DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the AIA first-to-invent provisions.
Claims Status
Claims 4, 9, 11, 16 and 20 are cancelled. Claims 1-3, 5-8, 10, 12-15, 17-19 and 21-32 are under examination on their merits.
Priority
Claims 1-3, 5-8, 10, 12-15, 17-19 and 21-32 in this application are under a National Stage application of International Patent Application No. PCT/US2022/020676, as filed March 17, 2022, which claims the benefit of priority to U.S. Provisional Patent Application No. 63/163,079, filed March 19, 2021.
Specification
The disclosure is objected to because of the following informalities: the specification recites two different abbreviated names for a recombinant Dengue virus serotype 3 “rDENV3Δ30/Δ31” and “rDENV3Δ30/31” (see page 4 of specification). Based on the application’s disclosure, it appears that these names refer to the same recombinant virus. It is suggested that applicants review which name is the proper form for identifying that recombinant Dengue virus serotype 3.
Appropriate correction is required.
Claim Objections
Claims 23 and 25 are objected to as containing inconsistent terminology. Specifically, these claims recite their recombinant Dengue virus serotype 3 as “rDENV3Δ30/Δ31” in Claim 23 and then as “rDENV3Δ30/31” in Claim 25, which is inconsistent. Applicant is advised to amend these claims by using consistent nomenclature for the virus in Claims 23 and 25.
Claim Rejection under 35 U.S.C. §112(b)
The following is a quotation of 35 U.S.C. §112(b) which forms the basis for indefiniteness rejections set forth in this Office action:
(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.
Claim 27 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
For Claim 27, cell “incubation and growth” time is recited on nine occasions as “about 120 ± 12 hours” in steps a), c), e), g) and i); “about 48 ± 12 hours” in steps b) and d), and “about 24 ± 12 hours” in steps f) and h). For each case, “±” already implies a range, so the word “about” simply adds confusion. The definition of “about” (“a variation of ± 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%”), as disclosed in Specification (lines 20-21 on Pg. 13) is also confusing here because “about” could be applied to the range of temperature or a midpoint for each range. It is also unclear if “about” further applies to other variables (temperature and CO2 concentration) cited after each time interval. Applicant is advised that amendments to Claims 27 need to clearly define the three variables (duration of culture, temperature and CO2 concentration). For compact prosecution, examiner takes the actual range in each variable (“x ± y”) as the definitive setting.
Claim Rejections under 35 U.S.C. §102(a)
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 24-25 are rejected under 35 U.S.C. §102(a)(1) as being anticipated by Frazatti Gallina 2017 (US 20170065701 A1, published 03/09/2017).
MPEP §2113 states, “[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) …”.
Claim 24 recites a “vaccine manufactured by the process” of Claim 1, which is drawn to “a closed-system manufacturing process for the production of dengue virus.” The process begins with an expansion of adherent host cells in serum-free medium, followed by transfer of host cells to bioreactors for virus inoculation and propagation, and ending with the harvest of dengue virus toward a vaccine formulation. Claim 25 limits Claim 24 by disclosing that “the dengue virus vaccine of Claim 24… is quadrivalent and comprises… four genetically attenuated viral strains rDENV1Δ30, rDENV2/4Δ30, rDENV3Δ30/31, and rDENV4Δ30.”
Therefore, in view of the MPEP 2113, if the prior art teaches a dengue virus vaccine or composition comprising genetically attenuated viral strains rDENV1Δ30, rDENV2/4Δ30, rDENV3Δ30/31, and rDENV4Δ30, the prior art would anticipate the claimed invention.
