DETAILED ACTION
All objections and rejections raised in prior Office Actions are withdrawn unless restated below.
Claims 8-9 and 17-19 remain withdrawn.
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 .
Claim Interpretation
Claims 1 and 22 are recites the open transitional phrase comprising such that the methods of the claims are open to unrecited steps.
Claims 1 and 22 recites no requirement regarding a beginning weight average molecular weight prior to a hydrolysis reaction. As such, starting with a poly(3-hydroxyalkanoate) (PHA) with a weight average molecular weight of 500,000 Da to produce a PHA with weight average molecular weight of 475,000 Da by hydrolysis as recited, for example, meets the claim feature of a “method for producing poly(3-hydroxyalkanoate) having a weight average molecular weight of from 100,000 to 700,000 Da” and “a weight average molecular weight of the poly(3-hydroxyalkanotate) in the microbial cell is adjusted to 100,000 to 700,000 by the hydrolysis reaction.”
Claim Objections
Claims 1 and 22 are objected to because of the following informalities:
In claim 1, line 5, “the pH” should preferably omit a definite article “the.” While the culture solution is considered to inherently have a pH, it is preferably for a definite article to be omitted upon first recitation of a claim term.
In claim 22, units are required for the recited weight average molecular weight, which is understood to be Daltons.
Appropriate correction is required.
Duplicate Claim Warning
Applicant is advised that should claim 10 be found allowable, claim 12 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). Claims 10 and 12 have minor verbal differences, such as, “wherein the enzyme treatment in (b)” in claim 10 and “a time for the enzyme treatment in (b)” in claim 12. Regardless, of such slight difference in wording, the claims both cover the same subject matter of the enzyme treatment step (b) being performed from 1 to 8 hours as to be so close in content that they both cover the same thing.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 21 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 21 depends from claim 1. Claim 1 requires a hydrolysis reaction be performed on the PHA. Any hydrolysis reaction will necessarily decrease the molecular weight of PHA including weight average molecule weight of the PHA by some amount, even if of a small magnitude. That is, weight average molecular weight is the average molecular weight of a polymer sample, wherein any hydrolysis of such sample will necessarily lower the average molecular weight regardless of which polymer species among a mixture are hydrolyzed. As such, claim 21 does not further limit the subject matter of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
Claim(s) 1, 7, 10-12, 15 and 21-22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yanagita et al. (WO 2010/116681 A1) further in view of Maruyama (WO 2008/010296 A1), Jiang et al. (Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture, AMB Express 5, 2015, 5) and Yu et al. (Characterization of low molecular weight poly(beta-hydroxybutyrate)s from alkaline and acid hydrolysis, Polymer 41, 2000, 1087-98). Machine translations of WO 2010/116681 A1 and WO 2008/010296 A1 are provided and cited herein.
Yanagita, abstract, states:
A high-purity PHA (polyhydroxyalkanoate) is separated from PHA-containing microbial cells by a commercially suitable, simple and low-cost method with a reduced number of processes, without causing a significant decrease in the molecular weight. Specifically disclosed is a method for collecting a polyhydroxyalkanoate, which comprises: (a) a step wherein an enzymatic treatment is performed by adding an enzyme and an alkali and/or a surfactant into an aqueous suspension of polyhydroxyalkanoate-containing microbial cells, thereby obtaining an enzymatic treatment liquid; (b) a step wherein the enzymatic treatment liquid is subjected to a physical crushing process under basic conditions in the presence of a surfactant, so that the cells are crushed and cell substances other than polyhydroxyalkanoate in the cells are solubilized or emulsified, thereby obtaining a polyhydroxyalkanoate suspension; and (c) a step wherein the polyhydroxyalkanoate is separated from the polyhydroxyalkanoate suspension.
