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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 08/20/2025 has been entered.
Election/Restrictions
Applicant’s election of Group I (i.e., claims 1, 2, 4, 5, 8-11, 14, 16-18, 23, 26, 27, 30, 36, 38, 39, 41, 42, 44, and 56 drawn to a method of isolating recombinant spider silk protein from a host cell ) and Species A (i.e., the isolated spider silk protein comprises at least 30% full length protein (claim 5); alkaline pH range of 11 to 12 (claim 2); purity of at least 65% (claim 56) and yield of at least 60-65% (claim 9); structure 18B, i.e., SEQ. ID NO. 38 (claim 41); incubation period of at least 15 minutes (claim 23); and centrifugation (claims 27 and 30)) in the reply filed on March 26, 2024 are acknowledged. Claims 1-2, 4-5, 8-11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42, 44, and 56 are under consideration as claim 49 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claims. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim 49 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claims. Election was made without traverse in the reply filed on March 26th 2024.
Status of Claims
Claims 1-55 were originally filed on May 27th, 2021.
The amendment received on March 11th, 2022 amended claims 2, 4, 8-11, 14, 16-18, 23, 26, 30, 36, 38-39, and 41-42; and cancelled claims 3, 6, 7, 12-15, 19-22, 14-15, 18-19, 31-35, 37, 40, 43, 45-48, and 50-55.
The amendment received on March 26th, 2024 added claim 56.
The amendment received on July 22nd 2025, amended claims 1, 11 and 44; and cancelled claim 10.
Claims 1-2, 4-5, 8-9, 11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42, 44, and 56 are currently pending and under consideration.
Priority
The present application claims status as a 371 (National Stage) of PCT/US2019/063208 filed on November 26th 2019 which claims benefit of provisional Application 62/772, 588 filed on November 28th 2018.
Claim Interpretation
For purposes of applying prior art, the claim scope has been interpreted as set forth below per the guidance set forth at MPEP § 2111. If Applicant disputes any interpretation set forth below, Applicant is invited to unambiguously identify any alleged misinterpretations or specialized definitions in the subsequent response to the instant action. Applicant is advised that a specialized definition should be properly supported and specifically identified (see, e.g., MPEP § 2111.01(IV), describing how Applicant may act as their own lexicographer).
For claim 1, regarding “recombinant spider silk protein,” it is noted that the instant specification does not define whether the “recombinant spider silk protein” is a full-length recombinant spider silk protein or a fragment thereof. As such, the Examiner is interpreting the scope of the “recombinant spider silk protein” as encompassing a full-length recombinant spider silk protein or a fragment thereof where the fragment encompasses any length fragment.
For claim 1 step c and claim 44 step c, regarding “alkaline conditions,” it is noted that the instant specification does not provide a definition for the phrase “alkaline conditions” rather the specification teaches that in certain embodiments the alkaline conditions comprise an alkaline pH from 9 to 14, and in one embodiment, the alkaline pH is from 11 to 12 (see instant specification at pg. 2, para[0008]). Pursuant to MPEP 21111.01, under a broadest reasonable interpretation, words of the claim must be given their plain meaning, unless such meaning is inconsistent with the specification. The plain meaning of a term means the ordinary and customary meaning given to the term by those of ordinary skill in the art at the time of the invention. The Khan Academy defines anything above pH 7.0 as alkaline, or basic (see Khan Academy, The pH scale available online at https://www.khanacademy.org/science/biology/water-acids-and-bases/acids-bases-and-ph/a/acids-bases-ph-and-bufffers accessed on 05/04/2024). As such, the Examiner is interpreting the scope of the “alkaline conditions” as conditions requiring a pH above 7.0 in order to solubilize a recombinant silk protein.
Response to Arguments
1. Applicant’s arguments, see Response, filed 07/22/2025, with respect to 35 U.S.C. 103 as being unpatentable over WO 2011/120690 A2 (Amsilk GMBH) International Publication Date October 6th 2011 (Cited in the IDS filed on 12/01/2022) (herein after “Amsilk”), ResearchGate, Solvent for Silk, Nov. 10th 2014, pp. 1-8 (Cited in the IDS filed on 12/01/2022) (herein after “Research Gate”); US 2016/0222174 A1 (Widmaier et al.) published on August 4th 2016 (herein after “Widmaier”); and Sigma Aldrich (Product Information, Ammonium Sulfate Solution (A5479), 2004, pp. 1-4 (herein after “Sigma Aldrich”), have been fully considered but they are not persuasive. The 35 U.S.C. 103 rejection to claims 1-2, 4-5, 8-9, 11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42, 44 and 56 is maintained.
