The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 July 16, 2026, has been entered.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claims 4, 8, 10-11, 13, 16-17, 20-21, 24, 28, 31 are canceled. Claims 25-27, 29-30 are withdrawn. Claims 1-3, 5-7, 9, 12, 14, 15, 18-19, 22-23, 32 are under consideration.
Priority: This application is a 371 of PCT/SG2021/050205, filed April 13, 2021, which claims benefit to foreign application SG 10202003536S. A copy of the foreign priority document has been received in the instant application on March 16, 2023, and is in the English language.
Objections and Rejections
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 1-3, 5-7, 9, 12, 14, 15, 18-19, 22-23, 32 are rejected under 35 U.S.C. 103 as being unpatentable over Shuai (2014 Human Hair Keratin Hydrogel: Fabrication, Characterization and Application Thesis, 138 pages; previously cited), Bowden et al. (1984 Eur J Biochem 142: 29-36; previously cited) and in view of the combined teachings of G-Biosciences (G-Biosciences - The Protein Man’s Blog | A Discussion of Protein Research: Dialysis – Knot the Way to Do It, posted on April 17, 2015, 5 pages) and Eichner et al. (1986 The Journal of Cell Biology 102: 1767-1777) on dialysis.
Shuai discloses a method for forming a keratin-based gel network comprising adding a salt CaCl2 or reducing pH to about 4 using citrate buffer/PBS to a soluble keratin solution (at least p. 61, 66-70, 91). Shuai discloses lyophilized keratin gels (at least p. 68-69). Shuai discloses extracting keratin to obtain the keratin solution for inducing keratin gel formation (at least p. 47-48). Shuai discloses methods for extracting keratin including a dialysis step, which removes unwanted chemicals, to obtain a keratin solution (at least p. 10, 11-12, 35). Shuai discloses that the overall procedure of all keratin extraction methods is similar; they all involve pretreatment, incubation with chemical treatments, filtration and dialysis (at least p. 10). Shuai discloses keratin solutions can be used directly for downstream processes or lyophilized into powder (at least p. 10). Therefore, Shuai reasonably discloses a method for forming a keratin filament network, comprising dialyzing a solubilized keratin solution in a buffer solution to obtain a purified keratin solution, mixing the purified keratin solution with a salt in an acidic buffer solution, and drying the acidic keratin solution, and forming a keratin filament network. Shuai does not teach dialyzing at a pH of about 2.5 to about 5.5 by stepwise dialysis.
Bowden et al. disclose that keratin can also be extracted by soaking the keratin protein source in Tris/HCl, pH 7.5, containing urea and mercaptoethanol (Tum buffer) for 16-24 h at 6º C; homogenized, sonicated, centrifuged, and then where the supernatant is dialyzed against citrate buffer (pH 2.65; citric acid and trisodium citrate) to obtain a keratin solution (p. 30) or against Tris/HCl pH 7.5 (at least p. 30). Bowden et al. disclose that keratins purified from Tum buffer extracts, by dialysis against citrate buffer, had identical two-dimensional profiles to those prepared by dialysis against low-ionic-strength neutral Tris, which conditions filament reassembly takes place (at least p. 32). Bowden et al. do not explicitly teach stepwise dialysis of the keratin against the citrate buffer pH 2.65 (acidic solution).
As already noted above, it is known that the purpose of dialysis is to remove unwanted chemicals (Shuai at least p. 10). G-Biosciences also discloses that dialysis is performed to ensure that all unwanted small molecular weight substances (such as reducing agents, nonreacted crosslinking or labeling reagents, and preservatives) are removed (at least p. 1). G-Biosciences discloses that for best results, perform a serial dialysis using buffers with decreasing concentration of solutes (p. 2). For instance, Eichner et al. disclose that keratin dialysis can be performed by stepwise dialysis against buffer containing decreasing concentrations of urea (denaturing agent) (p. 1768).
“[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.
