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 .
Support for the amendments is within the instant application specification.
Applicant’s amendment to the claims filed on 9/12/2025 in response to the Non-Final Rejection mailed on 7/14/2025 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 1-3, 5-8, 11-13, 15-18, 20, 25-28 are pending and examined on the merits.
Claims 4, 9-10, 14, 19, 21-24 are cancelled.
Applicant’s remarks filed on 9/12/2025 in response to the Non-Final Rejection mailed on 7/14/2025 have been fully considered and are deemed persuasive to overcome at least one of the rejections and/or objections as previously applied.
The text of those sections of Title 35 U.S. Code not included in the instant action can be found in the prior Office Action.
Withdrawn Objections
The objection to claims 7-8, 25-28 because of the following informalities: the term “conditions” is plural is withdrawn in view of Applicant’s amendment of claims to change “conditions” to “condition”.
Withdrawn Rejections
The rejection of claim 21 under 35 U.S.C. 103 as being unpatentable over Koda et al (2000, Cryobiology, cited on PTO-892 dated 7/14/2025) {herein Koda} is withdrawn in view of Applicant’s cancellation of claim 21.
Objection
Claims 1 is newly objected to for the following informalities: the recitation of ‘that constitutively expresses a recombinant protein.’ The claim should recite ‘that constitutively expresses the functional recombinant protein.’ Appropriate correction is suggested.
Maintained 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.
The rejection of claims 1-3, 5-8, 11, 13, 15-18, 20, 25-28 under 35 U.S.C. 103 as being unpatentable over Koda et al (2000, Cryobiology, cited on PTO-892 dated 7/14/2025) {herein Koda} is maintained. The rejection has been modified in view of Applicant’s amendment to claims 1 and 13 to recite ‘a functional recombinant protein.’
Claims 1-3, 5-8, 11 are drawn to a method of recovering a functional recombinant protein extracted from bacterial biomasses, the method comprising: providing two or more bacterial biomasses comprising a plurality of bacterial cells transformed with a plasmid that constitutively expresses a recombinant protein; storing each biomass in ultra-low temperature (ULT) for 10 minutes to 120 minutes, wherein the ULT is -80°C to -20°C, wherein each biomass is stored for the same time and at the same temperature, and wherein each biomass is stored as a cell pellet or in growth media that does not include a cryoprotectant; lysing each plurality of bacterial cells to produce two or more cell lysates after storage in ULT conditions; and extracting the functional recombinant protein from each cell lysate, wherein the mass of the functional recombinant protein isolated from each biomass stored in ULT is greater than the mass of the functional recombinant protein if each biomass has not been stored in ULT.
Claims 13, 15-18, 20, 27-28 are drawn a method of lysing bacterial cell biomasses, the method comprising: providing two or more bacterial biomasses comprising a plurality of bacterial cells transformed with a plasmid that constitutively expresses a recombinant protein; storing each biomass in ultra-low temperature (ULT) conditions for 10 minutes to 120 minutes, wherein the ULT is -80°C to -20°C, wherein each biomass is stored for the same time and at the same temperature, and wherein each biomass is stored as a cell pellet or in growth media that does not include a cryoprotectant; and lysing each plurality of bacterial cells to produce two or more cell lysates after storage in ULT conditions, wherein storing each biomass in ULT conditions prior to lysing the plurality of bacterial cells increases the mass of a recombinant protein compared to lysing the plurality of bacterial cells without storing the biomass in the ULT conditions prior to the lysing step.
