The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-17 are pending and under consideration.
Priority: This application is a 371 of PCT/CN2021/082346, filed March 23, 2021, which claims foreign priority to CN 202110246672.0, filed March 5, 2021. A copy of the foreign priority document has been received in the instant application on September 5, 2023, and is not in the English language.
Failure to Comply with Sequence Rules
Where the description of a patent application discusses a sequence of 4 or more amino acids or a sequence of 10 or more nucleic acids, reference must be made to the sequence by use
of the sequence identifier preceded by “SEQ ID NO:” in the text of the description even if the
sequence is also embedded in the text of the description of the patent application (see 37 CFR
1.821, especially paragraphs (a)-(d)). The sequence identifier may be used in either the drawing
or the Brief Description of Drawings (see MPEP 2422).
Objection to the Specification:
The specification is objected to for failure to comply with the sequence rules for the
reasons as given above. The specification refers to sequences without identifiers at: see at least
paragraph 0073 (of the application publication US 20240150742).
The sequences must be in computer readable form (CRF) for search. See also MPEP
2422 for sequence compliance requirements. Appropriate correction is required.
Claim Objections
Claims 8, 10 are objected to because of the following informalities: in claim 8, the term “BMMY” should be spelled out the first time that it is recited in the claims; in claim 10, the term “BSM” should be spelled out the first time that it is recited in the claims. Appropriate correction is required.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 17 provides for the use of the recited protein in the preparation of hyaluronic acid-containing adjuvants, foods and cosmetics, but since the claim does not set forth any steps involved in the method/process, it is unclear what method/process applicant is intending to encompass. A claim is indefinite where it merely recites a use without any active, positive steps delimiting how this use is actually practiced.
Claim 17 is rejected under 35 U.S.C. 101 because the claimed recitation of a use, without setting forth any steps involved in the process, results in an improper definition of a process, i.e., results in a claim which is not a proper process claim under 35 U.S.C. 101. See for example Ex parte Dunki, 153 USPQ 678 (Bd.App. 1967) and Clinical Products, Ltd. V. Brenner, 255 F. Supp. 131, 149 USPQ 475 (D.D.C. 1966).
Claims 1-2 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claimed invention is directed to a judicial exception (i.e., a law of nature, a natural phenomenon, or an abstract idea) without significantly more.
Claims 1-2 recite a nature-based product, i.e. a gene for efficiently expression hyaluronic acid hydrolase, having a nucleotide sequence as shown in SEQ ID NO: 4 and a protein encoded by the gene, having a sequence as shown in SEQ ID NO: 5. In view of the grammatically indefinite article “a”, the recitation of “a nucleotide sequence” encompasses all genes that comprise any two or more contiguous nucleotides of SEQ ID NO: 4 and the recitation of “a sequence” encompasses all proteins that comprise any two or more contiguous amino acid residues of SEQ ID NO: 5. The genes and proteins recited in the instant claims are not markedly differently from naturally occurring nucleic acids that have a nucleotide sequence as shown in SEQ ID NO: 4 and naturally occurring proteins that have a sequence as shown in SEQ ID NO: 5. The claims are directed to a statutory category, i.e. a composition of matter (step 1: YES).