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 forming a keratin filament network, comprising stepwise dialysis of a solubilized keratin solution in a buffer solution at a pH 2.5 to about 5.5 comprising citric acid and decreasing concentrations of urea (a denaturing agent), to obtain a purified keratin solution, mixing the purified keratin solution with a salt in an acidic buffer solution to form a keratin-based gel network, and drying the acidic keratin solution, to obtain a keratin filament network (instant claims 1, 32). The motivation to do so is given by the prior art. Shuai discloses a method for forming a keratin filament network, comprising dialyzing a solubilized keratin solution in a buffer solution to obtain a purified keratin solution, mixing the purified keratin solution with a salt in an acidic buffer solution, and drying the acidic keratin solution, and forming a keratin filament network. Bowden et al. disclose that a keratin solution can also be obtained by dialyzing in a citrate buffer at low pH ~2.65. Bowden et al. disclose subjecting keratin to denaturing and reducing agents (urea and mercaptoethanol) before dialysis with citrate buffer pH 2.65 (p. 30). It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Therefore, one of ordinary skill would have reasonable motivation to incorporate the acidic dialyzing step of Bowden et al. for the keratin extraction or dialyzing step for obtaining the keratin solution that is mixed with the acidic salt buffer solution in the method of Shuai for forming a keratin filament network, where the acidic dialysis is performed by stepwise dialysis against the acidic citrate in Bowden et al. comprising decreasing concentrations of a denaturing agent (urea) because alternative keratin extraction and dialyzing steps are known in the prior art and there is interest in forming materials comprising a keratin-based network. One of ordinary skill would have a reasonable expectation of success because method steps for extracting keratin and forming a keratin-based material are known as disclosed in the prior art.
Regarding instant claims 1, 3, 5, Bowden et al. disclose subjecting keratin to denaturing and reducing agents (urea and mercaptoethanol) before dialysis with citrate buffer pH 2.65 (p. 30). Shuai also discloses that denaturing agents (urea) can be incorporated into the extraction methods for keratin (p. 11-12). It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Therefore, it would have been obvious to include a denaturing agent, such as urea, into the citrate buffer pH 2.65 for stepwise dialysis of the keratin solution because its incorporation into the citrate buffer pH 2.65 will have the same purpose of removing urea (unwanted chemicals) and producing a keratin solution.
Regarding instant claims 2, 14-15, 32, Shuai discloses keratin gelation (or formation of a keratin network) is induced by calcium salt or PBS/citrate buffer pH 3 (at least p. 61, 66-70, 91). Therefore, Shuai can be deemed to disclose self-assembly of a keratin filament network when mixing the keratin solution with PBS/citrate buffer pH 3.
Regarding instant claim 6, Bowden et al. disclose a urea concentration of 8 M (p. 30) and Shuai also discloses urea concentrations including 8 M (p. 11-12).
Regarding instant claim 7, it is known that “where 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 Bowden et al. disclose extracting keratin with a denaturing agent (16-24 h) and then dialyzing in a citrate buffer pH 2.65 (p. 30). It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Eichner et al. disclose stepwise dialysis against decreasing concentrations of the denaturing agent (urea) for 2-3 hrs. for each reduced concentration (p. 1768). Therefore, it would have been obvious to arrive at a dialyzing time of at least two hours to remove residual molecules, including the denaturing agents, by routine optimization.
Regarding instant claim 9, the prior art Bowden et al. disclose extracting keratin with a denaturing agent (16-24 h) where the urea is 8M and then dialyzing in a citrate buffer pH 2.65 (p. 30). As noted above, it would have been obvious to include a denaturing agent, such as urea, into the citrate buffer pH 2.65 for stepwise dialysis of the keratin solution because its incorporation into the citrate buffer pH 2.65 will have the same purpose of removing urea (unwanted chemicals) and producing a keratin solution. It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Eichner et al. disclose stepwise dialysis against decreasing concentrations of the denaturing agent (urea) for 2-3 hrs. for each reduced concentration, 6.5 M, 4.5 M, 3.5 M, 0 M (p. 1768). MPEP 2144.04 notes that duplicating and/or rearranging parts is obvious. It is further known that “where 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. It would have been further obvious to perform the stepwise dialysis by reducing concentrations of the urea by about half the concentration by routine optimization because repeating the dialysis step (stepwise dialysis) and reducing the concentration of the urea in each subsequent dialysis step will still result in producing a keratin solution.
