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 claims benefit of priority to Provisional Application 62/960,831 filed on 01/14/2020 and is also a 371 of PCT/US2021/013171 filed on 01/13/2021. For the purposes of applying prior art, the effective filing date of the claimed invention is 01/14/2020.
Amendments and Claim Status
In the reply filed on 01/23/2026, Applicant amended claims 1, 21, 27, 38, 40, 45, 50, 54-57 and 61 and canceled claims 6, 7, 42-44 and 47.
Claims 1-2, 4, 11, 13-16, 18-19, 21, 24-27, 30, 38, 40, 45, 49-58 and 60-61 are currently pending and under examination.
Maintained Rejections (with modifications as necessitated by claim amendment)
Claim Objections
Claim 16 is objected to because of the following informalities: The ‘is’ should be ‘are.’ The claim is grammatically incorrect as-amended. Appropriate correction is required.
Claim 61 is objected to because of the following informalities: The claim recites “wherein the first carbon source is glucose” in line 5 of the claim and then subsequently recites “wherein the first carbon source and the second carbon source are glucose” in lines 12-13. The second recitation of glucose being the first carbon source is redundant and unnecessary. Appropriate correction is required.
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 1-2, 4, 11, 13-16, 18-19, 21, 24-27, 30, 38, 40, 45, 49-58 and 60-61 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 1 recites the limitation "for the remainder of the fermentation" in lines 12-13. There is insufficient antecedent basis for this limitation in the claim. The claim does not recite ‘fermentation’ prior to the above recited limitation. The claim recites culturing and culture, but not fermentation. Thus, there is insufficient antecedent basis for this limitation in the claim.
Claim 27 recites the limitation “"for the remainder of the fermentation" in line 14. There is insufficient antecedent basis for this limitation in the claim. The claim does not recite ‘fermentation’ prior to the above recited limitation. The claim recites culturing and culture, but not fermentation. Thus, there is insufficient antecedent basis for this limitation in the claim.
Claim 61 recites the limitation “"for the remainder of the fermentation" in lines 17-18. There is insufficient antecedent basis for this limitation in the claim. The claim does not recite ‘fermentation’ prior to the above recited limitation. The claim recites culturing and culture, but not fermentation. Thus, there is insufficient antecedent basis for this limitation in the claim.
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 1-2 ,4, 11, 13-16, 18-19, 21, 24-27, 30, 38, 40, 45, 49-55, 57-58 and 60-61 are rejected under 35 U.S.C. 103 as being unpatentable over Franano et al. (US 2009162343 A1, 12/03/2008) in view of Files et al. (10/04/2001, Enzyme and Microbial Technology) and further in view of Paulova et al. (Journal of Biotechnology, 11/22/2011) (Of Record).
Regarding claims 1-2 and 54-55, it is noted claim 1 recites ‘comprising the steps of’ which is open language, meaning the method allows for incorporation of unclaimed steps or components.
Franano et al. disclose recombinant methods for producing therapeutically useful elastase proteins (See entire document, more specifically, the Abstract). Further, a Pichia pastoris host cell genetically engineered to encode an open reading frame comprising a yeast α-factor signal peptide or a porcine elastase signal peptide operably linked to a human type I elastase proenzyme sequence, reading on a yeast cell genetically modified to express a recombinant protein (Paragraph [0060]). The invention further provides methods for producing an immature or mature elastase protein comprising culturing a host cell (Paragraph [0061]). The fermentation procedure started with a fed-batch of glycerol (i.e., first carbon source) at a pH of 5.7 (i.e., first pH) at 28°C (i.e., first temperature) with the pH being controlled by 10% phosphoric acid, reading on phosphate ions, and 30% ammonium sulfate, reading on ammonium, solutions (Paragraph [0399]). After the glycerol batch was depleted, 50% glycerol was fed at 131 g/h until the wet cell weight reached between 200 g/L to 300 g/L (Paragraph [0399]). Once the desired wet cell weight was reached, induction was initiated with a methanol (i.e., second carbon source) bolus (Paragraph [0399]). After the methanol bolus was depleted, induction was continued by the addition of limiting amounts of methanol with a constant feed rate and the pH of the fermentation broth was changed to 5.5 and the temperature changed to 22°C (Paragraph [0399]).
