DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 12 and 14-24 and the following species: a combination of glucose and glycerol as the carbon source, and a combination of corn steep liquor and bean cake powder as nitrogen source for claims 12-14; potassium dihydrogen phosphate, magnesium sulfate and zinc sulfate for claim 15; a combination of glucose, glycerol, bean cake powder, and corn steep liquor as the carbon and the nitrogen source, and a combination of sulfate, phosphate, and chloride as the inorganic salt for claims 18 and 24 in the reply filed on 11/19/2025 is acknowledged.
Status of the Claims
Claims 12, 14, 15, 18 and 24 are amended. Claim 25 is cancelled.
Claims 12 and 14-24 are pending (claim set filed 11/19/2025) and are examined on the merits herein.
Priority
This application is a 371 of PCT/CN2021/141025 filed 12/24/2024. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d) to CN 202110168751.4 filed 02/07/2021, CN 202110337621.9 filed 03/30/2021 and CN 202110678427.7 filed 06/18/2021. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 08/04/2023 complies with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
The Specification recites: “Polyether GPE defoamer” (p. 5 and throughout the Specification), however does not define or provide description of GPE.
The use of the term “GPE”, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 16, 18-24 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 16 and 24 recite: “polyether GPE defoamer”. Claims 16 and 24 contain the trademark/trade name GPE. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe defoamer and, accordingly, the identification/description is indefinite.
Claim 18 recites in line 11: “the fermentation tank culture” and in line 12: “the fermentation tank”. There is insufficient antecedent basis for this limitations in the claim since claim 12, from which claim 18 depends, does not recite “a fermentation tank culture” and “a fermentation tank”. The scope and boundaries of claim 18 are not certain making claim 18 indefinite.
Claims 19-21 and 24, dependent on claim 18, do not resolve the issue mentioned above and are rejected.
Claim 22 recites in lines 6-8: “adjusting the seed obtained in step a to pH 6.0-7.5, sterilizing, incubating the bacterial solution for 12-36 hours at a tank temperature of 20-30 °C to obtain a seed bacterial solution”. Claim 22 has the following issues:
There is insufficient antecedent basis for the limitation “the seed” in the claim. It is not
clear what “seed” claim refers to since “a shake flask seed” and “a seed shake flask” were recited prior to the recitation “the seed” in line 6 and “a seed” was not recited.
Assuming that “the seed” refers to “the shake flask seed”, is not clear how the seed
culture can be first sterilized and not destroyed and then cultured to obtain bacterial solution. Step (b) has the same issue.
It seems that the step of inoculating the shake flask seed bacterial solution into the seed
tank is missing since the previous step describes culturing in the flask and not tank and Specification describes on p. 12: “The shake flask seed bacterial solution prepared in the previous step was inoculated into the seed tank.”
The scope and boundaries of claim 22 are not certain making claim 22 indefinite.
Claim 23, dependent on claim 22, do not resolve the issue mentioned above and is rejected.
Claim 22 is interpreted as directed to three types of culture: shake flask seed culture, seed tank culture and fermentation culture, wherein the shake flask seed bacterial solution is inoculated into a tank to obtain a seed bacterial solution and the seed bacterial solution is inoculated into a fermenter for fermentation culture and sterilization of culture medium is performed prior to inoculation.
Claim 23 recites: “seed bacterial solution … is obtained by 1-2 levels of seed cultures”. It is not clear if levels of seed culture include repeat of step (a) or repeat step (b) of claim 22 or step (a) is considered level 1 and step (b) is considered level 2. The scope and boundaries of claim 23 are not certain making claim 23 indefinite.
For examination claim 23 is interpreted as directed to 1-2 levels of seed cultures wherein shake flask seed culture of step (a) of claim 22 is considered level 1 seed culture and seed tank culture is considered level 2 seed culture.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 18-21 and 24 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for the composition of the fermentation medium, the time and pH of the fermentation, does not reasonably provide enablement for the feeding volume and temperature of the fermentation. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
Claim 18 is directed to the fermentation method for mupirocin wherein a medium comprises the recited ingredients and their concentrations, the pH of the fermentation culture is 6.0-7.5, the time of fermentation is 48-144 h, the feeding volume is 0-20000 L and the temperature of the fermentation tank is 10-37°C.
