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 6, 8-9, 13-14, 16, 21-25 and 35 are pending following the Reply filed on 03/24/2026. Claims 2, 4, 7, 10-12, 15, 17-18 and 20 have been cancelled. Claims 6, 8, 13-14, 16, 21-25 and 35 have been amended without adding new matter. Claims 6, 8-9, 13-14, 16, 21-25 and 35 are presently considered.
Withdrawn
Any rejection of claims 2, 4, 7, 10-12, 15, 17-18 and 20 is moot because the claims have been cancelled.
Maintained Rejections and New Rejections Necessitated by Amendment
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) 6, 8-9, 13-14, 16, 21, 23-25 and 35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Curtis (U.S. Patent No. 9,826,743 B2; cited in the IDS filed 07/25/2023), and further in view of Chen (previously cited), Khedher (cited in the IDS filed 05/20/2022) and Posada-Uribe (previously cited).
Regarding claim 21, from which the other claims depend, Curtis teaches a method for enhancing plant growth and promoting plant health by use of a composition comprising recombinant exosporium-producing Bacillus cells that express a fusion protein comprising at least one plant growth stimulating protein or peptide and a targeting sequence that localizes the fusion protein to the exosporium of the Bacillus cells (see Abstract). Curtis teaches that the fusion protein may alternatively comprise an exosporium protein or exosporium protein fragment (see col. 6, lines 27-32). Curtis teaches that the compositions of the invention contain fermentation products produced in a culture broth (see col. 43, lines 53-55 & 63-66) comprising the microorganism and any bacterial cells, spores and metabolites resulting from the fermentation (see col. 43, lines 46-48 & 55-58). Curtis teaches that the fusion protein may further comprise a biological control agent that has activity against insects and phytopathogens that cause damage to plants (see col. 1, lines 31-43). Curtis teaches that the composition may further comprise a Bacillus subtilis or Bacillus amyloliquefaciens strain as an additional biological control agent (see col. 3, lines 18-24) and col. 4, lines 30-31). In Curtis’ Examples, whole broth cultures of a recombinant Bacillus cereus family member (Bacillus thuringiensis BT013A) expressing phospholipase C on its exosporium were obtained by inoculating brain heart infusion media (BHI) with the Bacillus expressing phospholipase C to be grown and later aliquoted to a yeast extract-based media until sporulation of the Bacillus was at least 95% complete (see col. 69, lines 50-53; col. 70, lines 3-11).
Curtis teaches that Bacillus cereus family bacteria undergo sporulation and form oval endospores, wherein the outermost layer of the endospores is known as the exosporium (see col. 12, lines 43-48). Curtis teaches that the recombinant exosporium-producing Bacillus cells can be exposed to conditions which will induce sporulation, and suitable conditions for inducing sporulation are known in the art (see col. 27, lines 43-47).
Hence, Curtis teaches a method of producing a fermentation product from recombinant exosporium-producing Bacillus cells that express a fusion protein, comprising culturing the recombinant exosporium-producing Bacillus cells that express a fusion protein in a medium comprising yeast extract, wherein the recombinant exosporium-producing Bacillus cells are cells of a Bacillus cereus family member, wherein the fusion protein comprises a protein or peptide of interest and a targeting sequence, and wherein culturing occurs until sporulation of the Bacillus cells is at least 95% complete.
Curtis does not explicitly teach the culture medium comprising limitations a-f, wherein the CFU counts and fusion protein expression per spore are higher than those from a medium that contains yeast extract as the principal source of carbon and nitrogen.
Chen’s disclosure relates to the optimization of nutritional conditions for the production of Bacillus subtilis spores (see Abstract). Chen teaches that B. subtilis spores have the potential to be developed as biocontrol agents for suppressing various plant disease and have superseded many chemical germicides, known for their harmful effects on humans and the environment, but the current high costs of spore production have hindered its widespread use in agriculture, especially in developing countries (see pg. 1353, col. 2, lines 24-33). Chen teaches that corn steep liquor, soybean flour, and yeast extract were selected as candidate nitrogen sources, while maintaining D-glucose monohydrate (glucose) as the constant variable for carbon nutrition (see pg. 1356, col. 2, para. 4) in the amount of 11.01 g/L (see pg. 1355, Table 2). Chen teaches that the final optimized medium contained 8.14 g/L of yeast extract and 17.53 g/L of soybean flour as key components in achieving high spore yields (see Abstract, lines 12-14). Chen also teaches that metal elements, including calcium chloride (CaCl2), were included due to their important functions in Bacillus growth and sporulation (see pg. 1355, col. 2, para. 2).
