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 .
Priority
This application is a 371 of PCT/EP2020/054172 filed 02/18/2020. 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) based on EP19215651.1, filed on 12/12/2019 and EP19158372.3 filed on 02/20/2019. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Status of the Claims
Claims 1, 10-23 and 25-30 are pending. Claims 1 and 25-30 are amended. Claims 2 and 24 are cancelled. Claims 21 and 22 are withdrawn.
Claims 1, 10-20, 23 and 25-30 (claim set as filed on 11/06/2025) are examined of the merits herein.
Withdrawal of Rejections
The response and amendment filed on 11/06/2025 and Declaration under 37 C.F.R. 1.132 of Dr. Daub filed on 11/06/2025 are acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
Maintained/Modified Rejections
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 10-20, 23, 26 and 28-30 are rejected under 35 U.S.C. 103 as being unpatentable over Wenzel (Wenzel et al. Appl. Environ. Microbiology, 2011, 77, 6419-6425 on record in IDS) in view of Bongiorni (US 8293499 B2).
Regarding claim 1, Wenzel teaches expression system for heterologous protein (eGFP) production in Bacillus subtilis (Abstract). Expression of eGFP is performed in a chemically defined fermentation media in a fed-batch fermentation. Wenzel describes that the chemically defined fermentation batch media (p. 6421, right column, 2nd paragraph) is inoculated with the preculture of Bacillus subtilis (p. 6422, left column, 1st paragraph) containing plasmid for expression of eGFP (Figure 3). Two feeding solutions are added to batch culture for 30-liter fermenter: “In the case of a 30-liter D598 laboratory fermenter (LF; Bioengineering AG, Wald, Switzerland), the batch volume was 8.0 liters, the glucose feed medium 4.2 liters, and the ammonia feed medium 1.0 liter (p. 6422, left column, 2nd paragraph). Wenzel discloses the composition of feeding solutions: “ The first medium contained 654.76 g liter-1 glucose x H2O, 23.5 g liter-1 MgSO4 x 7 H2O, 120 ml liter-1 TES, and the second 396 g liter-1 (NH4)2HPO4.” (p. 6421, right column, 2nd paragraph). Considering dilution with feed medium, the amount of glucose (carbon source) is above 200 g per liter of initial fermentation medium and the amount of magnesium ions is higher than 0.1 g/l that reads on claim 1 limitations. To remove dependency of protein of interest expression on the inducer, mannose, Wenzel mutated genes manA (responsible for mannose metabolism) and manP (mannose uptake) in mannose operon. Wenzel discloses that creation of a Bacillus strain with mutation TQ356 of manP gene allowed to develop of inducer-independent self-induction system: “An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system…” (Abstract).
Wenzel does not teach the fermentation process wherein the expression of the gene of interest is under the control of aprE promoter and wherein the Bacillus cell has not been genetically modified in its ability to take up or metabolize the inducer molecule.
Bongiorni teaches host Bacillus cells that have been genetically manipulated to have an enhanced capacity to produce proteins of interest (Abstract). The genetic manipulation involved inactivation of phr or rap genes involved in cellular response to changes in cell density and not related to induction of protein of interest expression with inducer molecule (column 21, lines 39-41, 54-56). Bongiorni discloses expression of the protein of interest under the control of an aprE promoter: “The recombinant nucleic acid comprises a promoter that is operably linked to the polynucleotide sequence that encodes the protein of interest. Preferably the promoter is the wild-type or mutant aprE promoter.” (column 2, lines 19-22). Bongiorni teaches that host cells can be cultured under different fermentation conditions including fed-batch condition: “… the host cells are cultured under batch, fed-batch or continuous fermentation conditions” (column 43, lines 32-34). Bongiorni describes that in the “fed-batch” fermentation system nutrients are added when their concentration in culture falls below a threshold (column 43, lines 49-53). Bongiorni teaches using different medium for growing host cells including minimal medium: “The medium used to culture the cells comprises any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements.” (column 43, lines 26-29). Bongiorni describes expression construct PaprE-FNA, containing aprE promoter for expression of FNA protease in Example 1 and successful expression of FNA in Bacillus subtilis in Example 3. The expression is performed without induction and hence it is inducer-independent.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine methods of Wenzel and Bongiorni for inducer-independent production of protein of interest and express protein of interest under control of aprE promoter in Bacillus cell that has not been genetically modified in its ability to take up or metabolize an inducer molecule as described by Bongiorni in a chemically defined medium and fed-batch fermentation described by Wenzel. One would have been motivated to do so because Bongiorni described successful production of a protein of interest under control of aprE promoter in Bacillus subtilis cells and Bongiorni and Wenzel teach fed-batch mode of cell culturing and application of minimal media. A skilled artisan would have reasonably expected success in the combination since Wenzel and Bongiorni teach production of protein of interest in inducer-independent systems and in Bacillus subtilis as a host. Thus, combination of Wenzel and Bongiorni teachings renders claim 1 obvious.