Frazatti Gallina 2017 taught a “process for preparing an attenuated tetravalent Dengue vaccine” (title) against “dengue virus serotypes 1, 2, 3 and 4” (Claim 1 on Pg. 27) using “attenuated virus strains rDENΔ30-1545 (SEQ ID NO:1) and variants thereof; rDEN2/4Δ30(ME)-1495,7163 (SEQ ID NO:2) and variants thereof; rDEN3Δ30/31-7164 (SEQ ID NO:3) and variants thereof; and rDEN4Δ30-7132,7163,8308 (SEQ ID NO:4) and variants thereof” (¶[0009]). The process starts with the “amplification” of “Vero cells in culture to produce Master and Working banks,” and “the Vero cells are adapted for growth in serum-free medium… sub-cultured in 225 cm2 Tissue Culture (TC)-flasks and later in a Cell Factory System™ (CFS)” before “infecting Vero cells” (Claim 1 on Pg. 27). This prior art also noted that “virus culture can be repeated up to seven times” (Claim 3) for preparation of “dengue vaccine 1,2,3,4 (attenuated)” (¶[0010]), including “formulation of monovalent and tetravalent vaccines” (¶[0010]). Although Frazatti Gallina 2017 did not use “a closed system” in the manufacturing process, it fully anticipated the product of Claims 24-25 in the instant application because the monovalent and quadrivalent (tetravalent) vaccines of Claims 24-25 are the same genetically modified dengue viruses taught by Frazatti Gallina 2017, as defined by Δ30 deletion in the 3’ untranslated region for dengue virus serotypes 1, 2, 3 and 4 (Frazatti Gallina 2017, ¶¶[0031]-[0034]). Therefore, Fazatti Gallina 2017 anticipates the instant invention.
Claim Rejections under 35 U.S.C. §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 the following:
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.
Claims 1-3, 5, 7-8, 10, 21-23 and 26 are rejected under USC §103 as being unpatentable over Frazatti Gallina 2017 (US 20170065701 A1, published 03/09/2017) in view of Kapre 2015 (US 2015/0064768 A1, published 03/05/2015).
Claim 1 is drawn to “a closed-system manufacturing process for the production of dengue virus”, which begins with an expansion of adherent host cells in serum-free medium, followed by transfer of host cells to bioreactors for virus inoculation and propagation, and ending with the harvest of dengue virus toward a vaccine formulation. Claims 2-3, 5, 7-8 and 10 go on to disclose several limitations of Claim 1, reciting the use of “Vero cells “ (Claim 5) and “serum-free medium” for “one or more cell passages” (Claim 2) or “at least 4 cell passages” (Claim 3) over a period of “2-20 days” (Claim 7) “at a temperature of 37 ± 1°C and at 5% ± 1% CO2” (Claim 8), with Vero cell culture done in “dextran microcarriers” (Claim 10). Claims 21-23 and 26 further extend Claim 1 by adding limitations related to available “dengue virus selection” (Claim 21), coupled with four virus serotypes (“DENV1, DENV2, DENV3 and DENV4”) (Claim 22) and four genetically modified (attenuated) forms (“rDENV1Δ30, rDENV2/4Δ30, rDENV3Δ30/31 and rDENV4Δ30”) (Claim 23), as well as procedures for the “harvest of dengue virus” (Claim 26).
Frazatti Gallina 2017 taught a “process for preparing an attenuated tetravalent Dengue vaccine” (title) using “attenuated virus strains rDENΔ30-1545 (SEQ ID NO:1) and variants thereof; rDEN2/4Δ30(ME)-1495,7163 (SEQ ID NO:2) and variants thereof; rDEN3Δ30/31-7164 (SEQ ID NO:3) and variants thereof; and rDEN4Δ30-7132,7163,8308 (SEQ ID NO:4) and variants thereof” (¶[0009]). Specifically, the process starts with the “amplification” of “Vero cells in culture to produce Master and Working banks,” and “the Vero cells are adapted for growth in serum-free medium… sub-cultured in 225 cm2 Tissue Culture (TC)-flasks and later in a Cell Factory System™ (CFS)” before “infecting Vero cells… with dengue virus serotypes 1, 2, 3 and 4… in separate cultures… at 36.5°C (±1 °C) for 10 to 20 days” (Claim 1 on Pg. 27). This prior art also noted that “virus culture can be repeated up to seven times” (Claim 3) for preparation of “a vaccine against… four types of dengue virus” (¶[0009]).