Duplicate Claim Warning
Applicants are advised that claim 44 includes an identical recitation of the claim limitations recited in independent claim 1; and should claim 1 be found allowable, claim 44 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). In particular, lines 1-13 in claim 44 recite duplicate limitations of the limitations recited in lines 1-13 in claim 1; therefore, claim 44 is found to be a duplicate of claim 1.
New Objections/Rejections
Claim Objections
Claim 11 is objected to because of the following informalities: punctuation. The claim lacks a pause (i.e., “,”) between the terms lowered pH and from 4 to 10. Appropriate correction is required.
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.
1. Claims 1-2, 4-5, 8-9, 11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42, 44, and 56 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 claims 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. This is a new matter rejection.
Claims 1 and 44 are independent claims and were amended to recite “incubating said portion of said cell culture in an aqueous solution under alkaline conditions, thereby solubilizing at least 30% of said recombinant spider silk protein in said aqueous solution.” Applicants assert that support for the amendments can be found throughout the specification, for instance, at pp. 44-46, para[0183]-[0189] (see Remarks, filed 07/22/2025, pg. 5, paragraph 2). Further, Applicants stated that “no new matter has been added” (see Remarks, filed 07/22/2025, pg. 5). However, the evidence (i.e., para[0183]-[0189]) provided by Applicants does not provide support that the subject matter pertaining to solubilizing at least 30% of said recombinant spider silk protein, in claims 1 and 44 was previously presented. The specification is void of literal and inherent support that would clearly provide support for the instant amendment. The specification does not teach solubilizing at least 30% of said recombinant spider silk protein. Examination of the instant support shows Fig. 4B, Fig. 5, Fig. 7A, and Fig. 8 (see instant specification, pg. 6, para[0038]-[0039], [0041] and [0042]), which depict the % area of 18B monomer obtained by size exclusion chromatography. In particular Fig. 4B shows a comparison of the amount of purity of 18B spider silk as purified using a urea extraction method or the alkaline extraction method (see instant and Fig. 4B), where it can be noted that the percent area of 18B monomer under alkaline conditions was 34.215. However, the percent area calculated by size exclusion chromatography is not equivalent to solubilizing at least 30% of said recombinant spider silk protein. As such, the amendment to claims 1 and 44 has not been adequately supported. Please note that claims 2, 4-5, 8-9, 11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42 are also rejected because they depend upon a rejected claim.
Maintained/Modified Rejections in light of Amendment
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
103 - KSR Examples of 'Rationales' Supporting a Conclusion of Obviousness
(Consistent with the "Functional Approach" of Graham)
Further regarding 35 USC 103(a) rejections, the Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S. 398, 127 S. Ct. 1727, 82 USPQ2d 1385, 1395-97 (2007) (KSR) identified a number of rationales to support a conclusion of obviousness which are consistent with the proper "functional approach" to the determination of obviousness as laid down in Graham. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit.
Exemplary rationales that may support a conclusion of obviousness include:
(A) Combining prior art elements according to known methods to yield predictable results;
(B) Simple substitution of one known element for another to obtain predictable results;
(C) Use of known technique to improve similar devices (methods, or products) in the same way;
(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;
(E) "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;
(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;
(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.
Note that the list of rationales provided is not intended to be an all-inclusive list. Other rationales to support a conclusion of obviousness may be relied upon by Office personnel.
Also, a reference is good not only for what it teaches by direct anticipation but also for what one of ordinary skill in the art might reasonably infer from the teachings. (In re Opprecht 12 USPQ 2d 1235, 1236 (Fed Cir. 1989); In re Bode 193 USPQ 12 (CCPA) 1976).