Regarding claims 10 and 11, Wenzel teaches addition of 4.2 liters of feeding solution containing 23.5 g/l of MgSO4 x 7 H2O to 8 l of fermentation batch solution (p. 6421, right column, 2nd paragraph and p. 6422, left column, 2nd paragraph) as described above that results in 8.1 g/l of magnesium ions provided by magnesium salt. That reads on claims 10 and 11 limitations. Thus, Wenzel and Bongiorni teachings render claims 10 and 11 obvious.
Regarding claims 12 and 13, Wenzel teaches addition of trace element solution (TES) containing iron (elected species) and cobalt with feeding solution: “TES contains 0.5 g liter-1 CaCl2, 0.18 g liter-1 ZnSO4 x 7 H2O, 0.1 g liter-1 MnSO4 x H2O, 10.05 g liter-1 Na2-EDTA, 8.35 g liter-1 FeCl3, 0.16 g liter-1 CuSO4 x 5 H2O, and 0.18 g liter-1 CoCl2 x 6 H2O.” (p. 6421, right column, 2nd paragraph). The amount of TES in feed solution is 120 ml/l and 4.2 liters of feed solution is added to 8 liters of batch solution (p. 6421, right column, 2nd paragraph) as described above for claim 1. The final concentrations of iron and cobalt cover limitations of claims 12 and 13. Thus , Wenzel and Bongiorni teachings render claims 12 and 13 obvious.
Regarding claim 14, Wenzel teaches addition of glucose as a carbon source as described above for claim 1. Thus, Wenzel and Bongiorni teachings render claim 14 obvious.
Regarding claim 15, Wenzel teaches addition of chemically defined nitrogen source, i.e. (NH4)2HPO4:“ The first medium contained 654.76 g liter-1 glucose x H2O, 23.5 g liter-1 MgSO4 x 7 H2O, 120 ml liter-1 TES, and the second 396 g liter-1 (NH4)2HPO4.” (p. 6421, right column, 2nd paragraph). Thus, Wenzel and Bongiorni teachings render claim 15 obvious.
Regarding claim 16, Wenzel teaches adjustment of pH to 7.0 which is above 6.0 (elected species): “The pH was adjusted to 7.0 with 24% (vol/vol) NH4OH and 20% (vol/vol) H3PO4.” (p. 6422, left column, 3rd paragraph). Thus, Wenzel and Bongiorni teachings render claim 16 obvious.
Regarding claim 17, Wenzel teaches production of 10 g/l of eGFP: “Nearly 10 g eGFP liter-1 were produced, corresponding to a product yield of 14.6% … without any addition of inducer.” (p. 6423, right column, 2nd paragraph). Thus, Wenzel and Bongiorni teachings render claim 17 obvious.
