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 .
Application Status
The Amendments and Remarks filed 25 February 2026 in response to the Office Action dated 02 December 2025 are acknowledged and have been entered. Claims 1-18 are pending and being examined on the merits.
Priority
The current application is a 371 PCT of application PCT US2019/035148 which claims priority to application 62/682/059.
Claim Rejections - 35 USC § 103
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 1-16 are rejected under 35 U.S.C. 103 as being unpatentable over Mattanovich (WO 2014/139608, published 9/18/2014) in view of Xu (Xu et al. Yeast. 2018; 35: 379–385). This rejection is modified to clarify the motivation and reasonable expectation of success in view of Applicant’s arguments, does not constitute a new ground of rejection, and continues to rely on the same references, findings, and combination set forth in the prior Office Action.
Regarding claims 1, 2 and 6-9, Mattanovich teach materials and methods used to identify a new promoter and to analyze its expression properties in Pichia pastoris (i.e., a K. phaffii ) [pg. 40, lines 16-17]. Mattanovich teach fermenting yeast cells in glycerol and glucose medium (i.e., medium without methanol) [pg. 40, line 19 - pg. 41, line 10]. Mattanovich teach collecting samples at different times by teaching that samples were taken close to the end of the first batch phase, and in limited growth conditions, e.g., using a limited amount of supplemental carbon source [pg. 27, line 30-32]. Mattanovich teaches microarray analysis (i.e., determining mRNA levels) of the samples to determine the strongly expressed genes [pg. 41, line 31-pg. 44]. Mattanovich teaches that the CS1 gene was identified by analysis of transcription strength (by measuring mRNA levels) in production process conditions as the strongest transcribed gene, thereby teaching that transcription strength of other genes were also measured [pg. 27, lines 10-12]. Mattanovich teaches that the pCS1 promoter sequence was identified as a surprisingly strong promoter at high and low growth rates [pg. 28, lines 1-2]. Mattanovich teaches comparative promoter activity studies of the pCS1 promoter in different P. pastoris cells using porcine carboxypeptidase B (CpB) as extracellular expressed reporter gene [example 6]. Mattanovich teaches that the pCS1 promoter was cloned into the pPUZZLE expression vector pPM1aZ30_aMF CpB, resulting in pPM1aZ30_pCS1_aMF CpB [pg. 56, lines 10-11]. Mattanovich teaches culturing the cells in rich medium containing glycerol as carbon source [pg. 55, lines 23-24]. Mattanovich teaches that genes and respective promoters with high transcription strength at high and low growth rate were identified by browsing the microarray data for genes with high signal intensities in both, high and low growth rate conditions [pg. 64, lines 10-13]. Mattanovich teaches that growth and/or production can suitably take place in batch mode, fed-batch mode or continuous mode [pg. 28, lines 30-31]. Mattanovich teaches that any suitable bioreactor can be used, including batch, fed-batch, continuous, stirred tank reactor, or airlift reactor [pg. 28, lines 31-32]. Mattanovich teaches that it is advantageous to provide for the fermentation process on a pilot or industrial scale where the industrial process scale would preferably employ volumina of at least 10 L, specifically at least 50 L, preferably at least 1 m3, preferably at least 10 m3, most preferably at least 100 m3. Mattanovich teaches that production conditions in industrial scale are preferred which refer to e.g. fed batch cultivation in reactor volumes of 100 L to 10 m3 or larger, employing typical process times of several days, or continuous processes in fermenter volumes of approximately 50 - 1000 L or larger [pg. 29, lines 3-6]. Mattanovich teaches that a transformant host cell according to the invention obtained by transforming the cell with the regulatory elements according to the invention and/or the POI genes may preferably first be cultivated at conditions to grow efficiently to a large cell number [pg. 39, lines 18-20].
Mattanovich do not teach making expression constructs comprising multiple promoters and a gene encoding a marker protein and comparing the marker protein expression driven by each of the putative promoters. Mattanovich do not specifically teach culturing the yeast cells comprising the expression constructs in a fermenter in the absence of methanol.
Xu teaches the identification of promoters in Pichia pastoris that could possibly be applied in recombinant protein expression [abstract]. Xu teaches the analysis of data where Pichia pastoris cells were cultured (fermenting) in medium conditions containing different carbon sources (glucose, glycerol and methanol) [see 3.1]. Xu teaches that mRNA levels of each gene was measured under each condition [see. 3.1]. Xu teaches that promoter candidates whose gene expression differed largely under any two carbon sources were selected [see 3.1]. Xu teaches that 16 potentially effective promoter candidates were selected to include 10 non‐methanol‐inducible promoters (glucose‐induced or glycerol‐induced) and one constitutive promoter (see 3.1, Table 1). Xu teaches that each promoter candidate was examined by the expression of intracellular reporter GFP [see 3.2; Fig. 1]. Xu teaches the selection of five promoter candidates for further study where the promoter strengths were compared to identify promoters that are useful in driving protein expression independently of methanol in yeast or promoters that are useful in driving constitutive or inducible protein expression in yeast [see 3.2-3.4].