Since the claims recite a nature-based product, i.e. nucleic acid molecules encoding hyaluronidase and hyaluronidase proteins, the claims are analyzed to determine whether it is directed to any judicial exception. The markedly different characteristics analysis is performed by comparing the nature-based product limitation in the claim to its naturally occurring counterpart to determine if it has markedly different characteristics from the counterpart. The recited nucleic acid molecules and proteins are compared to their closest naturally occurring counterparts to determine if they have markedly different characteristics. Here, the closest natural counterparts can be naturally occurring nucleic acid molecules encoding hyaluronidase and naturally occurring hyaluronidase proteins from humans and/or mammals. When the claimed nucleic acid molecules and proteins are compared to these counterparts, the comparison indicates that there are no differences in structure, function, or other characteristics. There is no indication that the claimed genes that comprise any two or more contiguous nucleotides of SEQ ID NO: 4 and the proteins that comprise any two or more contiguous amino acid residues of SEQ ID NO: 5 are nucleic acid molecules and proteins having any structural and/or functional characteristics that are different from the naturally occurring nucleic acid molecules encoding hyaluronidase and naturally occurring hyaluronidase proteins in their natural state. Therefore, the claimed nucleic acid molecules and proteins do not have markedly different characteristics from what occurs in nature and is a “product of nature” exception. Accordingly, the claim is directed to an exception (step 2A: YES). Next, the claims as a whole are analyzed to determine whether any additional element, or combination of elements, is sufficient to ensure that the claim amounts to significantly more than the exception(s). The claims do not recite anything significantly different than the natural product, i.e. the claims do not recite elements or features that demonstrate that the claimed nucleic acid molecules comprising any two or more contiguous nucleotides of SEQ ID NO: 4 and the proteins comprising any two or more contiguous amino acid residues of SEQ ID NO: 5 are markedly different from what exists in nature. Therefore, the claims as a whole add nothing significantly more to the “product of nature” itself (Step 2B: NO), and thus, the claims do not qualify as eligible subject matter.
See also Funk Brothers Seed Co., V. Kalo Inoculant Co., 333 U.S. 127, (1948) and Association for Molecular Pathology v. Myriad Genetics, Inc. 569 U.S., 133 S. Ct. 2107, 2116, 106 USPQ 2d. 1972 (2013).
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.2.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claim 1 is drawn to a gene for efficiently expression hyaluronic acid hydrolase, having a nucleotide sequence as shown in SEQ ID NO: 4.
Claim 2 is drawn to a protein encoded by the gene of claim 1, having a sequence as shown in SEQ ID NO: 5.
Claims 3-4 are drawn to a recombinant expression vector, comprising the nucleotide sequence of claim 1.
Claim 5 is drawn to a Pichia pastoris, comprising the expression vector of claim 3.
Claims 6-16 are dependent on claim 1 and are drawn to a method for producing hyaluronic acid hydrolase, comprising the step(s) and features listed in the claims.
Claim 17 is drawn to a use of the protein of claim 2 in the preparation of hyaluronic acid-containing adjuvants, foods and cosmetics.
Regarding claim 1, in view of the grammatically indefinite article “a”, the recitation of “a nucleotide sequence” encompasses all genes that comprise any two or more contiguous nucleotides of SEQ ID NO: 4.
Regarding claim 2, in view of the grammatically indefinite article “a”, the recitation of “a sequence” encompasses all proteins that comprise any two or more contiguous amino acid residues of SEQ ID NO: 5.
Accordingly, the claims encompass a significantly large genus of nucleic acids encoding a hyaluronic acid hydrolase and a significantly large genus of proteins that may or may not have hyaluronic acid hydrolase activity.
The specification discloses representative species of the respective genus of genes encoding a hyaluronic acid hydrolase, i.e. a gene comprising the nucleotide sequence of SEQ ID NO: 4, which encodes a hyaluronic acid hydrolase, and representative species of the respective genus of hyaluronic acid hydrolase proteins, i.e. a protein comprising the amino acid sequence of SEQ ID NO: 5. Other than these representative species of the genus, the specification does not appear to disclose any other genes that encode a hyaluronic acid hydrolase or other hyaluronic acid hydrolase proteins. Moreover, the specification and prior art fail to disclose a correlation between a sequence of SEQ ID NO: 4 and encoding a polypeptide with hyaluronic acid hydrolase activity or a sequence of SEQ ID NO: 5 having hyaluronic acid hydrolase activity. In this case, the genus of genes encoding a hyaluronic acid hydrolase is widely variant, encompassing all genes comprising at least 2 contiguous nucleotides of SEQ ID NO: 4, and the genus of hyaluronic acid hydrolase proteins is also widely variant, encompassing all proteins comprising at least 2 contiguous amino acid residues of SEQ ID NO: 5. However, the disclosed representative species of the genus of genes encoding a hyaluronic acid hydrolase and hyaluronic acid hydrolase protein fail to reflect the wide variation among each genus. As such, one of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the recited genus. The claimed subject matter is not supported by an adequate written description because a representative number of species has not been described.