Regarding claims 18, 19 and 20, Bongiorni teaches the protein of interest to be an enzyme: “The protein of interest is an enzyme, and preferably, a protease (e.g., a subtilisin).” (column 1, lines 58-59). Bongiorni described production of AprE protease and FNA protease in Examples 2 and 3, respectively. Bongiorni teaches that the produced protein of interest can be secreted and purified from the culture media by different methods including filtration, salt precipitation or chromatographic purification: “The protein of interest produced by a modified host cell overexpressing ymaH according to the present invention is secreted into the culture media. In some embodiments, the protein of interest (e.g., a protease), produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.). Thus, any method suitable for recovering the protease(s) of the present invention finds use in the present invention. Indeed, it is not intended that the present invention be limited to any particular purification method.” (column 42, lines 59-67, column 43, lines 1- 5).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce enzyme as protein of interest secreted into medium and purify it from the medium as described by Bongiorni using fed-batch fermentation in chemically defined medium described by Wenzel. One would have been motivated to do so because Bongiorni described successful production of two proteases in Bacillus subtilis cells and because method of protein production by secretion into media described by Bongiorni provides efficient purification of produced protein from media and allows continuous fermentation of Bacillus cells. A skilled artisan would have reasonably expected success in the modification since Wenzel and Bongiorni provide methods of production of protein of interest by Bacillus subtilis fermentation. Thus, Bongiorni and Wenzel teachings render claims 18, 19 and 20 obvious.
Regarding claim 23, Wenzel teaches that inducer-independent expression system improved product yield: “The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.” (Abstract). Table 2 demonstrates that production of recombinant eGFP increased from 0.2-2.0 g/l for mannose inducible system to 9.8 g/l for inducer-independent system for 30-liter fermenter. Thus, Wenzel and Bongiorni teachings render claim 23 obvious.
Regarding claims 26 and 28-30, Bongiorni teaches production of protein of interest under the control of an aprE promoter (column 2, lines 19-22). Bongiorni describes aprE promoter as a natural promoter of Bacillus subtilis: “The term "aprE promoter" herein refers to the polynucleotide promoter sequence that naturally drives the expression of subtilisin in B. subtilis…” (column 16, lines 56-58). The nucleotide sequence of aprE promoter in Bongiorni teaching of SEQ ID NO: 19 (column 45) is 100% identical to instant aprE promoter sequence of SEQ ID NO: 8. Bongiorni teaches that aprE promoter can comprise sigma A factor and several regulators, including DegU (elected species): “… the expression of the protein of interest in a host cell is driven by the aprE promoter of the aprE gene from which the B. subtilis subtilisin is naturally transcribed. The aprE gene is transcribed by sigma A (σA) factor and its expression is highly controlled by several regulators, such as: DegU/DegS, AbrB, Hpr and SinR…” (column 30, lines 14-19).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce recombinant protein under control of aprE promoter from Bacillus subtilis with SEQ ID NO:19 (identical to instant SEQ ID NO: 8) and comprising sigma A factor and DegU regulators as described by Bongiorni in fed-batch fermentation in chemically defined medium as described by Wenzel. One would have been motivated to do so because Bongiorni described successful production of a protein of interest under control of aprE promoter with SEQ ID NO: 19 in Bacillus subtilis cells. A skilled artisan would have reasonably expected success in the modification since Wenzel and Bongiorni teach methods of increased inducer-independent protein production in Bacillus subtilis. Thus, Bongiorni teaching in combination with Wenzel teaching renders claims 26 and 28-30 obvious.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Wenzel (Wenzel et al. Appl. Environ. Microbiology, 2011, 77, 6419-6425 on record in IDS) in view of Bongiorni (US 8293499 B2) as applied to claim 1 above, and further in view of Jarmer (Jarmer Microbiology, 2001, 147, 2417- 2424).
Wenzel and Bongiorni teachings have been set forth above.
Wenzel and Bongiorni do not teach the aprE promoter sequence with HMM-score above 50.