Hence, Chen teaches a method for increasing the production of spores in Bacillus strains by culturing the Bacillus in a culture medium comprising (a) yeast extract, (b) glucose, (c) soy flour, and (e) CaCl2, the medium specifically comprising (a) 8.14 g/L yeast extract and (c) 17.53 g/L of soybean flour.
Chen does not explicitly teach the culture medium comprising (b) glucose at a concentration of about 20 g/L to about 35 g/L, (d) K-2HPO4 at a concentration of about 1 g/L to about 5 g/L and KH-2PO4 at a concentration of about 0.5 g/L to about 2 g/L, (e) CaCl2*2H2O at a concentration of about 0.015 g/L to about 0.80 g/L, and MgSO4*7H2O at a concentration of about 0.10 g/L to about 0.80 g/L.
Khedher, in the same field of endeavor, teaches that the increasing trend of limiting the use of chemical agents in pest management has generated considerable interest in the use of natural alternatives for urban, agriculture and forestry control, and that different strains of Bacillus thuringiensis are commonly employed in bioinsecticides (see pg. 705, col. 1, lines 1-6). Khedher teaches that improved culture conditions have been found to increase the production of both spores and delta-endotoxin (see pg. 711, col. 1, lines 26-29) and teaches an optimized medium shown to be suitable for overproducing delta-endotoxins using both sporeless and sporulating strains (see Abstract). Khedher’s optimized medium also used 1 g/L of KH2PO4, 1 g/l of K2HPO4 and 0.3 g/l of MgSO4 (see pg. 706, col. 1, para. 4). Khedher also teaches that optimal culture conditions, such as pH, should be maintained (pg. 712, col. 1, para. 2). In view of the instant specification, KH2PO4 and K2HPO4 are a buffer (see instant specification at pg. 2, para. [0009]). Khedher teaches that the availability of carbon sources can influence the yield of viable cells, spores and toxins in the B. thuringiensis production process (see pg. 709, col. 1, lines 7-9) and teaches that the optimum culture medium consisted of 22.5 g/L of glucose.
Hence, Khedher teaches that the availability of carbon sources can influence the yield of viable cells and spores when culturing B. thuringiensis (a Bacillus cereus family member), and discloses an optimum culture medium comprising (b) 22.5 g/L glucose. Khedher also discloses that the optimized medium comprised (d) 1 g/L of KH2PO4 and 1 g/l of K2HPO4 which would be expected to act as a buffer to maintain pH during culturing, as well as (f) 0.3 g/L of MgSO4.
Chen and Khedher do not explicitly teach the culture medium comprising (e) CaCl2*2H2O at a concentration of about 0.015 g/L to about 0.80 g/L. Khedher also does not explicitly disclose the MgSO4 to be in its heptahydrate form, MgSO-4*7H2O.
Posada-Uribe, in the same field of endeavor, teaches the effects of medium components and culture conditions in Bacillus subtilis EA-CB0575 spore production with the aim of achieving high spore cell densities and sporulation efficiencies in fermentation, and reports that two key medium components, glucose and MgSO4·7H2O, were optimized to achieve a high spore density and sporulation efficiency (see Abstract). Posada-Uribe teaches that aerobic endospore-forming bacteria, especially some species of Bacillus, have received growing attention because of their industrial potential in the development of biopesticides and biofertilizers and that spore production is a key step in bio-product development (see pg. 1879, col. 2, para. 2). Posada-Uribe teaches that each particular strain has its own requirements and optimum conditions, and elements such as Ca, Mn, Mg, Fe and Zn in appropriate concentration are essential in sporulation since they are present in the spore layers and enable resistance to high temperatures (see pg. 1880, col. 1, para. 2). Posada-Uribe used a full factorial design to find the optimization area for MgSO4·7H2O, which was found to have a significant effect on spore cell density and sporulation efficiency (see pg. 1884, col. 1, para. 2), resulting in the highest spore cell density and sporulation efficiency at a concentration of 0.5 g/L MgSO4·7H2O (See Table 2 on pg. 1884). Posada-Uribe also teaches that the optimized medium contained 9.9 mL/L of CaCl2 0.1 M (see pg. 1880, col. 2, para. 1) which is equivalent to 0.1 g/L of CaCl2.