2. Claims 1-2, 4-5, 8-9, 11, 14, 16-18, 23, 26-27, 30, 36, 38-39, 41-42, 44 and 56 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2011/120690 A2 (Amsilk GMBH) International Publication Date October 6th 2011 (Cited in the IDS filed on 12/01/2022) (herein after “Amsilk”), in view of EP 2243792 A1 (Spiber Technologies AB) Date of Publication Oct. 27th 2010 (herein after “Spiber”), and US 2016/0222174 A1 (Widmaier et al.) published on August 4th 2016 (herein after “Widmaier”). Regarding claims 1 and 44, Amsilk teaches a method of isolating an insoluble target protein from a suspension of intact or disrupted host cells comprising the steps of : a) providing a suspension of intact host cells comprising an insoluble target protein and insoluble host cell parts (see Amsilk, pg. 3, lines 9-12). The term “host cells” refers to cells that comprise a target protein of interest, e.g. a silk protein such as a spider silk protein or a variant thereof (see Amsilk, pg. 6, lines 33-34). The host cells may be used for amplification and expression of a polynucleotide that codes for a target protein of interest, e.g. a silk protein such as a spider silk protein or a variant thereof (see Amsilk, pg. 7, lines 4-6). The target protein which is encoded by said recombinant polynucleotide may be designated as a recombinant target protein (see Amsilk, pg. 7, lines 16-17). The term “recombinant polynucleotide” refers to a polynucleotide synthesized or otherwise manipulated in vitro (see Amsilk, pg. 7, lines 14-16). A host cell comprising said recombinant polynucleotide and/or said recombinant target protein may be designated as a recombinant host cell (see Amsilk, pg. 7, lines 17-19). The term "a suspension of intact host cells" encompasses intact, generally non-lysed host cells, e.g. microbial host cells, plant host cells or insect host cells, suspended in a fluid, e.g. in an aqueous solution (e.g. a buffered aqueous solution), technical water, deionized water, culture medium or fermentation medium (see Amsilk, pg. 9, lines 8-11). The buffered aqueous solution may be, for example, Tris/HCl the pH of the buffered aqueous solution may be between pH 5.0 and pH 9.0 (see Amsilk, pg. 8, lines 25-27). Thus, constituting a method of isolating recombinant spider silk protein from a host cell culture, where the obtained cell culture comprises a host cell in a growth medium which expresses recombinant spider silk protein as recited in instant claims 1a and 44a. It is noted that steps a. and b. of instant claims 1 and 44 do not require means of collection, and thus, obtaining a cell culture and collecting a portion of it (note: a “portion” encompasses any portion including the whole culture as long as the protein is contained within) corresponds to or is synonymous with providing a suspension of the cell culture because by providing the cell culture it must necessarily result in obtaining and collecting it. Therefore, providing a suspension of intact host cells comprising an insoluble target protein and insoluble host cell parts as taught by Amsilk, correlates to collecting a portion of said cell culture comprising said recombinant spider silk as recited in instant claims 1 and 44, step b.
Amsilk’s method of isolating an insoluble target protein from a suspension of intact or disrupted host cells further comprises step b) adding an aqueous solution of at least one base, for example a metal hydroxide and/or ammonia such as sodium hydroxide (NaOH), potassium hydroxide (KOH) and/or calcium hydroxide (CaOH) (see Amsilk, pg. 16, lines 10-12), to said suspension in an amount which is sufficient to disrupt said host cells and to solubilize said insoluble host cell parts (see Amsilk, pg. 5, lines 28-30). The term "insoluble host cell parts” refers to insoluble host cell proteins, cell walls, cell membranes, cell wall parts, cell membrane parts, cytoskeleton parts, host cell debris, and/or insoluble cytoplasmic inclusions (e.g. crystals of calcium oxalate or silicon dioxide, granules of energy-storage materials such as starch, glycogen or polyhydroxybutyrate), which are parts of the host cells, e.g. microbial, plant or insect host cells, or of the expression system used to express the target protein (see Amsilk, pg. 11, lines 25-30). This term does not encompass the insoluble target protein of the present invention (see Amsilk, pg. 11, lines 30-31). The term "the target protein remains insoluble" used by Amsilk refers to the target protein as insoluble in a suspension, e.g. in an aqueous solution (e.g. buffered aqueous solution), in a culture medium, in a fermentation medium, in technical water, or in deionized water, and stays insoluble, e.g. to at least 80%, preferably to at least 90%, and more preferably to at least 95%, or even to 100%, for example, to at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9%, or 100%, in said suspension, e.g. in said aqueous solution (e.g. buffered aqueous solution), culture medium, fermentation medium, technical water, or deionized water, comprising a base over a time period of at least 5 minutes, preferably of at least 10 minutes, more preferably of at least 90 minutes and most preferably of at least 180 minutes (see Amsilk, pg. 15, lines 1-11); thereby corresponding to incubating said portion of said cell culture in an aqueous solution under alkaline conditions, as recited in step c. of instant claims 1 and 44.
Pursuant to MPEP 2144.05 (I), a prima facie case of obviousness exists where the claimed ranges and prior art ranges do not overlap but are close enough that one skilled in the art would have expected them to have the same properties. In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%). Therefore, the claimed solubilization of at least 30% of said recombinant spider silk protein would have been suggested to one skilled in the art.
Amsilk also teaches that the skilled person can easily access which amount of an aqueous solution comprising a base is sufficient/required to disrupt host cells, for example, by (i) performing a dilution series of a selected base, e.g. NaOH, adding the selected base (e.g. NaOH) at several concentrations to a suspension of intact or disrupted host cells, (iii) separating the insoluble target protein from the completely or partially solubilized insoluble host cell parts, e.g. by centrifugation or filtration, (iv) evaluating the portion of insoluble host cell parts within the separated insoluble target protein portion, e.g. in the centrifugate or retentate, and (v) determining the amount of the base which is sufficient/required in order to solubilize at least 80%, preferably at least 90%, more preferably at least 95%, or even 100% of the insoluble host cell parts (see Amsilk, pg. 14, lines 9-24).