Regarding instant claims 3, 12, Bowden et al. disclose subjecting keratin to denaturing and reducing agents (urea and mercaptoethanol) before dialysis with citrate buffer pH 2.65 (p. 30), at 2% mercaptoethanol (p. 30). Shuai also discloses that denaturing agents (guanidine) and reducing agent (DTT, Na2S) can be incorporated into the extraction methods for keratin (p. 11-12). It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Eichner et al. disclose stepwise dialysis against decreasing concentrations of the denaturing agent (urea) for 2-3 hrs. for each reduced concentration, 6.5 M, 4.5 M, 3.5 M, 0 M , and 1 mM DTT (p. 1768). Therefore, it would have been obvious to include a reducing agent, such as mercaptoethanol or DTT, into the citrate buffer pH 2.65 for dialysis of the keratin solution because its incorporation into the citrate buffer pH 2.65 will have the same purpose of obtaining a keratin solution.
Regarding instant claim 18, Shuai discloses adding the keratin solution to PBS/citrate buffer pH 3 for inducing network formation and monitoring at time intervals from 0 h to 8 hr (p. 26-27).
Regarding instant claim 19, Shuai discloses keratin solutions, including 3 ml of 15 mg/ml, mixed with the PBS/citrate buffer pH 3 for inducing network formation (at least p. 26).
Regarding instant claim 22, Shuai also discloses that denaturing agents (guanidine) and reducing agent (DTT, Na2S) can be incorporated into the extraction methods for keratin before dialysis (p. 11-12). Bowden et al. also disclose subjecting keratin to denaturing and reducing agents (urea and mercaptoethanol) before dialysis with citrate buffer pH 2.65 (p. 30). Therefore, it would have been obvious to include an extraction step before the dialysis step in the method for forming a keratin network of Shuai in view of Bowden et al. noted above.
Regarding instant claim 23, Shuai discloses that a source of keratin protein is hair (at least p. 91).
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining the 103 rejection are the same as previously noted and are incorporated herein.
Applicants assert that amended claim 1 is directed to a more specific step-down denaturing agent dialysis sequence that yields purified keratin which subsequently self-assembles into a keratin filament upon mixing with a salt in acidic buffer. Applicants assert that Shuai does not dialyze against an acidic buffer at all. Applicants assert that Shuai expressly dialyses the filtered keratin solution against deionized water (Shuai p. 21).
Applicants’ remarks are not persuasive. Instant claim 1 is amended to recite that the method comprises repeating the dialysis step (stepwise dialysis) against an acidic buffer solution, pH 2.5-5.5, comprising citric acid and a denaturing agent (i.e. urea) and reducing the concentration of the urea in each subsequent dialysis step.
As previously noted, Shuai discloses the method for forming a keratin-based gel network comprises adding a salt CaCl2 or reducing pH to about 4 using citrate buffer/PBS to a soluble keratin solution, obtained from dialysis (at least p. 61, 66-70, 91) and lyophilizing keratin gels (at least p. 68-69). Therefore, Shuai discloses the same steps recited in the instant claims 1(ii) and 1(iii) for forming a keratin filament network and therefore, Shuai can be deemed to disclose a keratin-based gel network.
Shuai discloses extracting keratin to obtain the keratin solution for inducing keratin gel formation (at least p. 47-48). Shuai discloses methods for extracting keratin including a dialysis step, which removes unwanted chemicals, to obtain a keratin solution (at least p. 10, 11-12, 35). Shuai discloses that the overall procedure of all keratin extraction methods is similar; they all involve pretreatment, incubation with chemical treatments, filtration and dialysis (at least p. 10).
Therefore, Shuai discloses performing dialysis on the keratin before inducing keratin gel formation. While Shuai does not explicitly disclose dialysis against an acidic buffer solution, this deficiency is remedied by Bowen et al., which disclose that extracted keratin can be dialyzed against citrate buffer (pH 2.65; citric acid and trisodium citrate) to obtain a keratin solution (p. 30) (see teachings of Bowen et al. above).
