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 .
Status of Claims
Receipt of Arguments/Remarks filed on 12/5/2025 is acknowledged. Claims 1-6, 8-14, and 16-21 are pending. Claims 1 and 14 were amended. New claims 17-21 were added.
Withdrawn Objections
The objections to the specification and to claims 1 and 14 are withdrawn.
New and modified rejections necessitated by amendment
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 17-21 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 17-19 recite limitations directed to measuring the high degree of lysis. Claim 1, upon which claims 17-19 depend, is directed to a method of recovering a protein. The limitation “wherein bacterial cells in the fermentation broth exhibit a high degree of lysis,” in claim 1 is a characterization of the bacterial cells. Cell lysis or measurement of cell lysis is not a positively recited active method step in claim 1. Therefore, it is unclear if the limitations of claim 17-19, which refer to “the high degree of lysis”, are reciting active method steps as part of the method of recovering a protein, or if they are directed to the characterization of the fermentation broth wherein the cells are lysed that is not part of the claimed method of recovering. Additionally, if claims 17-19 are directed to active method steps, it is unclear if these steps occur before or after steps a)-c), as cell lysis is not an active method step of claim 1. The claims should be amended to clarify whether these limitations recite active method steps in the method of recovering a protein.
Regarding claim 18, the time interval for the two measurements is unclear. For example, it is not clear whether “between onset of fermentation and 24 hours thereafter” means that the first measurement is taken at onset of fermentation and the second measurement is taken at 24 h, or if this means that the measurements are taken at any time points that fall between onset of fermentation and 24 h. This is also the case for “between 24 and 48 hours after onset of fermentation”.
Regarding claim 19, the time interval for these two measurements is also unclear. Given the phrase “within an interval”, it is not clear if this limitation means that the interval between measurements is within a time period of 24 h, meaning that they can be taken at any time from 0-24 h, or if the measurements must be taken 24 h apart. This is also true of the interval of 48 h. The use of “and/or” in this claim is also unclear. It appears that the claim is referring to only two measurements, and thus there would only be a single interval between measurements. If there are only two measurements with a single interval between, it is unclear how there can be both 24 h and 48 h intervals between the two measurements.
Claim 20 recites that there is no step of lysing the bacterial cells before step a). Under broadest reasonable interpretation, “no step of lysing” reads on a method wherein cells have not been lysed prior to step a) at all; meaning there are no lysed cells in the broth. However, claim 1 requires some lysis in order for recovery of the protein. It is unclear if the limitation in claim 20 means that there is no active step of lysis that is performed before step a), i.e. the cells have naturally lysed in the fermentation broth before performing step a), or that no lysis has occurred.
Claim 21 recites “wherein the high degree of lysis is a decrease in OD600 value”. However, claim 21 is directed to a method of recovering a protein. The limitation “wherein bacterial cells in the fermentation broth exhibit a high degree of lysis” in claim 21 is a characterization of the bacterial cells and is not a positively recited, active method step. Therefore, it is unclear if the limitation “wherein the high degree of lysis is a decrease in OD600 value” is an active method step of measuring lysis that is part of the method of recovering a protein, and if it is an active step, whether it occurs before or after steps a)-c). The claims should be amended to clarify whether these limitations recite active method steps in the method of recovering a protein.
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 6 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 6 recites “a concentration in a range of 0.5 to 25 g/L”. Claim 1, upon which claim 6 depends, recites that the flocculant is added in a range of 5 to 50 g/L. Thus, claim 6 does not further limit claim 1, as this concentration range includes values outside of the concentration range recited in claim 1 (i.e. 0.5 to 5 g/L).
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
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 1-6, 8-14, 16, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Chatel et al., US 2015/0368292 A1 in view of Nielsen et al., WO 96/38469, and as evidenced by Seitz K Series Depth Filter Sheets: Data Sheet.
