Ex Parte Huisman et alDownload PDFPatent Trial and Appeal BoardDec 29, 201611925482 (P.T.A.B. Dec. 29, 2016) Copy Citation United States Patent and Trademark Office UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O.Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO. MBX 033 CON (3) 3056 EXAMINER POPA, ILEANA ART UNIT PAPER NUMBER 1633 MAIL DATE DELIVERY MODE 11/925,482 10/26/2007 116248 7590 Pabst Patent Group 1545 Peachtree Street Atlanta, GA 30309-2492 12/29/2016 GJALT W. HUISMAN 12/29/2016 PAPER Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte GJALT W. HUISMAN, OLIVER P. PEOPLES, and FRANK A. SKRALY1 Appeal 2014-005598 Application 11/925,482 Technology Center 1600 Before TAWEN CHANG, RACHEL H. TOWNSEND, and DEVON ZASTROW NEWMAN, Administrative Patent Judges. CHANG, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134(a) involving claims to a genetically engineered microorganism, which have been rejected as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We AFFIRM. 1 In the Appeal Brief, Appellants identify the Real Party in Interest as Metabolix, Inc. (Appeal Br. 2.) During oral argument on December 8, 2016 (hereinafter “Tr.”), Appellants state that the assignee of record is now CJ Research Center LLC. (Tr. 2:14.) Appeal 2014-005598 Application 11/925,482 STATEMENT OF THE CASE “Poly(3-hydroxyalkanoates) (PHAs) are biological polyesters synthesized by a broad range of bacteria.” (Spec. 1:12—13.) An example of PHA is poly-3-hydroxybutyrate (PHB). (Id. at 1:16—19.) According to the Specification, “[gjenetic engineering . . . allows for the improvement of wild type PHA producing] microbes to improve the production of specific copolymers or to introduce the capability to produce different PHA polymers by adding PHA biosynthetic enzymes having different substrate- specificity or even kinetic properties to the natural system.” (Id. at 2:23—27.) The Specification suggests, however, that “one of the challenges of producing [PHAs such as] P(3HB) in recombinant organisms is the stable and constant expression of the [relevant] genes during fermentation.” (Id. at 5:17—18.) The Specification states that it is an object of the invention to “provide transgenic microbial strains for enhanced production of [PHAs].” (Id. at 7:24—25.) Claims 1—13 and 18—21 are on appeal. Claims 1, 4, and 13 are representative and reproduced below: 1. A genetically engineered microorganism having an operon comprising multiple genes encoding enzymes in a pathway for the production of a polymer, wherein the multiple genes are integrated into the chromosome, wherein the level of polymer produced by the microorganism with the integrated genes is higher than the microorganism expressing the same genes not integrated into the chromosome as an operon. 4. The microorganism of claim 1 comprising all of the genes required for synthesis of polyhydroxyalkanoate (PHA) integrated into the chromosome as an operon. 2 Appeal 2014-005598 Application 11/925,482 13. The microorganism of claim 1 wherein the genes are integrated as a single copy on the chromosome of the microorganism. (Appeal Br. 29—31 (Claims App’x).) The Examiner rejects the claims on the following grounds: 1. Claims 1—13 and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—9 and 12 ofU.S. Patent No. 7,229,804 B2 (Ans. 3); 2. Claims 1—13 and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—9 and 12 ofU.S. Patent No. 6,689,589 B2 in view of Slater2 (Id. at 4); 3. Claims 1—7, 9—13, and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1^4 of U.S. Patent No. 7,455,999 B2 in view of Slater (Id. at 5); 4. Claims 1—13 and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—13 and 16—18 ofU.S. Patent No. 6,913,911 B2 (Id. at 6); 5. Claims 1—7, 9—11, 13, and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—7 of U.S. Patent No. 8,071,355 B2 in view of Slater (Id. at 7); 6. Claims 1—13 and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—8 of U.S. Patent No. 6,316,262 B1 in view of Slater (Id. at 8); 2 Steven Slater et al., Production of Poly-(3-Hydroxybutyrate-Co-3- Hydroxyvalerate) in a Recombinant Escherichia coli Strain, 58 Applied and Environmental Microbiology 1089 (1992) (“Slater”). 3 Appeal 2014-005598 Application 11/925,482 7. Claims 1—13 and 18—21 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 1—12 and 17-19 ofU.S. Patent No. 6,593,116 B1 (Id. at 9); 8. Claims 1—6, 10—13, and 18—21 under 35 U.S.C. § 103(a) as being unpatentable over Slater and Peredelchuk3 (Id. at 11); and 9. Claims 1—13 and 18—21 under 35 U.S.C. § 103(a) as being unpatentable over Slater, Peredelchuk, Ben-Bassat,4 and Hirata5 (Id. at 14). I. Appellants do not present the obviousness-type double patenting rejections (Rejection Nos. 1—7) for review in this Appeal. (Appeal Br. 6; Reply Br. 3.) Instead, Appellants state that they “will address the rejections when the current claims are otherwise found allowable.” (Id.) Therefore, the obviousness-type double patenting rejections (Rejection Nos. 1—7) are summarily affirmed. See Manual of Patent Examining Procedure § 1205.02 (“If a ground of rejection stated by the examiner is not addressed in the appellant’s brief, appellant has waived any challenge to that ground of rejection and the Board may summarily sustain it.”). Issue II. The Examiner has rejected claims 1—6, 10-13, and 18—21 under 3 Michael Y. Peredelchuk et al., A method for construction ofE. coli strains with multiple DNA insertions in the chromosome, 187 Gene 231 (1997) (“Peredelchuk”). 4 Ben-Bassat et al., U.S. Patent No. 5,268,274, issued Dec. 7, 1993 (“Ben- Bassat”). 