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 .
Claim status
Claims 1-20 are pending.
Claims 12-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species/invention.
Claims 1-11 are under examination.
Election/Restrictions
Applicant's election without traverse of invention group 1, drawn to a process of producing a refined protein of interest, claims 1-11 in the reply filed on 4/13/2026 is acknowledged.
Claim objections.
Regarding claim 9, there should not be internal periods. The periods after the letters denoting the steps need to be replaced with a parenthesis. For example, in claim 9 "a." should be corrected to "a)" in line 1 and etc., for the rest of the steps a-h.
In other words, a claim should only contain one period. Please see MPEP 608.01(m).
Appropriate correction is appreciated.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 3 recites the phrase ‘plasmid is cloned with a 6x His-tag at a C terminal’ which renders the claim indefinite, because plasmids don’t have a c-terminal end. The clarity of the claim can be improved with the following claim language, ‘the plasmid comprises a nucleotide sequences encoding a His-tag’
Claim 4 recites the phrase ‘plasmid is cloned with one or more identified selection markers’ which implies that proteins are cloned in the plasmid, rendering the claim indefinite. The clarity of the claim language can be improved with the following claim language, ‘the plasmid comprises nucleotide sequences encoding one or more identified selection markers’.
Claim 7 contains the trademark names OptiPRO ™ SFM and ExpiFectamin™ . Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify transfection/expression media and, accordingly, the identification/description is indefinite.
Claim 7 also recites the limitation "the step of transfection" in line 1 of the claim. The claim 7 is dependent on the claim 1 which does not recite a ‘step of transfection’. Therefore, there is insufficient antecedent basis for this limitation in the claim, it is not understood which step of transection the claim is referring to. Therefore, the claim is indefinite.
Claim 8 recites the limitations:
" the crude protein " and “the mammalian cells obtained” in step 4,
“the solution” in step 5,
“the fraction” in step 7.
The claim 8 is dependent on the claim 1. Claims 1 or 8 do not recite these limitations previously. Therefore, there is insufficient antecedent basis for these limitations in the claim. It is not understood which ‘crude protein’, ‘mammalian cells obtained’, ‘ solution’ or ‘’fraction’ the claim is referring to, in the recited steps. Therefore the claim is indefinite.
Claim 9 recites “M-per” in step C. it’s not clear what “M-per” means. Specification does not provide a definition for “M-per”. Therefore, the claim is indefinite, due to not particularly pointing out and distinctly claiming the subject matter.
Also, claim 9 step (a) recites ‘thawing the harvested cells’, however there’s no freezing step recited in the claims from which claim 9 depends (step 4 of claim 8 or claim 1) which renders the claim unclear. Therefore the claim is indefinite.
Also, “cells harvested” in step (a) of claim 9 has no prior mention in claim 9, step 4 of claim 8 or in claim 1. Therefore, there is insufficient antecedent basis for this limitation in the claim. It is not understood which “harvested cells”, the claim is referring to. Therefore the claim is indefinite.
Claim 10 is not clearly written, it is not understood what solution is to be concentrated, it is not clear if the claim is referring to the dialyzed solution, or the solution obtained by eluting. Therefore, the claim is indefinite as written.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1, 3-7 are rejected under 35 U.S.C. 102 (1) as being anticipated by Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808, as evidenced by www.thermofisher.com.
Regarding claim 1, Gracia-Cordero teaches a process of producing a purified protein of interest, comprising the steps of:
Culturing isolated mammalian cells, i.e., Expi239F cells (materials and methods 2.2.1, page 3) and HEK293T cells ( results 3.2, page 6) transfected with mammalian expression vector or plasmid pcDNA 3.1(from the vendor Invitrogen), encoding the epitopes and subunits of SARS-CoV-2; nucleocapsid protein (N), structural spike protein (S1) , and receptor binding domain (RBD), in Expi293 media. The said antigens of SARS-CoV-2 showed immunogenic capabilities in mice (abstract, page 1, material and methods 2.3 and 2.4 page 3) .
