DETAILED OFFICE ACTION
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s preliminary amendment to the claims, filed on June 13, 2023, is
acknowledged. This listing of the claims replaces all prior versions and listings of the
claims.
Claims 1-14 and 26-31 are pending and are being examined on the merits.
Claims 15-25 are cancelled.
Applicant’s amendment to the specification, filed June 13, 2023, is acknowledged.
Priority
This application is filed under 35 U.S.C. 371 as a national stage of international
application PCT/EP2021/059342, which claims foreign priority under 35 U.S.C. 119(a)-(d) to a foreign patent application EP20169148.2 filed on April 9, 2020, EP20169147.4 filed on April 9, 2020, EP20169642.4 filed on April 15, 2020, EP20187283.5 filed on July 22, 2020, and EP20202816.3 filed on October 20, 2020. A certified copy of the foreign priority applications have been filed in this application on October 5, 2022.
Restrictions/Election
Applicants’ election of species (see below) without traverse, filed September 29, 2025 is acknowledged.
The elected species are:
Species Group 1: as the lipidation type, one fatty acid and a glycerol substituted with two fatty acids; and as the HPLC profile: peaks P1+P2, P3, P4 and P5+P6 comprise 23±10%, 41±10%, 25±10% and 12±10% of the total lipidated protein;
Species Group 2: a volume of at least 100 L; and
Species Group 3: an OspA heterodimer, SEQ ID NO: 1.
Therefore, claims 29-30 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected species.
Claims 1-14, 26-28, and 31 are being examined on the merits to the extent the claims read on the elected species.
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on June 13, 2023 and September 29, 2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS have been considered by the examiner and those references therein have been indicated as such.
Foreign Patent Document JP 4810428 of the IDS filed June 16, 2023 is lined through because there is no copy of the references in the application file. Foreign Patent Document WO 2021/205017, WO 2021/205022, and WO 2021/207615 of the IDS filed September 29, 2023 are lined through because there is no copy of the references in the application file.
Claim Objections
Claim 1 and 7 are objected to because of the following informalities:
Claim 1 is objected to because of the recitation of “a) E coli” without first writing out the full phrase for which the abbreviation “E coli” is used. In the interest of improving claim form, it is suggested that the noted phrase be amended to ““a) Escherichia coli (E. coli).”
Claim 7 is objected to because of the recitation of “iron(III)chloride” and “cobalt(II)chloride”. In the interest of improving claim form, it is suggested that the noted phrases be amended to “iron (III) chloride” and “cobalt (II) chloride,” respectively.
Claim Rejections - 35 USC § 112(b)
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.
Claims 1-14, 26-28, and 31 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1 (claims 2-14, 26-28, and 31 dependent therefrom), 8, and 14 are rejected as indefinite because of the use of exemplary claim language. The exemplary claim language of “particularly” is used in lines 3, 5, and 19 of claim 1 as well as lines 3-4 of claim 14(b). The exemplary claim language of “preferably” is used in line 6 of claim 1 as well as lines 3 and 4 of claim 8. The exemplary claim language of “such as” is used in line 4 of claim 8. The phrases "particularly", “preferably”, and “such as” render the claims indefinite because it is unclear whether the limitations following the phrases are part of the claimed invention. Description of examples or preferences is properly set forth in the specification rather than the claims. If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim. See MPEP § 2173.05(d). For the purpose of compact prosecution, claim limitations referred to by the exemplary claim language are not considered as required by the claims. The Office suggests amending the claims to remove exemplary language.
Claims 1 (claims 3-14, 26-28, and 31) and 2 are rejected as indefinite because the claims recite the phase "additional headspace pressure" which lacks antecedence. Prior to its use in the final line of claim 1, there is no recitation of the phrase “headspace pressure.” Therefore, there is insufficient antecedent basis for this limitation and therefore "additional headspace pressure" lacks antecedence. For the purpose of compact prosecution, the phrase "additional headspace pressure" is reconstrued to mean “headspace pressure above atmospheric pressure."
