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 the Application
1. Claims 1-9 are pending and subject to examination on the merits. Claims 1-9 are currently under examination.
Priority
2. Acknowledgement is made of applicant’s claim for foreign priority based on an application filed in EP (EP20212860.9) on 09 December 2020. Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Withdrawn Objections/Rejections
3. The objection to the drawings is withdrawn, since the drawings were amended to make the symbols more clearly distinguishable between the samples.
4. The 35 U.S.C. 112(a) written description and enablement rejections are withdrawn, since claim 1 was amended to recite “wherein the biological cells are bacterial cells or yeast cells, and wherein the electrically conductive liquid includes water, glycerol, a magnesium salt, a sulfate salt, and trace metals,” making it both enabled and in compliance with written description.
5. The 35 U.S.C. 112(b) indefiniteness rejection of claims 1-2 and 4-6 is withdrawn, since claim 1 was amended to delete the phrase “preferably from.”
6. The 35 U.S.C. 112(b) indefiniteness rejection of claim 3 is withdrawn, since claim 3 was amended to delete the phrase “preferably from.”
7. The 35 U.S.C. 112(b) indefiniteness rejection of claims 7-9 is withdrawn, since claim 7 was amended to delete the phrase “preferably from.”
8. The 35 U.S.C. 102 anticipation rejection of claims 1-3, 5, and 7-8 is withdrawn, since claim 1 was amended to add the limitation, “wherein the biological cells are bacterial cells or yeast cells, and wherein the electrically conductive liquid includes water, glycerol, a magnesium salt, a sulfate salt, and trace metals.” It is replaced with the instant rejection found below.
9. The provisional non-statutory double patenting rejection of claims 1 and 3 of copending Application No. 17797853 is withdrawn, since the application has been abandoned.
10. The provisional non-statutory double patenting rejection of claims 1 and 3 of copending Application No. 17797865 is withdrawn, since the application has been abandoned.
Claim Objections
11. Claim 13 is objected to because of the following informalities: “Na2MoO4” should be “Na2MoO4.” Appropriate correction is required.
12. Claim 14 is objected to because of the following informalities: “Na2MoO4” should be “Na2MoO4.” Appropriate correction is required.
13. Claim 15 is objected to because of the following informalities: “Na2MoO4” should be “Na2MoO4.” Appropriate correction is required.
New/Modified Rejections—necessitated by amendments
Claim Rejections - 35 USC § 103
14. 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.
15. 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.
16. Claims 1, 3, 5, 7, 10-15, 17, and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Wolfgang et al (Wolfgang et al., 2011, EP2308969A1—cited previously), as evidenced by Zhu and Thompson (Zhu and Thompson, 2019, Nat Rev Mol Cell Biol—cited previously), in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_popularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023). Regarding claims 1, 3, 5, and 7, drawn to an in vitro method of increasing metabolic activity and/or stimulating cell proliferation (claims 3 and 7) of biological cells, wherein said cells are yeast (claim 3), comprising (a) suspending the biological cells in an electrically conductive liquid, (b) positioning the suspension between two electrodes, and (c) applying 1-100 pulses of electricity between the electrodes, characterized in that the voltage increase between the electrodes is 10-90% of a target voltage, wherein the electric field strength is 0.5 kV/cm-50 kV/cm, pulse timing within 0.1-100 ns, and pulse duration of 5-5000 ns, wherein step (c) is carried out in a continuous recirculation process (claims 5 and 7), Wolfgang et al. teaches accelerating the cell proliferation of biological cell material, comprising (a) providing a cell material and suspending said material in an electrically conductive fluid between two electrodes, and (b) discharging at least one electric field pulse between the electrodes with a rise in voltage between 10-90% of a target voltage within a time period of 0.1-100ns (abstract). Wolfgang et al. continues to teach that the pulse duration is 5-5000 ns (p. 3, paragraph 8), wherein the field strength of the electric field strength pulse is between 0.5kV and 50kV/cm (p. 3, paragraph 10). Further, Wolfgang et al. teaches that the use of cell cultures in cell medium by fermenters, particularly microalgae, bacteria or yeast for the production of active ingredients, foods, or for the production of energy sources, where the suspension may be pumped during rearing, where then the treatment of cells can take place in continuous operation (p. 4, paragraph 4). Zhu and Thompson evidence that cell proliferation is a product of increased metabolic activity, specifically where cell proliferation requires the accumulation of intracellular biomass, such as proteins and lipids to produce daughter cells, where the biosynthesis of these molecules is due through a network of cellular metabolic pathways (p. 436, Introduction). Regarding claim 21, drawn to the electrically conductive liquid further including a potassium salt, Wolfgang et al. teach a treatment buffer of 40mM KCl (p.4, Example 1, paragraph 2).