Franano et al. further disclose culture conditions for producing the immature and mature proteins of the invention, particularly for the host cell Pichia pastoris, include a period of growth at a low pH. In specific embodiments the low pH is a pH of 2-6, a pH of 2-5, and pH of 3-6, a pH of 3-5, and pH of 4-6, a pH of 3-4 or any range whose upper and lower limits are selected from any of the foregoing values. At the end of the culture period, the pH of the culture can be raised, preferably to a pH of 7-11, most preferably to a pH of 8 (Paragraph [0214]). If a methanol-inducible promoter is being used, conditions for producing the protein may comprise a period of methanol induction (Paragraph [0215]). When it is desired to circumvent protein degradation, culture conditions for proelastase expression comprise a period of growth and induction at the lower end of the temperature range for the host cell, however, the growth and induction do not have to be performed at the same temperature, growth can be performed at 28°C (i.e., a first temperature) and induction can be performed at 22°C (i.e., a second temperature) (Paragraph [0218]). The activation of an auto-activated proelastase protein may be initiated by raising the pH of the solution containing the auto-activated proelastase protein to a pH between 7 and 11, most preferably 8 (Paragraph [0219]).
Franano et al. do not explicitly disclose changing the first pH to a second pH at the time the feeding of step (b) is initiated, wherein the second pH is 0.5 to 2 pH units higher than the first pH.
However, it appears when Franano et al. disclose raising the pH to a pH between 7 and 11, most preferably 8, to activate the proelastase protein, it is done at the beginning of the induction phase and not after the entire culturing process is complete. It appears when Franano et al. say ‘the end of the culture phase’, they mean the end of the growth phase and the beginning of the induction phase. Considering the induction phase is geared towards promoting production of the product of interest, this reaffirms the pH change disclosed by Franano et al. would be at the end of the growth phase and the beginning of the induction phase. This is further supported by Franano et al. disclosing a pH change from 5.7 to 5.5 for the induction phase as that reveals if a pH change were to occur, it would be after the growth phase, at the beginning of the induction phase (Paragraph [03999]). As the initial pH of the fermentation was 5.7, raising the pH to between 7 and 11, reads on the limitation of the second pH being 2.0 units higher than the first pH.
Additionally, Files et al. disclose a fermentation process for producing high-levels of recombinant human cystatin-C, reading on a recombinant protein, in Pichia pastoris (See entire document, more specifically, the Abstract). It is known that P. pastoris can grow over a wide pH range, from 3 to 7, with only a minimal effect on growth rate, but, pH has been seen to have a significant effect on secretion of recombinant proteins due to protease activity in the fermentation broth (Page 337, Right Column, Last Paragraph). Files et al. conducted multiples experiments to determine which pH, between 5 and 7, allowed for the production of the highest amount of recombinant protein (Page 338, Left Column, First Paragraph). Files et al. further disclose cultures of the P. pastoris were maintained at a pH of 5.0 for the batch phase of growth, which lasted about 22 hours, and then during the glycerol fed-batch phase, the pH was adjusted to the desired value and maintained at that pH for the remainder of the experiment (Page 338, Left Column, Paragraph 2). The glycerol fed-batch was the start of the induction period, as glycerol and methanol were used during the induction (Page 338, Left Column, Last Paragraph). Files et al. found the optimum pH for expression was 6.0 (Page 339, Right Column, Conclusions).
Thus, even if Franano et al. did not mean the end of the growth phase and the beginning of the induction phase, it would have been obvious to one of ordinary skill in the art to raise the first pH 0.5 to 2.0 pH units to a second pH at the time of feeding the second carbon source, the beginning of the induction period, in the method of Franano et al. since it was known in the art to change the pH at the beginning of the induction phase as taught by Files et al.
Regarding the new limitations added to claim 1 that were limitations of dependent claims 6-7, Franano et al. do not disclose the first temperature is changed to the second temperature linearly over a period of 30 minutes to two hours.
However, as discussed above, Franano et al. disclose when it is desired to circumvent protein degradation, culture conditions for proelastase expression comprise a period of growth and induction at the lower end of the temperature range for the host cell.