Example 1 of Yanagita states:
Ralstonia eutropha KNK-005 described in [0049] of WO 08/010296 A1 is cultured by the method described in [0050] to [0053], and 3-hydroxybutyrate and 3-hydroxyhexanoate are cultured. A cell culture broth containing polyhydroxyalkanoate (PHBH) composed of an ate copolymer was obtained. The weight average molecular weight of PHBH at the end of the culture was 1,840,000. Ralstonia and Eutropha are now classified as Capriavidas Necka.
1000 g of the obtained bacterial cell culture solution was sterilized at 70 to 75 ° C. for 1 hour, cooled to 50 ° C., 4.0 g of sodium dodecyl sulfate was added, and sodium hydroxide was added so that the pH was 8.5 [i.e. (a) adding sodium hydroxide to a culture solution comprising microbial cells comprising poly(3-hydroxybutyrate-co-3-hydroxyhexanote) to adjust the pH of the culture solution to pH 8.5]. Alcalase 2.5L (Novozymes) was added to 1.0 wt% of the amount of PHBH contained in the cell culture solution, and the mixture was stirred for 4 hours while controlling the pH at 8.5 [i.e. (b) adding an alkaline proteolytic enzyme being Alcalase™ to the culture solution obtained in (a) to subject the microbial cells to an enzyme treatment]. Then, after cooling to 25 ° C and adding sodium hydroxide to pH 12.0, the temperature was adjusted to 25 with a high-pressure crusher (high-pressure homogenizer model “PA2K type” manufactured by Nirosoavi) at a pressure of 45 to 55 MPa [i.e. (c) adding sodium hydroxide to the culture solution obtained in (b) to adjust the pH of the culture solution to 12]. While controlling at ~ 35 ° C, high-pressure crushing was performed three times. The crushed liquid was adjusted to a temperature of 30 ° C. and a pH of 12.0 and stirred for about 10 minutes, and then PHBH was recovered by centrifugation, and water was added to suspend PHBH. The operation from pH adjustment of the crushed liquid to suspension of PHBH was repeated three times. Thereafter, the pH of the suspension was adjusted to 4.5 to 5.0 and stirred for about 1 hour. PHBH was recovered from this adjusted solution by centrifugation. The obtained PHBH was dried under reduced pressure at 40 ° C. for 12 hours to obtain purified PHBH. The recovery rate of PHBH was 98%.
“As shown in Examples, the microbial strain obtained by substituting the polyhydroxyalkanoic acid synthase gene on the chromosome of the Ralstonia ′ Urea H16 strain with the polyhydroxyalkanoic acid synthase mutant gene derived from s was cultured for 48 hours to produce P (3 HB-co -3HH) having a bacterial cell production amount of 10.10 g/L and a polyester content of 733.8 Wt %. This productivity greatly exceeded the productivity of P (3 HB-co -3 HH) reported so far. It was also found that this strain does not require an antibiotic or any other selected pressure in all the steps of culture, and the cells in the base substantially accumulate P (3 HB-co -3 HH) at the end of the culture.” WO 08//010296, para. [0020]. Ralstonia eutropha KNK-005 described in [0049] of WO 08/010296 A1 produces P (3 HB-co -3 HH), which is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).
Regarding that recitation that sodium hydroxide is added as an aqueous solution, Yanagita, page 5, states “Alkali means a substance whose aqueous solution is basic when dissolved in water.” An ordinarily skilled artisan at the time of filing would have recognized that sodium hydroxide is a solid (at room temperature and atmospheric pressure) such that “sodium hydroxide was added” is suggestive of adding sodium hydroxide as an aqueous alkaline solution. Regardless, since sodium hydroxide is added to an aqueous cell culture/solution, an ordinarily skilled artisan at time of filing would have been motivated to add sodium hydroxide as taught by Yanagita as an aqueous alkaline solution as a convenient means of adding a controlled or specific amount of sodium hydroxide wherein Yanagita, page 5, directly teaches that alkali including sodium hydroxide as taught therein is a “substance whose aqueous solution is basic,” which directly teaches that it is appropriate to formulate such alkali/sodium hydroxide as an aqueous basic solution.