5 Hirata et al., U.S. Patent No. 4,861,718, issued Aug. 29, 1989 (“Hirata”). 4 Appeal 2014-005598 Application 11/925,482 35 U.S.C. § 103(a) as obvious over Slater and Peredelchuk. (Ans. 11.) The Examiner has also rejected claims 1—13 and 18—21 under 35 U.S.C. § 103(a) as obvious over Slater, Peredelchuk, Ben-Bassat, and Hirata. (Id. at 14.) With respect to the latter rejection, the Examiner finds that the teachings of Slater and Peredelchuk apply in the same way to claims 1—6, 10-13, and 18— 21 as the rejection of those claims over the two references alone, while Ben- Bassat and Hirata teach the additional limitations of claims 7—9. (Id. at 14— 15.) Appellants do not separately argue claims 7—9. Accordingly, the same issues are dispositive for both appeals and we discuss them together. The Examiner finds that Slater teaches “a method of producing PHAs by using E. coli transfected with a plasmid encoding the A. eutrophus PHB operon operably linked to a selection marker such [as] an antibiotic resistance gene.” (Ans. 12.) The Examiner finds that Slater does not teach “transposon-mediated integration of the operon into the host chromosome.” (Id.) The Examiner finds that Peredelchuk teaches that microorganisms transformed using plasmids “lose the heterologous genes due to plasmid segregational and structural instability.”6 (Id.) The Examiner finds that, “[w]hile Slater . . . use[s] antibiotics to improve the segregational instability, Peredelchuk . . . teach[es] that antibiotics could not overcome loss of heterologous genes due to structural instability” in plasmids and that “it is 6 Segregational instability results from loss of plasmids during cell division because of unequal plasmid partitioning, which is “aggravated by the fact that plasmid-free cells grew faster than recombinant cells, [causing] the take over of cell population by the former.” S.Y. Lee et al., Construction of plasmids, estimation ofplasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli, 32 J. Biotechnology 203, 209 (1994) (“Lee”). Structural instability refers to, e.g., “rearrangements and deletions within plasmids.” (Ans. 12.) 5 Appeal 2014-005598 Application 11/925,482 . . . chromosomal integration that overcomes such loss and stabilizes the heterologous genes in bacteria.” (Id.) The Examiner further finds that Slater teaches metabolically engineering the PHA pathway in E. coli to produce PHAs, while Peredelchuk teaches that, “for metabolic engineering, chromosomal integration allows the construction of industrially useful strains.” (Id. at 12—13.) The Examiner concludes that [i]t would have been obvious to one of skill in the art, at the time the invention was made, to modify the bioengineered bacterium of Slater et al. by integrating their PHA operon into the bacterial chromosome, with a reasonable expectation of success. One of skill in the art would have been motivated to do so in order to obtain microorganisms capable of high and constant PHA accumulation throughout the production process. One of skill in the art would have reasonably expected to be successful in doing such because the prior art teaches the integration of heterologous genes into the genome of microorganisms can be successfully achieved. (Id. at 13.) The Examiner further finds that, since the bacterial cell suggested by the combination of Slater and Peredelchuk “contains a chromosomally integrated operon, [it] must necessarily produce higher PHA levels than bacterial cells with separately integrated genes.” (Id.) With respect to claims 1—3, 5—6, 10-11, 18, and 20, Appellants contend that the subject matter of the claims show unexpected results and that the Examiner impermissibly combined the prior art before considering such expected results. (Appeal Br. 12—15.) Appellants contend that there is no expectation of success in integrating an operon into the chromosome or with respect to higher level of polymer production where the operon is so integrated, and that there is thus no motivation to combine the teachings of Peredelchuk and Slater. (Id. at 15—19, 24.) Appellants contend that the 6 Appeal 2014-005598 Application 11/925,482 Examiner engaged in selective analysis of the prior art based on hindsight and further argue that the prior art teaches away from the invention. (Id. at 22—24.) With respect to claims 4, 12, and 21, Appellants further argue that a skilled artisan “would not modify Slater with Peredelchuk with an expectation of success in obtaining higher levels of PHA accumulation” as recited in those claims. (Id. at 26.) Likewise, with respect to claim 13, Appellants further argue that a skilled artisan would not have modified the expression system disclosed in Slater with Peredelchuk “to provide a single gene copy integrated into the chromosome[] and expect high polymer accumulation.” (Id.) The issues with respect to these rejections are whether there is reason for a skilled artisan to combine the disclosure of Slater and Peredelchuk to arrive at the claimed invention, with a reasonable expectation of success; and, if so, whether Appellants have provided evidence of unexpected results that, when weighed with the evidence supporting obviousness, shows that the claimed microorganisms and methods would not have been obvious. Findings of Fact 1. Slater teaches the construction of an E. coli strain that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HB-co- HV)) by placing a plasmid carrying the A. eutrophus PHB operon into an E. coli mutant and culturing the strain in a particular medium. (Slater Abstract, 1090.) 