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method of Mattanovich with the teachings of Xu and where the method comprises making expression constructs comprising multiple promoters and a gene encoding a marker protein and comparing the marker protein expression driven by each of the putative promoters to arrive at the claimed invention. This approach applies the promoter-screening methodology of Xu using the fermentation systems taught by Mattanovich. One of ordinary skill would be motivated to make the modification for the advantage of identifying multiple promoters capable of driving large recombinant protein expression because Mattanovich teaches fermentation-based production environments in which such promoters are intended to function. Furthermore, it would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to culture the yeast cells comprising the expression constructs in a fermenter in the absence of methanol. One of ordinary skill would be motivated to make the modification with a reasonable expectation of success given Mattanovich explicit teaching that production conditions in industrial scale are preferred, which in includes fermentation, and that the transformant host cell according to the invention obtained by transforming the cell with the regulatory elements according to the invention and the POI genes may preferably first be cultivated at conditions to grow efficiently to a large cell number. Furthermore, once promoter-reporter constructs are evaluated under an initial cultivation condition as taught by Xu, it would have been obvious to measure reporter expression under the fermentation conditions taught by Mattanovich and compare the resulting expression values. Such comparison merely represents the use of known analytical techniques to evaluate promoter performance under different cultivation conditions and would have been a routine aspect of characterizing promoter suitability for recombinant protein production. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Mattanovich and Xu are directed to identifying promoters suitable for recombinant protein expression in the same host organism, Pichia pastoris, and for the same purpose of improving recombinant protein production.
Regarding claim 3, Mattanovich teaches that first a batch culture on a basal carbon source, such as glycerol, was employed, followed by a fed batch with limited feed of a supplemental carbon source, such as glucose [pg. 27, line 28-30]. Xu teaches fermenting using glucose and glycerol [see 3.1].
Regarding claim 4, Mattanovich teaches that transcriptome analysis with DNA microarrays revealed specific genes that are strongly active on the supplemental carbon source and in the presence of surplus carbon, i.e., the basal carbon source in excess amount [pg. 27, line 32-34]. Xu teaches that mRNA levels of each gene was measured under each condition [see. 3.1].
Regarding claim 5, Mattanovich teaches that the promoter sequences were verified by sequencing [pg. 46, lines 14-15]. Furthermore, Mattanovich teaches that transcription analysis is quantitative or semi-quantitative, preferably employing qRT-PCR, DNA microarrays, RNA sequencing and transcriptome analysis [pg. 23, lines 29-31].
Regarding claims 10 and 12-14, Mattanovich teaches that typical fermentation times are about 24 to 120 hours [pg. 30, line 33]. Mattanovich teaches that the samples were taken at the end of the glycerol batch phase and in steady state conditions of the glucose chemostat [pg. 41, lines 28-29]. Mattanovich teaches that the batch phase of approximately 25 h reached a dry biomass concentration of approximately 20 g/L, it was followed by a 10 h exponential fed batch with glucose medium, leading to a dry biomass concentration of approximately 50 g/L. Mattanovich teaches that the volume was then reduced to 1.5 L and the chemostat cultivation was started with a feed/harvest rate of 0.15 L h-1, resulting in a constant growth rate of μ= 0.1. The fermentation was terminated 50 h after the chemostat start [pg. 41, lines 16-22].
Regarding claim 11, Mattanovich and Xu do not teach where the different fermentation conditions comprise different media pH as claimed. Mattanovich teaches that the fermentation preferably is carried out at a pH ranging from 3 to 7.5 [pg. 30, line 32]. Mattanovich also teaches the use of medium with different pH values [pg. 50, lines 7-25]. It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method of Mattanovich where the different fermentation conditions comprise different media pH. One of ordinary skill would be motivated to make the modification since Mattanovich teaches that the fermentation preferably is carried out at a pH ranging from 3 to 7.5.
Regarding claim 15, Mattanovich do not specifically teach where the identified putative promoter in the expression construct is 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000 or 5000 bases upstream from a translational start site of the gene, or any number of bases in between. Mattanovich teaches that the promoters are inserted upstream of the start codon of the CpB gene [pg. 56, lines 14-15]. Mattanovich also teaches a promoter fragment that is approximately 1000 bps upstream of the start codon ATG of the respective gene [pg. 66, lines 16-17]. It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method of Mattanovich where the identified putative promoter is 1000 bases upstream from a translational start site of the gene. One of ordinary skill would be motivated to make the modification since Mattanovich teaches promoter fragments 1000 bps upstream of the start codon ATG of the respective gene.
Regarding claim 16, Mattanovich teaches that CpB concentration in the culture supernatant is quantified by an enzymatic assay, based on the conversion of hippuryl-L-arginine to hippuric acid by the CpB [pg. 56, lines 32-33].