With respect to claims 1-3, 5-8, 13, 15-18, 25-28 Koda teaches a method wherein a plurality of bacterial cells (Pantoea agglomerans (Erwinia herbicola), Xanthomonas campestris pv. Translucens, and Pseudomonas syringae)) stored in growth media at -20C (Examiner interpreted ULT) for 24 hrs (page 197, column 2, para 2). Said cells have an increase in intracellular proteins referred to as cold acclimation protein or cold shock proteins (page 196, column 1, para 2; page 197, column 1, para 1, column 2, para 2; page 201, column 1, para 2; page 201, column 2, para 3). Examiner is interpreting said proteins to be functional as they function in protecting cells against cold shock (page 195, column 2, para 1). Examiner is interpreting the plurality of bacterial cells taught by Koda to be two or more bacterial biomasses as they are comprised of different species of bacteria. Additionally, Examiner is interpreting each biomass to be stored for the same time and at the same temperature as said method is performed for each bacterial biomass (page 196, column 1, para 2). Examiner is interpreting the growth media at which the bacterial biomasses are stored at -20C to not include a cryoprotectant as Koda does not teach the addition of a cryoprotectant. In fact, Koda is measuring the production of naturally produced cryoprotectant proteins, cold shock proteins, in microbial biomasses stored at -20C (page 201, column 2, para 3). Koda further teaches to analyze the intracellular protein within the microbial biomass, said biomass was ultrasonically disrupted causing the cells to break open (page 196, column 1, para 3). Examiner is interpreting the teaching of ultrasonically disrupting the cells taught by Koda to be lysing the cells as the result is said cells breaking open. Upon analysis of the intracellular protein by SDS-Page, Koda found that some bands increased after storage at -20C (page 201, column 1, para 2). It would be obvious to one of ordinary skill in that art that after storage at -20C, protein is extracted as said step is a requirement for SDS page analysis as SDS page analysis involves the extraction of protein from samples. As such, Examiner is interpreting said samples to be stored at -20C, followed by the extraction of protein and analysis by SDS-Page. Furthermore, based on the SDS-Page analysis, Examiner is interpreting there to be an increase in intracellular protein in cells stored at -20C versus cells not stored at -20C as it is known to those of ordinary skill in the art that an increase in the intensity of bands within an SDS-Page gel is indicative of an increase in protein expression. Koda discloses the claimed invention except for ‘storing each biomass in ultra-low temperature for 10 minutes to 120 minutes (claims 1, 5-6, 15, 18, 25-28); ‘wherein the ULT is -80C’ (claim 3); ‘wherein the ULT condition is -80C and the biomass is stored in ULT conditions for 120 minutes’ (claims 7, 17) or ‘10 minutes’ (claims 8, 16). It would have been obvious to one having ordinary skill in the art at the time the invention was made to modify the temperature and the time at which the biomass is stored at said temperature, depending upon the particular application, since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. In re Aller, 105 USPQ 233.
With respect to claims 11, 20, Koda teaches Escherichia coli synthesizes cold shock proteins (page 195, column 1, para 2). As such, Koda provides the motivation for one of ordinary skill in the art to utilize E. coli in a method for the production of intracellular protein at -20C, as said proteins have been shown to be a vital necessity for the survival of E. coli at low temperatures (page 195, column 2, para 1). Examiner is interpreting the teaching of ‘low temperatures’ by Koda to include -20C.
However, Koda does not teach a method of recovering a functional recombinant protein (claims 1). The method of claim 3, wherein the ULT is -80°C (claim 3). The method of claim 5, wherein the biomass is stored in ULT conditions for 10 minutes (claim 5). The method of claim 6, wherein the biomass is stored in ULT conditions for 120 minutes (claim 6). The method of claim 7, wherein the ULT condition is -80°C and the biomass is stored in ULT conditions for 120 minutes (claims 7, 17). The method of claim 8, wherein the ULT condition is -80°C and the biomass is stored in ULT conditions for 10 minutes (claims 8, 16). The method of claim 13, transformed with a plasmid that constitutively expresses a recombinant protein (claim 13). The method of claim 15, wherein the biomass is stored in ULT conditions for 10 minutes (claim 15). The method of claim 18, wherein the biomass is stored in ULT conditions for 120 minutes (claim 18). The method of claim 25, wherein the ULT condition is -20°C and the biomass is stored in ULT condition for 120 minutes (claim 25). The method of claim 26, wherein the ULT condition is -20°C and the biomass is stored in ULT condition for 10 minutes (claim 26). The method of claim 27, wherein the ULT condition is -20°C and the biomass is stored in ULT condition for 120 minutes (claim 27). The method of claim 28, wherein the ULT condition is -20°C (claim 28).
