DETAILED ACTION
Applicant’s amendment filed on 12/16/2025 is acknowledged. Claims 2-9 and 11-21 remain pending in the instant application.
Claims 2-4, 6, 8-9, 11, 13-14, and 16-18 are currently amended. Claims 1 and 10 are canceled. Claims 19-21 are newly added.
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/16/2025 has been entered.
Priority
The instant application is a U.S. National Stage of PCT/EP2020/068335 filed on 6/30/2020 and claims foreign priority to EP20170663.7 filed on 5/5/2020 and EP19184580.9 filed on 7/5/2019. Receipt is acknowledged of certified copies of the foreign priority documents. Thus, the effective filing date of the claimed invention is 7/5/2019.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-9 and 11-21 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.
Claim 11 recites “providing a pre-culture of Bacillus cells producing said protein of interest, wherein the pre-culture comprises at least two stages and at least the last stage of the at least two stages is performed in fed-batch mode” in lines 3-5. It is not clear if this limitation requires an active pre-culturing step that comprises at least two stages or if the step can be satisfied by providing pre-cultured Bacillus cells that were purchased, etc. Therefore, the metes and bounds of the claim are unclear. Applicant may amend step (a) of claim 11 to recite:
“(a) pre-culturing Bacillus cells producing said protein of interest comprising at least two stages, wherein at least the last stage of the at least two stages is performed in fed-batch mode”.
Claims 2-9 and 12-21 are also rejected for being dependent on a rejected base claim and failing to remedy the issue set forth above.
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 2-9 and 11-21 are rejected under 35 U.S.C. 103 as being unpatentable over CN103805582 to Li et al. (citations corresponding to English translation; of record) in view of Huber et al. (Biotechnol. Bioeng., 2009, Vol. 103(6), p.1095-1102; of record in IDS filed 4/22/2022).
Regarding claims 2-7, 11, 13-15, and 20, Li teaches a method for the production of beta-glucanase in Bacillus licheniformis 10-25 (see Abstract). Li teaches the preparation method of beta-glucanase comprises a liquid seed expansion culture which comprises a primary seed culture, a secondary seed culture, and tertiary seed culture (see paragraphs [0046]-[0053]). The primary seed culture comprised inoculating 2 loops of B. licheniformis in a 500 mL shake flask having 100 mL volume of culture medium (see paragraph [0049]). The secondary seed culture comprised inoculating a second 500 mL shake flask with 10% of the primary seed culture (see paragraph [0050]). The tertiary seed culture comprised inoculating a 5000 mL shake flask with 10% of the secondary seed culture (see paragraph [0051]). 10% of the tertiary seed culture is inoculated in a primary seed tank culture with a total volume of 150 L and having 100 L culture medium (see paragraphs [0052]-[0053]). A fermentation tank (production bioreactor) was inoculated with 3% of the primary seed tank broth containing 3 L of fermentation medium and cultured at 37°C, agitation speed 250 r/min, aeration (V/V) 1: 1-3, incubation time 12 h; 1 L of fermentation medium sterilized at 121°C for 20 min and at a temperature of 10°C was then fed into the fermenter and allowed to incubate for 18 h when the temperature was raised to 37°C; at this point, the primary seed tank fermentation broth was added to the fermenter at 2% inoculum for isothermal incubation for 12 h; 1 L of fermentation medium sterilized at 121°C for 20 min and at a temperature of 10°C was continued to be fed into the fermenter and incubated for 12 h when the temperature was raised to 37°C (see paragraph [0055]). Thus, the fermentation tank is run on batch mode as defined in the specification at p.7, lines 24-26. Therefore, Li teaches a process for culturing the enzyme beta-glucanase in Bacillus licheniformis using a total of 4 stages of pre-culturing before entering the production phase in a production bioreactor.
Li does not teach wherein the last stage of the at least two stages is performed in fed-batch mode, wherein the time until harvest is the period between inoculation of the production bioreactor and the time point at which the titer of the protein of interest reaches at least 5 g product/kg fermentation broth, and wherein the time until harvest of the protein of interest is reduced compared to a process where the pre-culture step is performed in batch mode.