Claim 1 (claims 2-14, 26-28, and 31 dependent therefrom) is indefinite in the recitation of the approximation of "about." The examiner has reviewed the specification and can find no examples or teachings that can be used for ascertaining the variance intended by the term "about." Moreover, there is nothing in the specification or prior art of record to indicate that one of ordinary skill in the art could have ascertained the scope of the term "about." It is suggested that applicant clarify the meaning of the term "about." See MPEP 2173.05(b).III.A regarding the term "about."
Claim 7 (claim 8 dependent therefrom) is rejected as indefinite because the recitation of the phrase “16.7 mL/L hydrochloric acid” is unclear as to the intended meaning of the phrase because no concentration of hydrochloric acid is disclosed. The Offices suggests that the applicant clarify the meaning by amending the claim to just include the concentration of hydrochloric acid present in the composition. For the purpose of compact prosecution, the recited limitation is interpreted as the trace element solution comprising any volume and/or concentration of hydrochloric acid.
Claim 8 is rejected as indefinite because the claim recites the phase "the batch phase" which lacks antecedence. Claims 1 and 7 do not recite the "the batch phase.” Since claim 8 depends upon claim 7 and claim 7 depends upon claim 1, there is insufficient antecedent basis for this limitation and therefore "the batch phase" lacks antecedence. For the purpose of compact prosecution, “the TE solution is added in the bat phase” is interpreted as "the TE solution is added during culturing step a).”
Claims 10 (claims 11-12 dependent therefrom) and 12 are rejected as indefinite because of the use of parentheses in the phrases “exponential phase of E. coli growth feed phase)” and “continuously during the feed phase (exponentially)”, respectively. It is unclear whether the content of the parentheses is a required or merely exemplary. See MPEP § 2173.05(d). The Office suggests amending the claims to clearly indicate the required limitations. For the purpose of compact prosecution, the phrases “exponential phase of E. coli growth (feed phase)” and “continuously during the feed phase (exponentially)” are construed as phrases “exponential phase or feed phase of E. coli growth” and “continuously during the exponential phase or feed phase”, respectively.
Claim 31 is rejected as indefinite because the claim recites the phase "the induction" which lacks antecedence. Prior to its use in the final line of claim 31, there is no recitation of the phrase “induction.” Therefore, there is insufficient antecedent basis for this limitation and therefore "the induction" lacks antecedence. For the purpose of compact prosecution, the claim 31 is interpreted as “The method of claim 31 wherein the
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.
Claim 1-3 and 26-27 are rejected under 35 U.S.C. 103 as being unpatentable over Leng et al. (US 2009/0176273 A1; cited on the attached Form PTO-892; hereafter “Leng”) in view of Madurawe et al. (Biotechnol. Prog., published 2000, Vol. 16, p. 571-576; cited on the attached Form PTO-892; hereafter “Madurawe”), Knabben et al. (Journal of Biotechnology, published October 1, 2010, Vol. 150, No. 1, p. 73-79; cited on the attached Form PTO-892; hereafter “Knabben”).
Regarding claims 1-3 and 26-27, Leng teaches a method of producing a lipidated protein in E. coli, comprising providing a host E. coli cell transformed with an expression plasmid including a nucleotide sequence that encodes a naturally lipidated protein, wherein the host E. coli cell is adapted to membrane protein expression, culturing the host E. coli cell to allow expression of the protein in lipidated form, and isolating the protein (Claim 10). Leng teaches the method can produce a lipoprotein wherein the lipid moiety is N-acyl-5-diacylglyceryl moiety attached to a Cys residue, or wherein lipid moiety is as seen below wherein carbon chains are unsaturated or saturated and usually 16-20 carbons in length (para [0025, 0052]; Figure 7):
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Leng teaches wherein the lipidated protein of interest is harvested with the detergent Triton-100 (para [0013, 0025, 0037]). Leng teaches the inclusion of Triton-1000 during cell disruption increases protein yields (para [0037]).
The differences between Leng’s method of producing a lipidated protein and the claimed invention is that Leng does not explicitly teach wherein the method comprises culturing E. coli cells in culture conditions wherein the pH is in the range of 6.7 to 7.3, there is additional headspace pressure, and the volume of cultured E. coli cells is at least 40 L.