Wolfgang et al. as evidenced by Zhu and Thompson does not teach the electrically conductive liquid including water, glycerol, a magnesium salt, a sulfate salt, and trace metals, where the trace metals include iron, cobalt, manganese, molybdenum, aluminum, zinc, copper, and nickel (claims 10-12), and further, wherein the trace metals include Fe(III) citrate, FeSO4, CoCl-2, MnCl2, MnSO4, Na2MoO4, AlCl3, ZnSO4, ZnCl2 CuSO4, NiCl2 (claims 13-15). Additionally, Wolfgang et al. as evidenced by Zhu and Thompson does not teach the electrically conductive liquid further including a sodium chloride (claim 17).
Regarding the utilization of glycerol and an anti-foaming agent (claim 20), Kroemer teaches a protocol to prepare cells for electroporation, which includes the utilization of glycerol to help prevent arcing and give high transformation efficiency (p. 6, last paragraph), which can be used as an anti-foaming agent, as evidenced by Francis et al (Title, abstract).
Regarding the utilization of magnesium salts, sulfate salts, and sodium chloride (claim 17), Sherba et al. teaches the utilization of many different electroporation buffers of various conductivities using K+, Na+, Cl-, and SO42- ions and found that Na+ and K+ based buffers conferred similar viabilities at lower conductivities to cells upon electroporation (p.2, full paragraph 2). To further investigate the effect of conductivity, Sherba et al. teaches the specific utilization of MgSO4 in one of the electroporation buffers (Table 1).
Teknova teaches a trace metal solution to support microbial growth, fermentation, and the preparation of defined media, where the trace metals include iron, zinc, manganese, copper, cobalt, and molybdenum (p. 5), wherein the trace metals include CoCl-2, MnSO4, Na2MoO4, and ZnCl2 (p. 9).
Therefore, it 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 combine the teachings of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt, and trace metals to introduce DNA to cells by transformation as taught by Kroemer (p. 1, lines 1-2). One would be motivated to combine these teachings to arrive at the instant claims to accelerate biological cell material proliferation as taught by Wolfgang et al (abstract). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of Wolfgang et al. as evidenced by Zhu and Thompson, Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt, and trace metals, since Wolfgang et al. teaches a general method for electroporation of bacterial or yeast cells.
17. Claims 4, 6, and 9 are rejected under 35 U.S.C. 103 as being unpatentable over Wolfgang et al (Wolfgang et al., 2011, EP2308969A1—cited previously), as evidenced by Zhu and Thompson (Zhu and Thompson, 2019, Nat Rev Mol Cell Biol—cited previously) in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_popularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023) as applied to claims 1, 3, 5, 7, 10-15, 17, and 20-21 above, and further in view of Liu et al (Liu et al., 2019, Critical Reviews in Biotechnology—cited previously).
The teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of Sherba et al. are discussed above and incorporated into the instant rejection.
Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer, in view of Teknova, and in view of Sherba et al. do not teach the batch fermentation (claim 6) of the yeast, P. pastoris (claims 4 and 9). Liu et al. teach the fed-batch fermentation of P. pastoris in a high-cell density fermentation reaction (p. 258; title, abstract), where specifically Liu et al. focus on various stat-induction strategies, co-feeding, and the limited induction strategy.