Prior art of Tucaliuc (Tucaliuc et al. Biotechnol. Lett., 2018, 41, 495-502) teaches fermentation for production of mupirocin at 24-26°C and pH = 5.3-7 and feeding of dextrose without specifying the feeding volume (p. 499, left column, last paragraph). Zhang (CN 108949845A on record in IDS) teaches fermentation for mupirocin at 25-30°C for 60-120 h and at pH 6.0-7.0 (paragraphs 0023, 0062). Ge (CN 111996136A on record in IDS) teaches fermentation for mupirocin at 25°C for 120 h and at pH 6.5 (paragraph 0045, 0047).Thus, the prior art does not provide information about the feeding volume and fermentation at temperature lower than 24°C and higher than 30°C.
The Specification describes conditions for the fermentation culture including temperature of the fermenter of 20-30°C and pH 6.8-7.5 (p. 9, last paragraph). The Specification provides Examples 5 and 6 investigating different ingredients and their concentrations in the fermentation medium and in the feeding medium. The protocols were based on Examples 1-4 which mentioned the following parameters: pH 6.0-6.5 and temperature of the fermenter of 20-30°C (p. 12, lines 2 and 4). The incubation in the fermenter is described as performed for 6 days (p. 17, line 14) that supports 48-144 h limitation. However, the feeding volume was not specified. Example 7 describes the scale-up fermentation process in a 4-ton fermenter. The pH during fermentation was controlled at 5.6-6.5, the temperature was 20-30°C and the feeding volume 800 L (p. 20, lines 26, 27 and 31). Thus, while the Specification supports the pH 6.0-7.5 and the time of fermentation of 48-144 h, the limitations for 0-20000 L feeding volume and temperature 10-37°C are not supported by the Specification because the highest supported feeding volume is 800L and the temperature of the fermentation lower than 20°C and higher than 30°C was not described or used in the working examples and cannot be extrapolated.
Based on the unpredictability taught by the prior art and absence of working examples and directions provided by inventors, one of ordinary skill in the art would have to undergo undue experimentation to practice the full scope of the invention. Therefore, claim 18 is rejected under 35 U.S.C. 112(a) for failing to disclose sufficient supporting information for the 0-20000 L feeding volume and the fermentation temperature of 10-37°C to enable a person of skill in the art to produce mupirocin by fermentation method.
Claims 19-21 and 24 do not resolve the issue mentioned above and are rejected.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 12, 14-19 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Tucaliuc (Tucaliuc et al. Biotechnol. Lett., 2018, 41, 495-502) in view of Poontawee (Poontawee and Limtong Microorganisms 2020, 151, 1-19), Wang (CN 110747188A) and Xiong (CN 109929890A).
Regarding claim 12, Tucaliuc teaches that mupirocin is an antibiotic which can be obtained as a mixture of pseudomonic acids by fermentation of Pseudomonas fluorescens (Abstract). Tucaliuc describes different fermentation medium for production of mupirocin having glucose as the main carbon source for up-to 6% and glycerol at 1% (p. 500, Table 3). Tucaliuc discloses nitrogen source comprising corn steep liquor (0.5%) and soy bean meal (2%) (p. 500, Table 3). The inorganic salts include sulfates, phosphates and chlorides and in particular, KH2PO4 at 0.15%, (NH4)2SO4 at 0.5%, MgSO4 x7H2O at 0.05% and KCl at 0.1% (p. 500, Table 3). Production is performed for 50 hours, at 24-26°C and pH = 5.3-7 (p. 499, left column, last paragraph). Tucaliuc mentions feeding 50% glucose throughout the process (p. 499, left column, last paragraph) and describes feeding the dextrose solution to keep glucose at 0.5% level (p. 499, right column, 1st paragraph), however does not provide the description of the feeding medium.
Tucaliuc does not teach the complete composition of the medium for feed culture.
Poontawee teaches feeding strategies for microbial fermentation (Abstract). Poontawee discloses that during fed-batch fermentation the essential nutrients for cell growth or product formation are intermittently or continuously added to the culture vessel during cultivation. That allows to keep the concentration of limiting nutrients at an optimal level that shortens fermentation time, achieves high cell concentration and increases productivity (p. 2, 3rd paragraph).