While Frazatti Gallina 2017 teaches propagating dengue viruses, such as the recombinant dengue viruses of the tetravalent compositions presently claimed, it falls short of teaching the use of the presently claimed closed system for cell expansion and virus propagation.
Kapre 2015, on the other hand, taught “an integrated, closed cell culture system for the bulk growth of cells and… virus-infected cells in tissue culture and the subsequent collection of virus particles or viral components and, if desired, their inactivation” (¶[0037]). Equipped with a closed, medium exchange “tubing” (¶[0037]) and “transfer piping… in-line… with sterile connectors” (¶[0040]), the system is “scalable through a wide range of volumes and adjustable due to the modular design” (¶[0037]), requiring “minimal to no handling of live virus” within a “self-contained” environment “preferred for the manufacture of bio-safety level 3 virus (BSL 3 virus), BSL 4 virus, and/or BSL 5 virus and combinations thereof” (¶[0053]). The “incubation chambers have one or more porous or mesh surfaces with openings,” and they are “capable of supporting cellular attachment and growth in a nutrient media rich environment” (¶[0038]). Moreover, “cells that can be used in the systems and methods… include suspension cells, adherent cells and partially adherent cells” (¶[0053]). For virus infection, “retroviral cell line PM 3218 was introduced to the media tank through an injection port into the media tank and allowed to flow into the incubation chambers” ((¶[0056). Examples were given for the culture of “population of adherent cells” (Kapre 2015, Claim 3) for “about two days to about four weeks” (Kapre 2015, Claim 30), and inherent to Cytodex1, the “Cytodex 1 microcarrier beads” in Kapre 2015 (¶[0055]) are the equivalent of dextran microcarriers (Page 3, lines 9-10 in instant application). Thus, other than the specific use for culturing Vero cells and propagation of Dengue virus serotypes, Kapre 2015 teaches the closed system of Claims 1-3, 5, 7-8 and 10 of the instant application.
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Frazatti Gallina 2017 in order to propagate Vero cells and dengue viruses, such as the tetravalent recombinant dengue viruses presently rejected, in a closed system as presently claimed. One would have been motivated to do so, given the suggestion by Frazatti Gallina that tissue culture techniques (which one of ordinary skill in the art would perform aseptically) are used to propagate Vero cells and produce the claimed tetravalent dengue virus composition (see Claim 23 of instant application). There would have been a reasonable expectation of success, given the knowledge that another cell and virus culture process was previously taught by Kapre 2015, which was capable of supporting cell culture and virus production and is an example of the presently claimed closed system. Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Claim 6 is rejected under 35 U.S.C. §103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claim 1 above, and further in view of Li et al. 2020 (US 2020/0392468 A1, published 12/17/2020).
Regarding Claim 6, it discloses one limitation of Claim 1: “wherein the closed-system containers are selected from closed-system static cell culture containers.”
Based on the above rejection, Claim 1 is rendered obvious by the combined teachings of Frazatti Gallina 2017 and Kapre 2015. The “225 cm2 TC-flasks” used for “amplifying Vero cells” in Frazatti Gallina 2017 (¶[0030]) are the equivalents of “static cell culture containers,” although they fell short of calling them as such or mentioning other containers like “CellSTACKS and HYPERStacks” (Pg. 2 of instant application).
This deficiency is taught by Li et al. 2020, as they describe “an efficient process for culturing viruses… for producing vaccines, typically from live attenuated viruses” (Abstract). For “propagation of 4 dengue virus serotypes” (¶[0032]), “African green monkey kidney derived Vero... cells may be grown adherently on static plastic surfaces, such as in roller bottles, flasks, cell factories, or may be grown adherently on microcarriers” (¶[0027]), and “examples of static plastic surfaces include, but are not limited to, cell stacks, cell factory systems, T-flasks, HYPERFlasks™, HYPERStacks™, and the like” ([0025]).
