The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-10 are pending and under consideration.
Priority: This application is a 371 of PCT/CN2021/073745, filed January 26, 2021, which claims benefit to foreign application CN 202010795389.9, filed August 10, 2020. A copy of the foreign priority document has been received in this application on February 9, 2023 and is not in the English language.
Claim Rejections - 35 USC § 112
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.
Claims 1-10 are 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.
The claims are generally narrative and indefinite, failing to conform with current U.S. practice. They appear to be a literal translation into English from a foreign document and are replete with grammatical and idiomatic errors.
For instance, in claim 1, the term “MANNase” should be spelled out in full or clarified that the enzyme is “mannanase” the first time that it is recited in the claims. Similarly, the term “GLP-1” should be spelled out in full the first time that it is recited in the claims. Claim 1 further recites MANNase and homologues thereof. It is not clear what is meant by homologues of MANNase, i.e. enzymes having the same structure, enzymes having the same activity, etc. Further clarification and/or correction is requested.
Claim 1, steps 2-3 recite, to construct “recombinant engineering yeast”. It is not clear if the claim means to recite that the yeast is “a recombinant engineered yeast.” Further clarification and/or correction is requested.
Claim 1, step 4, recites the limitation “supernatant” in the claim. There is insufficient antecedent basis for this limitation in the claim. Further, it is not clear what is meant by the supernatant purified, concentrated and dried. Further clarification and/or correction is requested.
Claim 2 recites the sequences of the genes of encoding GLP-1 and mannanase are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. As currently written, the claim language reciting “the sequences…are shown in SEQ ID NO: X”, appears to mean that “the sequence shown in SEQ ID NO: X” encompasses all sequences that comprise any two or more contiguous nucleotides within the recited SEQ ID NO. It is not clear if the claim’s meaning is that the genes encoding GLP-1 and mannanase comprise the nucleic acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Further clarification and/or correction is requested.
Claim 3 is dependent on claim 1 and should recite that the method of claim 1, step 1, further comprises rather than that step 1 was done or performed in the past tense. Claim 3 recites the limitations "the target fragments…" and “the obtained gene sequences…” in the claim. There is insufficient antecedent basis for these limitations in the claim. Further correction is requested.
In claims 4-5, as similarly noted above for claim 2, the claim language reciting “the sequences…are shown in SEQ ID NO: X”, appears to mean that “the sequence shown in SEQ ID NO: X” encompasses all sequences that comprise any two or more contiguous nucleotides or any two or more contiguous amino acid residues within the recited SEQ ID NO. It is not clear if the claim’s meaning is that the primers comprise the nucleic acid sequence of the recited SEQ ID NO. and/or that the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. Further clarification and/or correction is requested.
In claim 6, the abbreviation “YPD” should be spelled out in full the first time that it is recited in the claims. Claim 6, step 3, recites adding the secondary seed solution into the fermentation culture medium. It is not clear if the fermentation culture medium is referring to the previous YPD medium or to a different culture medium. Further clarification and/or correction is requested. Claim 6 recites the limitations "the induction agent", “the OD600”, “the fermentation broth”, “the jar” in the claim. There is insufficient antecedent basis for these limitations in the claim. Claim 6, step 3, recites collecting the yeast by centrifugation. It is not clear if “the yeast” is referring to the recombinant engineered yeast or to yeast that has not been transformed. Further clarification and/or correction is requested.
Claim 7 is dependent on claim 5, where claim 7 recites “in step S3”. However, claim 5 does not recite a “step S3”. Further clarification and/or correction is requested. Claim 7 recites the limitations "the fermentation culture”, “the inducer”, “the volume percentage” in the claim. There is insufficient antecedent basis for these limitations in the claim.
Claim 8 is dependent on claim 5, where claim 8 recites “in step S4”. However, claim 5 does not recite a “step S4”. Further clarification and/or correction is requested. Claim 8 recites the limitations "the initial fermentation temperature”, “the stirring speed”, “the aeration rate”, “the pH” in the claim. There is insufficient antecedent basis for these limitations in the claim. Claim 8 recites when “carrying out the fermentation culture.” It is not clear what is meant by “carrying out” the fermentation culture. Further clarification and/or correction is requested.
Claim 9 recites the limitation "the filtrate" in the claim. There is insufficient antecedent basis for this limitation in the claim.
Claim 10 recites application of the recombinant fusion protein of MANNase and homologues thereof and GLP-1 in the preparation of drugs for the treatment of diabetes or high-fat, high-glucose diet-induced metabolic syndrome. Claim 10 recites the limitation "the fusion protein of…" in the claim. There is insufficient antecedent basis for this limitation in the claim. Further, claim 10 is incomplete for omitting essential steps and/or elements, such omission amounting to a gap between the steps/elements. See MPEP § 2172.01. The omitted steps and/or elements are the steps and elements required for preparing and/or a preparation of drugs for the treatment of diabetes or high-fat, high-glucose diet-induced metabolic syndrome with the fusion protein. Further clarification and/or correction is requested.