Regarding recitation in claim 1 of producing a poly(3-hydroxyalkanoate) (e.g. poly(3-hydroxybutyrate-co-3-hydroxyhexanote)) having a weight average molecular weight from 100,000 to 700,000 Da, “In order to put PHA into practical use, it is necessary to show the physical properties that the processed product can withstand. From this point of view, the weight average molecular weight of PHA using polystyrene as a molecular weight standard is required to be 10,000 or more by gel chromatography. It is preferably 50,000 or more, more preferably 100,000 or more, further preferably 200,000 or more.” Yanagita, page 4.
Yanagita does not make it clear whether the molecular weight of PHA (poly(3-hydroxyalkanoate)) referenced is weight average molecular weight before or after extraction from cells by the process of Yanagita, wherein it is understood that extraction will decrease molecular weight to some degree. “In particular, since the copolymer tends to decrease in molecular weight due to hydrolysis due to heating or the like, the present invention having the advantage that the molecular weight hardly decreases as will be described later is significant in recovering the copolymer.” Yanagita, page 4.
“In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” MPEP 2144.05(I). Here, Yanagita teaches that PHA polymers with a weight average molecular weight of 100,000 or more or 200,000 or more are particular understood to be desirable and functional within the methods of Yanagita. Even in view of the possibility that “the molecular weight hardly decreases” by purification/extraction as taught by Yanagita, there is substantial overlap between the ranges of weight average molecular weight taught by Yanagita and the broad range of 100,000 to 700,000 Da recited in claims 1 and 22 such that a prima facie case of obviousness exists.
Regarding recitation in claim 1 of “subjecting the microbial cells to a hydrolysis reaction for a period of time to hydrolyze the poly(3-hydroxyalkanoate) in the microbial cells and “wherein a reaction time in (a) is form 4 hours to 30 hours,” the cited claim language is understood as reference to a time period that the solution of microbial cells are maintained at a pH of 8.0 to 12.0 prior to addition of any enzyme as stated in part (b) of claim 1.
“In particular, since the copolymer tends to decrease in molecular weight due to hydrolysis due to heating or the like, the present invention having the advantage that the molecular weight hardly decreases as will be described later is significant in recovering the copolymer.” Yanagita, page 4. “In the step (a) of the present invention, the temperature of the aqueous suspension when adding alkali to the aqueous suspension is desirably around the optimum temperature of the enzyme used. Specifically, a range of 20 to 60 ° C. is preferable. When alkali is added at a temperature higher than 60 ° C., the molecular weight of PHA may be significantly reduced. If an alkali is added at a temperature lower than 20 ° C., the viscosity of the aqueous suspension may be improved and stirring may be difficult.” Yanagita, page 6.
It is noted that in Example 1 of Yanagita (adjustment to pH 8.5 by addition of NaOH) is at 50[Symbol font/0xB0]C. From the description of Yanagita, any reduction in molecular weight of PHA is due to hydrolysis. Yanagita is focused on minimizing hydrolysis, and therefore teaches addition of alkali at lower temperatures to reduce hydrolysis. However, from the description of Yanagita, “the molecular weight hardly decreases” is understood as a description that some minimal amount of hydrolysis nevertheless occurs, wherein the occurrence of any hydrolysis meets the claim feature of subjecting the microbial cells to a hydrolysis reaction to hydrolyze PHA in the microbial cells.
That is, Yanagita teach that PHA as found in a cell as taught therein is subject to hydrolysis and reduction in molecular weight depending upon conditions, e.g. temperature, that promote such hydrolysis. It is noted that step (a) of Yanagita is addition of NaOH to adjust pH of a cell culture followed by addition of an Alcalase® enzyme.