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Frazatti Gallina 2017 in order to propagate Vero cells and dengue viruses, such as the tetravalent recombinant dengue viruses presently rejected, in a closed system using closed-system static cell culture containers. One would have been motivated to do so, given the suggestion by Frazatti Gallina that tissue culture techniques that use tissue culture flasks (which one of ordinary skill in the art would perform aseptically) are used to propagate Vero cells and produce the claimed tetravalent dengue virus composition (see Claim 23 of instant application). There would have been a reasonable expectation of success, given the knowledge that another cell and virus culture process was previously taught by Kapre 2015, which was capable of supporting cell culture and virus production and is an example of the presently claimed closed system, and also given the knowledge that Li et al. 2020 teach the use of closed-system static cell culture containers, such as HYPERStacks and HYPERFlasks, for culturing cells and for virus propagation. Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Claims 12 is rejected under 35 U.S.C. §103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claim 1 above, and further in view of Calvosa et al. 2011 (US 2011/0151506 A1, published 06/23/2011).
Claim 12 extends Claim 1 by reciting another limitation – “the medium contained in the closed-system bioreactor is supplemented with Poloxamer 188.”
Based on the above rejection, Claim 1 is rendered obvious by the combined teachings of Frazatti Gallina 2017 and Kapre 2015. However, these references do not render Claims 12 obvious because neither of them teaches the supplementation of culture medium with Poloxamer 188.
However, Calvosa et al. 2011 taught a “process for culturing adherent sells” using “dextran based microbeads (CytodexTM 1, 2 or 3)” as a “microcarrier” and “poloxamer 188” as one of two "preferred cell protection agents" (¶[0041] & ¶[0095]).
A person having ordinary skill in the art and interested in using the invention of Claim 1 could readily apply “poloxamer 188” from Calvosa et al. 2011 to a closed-system manufacturing process for the production of dengue virus (as taught by Frazatti Gallina 2017 and Kapre 2015) because the addition of a cell protection agent like poloxamer 188 is necessary when “culture medium is free of animal serum or of serum protein or when its protein content is very low” (Calvosa et al. 2011, ¶[0095]). Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was filed.
Claims 13-15 are rejected under 35 U.S.C. §103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claim 1 above, and further in view of Thomassen et al. 2014 (Vaccine 32: 2782–2788).
Claims 13-15 further limit Claim 1 by disclosing that “wherein the final cell expansion in the closed-system bioreactor occurs over 120 ± 12 hours” (Claim 13) “at a pH range of 7.05 to 7.55” (Claim 14) and “at a temperature of 37 ± 1°C” (Claim 15).
Based on the rejections cited above, Claim 1 is rendered obvious by the combined teachings of Frazatti Gallina 2017 and Kapre 2015. However, they left out several specifics regarding the condition of cell expansion.
These deficiencies were overcome by Thomassen et al. 2014, as they taught “the use of animal-component-free cell and virus culture media” (Abstract) in optimizing “Vero cell cultivation methods” for the production of poliovirus vaccines. When Vero cell “cultures were maintained at pH 7.2, 37oC, 50% dissolved oxygen” (section 2.2.2 on Pg. 2783), cell “growth rate” differed by over 50-fold, depending on “cultivation methods” (Table 1 on Pg. 2784) and virus types (Fig. 5 on Pg. 2786). These technical details provided a framework that can “be further improved and… implemented in… manufacturing facilities” (Conclusions on Pg. 2787). A person having ordinary skill in the art and interested in using the invention of Claim 1 in this instant application could arrive at a desirable Vero cell culture condition by adjusting pH, temperature, gas concentration and duration of culture, because optimization of such conditions, as taught by Thomassen et al. 2014, can lead to “improved yields” (title) and help with “modernization of human viral vaccine manufacturing” (Abstract). Thus, the invention of Claims 13-15 is an example of rationale (C) of MPEP §2143: use of known technique in Thomassen et al. 2014 to improve similar methods in the same way for Vero cell culture and Dengue virus production.