Claims 1-17 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a gene that encodes a hyaluronic acid hydrolase, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 4, a hyaluronic acid hydrolase protein encoded by the noted gene, comprising the amino acid sequence of SEQ ID NO: 5, wherein the gene that encodes hyaluronic acid hydrolase is able to provide a Pichia pastoris cell with the ability to express hyaluronic acid hydrolase protein and a method for producing hyaluronic acid hydrolase comprising culturing the Pichia pastoris cell that comprises the gene comprising the nucleotide sequence of SEQ ID NO: 4, does not reasonably provide enablement for all genes comprising any two or more contiguous nucleotides of SEQ ID NO: 4 and encoding a hyaluronic acid hydrolase, a method for producing a hyaluronic acid hydrolase comprising culturing a Pichia pastoris cell comprising all genes encoding a hyaluronic acid hydrolase, all proteins comprising any two or more contiguous amino acid residues of SEQ ID NO: 5 having hyaluronic acid hydrolase activity. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
“The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) as follows: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The Factors considered to be most relevant to the instant rejection are addressed in detail below.
Claim 1 is drawn to a gene for efficiently expression hyaluronic acid hydrolase, having a nucleotide sequence as shown in SEQ ID NO: 4.
Claim 2 is drawn to a protein encoded by the gene of claim 1, having a sequence as shown in SEQ ID NO: 5.
Claims 3-4 are drawn to a recombinant expression vector, comprising the nucleotide sequence of claim 1.
Claim 5 is drawn to a Pichia pastoris, comprising the expression vector of claim 3.
Claims 6-16 are dependent on claim 1 and are drawn to a method for producing hyaluronic acid hydrolase, comprising the step(s) and features listed in the claims.
Claim 17 is drawn to a use of the protein of claim 2 in the preparation of hyaluronic acid-containing adjuvants, foods and cosmetics.
Regarding claim 1, in view of the grammatically indefinite article “a”, the recitation of “a nucleotide sequence” encompasses all genes that comprise any two or more contiguous nucleotides of SEQ ID NO: 4.
Regarding claim 2, in view of the grammatically indefinite article “a”, the recitation of “a sequence” encompasses all proteins that comprise any two or more contiguous amino acid residues of SEQ ID NO: 5.
Accordingly, the claims encompass a significantly large genus of nucleic acids encoding a hyaluronic acid hydrolase and a significantly large genus of proteins that may or may not have hyaluronic acid hydrolase activity.
(B) The nature of the invention: The nature of the invention is engineering nucleic acids and/or polypeptides for optimized expression of a hyaluronic acid hydrolase protein in Pichia pastoris.