Regarding claim 1, Chatel teaches a method of producing a recombinant protein by harvesting a microbial cell broth, i.e. fermentation broth (Chatel “Abstract”). Chatel teaches that the bacterial cells in the broth can exhibit a high degree of lysis, in some cases with 50% or more of the cells in the harvest having autolysed (Chatel p. 5 para. 99). Chatel teaches adding a flocculant to a microbial cell broth that expresses the recombinant protein (Chatel p. 1 para. 13-14). Chatel teaches that the amount of flocculant (PEI) added is between 0.01-2% w/v, with an optimum amount of PEI being 0.5% w/v (Chatel p. 1 para. 28; p. 10 para. 178). 0.5% w/v is equal to 0.5g/100mL, or 5 g/L, which is within the claimed range of 5-50 g/L. Chatel further teaches separating the protein of interest form the bacterial cells, or clarifying the flocculated harvest (Chatel p. 1 para. 15, para. 19). Chatel teaches that “clarifying” can comprise a filtration step, and that filtration may be used as the sole clarification process (Chatel p. 6 para. 116 and 119). Chatel teaches that the recombinant protein is then purified, or obtained, from the clarified flocculated harvest, i.e. after filtration (Chatel p. 1 para. 20). Chatel teaches that the bacteria can include Bacilli such as Bacillus subtilis (Chatel p. 4 para. 81).
Regarding claim 2, Chatel teaches that the flocculent can be cationic or anionic agents, including polyethyleneimine (PEI), cationic polyacrylamide, poly(diallyldimethylammonium chloride), natural polymers from organisms, or chemical flocculants such as aluminum sulfate (Chatel p. 5 para. 104).
Regarding claim 3, Chatel teaches that cell lysis is measured using DNA quantification because there should be little DNA in the supernatant of intact, or non-lysed, cells (Chatel p. 11 para. 190-191, Fig. 8). Chatel also teaches that cell lysis can be measured using a capacitance probe (Chatel p. 12 para. 198, Table 4).
Regarding claim 4, Chatel teaches that the bacterial fermentation broth used in the method of obtaining a protein of interest contains a high proportion of lysed cells, over 50%, as discussed above, as well as suitable methods for measuring cell lysis such as DNA quantification. The instant claims are directed to a method of obtaining a protein of interest. The limitation in claim 1, “wherein bacterial cells in the fermentation broth exhibit a high degree of lysis” is a characterization of the cells used in the method, and is not an active method step. Claim 4 is similarly directed to a characterization of the bacterial cells and does not recite an active method step. Therefore, the method of Chatel, which teaches the active method steps a)-c), reads on claim 4.
Regarding claim 6, Chatel teaches that the amount of flocculant (PEI) added is between 0.01-2% w/v, with an optimum amount of PEI being 0.5% w/v, which is equal to 5 g/L, within the range of 0.5-25 g/L (Chatel p. 1 para. 28; p. 10 para. 178).
Regarding claim 9, Chatel teaches that step (a), adding a flocculant, is performed before step (b), filtration (Chatel p. 1 para. 18-19, steps (b) and (c)).
Regarding claim 11, Chatel teaches filtration using a Pall Seitz-EKS 60D 0.2 μm filter (depth filter with nominal pore size 0.05-0.2 μm) (Chatel p. 11 para. 193). The instant specification states that a high-porosity filter aid includes diatomite or perlite precoat (Specification p. 12 lines 30-35). Pall Seitz-EKS filters, such as the one taught by Chatel, are made with diatomaceous earth, or diatomite, and perlite (see Seitz K Series Depth Filter Sheets Data Sheet). Therefore, Chatel teaches the use of a high porosity filter aid.
Regarding claim 12, Chatel teaches that clarification/filtration is followed by cation-exchange chromatography for protein purification (Chatel p. 6 para. 121). This would be prior to step (c), obtaining the protein of interest.
Regarding claim 13, Chatel teaches that prior to step (a) of adding the flocculant, bacterial cells are cultured in fermentation conditions before harvesting the cell broth to add the flocculant (Chatel p. 5 para. 93-94).
Regarding claim 14, Chatel teaches that the Bacillus cells can include Bacillus subtilis (Chatel p. 4 para. 81).
Regarding claim 16, Chatel teaches that the cell harvest is filtered with a 2 µm pore size filter (Chatel p. 11 para. 193). Microfiltration as commonly understood in the art refers to filtration using a pore size of 0.1 to 10 µm, and the instant specification exemplifies microfiltration using a membrane with a 0.1 to 0.2 µm pore diameter (instant specification p. 12 lines 39-42). Therefore, Chatel teaches microfiltration.