Before the effective filing date of the claimed invention, it would have been obvious to one having ordinary skill in the art at the time the invention was made to substitute a constitutively expressed functional recombinant protein for a functional intracellular biomolecule extract (cold shock protein) as a functional recombinant protein constitutively expressed from a plasmid that is transformed into a bacterial cell would yield predictable results of an increase yield (mass) of the recombinant protein, due to said recombinant protein being constitutively expressed (claims 1, 13). Additionally, said construct would allow for tighter control of protein expression due to said protein being expressed from a plasmid. Since it has been held to be within the general skill of a worker in the art to select a known material on the basis of its suitability for the intended use as a matter of obvious design choice. In re Leshin, 125 USPQ 416. Although the reference of Koda does not explicitly teach the limitations of claims 1, 13 (ULT for 10 minutes to 120 minutes); claim 3 (ULT is -80C); claims 5, 15 (biomass is stores in ULT conditions for 10 minutes); claims 6, 18 (biomass is stored in ULT conditions for 120 minutes); claim 7 (ULT conditions is -80C and the biomass is stored in ULT conditions for 120 minutes); claim 8 (ULT condition is -80°C and the biomass is stored in ULT conditions for 10 minutes); claim 16 (ULT condition is -80°C and the biomass is stored in ULT condition for 10 minutes); claim 17 (ULT condition is -80°C and the biomass is stored in ULT condition for 120 minutes); claims 25, 27 (ULT condition is -20°C and the biomass is stored in ULT condition for 120 minutes); claim 26 (ULT condition is -20°C and the biomass is stored in ULT condition for 10 minutes), MPEP 2144.05 states"[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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to modify the time and temperature at which the recombinant protein is stored depending on the particular application. It would be routine for one to arrive at the time and temperature for the application they intend on using the functional recombinant protein. Therefore, the above invention would have been prima facie obvious. As such, it is the Examiner’s position that it would be obvious to one of ordinary skill in that art to modify the temperature (ULT is -80C) and the time (10min and 120min) at which the biomass is stored at ULT for the accumulation of recombinant protein (claims 1, 3, 5-8, 15-18, 25-27).
One of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would be motivated to utilize a constitutively expressed functional recombinant cold shock protein as opposed to a functional internal cold shock protein, as taught by Koda, because the constant (constitutive) expression and molecular control over said functional recombinant cold shock protein may allow for better survival of bacteria at ULT and for the selection of specific protein for production and isolation. One would be motivated to adjust the temperature and time at which the biomass is stored at ULT to between 10 minutes to 120 minutes because doing so would help to ensure the recombinant protein is not damaged or degraded while at ULT. Furthermore, one of ordinary skill in the art would be motivated to adjust the temperature of the biomass to -80C because Koda teaches a method wherein a plurality of bacteria stored in growth media at ULT exhibited an increase in protein expression (page 196, column 1, para 2; page 197, column 1, para 1, column 2, para 2; page 201, column 1, para 2; page 201, column 2, para 3), thereby providing the motivation for one of ordinary skill in the art to try storing the bacteria at -80C. MPEP 2143.I.E. states “The rationale to support a conclusion that the claim would have been obvious is that "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense. Furthermore, one of ordinary skill in the art would expect similar results at -80C as at -20C since both -20C and -80C are considered by those of ordinary skill in the art to be ULT. Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Applicant's arguments filed 7/14/2025 have been fully considered but they are not persuasive. Beginning on p. 6 of Applicants’ remarks, Applicants in summary contends that the documents cited by the Examiner, whether considered alone or in combination with each other, do not teach, suggest, or render obvious at least the features of "providing two or more bacterial biomasses comprising a plurality of bacterial cells transformed with a plasmid that constitutively expresses a recombinant protein; storing each biomass in ultra-low temperature (ULT) for 10 minutes to 120 minutes, wherein the ULT is -80°C to -20°C, wherein each biomass is stored for the same time and at the same temperature, and wherein each biomass is stored as a cell pellet or in growth media that does not include a cryoprotectant."