It is apparent that the only difference between CaCl2 (anhydrous calcium chloride) and CaCl2*2H2O (calcium chloride dihydrate) is the water content, and these nutritional ingredients are otherwise equivalent, having the function of providing calcium chloride (CaCl2) to the medium. Both of these forms are discussed in Applicant’s embodiments (see specification at pg. 9, para. [0050]) as being sources of calcium chloride, and there is no statement to suggest that one serves a different purpose than the other. Furthermore, a person of skill would have recognized that the anhydrous form (CaCl2) is slightly more concentrated (because it does not contain 2H2O) and any adjustment in concentration of the dihydrate form (CaCl2*2H2O) in the culture broth would reasonably fall within the claimed range. For example, even if the amount of CaCl2*2H2O were quadrupled (i.e., to 0.4 g/L) to make up for the difference, it would still fall well within the claimed range. However, a person of skill would have also recognized that the difference in concentration between the two calcium chloride sources is smaller than this example, and would not require quadrupling the amount. Therefore, any reasonable adjustment when using CaCl2*2H2O as an equivalent to 0.1 g/L CaCl2 would have necessarily fallen within the claimed range, and it is considered to be within the ordinary skill in the art to have added CaCl2 in either form, while accounting for their minor differences, to achieve the same effect.
Hence, Posada-Uribe teaches an optimized culture medium for increasing spore cell density and sporulation efficiency in spore-forming Bacillus strains comprising 0.5 g/L MgSO4*7H2O and CaCl2 in an amount equivalent to CaCl2*2H2O within the claimed range.
In view of the instant specification, “media prototypes” were developed with titer yields ranging from 1 x 109 spores/mL to 3.5 x 109 spores/mL compared to a “base medium”, which yielded about 1 x 108 spores/mL (see pg. 22, para. [00113]). The inventors disclose that the base medium was derived from a laboratory-scale medium and used a low concentration of yeast extract as its principal source of carbon and nitrogen (see pg. 23, para. [00113]). Conversely, the media prototypes used in Applicant’s experiments comprised 3-25 g/L yeast extract, 25-30 g/L glucose, 0-30 g/L soy flour, 0.2-0.725 g/L CaCl2*2H2O, 1-3 g/L K--2HPO--4, 0.8-2.6 g/L KH2PO4, and 0.2-1.1 g/L MgSO4*7H2O (see pg. 25, Table 2). Hence, the amounts of yeast extract (3-25 g/L) and soy flour (0-30 g/L) varied considerably in the media found to have improved results over the base medium. Furthermore, there is no evidence in the disclosure to show, for example, that high concentrations of glucose (20-25 g/L) led to these results, because there are no results disclosed using lower concentrations, other than those reported using the “base medium” (i.e., 0 g/L glucose).
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 re Williams, 36 F.2d 436, 438, 4 USPQ 237 (CCPA 1929) ("It is a settled principle of law that a mere carrying forward of an original patented conception involving only change of form, proportions, or degree, or the substitution of equivalents doing the same thing as the original invention, by substantially the same means, is not such an invention as will sustain a patent, even though the changes of the kind may produce better results than prior inventions.").
In this case, all of the claimed culture media ingredients were known in the prior art and are taught by said prior art to have been optimized for achieving higher sporulation efficiency at concentrations that fall within the ranges of the claim. As these components and their concentrations have been taught to achieve the same desired effect in Bacillus species, a skilled artisan would have reasonably concluded that a more robust culture media, comprising more than just yeast extract at a low concentration, would have led to increased spore-forming productivity in a Bacillus strain compared to a base medium only containing a low concentration of yeast extract (and nothing more). Furthermore, one would have recognized the concentrations taught by Chen, Khedher and Posada-Uribe to have been result-effective, and it would have required no more than routine experimentation to have combined these elements. Therefore, it was also well within the ordinary skill in the art to have combined these elements using known methods to have arrived at a culture medium that was within the scope of the claims.
Furthermore, all Applicant has shown is that various concentrations of glucose, yeast extract, soy flour, etc. may improve spore production compared to a base medium comprising only a low concentration of yeast extract. However, it is not apparent in view of Applicant’s disclosure which specific combination of these factors and their respective concentrations were critical to the results achieved by the inventors. Moreover, these results do not appear to rise to the level of being “unexpected”, because, in view of the prior art, an ordinary artisan would have expected a culture medium within these parameters to be more sufficient for increasing sporulation compared to a base medium containing only a low concentration of yeast extract (and nothing more).
Regarding the limitation, “wherein the CFU counts and fusion protein expression per spore are higher than those from a medium that contains yeast extract as the principal source of carbon and nitrogen”, this is a functional limitation which is presumed to be an inherent effect of the claimed method. See Persion Pharms. LLC v. Alvogen Malta Operations LTD., 945 F.3d 1184, 1191, 2019 USPQ2d 494084 (Fed. Cir. 2019), where the court stated that a proper finding of inherency does not require that all limitations are taught in a single reference, and that inherency may meet a missing claim limitation when the limitation is "the natural result of the combination of prior art elements." (emphasis in original). The court found that pharmacokinetic limitations of the asserted claims were inherently met by combining prior art references because the limitations were necessarily present in the prior art combination. Id.