Huber teaches “the non-parallel growth in precultures can have a tremendous effect on performance of bioprocesses” (see p.1095, right column, 1st paragraph) wherein “[a] new approach to circumvent the problem of unequal growth kinetics in main cultures is the application of fed-batch mode in precultures” (see p.1095, Abstract). Through the use of this technique, the prior art teaches a self-regulating system wherein “the [preculture] cells are continuously supplied with substrate and are in a defined metabolic state” (see p.1101, right column, 1st passage) allowing for a more consistent inoculum for the main cultures. Huber teaches “[t]he concept worked for pro[k]aryotic and eu[k]aryotic microorganisms” and “this technique seems to be of general applicability” although the system was only tested with E. coli and Hansenula polymorpha (see p.1101, left column, 1st paragraph and 2nd passage). Huber further teaches their work presents a novel method for equalizing growth kinetics in high-throughput precultures in shake-flasks applying fed-batch mode (see p.1096, right column, 1st paragraph).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have performed the liquid seed culture stages of Li in fed-batch mode, as taught by Huber, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to incubate the preculture in fed-batch mode as taught by Huber with the Bacillus licheniformis cells for the production of beta-glucanase as taught by Li to permit for a more consistent, timely, and productive inoculum in the main cultures. One of ordinary skill in the art would have had a reasonable expectation of success since Huber teaches their method equalizes growth kinetics in high-throughput shake-flasks and Li teaches shake-flask seeding.
With respect to the limitations “wherein the time until harvest is the period between inoculation of the production bioreactor and the time point at which the titer of the protein of interest reaches at least 5 g product/kg fermentation broth, and wherein the time until harvest of the protein of interest is reduced compared to a process where the pre-culture step is performed in batch mode,” the method of Li in view of Huber as set forth above teaches each and every active method step of the claimed invention. Thus, the method of culturing obviated by Li in view of Huber is expected to reduce the time until harvest as required by the claim, absent evidence to the contrary.
Regarding claim 8, Li teaches the beta-glucanase is filtered from the fermentation liquid and thus is secreted by the B. licheniformis into the fermentation broth (see paragraph [0059]).
Regarding claim 9, Li teaches a harvest enzyme activity of 10,000 u/mL of broth (see paragraph [0060]). It would have been obvious to harvest the enzyme at the highest concentration possible and one of ordinary skill in the art would have been motivated to arrive at the claimed invention.
Regarding claim 12, Li in view of Huber teach each and every active method step of claim 11. With regards to the limitation “wherein the time until harvest of the protein of interest is reduced by 5% to 30% compared to a process where the preculture step is performed in batch mode”, this limitation does not further limit the active method steps of the claimed process recited in claim 11. Therefore, the equivalent process taught by Li in view of Huber is expected to reduce the time until harvest of beta-glucanase by 5% to 30% compared to a process where the preculture step is performed in batch mode, absent evidence to the contrary.
Regarding claims 16-18, Li in view of Huber teach wherein the pre-culture comprises four stages wherein the first three stages are conducted in shake flask and the fourth stage is conducted in a seed fermenter, and wherein all stages are conducted in fed-batch mode.
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have routinely optimized the pre-culturing process of Li in view of Huber, testing which stages are necessary and which culturing conditions provide the most optimized cultures for producing beta-glucanase, and arriving at the pre-culturing conditions of claims 16-18. One of ordinary skill in the art would have been motivated to increase the production of beta-glucanase by B. licheniformis by optimizing the number of stages of pre-culturing necessary, the equipment used in these stages, and the culturing modes used in the optimized pre-culturing stages. Thus, claims 16-18 are prima facie obvious.
Regarding claim 19, Li in view of Huber teach each and every active method step of claim 11. With regards to the limitation “wherein the time until harvest is the period between inoculation of the production bioreactor and the time point at which the titer of the protein of interest reaches 20 g product/kg fermentation broth”, this limitation does not further limit the active method steps of the claimed process recited in claim 11. Therefore, the equivalent process taught by Li in view of Huber is expected to reduce the time until the protein of interest reaches 20 g product/kg fermentation, absent evidence to the contrary.
Regarding claim 21, Li teaches beta-glucanase is prepared in cultured Bacillus licheniformis to be supplied in a pig feed (see paras [0008]-[0009] and [0065]-[0072]). Li teaches the feed also comprises alpha-amylase (see paras [0008]-[0009] and [0065]-[0072]).
It would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have prepared alpha-amylase in cultured Bacillus licheniformis according to the method of Li in view of Huber. One of ordinary skill in the art would have been applying recognized preparation and culturing methods to obtain alpha-amylase for the pig feed, yielding predictable results.