(C) The state of the prior art; (D) The level of one of ordinary skill; and (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure: According to MPEP 2164.03, “…what is known in the art provides evidence as to the question of predictability.” Regarding the scope of genes encoding hyaluronic acid hydrolase and the hyaluronic acid hydrolase proteins recited in the claims, it is noted that practice of the claimed invention would require undue experimentation by one of ordinary skill to ascertain which nucleic acid fragments and derivatives encode a protein having hyaluronic acid hydrolase activity and can be provided for efficient expression of a hyaluronic acid hydrolase protein in Pichia pastoris, and which polypeptide fragments and derivatives have hyaluronic acid hydrolase activity. Thus, there could be hundreds of variants which contain substitutions, deletions, substitutions, etc. In the instant case the quantity of experimentation would be large since there are myriad substitutions, deletions or insertions to choose from. The amount of guidance in the specification is zero with regard to which nucleotides in genes are essentially for encoding hyaluronic acid hydrolase and to which amino acids in a hyaluronic acid hydrolase protein are essentially for activity. The specification discloses representative species of the respective genus of genes encoding a hyaluronic acid hydrolase, i.e. a gene comprising the nucleotide sequence of SEQ ID NO: 4, which encodes a hyaluronic acid hydrolase, and representative species of the respective genus of hyaluronic acid hydrolase proteins, i.e. a protein comprising the amino acid sequence of SEQ ID NO: 5. No working examples are present of fragments or derivative nucleic acid molecules encoding a hyaluronic acid hydrolase or fragments or derivative proteins having hyaluronic acid hydrolase activity. The nature of the invention is such that many different nucleic acid molecules that are substantially similar may or may not encode a hyaluronic acid hydrolase and/or that many different proteins that are substantially similar to hyaluronic acid hydrolase may or may not have biological activity. The state of the prior art is that even proteins that are highly similar to the wild-type protein are at times not fully active. The relative level of skill in this art is very high. The predictability as to what substantially similar protein will have which activity is zero.
In view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, and the high degree of unpredictability as evidenced by the prior art, undue experimentation would be necessary for a skilled artisan to make and use the entire scope of the claimed invention. Applicants have not provided sufficient guidance to enable one of ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of having the desired biological characteristics is unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
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 4, 6-17 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.
Regarding claim 4, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 6 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps and/or elements, such omission amounting to a gap between the steps and/or elements. See MPEP § 2172.01. The omitted steps and/or elements are: the active and/or positive steps and conditions that are needed to produce the hyaluronic acid hydrolase. Claim 6 recites using the recombinant Pichia pastoris strain; however, it is not clear how the recited Pichia pastoris strain is used to produce hyaluronic acid hydrolase. Further clarification and/or correction is requested.
Claims 7-16 are included in this rejection because they are dependent on claim 6 and fail to cure its defects.
Claim 17 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps and/or elements, such omission amounting to a gap between the steps and/or elements. See MPEP § 2172.01. The omitted steps and/or elements are: the active and/or positive steps and conditions that are needed or required to prepare a hyaluronic acid-containing adjuvant, food, or cosmetic from the hyaluronic acid hydrolase. Further clarification and/or correction is requested.
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.
Claims 1-7, 17 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Reitinger et al. (2008 Protein Expression and Purification 57: 226-233). In view of the grammatically indefinite article “a”, the recitation of “a nucleotide sequence” encompasses all genes that comprise any two or more contiguous nucleotides of SEQ ID NO: 4 (instant claim 1) and the recitation of “a sequence” encompasses all proteins that comprise any two or more contiguous amino acid residues of SEQ ID NO: 5 (instant claim 2).
Reitinger et al. teach high-yield expression of honey bee hyaluronidase in Pichia pastoris (at least p. 226). Reitinger et al. teach nucleic acid molecules encoding bee hyaluronidase and expression of the bee hyaluronidase in a Pichia pastoris strain (at least p. 227-228). Therefore, Reitinger et al. can be deemed to anticipate at least instant claims 1-2.
Regarding instant claims 3-5, Reitinger et al. teach a pPIC9-Hya comprising nucleic acid molecules encoding bee hyaluronidase and transformed into the Pichia pastoris strain GS115 (at least p. 227).
Regarding instant claims 6-7, Reitinger et al. teach a method for expressing the bee hyaluronidase comprising culturing the Pichia pastoris strain GS115 transformed with the pPIC-Hya in medium to express the recombinant bee hyaluronidase (at least p. 227).
Regarding instant claim 17, Reitinger et al. teach hyaluronidases have been used in anti-cancer regimens, including as a hyaluronidase adjuvant (at least p. 227, 233).
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.
Claims 1-7, 8-9, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Reitinger et al. (2008 Protein Expression and Purification 57: 226-233) in view of Invitrogen (Invitrogen EasySelect™ Pichia Expression Kit User Manual 2010: 95 pages). The teachings of Reitinger et al. over at least instant claims 1-7, 17 are noted above.