Gracia-Cordero teaches that the sequences encoding S1 and N proteins were based on GenBank seq ID MN908947.3 (Materials and methods, Gene synthesis and plasmid construction, page 3). Said Genbank Seq ID was searched on NCBI database by the examiner, the sequence of the ‘surface glycoprotein’ encoded by the gene ’s’ with protein-id ‘QHD43416.1’ was a 100% sequence match to the protein sequence set forth by the Seq ID:1 of the instant application.
The amino acid sequence alignment between the Genbank surface glycoprotein of SARS-CoV-2 isolate, Whuhan-Hu-1 and the Seq ID 1 of the instant application is shown below. Sequence alignment was obtained from https://blast.ncbi.nlm.nih.gov/Blast.cgi.
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Gracia-Cordero teaches, (2) expressed and (3) harvested the proteins of interest from the mammalian cells and the culture media (materials and methods, 2.5 protein production and purification, page 4).
Regarding claim 3, Gracia-Cordero teaches that the plasmid in claim 1 rejection is cloned with a 6x His tag, and the resultant proteins were labeled with 6x His at the c-termini (Results, 3.1 construction of S1 and N recombinant plasmids, page 5, fig 1A).
Regarding claims 4 and 5, Gracia-Cordero teaches the expression vector pcDNA 3.1 which was purchased from the vendor, Invitrogen (material and methods 2.3, page 3). The said vector is constructed with identified selection markers, neomycin and ampicillin, as evidenced by the vector map published on the vendors’ website (https://documents.thermofisher.com/TFS-Assets/vectors/pcdna3.1cat.pdf).
Regarding claim 6, Gracia-Cordero teaches expressing the SARS-CoV-2 proteins in human embryonic kidney (HEK)-293T cells (3.2 Expression of recombinant S1 and N proteins in mammalian cells, page 6).
Regarding claim 7, Gracia-Cordero teaches transfection of Expi293F cells (Expi293f cells are commercially available, HEK293Tderived mammalian cell line), (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-expression/mammalian-protein-expression/transient-mammalian-protein-expression/expi293-expression-system.html) with SARS-CoV-2 constructs, and treating the constructs with Opti-MEM and Expi293 expression media (ExpiFectamin™ 293 expression media) prior to mixing with the mammalian cells ( materials and method, 2.4 transfection of Expi293F cells with SARS-CoV-2 constructs, page 3-4).
Therefore, the claimed invention is anticipated by the reference.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 1- 2 are rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808, and in view of Bal Ram Singh et al., 2019, US 2019/0076518 A1.
The applied reference has a common joint inventor with the instant application. Based upon the earlier effectively filing date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
Regarding claim 1, Gracia-Cordero teaches a process of producing a purified protein of interest, comprising the steps of:
(1) Culturing isolated mammalian cells, i.e., Expi239F cells (materials and methods 2.2.1, page 3) and HEK293T cells ( results 3.2, page 6) transfected with mammalian expression vector or plasmid pcDNA 3.1(from the vendor Invitrogen), encoding the epitopes and subunits of SARS-CoV-2; nucleocapsid protein (N), structural spike protein (S1) , and receptor binding domain (RBD), in Expi293 media. The said antigens of SARS-CoV-2 showed immunogenic capabilities in mice (abstract, page 1, material and methods 2.3 and 2.4 page 3) .
Gracia-Cordero teaches that the sequences encoding S1 and N proteins were based on GenBank seq ID MN908947.3 (Materials and methods, Gene synthesis and plasmid construction, page 3). Said Genbank Seq ID was searched on NCBI database by the examiner, the sequence of the ‘surface glycoprotein’ encoded by the gene ’s’ with protein-id ‘QHD43416.1’ was a 100% sequence match to the protein sequence set forth by the Seq ID:1 of the instant application.