Jiang has teachings that are duplicative of Yanagita teaching that the lysis conditions (chemical treatment) applied to a cell culture can affect hydrolysis of PHA within such cells. Jiang, page 2, right col., teaches:
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The thermal instability of PHB purified by alkalis based method could be due to PHB hydrolysis. As it has been reported . . . , abiotic hydrolysis of PHB by alkalis was observed in this study as well. Both HB monomer and CA were found as PHB hydrolysis products. Our data showed that the PHB degradation by NaOH in the fresh biomass was dependent on the treatment time and NaOH concentration. The hydrolysis products concentration showed linear relation with NaOH treatment time (Figure 2), while the relation between the NaOH concentration and the hydrolyzed products concentration is non-linear (Figure 3). In the tested NaOH concentration range, the HB monomer decreased with the increasing NaOH concentration before 0.1 M NaOH and then increased with NaOH concentration.” Jiang, page 5, right col.
As such, both Yanagita and Jiang teach that PHB as found within a cell culture or fresh biomass can be hydrolyzed as follows:
Yanagita teaches that increased temperature promotes hydrolysis.
Jiang teaches that increased NaOH concentration (i.e. increased pH) and treatment time promote hydrolysis.
“Disclosed examples and preferred embodiments do not constitute a teaching away from a broader disclosure or nonpreferred embodiments.” MPEP 2123(II). Here, both Yanagita and Jiang teach preferred embodiments of reducing hydrolysis and molecular weight rejection of PHA. Nevertheless, Yanagita and Jiang do not teach that conditions wherein moderate molecular weight reduction occurs due to hydrolysis due to exposure of appropriate conditions of pH, temperature and time are non-function so far as PHA is still produced and recovered with significant purity. Further, applications are known in the prior art wherein lower molecular weight PHA’s are desirable. “Low molecular weight PHB or PHB oligomers are useful for the preparation of specialty graft and block copolymers.” Yu, page 1088. Yanagita, page 4, directly teaches applicability to “homopolymer of 3HB,” i.e. PHB or poly(3-hydroxybutyrate).
In view of the above, in applications where a lower weight PHA/PHB is desirable as taught by Yu, an ordinarily skilled artisan at time of filing would have been motivated to expose a cell culture or fresh biomass containing PHA to conditions taught by Yanagita and Jiang as discussed above to promote hydrolysis and lower molecular weight, such conditions being increased NaOH concentration or pH, increased temperature and/or increased treatment time. As such, at the time of filing an ordinarily skilled artisan would have been motivate to modify step (a) of Yanagita wherein the pH of cell culture with PHA is adjusted to pH 8.5 followed by Alcalase® treatment to conditions that promote hydrolysis with a predictable effect in reducing molecular weight of PHA including treatment for a time at pH 8.5 at an elevated temperature to promote hydrolysis. Since such elevated temperature may not be compatible with Alcalase®, an ordinarily skilled artisan at time of filing would have recognized that such treatment for a period of time at pH 8.5 at an elevated temperature to promote hydrolysis can be followed by Alcalase® treatment at 50[Symbol font/0xB0]C as taught by Yanagita, wherein Alcalase® treatment is taught to increase purity of PHA recovered. While Yu focuses on PHB with smaller molecular weight, Yanagita and Jiang both expressly teach that all PHA’s are subject to hydrolysis under appropriate conditions such that it is not considered to be inventive simply to observe and implement the hydrolysis and reduction in molecular weight expressly taught by Yanagita and Jiang regardless of any specific application for lower weight PHA’s including copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate as recited in for example claims 7 and 11.
Regarding a specific treatment time of 8 to 20 hours as recited, "[W]here 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." MPEP 2144.05(II)(A). As discussed, at least Jiang teaches that treatment time directly relates to hydrolysis and reduction in molecular weight of PHA such that it is not inventive to discover the optimum or workable ranges of incubation/treatment time such as 8 hours, 10 hours, etc.
The above addresses the features of at least claims 1, 7, and 21-22.
Regarding claim 4, Alcalase® is understood to be a serine-specific proteolytic enzyme, wherein page 22 of specification indicates Alcalase® as being a subtilisin and page 21 of the specification indicates subtilisin as a serine-specific protease.