2. Slater discloses that a fragment carrying the PHB operon and plasmid origin of replication is 6.5 kb in length. (Id. at 1090.) 3. Peredelchuk teaches that 7 Appeal 2014-005598 Application 11/925,482 [t]he industrial use of genetically engineered bacteria depending on the presence of autonomously replicating plasmids has some drawbacks. A major one is the difficulty in maintaining these plasmids without loss due to their segregational instability. The problem is mostly solved by introducing antibiotic resistance genes into plasmids. Another problem is rearrangements and deletions within expression plasmids. Loss of the genes to be expressed often imparts a cell growth advantage, and these cells will rapidly overgrow their parents. The pressures exerted on autonomous replicons appear to be absent or reduced when genes are carried as fragments inserted into the bacterial chromosome. Therefore, integration of DNA into bacterial genomes would be a feasible approach for the stabilization of heterologous genes in industrially important bacteria. (Peredelchuk 231.) 4. Peredelchuk discloses a method of using “a pair of systems for site specific recombination: one of them with low site specificity to achieve random integration of the gene of interest and a drug-resistant marker into the chromosome and the second one with stringent specificity for recombination sites to carry out excision of the drug marker from the chromosome.” (Id. at 233.) 5. Ben-Bassat teaches “[njucleic acid sequences encoding the bacterial cellulose synthase operon derived from Acetobacter.” (Ben-Bassat Abstract.) 6. The cellulose synthase operon is approximately 9217 base pairs (bp) in length and comprises four genes. (Id. at 3:5—7.) 7. Ben-Bassat teaches that constructions containing the cellulose synthase operon “may be inserted into a suitable, cellulose-producing microorganism and either replicated independently using an appropriate expression vector or, if plasmid instability is thought to be a problem, the 8 Appeal 2014-005598 Application 11/925,482 promoter-gene construct may be integrated directly into the chromosome of the host microorganism.” (Id. at 9:33—39.) 8. Ben-Bassat teaches that [Replacement of the chromosomal cellulose synthase operon promoter with heterologous bacterial promoters has several advantages over a plasmid system designed to overexpress cellulose synthase. Chromosomal promoter replacement avoids any potential problems which may be due to plasmid instability. It also removes the need for an antibiotic to maintain plasmid selection. Lastly, chromosomal promoter replacement removes the control of the operon from Acetobacter, allowing for a stronger, constitutive promoter or providing for control using a regulated promoter. (Id. at 9:49-59.) 9. Rong7 teaches “[a]n effective DNA replacement system ... for engineering large fragment insertions into the chromosome of Escherichia coliE (Rong Abstract.) 10. Rong teaches that “[tjransposon can introduce stable integrations but the insertion sites are randomly located. The site-specific integration systems developed so far are mostly suitable for single gene insertion at one step. Small fragment insertions are favorable, particularly for PCR-based insertion preparation due to the size limitation of the PCR products. The size of the insertions reported to date is less than 4 kb for PCR-mediated integration . ... A 4.6-kb passenger fragment has been described ... in a plasmid-mediated integration. (Rong 74, bridging paragraph.) 11. Cardenas8 teaches that, 7 Rui Rong et al., Engineering large fragment insertions into the chromosome o/Escherichia coli, 336 Gene 73—80 (2004). 8 Lucia Cardenas et al., Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors, 11 Vaccine 126 (1993) (“Cardenas”). 9 Appeal 2014-005598 Application 11/925,482 [i]n general, . . . plasmids [containing foreign antigen genes are] unstable in vivo and also in vitro in the absence of a stabilizing antibiotic .... Different strategies have been proposed to address the issue of plasmid instability and to avoid the necessity for a stabilizing antibiotic. . . . An . . . approach to obtaining] stable antigen expression is to integrate theforeign genes into the chromosome of the carrier strain. (Cardenas at 126.) 12. Cardenas compares the expression level and stability of a bacterial “strain carrying [an antigen] gene chromosomally integrated and an isogenic strain carrying the same gene on a multicopy plasmid.” {Id. at Abstract.) 13. Cardenas teaches that the initial levels of expression of [antigen] by the strain carrying the [antigen] gene on the chromosome . . . and the plasmid-bearing strain grown in the absence of antibiotic were similar. . . . The amount of [antigen] produced by the cointegrate strain . . . was approximately 20-fold less than that produced by the strain carrying the multicopy plasmid when the antibiotic was supplied in the growth medium. {Id. at 133.) 14. However, Cardenas also teaches that, due to plasmid loss, “expression by the plasmid-containing strain was reduced to negligible levels following the first subculture when the stabilizing antibiotic was omitted from the growth medium.” {Id.) Cardenas further teaches that, “[e]ven in the presence of the stabilizing antibiotic, the plasmid-bearing strain exhibited significant plasmid loss” such that only 20% of the isolates retained the recombinant plasmid after 24 hours. {Id.) “By contrast, 100% of the recovered cointegrate isolates retained the ability to express the antigen throughout the 21 days of the experiment.” {Id. at Abstract.) 