In the instant case, the higher CFU counts and fusion protein expression are inherent effects of performing the claimed method, that do not affect the structure or steps of said method, and would necessarily be present in the prior art combination. It should also be noted that an ordinary artisan would have recognized the additional ingredients in the “media prototypes” of Applicant’s Examples to be known in the art as providing nutrients necessary for the growth and metabolic activities of the bacterium. Therefore, this inherent result does not appear to have been “unexpected”, because an ordinary artisan would have expected a culture medium within these parameters to be more sufficient for the growth of the bacterium and the production of fermentation products (e.g., fusion proteins) compared to a base medium containing only a small concentration of yeast extract (and nothing more).
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 Curtis, Chen, Khedher and Posada-Uribe because each reference teaches the formulation of fermentation products used to protect and enhance the growth of plants by the culturing of Bacillus. A person of skill would have recognized that Curtis teaches a method for producing fermentation products that enhance plant health and/or protects plants from pests which relies on the sporulation activity of the bacterium, which may be further enhanced by an optimized culture medium as taught by Chen, Khedher and Posada-Uribe. As each reference teaches the use of a spore-forming Bacillus species, one would have recognized that these teachings could be combined and that the results of the combination would have led to reasonably predictable results. Furthermore, both Chen and Posada-Uribe teach the importance of including sources of Mg2+ and Ca2+ in the culture medium, and Posada-Uribe further teaches that including sources of these elements in appropriate amounts is essential to sporulation. One would have also recognized that each reference teaches common nutritional factors affecting the growth and sporulation of Bacillus (i.e., nitrogen, carbon, inorganic salts), the common sources of these factors (i.e., yeast extract, glucose, soy flour, MgSO4, CaCl2), the effective culture conditions (i.e., pH), the means to control these conditions (i.e., KH2PO4/K2HPO4 buffer), and, collectively, the amounts of these ingredients as they have been applied successfully in the prior art. 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 6, Chen teaches the use of cotton seed flour as a source of nitrogen in the culture medium at a concentration of 4.31 g/L (see pg. 1355, Table 2).
Regarding claim 8, Chen teaches that during batch fermentation, the liquid medium pH was maintained at 6.8-7.2 (see pg. 1354, col. 2, “Bioreactor fermentation”).
Regarding claim 9, Chen teaches that during batch fermentation, the liquid medium pH was maintained at 6.8-7.2 by adding HCl (an acid) or NH3OH (a base) (see pg. 1354, col. 2, “Bioreactor fermentation”).
Regarding claim 13, Chen teaches that during batch fermentation, the cultivation temperature was maintained at 30°C (see pg. 1354, col. 2, “Bioreactor fermentation”).
Regarding claim 14, Chen teaches that spore production by batch fermentation was performed by incubating the liquid medium for 48 hours (see pg. 1354, col 2, “Shake flask cultivation”).
Regarding claim 16, Chen teaches the improved medium produced spores as high as 1.52 ± 0.06 x 1010 spores/mL under flask cultivation conditions (see Abstract).
Furthermore, the further limitation of the claim is directed to a result of using the claimed method which is an inherent property that does not affect the structure or steps of the claimed invention. See Persion Pharms. LLC v. Alvogen Malta Operations LTD., 945 F.3d 1184, 1191, 2019 USPQ2d 494084 (Fed. Cir. 2019), where the court stated that a proper finding of inherency does not require that all limitations are taught in a single reference, and that inherency may meet a missing claim limitation when the limitation is "the natural result of the combination of prior art elements." (emphasis in original). The court found that pharmacokinetic limitations of the asserted claims were inherently met by combining prior art references because the limitations were necessarily present in the prior art combination. Id. See also MPEP 2112.
In the instant case, the claim does not recite any further method steps, and the functional limitation of the claim is necessarily present in the method of claim 21. Therefore, claim 16 is directed to an inherent property and is obvious for the same reasons as claim 21.
Regarding claim 23, Curtis teaches the composition comprising at least one plant growth stimulating protein or peptide and a protein or peptide that protects a plant from a pathogen or a pest (see, e.g., col. 2, lines 43-49).
Regarding claim 24, Curtis teaches the targeting sequence comprising amino acids 20-35 of SEQ ID NO: 1 (see col. 3, lines 30-31). See SEQ ID NO: 1 of Curtis below:
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217
531
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Hence, Curtis teaches the targeting sequence A-F-D-P-N-L-V-G-P-T-L-P-P-I-P-P, which meets the claim.