Therefore, it 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 to combine the teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of Sherba et al., and in further view of Liu et al. to devise a method to increase cell proliferation and/or metabolic activity of P. pastoris by (a) suspending the biological cells in an electrically conductive liquid, (b) positioning the suspension between two electrodes, and (c) applying 1-100 pulses of electricity between the electrodes, characterized in that the voltage increase between the electrodes is 10-90% of a target voltage, wherein the electric field strength is 0.5 kV/cm-50 kV/cm, pulse timing within 0.1-100 ns, and pulse duration of 5-5000 ns, wherein step (c) is carried out in a batch process because utilization of pulsed electric field is as versatile as possible as taught by Wolfgang et al. (p. 3, paragraph 3). One would be motivated to combine the teachings to arrive at the instant claims because the method according to Wolfgang et al. is an economically sensible method, since a significantly higher yield of cell material is obtainable with relatively low energy input in the form of electric field strength pulses (p. 6, paragraph 5). Additionally, one would be further motivated to combine the teachings because P. pastoris is utilized extensively to produce various heterologous proteins and industrial enzymes have been successfully produced by fed-batch-high-cell-density fermentation as taught by Liu et al (p. 258, abstract). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of Sherba et al. with those of Liu et al., since Wolfgang et al. teach the utilization of yeast in a pulse electric field method, and a common yeast utilized in industrial applications is P. pastoris as taught by Liu et al.
18. Claims 16 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Wolfgang et al (Wolfgang et al., 2011, EP2308969A1—cited previously), as evidenced by Zhu and Thompson (Zhu and Thompson, 2019, Nat Rev Mol Cell Biol—cited previously), in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_p--opularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023), as applied to claims 1, 3, 5, 7, 10-15, 17, and 20-21 above, and further in view of Lamichhane et al (lamichhane et al., 2015, molecular pharmaceutics—cited herein).
The teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of Sherba et al. are discussed above and incorporated into the instant rejection.
Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer, in view of Teknova, and in view of Sherba et al. do not teach a phosphate component (claim 16) or the chelating agent (claim 18), EDTA (claim 19).
Lamichhane et al. teaches the utilization of the electroporation buffer containing potassium phosphate with 1mM EDTA to electroporate EVs with DNA (p. 3651, “DNA Loading into EVs by Electroporation”).
Therefore, it 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 to combine the teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of Sherba et al., and further in view of Lamichhane et al. to electroporate a bacterial or yeast cell in a phosphate and EDTA containing buffer to increase proliferation as taught by Wolfgang et al. One would be motivated to combine these teachings to arrive at the instant because the method according to Wolfgang et al. is an economically sensible method, since a significantly higher yield of cell material is obtainable with relatively low energy input in the form of electric field strength pulses (p. 6, paragraph 5). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of combine the teachings of Wolfgang et al., as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al., in view of Teknova, and in view of iSherba et al., and further in view of Lamichhane et al. to electroporate a bacterial or yeast cell in a phosphate and EDTA containing buffer, since Lamichhane et al. teaches the utilization of a phosphate and EDTA containing buffer during electroporation.
Double Patenting
19. 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.
20. Claims 1 and 3 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 26, 27, and 31 of copending Application No. 17797857 in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_popularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023).
The instant claims in their broadest are drawn to an in vitro method of increasing metabolic activity and/or stimulating cell proliferation (claim 3) of biological cells, comprising (a) suspending the biological cells in an electrically conductive liquid, (b) positioning the suspension between two electrodes, and (c) applying 1-100 pulses of electricity between the electrodes, characterized in that the voltage increase between the electrodes is 10-90% of a target voltage, wherein the electric field strength is 0.5 kV/cm-50 kV/cm, pulse timing within 0.1-100 ns, and pulse duration of 5-5000 ns.
The claims in the ‘857 application in their broadest are drawn to a method for treating cells for targeted inactivation, the extraction of bioactive compounds, and the stimulation of cell growth and/or cellular compounds, comprising the steps a) applying an electric field to a treatment space in a treatment unit that has a geometric shape which allows a non-contact passage of cell material, b) introducing cell material through an inlet of the treatment unit into the treatment space, c) passing of the cell material without contact through the treatment space wherein there are no interactions of the cell material with boundary walls of the treatment space during the movement, and the electric field penetrating the treatment space to an outlet of the treatment unit, and supplying the cell material moved through the treatment space to the outlet by means of a receiving device that is provided in the treatment unit, wherein said receiving device is funnel-shaped, and wherein the diameter of the treatment space is wider than the diameter of the inlet, outlet and the receiving device, wherein an electric field is applied with such electric pulses, so that a voltage increase takes place between two electrodes or plates of a capacitor of 10% to 90% of a target voltage of the electric pulses within a period of 0.1 to 1000 ns, the electric pulses have a pulse duration of 5 ns to 50000 ns, and the electric pulses, upon reaching the target voltage, have an electric field strength of 0.5 kV/cm to 100 kV/cm.