Therefore, as long as the manipulative step is met, i.e., incubating said portion of said cell culture in an aqueous solution under alkaline conditions, as discussed in the claimed claim interpretation above, only requires a pH above 7.0, will necessarily result in solubilizing the protein. Moreover, it is noted that the level of solubilization in instant claim 1, is limited to at least 30%, thereby encompassing partial solubilization (i.e., 30%) of the recombinant spider silk protein or total solubilization (i.e., 100%). As such, since Amsilk teaches that 80% of the silk protein remains insoluble, that would necessarily correlate to 20% of the silk protein being soluble.
Additionally, it is noted that since Amsilk teaches that a skilled person can easily access which amount of an aqueous solution comprising a base is sufficient/required to disrupt host cells, for example by performing a dilution series of a selected base, e.g. NaOH, (ii) adding the selected base (e.g. NaOH in final concentrations of 0.01 M NaOH, 0.02 M NaOH, 0.03 M NaOH, 0.04 M NaOH, 0.05 M NaOH, 0.06 M NaOH, 0.07 M NaOH, 0.08 M NaOH, 0.09M NaOH, 0.1 M NaOH, 0.2 M NaOH, 0.3 NaOH, 0.4 M NaOH, 0.5 M NaOH, 0.6 M NaOH, 0.7 M NaOH, 0.8 M NaOH, 0.9 M NaOH, or 1 M NaOH), (iii) separating the insoluble target protein from the completely or partially solubilized insoluble host cell parts, (iv) evaluating the portion of insoluble host cell parts within the separated insoluble target protein portion, e.g. in the centrifugate or retentate, and (v) determining the amount of the base which is sufficient/required in order to solubilize at least 80%, preferably at least 90%, more preferably at least 95%, or even 100% of the insoluble host cell parts (see Amsilk, pg. 16, lines 9-24).
Amsilk adds that “the target protein remains insoluble" refers to a target protein which is insoluble in a suspension, e.g. in an aqueous solution ( e.g. buffered aqueous solution), in a culture medium, in a fermentation medium, in technical water, or in deionized water, and which stays insoluble, e.g. to at least 80%, preferably to at least 90%, and more preferably to at least 95%, or even to 100%, for example, to at least 80-99.9%, or 100%, in said suspension, e.g. in said aqueous solution (e.g. buffered aqueous solution), culture medium, fermentation medium, technical water, or deionized water, comprising a base, e.g. over a certain period of time and/or at a specific temperature, and, thus, allows the separation and isolation of said target protein, e.g. by filtration and/or centrifugation (see Amsilk, pg. 14, lines 25-34). As such, if the target molecule and the insoluble host cell parts are in the same aqueous solution, it must follow that "the target protein remains insoluble" to at least 80%, in said aqueous solution (e.g. buffered aqueous solution), culture medium, fermentation medium, technical water, or deionized water, comprising a base over a time period of at least 5 minutes, preferably of at least 10 minutes, more preferably of at least 90 minutes and most preferably of at least 180 minutes (see Amsilk, pg. 15, lines 1-11). Then the partial or complete solubilization of said recombinant spider silk protein in said aqueous solution is clearly a result specific parameter that a person of ordinary skill in the art would routinely optimize. Optimization of parameters is a routine practice that would be obvious for a person of ordinary skill in the art to employ. It would have been customary for an artisan of ordinary skill to determine the optimal alkaline conditions needed to achieve the desired results (i.e., solubilizing at least 30% of said recombinant spider silk protein). Thus, an ordinary skilled artisan would have been motivated to modify the aqueous solution comprising a base as taught by Amsilk, for solubilizing insoluble host cell parts to such that at least 80% or more of the insoluble host cell parts are solubilized and for the target protein to stay insoluble for at most 80% thereby resulting in solubilizing at least 30% of the recombinant spider silk protein, because an ordinary skilled artisan would have been able to utilize the teachings of Amsilk to obtain various incubation parameters (i.e., NaOH concentration and incubation time) with a reasonable expectation of success. Thus, absent some demonstration of unexpected results from the claimed parameters as recited in step c. of claims 1 and 44; the optimization of the aqueous solution under alkaline conditions would have been obvious before the effective filing date of Applicants’ invention. Therefore, the claimed invention, as a whole, would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made, because the combined teachings of the prior art are fairly suggestive of the claimed invention.