Regarding Applicants’ remarks that Shuai expressly dialyses the filtered keratin solution against deionized water, the remarks are not persuasive. As previously noted, while Shuai discloses that keratin can be dialyzed against deionized water, at the time of the invention, alternative keratin extraction and dialyzing steps were known in the prior art, including citrate buffer dialysis at pH 2.65 as disclosed in Bowden et al. Bowden et al. disclose that keratin can also be extracted by soaking the keratin protein source in Tris/HCl, pH 7.5, containing urea and mercaptoethanol (Tum buffer) for 16-24 h at 6º C; homogenized, sonicated, centrifuged, and then where the supernatant is dialyzed against citrate buffer (pH 2.65; citric acid and trisodium citrate) to obtain a keratin solution (p. 30).
Further, Shuai is cited with new reference G-Biosciences, which discloses to avoid dialysis against pure deionized water since this will cause membrane rupture (at least p. 2).
Bowden et al. disclose that keratins purified from Tum buffer extracts, by dialysis against citrate buffer, had identical two-dimensional profiles to those prepared by dialysis against low-ionic-strength neutral Tris, which conditions filament reassembly takes place (at least p. 32).
Therefore, the prior art reasonably discloses that keratin can be dialyzed against an acidic buffer solution pH 2.65 as suggested in Bowen et al. prior to further downstream processing or utilization.
Applicants assert that the citrate buffer in Bowden et al. contains no denaturing agent and that dialysis in Bowden et al. is performed for the very purpose of removing the denaturant (i.e. urea) introduced during extraction.
Applicants’ remarks are not persuasive. Bowden et al. is cited with new references G-Biosciences and Eichner et al., which reasonably disclose stepwise dialysis of the protein against buffer comprising decreasing concentrations of the unwanted chemical (i.e. denaturant) is an obvious alternative to one-step dialysis.
Bowden et al. disclose dialysis (or one-step dialysis) against citrate buffer pH 2.65 of the solubilized keratin solution extracted by soaking the keratin protein source in Tris/HCl, pH 7.5, containing urea and mercaptoethanol (Tum buffer) (p. 30).
As already noted above, it is known that the purpose of dialysis is to remove unwanted chemicals (Shuai at least p. 10). G-Biosciences also discloses that dialysis is performed to ensure that all unwanted small molecular weight substances (such as reducing agents, nonreacted crosslinking or labeling reagents, and preservatives) are removed (at least p. 1). G-Biosciences discloses that for best results, perform a serial dialysis using buffers with decreasing concentration of solutes (p. 2). For instance, Eichner et al. disclose that keratin dialysis can be performed by stepwise dialysis against buffer containing decreasing concentrations of urea (denaturing agent) (p. 1768).
Therefore, one of ordinary skill would have reasonable motivation to incorporate the acidic dialyzing step of Bowden et al. for the keratin extraction or dialyzing step for obtaining the keratin solution that is mixed with the acidic salt buffer solution in the method of Shuai for forming a keratin filament network, where the acidic dialysis is performed by repeating the dialysis step (stepwise dialysis) and reducing the concentration of the urea in each subsequent dialysis step because alternative keratin extraction and dialyzing steps are known in the prior art and there is interest in forming materials comprising a keratin-based network. One of ordinary skill would have a reasonable expectation of success because method steps for extracting keratin and forming a keratin-based material are known as disclosed in the prior art.
Regarding Applicants’ remarks that Bowden et al.’s citrate dialysis is followed by isolation through isoelectric precipitation and analytical characterization, not by salt-induced self-assembly or formation of any keratin filament network, the remarks are not persuasive. In this instance, Bowden et al. is cited with Shuai, which discloses that alternatively, after dialysis of keratin to remove unwanted chemicals, keratin protein can be utilized to form a keratin-based gel network by adding a salt CaCl2 or reducing pH to about 4 using citrate buffer/PBS to a soluble keratin solution, obtained from dialysis (Shuai at least p. 61, 66-70, 91).
Regarding Applicants’ remarks that neither Shuai nor Bowden et al. disclose a dialysis buffer that comprises both an acid and a denaturing agent, as required by amended claim 1, the remarks are not persuasive for the reasons already noted above.
Applicants assert that Shuai reinforces the same conclusion. Applicants assert that Shuai compares Na2S extraction with BME extraction and reports that proteins extracted using the BME method, which involved strong denaturing conditions due to urea and thiourea, tended to precipitate after dialysis, whereas proteins extracted using Na2S extraction remained soluble. Applicants assert that therefore, Shuai selected the Na2S method because protein solubility after dialysis was critical for downstream applications. Applicants assert that thus, Shuai would not have led a person of ordinary skill to introduce a denaturing agent into the dialysis buffer, much less to use repeated acidic dialysis steps with progressively decreasing denaturing agent concentrations to condition keratin for subsequent salt-induced self-assembly.