The amino acid sequence alignment between the Genbank surface glycoprotein of SARS-CoV-2 isolate, Whuhan-Hu-1 and the Seq ID 1 of the instant application is shown below. Sequence alignment was obtained from https://blast.ncbi.nlm.nih.gov/Blast.cgi.
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Gracia-Cordero teaches, (2) expressed and (3) harvested the proteins of interest from the mammalian cells and the culture media (materials and methods, 2.5 protein production and purification, page 4).
Garcia-Cordero doesn’t teach the SARS-CoV-2 epitope set forth by the Seq ID: 1 of the instant application being embedded in the backbone of the nucleotide sequence of detoxified recombinant tetanus toxin (DrTeNT).
However, before the effective filling date of the claimed invention, vectors containing the backbone of the nucleotide sequence of DrTeNT were known in the art.
Regarding claim 2, Bal Ram Singh teaches a plasmid vector containing DrTeNT backbone and several restriction enzyme digestion sites (fig.7, page 10), this vector was designed to produce a neurotoxin associated protein, for oral or nasal delivery of tetanus vaccine in combination with other drugs (abstract, technical field (0001), page 1).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the above mentioned teachings of Garcia-Cordero and Singh to generate a nucleotide sequence encoding an epitope of SARS-CoV-2 in a backbone of the nucleotide sequence encoding detoxified DrTeNT. One of ordinary skill in the art would have been motivated to combine the teachings of Garcia-Cordero and Singh to produce a nucleotide sequence encoding an epitope of SARS-CoV-2 in a backbone of the nucleotide sequence of detoxified DrTeNT, in order to generate a both proteins in one vector that may have immunogenic or therapeutic value such as a combination vaccine for SARS-CoV-2 and tetanus, because as Singh suggest that their invention relates to a composition of vaccines in combination with neurotoxin associated proteins such as tetanus. There would be a reasonable expectation of success to combine the teachings of Garcia-Cordero and Singh to produce the aforementioned combination vaccine, because both references teach functional expression vectors that produce their respective recombinant proteins
Hence, the claimed invention as a whole was prima facie obvious.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808 and Bal Ram Singh et al., 2019 as applied to claims 1-2 above, further in view of Grodzki, et al., NJ: Humana Press, (2009) 27-32, as evidence by Madadlou, et al., 2011.
Teachings of Garcia-Cordero and Bal Ram Singh are as relied on above.
Regarding claim 8, Garcia-Cordero teaches the following protein purification steps:
Dialysis of the harvested culture supernatants with buffer A which is maintained at pH 6.4 for S1 protein purification (step 4 and 5 of claim 8), subsequently subjecting the dialyzed solution to fast protein liquid chromatography (FPLC) while still in buffer A which was maintained at pH 6.4 which contains 500mM NaCl. FPLC step was performed to further purify proteins. FPLC is a high performance chromatography that most commonly use ion exchange of proteins, as evidenced by Madadlou, et al., 2011 (step 6 of claim 8). Finally the concentrated supernatant was injected through His trap column to remove contaminants, eluted through a concentration gradient, and further concentrated to 1ml by centrifugation which teaches step 7 of claim 8 ( materials and methods, 2.5 protein production and purification, page 4).
Garcia-Cordero doesn’t teach eluting protein containing solutions through DEAE A50 ion-exchange columns.
However, before the effective filling date of the claimed invention, eluting protein containing solutions through DEAE A50 ion-exchange columns was known in the art.