Regarding instant claims 8-9, Reitinger et al. disclose that the method for expressing the bee hyaluronidase comprising culturing the Pichia pastoris strain GS115 transformed with the pPIC-Hya includes incubation at 30º C, suspending the Pichia pastoris cells in buffered methanol minimal medium (BMM, 100 mM potassium phosphate, 0.34% yeast nitrogen base) containing 1% methanol; incubating the yeast cells for 3 days and adding methanol (1% v/v) daily (at least p. 227). Reitinger et al. do not explicitly teach BMMY medium. Invitrogen discloses that for expressing recombinant Pichia strains, alternative mediums include BMMY (Buffered Methanol-complex Medium) containing yeast extract and peptone to stabilize secreted proteins and to prevent or decrease proteolysis of secreted proteins (at least p. 38, 58). Therefore, it would have been obvious to one of ordinary skill to incorporate the BMMY of Invitrogen for the BMM in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells of Reitinger et al. noted above (instant claims 8-9). The motivation to do so is given by the prior art Invitrogen, which disclose BMMY is also a recognized medium for culturing or fermenting Pichia pastoris cells. It would have been further obvious to arrive at the recited fermentation conditions of 25º-30º C, 1% (v/v) methanol induction every 24 hours for 96 hours, by routine optimization, because they are similar to the conditions disclosed in Reitinger et al. for expressing recombinant Pichia pastoris cells. It is known that “[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. One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including hyaluronidase, in Pichia pastoris is known in the prior art.
Claims 1-7, 10-15, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Reitinger et al. (2008 Protein Expression and Purification 57: 226-233) in view of Zhang et al. (2005 Biotechnol Prog 21(2): 386-393). The teachings of Reitinger et al. over at least instant claims 1-7, 17 are noted above.
Regarding instant claim 10, Reitinger et al. disclose that the method for expressing the bee hyaluronidase comprising culturing the Pichia pastoris strain GS115 transformed with the pPIC-Hya includes incubation at 30º C, suspending the Pichia pastoris cells in buffered methanol minimal medium (BMM, 100 mM potassium phosphate, 0.34% yeast nitrogen base) containing 1% methanol; incubating the yeast cells for 3 days and adding methanol (1% v/v) daily (at least p. 227). Reitinger et al. do not explicitly teach BSM medium. Zhang et al. disclose culturing or fermenting recombinant P. pastoris GS115 strains also expressing an enzyme (at least p. 387). Zhang et al. disclose fermentation with BSM (basal salts medium) and methanol induction (at least p. 387). Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate the BSM of Zhang et al. in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells of Reitinger et al. noted above (instant claim10). The motivation to do so is given by the prior art Zhang et al., which disclose BSM is also a recognized medium for culturing or fermenting Pichia pastoris cells. One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including an enzyme protein, in Pichia pastoris is known in the prior art.
Regarding instant claim 11, Reitinger et al. disclose inoculating the recombinant Pichia pastoris cells into YPD medium, inoculating the Pichia pastoris into BMM medium, incubating the Pichia pastoris cells for 3 days, and adding methanol daily (at least p. 227). Therefore, Reitinger et al. can be deemed to teach fermentation stages including an initial fermentation, feeding, starvation, ad methanol induction. Similarly, Zhang et al. also disclose inoculating the recombinant Pichia pastoris cells into a yeast medium, inoculating the inoculum into BSM, fermenting at 30º C, pH 5.0, feeding the Pichia pastoris cells at a feed rate, decreasing the glycerol feed rate during transition to methanol as the sole carbon source, feeding or inducing methanol under control of a methanol sensor (at least p. 387). Therefore, Zhang et al. can also be deemed to teach fermentation stages including an initial fermentation, feeding, starvation, ad methanol induction. “[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. Therefore, it would have been obvious to one of ordinary skill to incorporate the BSM of Zhang et al. in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells of Reitinger et al. noted above, where the method comprises steps of inoculating the recombinant Pichia pastoris cells into a yeast medium, inoculating the inoculum into BSM, fermenting at 30º C, pH 5.0, feeding the Pichia pastoris cells at a feed rate, decreasing the feed rate during transition to methanol as the sole carbon source, feeding or inducing methanol (instant claims 11-12). The motivation to do so is given by the prior art Zhang et al., which disclose BSM is also a recognized medium for culturing or fermenting Pichia pastoris cells. One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including an enzyme protein, in Pichia pastoris is known in the prior art.