Claims 17-18 are rejected under 35 U.S.C. §103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claim 1 above, and further in view of Meyer et al 2003 (US 2003/0108861 A1, published 06/12/2003).
MPEP § 2144.05 (II) (A) states, “…Optimization Within Prior Art Conditions or Through Routine Experimentation…“[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to dis-cover the optimum or workable ranges by routine experimentation…”.
Regarding Claims 17-18, the claimed invention further requires that “wherein the production of dengue virus occurs at a pH range of 6.75 to 7.35 (Claim 17) and “at a temperature of 34 ± 1°C” (Claim 18).
While the combined teachings of Frazatti Gallina 2017 and Kapre 2015 render Claim 1 obvious, they do not teach wherein the production of dengue virus occurs at a pH range of 6.75 to 7.35 and at a temperature of 34 ± 1°C.
Meyer et al 2003, on the other hand, detailed the methods of using “a serum free cell culture of VERO cells bound to a microcarrier” for “large-scale production of hepatitis A virus (HAV) by “infecting said serum free cell culture of VERO cells with HAV, incubating said cell culture infected with HAV to propagate said HAV, whereby HAV is continuously released into the cell culture medium” for “harvesting” (¶[0017]). When “VERO cells bound to a microcarrier are grown under serum free media conditions” (¶[0018]), using “microcarriers based on dextran, collagen, plastic, gelatine and cellulose…” (¶[0019]), the “parameters for optimal cell culture conditions are a pH between about 6.5 and about 8.0, a O2 concentration between about 15% and about 40%, and a temperature at 34°C ± 0.2°C or 37°C ± 0.2°C” (¶[0023]). “HAV strain HM175/7 replicates on VERO cells better at lower temperature of 34°C than at 37°C” (¶[0040]), so “temperature is reduced to about 34°C prior to infection” (Meyer et al 2003, Claim 3). “Propagation of infected cells at 34°C” lasted “up to 350 days” (¶[0051]) or “at least 60 days” (Meyer et al 2003, Claim 20). Thus, the procedures for optimizing pH, gas concentration and temperature for VERO (Vero) cell-based virus propagation in a serum-free culture system have been established before the invention of Claims 17-18.
A person having ordinary skill in the art and interested in optimizing the dengue virus production method based on the combined teachings of Frazatti Gallina and Kapre et al. would be motivated to adjust parameters of the production method through routine optimization (see MPEP 2144.05 (II) (A)). Such as adjusting the pH, temperature and gas (including O2 and CO2) concentration to arrive at the combinations which would include those presently claimed. Moreover, because the ranges stated in Meyer et al 2003 are narrow and because cell growth and virus propagation can be monitored on a regular basis (e.g., “days 7, 14, 21 and 28 after infection”) over an extended time period, as taught by Meyer et al 2003 (¶[0023] & ¶[0051]), one of ordinary skill in the art would be motivated to adapt and have a reasonable expectation of success. Lastly, the invention of Claims 1 and 17-18 is an example of rationale (D) of MPEP §2143: applying a known procedure for optimizing HAV propagation and large-scale HAV vaccine production (from Meyer et al 2003) to the method of Dengue vaccine production (Frazatti Gallina 2017) in a closed system (Kapre 2015) to yield predictable results – optimized conditions for propagating Dengue viruses over a desired time period.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claim 1 above, and further in view of Putnak et al 1996 (The Journal of Infectious Diseases 174:1176-84).
Claim 19 depends on Claim 1 and recites a limitation that “the harvest of dengue virus occurs at a temperature of 5 ± 3°C”.
Based on the above rejection, Claim 1 is rendered obvious by the combined teachings of Frazatti Gallina 2017 and Kapre 2015. However, these references do not teach the harvesting of dengue virus at a temperature of 5 ± 3°C.