Regarding claim 8, step 6, Grodzki teaches a protocol for antibody purification, using DEAE A50 ion-exchange column (page 29, 2 materials). Grodzki also teaches that this technique is powerful and can separate biomolecules that have minor differences in their net charge (abstract, page, 27).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the above mentioned teachings of Garcia-Cordero and Grodzki to express and purify antigens of SARS-CoV-2. One of ordinary skill in the art would have been motivated to combine the teachings of Garcia-Cordero and Grodzki to separate the SARS-CoV-2 antigens, depending on their net charge by eluting through DEAE A50 ion-exchange columns, as taught by Grodzki. There would be a reasonable expectation of success to combine the teachings of Garcia-Cordero and Grodzki to produce and purify the antigens of SARS-CoV-2, because the ion exchange chromatography is a known technique in the art to have the capability to separate proteins based on minor charge differences, as suggested by Grodzki.
Hence, the claimed invention as a whole was prima facie obvious.
Claims 9 is rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808 and Bal Ram Singh et al., 2019, as applied to claims 1-2 above, and further in view of Litovchick, et al., Cold Spring Harbor Protocols 2019.7 (2019): pdb-prot098541, as evidenced by https://attheu.utah.edu/research/why-does-ice-form-at-a-range-of-temperatures, and thermofisher.com.
Teachings of Garcia-Cordero and Bal Ram Singh are as relied on above.
Garcia-Cordero doesn’t teach one or more of the steps listed in claim 9.
However, before the effective filling date of the claimed invention, one or more of the steps listed in claim 9 were known in the art.
Regarding claim 9, Litovchick teaches thawing harvested and frozen cultured cells on ice prior to immunoprecipitation. Temperature of ice/ice bath is about 4-10 0C, as evidenced by https://attheu.utah.edu/research/why-does-ice-form-at-a-range-of-temperatures/. Keeping the protein containing solution at 4 0C helps to stabilize proteins as suggested by https://documents.thermofisher.com/TFS-Assets/LSG/Application-Notes/TR0043-Protein-storage.pdf.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the above mentioned teachings of Garcia-Cordero and Litovchick to express antigens of SARS-CoV-2 in mammalian cells and thaw the frozen cells on ice. One of ordinary skill in the art would have been motivated to combine the teachings of Garcia-Cordero and Litovchick to thaw cells on ice or at 4-10 0C to stabilize the proteins as evidenced by thermofisher.com. There would be a reasonable expectation of success to combine the teachings of Garcia-Cordero and Litovchick to express antigens of SARS-CoV-2 in mammalian cells, and to thaw the frozen cells on ice prior to further purification, because thawing cells at 4-10 0C, prior to protein isolation is well known in the art and also suggested by thermofisher.com.
Hence, the claimed invention as a whole was prima facie obvious.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808 and Bal Ram Singh et al., 2019, as applied to claims 1-2 above, and further in view of Duong-Ly et al.," Methods in enzymology. Vol. 541. Academic Press, 2014. 85-94.
Teachings of Garcia-Cordero and Bal Ram Singh are as relied on above.
Garcia-Cordero doesn’t teach concentrating the dialyzed protein solution to obtain the protein of interest using salting out method.
However, before the effective filling date of the claimed invention, concentrating the protein solution to obtain proteins of interest using salting out method was known in the art.
Regarding claim 10, Duong-Ly teaches concentrating the cell lysate using an optimized amount of ammonium sulphate to precipitate the protein of interest in a salting-out method which provides a low cost method of isolating proteins (theory, page 86, 4. Protocol, pages 88-92).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the above mentioned teachings of Garcia-Cordero and Duong-Ly to express and precipitate antigens of SARS-CoV-2. One of ordinary skill in the art would have been motivated to combine the teachings of Garcia-Cordero and Duong-Ly to produce and isolate antigens of SARS-CoV-2 according to the salting-out method taught by Duong-Ly, for the benefit of low-cost associated with the method as suggested by Duong-Ly. There would be a reasonable expectation of success to combine the teachings of Garcia-Cordero and Duong-Ly to obtain the antigens of SARS-CoV-2 via salting out method, because ammonium sulphate/anion based salting-out method is ideal for small protein molecules as suggested by Duong-Ly (theory, page 86).
Hence, the claimed invention as a whole was prima facie obvious.