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.
Claims 1-8, 10 are rejected under 35 U.S.C. 103 as being unpatentable over CN 1927888 (CN ‘888) (IDS 02.09.23) (translation provided by Google Patents cited herein) in view of CN 1834237 (‘237) (translation provided by Google Patents cited herein) and Zhou et al. (2019 J Sci Food Agric 99:6981-6988; IDS 02.09.23).
CN ‘888 discloses GLP-1 is a blood sugar reducing peptide and further discloses a fusion protein comprising GLP-1 and an enzyme (lysozyme), for lowering blood glucose effectively (at least p. 1-2). CN ‘888 discloses a method for expressing and purifying a recombinant fusion protein comprising lysozyme and GLP-1, the method comprising cloning genes encoding GLP-1 and lysozyme into a PIC plasmid to obtain a recombinant expression vector; transforming the recombinant expression vector into a Pichia yeast cell to obtain a recombinant engineered Pichia yeast cell; fermenting and culturing the recombinant engineered Pichia yeast cell to induce expression of the fusion protein comprising lysozyme and GLP-1; centrifuging and purifying the fermentation broth and concentrating the liquid to obtain the fusion protein comprising lysozyme and GLP-1 (at least embodiments 1-16). CN ‘888 differs from the claimed fusion protein by not teaching a mannanase enzyme.
CN ‘237 discloses engineering a Pichia pastoris yeast cell to express a beta-mannanase enzyme at high efficiency and low cost (at least p. 1). CN ‘237 discloses cloning a gene encoding beta-mannanase into a plasmid pPICZα to obtain a recombinant expression vector; transforming the recombinant expression vector into a Pichia pastoris yeast cell; fermenting and culturing the recombinant Pichia pastoris yeast cell to express the mannanase (at least p. 1-3).
Zhou et al. disclose that a composition comprising a yeast cell supernatant treated with beta-mannanase, led to blood glucose reduction and improving diabetic syndromes (at least p. 6980).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of the prior art and arrive at the claimed method for expression and purification of a recombinant fusion protein comprising a mannanase and a GLP-1, the method comprising cloning genes encoding GLP-1 and mannanase into a pPICZα plasmid to obtain a recombinant expression vector; transforming the recombinant expression vector into a Pichia pastoris yeast cell to obtain a recombinant engineered Pichia pastoris yeast cell; fermenting and culturing the recombinant engineered Pichia pastoris yeast cell to induce expression of the fusion protein comprising mannanase and GLP-1; centrifuging and purifying the fermentation broth and concentrating the liquid to obtain the fusion protein comprising mannanase and GLP-1 (instant claim 1). The motivation to do is given by the prior art. CN ‘888 discloses a method for expressing and purifying a fusion protein comprising an enzyme and GLP-1 from a Pichia yeast expression system. CN ‘237 discloses expressing and purifying a beta-mannanase enzyme from a Pichia yeast expression system. Zhou et al. disclose that a beta-mannanase enzyme incorporated into a yeast cell supernatant results in a composition that reduces blood glucose and improves diabetic syndromes. Therefore, one of ordinary skill would have reasonable motivation to incorporate the GLP-1 gene of CN ‘888 with the beta-mannanase gene in the expression system and methods disclosed in CN ‘237 to arrive at a method for expressing and purifying a yeast supernatant comprising a fusion protein comprising mannanase and GLP-1 from a Pichia pastoris expression system because there was interest in expressing products that treat diabetic syndromes and it was recognized that GLP-1 and mannanase have blood glucose reducing functions. One of ordinary skill would have a reasonable expectation of success because methods for producing target proteins, including fusion proteins, in Pichia yeast are known and expression vectors for Pichia yeast are also known.
It would have been further obvious to dry the concentrated supernatant comprising the mannanase-GLP-1 fusion protein because it is routine to dry and/or lyophilize a purified protein composition for storage in the protein and pharmaceutical arts (instant claim 1).
Regarding instant claim 3, CN ‘888 discloses primer pairs for the GLP-1 gene, and PCR amplification and cloned into a vector carrier (at least embodiments 2-20) and CN ‘237 discloses primer pairs for the beta-mannanase gene, with PCR amplification and cloned into the vector carrier pPICZα (at least p. 4-5). Therefore, it would have been obvious to arrive at the recited steps for encoding GLP-1 and mannanase using primer pairs, PCR amplification and double digestion, and ligation into a pPICZα plasmid to arrive at an expression vector encoding a fusion protein comprising mannanase and GLP-1, by routine optimization.