The ‘857 claims do not teach he electrically conductive liquid including water, glycerol, a magnesium salt, a sulfate salt, and trace metals.
Regarding the utilization of glycerol, Kroemer teaches a protocol to prepare cells for electroporation, which includes the utilization of glycerol to help prevent arcing and give high transformation efficiency (p. 6, last paragraph).
Regarding the utilization of magnesium salts and sulfate salts, Sherba et al. teaches the utilization of many different electroporation buffers of various conductivities using K+, Na+, Cl-, and SO42- ions and found that Na+ and K+ based buffers conferred similar viabilities at lower conductivities to cells upon electroporation (p.2, full paragraph 2). To further investigate the effect of conductivity, Sherba et al. teaches the specific utilization of MgSO4 in one of the electroporation buffers (Table 1).
Teknova teaches a trace metal solution to support microbial growth, fermentation, and the preparation of defined media (p. 5).
Therefore, it 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 combine the teachings of the ‘857 claims in further view of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt in a treatment space to introduce DNA to cells by transformation as taught by Kroemer (p. 1, lines 1-2). One would be motivated to combine these teachings to arrive at the instant claims to accelerate biological cell material proliferation as taught by Wolfgang et al (abstract). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt, and trace metals, since Wolfgang et al. teaches a general method for electroporation of bacterial or yeast cells.
This is a provisional nonstatutory double patenting rejection.
21. Claims 1 and 3 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 27-29 of U.S. Patent No. 12559709 (formerly US application 17797866) in view of Wolfgang et al (Wolfgang et al., 2011, EP2308969A1—cited previously), as evidenced by Zhu and Thompson (Zhu and Thompson, 2019, Nat Rev Mol Cell Biol—cited previously), in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_popularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023).
The instant claims in their broadest are drawn to an in vitro method of increasing metabolic activity and/or stimulating cell proliferation (claim 3) of biological cells, comprising (a) suspending the biological cells in an electrically conductive liquid, (b) positioning the suspension between two electrodes, and (c) applying 1-100 pulses of electricity between the electrodes, characterized in that the voltage increase between the electrodes is 10-90% of a target voltage, wherein the electric field strength is 0.5 kV/cm-50 kV/cm, pulse timing within 0.1-100 ns, and pulse duration of 5-5000 ns.
The claims in the ‘709 patent in their broadest are drawn to a method for treating cells for targeted inactivation, the extraction of bioactive compounds, and/or the stimulation of cell growth and/or cellular compounds, performed in a device, comprising the steps: a) introducing cell material through an inlet of a treatment unit into a treatment space; b) applying electric pulses to the treatment space in the treatment unit; and c) passing the cell material through the treatment space and the electric pulses penetrating the treatment space to an outlet of the treatment unit, wherein an electric field is applied with such electric field strength pulses that a voltage increase takes place between the two electrodes or plates of a capacitor of 10% to 90% of a target voltage of the electric field strength pulses within a period of 0.1 to 1000 ns, the electric field strength pulses have a pulse duration of 5 ns to 50000 ns, and the electric field strength pulses, upon reaching the target voltage, have an electric field strength of 0.5 kV/cm to 100 kV/cm.
The ‘709 claims do not teach he electrically conductive liquid including water, glycerol, a magnesium salt, a sulfate salt, and trace metals.
Regarding the utilization of glycerol, Kroemer teaches a protocol to prepare cells for electroporation, which includes the utilization of glycerol to help prevent arcing and give high transformation efficiency (p. 6, last paragraph).
Regarding the utilization of magnesium salts and sulfate salts, Sherba et al. teaches the utilization of many different electroporation buffers of various conductivities using K+, Na+, Cl-, and SO42- ions and found that Na+ and K+ based buffers conferred similar viabilities at lower conductivities to cells upon electroporation (p.2, full paragraph 2). To further investigate the effect of conductivity, Sherba et al. teaches the specific utilization of MgSO4 in one of the electroporation buffers (Table 1).