Jarmer teaches a hidden Markov model of σA RNA polymerase cofactor recognition sites in Bacillus subtilis: “We have used hidden Markov models (HMMs) and trained them to recognize σA - binding sites in B. subtilis from existing experimentally generated data.” (p. 2418, left column, 3rd paragraph). Jarmer discloses that: “The central idea of an HMM is to embed the statistics of a motif in a set of states with transitions between them.” (p. 2418, left column, 4th paragraph). The training of a model is performed based on the prior knowledge of signal associated with the sequence motif (p. 2418, left column, 5th paragraph). Jarmer teaches that the developed trained model can be used to search for new sequences: “The trained model is then used to analyse sequences not included in the training set.” (p. 2418, right column, 3rd paragraph). By using HMM-based prediction tool Jarmer predicted Bacillus subtilis genome to have 2538 σA – binding sites, from which 1127 binding sites were located within the 400 bp sequences upstream of predicted genes. Jarmer confirmed the HMM predictions on the example of response regulator aspartate phosphatase (rap) promoter since site-directed mutagenesis of the predicted σA – binding sites resulted in the decrease in expression of rap genes. (p. 2423, left column, 3rd paragraph). Jarmer teaches that the probability score in HMM model depends on the motif and signals associated with the motif: “The better the motif and its contextual signals fit the model, the higher the probability score, and the more confidence will be placed in the prediction.” (p. 2418, right column, 6th paragraph).
Jarmer does not explicitly teach an HMM-score above 50 for the aprE promoter, however, Jarmer teaches that the predictability score of HMM model depends on the motif itself and its training based on prior knowledge about signals associated with the motif. Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that the HMM score is the result effective variable that can be determined by motif of interest. One would have been motivated to determine the HMM score with reasonably expected success for the application to aprE promoter based on set of corresponding sequences used to train HMM.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add HMM prediction model for aprE promoter as described by Jarmer for σA - binding sites in B. subtilis promoters to the combined method of production of protein of interest in chemically defined medium described by Wenzel under control of aprE promoter as described by Bongiorni. One would have been motivated to do so because Jarmer described development of HMM model used to identify 2538 σA – binding sites in Bacillus subtilis cells and HMM model trained on aprE promoter sequence can be applied for search of new genes regulated by aprE promoter. A skilled artisan would have reasonably expected success in the modification since Wenzel and Bongiorni provided method of production of protein of interest under the control of aprE promoter in Bacillus subtilis using chemically defined fermentation medium, Bongiorni teaches that aprE promoter includes σA – binding site as described for claim 28 above and Jarmer provided method of developing HMM model for σA - binding region in Bacillus subtilis promoters. Thus, combination of Wenzel, Bongiorni and Jarmer teachings renders claim 25 obvious.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Wenzel (Wenzel et al. Appl. Environ. Microbiology, 2011, 77, 6419-6425) in view Bongiorni (US 8293499 B2) of as applied to claim 1 above, and further in view of Wieland (US 7510859 B2).
Wenzel and Bongiorni teachings have been set forth above.
Bongiorni provides examples of different subtilisins produced by invention method, including Carlsberg subtilisin (column 28, line 20), and examples of promoters including promoter for subtilisin Carlsberg gene: “… the promoter of the Bacillus licheniformis alkaline protease gene (subtilisin Carlsberg gene)…” (column 38, lines 14-16). However, Wenzel and Bongiorni do not teach the sequence of subtilisin Carlsberg protease.
Wieland teaches novel perhydrolases, subtilisin variants, derived from Carlsberg protease and methods of production of novel perhydrolases (Abstract). The subtilisin Carlsberg protease in Wieland teaching with SEQ ID NO: 23 has 100% identity to instant subtilisin Carlsberg protease with SEQ ID NO:2 (elected species). Wieland discloses that expression of protease can be regulated either by natural or genetically modified promoter: “Thus expression can be carried out by the natural promoter originally located outside this gene, and also via genetically engineered fusion both by a host cell promoter on the expression vector and also by a modified or completely different promoter from another organism. ” (column 9, lines 22- 27). Wieland teaches host cells for perhydrolases fermentation to preferably be Bacillus cells, including Bacillus licheniformis from which subtilisin Carlsberg protease originates (column 10, lines 37-43). Wieland discloses that modification of subtilisin Carlsberg protease can generate perhydrolases with improved properties for bleaching as washing and cleaning agents (column 2, lines 8- 16).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine method of production of protein of interest in chemically defined medium described by Wenzel, method of production of subtilisin proteases under control of aprE promoter as described by Bongiorni with the method of production of subtilisin protease with improved efficiency based on modified Carlsberg protease as described by Wieland. One would have been motivated to do so because Wieland teaches modification of subtilisin Carlsberg protease which can be produced in Bacillus cells and which has better bleaching properties to serve as perhydrolase in washing and cleaning agents. A skilled artisan would have reasonably expected success in the modification since Wenzel, Bongiorni and Wieland provide methods of production of protein of interest in Bacillus fermentation. Thus, combination of Wenzel, Bongiorni and Wieland teachings renders claim 27 obvious.