This deficiency was overcome by Putnak et al 1996 in their evaluation of “a monotypic, purified, inactivated vaccine” candidate for “DEN-2 virus” (Abstract). By their method, Vero cell “culture supernatants were pooled for virus” and then “concentrated by tangential flow ultrafiltration… at 4°C using a flow rate of 400 mL/min” and “samples were held briefly at 4°C prior to sucrose gradient ultracentrifugation” (1st and 2nd para. on Pg. 1177). The harvested “virus retained its antigenicity and was immunogenic in mice and rhesus monkeys, in which it elicited high titers of DEN-2 virus-neutralizing antibody” (Abstract).
A person having ordinary skill in the art could readily follow the teaching of Putnak et al 1996 to arrive a temperature for the harvest of dengue virus (under refrigeration) and for downstream procedures before adapting this harvest step with the combined teachings of Frazatti Gallina and Kapre et al. Thus, the invention of Claims 1 and 19 is an example of rationale (D) of MPEP §2143: applying a known procedure for optimizing DENV propagation and harvest (from Putnak et al 1996) to another method of Dengue vaccine production (Frazatti Gallina 2017) in a closed system (Kapre 2015) to yield predictable results.
Claims 27-32 are rejected under 35 U.S.C. §103 as being unpatentable over Frazatti Gallina 2017 (supra) and Kapre 2015 (supra) as applied to Claims 1-3, 5, 7-8, 10, 21-23 and 26 above, and further in view of Thomassen et al. 2014 (supra).
Similar to Claim 1 and its dependent Claims 2-3, 5, 7-8, 13, 15, 19 and 21, Claim 27 is drawn to “a closed-system, manufacturing process for the production of dengue virus”, “comprising” 13 steps as: “a) incubation and growth of Vero cells in one or more closed-system containers for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; b) medium exchange and continued incubation and growth of the Vero cells in the one or more closed-system containers for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; c) harvest and plant of the Vero cells into one or more closed-system containers for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth of the Vero cells; d) medium exchange and continued incubation and growth of the Vero cells in the one or more closed-system containers for about 48 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; e) harvest and plant of the Vero cells into one or more closed-system containers for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth of the Vero cells; f) medium exchange and continued incubation and growth of Vero cells in the one or more closed-system containers for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; g) harvest and plant of the Vero cells into one or more closed-system containers for about 120 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2 for continued incubation and growth of the Vero cells; h) medium exchange and continued incubation and growth of the Vero cells in the one or more closed-system containers for about 24 ± 12 hours at 37 ± 1°C at 5% ± 1% CO2; i) harvest and plant of the Vero cells into one or more closed-system bioreactors, each closed-system bioreactor containing microcarriers and medium, for about 120 ± 12 hours for continued incubation and growth of the Vero cells; j) medium exchange in preparation for dengue virus infection of the Vero cells; k) addition of dengue virus to the one or more bioreactors and infection of the Vero cells; l) production of the dengue virus; and m) harvest of the dengue virus.” Claims 28-32 then extend Claim 27 by disclosing “a closed-system environment” where “Vero cells are harvested and planted” as set forth in Claim 27 as “steps c), e), and g)” (Claim 28) or “step i)” (Claim 29); “the addition of dengue virus occurs” (Claim 30); “the production of the dengue virus occurs (Claim 31); and the harvest of the dengue virus occurs” (Claim 32).