Amsilk teaches a method of isolating an insoluble target protein from a suspension of intact or disrupted host cells comprising the steps of: c) separating the insoluble target protein from the solubilized insoluble host cell parts (see Amsilk, pg. 5, lines 31-32), by centrifuging between 3000 x g and 12000 x g, i.e. the insoluble target protein is present as precipitate and the solubilized insoluble
bacterial host cell parts are in the supernatant which can be discarded (see Amsilk, pg. 31, lines 22-24). However Amsilk does not expressly teach isolating the solubilized recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein sample, wherein isolating the solubilized recombinant spider silk protein comprises precipitating the recombinant spider silk protein by altering said alkaline conditions of said aqueous solution, as recited in instant claim 1, step d; nor adjusting the aqueous solution to a non-alkaline pH, thereby precipitating the said solubilized recombinant spider silk protein; and e. isolating the recombinant spider silk protein from said portion of cell culture, thereby producing an isolated recombinant spider silk protein, as recited in instant claim 44.
Spiber teaches a method of producing polymers of an isolated spider silk protein consisting of from 170 to 600 amino acid residues involves providing a solution of said spider silk protein in a liquid medium at pH 6.4 or higher and/or an ion composition that prevents polymerization of the spider silk protein (see Spiber, Abstract). The properties of the liquid medium are adjusted to a pH of 6.3 or lower and an ion composition that allows polymerization of the spider silk protein (see Spiber, Abstract); thereby constituting precipitating the recombinant spider silk protein by altering said alkaline conditions of said aqueous solution as recited in step d. of instant claim 1; and constituting adjusting the aqueous solution to a non-alkaline pH, thereby precipitating the said solubilized recombinant spider silk protein as recited in step d. of instant claim 44. Spiber also teaches that the spider silk protein is allowed to form solid polymers in the liquid medium, and the resulting solid spider silk protein polymers are isolated from the liquid medium (see Spiber, Abstract); thereby corresponding to isolating the solubilized recombinant spider silk protein from said aqueous solution as recited in step d. of instant claim 1, and also constituting isolating the recombinant spider silk protein from said portion of cell culture, thereby producing an isolated recombinant spider silk protein as recited in step e. of instant claim 44. Spiber teaches that it has surprisingly been found that although the presence of the NT (i.e., NT is a protein fragment having from 100 to 160 amino acid residues, which fragment is a N-terminal fragment derived
from a spider silk protein (see Spiber, pg. 4 para[0028])) fragment improves solubility of the spider silk protein at a pH of 6.4 or higher and/or an ion composition that prevents polymerization of the spider silk protein, it accelerates polymer formation at a pH of 6.3 or lower when the ion composition allows polymerization of the spider silk protein (see Spiber, pg. 16, para[0085]). The resulting polymers are solid and macroscopic, and they are formed in the liquid medium having a pH of 6.3 or lower and an ion composition that allows polymerization of the spider silk protein (see Spiber, pg. 16, para[0085]). Spiber adds that the inventive insights that the N-terminal non-repetitive fragment of spider silk proteins is involved in polymerization of these proteins and that the formation of polymers involving this fragment can be tightly controlled by varying certain parameters have also been developed into a novel method of reversibly assembling a polymer or oligomer of molecules carrying the N-terminal non-repetitive spidroin fragment (see Spiber, pg. 18, para[0101]). Additionally the method is applicable to any similar protein for the purpose of producing polymers or oligomers (see Spiber, pg. 18, para[0101]). Without desiring to be limited to any particular theory, the polar and unbalanced charge distribution of NT is ideally suited for generation of a polymerizable module that can be simply controlled by pH and salt concentration, thus allows NT to regulate silk assembly by preventing premature aggregation and triggering polymerization as the pH is lowered, similar to what is perceived to occur along the spider's silk extrusion duct (see Spiber, pg. 20, para[0125]).
From the teachings of the references, the Examiner recognizes that it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of isolating an insoluble target protein from a suspension of intact or disrupted host cells of Amsilk and incorporate the teachings of Spiber in order to arrive at the claimed method of isolating recombinant spider silk protein from a host cell culture comprising: d. isolating the solubilized recombinant spider silk protein from said aqueous solution, wherein isolating the solubilized recombinant spider silk protein comprises precipitating the recombinant spider silk protein by altering said alkaline conditions of said aqueous solution.
One of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so because it was known that adjusting the properties of the liquid medium to a pH of 6.3 or lower and an ion composition allows polymerization of spider silk proteins; because it was known that the N-terminal non-repetitive fragment of spider silk proteins is involved in polymerization of spider silk proteins and that the formation of polymers involving this fragment can be tightly controlled by varying certain parameters such as pH and salt concentration; and because it was known that NT regulates silk assembly by preventing premature aggregation and triggering polymerization as the pH is lowered, similar to what is perceived to occur along the spider’s silk extrusion duct as taught by Spiber. One of ordinary skill in the art before the effective filing date of the claimed invention would have had a reasonable expectation of success given that the method of producing polymers of Spiber produced spider silk protein consisting of from 170 to 600 amino acid residues, and given that Spiber’s method is applicable to any similar protein for the purpose of producing polymers or oligomers. Therefore, a method of isolating an insoluble target protein from a suspension of intact or disrupted host cells as taught by Amsilk, by adjusting the properties of the liquid medium to a pH of 6.3 or lower and an ion composition that allows polymerization of spider silk proteins as taught by Spiber; would support a method of isolating recombinant spider silk protein from a host cell culture comprising precipitating the recombinant spider silk protein by altering said alkaline conditions of said aqueous solution; by constituting some teaching, suggestion or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention/and or the use of a known technique to improve similar devices (methods, or products) in the same way and/or the application of a known technique to a known device (method, or product) ready for improvement to yield predictable results pursuant to KSR.