Wang teaches method for producing keratinase by bacterial fermentation (Abstract). Wang describes a fermentation medium: “Corn starch 2.5%, corn flour 3.5%, feather flour 1.5%, soy flour 3.5%, corn pulp 3%, sucrose 3.2%, potassium dihydrogen phosphate 1.7%, magnesium sulfate 0.27%, pH 6.5” (p. 2, lines 38-39) and feed medium: “Corn starch 25%, soybean cake flour 5.2%, corn pulp 2.3%, feather flour 1.5%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.2%, pH 6.5” (p. 3, lines 6-7). Fermentation and feed medium have similar components of carbon and nitrogen source and inorganic salts, however, the amount of carbon source, corn starch, is increased from 2.5% to 25% in feed medium. Wang mentions that the method provided higher fermentation activity, higher extraction yield and lower manufacturing cost (p. 3, lines 31-32).
Xiong teaches microbial fermentation and production of mycophenolic acid (Abstract). Xiong describes the composition of the fermentation medium which contains GPE defoamer at 0.4-0.6 g/L (paragraph 0020) which corresponds to 0.04-0.06%. Xiong mentions that the fermentation medium contains the sunflower seed cake powder which not only provides nutrients, but also plays antifoaming role and allows to reduce the amount of GPE (paragraph 0032). Xiong mentions that foam produced during microbial fermentation poses a risk of bacterial contamination (paragraph 0007).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Tucaliuc and Poontawee and apply fed-batch fermentation strategy described by Poontawee to fermentation of Pseudomonas fluorescence for production of mupirocin as taught by Tucaliuc. One would have been motivated to make this combination since Poontawee teaches that feeding essential nutrients shortens fermentation time, achieves high cell concentration and increases productivity. A skilled artisan would have reasonably expected success in this combination because Tucaliuc and Poontawee teach microbial fermentation.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Wang teaching and prepare feeding medium based on fermentation medium with increase in the main carbon source, glucose, and use it for fermentation based on Tucaliuc and Poontawee teachings. One would have been motivated to do so since Tucaliuc teaches feeding 50% glucose, the main carbon source, throughout the fermentation process and the dextrose solution to keep glucose at 0.5% level, Poontawee teaches feeding essential nutrients and Wang describes feed medium containing similar to fermentation medium components and 10-fold increase in the amount of main carbon source in feed medium. A skilled artisan would have reasonably expected success in that because Tucaliuc, Poontawee and Wang teach microbial fermentation.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Xiong teaching and include GPE defoamer in the feeding medium based on Tucaliuc, Poontawee and Wang teachings. One would have been motivated to do so with reasonably expected success since Xiong teaches that foam produced during microbial fermentation poses a risk of bacterial contamination and describes using GPE in the fermentation medium.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to optimize the concentration of GPE defoamer in the feeding medium and increase the concentration over that of 0.04-0.06% in Xiong teaching. One would have been motivated to do so with reasonably expected success since Xiong describes that presence of sunflower cake powder in the medium allows to reduce the amount of GPE and hence the amount of GPE can be optimized based on the composition of the medium and it is within the skills of the artisan in the field to optimize the concentration of the ingredient in the fermentation medium.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to optimize the concentration of other ingredients in the feeding medium. One would have been motivated to do so to achieve the high productivity level of mupirocin and efficiency of the fermentation process. A skilled artisan would have reasonably expected success with that since it is within the skills of the artisan in the field to optimize the concentration of the ingredients in the fermentation medium.
Although Tucaliuc does not explicitly teach bean cake powder as nitrogen source it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that soy bean meal and soybean flour used as nitrogen source in Tucaliuc teaching can be replaced with bean cake powder. One would have been motivated to do so with reasonably expected success since bean cake powder originates from the same plant, the same part of the plant, i.e. beans, has the same powder form and hence will provide the same nutrition to bacterial culture.
Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 12 obvious.
Regarding claim 14, Tucaliuc teaches feeding 50% glucose (p. 499, left column, last paragraph). As discussed above for claim 12, the other components in the feeding medium can be similar to fermentation medium. Tucaliuc describes fermentation medium to include glycerol at 1% (Table 3), corn steep liquor (0.5%) and soy bean meal (2%) (p. 500, Table 3). Wang teaches 5.2% soybean cake flour and 2.3% corn pulp for the nitrogen source (p. 3, lines 6-7). Thus, the amount of glucose and glycerol in Tucaliuc teaching reads on claim 14 limitations and amount of corn steep liquor in Tucaliuc teaching and soybean cake flour in Wang teaching is close to claim 14 limitations.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to prepare feeding medium based on Tucaliuc and Wang teachings and optimize the concentration of ingredients in the feeding medium. One would have been motivated to do so to increase efficiency of mupirocin fermentation with reasonably expected success since it is within the skills of the artisan in the field to optimize the concentration of the ingredients in the fermentation medium. Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 14 obvious.