As such, since temperature affects protein degradation, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed temperature change rate and time motivated by the desire to circumvent protein degradation as taught by Franano et al. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Regarding claims 4 and 38, Franano et al. do not disclose wherein the first temperature is 30°C and the second temperature is 26°C.
However, as discussed above, Franano et al. disclose a first temperature of 28°C and a second temperature of 22°C. The claimed first and second temperature would have been obvious since the claimed temperatures are so close to the first and second temperature disclosed in the prior art that one would have expected them to have the same properties. A prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. See MPEP 2144.05(I).
Regarding claim 11, Franano et al. do not disclose wherein the first pH is 5.0 and the second pH is 6.0.
However, as discussed above, Files et al. disclose the initial batch phase (i.e., the growth phase) was maintained at the pH of 5.0 and at the beginning of the induction phase was adjusted to 6.0 (Page 338, Left Column, Paragraph 2). Therefore, it would have been obvious to one of ordinary skill in the art to have a first pH of 5.0 for the growth period and a second pH of 6.0 for the induction period in the method of Franano et al. because these two values were known and effective pH values for growth and induction for producing high levels of recombinant proteins as taught by Files et al.
Regarding claims 13-14, Franano et al. do not disclose wherein the first pH is changed to the second pH linearly over a period of time, or wherein that time period is 30 minutes to two hours.
However, as discussed above, Franano et al. disclose a first pH of 5.7 and second pH between 7 and 11. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal pH ranges because pH is an art-recognized result-effective variable having the ability to circumvent autoactivation of proelastases or activate elastase proteins as disclosed by the prior art above. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed pH motivated by the desire to circumvent autoactivation or to activate the proteins as taught by Franano et al. Additionally, since pH affects activation, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed pH change rate and time motivated by the desire to circumvent autoactivation of proelastases or activate elastase proteins as taught by Franano et al. Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Regarding claims 15-16 and 49-50, Franano et al. do not disclose the first and second carbon source are the same or wherein the carbon source is glucose.
However, Paulova et al. disclose a method for producing recombinant trypsinogen in Pichia pastoris (See entire reference, More specifically, Title). Paulova et al. further disclose a defined mineral medium contained (11.7 ± 1.1) g of carbon source (i.e., only glucose for the starting batch cultures, and only glucose, only methanol, or a mixture of glucose and methanol (60:40) for continuous cultures) (Paragraph 2.2, Page 181).
Generally, it is prima facie obvious to select a known material for incorporation into a composition, based on its recognized suitability for its intended use. See MPEP 2144.07. As such, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have used glucose in the batch and induction phases in the method of Franano et al./Files et al. since the method does not require a specific carbon source and glucose is a known and effective carbon source for both phases as taught by Paulova et al.
Regarding claims 18-19, Franano et al. do not disclose wherein a concentration of potassium ions is higher than a concentration of sulfate ions and/or phosphate ions in the culture medium or wherein a molar ratio of potassium ions to phosphate ions is 1.5:1 or 2.8:1 and a molar ratio of potassium ions to sulfate ions is 2:1 to 4:1.
However, Franano et al. disclose a BKGY solution comprising 10 g/L glycerol, 13.4 g/L yeast nitrogen base with ammonium sulfate, 20 g/L soy peptone, 10 g/L yeast extract, 0.4 mg/L biotin in 0.1 M potassium-phosphate buffer (Paragraph [0371]).
“Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal concentrations of potassium ions, sulfate ions and phosphate ions because all are art-recognized result-effective variables that comprise culture mediums. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed concentrations and molar ratios of potassium ions, sulfate ions and phosphate ions motivated by the desire to create an optimum culture medium as potassium ions, sulfate ions and phosphate ions are all components of culture mediums as taught by Franano et al.
Regarding claim 21, Franano et al. do not disclose wherein an ammonium concentration in the first and second culture is 5 mM to 80 mM, a calcium concentration in the first and second culture is 0.5 mM to 3 mM or a magnesium concentration in the first and second culture is 5 mM to 30 mM.