Teknova teaches a trace metal solution to support microbial growth, fermentation, and the preparation of defined media (p. 5).
Therefore, it 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 combine the teachings of the ‘709 claims in further view of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt in a device to introduce DNA to cells by transformation as taught by Kroemer (p. 1, lines 1-2). One would be motivated to combine these teachings to arrive at the instant claims to accelerate biological cell material proliferation as taught by Wolfgang et al (abstract). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt, and trace metals, since Wolfgang et al. teaches a general method for electroporation of bacterial or yeast cells.
22. Claims 1 and 3 provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 12 and 15 of copending Application No. 18252834 in view of Wolfgang et al (Wolfgang et al., 2011, EP2308969A1—cited previously), as evidenced by Zhu and Thompson (Zhu and Thompson, 2019, Nat Rev Mol Cell Biol—cited previously), in view of Kroemer (Kroemer, 2010, downloaded on 15 July 2026 from <https://www.goldbio.com/blogs/articles/understanding-competent-cells-for-bacterial-transformation> and provided as a PDF—cited herein) as evidenced by Francis et al (Francis et al., 1969, US 3458567—cited herein), in view of Teknova (Teknova, 2010, downloaded on 15 July 2026 as a PDF from <https://www.teknova.com/en/products/category-page.html/buffers-and-reagents/additives/trace-metalssolutions.html?page=1&resultsPerPage=15&sort_key=netsuite_popularity&sort_order=desc&productView=list#accordion-00cd508dff-item-53383d5722e> --cited herein), and in view of Sherba et al (Sherba et al., 2020, Scientific Reports—cited on the IDS dated 09 June 2023).
The instant claims in their broadest are drawn to an in vitro method of increasing metabolic activity and/or stimulating cell proliferation (claim 3) of biological cells, comprising (a) suspending the biological cells in an electrically conductive liquid, (b) positioning the suspension between two electrodes, and (c) applying 1-100 pulses of electricity between the electrodes, characterized in that the voltage increase between the electrodes is 10-90% of a target voltage, wherein the electric field strength is 0.5 kV/cm-50 kV/cm, pulse timing within 0.1-100 ns, and pulse duration of 5-5000 ns.
The claims in the ‘834 application in their broadest are drawn to method for treating cells for targeted inactivation, the extraction of bioactive compounds, and the stimulation of cell growth and/or cellular compounds, performed in a device, comprising the steps of:a) applying electric pulses to a treatment spacepassing of the cell material through the treatment space and the electric pulses penetrating the treatment space and conveying said cell material back into said or optionally another unit for taking up a fluid, wherein an electric field is applied with such electric field strength pulses, so that a voltage increase takes place between the two electrodes or plates of 10% to 90% of a target voltage of the electric field strength pulses within a period of 0.1 to 1000 ns, the electric field strength pulses have a pulse duration of 5 ns to 50000 ns, and the electric field strength pulses, upon reaching the target voltage, have an electric field strength of 0.5 kV/cm to 100 kV/cm.
The ‘857 claims do not teach he electrically conductive liquid including water, glycerol, a magnesium salt, a sulfate salt, and trace metals.
Regarding the utilization of glycerol, Kroemer teaches a protocol to prepare cells for electroporation, which includes the utilization of glycerol to help prevent arcing and give high transformation efficiency (p. 6, last paragraph).
Regarding the utilization of magnesium salts and sulfate salts, Sherba et al. teaches the utilization of many different electroporation buffers of various conductivities using K+, Na+, Cl-, and SO42- ions and found that Na+ and K+ based buffers conferred similar viabilities at lower conductivities to cells upon electroporation (p.2, full paragraph 2). To further investigate the effect of conductivity, Sherba et al. teaches the specific utilization of MgSO4 in one of the electroporation buffers (Table 1).
Teknova teaches a trace metal solution to support microbial growth, fermentation, and the preparation of defined media (p. 5).