Regarding the pH of the cell culture, Madurawe teaches production of a recombinant lipoprotein in E. coli wherein the cells are cultured in pH 7.0 media (p. 571, col 2, para 3 – p. 572, col 1, para 2).
In view of the combined teachings of Leng and Madurawe, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by Leng such that the E. coli is cultured in pH 7.0 media as taught by Madurawe.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by Leng such that the E. coli is cultured in pH 7.0 media since Madurawe taught producing a recombinant lipoprotein in E. coli wherein the cells cultured in pH 7.0 media.
Regarding cell culture comprising additional headspace pressure, Knabben teaches using a pressurized 50 L bioreactor with elevated headspace pressure to overcome oxygen transfer growth limitations in order to allow high growth conditions and high-density fed-batch culture (Abstract; p. 73, col 2, para 1; p. 75, col 1, para 3; Figure 1). Knabben teaches altering the headspace pressure in a range from zero to 4.8 bar, and increasing the headspace overpressure increases oxygen transfer rate (Abstract; p. 74, col 2, para 1; Figure 3). Knabben reports increasing the headspace pressure up to 10 or 11 bar (p. 75, col 2, para 1; p. 75, col 1, para 3).
In view of the combined teachings of Leng, Madurawe, and Knabben, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng and Madurawe such that the culturing of E. coli occurs in a fed-batch manner in the 50 L bioreactor with headspace overpressure as taught by Knabben and wherein through routine optimization the headspace overpressure is between 0.4-1 bar.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng and Madurawe such that the culturing of E. coli occurs in a fed-batch manner with headspace overpressure since Knabben taught using fed-batch culture and a pressurized bioreactor with elevated headspace pressure to overcome oxygen transfer growth limitations in order to allow high growth conditions and high-density culture. Knabben also discloses wherein the headspace pressure is 0-4.8 bar as well as the possibility of the headspace pressure being as high as 11 bar. Since Knabben taught that cell culture is limited by oxygen transfer rate in the culture system and that elevated headspace pressure increases oxygen transfer rate, it would have been obvious for one of ordinary skill to discover the optimum workable extraction temperature disclosed by the prior art by normal optimization procedures known in the art and arrived at a headspace pressure of 0.4-1 bar.
Since the combination of Leng, Madurawe, and Knabben teaches the actively recited process steps of claim 1, it is the Examiner’s position that the lipidation profiled required in claims 1 and 26-27 would inherently result from the method taught by the combination of Leng, Madurawe, and Knabben. Since the Office does not have the facilities for examining and comparing applicant’s method of producing a lipidated protein with the method of producing a lipidated protein of the prior art, the burden is on the applicant to show a novel or unobvious difference between the recited product and the recited method of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594. As a result, the combination of Leng, Madurawe, and Knabben as described above arrive at the invention of claims 1-3 and 26-27.
Consequently, the invention of claims 1-3 and 26-27 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Leng in view of Madurawe and Knabben as applied to claims 1-3 and 26-27 above, and further in view of Khan et al. (US 2017/0356022 A1; cited on the attached Form PTO-892; hereafter “Khan”).
The relevant teachings of Leng, Madurawe, and Knabben as applied to claims 1-3 and 26-27 are discussed above and incorporated herein.
Regarding claim 4, the combination of Leng, Madurawe, and Knabben does not explicitly teach wherein cell culture volume is at least 100 L.
Khan taught a method producing a protein from a recombinant host cell such as E. coli (Abstract; para [0406]; Claim 1). Khan teaches the method comprises a bioreactor configured to comprise or provide a headspace above the culture in order to aid increasing the solubility of gasses, like oxygen, into the liquid phase (para [0014, 0024, 0060, 0162, 0223]; Claim 41). Khan teaches the bioreactor used to execute the method can have a volume between up to 50,000 L, including a 100 L, 200 L, or 300 L bioreactors (para [0162, 0168]).