Regarding claim 20, Chatel teaches that the cells undergo autolysis, meaning they are naturally lysed (Chatel p. 5 para. 99). Chatel does not teach an active method step of lysing the bacterial cells before step a). Therefore, it is considered that Chatel teaches a method wherein there is no step of lysing the bacterial cells before step a).
Chatel does not teach that the protein of interest is an enzyme (claims 1, 8), adding a divalent cation prior to step b (claim 5) or a filtration step using a Nutsche pressure filter or cross-flow microfiltration (claim 10).
Regarding claim 1, Nielsen teaches a method for recovering proteins from a fermentation broth which is treated with a flocculating agent (Nielsen p. 1 “Technical Field”; p. 9 lines 25-34). Nielsen teaches that the protein is then separated from bacterial cells by filtration and the protein of interest is obtained (Nielsen p. 14 lines 5-10). Nielsen teaches that the cells are Bacillus cells and that the protein of interest is an enzyme (Nielsen p. 3 lines 27-34, p. 13 line 19).
Regarding claim 5, Nielsen teaches that, prior to filtration, a divalent cation (calcium chloride) is added to the fermentation broth in addition to flocculating agents (Nielsen pg. 14 lines 24-30; Table 7). Nielsen teaches that calcium chloride is a useful flocculating agent (Nielsen p. 8 lines 21-26).
Regarding claim 8, Nielsen teaches that the protein of interest is an enzyme, including proteases (pg. 3 lines 27-34), lipases (pg. 4 lines 10-12), amylases (pg. 5 lines 5-13), cellulases (pg. 5 lines 14-23), laccase (pg. 6 line 8), catalase (pg. 6 line 9), peroxidase (pg. 6 line 9), or oxidase (pg. 6 line 10).
Regarding claim 10, Nielsen teaches filtration using a Nutsche pressure filter (Nielsen pg. 14 lines 6-7).
It would have been obvious for a skilled artisan to utilize the method of Chatel to recover an enzyme, such as those taught by Nielsen, from a fermentation broth. Both Nielsen and Chatel teach methods involving adding a flocculant and filtering a fermentation broth to recover a protein. A skilled artisan would have found it obvious to substitute a fermentation broth comprising an enzyme as the protein of interest in the method of Chatel, given the similar method taught by Nielsen to recover an enzyme.
An ordinary artisan would have been motivated to use an enzyme in the method of Chatel, because enzymes such as proteases and amylases, as taught by Nielsen, have many beneficial commercial uses and it would therefore be of interest to use Chatel’s method of protein recovery to recover an enzyme (Nielsen pp. 4-6 describing commercial enzymes). There would have been a reasonable expectation of success in making this substitution because Chatel and Nielsen teach many similar steps in their methods of recovering a protein, and Nielsen teaches that enzymes can be successfully recovered using flocculants and filtration techniques similar to those used in the method of Chatel.
An ordinary artisan would have further found it obvious to add a divalent cation to the fermentation broth in the method of Chatel, as Nielsen teaches the use of divalent cations such as calcium chloride in a similar method for protein recovery. A person of ordinary skill in the art would have been motivated to incorporate a divalent cation such as calcium in the method of Chatel because Nielsen teaches that calcium chloride has utility as a flocculating agent and is added in addition to other flocculating agents such as sodium aluminate (Nielsen p. 8 lines 21-26). It would therefore would be expected that adding a divalent cation in addition to other flocculants in the method of Chatel would be beneficial and would improve the flocculation step. A skilled artisan would have had a reasonable expectation of success in incorporating a divalent cation in the method of Chatel given the successful addition of a divalent cation for recovering an enzyme in a similar method as taught by Nielsen.
It would have additionally been obvious to an ordinary artisan that a Nutsche pressure filter, as taught by Nielsen, could be substituted in the method of Chatel. Both of these methods are directed to recovering proteins by adding a flocculant to a fermentation broth and filtering. It would have been obvious to substitute the filtration method of Chatel with the filtration method of Nutsche as both are used to achieve the same purpose, filtering a protein after adding a flocculant. It is considered a simple substitution of one known element for another with an expectation of predictable results, as both the Nutsche pressure filter of Nielsen and the high porosity filter aid of Chatel achieve the same result of filtering a fermentation broth to obtain a protein of interest in the filtrate. A skilled artisan would have recognized that both of these methods are known techniques in the art for achieving the end result of protein recovery, and would have found it obvious to substitute one known filtration technique for another.