Applicants’ remarks are not persuasive. Shuai discloses that the overall procedure of all keratin extraction methods is similar; they all involve pretreatment, incubation with chemical treatments, filtration and dialysis (at least p. 10). While Shuai compares keratin extraction by Na2S and BME and selects Na2S as the keratin extraction method, it is submitted that the BME method reported in Shuai is not the extraction conditions disclosed in Bowden et al. Shuai discloses that the BME method contains concentrated urea and thiourea that denature keratins and resulting in denatured proteins that aggregate to form precipitates (Shuai p. 37). It is recognized in the prior art that keratin extraction with denaturing agents (urea, thiourea) do not necessarily lead to protein degradation (thereby forming precipitates). Fujii et al. disclose extracting keratins in Tris-HCl, thiourea, urea, DTT, etc. (which also contains denaturants urea and thiourea), but Fujii et al. disclose that no significant degradation of the protein components was observed (Fujii et al. p. 96).
As noted above, Bowden et al. disclose that keratin can also be extracted by soaking the keratin protein source in Tris/HCl, pH 7.5, containing urea and mercaptoethanol (Tum buffer) for 16-24 h at 6º C; homogenized, sonicated, centrifuged, and then where the supernatant is dialyzed against citrate buffer (pH 2.65; citric acid and trisodium citrate) to obtain a keratin solution (p. 30) or against Tris/HCl pH 7.5 (at least p. 30). Bowden et al. disclose that keratins purified from Tum buffer extracts, by dialysis against citrate buffer, had identical two-dimensional profiles to those prepared by dialysis against low-ionic-strength neutral Tris, which conditions filament reassembly takes place (at least p. 32).
It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.).
Therefore, Applicants’ remarks that Shuai would not have led a person of ordinary skill to introduce a denaturing agent into the dialysis buffer are not persuasive. In this instance, the prior art reasonably discloses that keratin can be successfully extracted by solutions comprising denaturants, including urea; dialyzed in acidic buffer solution (Bowden et al.) by repeating the dialysis step (stepwise dialysis) and reducing the concentration of the urea in each subsequent dialysis step will still result in producing a keratin solution (G-Biosciences; Eichner et al.).
Applicants assert that the office previously reasoned (with respect to claims 3 and 5) that it would have been obvious to add urea to Bowden's citrate dialysis buffer because urea's incorporation "will have the same purpose of producing a keratin solution" (Final Office Action, p. 5). Applicants assert that respectfully, that reasoning is contrary to Bowden's own protocol, in which the citrate dialysis step functions to remove the denaturant. A person of ordinary skill following Bowden would not re-introduce the denaturant into the dialysate, because doing so would defeat the purifying function that Bowden's dialysis step performs.
Applicants’ remarks are not persuasive. As noted above, Bowden et al. disclose dialysis (or one-step dialysis) against citrate buffer pH 2.65 of the solubilized keratin solution extracted by soaking the keratin protein source in Tris/HCl, pH 7.5, containing urea and mercaptoethanol (Tum buffer) (p. 30).
As also noted above, it is known that the purpose of dialysis is to remove unwanted chemicals (Shuai at least p. 10). G-Biosciences also discloses that dialysis is performed to ensure that all unwanted small molecular weight substances (such as reducing agents, nonreacted crosslinking or labeling reagents, and preservatives) are removed (at least p. 1). G-Biosciences discloses that for best results, perform a serial dialysis using buffers with decreasing concentration of solutes (p. 2). For instance, Eichner et al. disclose that keratin dialysis can be performed by stepwise dialysis against buffer containing decreasing concentrations of urea (denaturing agent) (p. 1768).
Therefore, it would be obvious that the acidic dialysis in Bowden et al. can be performed by repeating the dialysis step (stepwise dialysis) and reducing the concentration of the urea in each subsequent dialysis step because stepwise dialysis is a known alternative to single step dialysis for achieving the same purpose of obtaining a protein (keratin) solution with no unwanted chemicals.