In view of the combined teachings of Leng, Madurawe, Knabben, and Khan, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben such that the bioreactor culture volume used to culture the E. coli host is 100 L as taught by Khan, thereby arriving at the invention of claim 4.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben such that the bioreactor culture volume used to culture the E. coli host is 300 L because Khan taught a method producing a protein from a recombinant E. coli host cell in 300 L bioreactor configured to comprise or provide a headspace above the culture in order to aid increasing the solubility of oxygen into the liquid phase.
Consequently, the invention of claim 4 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Leng in view of Madurawe, Knabben, and Khan, as applied to claims 1-3 and 26-27 above, and further in view of Taberna et al. (WO 2019/126652 A1; cited on the attached Form PTO-892; hereafter “Taberna”), Kellerhals et al. (Enzyme and Microbial Technology, published 1999, Vol. 24, p. 111-116; cited on the attached Form PTO-892; hereafter “Kellerhals”), Weber et al. (Methods in Molecular Biology, Chapter 17, published 2017, Vol. 1498, p. 257-271; cited on the attached Form PTO-892; hereafter “Weber”), Bernard et al. (Current Protocols in Protein Science, published 1995, p. 5.3.1-5.3.18; cited on the attached Form PTO-892; hereafter “Bernard”), Mattanovich et al. (CN108350447; cited on the attached Form PTO-892), and Hartlep et al. (Appl Microbiol Biotechnol, published 2002, Vol. 50, p. 60-66; cited on the attached Form PTO-892; hereafter “Hartlep”). Reference is made to a machine translation of CN108350447 obtained from Espacenet; cited on the attached Form PTO-892; hereafter “Mattanovich”).
The relevant teachings of Leng, Madurawe, and Knabben as applied to claims 1-3 and 26-27, are discussed above and incorporated herein.
Regarding claims 5-7, the combination of Leng, Madurawe, and Knabben does not explicitly teach wherein a trace element solution is added during culturing of the cells.
Taberna teaches wherein trace elements are chemicals influence microbial growth and maintenance in culture (para [0083]). Taberna teaches a trace solution comprising 0.29 g/L FeCl3•6H2O, 0.006 g/L CoCl2•6H2O, 0.026 g/L ZnCl, and 6.84 g/L H2BO3 (Example 1; para [0083, 0086, 0099, 00107, 00111, 00121, 00164]). An ordinary artisan would immediately recognize H2BO3 as the chemical formula for boric acid.
Kellerhals teaches a microorganism fed-batch culture medium comprising a microelement solution comprising 4.0 g/L CoCl2•6H2O and 1.0 g/L CuCl2•2H2O (Abstract; p. 112, col 1, para 7). Kellerhals teaches optimizing the culture media metal contents in order to increase the increase the cell growth density of the microorganism (p. 114, col 1, para 3; Figure 2).
Weber teaches a trace element solution used in a bacterial medium comprising 0.010 g cupric chloride dihydrate (CuCl2•2H2O) (p. 257, para 1 - p. 261, para 1).
Bernard teaches a fed-batch method of for optimal protein production in E. coli comprising a trace element solution (Title; p. 5.3.8, text beginning with “PROTEIN PRODUCTION IN HIGH-CELL-DENSITY…” – p. 5.3.9, text ending with “Proceed to monitoring expression and harvesting (see Basic Protocol 1, steps 18 to 23) while continuing to match the rate of HCDF1 supplementation with growth.”; p. 5.3.12, “HCDF1 medium” – p. 5.3.13, “Trace element solution 2”). Bernard teaches trace element solution 2 used in the method comprising 0.3 g/L CuCl2•2 H2O, 0.02 g H3BO3, 0.8 g CoCl2⋅6 H2O as well as that the trace element solution 2 also comprises iron-, zinc-, molybdenum-containing chemicals (p. 5.3.14, “Trace element solution 2”). Bernard teaches the metal-containing chemicals are dissolved in minimal 10 N HCl prior to addition to the remaining water-based solution (p. 5.3.13, “Trace element solution 2”).
