. Oboh further establishes that residual externally supplied precursor remains in the harvested lipid.
Claim 28
Anderson expressly identifies yeast, including Saccharomyces, as a suitable ELOVL4 production host, ¶[0026], and expressly teaches extracted VLC-PUFA oil originating from yeast, ¶[0064]. Oboh actually performs ELOVL4 VLC-PUFA production in S. cerevisiae.
Claim 29
Anderson teaches a host cell containing and expressing an ELOVL4 nucleic-acid sequence for production of VLC-PUFAs and specifically includes yeast hosts. Anderson ¶¶[0025]–[0027]. Oboh expressly transforms S. cerevisiae with elovl4a and elovl4b coding sequences and demonstrates PUFA chain-elongation activity.
Claim 31
Claim 31 requires a non-mammalian ELOVL4 or a ≥90%-identity sequence encoding a protein having PUFA-chain-elongation activity.
Anderson expressly teaches ELOVL4 nucleic-acid sequences having at least 90% sequence identity and encoding ELOVL4 activity and teaches operable linkage to a functional promoter. Anderson ¶[0025].
Oboh provides the more specific first alternative of claim 31. The elovl4a and elovl4b genes are obtained from the African catfish Clarias gariepinus, i.e., a non-mammalian species, expressed in yeast, and experimentally shown to produce C30–C36 PUFAs.
It would have been obvious to employ Oboh's fish ELOVL4 in Anderson's yeast VLC-PUFA production system because Oboh establishes that the particular non-mammalian ELOVL4 possesses precisely the activity sought by Anderson and functions successfully in the contemplated yeast host.
Rejection II — Claim 30
Claim 30 is/are rejected under AIA 35 U.S.C. §103 as being unpatentable over Anderson in view of Oboh and US 2013/0046106 A1, as applied to claim 29 above, and further in view of Metz et al., U.S. Patent Publication No. US 2007/0245431 (“Metz”).
Claim 30 further requires expression of the PUFA-chain-elongase gene by an inducible promoter.
Anderson teaches that its ELOVL4 gene is operably linked to a promoter functional in the selected host. Anderson ¶[0025].
Oboh uses the pYES2 expression vector to express fish ELOVL4 in S. cerevisiae and adds galactose after the culture reaches OD600=1. Metz removes any possible ambiguity concerning the promoter associated with this yeast expression technique: Metz expressly states that genes carried by the pYES expression constructs were cloned behind the GAL1 promoter and that expression was induced by transferring the washed yeast cells into galactose-containing medium. Metz ¶[0429].
It would have been obvious to use the known GAL1 inducible promoter of Metz for expression of Anderson's ELOVL4 gene in yeast because Anderson expressly requires a promoter functional in the host, and Metz/Oboh establish GAL1/pYES as an operative inducible expression system for heterologous lipid-biosynthetic genes in S. cerevisiae. The modification constitutes use of a known expression technique for its established purpose, with Oboh providing direct evidence that a fish ELOVL4 functions in that yeast expression environment.
Rejection III — Claims 23–25
Claims 23–25 is/are rejected under AIA 35 U.S.C. §103 as being unpatentable over Anderson in view of Oboh and US 2013/0046106 A1, as applied to claims 16–17 above, and further in view of Bijl et al., U.S. Patent No. 6,727,373 (“Bijl”).
Claim 23
Claim 23 requires the combined cholesterol ester, phospholipid, glycolipid, monoglyceride, diglyceride and triglyceride content to be at least 70 wt.% of the crude oil.
Anderson teaches solvent extraction of VLC-PUFA-containing lipid from microbial biomass to recover oil before optional refining. Anderson ¶¶[0044]–[0047].
Bijl teaches the same type of microbial PUFA-oil recovery and expressly states that solvent-extracted microbial lipid is obtained in a crude form before refining. Bijl further teaches microbial oil having greater than 90% triglyceride. Id. More specifically, Example 23 reports crude oil prepared by fluidized-bed drying and hexane extraction containing 96.6, 96.5 and 96.6% triglyceride in three batches.
It would have been obvious to recover Anderson's ELOVL4-produced microbial oil using Bijl's conventional microbial-PUFA-oil extraction procedure because both references concern solvent extraction of intracellular microbial PUFA lipid from biomass. Bijl establishes the conventional triglyceride-rich composition of such crude microbial oil. Since triglyceride alone constitutes approximately 96.5–96.6% of Bijl's crude oil, the combined lipid classes recited by claim 23 necessarily exceed 70 wt.% based on Bijl's express quantitative disclosure, not an inherency assumption.
Claim 24
Claim 24 requires at least one constituent fatty acid of the recited lipid classes to be a C30/C32/C34/C36 PUFA.
Anderson expressly states that the VLC-PUFAs may occur or be provided as phospholipids and glyceride esters, including mono-, di- and triglycerides, Anderson ¶[0067]. Anderson separately teaches the C30/C32/C34/C36 VLC-PUFA species, ¶¶[0068]–[0069]. Bijl establishes that the recovered crude microbial oil is overwhelmingly triglyceride-containing.
It would therefore have been obvious for at least one of Anderson's C30–C36 VLC-PUFAs to occur as a constituent fatty-acid residue of the phospholipid or glyceride fraction in the extracted microbial oil, consistent with Anderson's express teaching of those chemical forms and Bijl's conventional triglyceride-rich crude microbial-oil matrix.
Claim 25
Claim 25 further requires the other constituent fatty acids to have 8–22 carbons.
Anderson expressly contemplates C28–C38 VLC-PUFAs in combination with ordinary PUFAs including linoleic acid, linolenic acid, ARA, EPA, DPA and DHA, which are C18–C22 fatty acids. Anderson ¶[0067]. Liang provides a concrete yeast-oil example containing C16:0, C16:1, C18:0, C18:1, C18:2 and multiple C18–C20 PUFAs in addition to EPA. Liang ¶[0159]. Bijl establishes that those microbial-oil fatty acids are predominantly present in the triglyceride-containing crude-oil fraction.
Thus, one of ordinary skill preparing the combined Anderson/Oboh microbial oil using a conventional yeast fatty-acid background would reasonably have expected the triglyceride and related cellular lipid pool to contain both the ELOVL4-derived C30+ PUFA residues and the ordinary C8–C22-range fatty acids expressly shown in microbial oils.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DEBORAH D CARR whose telephone number is (571)272-0637. The examiner can normally be reached Monday-Friday (10:30 am -6:30 pm).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Renee Claytor can be reached at 572-272-8394. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/DEBORAH D CARR/Primary Examiner, Art Unit 1691
Prosecution Insights