Regarding claims 10 and 12, Example 1 of Yanagita, Example 1, as discussed, teaches that 4 hours is an appropriate time period for enzyme treatment with Alcalase®.
Regarding claim 11, “Particularly, a copolymer having 3HH [3-hydroxyhexanoate] as a monomer unit is preferable, and PHBH is more preferable from the viewpoint of degradability as a biodegradable polymer and soft properties. At this time, the composition ratio of each monomer unit constituting PHBH is not particularly limited, but from the viewpoint of showing good processability, the content of 3HH units with respect to the total units is preferably 1 to 99 mol%, more preferably. 3 to 30 mol%.” Yanagita, page 4. As discussed above, Yanagita directly cites WO 08/010296 A1 producing poly(3-hydroxybutyrate-co-3-hydroxyhexanote). The content of 3HH units with respect to the total units is preferably 1 to 99 mol% is a ratio from 1/99 to 99/1 of 3-hydroxyhexanoate to remaining copolymer monomer, which is illustrated to specifically be 3-hydroxybutyrate. “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists.” MPEP 2144.05(I). Here, Yanagita teaches that the fraction of a copolymer that is 3-hydroxyhexanoate substantially overlaps the recited range in claim 11 wherein the balance of monomers in a copolymer is or can be specifically 3-hydroxybutyrate such that a prima facie case of obviousness exits.
Regarding claim 15, Yanagita, in Example 1, reports “rate [i.e. yield] of PHBH was 98%,” wherein an ordinarily skilled artisan at time of filing would expect a similar yield (i.e. at least 95%) regardless of the weight average molecular weight of polymer recovered, or in the alternative Yanagita teach an active expectation to an ordinarily skilled artisan that yields greater than 95% are obtainable by the methods of Yanagita.
Claim(s) 1-5, 7, 10-13, 15 and 20-22 (all non-withdrawn claims) is/are rejected under 35 U.S.C. 103 as being unpatentable over Yanagita et al. (WO 2010/116681 A1), Maruyama (WO 08/010296 A1), Jiang et al. (Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture, AMB Express 5, 2015, 5) and Yu et al. (Characterization of low molecular weight poly(beta-hydroxybutyrate)s from alkaline and acid hydrolysis, Polymer 41, 2000, 1087-98) as applied to claims 1, 7, 10-12, 15 and 21-22 above, and further in view of Yasotha et al. (Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration, Biochem. Eng. J. 30, 2006, 260-68).
Regarding claims 2-5, 13 and 20, Yanagita, page 5, teaches: “The enzyme used in the step (a) is not particularly limited as long as it can be used for industrial products, but for the purpose of degrading the cell wall to obtain a higher crushing effect, a proteolytic enzyme (protease) Cell wall degrading enzymes are preferred. In terms of supply stability and cost, industrially, a proteolytic enzyme is more preferable. Only one type of enzyme may be used, or two or more types may be used in combination. One of proteolytic enzymes and cell wall degrading enzymes may be used, or both enzyme may be used in combination. Examples of proteolytic enzymes include alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, carboxypeptidase and the like. Examples of cell wall degrading enzymes include lysozyme, amylase, cellulase, maltase, saccharase, α and β-glycosinase.” From Yanagita, page 6: The enzyme treatment is preferably performed at the optimum temperature and pH of the enzyme used. For example, when using Alcalase (manufactured by Novozymes), temperature: 50-60 ° C. and pH: 8-9 are preferable . . . Preferably, when lysozyme is used, the temperature is preferably 40-50 ° C. and the pH is 6-7.
As discussed, Yanagita, Example 1, teach the application of Alcalase® without an additional enzyme. However, as discussed, Yanagita teaches that a proteolytic enzyme such as Alcalase can be used in combination with a cell wall degrading enzyme such as lysozyme. Yasotha et al. demonstrates how Alcalase and lysozyme can be applied for recovery of PHA from cells.
“Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell.” Yasotha, abstract.