Mattanovich teaches culturing a microorganism a trace salt stock solution comprising 20.0 g/L ZnCl2, 0.2 g/L Na2MoO4•2H2O, 0.02 g/L H3BO3, and 5.0 mL of an acid H2SO4 (p. 38, para 2-13; p. 4, paragraph beginning with “Herein, the pG1 promoter can serve…”; p. 11, para 2; p. 13, para 1).
Hartlep teaches cultivation of a microorganism comprising a culture media with a trace element solution containing 5 g/L FeCl3•6H2O (Abstract; p. 61, col 1, para 7).
In view of the combined teachings of Leng, Madurawe, Knabben, Taberna, Kellerhals, Weber, Bernard, Mattanovich, and Hartlep, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben such that a trace element solution is added to the cell culture comprising FeCl3•6H2O, CoCl2•6H2O, ZnCl, and H2BO3 as taught by Taberna, CuCl2•2H2O taught by Kellerhals and Weber, and 0.2 g/L Na2MoO4•2H2O and HCl as taught by Mattanovich, and wherein through optimization trace element components such that it comprises 1.6 g/L FeCl3•6H2O, 0.37 g/L CoCl2•6H2O, 0.2 g/L ZnCl, and 0.05 g/L H2BO3, thereby arriving at the invention of claim 5-7.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben such that a trace element solution is added to the cell culture comprising FeCl3•H2O, CoCl2•6H2O, ZnCl, and H2BO3 as taught by Taberna, CuCl2•2H2O taught by Kellerhals and Weber, and 0.2 g/L Na2MoO4•2H2O and HCl as taught by Mattanovich because Taberna taught wherein trace elements are chemicals influence microbial growth and maintenance in culture and the prior art taught the use of the recited trace elements in the culture of microorganisms. For the same reason, an ordinary artisan would have been motivated to and would have had a reasonable expectation of optimizing the concentration of the trace element solution such that through optimization it also comprises 1.6 g/L FeCl3•H2O, 0.37 g/L CoCl2•6H2O, 0.2 g/L ZnCl, and 0.05 g/L H2BO3. A range can be disclosed in multiple prior art references instead of in a single prior art reference depending on the specific facts of the case (see MPEP2144.05.I). Taberna and Hartlep taught trace element solutions for microorganisms comprising 0.29 g/L and 5 g/L FeCl3•6H2O, respectively. Taberna and Kellerhals taught trace element solutions for microorganisms comprising 0.006 g/L and 4.0 g/L CoCl2•6H2O, respectively. Weber, Bernard, and Kellerhals taught trace element solutions for microorganisms comprising 0.010 g/L, 0.3 g/L, and 1.0 g/L CuCl2•2H2O, respectively. Taberna and Mattanovich taught trace element solutions for microorganisms comprising 0.026 g/L and 20.0 g/L ZnCl, respectively. Bernard , Mattanovich, and Taberna taught trace element solutions for microorganisms comprising 0.2 g/L, 0.2 g/L, and 6.84 g/L H3BO3, respectively. An ordinary would have an operational expectation of success over the entire range of each trace element component taught in the prior art. Therefore, since the effectiveness of the trace element solution, would have been influenced by the result effective variable of the trace element concentrations, it would have been obvious for one of ordinary skill to discover the optimum workable trace element concentrations disclosed by the prior art by normal optimization procedures known in the art and arrived at wherein the trace element solution also comprises 1.6 g/L FeCl3•H2O, 0.37 g/L CoCl2•6H2O, 0.2 g/L ZnCl, and 0.05 g/L H2BO3.
Regarding claim 8, Taberna further teaches wherein the trace element solution is diluted 1/200 in the culture media (5 mL trace element solution/1000 mL total= 1/200; para [0086, 0099, 00107, 00111, 00121, 0164]).
Bernard further teaches wherein the trace element solution is diluted 7/100 in the culture media (70 mL trace element solution/1000 mL total= 7/100; p. 5.3.12, “HCDF1 medium” – p. 5.3.13, “HCDM1 medium”
Hartlep further teaches wherein the trace element solution is diluted 1/200 in the culture media (5 mL trace element solution/1000 mL total= 1/200; p. 61, col 1, para 7).