Regarding claim 25, Curtis teaches the targeting sequence comprises at least about 43% identity with amino acids 20-35 of SEQ ID NO: 1 (see col. 3, lines 25-27). As shown in the following alignment, instant SEQ ID NO: 2 (top) is identical to SEQ ID NO: 1 of Curtis (bottom), which includes amino acids 20-35:
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134
642
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Regarding claim 35, Curtis teaches a fermentation broth for producing a fermentation product, as discussed regarding claim 21.
Claim(s) 22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Curtis, Chen, Khedher and Posada-Uribe, as applied to claims 6, 8-9, 13-14, 16, 21, 23-25 and 35 above, and further in view of Sasaki (previously cited).
Regarding claim 22, as previously discussed, Chen teaches 8.14 g/L yeast extract and 17.53 g/L soy flour; Khedher teaches 22.5 g/L glucose, 1 g/L KH2PO4 and 1 g/l K2HPO4; and Posada-Uribe teaches 0.5 g/L MgSO4·7H2O and 0.1 g/L of CaCl2.
Regarding the limitation of “b) glucose at a concentration of about 25 g/L to about 30 g/L”, Khedher teaches that it was evident from statistical analysis that “the optimal glucose concentration for delta-endotoxin production was situated in the range of 20-25 g/L”; however, the growth of B. thuringiensis was affected at concentrations above 30 g/L, harming sporulation and toxin synthesis (see pg. 709 col. 1, line 13 to col. 2, line 1). Hence, in view of Khedher, it would have been obvious for one of ordinary skill to have selected a glucose concentration at the endpoint of the claimed range at 25 g/L glucose, while also being apprised of the negative effects when exceeding concentrations of 30 g/L.
Furthermore, in view of the instant specification, there is no apparent support showing the criticality of the claimed range. There is zero discussion in the present disclosure as to what considerations are taken into account when selecting a concentration of, for example, about 25 g/L of glucose as opposed to about 30 g/L of glucose. See instant specification at Table 2 on page 25 and the “Examples” from pages 26 to 28. In these examples, all media contained either 25 or 30 g/L of glucose. However, no results are specifically disclosed regarding three of the media containing the lower amount of 25 g/L glucose (M1, M3, and M4). The one medium containing 25 g/L of glucose with disclosed results (M0) lacked the claimed ingredient of soy flour, whereas all other media contained 15-30 g/L of soy flour. Hence, it is not apparent what difference in the medium composition attributed to the results. Furthermore, there is no data regarding media containing less than 25 g/L of glucose to support that lower concentrations would be less effective. Hence, no evidence of unexpected results within the claimed range has been disclosed on record. In accordance with MPEP 2144.05(I), an overlapping endpoint of the prior art range and the claimed range is sufficient to support a prima facie case of obviousness, particularly when there is no showing of criticality of the claimed range.
Chen, Khedher and Posada-Uribe do not explicitly teach a) yeast extract at a concentration of about 10 g/L to about 15 g/L.
Sasaki, in the same field of endeavor, teaches that bioinsecticides produced from Bacillus thuringiensis containing crystal toxins active against various species of mosquitoes have attracted considerable attention as possible replacements for chemical insecticides, and strains of the B. thuringiensis subspecies, Bacillus sphaericus and israelensis, have potential as biocontrol agents (see pg. 165, col. 1, para. 1). Note that Bacillus thuringiensis is also the same “Bacillus cereus family member” taught by Curtis. Sasaki teaches that production of toxins is closely related to spore formation, and spore formation may be significantly affected by the nutritional composition of the medium (see pg. 165, col. 1, para. 2). Sasaki teaches that yeast extract is frequently utilized for cultures of the Bacillus thuringiensis strains as it is an excellent source of amino acids and vitamins for both growth and sporulation (see pg. 165, col. 1, para. 2). In Sasaki’s experiments, Sasaki’s production media used 10 g per liter of yeast extract (see pg. 165, col. 2, para. 4), and relatively good growth and sporulation was observed (see pg. 166, col. 2, para. 2; Fig. 1(a)). Hence, Sasaki teaches the medium comprising 10 g/L of yeast extract which is at the lower endpoint of the claimed range.
In view of the instant specification, media prototypes were developed with titer yields ranging from 1 x 109 spores/mL to 3.5 x 109 spores/mL compared to a “base medium”, which yielded about 1 x 108 spores/mL (see pg. 22, para. [00113]). The inventors disclose that the base medium was derived from a laboratory-scale medium and used a “low concentration” of yeast extract as its principal source of carbon and nitrogen (see pg. 23, para. [00113]). Conversely, the optimized media used in Applicant’s experiments comprised 3-25 g/L yeast extract (see pg. 25, Table 2). However, none of the optimized media contained yeast extract in the range of 11 g/L to 24 g/L. 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). In the instant case, Applicant has shown that media comprising various components, including various concentrations of yeast extract (i.e., 3, 5, 10 or 25 g/L), may improve spore production compared to a base medium comprising only a “low concentration” of yeast extract (and nothing more). However, there is no apparent showing that the claimed range of “about 10 to about 15 g/L” of yeast extract was critical to these results, and the prior art of Sasaki teaches the culturing of a Bacillus cereus family member using yeast extract at a concentration of 10 g/L to have been effective for growth and sporulation.