Claims 4, 17-19, and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Chatel et al., US 2015/0368292 A1 in view of Nielsen et al., WO 96/38469 as applied to claims 1-6, 8-14, 16, and 20 above, and further in view of Ng PeerJ Preprints; 2018 Apr 13.
Chatel and Nielsen teach the method of claim 1 as set forth above. Regarding claim 21, Chatel and Nielsen teach a method of recovering a protein of interest comprising steps a)-c), wherein the protein is an enzyme and the bacterial cell is Bacillus, as discussed above with regard to claim 1.
These references do not teach that the high degree of lysis corresponds to a decrease in OD600 or increase in shear viscosity (claim 4), or a decrease in OD600 value of at least 10% between two measurements (claims 17-19 and 21).
The “wherein” clause in claim 4 is directed to a characterization of the bacterial cells and does not recite an active method step, and therefore the method of Chatel and Nielsen reads on claim 4 as discussed above. Regarding claims 17-19 and 21, it is unclear whether the “wherein” clause directed to the high degree of cell lysis recites an active method step, as discussed in the rejection under 35 U.S.C. § 112(b) above. However, in the interest of compact prosecution, the following rejections are included.
Regarding claim 4, Ng teaches measuring Bacillus subtilis optical density (OD600) and teaches that a decline in in OD600 corresponds to cell lysis (Ng “Abstract”, p. 2 para. 3).
Regarding claims 17 and 21, Ng teaches that a drastic decline in Bacillus OD600 corresponds to cell lysis (Ng p. 2 para. 3). Ng teaches measuring OD at appropriate time points during the culture (Ng p. 3 “Growth Experiments”). In one example, Ng measures a decline in OD of the Bacillus culture from 4.5 to 2.2 between two measurements, which is a 51% decrease, i.e., a decrease of at least 10%, and teaches that this decrease indicates cell lysis (Ng p. 3 “Results and Discussion”; Fig. 2).
Regarding claim 18, “between onset of fermentation and 24 hours thereafter” under broadest reasonable interpretation is considered to mean that the two measurements may fall anywhere in the time period of 0-24 h. Ng teaches taking OD measurements at appropriate intervals (Ng p. 3 “Growth Experiments”). For example, Ng teaches measuring OD of Bacillus grown in LB Lennox at 37°C at a time point of 22.5 h and a time point of 24 h (Ng Fig. 5a). Thus, Ng teaches that a suitable time interval is between onset of fermentation at 24 h, with both measurements being taken within that time frame.
Regarding claim 19, “within an interval of 24 h and/or 48 h between the two measurements” under broadest reasonable interpretation means that the interval between two measurements is within 24 h (i.e. any time below 24 h) or within 48 h (i.e. any time below 48 h). Ng teaches various examples of two measurements taken in these time frames, for example two measurements of OD of Bacillus grown in LB Lennox medium are taken at 23.5 h and 48 h (24.5 h between measurements) (Ng Fig. 2 LB Lennox).
It would have been obvious for a skilled artisan that OD600 measurements at suitable time points such as those taught by Ng could be used to measure the amount of lysis of Bacillus cells in a fermentation broth. Ng teaches measuring OD600 of Bacillus cultures at two time points and teaches that this corresponds to cell lysis. Chatel teaches that the lysis of Bacillus cells is assessed by measuring DNA concentration or capacitance (Chatel p. 5 para. 99). However, a skilled artisan would have been aware that there are numerous methods for assessing cell lysis, and would have found it obvious to substitute the method of Ng using OD600 measurements to assess Bacillus lysis in place of the methods used by Chatel. It is considered a simple substitution of one known element for another with an expectation of predictable results, as both measurement techniques are used for the same purpose of determining cell lysis over a period of time. As both of these techniques are known in the art as methods to provide a measurement of cell lysis over time, it would have been obvious to substitute one for the other with a reasonable expectation of success.
Response to Arguments
In light of amendments to the claims, the rejections under 35 U.S.C. § 102 have been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-6, 9-14, and 16-21 are made under 35 U.S.C. § 103 as set forth above. Given these new grounds of rejection, the arguments presented regarding claims rejected under 35 U.S.C. § 102 are moot.
Conclusion
Claims 1-6, 8-14, and 16-21 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653