Claim 11 is rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Cordero et al., Diagnostics 11.10 (2021): 1808 and Bal Ram Singh et al., 2019, as applied to claims 1-2 above, and further in view of Machuca et al., 2017, Journal of Visualized Experiments: Jove 130: 57092.
Teachings of Garcia-Cordero and Bal Ram Singh are as relied on above.
Garcia-Cordero also teach treating the harvested cell supernatant with 1X protease inhibitors to prevent protein degradation (materials and methods, 2.4, page 4).
Garcia-Cordero doesn’t teach dialysis of protein solution with Tris-buffer, urea and NaCl.
However, before the effective filling date of the claimed invention, dialysis of protein solutions with Tris-buffer, urea and NaCl was known in the art.
Regarding claim 11, Machuca teaches a method to recover and purify the bacterial protein, chemoreceptor ligand binding domain in E.Coli which includes dialyzing the protein solution containing Tris-buffer, urea and NaCl (abstract, page 1, protocol, page 2).
Denatured protein was added to buffer E (i.e. 3M urea, 100mM Tris-HCl at pH 8, and other components) which was subsequently added to the dialysis membrane clamped at one end and the dialysis was carried out in pre-cooled buffer A ( i.e. 10mM Tris-HCl, pH 8.0 and 150mM NaCl) (protocol, 3. protein refolding, page 2). Machuca also teaches that excess urea (3M urea added with buffer E) is gradually removed from the protein, allowing the protein to refold and a heavy protein precipitate can be observed at the end of dialysis (protocol, 3. protein refolding, page 2).
The concentrations of urea, Tris-buffer and NaCl taught by Machuca are not the same as those recited in claim 11. However, Machuca teaches that protocol optimization is likely needed for various proteins when using their method (discussion, page 8, first paragraph). Furthermore, one of ordinary skill in the art would recognize that the concentrations of various constituents in the dialysis buffer is a result effective variable, and determining the appropriate concentrations of the constituents would be a matter of routine optimization. Please see MPEP 2144.05 II routine optimization.
MPEP section 2143 teaches Examples of basic requirements of Prima Facie case of obviousness which states the following:
A. Combining Prior Art Elements According to Known Methods To Yield Predictable Results
To reject a claim based on this rationale, Office personnel must resolve the Graham factual inquiries. Then, Office personnel must articulate the following:
(3) a finding that one of ordinary skill in the art would have recognized that the results of the combination were predictable; and
(4) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness.
Dialysis for protein purification is a known technique in the art as evidenced by Machuca and Garcia-Cordero. One of ordinary skill in the art would have recognized that the concentrations of urea, Tris-HCl and NaCl used in Machuca, predictably need optimization to adjust to isolate SARS-CoV-2 proteins, and the dialysis method taught by Machuca lead to a high yield of purified protein (protocol, 3. protein refolding, page 2).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the above mentioned teachings of Garcia-Cordero and Machuca to express and purify antigens of SARS-CoV-2. One of ordinary skill in the art would have been motivated to combine the teachings of Garcia-Cordero and Machuca to produce and purify antigens of SARS-CoV-2 that have immunogenic capabilities as suggested by Garcia-Cordero, at high yield by the dialysis method taught by Machuca. There would be a reasonable expectation of success to combine the teachings of Garcia-Cordero and Machuca to express and purify the antigens of SARS-CoV-2 via dialysis, because the dialysis buffer taught by Machuca produces heavy precipitate of purified protein.
Hence, the claimed invention as a whole was prima facie obvious.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to HASHANTHI ABEYRATNE-PERERA whose telephone number is (571)272-6562. The examiner can normally be reached Monday-Friday 7:30 am- 5:00pm.
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/HASHANTHI KOMITIGE ABEYRATNE-PERERA/ Examiner, Art Unit 1632
/PETER PARAS JR/ Supervisory Patent Examiner, Art Unit 1632
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