Examiner contends that said recitation is obvious over the teachings of Koda. Examiner contends one of ordinary skill in the art would be motivated to utilize a constitutively expressed functional recombinant cold shock protein as opposed to a functional internal cold shock protein, as taught by Koda, because the constant (constitutive) expression and molecular control over said functional recombinant cold shock protein may allow for better survival of bacteria at ULT and for the selection of specific protein for production and isolation. One would be motivated to adjust the temperature and time at which the biomass is stored at ULT to between 10 minutes to 120 minutes because doing so would help to ensure the recombinant protein is not damaged or degraded while at ULT. Furthermore, one of ordinary skill in the art would be motivated to adjust the temperature of the biomass to -80C because Koda teaches a method wherein a plurality of bacteria stored in growth media at ULT exhibited an increase in protein expression (page 196, column 1, para 2; page 197, column 1, para 1, column 2, para 2; page 201, column 1, para 2; page 201, column 2, para 3), thereby providing the motivation for one of ordinary skill in the art to try storing the bacteria at -80C. Furthermore, Examiner contends that the additional limitation of ‘a functional recombinant constitutively expressed protein’ is routinely utilized in the art. Supporting the Examiner’s view is the reference of Rosano et al (2014, frontiers in Microbiology, Examiner cited), which is cited to demonstrate that there are many molecular tools and protocols at hand for the high level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies (abstract).
Applicant contends Koda does not teach storage of a biomass at - 20°C followed by extraction of proteins.
Examiner contends Koda teaches upon analysis of the intracellular protein by SDS-Page, some bands increased after storage at -20C (page 197, column 1, para 1 and page 201, column 1, para 2). Additionally, Examiner contends it would be obvious to one of ordinary skill in that art that after storage at -20C, protein is extracted as said step is a requirement for SDS page analysis as SDS page analysis involves the extraction of protein from samples. As such, absent evidence otherwise, it is the Examiner’s position that said samples are stored at -20C, followed by the extraction of protein and analysis by SDS-Page.
Applicant demonstrates that the time and temperature of biomass storage before lysis are critical to the yield of constitutively expressed recombinant proteins extracted from the biomass; thus, the effect of altering the time of storage is not predictable for recombinant proteins, which are not under the control of a promoter activated by cold temperature.
Examiner contends that Applicant has not sufficiently demonstrated the critically of the time and temperature of the biomass storage before lysis. Especially since Koda teaches an increase in protein expression when stored at -20C for 24 hrs (page 197, column 2, para 2), which is longer than Applicant’s recited 10 minutes to 120 minutes. In fact, cells were also viable after storage at -20C for 24 hrs (Koda: fig. 4). Examiner welcomes Applicant to submit a declaration of evidence supporting the Applicant’s positions or to amend the claims to reflect the criticality of the time at which cells are stored at ULT (10 minutes to 120 minutes) for the extraction of recombinant functional protein.
The rejection of claim 12 under 35 U.S.C. 103 as being unpatentable over Koda et al (2000, Cryobiology, examiner cited) {herein Koda} as applied to claims 1-3, 5-8, 11, 13, 15-18, 20, 25-28 in view of Gomes et al (2020, Available online 19 February 2020, Preventative Biochemistry & Biotechnology, cited on PTO-892 form dated 2/13/2024) {herein Gomes} is maintained. The rejection has been modified in view of Applicant’s amendment to claims 1 and 13 to recite ‘a functional recombinant protein.’