10 Appeal 2014-005598 Application 11/925,482 15. Cardenas further teaches that “the ability to express [antigen] was stably maintained in vivo by all colonies tested from the cointegrate strain . . . throughout the 21 days of the study,” whereas “expression of [antigen] from colonies recovered from animals inoculated with [plasmid bearing strains with or without ampicillin] was markedly reduced.” {Id.) In particular, Cardenas teaches that, from its experiments, it was evident that [the] strain [containing chromosomally integrated antigen gene] could persist in tissues and maintain the ability to express [the antigen] from the chromosomal integration site, while [the plasmid-bearing] strain . . . grown in the absence of antibiotic rapidly lost the plasmid in vivo and, consequently, the ability to express [the antigen]. When [the plasmid-bearing strain] was grown in the presence of the stabilizing antibiotic the plasmid was retained in vivo for a longer period of time. However, by day 21 none of the recovered colonies retained the plasmid or the ability to express [the antigen], {Id. at 131-132.) 16. Li9 teaches that, [f]or metabolic engineering, many rate-limiting steps may exist in the pathways of accumulating the target metabolites. Increasing copy number of the desired genes in these pathways is a general method to solve the problem, for example, the employment of the multi-copy plasmid-based expression system. However, this method may bring genetic instability, structural instability and metabolic burden to the host, while integrating of the desired gene into the chromosome may cause inadequate transcription or expression. (Li Abstract.) 17. In particular, Li teaches that 9 Mingji Li et al., A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli, 11:19 Microbial Cell Factories (2012) (“Li”). 11 Appeal 2014-005598 Application 11/925,482 [integration of the desired genes into the chromosome of the host seems able to circumvent. . . problems [of employing plasmids in metabolic pathway engineering, such as genetic instability, structural instability and metabolic burden] .... However, these approaches cannot obtain sufficient gene expression due to the inadequate strength of the promoter or the scant copy number of the target gene which was integrated into the chromosome. (Id. at 1.) 18. Li teaches “a strategy for obtaining gene overexpression by engineering promoter clusters [consisting] of multiple core-toopromoters (MCPtacw) in tandem.” (Id.) 19. Lee teaches E. coli transformed with high and medium copy plasmids (pSYLlOl andpSYL102) containing A. eutrophus PHB biosynthetic genes. (Lee Abstract.) 20. Lee teaches that both high and medium copy plasmids were unstable during subculturing without antibiotic pressure and that higher instability was observed when cells were accumulating PHB. (Id.) 21. Lee teaches derivative plasmids developed by cloning the parB locus of plasmid R1 into pSYL 101 and pSYL102, respectively. (Id.) Lee teaches that these derivative plasmids were stable during PHB synthesis and accumulation of 110 generations. (Id.) 22. Lee teaches that the final PHB concentration was lower using the medium copy plasmid, “which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.” (Id.) 23. Lee teaches that a PHB operon that is 5.2 kb in size. (Lee 205, Fig. 1.) 12 Appeal 2014-005598 Application 11/925,482 24. Madison10 describes a study similar to that described in Lee and states that, “[b]y using stabilized plasmids derived from either medium- or high-copy-number plasmids, it was shown that only high-copy-number vectors support substantial P(3HB) accumulation in E. coli [strain] XL1- Blue.” (Madison 42.) 25. Madison teaches that [o]ne of the challenges of producing P(3HB) in recombinant organisms is the stable and constant expression of the phb genes during fermentation. P(3HB) production by recombinant organisms is often hampered by the loss of plasmid from the majority of the bacterial population. Such stability problems may be attributed to the metabolic load exerted by the need to replicate the plasmid and synthesize P(3HB), which diverts acetyl-CoA to P(3HB) rather than to biomass. In addition, plasmid copy numbers often decrease upon continued fermentation because only a few copies provide the required antibiotic resistance or prevent cell death by maintaining parB. {Id.) Analysis Claim 1 Citing to Plantronics, Leo Pharamceutical, and Rambus, Appellants first contend that the Examiner “impermissibly combine[d] the prior art before considering [the] unexpected property of high product accumulation which the claimed microorganisms possess.” (Appeal Br. 13 (emphasis omitted).) Although we agree that “consideration of the objective indicia is part of the whole obviousness analysis, not just an afterthought” and that “all 10 Lara L. Madison et al., Metabolic Engineering of Poly (3- Hydroxyalkanoates): From DNA to Plastic, 63 Microbiology and Molecular Biology Reviews 21 (1999) (“Madison”). 