Regarding claim 2, Amsilk’s method of isolating an insoluble target protein from a suspension of intact or disrupted host cells further comprises step b) adding an aqueous solution of at least one base, for example a metal hydroxide and/or ammonia such as sodium hydroxide (NaOH), potassium hydroxide (KOH) and/or calcium hydroxide (CaOH) (see Amsilk, pg. 16, lines 10-12). Amsilk adds that it is preferred that the aqueous solution comprising a base in step b) has a pH of between 7 and 14, more preferably of between 9 and 13, and most preferably of between 10 and 12 (see Amsilk, pg. 23, lines 4-6). For example, the aqueous solution comprising a base in step b) has a pH of 7, 7 .5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14 (see Amsilk, pg. 23, lines 6-7), thereby constituting wherein the alkaline conditions comprise an alkaline pH from 11 to 12 as recited in instant claim 2. MPEP 2144.05(I) states that "[i]n the case where the claimed ranges "overlap or lie inside ranges discloses by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990) (The prior art taught carbon monoxide concentrations of "about 1-5%" while the claim was limited to "more than 5%". The court held that "about 1-5%" allowed for concentrations slightly above 5% thus the ranges overlapped.) Moreover, the Federal Circuit found that a prima facie case existed where a claim reciting thickness of a protective layer as falling within a range of "50 to 100 Angstroms and the prior art taught that "for suitable protection, the thickness of the protective layer should be not less than about 10 nm [i.e., 100 Angstroms]." In re Geisler, 116 F.3d 1465, 1469-82, 43 USPQ2d 1362, 1365-66 (Fed. Cir. 1997). Therefore, the claimed alkaline conditions would have been obvious to one of ordinary skill in the art since the prior art range (i.e., pH of between 7 and 14, more preferably between 9 and 13) lies within the claimed pH range (i.e., pH from 11 to 12).
Regarding claim 4, Amsilk teaches Fig. 6, which depicts the stability assay of purified recombinant spider silk protein C16 treated with 0.05 M sodium hydroxide as described in example 2 (see Amsilk, pg. 62, lines 12-13). No significant protein degradation could be observed after incubation with 0.05 M for a time period of between 5 min to 24 hours (see Amsilk, pg. 62, lines 13-14). Although Amsilk teaches that the recombinant spider silk protein was not degraded, Amsilk does not expressly teach that the recombinant spider silk protein is a full-length recombinant spider silk protein as recited in instant claim 4.
Widmaier teaches that spider's silk polypeptides are large (>150 kDa, >1000 amino acids) polypeptides that can be broken down into three domains: an N-terminal non-repetitive domain (NTD), the repeat domain (REP), and the C-terminal nonrepetitive domain (CTD) (see Widmaier, pg. 1, para[0004]). The NTD and CTD are relatively small (~150, ~100 amino acids respectively), well-studied, and are believed to confer the polypeptide’s aqueous stability, pH sensitivity, and molecular alignment upon aggregation (see Widmaier, pg. 1, para[0004]). Widmaier’ s invention provides compositions of proteinaceous block co-polymers capable of assembling into fibers, and methods of producing said co-polymers (see Widmaier, pg. 1, para[0009]). In some of Widmaier’ s embodiments, the co-polymer also includes an N-terminal non-repetitive domain between 75-350 amino acids in length, and a C-terminal non-repetitive domain between 75-350 amino acids in length (see Widmaier, pg. 2, para[0010]); in other embodiments the quasi-repeat domain is 500-5000, 119-1575, or 900-950 amino acids in length (see Widmaier, pg. 2, para[0010]); and the mass of the co-polymer is 40-400, 12.2-132, or 70-100 kDa (see Widmaier, pg. 2, para[0010]). Widmaier also teaches Example 6, Assembly and Assay of 4x Repeat R Sequences, R domains from SEQ ID NOs: 1-1398 that expressed and secreted well, were concatenated into 4x repeat domains using the assembly scheme shown in FIG. 12 (see Widmaier, pg. 22, para[0141]). The concatenation was performed as described in Example 4 and shown in FIGS. 7 and 8 (see Widmaier, pg. 22, para[0141]). Selected sequences from this ligation of R sequences are shown in Table 6 (see Widmaier, pg. 22, para[0141]). Sequences for these silk constructs include those full-length silk construct sequences of SEQ ID NOs: 1411-1468 (see Widmaier, pg. 22, para[0141]). Additionally, Table 10, depicts additional full-length block copolymer constructs with alpha mating factor, multiple repeat domains, and 3X FLAG domains (see Widmaier, pg. 29, Table 10), thereby constituting where the isolated recombinant spider silk protein is a full-length recombinant silk protein as recited in instant claim 4.