Regarding instant claim 13, Zhang et al. disclose programming the feed rate to 0 over 3 h (at least p. 387). Therefore, it would have been obvious to arrive at the recited starvation culture of 2-3 hrs. by routine optimization in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells in BSM of Reitinger et al. in view of Zhang et al. noted above (instant claim 13). One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including an enzyme protein, in Pichia pastoris is known in the prior art.
Regarding instant claim 14, Reitinger et al. disclose daily methanol induction for 3 days (at least p. 227). Zhang et al. disclose methanol induction at 30º C and at a controlled rate until the Pichia pastoris cells reached a desired density (at least p. 387). Therefore, it would have been obvious for one of ordinary skill to arrive at the recited methanol induction conditions of a temperature of 25º-30º C for 80-120 hours by routine optimization, because they are similar to the conditions disclosed in Reitinger et al. and Zhang et al. for expressing recombinant Pichia pastoris cells.
Regarding instant claim 15, Reitinger et al. disclose purifying the Pichia pastoris supernatant (at least p. 228). Therefore, it would have been obvious to one of ordinary skill to purify the bee hyaluronidase in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells in BSM of Reitinger et al. in view of Zhang et al. noted above. One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including an enzyme protein, in Pichia pastoris is known in the prior art.
Claims 1-7, 10-15, 16, 17 are rejected under 35 U.S.C. 103 as being unpatentable over Reitinger et al. (2008 Protein Expression and Purification 57: 226-233), Zhang et al. (2005 Biotechnol Prog 21(2): 386-393), Park et al. (2019 J Microbiol Biotechnol 29(8): 1310-1315) and Block et al. (2009 Methods in Enzymology 463: 439-473). The teachings of Reitinger et al. and Zhang et al. over at least instant claims 1-7, 10-15, 17 are noted above.
Regarding instant claim 16, Reitinger et al. disclose purifying the Pichia pastoris supernatant by cation exchange (at least p. 228). Reitinger et al. do not explicitly teach affinity chromatography. Park et al. disclose a chimeric hyaluronidase comprising a His-tag and a simplified purification with Ni2+ affinity chromatography (at least p. 1310). Block et al. disclose purification of recombinant His-tagged proteins with Ni-NTA and an imidazole gradient, including 4 → 250 mM imidazole (at least p. 446-447, p. 453 table 27.2), and elution at 500 mM imidazole (p. 464-465). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to add a His-Tag to the bee hyaluronidase and incorporate the nickel affinity chromatography and gradient elution with 100-500 mM imidazole buffer of Park et al. and Block et al. for the chromatographic purification of the bee hyaluronidase in the method for expressing bee hyaluronidase comprising culturing Pichia pastoris cells in BSM of Reitinger et al. in view of Zhang et al. noted above (instant claim 16). The motivation to do so is given by the prior art, which disclose nickel affinity chromatography is a known purification method for recombinant hyaluronic proteins. One of ordinary skill would have a reasonable expectation of success because expression of recombinant proteins, including an enzyme protein, in Pichia pastoris is known in the prior art, and protein purification by affinity chromatography is also known in the prior art.
The full-length nucleotide sequence of SEQ ID NO: 4 and the full-length amino acid sequence of SEQ ID NO: 5 appear to be free of the prior art.
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Marsha Tsay whose telephone number is (571)272-2938. The examiner can normally be reached M-F.
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/Marsha Tsay/Primary Examiner, Art Unit 1656