Frazatti Gallina 2017 taught “the development of a vaccine against four types of dengue virus” (¶[0009]) through a “process” divided into 12 steps (i through xii) (Claim 1): (i) “amplifying Vero cells in culture” as “Master and Working Banks” (first in “Tissue Culture (TC)-flasks and later in Cell factory System”), (ii) “infecting Vero cells… with dengue virus serotypes 1, 2, 3 and 4”, (iii) “incubating Cell Factory System… containing the Vero cells infected with each dengue virus at 36.5°C (± 1°C) for 10 to 20 days”, (iv) “harvesting the supernatants of each culture” before “filtering.. virus suspension”, (v) “filtering… virus suspension”, (vi) “preparing… virus bulk”, (vii) “formulating monovalent vaccines”, (viii) “formulating tetravalent vaccine”, along with four more downstream steps. These procedures covered Vero cell expansion in sub-culture, virus inoculation and propagation in a Cell Factory System, as well as the harvest and preparation of dengue virus for vaccine formulation. They also taught that “steps (iii) and (iv) are repeated one to about seven times” (Claim 3), while “serum free medium is added to the culture” after each harvest “in step (iv)” (Claim 2). Thus, Frazatti Gallina 2017 taught the core concepts and methods for Vero cell-based dengue vaccine development, with multiple steps that can be repeated to boost cell and virus yields, although this primary reference fell short of providing some of the cell culture conditions (pH and gas concentration) in a closed system (container and bioreactor) and a closed environment.
Kapre 2015 taught that “population of adherent cells” (Kapre 2015, Claim 3) can be cultured in “an integrated, closed cell culture system” and used “for the bulk growth of cells and… virus-infected cells in tissue culture and the subsequent collection of virus particles or viral components” (¶[0037]). Their system had closed, medium exchange “tubing” (¶[0037]) and “transfer piping… in-line… with sterile connectors” (¶[0040]), being “scalable through a wide range of volumes and adjustable due to the modular design” (¶[0037]), with “minimal to no handling of live virus” in a “self-contained” environment suitable “for the manufacture of bio-safety level 3 virus (BSL 3 virus), BSL 4 virus, and/or BSL 5 virus and combinations thereof” (¶[0053]). In one example, cell culture lasted for “about two days to about four weeks” (Kapre 2015, Claim 30). Thus, Kapre 2015 taught the key elements of virus production in a closed culture system and a “self-contained” environment capable of manufacturing BSL3, BSL4 and BSL5 pathogens, but this secondary reference still fell short of providing specific Vero cell culture conditions like temperature, pH, gas concentration and the timing of medium exchange and virus propagation.
The deficiencies noted for Frazatti Gallina 2017 and Kapre 2015 were overcome by methods of Thomassen et al. 2014. Specifically, this third reference taught ways to optimize “Vero cell cultivation” for the production of poliovirus vaccines. Vero cell “cultures were maintained at pH 7.2, 37oC, 50% dissolved oxygen” (section 2.2.2 on Pg. 2783), and cell “growth rate” differed by over 50-fold, depending on “cultivation methods” (Table 1 on Pg. 2784) and virus types (Fig. 5 on Pg. 2786). Their methods can “be further improved and… implemented in… manufacturing facilities” (Conclusions on Pg. 2787). One of ordinary skill in the art and interested in using the invention of Claims 27-32 in this instant application could monitor cell growth curves in response to pH, temperature, gas concentration, frequency of medium exchange and duration of culture, as taught by Thomassen et al. 2014, before arriving at an “improved” setting suitable for serum-free culture in a closed system that manufactures dengue virus vaccines. Thus, the invention of Claims 27-32 is an example of rationale (C) of MPEP §2143: use of known technique in Thomassen et al. 2014 to improve similar methods for Vero cell culture and dengue virus production (Frazatti Gallina 2017) in the same way when applied to a closed culture system in a self-contained environment (Kapre 2015).
Conclusion
No claim is allowed.
Additional Prior Art Cited but Not Applied
Whitehead et al. 2010 (US 2010/0104598 A1, published 04/29/2010). Development of dengue virus vaccine components.