Response to Arguments
Applicant's arguments filed 11/06/2025 and arguments of Dr. Daub in Declaration under 37 C.F.R. 1.132 filed on 11/06/2025 have been fully considered but they are not persuasive.
Applicant and Dr. Daub argue (addressing pages 7-8 of the Remarks and paragraphs 6-10 and 14 of the Declaration) that Wenzel describes a Bacillus cell that has been genetically modified in its ability to take up or metabolize an inducer molecule. Wenzel developed a strain that can use PmanP promoter but is independent on inducer mannose. Wenzel did not use aprE promoter known at that time. The system with PmanP promoter in Wenzel improved product yield and reduced cost and one of ordinary skill in the art would not have any reason to replace PmanP promoter. These arguments are not persuasive because:
In response to applicant’s argument that there is no teaching, suggestion, or 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). In this case, Bongiorni showed successful production of protein using aprE promoter (column 2, lines 19-22, Examples 1 and 3) and Bongiorni and Wenzel have many common features, such as Bacillus subtilis host, increased production of protein of interest, inducer-independent promoter, application of fed-batch fermentation and chemically defined medium providing motivation to combine these teachings and substitute genetically modified strain in its ability to use inducer in Wenzel teaching with the strain with inducer-independent aprE promoter from Bongiorni teaching and express protein of interest in a chemically defined medium and fed-batch fermentation as described by Wenzel.
Applicant and Dr. Daub argue (addressing pages 7-8 of the Remarks and paragraphs 13-15 of the Declaration), that there is no reason to combine Wenzel and Bongiorni since Bongiorni exemplified the use of aprE promoter in complex medium, mentioned suitable medium but did not specifically direct the skilled person to use aprE promoter with a minimal medium. Dr. Daub argues that it was generally accepted that the aprE promoter is activated only in non-growing stationary cells and when complex nitrogen sources are present and induction of aprE would not have been expected in chemically defined medium. Dr. Daub provides supporting publications in Exhibits A-E. Dr. Daub further argues (addressing paragraphs 17-20 of the Declaration) that references of Schaeffer, Jan and Espinosa-de-los-Monteros provided in the response to arguments section of the previous office action are not relevant to the instant claims and do not support rejection based of Wenzel and Bongiorni. Schaeffer investigates sporulation in defined medium, but does not involve expression of any protein of interest, does not employ nutrient feeding and does not show industrially relevant process. Jan conducted experiments in shake flasks, not in bioreactors, “does not address process optimization or scale-up relevant parameters such as feeding strategies, magnesium supplementation or higher amount of glucose added”. Regarding Espinosa-de-los-Monteros, Dr. Daub argues that: “Overall, due to the use of anaerobic conditions, sporulation-inducing media, low substrate concentrations, a skilled person would not consider this publication when designing an industrial recombinant protein production under aerobic conditions”. These arguments are not persuasive because:
Bongiorni teaches expression of protease under control of aprE as a preferable promoter (column 2, lines 19-22). Although the working examples described complex medium and continuous fermentation, the minimal medium and the fed-batch fermentation were not excluded (column 43, lines 26-29, 49-53). Bongiorni mentions that: “Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of nutrients in the medium” (column 43, lines 49-56).