Regarding claim 15, Tucaliuc teaches potassium dihydrogen phosphate and magnesium sulfate in the fermentation medium (p. 500, Table 3). Xiong teaches magnesium sulfate and zinc sulfate in the fermentation medium (paragraph 0020).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Xiong teaching and include zinc sulfate in the feeding medium for production of mupirocin based on Tucaliuc, Poontawee and Wang teachings. One would have been motivated to do so with reasonably expected success since Tucaliuc discloses that inorganic salts are necessary for mupirocin production and provides examples of using different inorganic salts, including potassium dihydrate phosphate and magnesium sulfate in the mupirocin fermentation and Xiong includes the same salt, magnesium sulfate and additional salt, zinc sulfate, during bacterial fermentation. Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 15 obvious.
Regarding claim 16, Xiong teaches GPE defoamer at 0.4-0.6 g/L (paragraph 0020) which corresponds to 0.04-0.06% in the fermentation medium as described above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Xiong teaching and include GPE defoamer in the feeding medium for production of mupirocin based on Tucaliuc, Poontawee and Wang teachings and optimize its concentration. One would have been motivated to do so since Xiong teaches that foam produced during microbial fermentation poses a risk of bacterial contamination and describes using GPE in the fermentation medium. One would have been motivated to optimize the GPE concentration to increase efficiency of mupirocin production and since and it is within the skill of the artisan in the field to optimize the concentration of the ingredient in the fermentation medium. A skilled artisan would have reasonably expected success with that because Tucaliuc, Poontawee, Wang and Xiong teach microbial fermentation. Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 16 obvious.
Regarding claim 17, Tucaliuc teaches pH 5.3-7.0 for fermentation production of mupirocin. Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 17 obvious.
Regarding claims 18, Tucaliuc teaches fermentation medium for production of mupirocin having glucose as the main carbon source for up-to 6% and glycerol at 1% (p. 500, Table 3). Tucaliuc discloses nitrogen source comprising corn steep liquor (0.5%) and soy bean meal (2%) or soybean flour (5%) (Table 3). The inorganic salts include sulfates, phosphates and chlorides and in particular, KH2PO4 at 0.15%, (NH4)2SO4 at 0.5%, MgSO4 x7H2O at 0.05% and KCl at 0.1% (p. 500, Table 3). Production is performed for 50 hours, at 24-26°C and pH = 5.3-7 (p. 499, left column, last paragraph). Since the limitation for the feeding volume includes “0”, fermentation without feeding reads on that limitation.
Tucaliuc does not teach GPE defoamer in the fermentation medium and does not explicitly teach bean cake powder.
Xiong teaches GPE defoamer at 0.4-0.6 g/L (paragraph 0020) which corresponds to 0.04-0.06% in the fermentation medium as described above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Xiong teaching and include GPE defoamer in the fermentation medium for production of mupirocin based on Tucaliuc, Poontawee and Wang teachings and optimize its concentration. One would have been motivated to do so with reasonably expected success since Xiong teaches that foam produced during microbial fermentation poses a risk of bacterial contamination and describes using GPE in the fermentation medium. One would have been motivated to optimize the GPE concentration to increase efficiency of mupirocin production and since and it is within the skills of the artisan in the field to optimize the concentration of the ingredient in the fermentation medium. A skilled artisan would have reasonably expected success with that because Tucaliuc, Poontawee, Wang and Xiong teach microbial fermentation.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that soy bean meal and soybean flour used as nitrogen source in Tucaliuc teaching can be replaced with bean cake powder. One would have been motivated to do so with reasonably expected success since bean cake powder originates from the same plant, the same part of plant, beans, has the same powder form and hence will provide the same nutrition to bacterial culture. Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 18 obvious.
Regarding claim 19, Tucaliuc teaches pH 5.3-7 for mupirocin fermentation (p. 499, left column, last paragraph). Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 19 obvious.