“In this study, shake flask experiments were conducted with
P. putida as the bacterial species. P. putida was cultivated in
nutrient-rich medium for 24 h in a shaker incubator at 30 ◦C
at 240 rpm. The culture 0.5% (v/v) were then transferred into
a 1000mL Erlenmeyer flask containing 300mL modified-R
medium plus 10 g/L of oleic acid as the carbon source. The
flask was incubated at 30 ◦C with a shaker speed of 240 rpm
for a fermentation period of 48 h. Cells were harvested by
centrifuging the broth and re-suspending the cells in water. This
suspension was then subjected to heat treatment by autoclaving
at 121 ◦C for 1 min prior to enzymatic treatments. The cell
weight was predetermined by drying a portion of cells at 80 ◦C
until constant weight was achieved. The harvested suspension
was initially subjected to digestion with Alcalase and SDS at pH
8.5 and temperature of 55 ◦C at the time duration, which was
optimized through Taguchi’s method (refer to Section 3). This
was followed by further treatments with EDTA and lysozyme
at pH 7 and temperature of 30 ◦C for 15 min.” Yasotha, page 261, right col.
As discussed, Yanagita teaches that it is appropriate to apply both Alcalase and lysozyme, wherein Yanagita directly teaches that Alcalase and lysozyme have different pH preferences. Yasotha teaches that in lysing cells with Alcalase and lysozyme to recover PHA, it is appropriate to first apply Alcalase at a time and at a pH appropriate for alcalase (e.g. pH 8.5), and then follow with a separate treatment at a different pH of 7.0 for treatment of lysozyme, which is consist with the pH preferences for Alcalase and lysozyme directly taught by Yanagita.
Since Yanagita directly teaches that it is appropriate to treat R. eutropha cells as taught therein with Alcalase and lysozyme, an ordinarily skilled artisan would have been motivated to do the same as directly suggested by Yanagita. Although Yasotha is directed towards a different cell type, Yasotha in confirmation and reinforcing the teachings of Yanagita teaches that a combination of Alcalase and lysozyme is particularly effective for cell lysis to recover PHA such that an ordinarily skilled artisan at the time of filing would have been motivated to apply the same to R. eutropha cells as taught by Yanagita. As emphasized above, Yanagita teaches that Alcalase is preferably applied at pH 8-9 and lysozyme is preferably applied at pH 6-7. Yasotha teaches that the different pH preferences of Alcalase and lysozyme can be accommodated by a treatment with Alcalase at a pH 8.5 as also taught by Yanagita. Yasotha then teaches that pH can be separately adjusted lower to pH 7.0 for a separate enzymatic treatment with lysozyme. At the time of filing an ordinarily skilled artisan would have been motivated to modify the methods of Yanagita to have an additional step of adding lysozyme enzyme (after treatment with Alcalase at pH 8.5) by adjusting pH of the culture solution to 7 for a separate treatment with lysozyme in view of Yanagita directly teaching that it is appropriate to treat R. eutropha cells as taught therein with Alcalase and lysozyme.
Regarding claims 2, 3, 4 and 13, “[S]election of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.” 2144.04(IV)(C). Performance of the following meets the features of claim 3:
-Alcalase treatment of the culture solution at pH 8.5;
-adjustment of pH to 7.0 and lytic enzyme/lysozyme treatment of the culture solution;
-adjustment of the culture solution back to pH 8.5 and a second Alcalase treatment of the culture solution; and
-adjustment of pH to 7.0 and a second lytic enzyme/lysozyme treatment of the culture solution.