Bongiorni describes expression of FNA protease in Examples 1 and 3 from the construct containing aprE promoter indicating that Bacillus cells are actively growing and do not undergo sporulation and aprE promoter is activated to express the gene encoding FNA protease. Additionally, Bongiorni describes that modification of the Bacillus cells by mutation degU(Hy)32 increases the level of secreted subtilisin by several fold (column 77, lines 2-6) indicating possibility of further activation of aprE promoter and increase in the production rate of protein of interest in Bacillus cells under control of aprE promoter. This mutation is not related to cells ability to take up or metabolize the inducer molecule since this is the inducer-independent fermentation. The regulation of aprE promoter by degU is taught by Bongiorni (column 30 , line 19) and was mentioned by Dr. Daub and in publications provided by Dr. Daub. For instance Lee in Exhibit A teaches that protein expression from the aprE promoter increased significantly in cells with the degU32 genetic background (p. 18, left column, 1st paragraph) and Kobayashi in Exhibit B teaches that degU mutation prolongs growth and prevents biofilm formation (Abstract).
The prior art of Schaeffer, Jan and Espinosa-de-los-Monteros was not included in the rejection and was used to support that Bacillus cells are able to grow in minimal media cited by Schaeffer and under control of aprE promoter. Jan teaches culturing Bacillus subtilis cells expressing β-galactosidase under aprE native or mutated promoter in Shaeffer’s sporulation media (p. 10, right column, 4th paragraph) and Espinosa-de-los-Monteros, though describes anaerobic culturing, describes fermentation under control of aprE promoter and mentions advantage of the fed-batch fermentation preventing sporulation (Abstract). These references support protein expression under control of aprE promoter in chemically defined medium. Although additional references do not teach certain features mentioned by Dr. Daub, such as bioreactor or industrially relevant process, these features are not within the scope of the claims and references teach fermentation in chemically defined medium and under aprE promoter indicating their relevancy.
In response to Applicant’s and Dr. Daub’s arguments that: “… the instant inventors successful use of the aprE promotor in a chemically defined medium was surprising and would not have been expected by a person of ordinary skill in the art.”, these arguments are not persuasive because:
MPEP 2145 states: “The fact that appellant 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." Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985). In instant case, Wenzel expressly teaches fermentation process for cultivating Bacillus cell involving instant steps and in chemically defined medium as in instant application in inducer-independent system and Bongiorni teaches successful protein production in inducer-independent system with aprE promoter and does not exclude the use of chemically defined medium motivating to substitute the inducer-independent system in Wenzel teaching with that of Bongiorni. Therefore, one of ordinary skill in the art would expect the Bongiorni system with aprE promotor to work in a chemically defined medium of Wenzel.
Assuming arguendo that Applicant has shown unexpected data, claims are not commensurate in scope with the unexpected results. MPEP 716.02: “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the "objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support." In instant case, results presented in Example 1 of the specification are based on a specific Bacillus strain, Bacillus licheniformis ATCC53926, very specific composition of the chemically defined medium including components of the composition such as citric acid, calcium sulphate, monopotassium phosphate, sodium hydroxide, ammonia and specific combination of trace elements and concentrations of these components. These components of the composition and their concentrations are not present in the claim 1 which only requires the Bacillus cell, above 200 g of carbon source and at least 0.1 g of magnesium ions and hence the claims are not commensurate in scope with the unexpected results.
Thus, the 35 U.S.C. 103 rejection is maintained and modified necessitated by amendment of claims.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 10-19 and 25-30 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1, 2, 10, 11, 13-20 and 26-32 of copending Application No. 17/432,203 (reference application) (claim set 05/19/2025). Although the claims at issue are not identical, they are not patentably distinct from each other because.
Claim 1 of instant application is drawn to a fermentation process for cultivating Bacillus cell in chemically defined medium, comprising the steps of (a) providing fermentation medium, (b) inoculating Bacillus cell comprising a gene encoding a protein of interest under the control of an inducer independent aprE promoter and (c) cultivating the Bacillus cell in the fermentation medium with addition of feed solutions containing above 200 g/l carbon source and at least 0.1 g/l magnesium ions wherein Bacillus cell which has not been genetically modified in its ability to take up or metabolize inducer molecule.