However, as discussed above, Franano et al. do disclose ammonium sulfate to aid in controlling pH (Paragraph [0399]). Additionally, Files et al. disclose their fermentation medium contained:
10 g l−1 yeast extract, 20 g l−1 peptone, and 20 g l−1 glycerol. 100 ml of inoculum was added to 1.0 liter of Basal Salts Medium (26.7 ml l−1 85% o-phosphoric acid, 0.93 g l−1 calcium sulfate·2H2O, 18.2 g l−1 potassium sulfate, 14.9 g l−1 magnesium sulfate·7H2O, 4.13 g l−1 potassium hydroxide, 40 g l−1 glycerol) and 4.35 ml l−1 of PTM1 Trace Salts (6.0 g l−1 cupric sulfate·5H2O, 0.08 g l−1 sodium iodide, 3.0 g l−1 manganese sulfate·H2O, 0.2 g l−1 sodium molybdate·2H2O, 0.02 g l−1 boric acid, 0.5 g l−1 cobalt chloride, 20.0 g l−1 zinc chloride, 65.0 g l−1 ferrous sulfate·7H2O, 5.0 ml sulfuric acid, and 0.2 g l−1 d-biotin) (Page 337, Left Column, Paragraph 2).
“Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal concentrations of ammonium, calcium and magnesium because all are art-recognized result-effective variables that are integral components of cultures. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed concentrations of ammonium, calcium and magnesium motivated by the desire to create an optimum culture as ammonium, calcium and magnesium are all components of cultures as taught by Files et al.
Regarding claim 24, as discussed above, Franano et al. disclose the methanol feed was a constant methanol feed (Paragraph [0399]).
Regarding claims 25-26 and 52-53, Franano et al. do not disclose wherein a feed rate of the feeding of the second carbon source is increased exponentially from an initial feed rate to a maximum feed rate between 15 to 22 g/l/h or wherein the feed rate is decreased linearly from the maximum feed rate to an intermediate feed rate between 5 to 8 g/l/h and then decreased to a final feed rate.
However, as discussed above, Franano et al. do disclose induction was initiated with methanol (i.e., second carbon source) bolus of 0.025 mL per gram of wet biomass (Paragraph [0399]). After the methanol bolus was depleted, induction was continued by the addition of limiting amounts of methanol with a constant feed rate of 0.0034 g methanol/g wet cell weight/hr (Paragraph [0399]).
Thus, while not explicitly stated, the bolus of methanol reads on the second carbon source being increased exponentially from an initial feed rate. Assuming an initial feed rate of 0, adding a bolus of methanol would be an exponential increase. After the addition of the bolus of methanol, methanol was added with a constant feed rate of 0.0034 g methanol/g wet cell weight/hr, reading on the feed rate being decreased from the maximum to an intermediate feed rate. The end of the induction phase would end the methanol feed, reading on being decreased to a final feed rate, which would be zero. Regarding the specific numerical values for the rates, “where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal feed rates of methanol because methanol is an art-recognized result-effective variable for inducing production of recombinant proteins. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed feed rates of methanol motivated by the desire to effectively produce recombinant proteins as taught by Franano et al. Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Regarding claims 27 and 61, Franano et al. disclose recombinant methods for producing therapeutically useful elastase proteins (See entire document, more specifically the Abstract). Further, a Pichia pastoris host cell genetically engineered to encode an open reading frame comprising a yeast α-factor signal peptide or a porcine elastase signal peptide operably linked to a human type I elastase proenzyme sequence, reading on a yeast cell genetically modified to express a recombinant protein (Paragraph [0060]). The invention further provides methods for producing an immature or mature elastase protein comprising culturing a host cell (Paragraph [0061]). The fermentation procedure started with a fed-batch of glycerol (i.e., first carbon source) at a pH of 5.7 (i.e., first pH) at 28°C (i.e., first temperature) with the pH being controlled by 10% phosphoric acid, reading on phosphate ions, and 30% ammonium sulfate, reading on ammonium, solutions (Paragraph [0399]). After the glycerol batch was depleted, 50% glycerol was fed at 131 g/h until the wet cell weight reached between 200 g/L to 300 g/L (Paragraph [0399]). Once the desired wet cell weight was reached, induction was initiated with a methanol (i.e., second carbon source) bolus (Paragraph [0399]). After the methanol bolus was depleted, induction was continued by the addition of limiting amounts of methanol with a constant feed rate and the pH of the fermentation broth was changed to 5.5 and the temperature changed to 22°C (Paragraph [0399]).