13 Appeal 2014-005598 Application 11/925,482 evidence pertaining to the objective indicia of nonobviousness must be considered before reaching an obviousness conclusion,” we are not persuaded by Appellants’ argument. See, e.g., Leo Pharm. Prods, Ltd. v. Rea, 726 F.3d 1346, 1357 (Fed. Cir. 2013); Plantronics, Inc. v. Aliph, Inc., 724 F.3d 1343, 1355 (Fed. Cir. 2013). As the Federal Circuit explained in Leo Pharmaceutical, “[w]hen an applicant appeals an examiner’s objection to the patentability of an application’s claims for obviousness, the PTO necessarily has the burden to establish a prima facie case of obviousness which the applicant then rebuts.” Id. Here, the Examiner properly combined the references as part of meeting the PTO’s burden of establishing a prima facie case.11 We next turn to the arguments in the Appeal Brief regarding the sufficiency of the prima facie case. During oral argument, Appellants conceded that the Examiner has established a prima facie case. (Tr. 7:20- 22.) Nevertheless, we address these arguments for the sake of completeness. Appellants first argue that the Examiner has not established that a skilled artisan would be motivated to “replace the stabilized plasmids in Slater with Peredelchuk’s method of chromosomal integration,” because (1) the loss of genes due to rearrangements and deletions within plasmids does not appear to have occurred and/or is of “no apparent consequence” in Slater’s 11 Of course, once the prima facie case has been established, all evidence, including any evidence of unexpected results, must be reweighed to determine whether the claim is obvious. In re Reinhart, 531 F.2d 1048, 1052 (Fed. Cir. 1976) (“When prima facie obviousness is established and evidence is submitted in rebuttal, the decision-maker must start over. . . . An earlier decision should not... be considered as set in concrete, and applicant’s rebuttal evidence then be evaluated only on its knockdown ability.”). 14 Appeal 2014-005598 Application 11/925,482 microorganisms; (2) “[g]ene expression by chromosomal integration is not devoid of drawbacks”; and (3) there are other methods of further stabilizing plasmids. {Id. at 21—22; see also Reply Br. 6, 8—11.) We are not convinced. “Non-obviousness cannot be established by attacking references individually where the rejection is based upon the teachings of a combination of references . . . .” In re Merck & Co., 800 F.2d 1091, 1097 (Fed. Cir. 1986). Here, even if Slater does not explicitly mention the loss of genes due to rearrangements and deletions within plasmids, the disclosure in Peredelchuk of such instability provides a reason for a skilled artisan to integrate the PHB operon disclosed in Slater into the bacterial chromosome. Neither are we persuaded by Appellants’ arguments that integration of genes into chromosomes has other drawbacks that would discourage the skilled artisan from pursuing this method and that the ordinary artisan would more likely pursue the available methods of stabilizing plasmids other than integrating genes into chromosomes. (App. Br. 21—22; Reply Br. 6, 8—11.) “[A] given course of action often has simultaneous advantages and disadvantages, and this does not necessarily obviate motivation to combine.”12 Medichem, S.A. v. Rolabo, S.L., 437 F.3d 12 Appellants cite In re Omeprazole Patent Litigation, 536 F.3d 1361 (Fed. Cir. 2008), for the proposition that claims are non-obvious “where one of ordinary skill in the art would have made a different modification had they recognized the problem.” (Reply Br. 8.) As an initial matter, Omeprazole involved a district court litigation and thus a different burden of proof for showing obviousness. Omeprazole Patent Litigation, 536 F.3d at 1380. Furthermore, in Omeprazole the only evidence of a recognized problem in the prior art came from an expert declaration, and there appeared to be little to no evidence suggesting the particular claimed water-soluble subcoating as a solution. Id. at 1380-138. In contrast, in the instant appeal, Peredelchuk discloses the problem of plasmid instability in the industrial use of 15 Appeal 2014-005598 Application 11/925,482 1157, 1165 (Fed. Cir. 2006) (citing Winner Int 7 Royalty Corp. v. Wang, 202 F.3d 1340, 1349 n. 8 (“The fact that the motivating benefit comes at the expense of another benefit. . . should not nullify its use as a basis to modify the disclosure of one reference with the teachings of another. [T]he benefits, both lost and gained, should be weighed against one another.”)). Here, we find that the benefits of integrating genes into the bacterial chromosome, as disclosed in prior art references such as Peredelchuk, would have provided a skilled artisan with a reason to integrate the PHB operon disclosed in Slater into the bacterial chromosome, despite the alleged drawbacks cited by Appellants. (FF3; see also FF7, FF8.) For the same reason, we are likewise unconvinced by Appellants’ apparent argument that the claims are non- obvious because, despite the availability of the technology to integrate heterologous genes into chromosomes, skilled artisans continued to “use plasmid expression systems and engineered ways to stabilize the plasmids.” (Appeal Br. at 24.) The existence of one possible solution to a problem in the field does not necessarily render all alternative solutions non-obvious. Appellants further argue that the Examiner has not shown that a skilled artisan would have had a reasonable expectation of success of arriving at the claimed invention in light of the prior art. (Id. at 15.) Appellants first argue that the Examiner has not established that “one would expect success in integrating an operon into chromosome based on the disclosure in Peredelchuk.” (Id.) In particular, Appellants rely on Rong, which Appellants argue “establishes that a method disclosed as suitable for integrating a single gene into the chromosome does not provide an genetically engineered bacteria and specifically suggested integration of the gene into the chromosome as a solution. (FF3.) 