Therefore, it 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 combine the teachings of the ‘857 claims in further view of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt in a treatment space to introduce DNA to cells by transformation as taught by Kroemer (p. 1, lines 1-2). One would be motivated to combine these teachings to arrive at the instant claims to accelerate biological cell material proliferation as taught by Wolfgang et al (abstract). There would be reasonable expectation of success, yielding no surprising results when combining the teachings of Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. to electroporate a bacterial or yeast cell in a buffer containing water, glycerol, a magnesium salt, a sulfate salt, and trace metals, since Wolfgang et al. teaches a general method for electroporation of bacterial or yeast cells.
This is a provisional nonstatutory double patenting rejection.
Applicant’s Arguments and Examiner’s Rebuttal:
The Applicant traverses the previous anticipation rejection of claims 1-3, 5, and 7-8 over Wolfgang et al, as evidenced by Zhu and Thompson. Additionally, the Applicant traverses the obviousness rejection of claims 4, 6, and 9 over Wolfgang et al, as evidenced by Zhu and Thompson and in further view of Liu et al. The anticipation rejection of claims 1-3, 5, and 7-8 was withdrawn in lieu of the claim amendments to claim 1 and replaced with the instant rejection found above of claims 1, 3, 5, 7-8, 10-15, 17, and 20-21 over Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. Additionally, the previous obviousness rejection of claims 4, 6, and 9 was modified to the obviousness rejection of claims 1, 3, 5, 7-8, 10-15, 17, and 20-21 over Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. and further in view of Liu et al. The new obviousness rejection of claims 16 and 18-19 over Wolfgang et al. as evidenced by Zhu and Thompson, in view of Kroemer as evidenced by Francis et al, in view of Teknova, and in view of Sherba et al. in further view of Lamichhane et al. was added due to the addition of these new claims. New claims 10-21 have been addressed in the above instant rejections.
First, the applicant argues that claims 1, 3, and 7 were amended to recite “(c) applying from 2 pulses to 12 pulses of electricity between the electrodes…wherein a voltage increase between the two electrodes from 10% to 90% of a target voltage of the pulses of electricity takes place within a time period of 3 ns to 15 ns; wherein the pulses of electricity have a pulse duration of between 90 ns and 125 ns; wherein the pulses of electricity, when reaching the target voltage, have an electric field strength of 0.5 kV/cm to 30 kV/cm,” which distinguishes the claims from the applied art, Wolfgang et al. The examiner respectfully disagrees. As recited in the above rejection, Wolfgang et al teaches “accelerating the cell proliferation of biological cell material, comprising (a) providing a cell material and suspending said material in an electrically conductive fluid between two electrodes, and (b) discharging at least one electric field pulse between the electrodes with a rise in voltage between 10-90% of a target voltage within a time period of 0.1-100 ns (abstract). Wolfgang et al. continues to teach that the pulse duration is 5-5000 ns (p. 3, paragraph 8), wherein the field strength of the electric field strength pulse is between 0.5kV and 50kV/cm (p. 3, paragraph 10). Although the applicant narrowed the recited ranges in the claims, the ranges are still obviated by Wolfgang et al., since the ranges overlap. The MPEP states: “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)” (See MPEP 2144.05, Section I).
Additionally, the applicant traverses the double patenting rejections of claims 1 and 3 over claims 26-27, and 31 of Application No. 17797853, over claims 26-27 and 30 of Application No. 17797857, over claims 16 and 18 of Application No. 17797865, over claims 27-29 of Application No. 17797866, and over claims 12 and 15 of Application No. 18252834. The double patenting rejections of claims 1 and 3 over claims 26-27 of Application Nos. 17797853 and 17797865 were withdrawn due to the applications being abandoned. The applicant argues that the amended claims are distinct of the claims recited in these applications. The Examiner respectfully disagrees The additional double patenting rejections were modified to include the secondary references addressing the buffer constituents and are found above.
The examiner does not find the arguments presented by the Applicant persuasive, and for these reasons, the rejections of record above apply.
Conclusion
23. All claims are rejected.
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.
24. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CIARA A MCKNIGHT whose telephone number is (703)756-4791. The examiner can normally be reached M-F 8:00am-4:30pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath Rao can be reached on (571) 272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CIARA A MCKNIGHT/Examiner, Art Unit 1656
/SUZANNE M NOAKES/Primary Examiner, Art Unit 1656
Prosecution Insights