Again, as discussed, the methods recite an open transitional phrase comprising such that the claims encompass performing (b) of claim 1 more than one time. As indicated, Yanagita and Yasotha teaches the application of separate Alcalase and lysozyme treatments with pH adjustment to the appropriate pH of 8.5 or 7.0 prior to each treatment as to suggest at least one cycle of Alcalase treatment at pH 8.5 followed by lysozyme treatment at pH 7, which meets the features of claims 4 and 13. Simply repeating this cycle is prima facie obvious in the absence of new or unexpected results as a selection of order of steps by selecting performing an enzymatic treatment more than one time. In the alternative, an ordinarily skilled artisan at the time of filing would have been motivated to repeat enzymatic treatment with alcalase followed lysozyme (such that four steps of enzymatic treatment are done) if the first round of treatment with alcalase followed by lysozyme did not yield complete enough cell lysis or PHA recovery wherein additional treatment with alcalase followed by lysozyme would be expected to yield additional PHA recovery.
Regarding claim 3 specifically, recitation of “further comprising between (a) and (b), (a’) adjusting the pH of the culture solution obtained in (a) to form 6.0 to 8.0” is met if such pH adjustment is done prior to at least one enzyme treatment if plural enzyme treatments are performed. “Carrying out a lytic enzyme treatment and an alkaline proteolytic enzyme treatment in this order” does not preclude the performance other additional enzyme treatments wherein claim 3 recites “further comprising.” For example, claim 13 recites carrying out proteolytic enzyme treatment and a lytic enzyme treatment “at least one each,” such that claim 1 and all claims depending therefrom are understood as encompassing an enzyme treatment as in (b) in claim 1 multiple times in the absence of specific negative claim limitations excluding the same, wherein such a negative claim limitation is not recited. The first performed lytic enzyme treatment followed by a second Alcalase enzyme treatment (wherein four total enzyme treatments are performed) as outlined above is “carrying out a lytic enzyme treatment and an alkaline proteolytic enzyme treatment in this order” as recited in claim 3. Regarding claim 2, performance of four enzymatic treatments as outlined above also satisfies the features of claim 2.
Regarding claims 5 and 20, Example 1 of Yanagita, Yanagita describes addition of SDS (sodium dodecyl sulfate/surfactant prior to addition of enzyme. “The use of anionic detergent such as sodium dodecyl sulfate (SDS) can decompose any insoluble matters such as protein and lipids and solubilise the components by incorporation in micelles. [A reference] found in their research that reaction of Alcalase and SDS simultaneously bore no synergistic effect at the optimum pH and temperature condition of Alcalase, and as such this leads to considerable time savings since the reactions can be carried out simultaneously.” Yasotha, page 261, left col. As such, addition of SDS (a surfactant) at some point in recovery of PHA is beneficial for solubilizing insoluble material. “[S]election of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.” Addition of SDS after the pH adjustment of step (c) of claim 1 (as taught by Yanagita as discussed above), either instead of at the beginning of the process or as and additional addition of SDS, is selection of performing process steps that is prima facie obvious in the absence of new or unexpected results. That is, the prior art cited teaches the addition of SDS wherein the point of addition of SDS in the process is selection of performing process steps that is prima facie obvious in the absence of new or unexpected results. Regarding claim 20, Example 1 of Yanagita, Example 1, states: 1000 g of the obtained bacterial cell culture solution was sterilized at 70 to 75 ° C. for 1 hour, cooled to 50 ° C., 4.0 g of sodium dodecyl sulfate was added. 4 g SDS in 1000 g of cell culture solution is 0.4% by weight relative to the culture solution. Regardless of at what point in the process SDS is added, an ordinarily skilled artisan at the time of filing would have been motivated to utilize the amount of SDS specifically taught by Yanagita, or stated in other words, Yanagita directly indicates 0.4% weight relative to the culture solution is an appropriate amount of surfactant.
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 1-5, 7, 10-13, 15 and 20-22 (all non-withdrawn claims) are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 3-7, 9, and 14-19 of copending Application No. 18/681,441 in view of Yanagita et al. (WO 2010/116681 A1), Maruyama (WO 08/010296 A1), Jiang et al. (Feasibility study of an alkaline-based chemical treatment for the purification of polyhydroxybutyrate produced by a mixed enriched culture, AMB Express 5, 2015, 5), Yu et al. (Characterization of low molecular weight poly(beta-hydroxybutyrate)s from alkaline and acid hydrolysis, Polymer 41, 2000, 1087-98) and Yasotha et al. (Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration, Biochem. Eng. J. 30, 2006, 260-68). Machine translations are provided and cited as indicated above.