Regarding claim 1, claim 1 of reference application recites a fermentation process for cultivating a Bacillus cell in a chemically defined medium comprising the same steps (a) and (b) as in instant application and step (c) of cultivating the Bacillus cell in the fermentation medium with addition of feed solutions containing above 200 g/L carbon source and one or more trace ions. Claim 13 of reference application is drawn to addition of at least 0.1 g/l magnesium ions to the fermentation medium. Reference claim 2 teaches the Bacillus cell which has not been genetically modified in its ability to take up or metabolize inducer molecule. AprE promoter is included in the promoters recited in reference claim 1 and reference claim 26 teaches the fermentation process where the inducer-independent promoter is aprE promoter. Thus, reference claims 1, 2, 13 and 26 anticipate instant claim 1.
Regarding claim 10, reference claim 13 teaches addition of at least 0.1 g/L of magnesium ions to the fermentation medium that reads on instant claim 10 limitation of 0.1-10 g/L magnesium ions added to the fermentation medium.
Regarding claim 11, reference claim 14 teaches magnesium ions provided by magnesium salts, magnesium hydroxide or by their combination, that corresponds to instant claim 11.
Regarding claim 12, reference claim 10 teaches addition of trace elements selected from the same amounts of iron, copper, manganese and zinc as in instant claim 12 and hence reference claim 10 reads on instant claim 12.
Regarding claim 13, reference claim 11 teaches addition of trace elements selected from the same amounts of cobalt, nickel and molybdenum as in instant claim 13 and hence reference claim 11 reads on instant claim 13.
Regarding claim 14, reference claim 15 teaches chemically defined carbon source comprising glucose, that corresponds to instant claim 14.
Regarding claim 15, reference claim 16 teaches chemically defined nutrient sources selected from the group of nitrogen source, sulfur source and potassium source, that corresponds to instant claim 15.
Regarding claim 16, reference claim 17 teaches pH of the fermentation broth adjusted at the same levels as in instant claim 16 and hence reference claim 17 reads on instant claim 16.
Regarding claim 17, reference claim 18 teaches the fermentation process providing a titer of at least 5g/l of protein of interest, that corresponds to instant claim 17.
Regarding claim 18, reference claim 19 teaches the protein of interest is an enzyme that corresponds to instant claim 18.
Regarding claim 19, reference claim 20 teaches the fermentation product secreted by the Bacillus cell into the fermentation broth, that corresponds to instant claim 19.
Regarding claim 25, reference claim 27 teaches the aprE promoter sequence with an HMM score above 50, that corresponds to instant claim 25.
Regarding claim 26, reference claim 28 teaches aprE promoter selected from the group of aprE promoters from various Bacillus species, that correspond to instant claim 26.
Regarding claim 27, reference claim 29 teaches the aprE promoter of the gene of the subtilisin Carlsberg protease with the same sequence identity as in instant claim 27 and hence reference claim 29 reads on instant claim 27.
Regarding claim 28, reference claim 30 teaches aprE promoter sequence comprising the sigma factor A core promoter, that corresponds to instant claim 28.
Regarding claim 29, reference claim 31 teaches the aprE promoter sequence comprising one or more binding motifs of regulatory factors, that corresponds to instant claim 29.
Regarding claim 30, reference claim 32 teaches aprE promoter sequence with the same identity as in instant claim 30 and hence reference claim 32 reads on instant claim 30.
Thus, since instant claims 1, 10-19 and 25-30 and reference claims 1, 2, 10, 11, 13-20 and 26-32 are directed to the same subject matter, they are rejected under obviousness double patenting.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Arguments
Applicant did not provide arguments for Double Patenting rejection. Since the 35 U.S.C. 103 rejection is maintained, the Double Patenting rejection is maintained and modified necessitated by amendment of instant claims (claim set filed 11/06/2025).
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/L.G.K./ Examiner, Art Unit 1653
/SHARMILA G LANDAU/ Supervisory Patent Examiner, Art Unit 1653