Therefore, Applicants’ remarks that a person of ordinary skill following Bowden would not re-introduce the denaturant into the dialysate, because doing so would defeat the purifying function that Bowden's dialysis step performs are not persuasive.
Applicants assert that MPEP 2144.05 does not apply here because the general conditions are not disclosed in the cited art. Applicants assert that new step (i-i) requires repeating the acidic dialysis more than once, wherein the denaturing-agent concentration is progressively lower in each successive repetition. Applicants assert that neither Shuai nor Bowden et al. disclose repeating a dialysis step at all, much less repeating it with a stepwise reduction of a denaturing agent that is present in the dialysate.
Applicants’ remarks are not persuasive. The repeating dialysis step (i-i) is essentially performing stepwise dialysis, which is recognized as an alternative to single step dialysis and for achieving the same purpose of removing unwanted chemicals or small substances from the protein solution. As noted above, G-Biosciences also discloses that dialysis is performed to ensure that all unwanted small molecular weight substances (such as reducing agents, nonreacted crosslinking or labeling reagents, and preservatives) are removed (at least p. 1). G-Biosciences discloses that for best results, perform a serial dialysis using buffers with decreasing concentration of solutes (p. 2). For instance, Eichner et al. disclose that keratin dialysis can be performed by stepwise dialysis against buffer containing decreasing concentrations of urea (denaturing agent) (p. 1768).
As already noted, Bowden et al. disclose subjecting keratin to denaturing and reducing agents (urea and mercaptoethanol) before dialysis with citrate buffer pH 2.65 (p. 30). Shuai also discloses that denaturing agents (urea) can be incorporated into the extraction methods for keratin (p. 11-12). It is disclosed that dialysis to remove unwanted chemicals or unwanted small molecular weight substances can alternatively be achieved by stepwise dialysis against the buffer, where the buffer contains decreasing concentrations of the unwanted chemical/solute, i.e. urea (G-Biosciences; Eichner et al.). Therefore, it would have been obvious to include a denaturing agent, such as urea, into the citrate buffer pH 2.65 for stepwise dialysis of the keratin solution because its incorporation into the citrate buffer pH 2.65 will have the same purpose of removing urea (unwanted chemicals) and producing a keratin solution. It would be obvious because stepwise dialysis with decreasing concentrations of the solute (urea) is a known and recognized technique in protein dialysis to remove unwanted solutes and the conditions for alternative keratin extraction and dialyzing steps are known in the prior art.
Applicants assert that moreover, MPEP § 2144.04 (duplication or rearrangement of parts) applies only where the duplication yields the same result the prior art already achieved. Applicants assert that the claimed step-down sequence is not the mere duplication of a dialysis step to achieve the same "keratin extraction"; it is a directional, ordered conditioning of the keratin that enables subsequent salt-induced self- assembly into a filament network - an outcome neither reference achieves or contemplates.
Applicants’ remarks are not persuasive. As noted above, the repeating dialysis step (i-i) is essentially performing stepwise dialysis, which is recognized as an alternative to single step dialysis and for achieving the same purpose of removing unwanted chemicals or small substances from the protein solution.
The advantages of stepwise dialysis are already noted above.
As already noted, Shuai discloses forming a keratin-based gel network comprises adding a salt CaCl2 or reducing pH to about 4 using citrate buffer/PBS to a soluble keratin solution, obtained from dialysis (at least p. 61, 66-70, 91) and lyophilizing keratin gels (at least p. 68-69). Therefore, Shuai discloses the same steps recited in the instant claims 1(ii) and 1(iii) for forming a keratin filament network and therefore, Shuai can be deemed to disclose a keratin-based gel network.
Therefore, the prior art (Shuai) also already recognize that dialyzed keratin solutions can be utilized to form a keratin-based gel network.
Therefore, Applicants’ remarks that the claimed step-down sequence is not the mere duplication of a dialysis step to achieve the same "keratin extraction" are not persuasive because the claimed repeating dialysis step (i-i) stepwise dialysis, which is recognized as an alternative to single step dialysis and for achieving the same purpose of removing unwanted chemicals or small substances from the protein solution.