Response to Arguments
Applicant's arguments filed 12/16/2025 have been fully considered but they are not persuasive.
The Declaration under 37 CFR 1.132 filed 12/16/2025 is insufficient to overcome the rejection of claims 2-9 and 11-21 based upon the 35 U.S.C. 103 rejection as set forth in the last Office action because Applicant’s arguments are not found to be persuasive for the following reasons.
In the Declaration, see 6th-8th statements, Declarant argues that a person of ordinary skill in the art would have no reason to combine the teachings of Li and Huber, since they are directed to distinct methods with distinct goals to produce distinct products. This is not found persuasive. Although, Huber is not expressly focused on the expression of any product directly, but on the growth equalization of precultures, this statement does not preclude the applicability of the teachings of Huber to product expression. The teachings of Huber regarding preculture growth equalization are found to be applicable to the beta-glucosidase production in Bacillus licheniformis taught by Li, since Li’s enzyme production involves a preculturing process. Specifically, Li teaches the beta-glucanase production includes steps of a seed train preculture to provide inoculum for a production bioreactor (see Li – paras [0046]-[0053]). Huber demonstrates growth equalization in main cultures by applying fed-batch mode in precultures (see Huber – p.1095, Abstract). Huber further teaches the equalization is applicable in high-throughput shake-flask precultures (see p.1096, right column, 1st paragraph). Li’s seeding steps are performed in shake-flask seed trains. Thus, one of ordinary skill in the art would have sought to apply fed-batch mode to the preculture of Li in order to equalize the growth to provide uniformity in the production bioreactor, i.e. the main culture, of the production Bacillus licheniformis strain. One of ordinary skill in the art would have had a reasonable expectation of success because Huber teaches the fed-batch preculture technique works for prokaryotic and eukaryotic microorganisms and seems to be of general applicability (see p.1101, left column, 1st paragraph and 2nd passage). Therefore, one of ordinary skill in the art would find the teachings of Huber regarding precultures applicable to the preculture steps in the beta-glucosidase production of Li.
In the Declaration, see 9th statement, Declarant argues that Li and Huber are directed to two unrelated fields. Declarant further emphasizes that Huber expressly admits that the focus of their publication is not the expression of any product directly, but on the growth equalization of precultures. Declarant further argues Li involves the industrial production of an enzyme, whereas Huber is limited to studying growth equalization in a microtiter plate on a microliter scale, not industrial production. Declarant further argues that the different scales require different considerations, even in a situation in which the strain, medium, and nominal process conditions are identical. Declarant further argues the differences of microorganisms used in Li and Huber would prevent a person of ordinary skill in the art from applying Huber’s microliter scale, growth equalization methods to the industrial process of making an enzyme product for pigs, as taught by Li. This is not found persuasive. The arguments regarding the teachings of Huber not being applicable to Li have been addressed above. Declarant’s arguments regarding differences in scales are not substantiated by evidence that the findings in Huber’s microliter scale cannot be applicable to production level scales. Furthermore, the argument regarding differences in microorganisms is not substantiated by evidence that would prevent a person of ordinary skill in the art from applying the teachings of Huber to Li.
In the Declaration, see 10th statement, Declarant argues that even if there was a reason to combine the teachings of Li and Huber, one of ordinary skill in the art would not expect a reduction in the time until harvest in the fermentative production of a protein of interest, wherein the time until harvest is the period between inoculation of the production bioreactor and the time point at which the titer of the protein of interest reaches at least 5 g product/kg fermentation broth, and wherein the time until harvest of the protein of interest reduced compared to a process where the preculture step is performed in batch mode. This is not found persuasive. Li in view of Huber teach each active method step of the claimed invention, and the equivalent process is expected to reduce the time until harvest as claimed, absent evidence to the contrary. As discussed in the rejection of claim 11, Huber’s fed-batch preculturing method allows for more consistent, timely, and productive inoculum in the main cultures.