Claims 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Mattanovich (WO 2014/139608, published 9/18/2014) in view Xu (Xu et al. Yeast. 2018; 35: 379–385), as applied to claim 1 and 16, and further in view of Vogl (US 2015/0011407 A1, published 1/8/2015). This rejection is maintained.
The teachings of Mattanovich and Xu are discussed above as applied to claims 1 and 16 and similarly apply to claims 17-18.
Mattanovich do not teach where the enzyme is a lipase and the method further comprises testing the enzyme for activity for determining proper folding of protein.
Vogl teaches method of screening or selecting and identifying a bidirectional promoter suitable for expressing at least two GOI in a Pichia pastoris host cell [abstract, 0048]. Vogl teaches that genes were used to optimize the expression of a gene pair with a set of bidirectional promoters [0211]. Vogl teaches that one gene is a lipase B [0211]. Vogl also teaches the use of a folding catalyst before the starting of the GOI expression to assist with protein folding.
It would have been obvious to one ordinary skilled in the art before the effective filing date of the claimed invention to modify the method of Mattanovich where the enzyme is a lipase and the method further comprises testing the enzyme for activity for determining proper folding of protein. The combination of prior art elements according to known methods to yield predictable results supports can support a conclusion of obviousness. See MPEP 2143(I). One of ordinary skill in the art would have a reasonable expectation of success since both Mattanovich and Vogl each teach identifying promoters in yeast cells using a GOI expression construct to analyze promoter function.
Response to Arguments
Applicant's arguments filed 25 February 2026 have been fully considered but they are not persuasive.
Applicant argues that Mattanovich teaches industrial-scale production rather than promoter identification. This argument is not persuasive. The rejection does not rely upon Mattanovich solely for industrial-scale protein production. Rather, Mattanovich is relied upon for teaching identification of highly expressed genes and corresponding promoters under fermentation conditions as well as cultivation of recombinant yeast cells in fermenters and bioreactors. Xu is relied upon for the promoter-screening methodology involving promoter-reporter constructs and comparative promoter analyses. The rejection is based upon the combined teachings of Mattanovich and Xu.
Applicant further argues that claim 1 recites a singular multi-stage method for identifying promoters under scale-relevant fermentation conditions and that the prior art does not teach such scalable systems. This argument is not persuasive. Claim 1 does not recite “scale-relevant fermentation conditions,” “industrial-scale fermentation,” “pilot-scale fermentation,” or any particular fermentation scale. Rather, claim 1 recites culturing yeast cells comprising expression constructs in a fermenter, determining marker protein expression, and comparing expression levels. Mattanovich teaches cultivation of recombinant yeast cells in fermenters and bioreactors, while Xu teaches promoter screening through reporter-gene expression analysis. The combination teaches or renders obvious the claimed method.
Applicant additionally argues that Mattanovich and Xu do not teach or suggest scalable systems or methods for identifying promoters. This argument is not persuasive because the claims do not require any particular scalable system beyond the recited fermenter cultivation step. Mattanovich expressly teaches fermentation processes performed in fermenters and bioreactors, and Xu teaches identifying and comparing promoter candidates using reporter-gene expression. Applying the promoter evaluation techniques of Xu under the fermentation conditions taught by Mattanovich would have been an obvious use of known techniques for their intended purpose.
Applicant further argues that paragraph [0054] of the specification states that promoter performance observed in shaker flask studies may not correlate with performance in scale-relevant fermentation conditions and therefore a person of ordinary skill in the art would not have had a reasonable expectation of success. This argument is not persuasive. The cited disclosure merely indicates that promoter performance may vary under different cultivation conditions. The claims do not require a particular expression level, promoter ranking, successful scale-up result, or any correlation between cultivation conditions. Rather, the claims merely require measuring and comparing expression levels. A reasonable expectation of success does not require predictability of the ultimate results, but only a reasonable expectation that the experiment can be successfully performed. Because promoter activity measurements and reporter-gene assays in fermenters were known in the art, one of ordinary skill in the art would have reasonably expected to obtain expression measurements and compare them as claimed.
Applicant further argues that neither Mattanovich nor Xu teach relevant experiments or scaling. This argument is not persuasive. The rejection does not require either reference to expressly disclose the precise sequence of experiments recited in the claims. Mattanovich teaches fermentation and bioreactor cultivation of recombinant yeast cells, while Xu teaches promoter screening using promoter-reporter constructs and comparative expression analysis. The claimed method represents the application of these known techniques in combination and would have been obvious to one of ordinary skill in the art.
With respect to claims 17-18, Applicant argues that Vogl does not cure the alleged deficiencies of Mattanovich and Xu. However, Vogl is relied upon only for the additional enzyme and enzyme activity limitations recited in claims 17-18. Applicant does not separately traverse the Examiner’s findings regarding Vogl. Therefore, because the rejection of claim 1 is maintained and Vogl teaches the additional limitations of claims 17-18, the rejection of claims 17-18 is likewise maintained.
Conclusion
No claims allowed.
THIS ACTION IS MADE FINAL. 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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/TIFFANY NICOLE GROOMS/Examiner, Art Unit 1637
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