From the teachings of the references, the Examiner recognizes that it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of isolating an insoluble target protein from a suspension of intact or disrupted host cells of Amsilk and incorporate the teachings of Widmaier in order to arrive at the claimed method of isolating recombinant spider silk protein from a host cell culture wherein the isolated recombinant spider silk protein is a full-length recombinant spider silk protein.
One of ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so because it was known that spider’s silk polypeptides can be broken down into three domains and the NTD (N-terminal non-repetitive domain) and CTD (C-terminal nonrepetitive domain) domains are relatively small, well-studied and are believed to confer the polypeptide’s aqueous stability, pH sensitivity, and molecular alignment upon aggregation as taught by Widmaier. One of ordinary skill in the art before the effective filing date of the claimed invention would have had a reasonable expectation of success given that Widmaier’ s invention provides compositions of proteinaceous block co-polymers capable of assembling into fibers that include an N-terminal non-repetitive domain between 75-350 amino acids in length, and a C-terminal non-repetitive domain between 75-350 amino acids in length; and given that Widmaier’ s sequences for these silk constructs include those full-length silk construct sequences of SEQ ID NOs: 1411-1468. Therefore, a method of isolating an insoluble target protein from a suspension of intact or disrupted host cells as taught by Amsilk, by assembling into fibers that include full-length silk construct sequences as taught by Widmaier; would support a method of isolating recombinant spider silk protein from a host cell culture comprising wherein the isolated recombinant spider silk protein is a full-length recombinant spider silk protein; by constituting some teaching, suggestion or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention/and or the use of a known technique to improve similar devices (methods, or products) in the same way and/or the application of a known technique to a known device (method, or product) ready for improvement to yield predictable results pursuant to KSR.
Regarding claim 5, The MPEP 2112-2112.02 states that when a reference discloses all the limitations of a claim except for a property or function, and the Examiner cannot determine whether or not the reference inherently possesses properties which render obvious the claimed invention but has basis for shifting the burden of proof to Applicant as in In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980). In the instant case, Amsilk teaches that the target protein which is insoluble in a suspension, e.g. in an aqueous solution ( e.g. buffered aqueous solution), in a culture medium, in a fermentation medium, in technical water, or in deionized water, (see Amsilk pg. 14, lines 25-28). Amsilk also teaches adding an aqueous solution of at least one base, for example a metal hydroxide and/or ammonia such as sodium hydroxide (NaOH), potassium hydroxide (KOH) and/or calcium hydroxide (CaOH) (see Amsilk, pg. 16, lines 10-12); with a pH of between 7 and 14, more preferably of between 9 and 13, and most preferably of between 10 and 12 (see Amsilk, pg. 23, lines 4-6). Spiber teaches that the presence of the NT fragment (i.e., protein fragment having from 100 to 160 amino acid residues, derived from a spider silk protein) improves solubility of the spider silk protein at a pH of 6.4 or higher and/or an ion composition that prevents polymerization of the spider silk protein (see Spiber, pg. 16, para[0085]). Therefore, as long as the manipulative step is met, i.e., incubating said portion of said cell culture in an aqueous solution under alkaline conditions, will necessarily result in solubilizing the recombinant silk protein. Additionally, as discussed in the “Claim Interpretation” section above, the term “alkaline conditions” is interpreted as only requiring a pH above 7.0 in order to solubilize a recombinant silk protein. The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether or not the isolated recombinant spider silk protein sample comprises at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% full-length recombinant spider silk protein as compared to total isolated recombinant spider silk protein. The cited art taken as a whole demonstrates a reasonable probability that the isolated silk constructs are either identical or sufficiently similar to the claimed isolated recombinant spider silk protein sample comprising at least 30% full-length recombinant spider silk protein as compared to total isolated recombinant spider silk protein and that whatever differences exists are not patentably significant. Therefore, with the showing of the reference, the burden of establishing non-obviousness by objective evidence is shifted to the Applicants.