16 Appeal 2014-005598 Application 11/925,482 expectation of success in using the same method to integrate large DNA fragments into the chromosome such as those involved in operon.” (Id. at 16; see also id. at 18 (“[T]he Examiner has cited no art that supports extrapolation of integration of one gene encoding one enzyme to integration of genes expressing enzymes involved in a pathway for [a] product.”).) We are not convinced. As the Examiner points out, Rong is post filing art (Ans. 24), and Appellants have not provided persuasive evidence that the alleged lack of expectation of success with respect to integrating multiple genes into the chromosome was known at the relevant time, i.e., the time of the invention. Bristol-Myers Squibb Co. v. Teva Pharm. USA, Inc., 752 F.3d 967, 974 (finding selection of a compound as lead compound to be proper for purpose of obviousness analysis despite later discovery that the compound is toxic). In addition, Appellants have not shown that Rong’s disclosure, which appears to suggest that site-specific integration systems are “mostly suitable for single gene insertion at one stop” and that “[s]mall fragment insertions are favorable,” is applicable to the method of chromosomal integration disclosed in Peredelchuk, which appears to integrate the gene of interest using a system with low site specificity. (Ans. 24; FF4.) Fikewise, to the extent that Rong’s disclosure of potential size limitations in chromosomal insertions is applicable to all methods of chromosomal integration, Appellants have not shown that the PHB operon is of a size that would render the expectation of success of integration into the chromosome unreasonable.13 13 The Examiner points out, for instance, that Ben-Bassat teaches integrating an operon with multiple genes (i.e., the cellulose synthase operon) into bacterial chromosome. (Ans. 16; FF7.) Ben-Bassat discloses that the cellulose synthase operon is approximately 9.2 kb in length. (FF6.) In 17 Appeal 2014-005598 Application 11/925,482 In light of the relevant evidence, therefore, we also do not agree that the Examiner improperly placed the burden of proving lack of reasonable expectation of success on Appellants. (Appeal Br. 16—17, 18—19.) Appellants have not established with relevant evidence that one of ordinary skill in the art would have understood Peredelchuk’s method of integrating genes into chromosomes is limited by the size of the gene. See also supra n. 13. Accordingly, we find that the Examiner has established a prima facie case that a skilled artisan would reasonably expect success in integrating a PHA operon into bacterial chromosome. Appellants further argue that, even if a skilled artisan had a reasonable expectation of success with respect to integrating the PHA operon into the chromosome, the Examiner has not established that such a person “would also expect success obtaining higher PHA accumulation,” because “[s]uccess in integrating a gene into the chromosomes does not translate invariably into success in high gene expression, much less high polymer production which involves not just production of enzymes, but utilization of substrates in a multi-step process.” (Appeal Br. at 15—16, 17, 23—24; Reply Br. 11—14.) Appellants argue that the Examiner failed to explain WHY one would have had any expectation of obtaining microorganisms producing higher PHB following chromosomal integration of the PHA biosynthetic genes using the method of Peredelchuk (which provides at most 3 gene copies) as compared to microorganisms where the operon is expressed from stabilized plasmids, as disclosed in Slater (using a method which provide a gene copy number of 500). (Appeal Br. 15.) contrast, Slater discloses a PHB operon less than 6.5 kb in length (FF2), while Lee teaches a PHB operon that is 5.2 kb in size (FF23). 18 Appeal 2014-005598 Application 11/925,482 We are not convinced. As an initial matter and as the Examiner points out (Ans. 13), the microorganism suggested by the combination of Slater and Peredelchuk is substantially identical to the claimed microorganism, and ‘“mere recitation of a newly discovered function or property, inherently possessed by things in the prior art, does not cause a claim drawn to those things to distinguish over the prior art.’” In re Best, 562 F.2d 1252, 1254 (CCPA 1977) (quoting In re Swinehart, 439 F.2d 210, 212—13 (CCPA 1971)). Neither are we persuaded by Appellants’ reliance on Cardenas, Fi, and Fee or Appellants’ argument that the Examiner has not shown why a skilled artisan would expect higher PHB production following chromosomal integration of PHA genes using Peredelchuk’s method as compared to Slater’s microorganisms where such genes are expressed from high copy plasmids. (Appeal Br. 17—18; Reply Br. 11—14.) Cardenas, Fi, and Fee at best suggest that, under some conditions, the amount of polymer produced by a bacterial strain carrying the relevant genes integrated into its chromosome may be less than the amount produced by a strain carrying such genes on a multicopy plasmid. (FF13, FF17, FF22.) However, claim 1 neither requires a particular level of polymer production, nor requires a level of polymer production higher than the level produced by a multicopy plasmid. It merely requires a higher level of polymer production than if the genes encoding enzymes for polymer production were not integrated into the chromosome as an operon, e.g., if the genes were introduced via a low copy plasmid. We agree with the Examiner that, given the teachings in the prior art that “chromosomal integration provides the advantage of stabilizing the transgenes in the bacterial cells and their progeny,” a skilled artisan would 19 Appeal 2014-005598 Application 11/925,482 