In view of the combined teachings of Leng, Madurawe, Knabben, Taberna, Kellerhals, Weber, Bernard, Mattanovich, and Hartlep, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, Knabben, Taberna, Kellerhals, Weber, Bernard, Mattanovich, and Hartlep such that a concentrated stock trace element solution is added to the batch culture medium, and through optimization wherein the trace element solution is diluted in the batch culture medium at a dilution range (trace element solution/total batch media) of 1/200 to 7/100, thereby arriving at the invention of claim 8.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, Knabben, Taberna, Kellerhals, Weber, Bernard, Mattanovich, and Hartlep such that a concentrated stock trace element solution is added to the batch culture medium because Taberna taught wherein trace elements are chemicals influence microbial growth and maintenance in culture and the prior art taught the use of the recited trace elements in the culture of microorganisms. Furthermore, an ordinary artisan would have immediately recognized that using a concentrated stock solution of the trace element dilution would limit the dilution of the culture media components. For the same reasons, an ordinary artisan would have been motivated to and would have had a reasonable expectation of optimizing amount of trace element solution diluted in the batch culture medium such that the dilution range (trace element solution/total media) is 1/200 to 7/100. A range can be disclosed in multiple prior art references instead of in a single prior art reference depending on the specific facts of the case (see MPEP2144.05.I). Since Taberna, Hartlep, and Bernard taught diluting trace element solution in culture medium (trace element solution/total batch media) at a ratio of 1/200, 1/200, and 7/200, respectively, an ordinary would have an operational expectation of success over the entire range taught in the prior art. Therefore, since the effectiveness of the trace element solution, would have been influenced by the result effective variable of the trace element concentrations, it would have been obvious for one of ordinary skill to discover the optimum workable dilution factor of the trace element solution in the batch media disclosed by the prior art by normal optimization procedures known in the art and arrived at wherein the dilution range (trace element solution/total media) is 1/200 to 7/100.
Consequently, the invention of claims 5-8 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 9-13 and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Leng in view of Madurawe, Knabben, and Khan as applied to claims 1-3 and 26-27, and further in view of Schuler (Dublin University Research Repository, originally published November 20, 2012, last modified July 19, 2018; cited on the attached Form PTO-892; hereafter “Schuler”).
The relevant teachings of Leng, Madurawe, and Knabben as applied to claims 1-3 and 26-27, are discussed above and incorporated herein.
Regarding claim 9-10 and 12-13, Khan further teaches high aeration required for high cell density cell cultivation can result in foam production and excessive foam can lead to stopping cell cultivation (p. 74, col 2, para 1; p. 78, col 2, para 4).
The combination of Leng, Madurawe, and Knabben does not explicitly teach wherein an anti-foam agent is present during cell cultivation.
Schuler teaches the use of polypropylene glycol 2000 as an antifoam agent fed-batch cell culture, and wherein the concentration of the polypropylene glycol 2000 is higher in the feed medium than the batch medium (Abstract; p. 78, para 5 – p. 79, para 1; Tables 4.1, 4.4, 5.1, 6.1, and 7.1).
In view of the combined teachings of Leng, Madurawe, Knabben, and Schuler, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben comprises the anti-foaming agent polypropylene glycol 2000 in the batch medium and in a higher amount in the feed medium as taught by Schuler, and arrived through routine optimization that the , thereby arriving at the invention of claim 9-10 and 12-13.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben comprises the anti-foaming agent polypropylene glycol 2000 in the batch medium and in a higher amount in the feed medium as taught by Schuler in order to control excessive foam accumulation during the bioreactor.
Regarding claims 11 and 31, the relevant teachings of Leng, Madurawe, Knabben, and Schuler as applied to claims 1-3, 9-10 and 12-13, and 26-27, are discussed above and incorporated herein.
Leng further teaches wherein the E. coli host cells are induced with isopropyl β-D-thiogalactoside (IPTG) in order to produce the recombinant protein of interest (para [0010, 0026, 0032]).
Knabben teaches higher levels of glucose in culture media increases the cell growth capacity of E. coli (Figure 3). Knabben further teaches wherein glucose is depleted during exponential cell growth (Figures 2-3*-9658).