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference.
The copending (i.e. reference) claims state:
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The maintaining the culture solution a pH of 10.5 as in reference claim 1 meets step (a) of claim 1; however, the reference claims does not state a time for maintaining. Regardless, maintain must be performed for a period of time wherein it is not inventive to discover that a maintenance time of, for example, 8 or 10 hours is effective or operative. See MPEP 2144.05(II)(A). As explained in MPEP 2144.05(II)(A), since the general condition of maintaining at pH 10.5 is recited it is not inventive to discover that 8 or 10 hours is a workable time period.
Reference claim 3 indicates enzymatic treatment of cells for recovery of PHA including with a lytic enzyme and an alkaline proteolytic enzyme (reference claim 8), including later adjustment to pH 10 and addition of surfactant as recited in claim 1, part(c). Co pending claim 6 directly indicates that a hydrolysis occurs such that PHA has weight average molecular weight of 100,000 and 800,000 that substantially overlaps the molecular weight range recited in the rejected claims such that it is apparent that a hydrolysis reaction occurs due to performance of the method of copending claim 1; adjusting of pH to 10.5 to 13.01 as recited in copending claim 1 is understood to be performed by addition of some alkaline material or solution. The reasons one having skill in the art at time of filing would have performed such enzymatic treatment is the manner recited in the rejected claims and to meet all limitations of the rejected claims are set forth above in the rejections under 35 USC 103 over Yanagita et al. (WO 2010/116681 A1), Maruyama (WO 08/010296 A1), Jiang, Yu and Yasotha et al.
This is a provisional nonstatutory double patenting rejection.
Response to arguments
The declaration under 37 CFR 1.132 (filed 4/27/2026) filed is insufficient to overcome the rejection of the claims based upon 35 U.S.C. 103 as set forth in the last Office action because:
Applicant argues:
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The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). "[A]ppellants have the burden of explaining the data in any declaration they proffer as evidence of non-obviousness." Ex parte Ishizaka, 24 USPQ2d 1621, 1624 (Bd. Pat. App. & Inter. 1992); MPEP 716.02(b). “An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e). "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II). “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.’" MPEP 716.02(d). “The nonobviousness of a broader claimed range can be supported by evidence based on unexpected results from testing a narrower range if one of ordinary skill in the art would be able to determine a trend in the exemplified data which would allow the artisan to reasonably extend the probative value thereof.” MPEP 216.02(d)(I).
The specification, para. [0146], describes a temperature of 75+/-2[Symbol font/0xB0]C as a temperature under which hydrolysis is performed, which is understood to be the same temperature utilized by examples contained with the declaration. The claims do not recite any limitation on temperature. As such, an embodiment of the claims is a hydrolysis reaction performed at ambient temperature (e.g. 20[Symbol font/0xB0]C) at a pH of 8, wherein there is no evidence of record that any significant hydrolysis occurs under such conditions.
"Expected beneficial results are evidence of obviousness of a claimed invention.” MPEP 716.02(c)(II). As discussed above, Yanagita teaches that elevate temperature above 60[Symbol font/0xB0]C is expected to promote hydrolysis of PHA contained within cells/biomass. Jiang teaches that increase in NaOH and increased treatment time is expected to promote hydrolysis of PHA contained within cells/biomass. As such, the observation that increased incubation time increases hydrolysis and reduction in molecular weight is an expected results explicitly taught by the prior art.
Conclusion
New grounds of rejection above are necessitied in part by amendment to claim 1 specifically required performance of a “hydrolysis reaction” not previously recited.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TODD M EPSTEIN whose telephone number is (571)272-5141. The examiner can normally be reached Mon-Fri 9:00a-5:30p.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
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