In the Declaration, see 12th-13th statements, Declarant argues Huber is silent with regards to the time until harvest in the fermentative production of a protein of interest. Declarant argues that Huber acknowledges that the equalization technique taught may result in the expression of toxic products in host cells which are de-repressed by low glucose concentrations during the fed-batch phase. Declarant further argues that Huber explains that “further investigations have to be performed to prove this method with different clone libraries and the impact of equalized precultures on product formation (e.g., recombinant proteins or amino acids) in subsequent main cultivations.” Finally, Declarant argues that there is nothing in Huber that suggests a reduction in time until harvest in any of the cited passages. This is not found persuasive. While Huber did not focus their study on fermentative production of a protein of interest, the teachings of Huber regarding performing precultures in fed-batch mode are still applicable to Li, since Li teaches steps of preculturing by seed fermentation for beta-glucanase production in Bacillus licheniformis. Thus, one of ordinary skill in the art would have found it obvious to modify the preculturing steps within Li’s production process to accord the benefits described in Huber pertaining to equalizing growth. Therefore, an equivalent process is arrived from the teachings of Li in view of Huber that would reflect the same claimed properties, absent evidence to the contrary. Huber’s mention of toxic products is speculative and does not mention which hosts toxic products may be expressed in to prevent one of ordinary skill in the art from applying the technique of Huber. Furthermore, Huber compares fed-batch mode and batch mode preculturing and observed benefits in fed-batch mode that were not realized in batch mode (see Huber – Fig. 1). Although Huber discloses further investigations are needed to prove the impact of equalized precultures on product formation in subsequent main cultivations, Huber’s growth equalization findings would provide motivation to one of ordinary skill in the art to perform precultures in fed-batch mode in order to equalize growth. Furthermore, Huber teaches that non-parallel growth in precultures can tremendously effect bioprocess performance, and provides a method to achieve parallel, equalized growth in precultures to mitigate those effects. Huber further teaches precultures methods are the crucial factor in providing defined starting conditions for production hosts (see paragraph bridging pp.1095-96). Even more, Huber teaches fed-batch mode is superior for producing biomass and product in main cultures (see p.1096, left column, 1st paragraph).
In the Declaration, see 14th statement, Declarant argues that Huber’s method of fed-batch preculture is not used or even intended or discussed to be used to reduce cultivation time until a certain product yield is achieved. This is not found persuasive. Arguments regarding to the reduction in time until harvest have been previously addressed in the replies above.
In the Declaration, see 15th-20th statements, Declarant argues that Example 1 of the instant application demonstrates a ~30% reduction in time until harvest for protease in Bacillus licheniformis and a ~20% reduction in time until harvest for amylase in Bacillus licheniformis, as compared to the method where the preculture was done completely in batch mode. Declarant argues that Example 4 of the instant application demonstrates a ~12% reduction in time until harvest for protease cultured in Bacillus subtilis as compared to the method where precultures were done completely in batch mode. Declarant further argues that time until harvest was reduced by about 33% when preculturing preceding the bioreactor was run in fed-batch mode compared to batch mode in Bacillus licheniformis cultured in two preculturing stages. Lastly, Declarant argues the data presented in the instant application and Declaration demonstrate the claimed process consistently reduces the time until harvest and was not expected in view of the combination of Li and Huber. This is not found persuasive. The steps performed in Examples 1 and 4 are not commensurate in scope with claimed invention because the examples demonstrate 3 steps of preculturing in either B. licheniformis or B. subtilis and only the production of protease or amylase, whereas the claims are not limited to such active steps and limitations. That is, the method of claim 11 is broadly directed to any protein of interest produced in any of B. subtilis, B. licheniformis, B. pumilus, or B. lentus, and requires at minimum two stages of preculturing, wherein the last stage is performed in fed-batch mode. Declarant’s demonstration with two stages of preculturing wherein the last stage is fed-batch is only performed in B. licheniformis cultivated for the production of protease. This demonstration is not commensurate in scope with the claimed invention which is not limited to the production of protease in B. licheniformis. Therefore, the reduction in time until harvest shown in the examples has not been demonstrated across all species of proteins of interest and claimed Bacillus species of the claimed invention.
For the above reasons, the Declaration is insufficient to overcome the 35 U.S.C. 103 rejection of claims 2-9 and 11-21 over Li in view of Huber.
In Applicant’s Remarks, see p.5, 4th paragraph,-p.6, 3rd paragraph, Applicant reiterates the arguments presented in the Declaration. The arguments presented in the Declaration are addressed in the replies above.
Conclusion
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/J.P.S./
Examiner, Art Unit 1657
/MELENIE L GORDON/