Regarding claim 21, Tucaliuc teaches 24-26°C for mupirocin fermentation (p. 499, left column, last paragraph). Thus, Tucaliuc, Poontawee, Wang and Xiong teachings render claim 21 obvious.
Claims 20 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Tucaliuc (Tucaliuc et al. Biotechnol. Lett., 2018, 41, 495-502) in view of Poontawee (Poontawee and Limtong Microorganisms 2020, 151, 1-19), Wang (CN 110747188A) and Xiong (CN 109929890A) as applied to claims 12 and 18 above, and further in view of Ge (CN 111996136A on record in IDS).
The teachings of Tucaliuc, Poontawee, Wang and Xiong have been set forth above.
Tucaliuc teaches fermentation medium for mupirocin production comprising 6% glucose, 1% glycerol, 2% soybean meal, 0.5% corn steep liquor, 0.5% ammonium sulfate, 0.15% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.1% potassium chloride (p. 500, Table 3). Xiong teaches 0.2-0.4% zinc sulfate and 0.04-0.06% GPE defoamer in the fermentation medium (paragraph 0020) as described above. Addition of GPE to the fermentation medium taught by Xiong was discussed above for claim 18.
Tucaliuc, Poontawee, Wang and Xiong do not teach fermentation time of 80-132h and do not teach urea in the fermentation medium.
Regarding claim 20, Ge teaches fermentation of Pseudomonas fluorescence producing mupirocin
(paragraphs 0002, 0004). Ge describes that fermentation was performed for 120 hours (paragraph 0047) and mentions that the cell activity increased continuously from 24 hours to 120 hours (paragraph 0057).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Ge teaching and perform fermentation for mupirocin based on Tucaliuc, Poontawee, Wang and Xiong teachings for 120 h. One would have been motivated to do so since Ge teaches that the cell activity increased continuously from 24 hours to 120 hours and hence fermentation for 120 hours will provide higher yield of mupirocin. A skilled artisan would have reasonably expected success with that because Tucaliuc and Ge describe microbial fermentation for production of mupirocin. Thus, Tucaliuc, Poontawee, Wang, Xiong and Ge teachings render claim 20 obvious.
Regarding claim 24, Ge teaches that addition of sodium molybdate and urea significantly improved cell activity of bacteria and the yield of mupirocin (paragraph 0011). Ge describes that urea was added at 0.1-1% (paragraph 0014). Ge used glucose and glycerol as carbon source and corn steep liquor as nitrogen source (paragraph 0015).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Xiong teaching and include 0.2-0.4% zinc sulfate in the feeding medium for production of mupirocin based on Tucaliuc, Poontawee and Wang teachings. One would have been motivated to do so with reasonably expected success since Tucaliuc discloses that inorganic salts are necessary for mupirocin production and provides examples of using different inorganic salts, including potassium dihydrate phosphate and magnesium sulfate in the mupirocin fermentation and Xiong includes the same salt, magnesium sulfate and additional salt, zinc sulfate, during bacterial fermentation.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to follow Ge teaching and include urea at 0.1-1% in the fermentation medium for production of mupirocin based on Tucaliuc, Poontawee, Wang and Xiong teachings. One would have been motivated to do so with reasonably expected success since Tucaliuc and Ge teach production of mupirocin and disclose the same components of the fermentation medium for the carbon source and nitrogen source and Ge showed that addition of urea improved cell activity of bacteria and the yield of mupirocin. Thus, Tucaliuc, Poontawee, Wang, Xiong and Ge teachings render claim 24 obvious.
Claims 22 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Tucaliuc (Tucaliuc et al. Biotechnol. Lett., 2018, 41, 495-502) in view of Poontawee (Poontawee and Limtong Microorganisms 2020, 151, 1-19), Wang (CN 110747188A) and Xiong (CN 109929890A) as applied to claim 12 above, and further in view of Zhang (CN 108949845A on record in IDS) and Ge (CN 111996136A on record in IDS).
The teachings of Tucaliuc, Poontawee, Wang and Xiong have been set forth above.
Regarding claims 22 and 23, Tucaliuc teaches fermentation for mupirocin at 24-26°C and pH = 5.3-7 (p. 499, left column, last paragraph). Tucaliuc mentions controlling pH during the fermentation process and feeding and controlling glucose to maintain it at 0.5% level (p. 499, left column, last paragraph, right column, 1st paragraph). Tucaliuc describes collection of the fermentation broth for isolation of the product, mupirocin (p. 499, right column, 2nd paragraph).