Regarding claim 8, Amsilk teaches that preferably, the insoluble target protein or insoluble target protein aggregate isolated/separated with the method has a purity of at least 50% or 60%, more preferably of at least 70% or 80%, and most preferably of at least 90%, 95%, or even of 100%, e.g. of at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84,85, 86, 87,88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9%, or 100% (see Amsilk, pg. 20, lines 3-8). Additionally, Amsilk’s claim 3, teaches that the isolated insoluble target protein has a purity of at least 80%, preferably of at least 90%, and more preferably of at least 95% (Amsilk, pg. 66, line 19-20), thereby constituting where the purity of the isolated recombinant spider silk protein is between 50% and 100% as recited in instant claim 8.
Regarding claim 9, Amsilk teaches that the addition of a denaturing agent, a kosmotropic agent, and/or detergent prior to step b), in step b) and/or subsequent to step b) can enhance the purity of the isolated insoluble target protein about up to 5 to 20%, e.g. up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% and/or the yield of the isolated insoluble target protein about up to 5 to 20%, e.g. up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%. For example, an isolated insoluble target protein having after the separation step c) a purity of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95% without the addition of a denaturing agent, a kosmotropic agent and/or a detergent prior to step b), in step b) and/or subsequent to step b), may have a purity of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% after the addition of a denaturing agent, a kosmotropic agent, and/or detergent prior to step b), in step b) and/or subsequent to step b) (see Amsilk, pg. 25, lines 10-21). Preferably, (i) the denaturing agent is urea (CH4N2O) or guanidinium hydrochloride (CH5N3 HCl), (ii) the kosmotropic agent is a phosphate salt or a sulfate salt, or (iii) the detergent is Tween20, Tween 60, Tween 80, TritonX-15, Triton X-45, Triton X-100, Triton X-102, ,Triton X-114, Triton X-151, TritonX-165, Triton X-200, Triton X-207, Triton X-301, Triton X-305, Triton X-405, Triton X-705, SDS, or Brij (see Amsilk, pg. 25, lines 22-26). The person skilled in the art is able to select further suitable denaturing agents, kosmotropic agents or detergents (see Amsilk, pg. 25, lines 26-27), thereby constituting where the yield of the isolated recombinant spider silk protein is at least between 85% to 100% as compared to recombinant spider silk isolated by urea as recited in instant claim 9.
Regarding claim 11, Spiber’s method of producing polymers of an isolated spider silk protein involves adjusting the properties of the liquid medium to a pH of 6.3 or lower and an ion composition that allows polymerization of the spider silk protein (see Spiber, Abstract); thereby constituting wherein altering said alkaline conditions comprises adjusting the alkaline pH of the portion of the cell culture to a lowered pH from 6 to 7, as recited in instant claim 11. Additionally, MPEP 2144.05(I) states that "[i]n the case where the claimed ranges "overlap or lie inside ranges discloses by the prior art" a prima facie case of obviousness exists. Therefore, the claimed pH range would have been obvious to one of ordinary skill in the art since the prior art pH (i.e., 6.3 or lower) lies within the claimed pH range (i.e., pH from 4 to 10 or a pH from 6 to 7).
Regarding claim 14, Spiber teaches that if the liquid medium wherein the spider silk protein is dissolved has a pH of 6.4 or higher, the pH is decreased to 6.3 or lower (see Spiber, pg. 16, para[0084]). The skilled person is well aware of various ways of achieving this, typically involving addition of a strong or weak acid (see Spiber, pg. 16, para[0084]); thereby constituting to wherein adjusting the alkaline pH comprises adding an acid to the aqueous solution, as recited in instant claim 14. If the liquid medium wherein the spider silk protein is dissolved has an ion composition that prevents polymerization, the ion composition is changed so as to allow polymerization (see Spiber, pg. 16, para[0084]). If required, this step involves both decreasing the pH of the liquid medium to 6.3 or lower and changing the ion composition so as to allow polymerization; however, it is preferred that the pH of the liquid medium is adjusted to 6.2 or lower, such as 6.0 or lower (see Spiber, pg. 16, para[0084]).
Regarding claim 16, Amsilk teaches that The term "a suspension of intact host cells" encompasses intact, generally non-lysed host cells, e.g. microbial host cells, plant host cells or insect host cells, suspended in a fluid, e.g. in an aqueous solution (e.g. a buffered aqueous solution), technical water, deionized water, culture medium or fermentation medium (see Amsilk, pg. 9, lines 8-11), and thus, constituting wherein the portion of the cell culture comprises a supernatant, a whole cell broth, or a cell pellet as recited in instant claim 16.
Regarding claim 17, Amsilk et al. teaches that the amount of soluble host cell parts and/or of other soluble ingredients is lower (i) in a cell sediment of intact or disrupted host cells having a moisture content of between 5 to 10%, produced by separating the intact or disrupted host cells from the culture medium or fermentation medium via centrifugation or filtration, or (ii) in an aqueous solution, wherein said cell s