The combination of Leng, Madurawe, Knabben, and Schuler does not explicitly teach wherein an anti-foam agent added in two boluses.
In view of the combined teachings of Leng, Madurawe, Knabben, and Schuler, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, Knabben, and Schuler such that the feed medium comprising polypropylene glycol 2000 is bolused first before bacterial induction by IPTG and the second is after induction, thereby arriving at the invention of claim 11 and 31.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, Knabben, and Schuler such that the feed medium comprising polypropylene glycol 2000 is bolused once before bacterial induction by IPTG and another after the induction.
Since Knabben taught higher levels of glucose in culture media increases the cell growth capacity of E. coli as well as that glucose is depleted during exponential cell growth, it would have been obvious for one of ordinary skill to discover the optimum workable frequency and timing of feed media boluses in order to optimize cell growth and arrived at where the feed medium comprising polypropylene glycol 2000 is bolused first before bacterial induction by IPTG and the second is after induction..
Consequently, the invention of claims 9-13 and 31 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 14 and 28 are rejected under 35 U.S.C. 103 as being unpatentable over Leng in view of Madurawe and Knabben as applied to claims 1-3 and 26-27 above, and further in view of Comstedt et al. (US 2018/0327460 A1; cited on the attached Form PTO-892; hereafter “Comstedt”).
The relevant teachings of Leng, Madurawe, and Knabben as applied to claims 1-3 and 26-27 are discussed above and incorporated herein.
Regarding claims 14 and 28, the combination of Leng, Madurawe, and Knabben does not explicitly teach wherein the lipidated protein comprises the amino acid sequence of SEQ ID NO: 1 or a variant thereof.
Comstedt teaches a lipidated OspA heterodimer Lip-S1D1-S2D1 that is identical to SEQ ID NO: 1 (SEQ ID NO: 186, see sequence alignment below; Table A-5).
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Comstedt teaches the outer surface protein A (OspA) is an abundant immunogenic lipoprotein of Borrelia has potential to be used as a vaccine candidate for Borrelia infection (Abstract; para [0018]). Comstedt teaches wherein the OspA lipoprotein can comprises the lipidation moiety comprising a glycerol group wherein it comprises one to one to three lipids such as C14-20 alkyl and/or C14-20 alkenyl attached to a glycerol and the N-terminal cysteine of the polypeptide of the invention, or preferably wherein Lip is a moiety of formula (I) below, in which one of R1, R2 or R3 is C14-C20 alkyl or alkenyl, and each of the others, independently is C14-C20 alkyl or C14-C20 alkenyl, and X is an amino acid sequence attached to the cysteine residue shown in Formula (I) (para [0018, 0023, 0238]; Formula I, see structure below). Comstedt teaches wherein the preferred lipid moiety comprises N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy) propyl cysteine (referred to as “Pam3Cys”) (para [0023, 0238]). Comstedt teaches lipidation of OspA with Pam3Cys enhances the immunogenicity of OspA (para [0018]).
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In view of the combined teachings of Leng, Madurawe, Knabben, and Comstedt, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben produces the lipidated OspA taught by Comstedt, thereby arriving at the invention of claim 14 and 28.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success of modifying the method of producing a lipidated protein in E. coli taught by the combination of Leng, Madurawe, and Knabben produces the lipidated OspA taught by Comstedt because Comstedt taught the outer membrane lipoprotein OspA as a vaccine for and also teaches that lipidation with Pam3Cys increases its immunogenicity of OspA. Furthermore, it is obvious to one of ordinary skill in the art that in a method of producing a lipidated protein E. coli adapted to membrane expression to substitute a known lipidated protein for another lipidated protein of interest with predictable results because they are both lipoproteins.
Consequently, the invention of claims 14 and 28 would have been obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
No claims are currently allowed for the reasons as stated above. Applicants must
respond to the objections/rejections in this Office action to be fully responsive in
prosecution.
The instant Office Action is non-final.
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/SCOTT E. MULDER/Examiner, Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656