Original claim 12 is drawn to the method of claim 1, wherein the plurality of cells is lysed with a solution comprising a hydrolyzing enzyme.
The teachings of Koda as applied to claims 1-3, 5-8, 11, 13, 15-18, 20, 25-28 in the 103 rejection above.
However, Koda does not teach the method of claim 12, wherein the plurality of cells is lysed with a solution comprising a hydrolyzing enzyme (claim 12).
With respect to claim 12, Gomes teaches a method wherein bacterial cells, such as Escherichia coli, are stored at temperatures between -15C and -80C and thawed for the recovery of biomolecules (page 642, column 1, para 3; page 639, column 1, para 3) via lysing cells with lysozyme (page 640, column 2, paragraph 3). Examiner is interpreting -15C and -80C to encompass -20C. Said method results in an improved yield of intracellular proteins (page 639, column 1, para 3-4; column 2, para 1).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Koda of a method for recovering functional protein extracted from bacterial biomasses stored at -20C or combine the teachings of Gomes because Gomes teaches a method wherein bacterial cells, such as Escherichia coli, are stored at temperatures between -15C and -80C and thawed for the recovery of biomolecules (page 642, column 1, para 3; page 639, column 1, para 3) via lysing cells with lysozyme (page 640, column 2, paragraph 3). Whereas Koda teaches a method wherein a plurality of bacterial cells (Pantoea agglomerans (Erwinia herbicola), Xanthomonas campestris pv. Translucens, and Pseudomonas syringae) stored in growth media at -20C have an increase in intracellular proteins referred to as cold acclimation protein (page 196, column 1, para 2; page 197, column 1, para 1, column 2, para 2; page 201, column 1, para 2; page 201, column 2, para 3) in the absence of cryoprotective agents.
One of ordinary skill in the art would be motivated to either use the teachings of Koda et al. by itself or combine the teachings of Gomes because Gomes provides the motivation for Koda to utilize lysozyme for the lysing of cells as lysozyme is widely used and can also be applied in large-scale (Gomes: page 640, column 2, para 2). One of ordinary skill in the art would be motivated to utilize lysozyme because lysozyme is the most known and the best described for the bacteria cell disruption (page 640, column 2, para 3). Additionally, lysozyme is commercially available at a reasonable cost (Gomes: page 640, column 2, para 3).
One of skill in the art would have a reasonable expectation of success to make and use the claimed method for recovering functional intracellular protein extracted from bacterial biomasses lysed by lysozyme and stored at ULT for 10 min to 120 min because Koda provides the method for recovering functional intracellular protein extracted from bacterial biomasses stored at ULT compared to a conventional temperature (page 196, column 1, para 2; page 197, column 1, para 1, column 2, para 2; page 201, column 1, para 2; page 201, column 2, para 3). While Gomes provides the teaching of utilizing lysozyme for the recovery of intracellular proteins (page 640, column 2, para 3). Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Applicant's arguments filed 7/14/2025 have been fully considered but they are not persuasive.
Examiner contends all claim limitations have been addressed in the 103 rejection. Examiner contends Koda, in view of Gomes teaches the claimed limitation of ‘the plurality of cells is lysed with a solution comprising a hydrolyzing enzyme’ (claim 12) with the teaching of ‘a method wherein bacterial cells, such as Escherichia coli, are stored at temperatures between -15C and -80C and thawed for the recovery of biomolecules (page 642, column 1, para 3; page 639, column 1, para 3) via lysing cells with lysozyme (page 640, column 2, paragraph 3). Examiner is interpreting -15C and -80C to encompass -20C.’
Conclusion
Status of claims
Claims 1-3, 5-8, 11-13, 15-18, 20, 25-28 are pending.
Claims 4, 9-10, 14, 19, 21-24 are cancelled.
Claims 1-3, 5-8, 11-13, 15-18, 20, 25-28 are rejected.
No claims are in condition for allowance.
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 ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656