Tucaliuc, Poontawee, Wang and Xiong do not teach shake flask culture and seed tank culture with the recited parameters.
Regarding claims 22 and 23, Zhang teaches preparation of mupirocin from fermentation medium of Pseudomonas fluorescens (Abstract, paragraph 0007). Zhang describes that fermentation medium contains a carbon source, comprising glucose and glycerol, a nitrogen source, comprising soybean meal powder and corn steep liquor, inorganic salts and other components (paragraphs 0012-0014). The method of Zhang includes steps of plate culture, slant culture, shake flask culture, seed culture and fermentation culture (paragraphs 0019-0023). The single colony of slant culture is inoculated into shake flask culture medium and incubated at 25-30°C for 10-15 hours to obtain shake flask seed culture solution (paragraph 0021). The shake flask seed culture is inoculated into seed culture medium and cultured until the logarithmic growth phase to obtain seed culture (paragraph 0022). The seed culture is inoculated into the fermentation medium and cultured at 25-30°C for 60-120 hours (paragraph 0023). The pH of the shake flask seed culture medium, the seed culture medium and the fermentation medium is 6.0-7.0 (paragraphs 0040, 0048, 0062). As discussed in 112(b) rejection above, claim 23 is interpreted as directed to 1-2 levels of seed cultures wherein shake flask seed culture of step (a) of claim 22 is considered level 1 seed culture and seed tank culture is considered level 2 seed culture and hence Zhang teaches 2 levels of seed culture. Zhang mentions that using processes in steps such as shake flask culture, seed culture and fermentation cultured resulted in mupirocin content of over 6000 µg/mL in the fermentation broth (paragraph 0065).
Regarding claims 22 and 23, Ge teaches fermentation of Pseudomonas fluorescence producing
mupirocin (paragraphs 0002, 0004). Method of Ge includes shake flask seed culture that is transferred to a seed tank after maturity and after the seed culture in the seed tank matures it is transferred to the fermentation tank (paragraph 0025). Ge describes sterilization of the fermentation medium for the recited cultures (paragraph 0026). Ge discloses conditions for the seed tank culture, i.e. 25°C, 18 hours culturing time and inoculation of 100 ml into 10L seed culture medium which corresponds to 1% inoculation amount (paragraph 0044). Ge provides example of inoculation of 300L of seed culture into 4000L of the fermentation medium (paragraphs 00564, 0055) that corresponds to 7.5% inoculation amount and reads on claim 23 limitation. Ge mentions that fermentation produced 6562 µg/mL of mupirocin (paragraph 0047).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Zhang teaching to method of mupirocin production based on Tucaliuc, Poontawee, Wang and Xiong teachings and include steps of shake flask seed culture and seed tank culture prior to fermentation step performed at pH 6-7 and at 25-30°C for 10-15 hours for shake flask seed culture as described by Zhang. One would have been motivated to do so since Zhang teaches that using steps such as shake flask culture, seed culture and fermentation cultured resulted in mupirocin content of over 6000 µg/mL in the fermentation broth. A skilled artisan would have reasonably expected success in that because Tucaliuc and Zhang teach fermentation method for mupirocin production.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add Ge teaching to method of mupirocin production based on Tucaliuc, Poontawee, Wang, Xiong and Zhang teachings and follow Ge instructions for the shake flask seed culture and seed tank culture including sterilizing culture medium for all steps, 25°C and 18 hours culturing time for the seed tank culture and inoculation volume of 7.5% for the fermentation culture. One would have been motivated to do so since Ge provides instructions for the fermentation steps and teaches that fermentation cultured resulted in mupirocin content of over 6000 µg/mL in the fermentation broth. A skilled artisan would have reasonably expected success in that because Tucaliuc, Zhang and Ge teach fermentation method for mupirocin production with Zhang and Ge following the same protocol steps and similar parameters and production yields.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the inoculation volume taught by Ge for the seed tank culture (1%) to the shake flask culture. One would have been motivated to do so with reasonably expected success since 100-fold dilution of the strain culture allows to maintain healthy growth of cells and to reach larger volume for the next step of inoculation.
Thus, Tucaliuc, Poontawee, Wang, Xiong, Zhang and Ge teachings render claims 22 and 23 obvious.
Conclusion
No claims are allowed.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653