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 above teachings with that of Sasaki, because each reference teaches the formulation of fermentation products used to protect and enhance the growth of plants by the culturing of Bacillus. A person of skill would have recognized that Curtis teaches a method for producing fermentation products that enhance plant health and/or protects plants from pests which relies on the sporulation activity of the bacterium, which may be further enhanced by an optimized culture medium as taught by Chen, Khedher, Posada-Uribe and Sasaki. As each reference teaches the use of a spore-forming Bacillus species, one would have recognized that these teachings could be combined and that the results of the combination would have led to reasonably predictable results. One of ordinary skill in the art could have cultured the Bacillus strain expressing a fusion protein as taught by Curtis by combining known elements (e.g., glucose, yeast extract, soy flour, etc.) as taught by the other references using known methods, with each element merely performing the same function as it does separately. 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).
Response to Arguments
Regarding the rejections under 35 U.S.C. 103, Applicant argues that the Examiner combines disclosure from four different references to arrive at the invention recited in the claims. “The Examiner asserts that there is motivation to combine because all references relate to strains of spore-forming Bacillus species and some of the references relate to formation of fermentation products from B. thuringiensis, but the Examiner does not provide specific motivation to combine certain elements of the references in order to achieve the enhanced spore count and fusion protein expression that is the crux of the presently claimed invention.”
Applicant’s allegation that the examiner fails to provide “specific motivation” appears to be related to the fact that several of the references used in the rejection are related to methods of culturing other spore-forming members of Bacillus, as opposed to being specifically related “Bacillus cereus family members” (e.g., B. thuringiensis).
Applicant’s arguments have been fully considered but they are not persuasive.
In response to applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
In response to applicant’s argument that there is no teaching, suggestion, or motivation (“specific motivation”) to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). "It's enough … to show that there was a known problem … in the art, that [another reference] … helped address that issue, and that combining the teachings of [the two references] wasn't beyond the skill of an ordinary artisan. Nothing more is required to show a motivation to combine under KSR." See Intel Corp. v. PACT XPP Schweiz AG, 61 F.4th 1373, 1380-81, 2023 USPQ2d 297 (Fed. Cir. 2023) (finding that both prior art references "address the same problem and that [the secondary reference’s] cache was a known way to address that problem is precisely the reason that there's a motivation to combine under KSR and our precedent.").
In this case, the Curtis reference clearly requires the culturing of a Bacillus cereus family member (i.e., Bacillus thuringiensis) for the production of fermentation products associated with the exosporium of the bacterium’s endospore. Curtis makes clear, as discussed in the rejection, that this production process requires sporulation in order to produce the endospores comprising the exosporium which comprises the desired product. Each of the additional references used in the rejections relate to prior art efforts to increase sporulation in species of Bacillus, including Bacillus thuringiensis (in the case of Khedher and Sasaki). While the prior art teachings of Chen and Posada-Uribe disclose experiments using other members of Bacillus (i.e., Bacillus subtilis) it is understood that this is a well-studied species of spore-forming Bacillus that is closely related to the species taught by the other references. Therefore, the “specific motivation” for a person of ordinary skill was to provide an optimized culture media for increasing sporulation, which has been reported in closely-related species, which would be presumed to have similar nutritional requirements for sporulation. The examiner has found no evidence that would have discouraged a skilled artisan from considering these teachings just because they are not directed to same “specific” species taught by Curtis, Khedher and Sasaki.
Applicant further argues that the Curtis reference describes recombinant exosporium-producing Bacillus cells that express a fusion protein and are grown in fermentation media that is yeast-extract based but does not disclose any of the other media components in the present claims nor any ranges of components at all. Applicant also points out that the Chen reference focuses on a fermentation media specifically for increasing spore yields in Bacillus subtilis, a Bacillus species that does not have an exosporium let alone a recombinant fusion protein expressed on such exosporium. Therefore, the effect of modifying Curtis with the teachings of Chen is not predictable, especially as to the production of the fusion protein.
Applicant’s arguments have been fully considered but they are not persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, Curtis discloses a Bacillus species that has an exosporium and a recombinant fusion protein expressed on the exosporium, and the other references disclose media components and concentrations within the claimed ranges.