This PGPub (the parent of US 8337860 B2, of record) teaches the use of genetically modified Dengue viruses, including “a Δ30 mutation that removes the TL-2 homologous structure in each of the dengue virus serotypes 1, 2, 3, and 4” (Abstract). “The Δ30 mutation deletes nt 174 to 145 of DEN1, nt 173 to 144 of DEN2, nt 173 to 143 of DEN3, and nt 172 to 143 of DEN4, with reverse-direction numbering system. The deleted region is indicated by the Δ symbol” (¶[0011]). This prior art also provides a long list of known Dengue virus strains (TABLE A), as well as growth curves (day 1 through day 7) for five recombinant viruses in Vero cell culture (Figure 19). In one example for serotype 3 alone, “viable viruses were analyzed for attenuation phenotypes in tissue culture, SCID mice transplanted with HuH-7 cells, and rhesus monkeys,” and “three mutant viruses (rDEN3Δ30/31, rDEN3Δ86, and rDEN3-3’D4Δ30) have preclinical phenotypes which suggest they may be safe and immunogenic in humans” (¶[0051]). Thus, well before the filing of this instant application, Whitehead et al. 2010 has already taught the rationale and choice of Dengue virus strains and genetically modified viruses toward attenuated vaccine constructs and their effective propagation in Vero cell culture.
Fielder & Gildea 2013 (US 2013/0189723 A1, published 07/25/2013). Automated cell culture system and process.
Fielder & Gildea 2013 invented an “automated cell culture system” (Figure 4) “consisting of a single or plurality of bioreactors” ¶[0116]) equipped with flexible “media exchange” and “microcarrier aspiration” (Figure 7 & ¶[0016]) that “eliminates the traditional manipulative steps of seeding, growing, feeding, splitting and assaying the cells or cell products” (¶[0006]). As such, cells “grow inside of and outside on the surface of… engineered microcarriers” (¶[0007]), and each “microcarrier has a mean diameter between approximately 100 nm and 500 µm" (Claim 10), while “a plurality of control systems can operate on a plurality of automated bioreactors” (¶[0062]) and can allow “free exchange of gases such as CO2 and O2” (¶[0067]). Since “bioreactors are operated for as long as 120 days” and require “optimal reactor conditions (pH, nutrient level, temperature, dissolved gas concentrations) (¶[0058]), “manipulation of microcarriers, fluids, gases, and bioreactor components” (¶[0066]) was automated to facilitate “continuous culture of cells” (¶[0066]), and cells grown in bioreactors can be either “stored” or “re-cultured” (Fielder & Gildea 2013, Claims 42-43). These teachings indicate that optimization of cell culture systems can even extend to automation, beyond the inventive concept for this instant application.
Fu et al. 2017 (CN 106676076 A, published 05/17/2017). Method for preparing rotavirus vaccine stock solution using serum-free Vero cells and for making a serum-free rotavirus vaccine product.
Fu et al. 2017 taught “a method for preparing serum-free rotavirus vaccine stock solution from Vero cells cultured in a bioreactor microcarrier, as well as a serum-free rotavirus vaccine product” (¶[0013]). From a Vero cell seed bank, “cells were resuscitated, cultured, passaged, and expanded using serum-free medium. These cells served as the base cells for culture in the bioreactor. After cell expansion, high-density Vero cells were cultured by continuous perfusion using a bioreactor and microcarriers in serum-free medium” (¶[0018]). Culture conditions were “37oC, pH 7.2-7.6 for 24 hours after cell seeding” (Claim 7). “Virus inoculation” was done when “the cell count reaches 0.9-1.1 × 10⁻² cells/ml on days 6-7” (Claim 7), and “cells and virus” were allowed to settle “for about 30-60 minutes at a temperature of 33-35°C” before virus propagation “at a temperature of 35°C” (¶[0026]). Pertinent to Claims 8, 13-15, 17-19 and 27 in the instant application, the teachings from Fu et al. 2017 made it clear that the conditions for initial Vero cell expansion/amplification and subsequent virus propagation/amplification can be optimized based on empirical data and regular monitoring of cell growth and signs of cytopathy (Figures 5-6).
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/JIANMING TANG/
Examiner, Art Unit 1671
/BENJAMIN P BLUMEL/ Primary Examiner, Art Unit 1671