Regarding instant claims 2, 4-5, in view of the claim language reciting “the sequences…are shown in SEQ ID NO: X” or “the amino acid sequence is shown in SEQ ID NO: X”, the recitation of “the sequence shown in SEQ ID NO: X” encompasses all sequences that comprise any two or more contiguous nucleotides or amino acid residues of the recited SEQ ID NO. Therefore, the CN ‘237 and CN ‘888 discloses genes for beta-mannanase and GLP-1 respectively, that comprise sequences having two or more contiguous nucleotides shown in recited SEQ ID NO: 2 and 1, respectively, primer sequences having two or more contiguous nucleotides show in recited SEQ ID NOS: 3-4, 5-6, and it would be further obvious that the mannanase-GLP-1 fusion protein of CN ‘237 and CN ‘888 noted above comprises two or more contiguous amino acid residues of recited SEQ ID NO: 7.
Regarding instant claim 6, CN ‘237 discloses steps for fermenting and culturing the Pichia pastoris yeast cell to express the target protein include, including preparing seed liquor comprising the engineered Pichia pastoris yeast cell to express the target protein, including inoculating an engineered Pichia pastoris yeast cell to a YPD medium containing Zeocin and incubation at 28-30º C, with shaking (250-300 rpm) for >24 hrs., monitoring a OD600, adding methyl alcohol (inducer) (at least p. 3-6), and CN ‘888 also discloses steps for fermenting and culturing Pastoris yeast including fermentation under methanol induction and then centrifuging the fermented liquid (at least p. 5). It is known that generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, the prior art references discloses expressing similar proteins using the same expression vectors recited and the same Pichia expression system recited and at similar fermentation and culture conditions. Therefore, it would have been obvious to one of ordinary skill to arrive at the recited fermenting and culturing steps by routine optimization.
Regarding instant claim 7, CN ‘237 discloses induction by adding methyl alcohol at a concentration 0.8-1.2% (at least p. 3) and CN ‘888 discloses fermentation expression under methanol induction of 1 mg/mL (1000mg/l) (p. 5). As similarly noted above, the prior art references discloses expressing similar proteins using the same expression vectors recited and the same Pichia expression system recited and at similar fermentation and culture conditions. Therefore, it would have been obvious to one of ordinary skill to arrive at the recited methanol concentration and high density culture by routine optimization.
Regarding instant claim 8, CN ‘237 discloses fermentation conditions including 28-30º C, 250-300 rpm, pH 5.5-6.0 (at least p. 5-6). As similarly noted above, the prior art references discloses expressing similar proteins using the same expression vectors recited and the same Pichia expression system recited and at similar fermentation and culture conditions. Therefore, it would have been obvious to one of ordinary skill to arrive at the recited methanol concentration and high density culture by routine optimization.
Regarding instant claim 10, as noted above, CN ‘888 discloses GLP-1 is a blood sugar reducing peptide and discloses a fusion protein comprising GLP-1 and an enzyme for lowering blood glucose effectively (at least p. 1-2). Zhou et al. disclose that a yeast supernatant extract comprising beta-mannanase leads to blood glucose reduction and improving diabetic syndromes (at least p. 6980). Therefore, it would have been obvious to one of ordinary skill to arrive at a preparation of the mannanase-GLP-1 fusion protein noted above for treating diabetes.
Claims 1-8, 9, 10 are rejected under 35 U.S.C. 103 as being unpatentable over CN 1927888 (CN ‘888) (IDS 02.09.23) (translation provided by Google Patents cited herein) in view of CN 1834237 (‘237) (translation provided by Google Patents cited herein), Zhou et al. (2019 J Sci Food Agric 99:6981-6988; IDS 02.09.23), and Sun et al. (EP 1408050). The teachings of CN ‘888, CN ‘237, and Zhou et al. over at least instant claims 1-8, 9, 10 are noted above.
Regarding instant claims 1, 9, CN ‘888 discloses purifying the yeast extract comprising the fusion protein, comprising centrifuging the yeast fermentation broth (or supernatant), performing ultrafiltration to concentrate the fermentation liquid, where the ultrafiltration membrane is a 5K film membrane (at least p. 5). Sun et al. disclose GLP-1 peptide compositions for treating diabetes (paragraphs 0001-0002). Sun et al. disclose purifying a GLP-1 composition including by ultrafiltration and then lyophilizing the GLP-1 composition (at least paragraphs 0107, 0110). “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). MPEP 2144.05. In this instance, the prior art references discloses expressing and purifying similar proteins using the same expression vectors recited and the same Pichia expression system recited and at similar fermentation and culture conditions. Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further concentrate and dry the yeast supernatant comprising the mannanase-GLP-1 fusion protein noted above and arrive at the recited conditions including ultrafiltration membranes through various sizes and freeze-drying the concentrated mannanase-GLP-1 fusion protein by routine optimization. The motivation to do so is given by the prior art, which disclose that compositions of GLP-1 products can be concentrated and lyophilized. One of ordinary skill would have a reasonable expectation of success because methods for concentrating protein compositions and lyophilizing (or freeze-drying) protein compositions are known and routinely performed.
No claim is allowed.
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/Marsha Tsay/Primary Examiner, Art Unit 1656