reasonably expect that integrating, e.g., the PHA operon, into the bacterial chromosome “would enhance polymer accumulation over the bacteria comprising the operon on a plasmid that is not integrated into the chromosome.” (Ans. 16.) “[Expectation of success need only be reasonable, not absolute.” Pfizer, Inc. v. Apotex, Inc., 480 F.3d 1348, 1364 (Fed. Cir. 2007). Finally, in light of the above, we find that the Examiner’s prima facie case is properly grounded in the disclosures of the cited prior art. Thus, we note, but are not convinced by, Appellants’ argument that the Examiner engaged in improper hindsight analysis. (Appeal Br. 20.) In sum, we find that the Examiner has established a prima facie case of obviousness. We next turn to Appellants’ arguments with respect to unexpected results and teaching away. As discussed below, we do not find Appellants’ proffered evidence sufficient to render the claim non-obvious when weighed with the evidence of obviousness. Appellants first argue that the claimed subject matter exhibits unexpected results: Examples 8, 9, and 21 [] of the [Specification show a dramatic differences in the levels of accumulation of PHA when genes encoding PHA biosynthetic enzymes are integrated into the chromosome as an operon (82% by weight) as compared to a maximum of 58% PHB of weight, when the genes are not incorporated [as] an [] operon, i.e., the accumulation of PHA differs dramatically, depending on whether the same genes, though integrated into the chromosome, are integrated as an operon or not. (Appeal Br. 12.) We are not persuaded. As an initial matter, as the Examiner points out, Slater already teaches the PHB operon. (Ans. 17; FF1.) Thus, this comparison does not compare Appellants’ invention to the 20 Appeal 2014-005598 Application 11/925,482 closest prior art. In re Baxter Travenol Labs., 952 F.2d 388, 392 (Fed. Cir. 1991) (“[W]hen unexpected results are used as evidence of nonobviousness, the results must be shown to be unexpected compared with the closest prior art.”). Moreover, while Appellants cite to data comparing a chromosomally integrated operon with genes that are separately integrated into the chromosome, Appellants’ allegation of unexpectedness does not relate to the difference between integration of the genes as an operon or not. Rather, Appellants contend that the results are unexpected because chromosomal integration provides low gene copy number when compared to high copy number plasmids '. Such high levels of polymer accumulation [when PHA genes are integrated into the chromosome as an operon] are unexpected and could not have been predicted from the cited art for at least the reason that chromosomal integration provides very low gene copy number, when compared to the gene copy number provided by high copy number plasmid gene expression. {Id. at 12—13.) Appellants do not suggest that the microorganisms in Examples 8 and 9 had a high gene copy number or that separately integrating the genes into the chromosome allows for incorporation of greater number of gene copies. Thus, Appellants have not explained why the level of polymer accumulation in Example 21 (where the genes are integrated as an operon) is unexpected when compared to that of Examples 8 and 9 (where the genes are separately integrated into the chromosome, i.e., not as an operon). Likewise, while Appellants claim that it would be unexpected for bacteria with a chromosomally integrated operon to accumulate higher levels 21 Appeal 2014-005598 Application 11/925,482 of polymer than bacteria containing a high number of gene copies, e.g., a bacteria containing a high copy number plasmid comprising the polymer producing genes, Appellants have provided no evidence in their Appeal or Reply Briefs that the claimed microorganism in fact accumulates polymer at a higher level than a microorganism containing polymer producing genes in high copy number plasmids.14 (Ans. 17—18.) Furthermore, Appellants do not persuasively explain why, regardless of gene copy number, bacteria containing a chromosomally integrated operon would not be expected to produce a higher amount of polymer over time during fermentation, in light of the disclosure in the prior art that chromosomally integrated genes are more stable than genes provided in the form of plasmids. (Ans. 15—16.) Finally, as the Examiner points out, Appellants’ unexpected results argument is not commensurate with the scope of the claim, because the claim is not limited to microorganisms with chromosomally integrated operons that produce more polymer than microorganisms containing high number of gene copies. (Ans. 17—18.) 14 During oral argument, Appellants argued that a comparison of the Specification and Slater shows that the microorganism of the claimed invention unexpectedly produced a comparable amount of PHB as Slater’s microorganisms containing high copy number plasmids. (Tr. 4:13—16, 8:1— 9, 12:1—13:19.) However, even assuming that this is not impermissible new argument, “[attorneys’ argument is no substitute for evidence.” Johnston v. IVAC Corp., 885 F.2d 1574, 1581 (Fed. Cir. 1989). This argument also does not address the disclosure in Slater, for instance, that “[t]he operon responsible for PHB production in A. eutrophus has been cloned and expressed in Escherichia coli. . . and in one laboratory has directed the accumulation of PHB to intracellular concentrations as high as 95% (polymer weight per cell dry weight. . .),” i.e., higher than the 82% disclosed in Example 21. (Slater 1089.) 