Regarding the argument that the spores produced by Bacillus subtilis do not have an exosporium, both Bacillus subtilis and Bacillus thuringiensis produce endospores by a process that the prior art references teach can be improved by using optimized media. Hence, a person of ordinary skill would have expected that increasing sporulation in a species that has this feature (i.e., an exosporium) would render a desirable result when applying the teachings of Curtis to produce such fusion proteins, because they require the production of said spores. Furthermore, Applicant is reminded that obviousness does not require absolute predictability, nor conclusive proof of efficacy to show a reasonable expectation of success. See OSI Pharm., LLC v. Apotex Inc., 939 F.3d 1375, 1385, 2019 USPQ2d 379681 (Fed. Cir. 2019). In the instant case, an expectation of success would have been reasonable, because a person of skill would have recognized that each reference teaches culture media comprising components that are known to provide the same common nutrients for growth and sporulation (e.g., carbon, nitrogen, amino acids, salts, trace metals, etc.), and each reference teaches a culture medium that was shown to be successful in achieving spore-production in spore-producing members of Bacillus.
Applicant also argues that the Chen reference mentions that previous studies for enhanced spore yields have been conducted in Bacillus subtilis but that the high rates achieved in other species, such as Bacillus thuringiensis, have not been achieved in Bacillus subtilis. See Chen, page 1354, column 1, first paragraph. Thus, the Chen reference focuses on a fermentation media specifically for increasing spore yields in Bacillus subtilis. The Examiner does not explain why this combination would be predictable, especially when Chen itself notes that there is a separate body of literature dedicated to Bacillus thuringiensis.
Applicant’s arguments have been fully considered but they are not persuasive.
“The use of patents as references is not limited to what the patentees describe as their own inventions or to the problems with which they are concerned. They are part of the literature of the art, relevant for all they contain." In re Heck, 699 F.2d 1331, 1332-33, 216 USPQ 1038, 1039 (Fed. Cir. 1983) (quoting In re Lemelson, 397 F.2d 1006, 1009, 158 USPQ 275, 277 (CCPA 1968)). A reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments. Merck & Co. v. Biocraft Labs., Inc. 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir. 1989), cert. denied, 493 U.S. 975 (1989). Disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments. In re Susi, 440 F.2d 442, 169 USPQ 423 (CCPA 1971). See MPEP 2123.
In the instant case, the fact that Chen acknowledges that increased sporulation has been previously achieved in Bacillus thuringiensis, and that such efforts in Bacillus subtilis have been more difficult, does not constitute a “teaching away” from considering the teachings of the disclosure as they may be applied to other Bacillus species, including Bacillus thuringiensis. Furthermore, it should be noted that the “body of literature” mentioned by Chen on page 1354, column 1, first paragraph, refers to only two articles, from 1992 and 1998, one of which was relied upon by the examiner in the rejections (Sasaki, 1998). Therefore, Chen’s mention of B. thuringiensis is not a summons for one to consider a completely separate and extensive body of literature over Chen’s disclosure, but is part of a broader summary of relevant prior art related to achieving enhanced spore production in species of Bacillus.
Regarding the “predictability of the combination”, both Bacillus subtilis and Bacillus thuringiensis are endospore-forming members of Bacillus and the prior art combination teaches optimized media for increasing sporulation in both species. An ordinary artisan would have had a reasonable expectation that an optimized culture medium comprising nutrients commonly known in the art for culturing spore-forming Bacillus species, in a formulation taught to increase sporulation in said Bacillus, would have had predictable results. Furthermore, the present rejections further rely on the teachings of Khedher and Posada-Uribe, and the predictability of this combination is discussed further in the rejection of claim 21.
Applicant argues that Khedher teaches improved culture conditions that increase the production of spores and delta-endotoxin in Bacillus thuringiensis. In contrast, the currently claimed method is focused on increasing the levels of a fusion protein expressed on the exosporium of a Bacillus strain and not on production of delta-endotoxins, as is described by Khedher.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). In the instant case, Curtis teaches a method that is focused on producing a fusion protein expressed on the exosporium of a Bacillus strain. As discussed in the present rejection, Khedher teaches that the availability of carbon sources can influence the yield of viable cells and spores when culturing B. thuringiensis.