Franano et al. further disclose culture conditions for producing the immature and mature proteins of the invention, particularly for the host cell Pichia pastoris, include a period of growth at a low pH. In specific embodiments the low pH is a pH of 2-6, a pH of 2-5, and pH of 3-6, a pH of 3-5, and pH of 4-6, a pH of 3-4 or any range whose upper and lower limits are selected from any of the foregoing values. At the end of the culture period, the pH of the culture can be raised, preferably to a pH of 7-11, most preferably to a pH of 8 (Paragraph [0214]). If a methanol-inducible promoter is being used, conditions for producing the protein may comprise a period of methanol induction (Paragraph [0215]). When it is desired to circumvent protein degradation, culture conditions for proelastase expression comprise a period of growth and induction at the lower end of the temperature range for the host cell, however, the growth and induction do not have to be performed at the same temperature, growth can be performed at 28°C (i.e., a first temperature) and induction can be performed at 22°C (i.e., a second temperature) (Paragraph [0218]). The activation of an auto-activated proelastase protein may be initiated by raising the pH of the solution containing the auto-activated proelastase protein to a pH between 7 and 11, most preferably 8 (Paragraph [0219]).
Franano et al. do not explicitly disclose the cell culture comprises potassium ions, sulfate ions and phosphate ions, changing the first pH to a second pH at the time the feeding of step (b) is initiated, wherein a concentration of the potassium ions in the culture medium of step (a) is higher than a concentration of the sulfate ions and/or the phosphate ions or wherein a molar ratio of potassium ions to phosphate ions is 1.5:1 to 2.8:1 and a molar ratio of potassium ions to sulfate ions is 2:1 to 4:1.
However, as disclosed above, Franano et al. disclose a BKGY solution comprising 10 g/L glycerol, 13.4 g/L yeast nitrogen base with ammonium sulfate (i.e., sulfate ions), 20 g/L soy peptone, 10 g/L yeast extract, 0.4 mg/L biotin in 0.1 M potassium-phosphate buffer (i.e., potassium and phosphate ions) (Paragraph [0371]).
“Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal concentrations of potassium ions, sulfate ions and phosphate ions because all are art-recognized result-effective variables that comprise culture mediums. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed concentrations and molar ratios of potassium ions, sulfate ions and phosphate ions motivated by the desire to create an optimum culture medium as potassium ions, sulfate ions and phosphate ions are all components of culture mediums as taught by Franano et al.
Additionally, as discussed above, it appears when Franano et al. disclose raising the pH to a pH between 7 and 11, most preferably 8, to activate the proelastase protein, it is done at the beginning of the induction phase, not once the entire culturing process is complete. It appears when Franano et al. say the end of the culture phase, they mean the end of the growth phase and the beginning of the induction phase. Considering the induction phase is geared towards promoting production of the product of interest, this reaffirms the pH change disclosed by Franano et al. would be at the end of the growth phase and the beginning of the induction phase. This is further supported by Franano et al. disclosing a pH change from 5.7 to 5.5 for the induction phase (Paragraph [03999]). As the initial pH of the fermentation was 5.7, raising the pH to between 7 and 11, reads on the limitation of the second pH being 2.0 units higher than the first pH.
Files et al. disclose a fermentation process for producing high-levels of recombinant human cystatin-C, reading on a recombinant protein, in Pichia pastoris (See entire document, more specifically, the Abstract). It is known that P. pastoris can grow over a wide pH range, from 3 to 7, with only a minimal effect on growth rate, but, pH has been seen to have a significant effect on secretion of recombinant proteins due to protease activity in the fermentation broth (Page 337, Right Column, Last Paragraph). Files et al. conducted multiples experiments to determine which pH, between 5 and 7, allowed for the production of the highest amount of recombinant protein (Page 338, Left Column, First Paragraph). Files et al. further disclose cultures of the P. pastoris were maintained at a pH of 5.0 for the batch phase of growth, which lasted about 22 hours, and then during the glycerol fed-batch phase, the pH was adjusted to the desired value and maintained at the pH for the remainder of the experiment (Page 338, Left Column, Paragraph 2). The glycerol fed-batch was the start of the induction period, as glycerol and methanol were used during the induction (Page 338, Left Column, Last Paragraph). Files et al. found the optimum pH for expression was 6.0 (Page 339, Right Column, Conclusions).