22 Appeal 2014-005598 Application 11/925,482 We are likewise unconvinced by Appellants’ argument that prior art such as Lee and Madison teach away from the claimed invention. (Appeal Br. at 22—24; Reply Br. 4—8.) These references teach that plasmids with high copy numbers and PHB genes accumulated higher levels of PHB than plasmids with low copy numbers. (FF19—FF22, FF24.) Such teaching does not “criticize, discredit, or otherwise discourage” the alternative solution of incorporating PHB genes into bacterial chromosomes to avoid the problem of plasmid instability. In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004) (“The prior art’s mere disclosure of more than one alternative does not constitute a teaching away from . . . alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed.”). Even assuming that Lee and Madison’s teachings may be considered a teaching away, “[w]here the prior art contains ‘apparently conflicting’ teachings (i.e., where some references teach the combination and others teach away from it) each reference must be considered ‘for its power to suggest solutions to an artisan of ordinary skill. . . . considering] the degree to which one reference might accurately discredit another.’” Medichem S.A. v. Rolabo S.L., 437 F.3d 1157, 1165 (Fed. Cir. 2006) (quoting In re Young, 927 F2d 588, 591 (Fed. Cir. 1991)). Given the clear and consistent teaching in the prior art that incorporating PHB genes into chromosome is a solution to the problem of plasmid instability in, e.g., the industrial use of genetically engineered bacteria, we find that Lee and Madison do not render the claimed invention non-obvious because there are other references (e.g., Peredelchuk) that encourage the skilled artisan to pursue chromosomal integration. (See, e.g., FF3, FF7, FF8.) 23 Appeal 2014-005598 Application 11/925,482 Finally, we note but are not convinced by Appellants’ argument in the Reply Brief that similar claims have previously been found patentable over Slater and Peredelchuk by the Patent Office. Appellants did not raise this issue in the Appeal Brief; in any event, decisions of an examiner during ex parte prosecution in applications not before us on review are not binding on the dispute before us. Claim 4 Appellants argue claims 4, 12, and 21 together. (Appeal Br. 25—26.) Thus, we choose claim 4 as representative for purposes of our analysis. Claim 4 depends from claim 1 and further requires the microorganism “compris[e] all of the genes required for synthesis of polyhydroxyalkanoate (PHA) integrated into the chromosome as an operon.” (Id. at 29 (Claims App’x).) Appellants argue that claim 4 is not obvious over the combination of Slater and Peredelchuk for at least the reasons discussed with respect to claim 1 and further argue that “where Lee expressly discloses that a high gene dosage is required to obtain high levels of PHB in E. coli, one of ordinary skill in the art would not modify Slater with Peredelchuk with an expectation of success in obtaining higher levels of PHA accumulation as recited in claim 4.” (Id. at 26.) As to the arguments Appellants made with respect to claim 1, we are not convinced for the reasons already discussed. As to Appellants’ argument specific to claim 4, we are unpersuaded for similar reasons: Lee’s suggestion that a high copy plasmid may be needed for high level of PHB accumulation does not obviate the reason a skilled artisan has for integrating the genes as an operon in the chromosome of the microorganism (e.g., to solve the problem of plasmid instability), and a skilled artisan would have a reasonable expectation that such a 24 Appeal 2014-005598 Application 11/925,482 microorganism would produce a higher level of polymer than “[a] microorganism expressing the same genes not integrated into the chromosome as an operon,” e.g., a microorganism expressing the same genes on an unstable plasmid. (FF3.) Claim 13 Claim 13 depends from claim 1 and further requires that “the genes are integrated as a single copy on the chromosome of the microorganism.” (Appeal Br. 30 (Claims App’x).) Appellants argue that claim 13 is not obvious over the combination of Slater and Peredelchuk for at least the reason discussed with respect to claim 1 and further argue that “in view of Madison, Lee and Cardenas . . ., one would not modify the expression system disclosed in Slater which provides a gene dosage of 500, with Peredelchuk to provide a single gene copy integrated into the chromosome, and expect high polymer accumulation.” {Id. at 26.) We are not convinced for the reasons already discussed. Claim 13, like claim 1, does not require “high” polymer accumulation or higher polymer accumulation than Slater’s microorganisms. It merely requires “higher [level of polymer production] than microorganism expressing the same genes not integrated into the chromosome as an operon.” {Id. at 29 (Claims App’x).) For the reasons enumerated above, we affirm the Examiner’s rejection of claim 1, 4, and 13 over Slater and Peredelchuk. Claims 2, 3, 5, 6, 10—12, and 18—21, which were not separately argued, fall with claims 1, 4, and 13. 37 C.F.R. § 41.37(c)(l)(iv). Likewise, we affirm the Examiner’s rejection of claims 1, 4, and 13 over Slater, Peredelchuk, Ben-Bassat, and Hirata. Claims 2, 3, 5—12, and 18—21, which were not separately argued, fall with claims 1, 4, and 13. Id. 25 Appeal 2014-005598 Application 11/925,482 SUMMARY For the reasons above, we affirm the Examiner’s decision rejecting claims 1—13 and 18—21. TIME PERIOD FOR RESPONSE No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). AFFIRMED 26 Copy with citationCopy as parenthetical citation