In response to applicant's argument that Khedher is not focused on increasing the levels of a fusion protein, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In the instant case, it is clear that the methods taught by Khedher use a high concentration of glucose which was demonstrated to be effective for growth and sporulation in B. thuringiensis. Furthermore, Khedher teaches that the sporulation process is widely related to delta-endotoxin synthesis (see pg. 712, col. 2, para. 1). Hence, while Khedher considers blocking sporulation to be a means for increasing delta-toxin production, this does not prevent an ordinary artisan from recognizing the teachings of Khedher’s broad disclosure, particularly as it relates to increased sporulation in spore-producing strains. The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Applicant further argues that Khedher's optimization is focused on culturing of sporeless B. thuringiensis strains. While the media in Khedher is also applied to a sporulating B. thuringiensis, the optimized medium does not significantly enhance CFU
counts at least for the sporogenic strain (although some increases are seen in oligosporogenic strains). See Table 8 of Khedher. In light of the lack of a specific reason to combine Khedher with the other references and the fact that Khedher itself does not provide improved spore counts for a sporogenic B. thuringiensis strain, Applicant submits that applying the teachings of Khedher to Chen and Curtis would not predictably achieve the higher CFU counts or the higher fusion protein levels recited in the presently amended claims.
Applicant’s arguments have been fully considered but they are not persuasive.
First, Applicant is reminded that a reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, and the preferred focus of said reference does not constitute a teaching away from a broader disclosure or nonpreferred embodiments. As discussed in the present rejection, Khedher teaches that improved culture conditions have been found to increase the production of both spores and delta-endotoxin and the availability of carbon sources can influence the yield of viable cells, spores and toxins in the B. thuringiensis production process. Khedher’s experiments demonstrate that B. thuringiensis was tolerant to high levels (20-25 g/L) of glucose, and Khedher reasonably suggests that this ingredient also influences spore count as a carbon source.
Second, in response to applicant's argument that Khedher does not teach “higher CFU counts or the higher fusion protein levels”, the fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). Furthermore, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In this case, Curtis is relied upon in the rejection for the production of a fusion protein, and the combination of Chen, Khedher and Posada-Uribe are relied upon for an optimized culture medium for increasing sporulation in Bacillus.
Finally, as discussed in the present rejection, the higher CFU counts and fusion protein expression are inherent effects of performing the claimed method, that do not affect the structure or steps of said method, and would necessarily be present in the prior art combination.
Applicant argues that while Posada-Uribe discloses a study of the effect of culture conditions on spore production in Bacillus subtilis and the use of MgSO4*7H2O to improve spore production, the Examiner provides no specific motivation to combine this teaching with the teachings of the other references, asserting only that all of the references relate to enhancing sporulation of Bacillus, in general, so the results of their combination would be predictable. Posada-Uribe, however, discloses, "The media with high quantities of glucose(> 11 g/L) did not produce spores ... " See page 1883 of Posada-Uribe. The claims as amended require glucose at a concentration of about 20 g/L to about 35 g/L and culturing until sporulation of the Bacillus cells is at least 90% complete.
Applicant’s arguments have been fully considered but they are not persuasive.
Applicant is reminded that a reference may be relied upon for all that it would have reasonably suggested to one having ordinary skill in the art, including nonpreferred embodiments, and disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments.
In this case, Table 1 of Posada-Uribe, which contains the data that Posada-Uribe is referring to on page 1883, shows that media with low quantities of glucose (2 g/L) and media containing mid-range levels of glucose (11 g/L) also did not produce spores (see pg. 1881, Table 1). In fact, there are a few instances where it appears there was little to no cell growth at all using 2 g/L (see M2 and M12 in Table 1). Therefore, it would be apparent to an ordinary artisan that media containing any level of glucose may not result in sporulation, or even growth, and these results have to be attributable to additional factors. It should also be noted that only four (out of 15) of the media in Posada-Uribe’s initial experiment (Table 1) actually produced spores and there were numerous other variables included in this experiment (i.e., MgSO4, MnCl2, KH2PO4, yeast extract, meat extract, peptone, ammonium sulfate). The remainder of Posada-Uribe’s experiments do not test the higher levels of glucose and instead focus on varying MgSO-4*7H2O concentrations while maintaining a low level of glucose. Furthermore, Posada-Uribe states that “[d]ifferent optimum culture media and culture conditions for AEFB [Aerobic Endospore-Forming Bacteria] sporulation have been reported, where each particular strain has its own requirements and optimum conditions… For example, the presence of glutamate in the medium benefits the growth of B. cereus while for other species of Bacillus this compound inhibits growth” (see pg. 1880, col. 1, para. 2; Emphasis added). In view of Khedher, one would have recognized that B. thuringiensis may be more tolerant to high concentrations of glucose compared to B. subtilis.
Furthermore, the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). In the instant case, an ordinary artisan would have recognized from Khedher, who also teaches culture media comprising MgSO4, that B. thuringiensis (a B. cereus family member) may be more tolerant to higher concentrations of glucose (e.g., 20-25 g/L), which Khedher suggests may increase the yield of viable cells and spores, as discussed in the present rejections of claims 21 and 22.
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