Thus, even if Franano et al. did not mean the end of the growth phase and the beginning of the induction phase, it would have been obvious to one of ordinary skill in the art to raise the first pH 0.5 to 2.0 pH units to a second pH at the time of feeding the second carbon source, the beginning of the induction period, in the method of Franano et al. since it was known in the art to change the pH at the beginning of the induction phase as taught by Files et al.
Regarding the new limitations to claims 27 and 61, Franano et al. do not disclose an ammonium concentration in the culture medium is 5 mM to 80 mM, a calcium concentration in the culture medium is 0.5 mM to 3 mM and a magnesium concentration in the culture medium is 5 mM to 30 mM, the first temperature is changed to the second temperature linearly over a period of time or wherein that period of time is 30 minutes to two hours or wherein the first pH is changed to the second pH linearly over a period of time, or wherein that time period is 30 minutes to two hours, or wherein the first and second carbon source are glucose.
Additionally, as discussed above, Franano et al. disclose a first pH of 5.7 and second pH between 7 and 11. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal pH ranges because pH is an art-recognized result-effective variable having the ability to circumvent autoactivation of proelastases or activate elastase proteins as disclosed by the prior art above. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed pH motivated by the desire to circumvent autoactivation or to activate the proteins as taught by Franano et al. Additionally, since pH affects activation, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed pH change rate and time motivated by the desire to circumvent autoactivation of proelastases or activate elastase proteins as taught by Franano et al. Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Additionally, as discussed above, Franano et al. disclose when it is desired to circumvent protein degradation, culture conditions for proelastase expression comprise a period of growth and induction at the lower end of the temperature range for the host cell.
Moreover, Paulova et al. disclose a method for producing recombinant trypsinogen in Pichia pastoris (See entire reference, More specifically, Title). Paulova et al. further disclose a defined mineral medium contained (11.7 ± 1.1) g of carbon source (i.e., only glucose for the starting batch cultures, and only glucose, only methanol, or a mixture of glucose and methanol (60:40) for continuous cultures) (Paragraph 2.2, Page 181).
Thus, since temperature affects protein degradation, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed temperature change rate and time motivated by the desire to circumvent protein degradation as taught by Franano et al. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Generally, it is prima facie obvious to select a known material for incorporation into a composition, based on its recognized suitability for its intended use. See MPEP 2144.07. As such, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have used glucose in the batch and induction phases in the method of Franano et al./Files et al. since the method does not require a specific carbon source and glucose is a known and effective carbon source for both phases as taught by Paulova et al.
Regarding claim 30, Franano et al. do not disclose wherein an ammonium concentration in the first and second culture is 5 mM to 80 mM, a calcium concentration in the first and second culture is 0.5 mM to 3 mM and a magnesium concentration in the first and second culture is 5 mM to 30 mM.
However, as discussed above, Franano et al. do disclose ammonium sulfate to aid in controlling pH (Paragraph [0399]). Additionally, Files et al. disclose their fermentation medium contained:
10 g l−1 yeast extract, 20 g l−1 peptone, and 20 g l−1 glycerol. 100 ml of inoculum was added to 1.0 liter of Basal Salts Medium (26.7 ml l−1 85% o-phosphoric acid, 0.93 g l−1 calcium sulfate·2H2O, 18.2 g l−1 potassium sulfate, 14.9 g l−1 magnesium sulfate·7H2O, 4.13 g l−1 potassium hydroxide, 40 g l−1 glycerol) and 4.35 ml l−1 of PTM1 Trace Salts (6.0 g l−1 cupric sulfate·5H2O, 0.08 g l−1 sodium iodide, 3.0 g l−1 manganese sulfate·H2O, 0.2 g l−1 sodium molybdate·2H2O, 0.02 g l−1 boric acid, 0.5 g l−1 cobalt chloride, 20.0 g l−1 zinc chloride, 65.0 g l−1 ferrous sulfate·7H2O, 5.0 ml sulfuric acid, and 0.2 g l−1 d-biotin) (Page 337, Left Column, Paragraph 2).
“Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges through routine experimentation.” See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). As such, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to determine all operable and optimal concentrations of ammonium, calcium and magnesium because all are art-recognized result-effective variables that are integral components of cultures. Therefore, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed concentrations of ammonium, calcium and magnesium motivated by the desire to create an optimum culture as ammonium, calcium and magnesium are all components of cultures as taught by Files et al.
Regarding claim 40, Franano et al. do not disclose wherein the first temperature is changed to the second temperature linearly over a period of time or wherein that period of time is 30 minutes to two hours.
However, as discussed above, Franano et al. disclose when it is desired to circumvent protein degradation, culture conditions for proelastase expression comprise a period of growth and induction at the lower end of the temperature range for the host cell.
As such, since temperature affects protein degradation, it would have taken no more than the relative skills of one of ordinary skill in the art through routine experimentation to have arrived at the claimed temperature change rate and time motivated by the desire to circumvent protein degradation as taught by Franano et al. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP 2144.05(II)(A). See also In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Additionally, it is noted, ‘linearly’ does not meaningfully limit the claim as the change would produce a line, be ‘linear,’ on a graph regardless of how fast or slow the change occurred.
Regarding claim 51, Franano et al. disclose a constant feed rate of methanol (Paragraph [0399]).
Regarding claim 57, as discussed above, Franano et al. disclose the use of a methanol promoter (Paragraph [0063]).
Regarding claims 58 and 60, as discussed above, Franano et al. disclose methods for producing an elastase (Abstract). Elastases are proteases.
Claims 1-2, 4, 11, 13-16, 18-19, 21, 24-27, 30, 38, 40, 45, 49-58 and 60-61 is are rejected under 35 U.S.C. 103 as being unpatentable over Franano et al. (US 2009162343 A1, 12/03/2008) in view of Files et al. (10/04/2001, Enzyme and Microbial Technology) and Paulova et al. (Journal of Biotechnology, 11/22/2011) (Of Record), and further in view of Lesnicki et al. (US 20190119360 A1, 04/25/2019).
The teachings of Franano et al., Files et al. and Paulova et al. are discussed above.
Regarding claim 56, neither Franano et al., Files et al. nor Paulova et al. disclose the recombinant protein is expressed under the control of a promoter which is not inducible by methanol.
However, Lesnicki et al. disclose methods for producing proteins with a high yield and purity in yeast cells, more specifically, Pichia pastoris (See entire reference, More specifically the Abstract). Further, suitable promoters for Pichia include CUP1 which is induced by the level of copper in the medium and also glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter which is a constitutive promoter (Paragraph [0287]).
Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized CUP1 or GAP as the promoter in the method of Franano et al./Files et al. because they are both known and effective promoters for producing protein in Pichia as taught by Lesnicki et al. with a reasonable amount of success.
USC § 103 - Response to Arguments
Applicant's arguments filed 01/23/2026 have been fully considered but they are not persuasive.
Applicant argued on Page 10 that the Examiner did not account for the synergistic effect achieved by the specific timing and coordinating of changing the temperature and pH at the time the second feeding was initiated. Applicant continued on Page 5 to argue that the claimed method produces unexpected results and points to paragraph [0005] and Table 1 of the instant Specification.
It is noted that paragraph [0005] simply states “The present inventors have found cell culture parameters which lead to a particularly high titer of recombinant protein expressed by yeast cell. Furthermore, these cell culture parameters enhance the scalability of the cell culture process” and Table 1 shows the amino acid substitutions or insertions of lipases suitable for the instant invention. It is unclear how Table 1 relates to synergistic effects or unexpected results. Also, paragraph [0005] does not provide evidence of synergy or an unexpected results as it is a simple statement with no corresponding data. A showing of unexpected results must be based on evidence, not argument or speculation. See MPEP 2145. Thus, Applicant has not provided any evidence of a synergistic effect or unexpected results.
The remainder of Applicant’s arguments state the dependent claims are novel and nonobvious because the independent claims they depend upon are novel and nonobvious.
The Examiner respectfully disagrees. It remains the Examiner’s position that the claims are obvious over the prior art of record for the reasons set forth in the rejection above.
Conclusion
Claims 1-2, 4, 11, 13-16, 18-19, 21, 24-27, 30, 38, 40, 45, 49-58 and 60-61 are rejected.
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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/A.T.W./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653