Applicants assert again that Shuai leads one away from denaturing conditions in the dialysis workflow, and there is no reasonable expectation of success. Applicants assert that Shuai expressly compared two extraction chemistries and selected sodium sulfide (Na2S) over beta-mercaptoethanol (BME) precisely because of post-dialysis solubility.
Applicants’ remarks are not persuasive for the reasons already noted above. Specifically, the BME method reported in Shuai is not the extraction conditions disclosed in Bowden et al. Further, it is recognized in the prior art that keratin extraction with denaturing agents (urea, thiourea) do not necessarily lead to protein degradation (thereby forming precipitates). Fujii et al. disclose extracting keratins in Tris-HCl, thiourea, urea, DTT, etc. (which also contains denaturants urea and thiourea), but Fujii et al. disclose that no significant degradation of the protein components was observed (Fujii et al. p. 96).
Therefore, Applicants’ remarks that Shuai teaches away from denaturing conditions are not persuasive.
Again, regarding Applicants’ remarks that one of ordinary skill would not replace the deionized water dialysis of Shuai with a repeated acidic dialysis, the remarks are not persuasive. As noted above, Shuai is cited with new reference G-Biosciences, which discloses to avoid dialysis against pure deionized water since this will cause membrane rupture (at least p. 2).
Bowden et al. disclose that keratins purified from Tum buffer extracts, by dialysis against citrate buffer, had identical two-dimensional profiles to those prepared by dialysis against low-ionic-strength neutral Tris, which conditions filament reassembly takes place (at least p. 32).
Therefore, the prior art reasonably discloses that keratin can be dialyzed against an acidic buffer solution pH 2.65 as suggested in Bowen et al. prior to further downstream processing or utilization.
MPEP 2141 notes that examples of rationales that may support a conclusion of obviousness include among others: combining prior art elements according to known methods to yield predictable results. Therefore, it would have been obvious that conditions for extracting keratin known in the prior art, including the citrate buffer dialysis at pH 2.65 of Bowden et al. can be incorporated for the keratin extraction or dialyzing step for obtaining the keratin solution that is mixed with the acidic salt buffer solution in the method of Shuai for forming a keratin filament network for the keratin extraction or dialyzing step for obtaining the keratin solution that is mixed with the acidic salt buffer solution in the method of Shuai for forming a keratin filament network, where the acidic dialysis is performed by repeating the dialysis step (stepwise dialysis) and reducing the concentration of the urea in each subsequent dialysis step because alternative keratin extraction and dialyzing steps are known in the prior art.
Applicants then assert that Shuai teaches away from dialysis at acidic pH. Applicants assert that Shuai explicitly discloses that keratin undergoes flocculation at lowered pH (Shuai p. 6-7, 63-64). Applicants assert that Shuai further discloses that the gelation process of keratin hydrogel formation involves two stages: keratin flocculation followed by gelation with stabilized keratin flocs.
Applicants’ remarks are not persuasive. In this instance, Shuai expressly discloses that the flocculation (or gelation process) is triggered by mixing keratin solution with PBS/citrate buffer at acidic pH (at least p. 26). Therefore, Shuai discloses that PBS (which contains a salt) is present with the citrate buffer to trigger gelation. Therefore, Shuai does not teach away from dialysis at an acidic pH 2.65 as disclosed in Bowden et al. during keratin extraction.
Applicants assert that Shuai is directed to developing keratin-based hydrogels as cell culture substrates for tissue engineering applications. Applicants assert that Bowden et al. is directed to investigating proteolytic modifications of acidic and basic keratins during terminal differentiation of mouse and human epidermis, not a method of forming any keratin-based material or network.
Applicants’ remarks are not persuasive. In this instance, both the Shuai and Bowden et al. references disclose keratin extraction or purification is needed in order to arrive at a keratin protein for further processing. While Shuai discloses the keratin after extraction is for gel formation and Bowden et al. disclose that the keratin after extraction is for further studies, both Shuai and Bowden et al. disclose the same understanding that keratin extraction is needed in order to obtain keratin protein for further utilization. Therefore, Applicants’ remarks that Shuai and Bowden et al. are directed to different technical problems in different fields are not persuasive. In this instance, Shuai and Bowden et al. disclose the same solution of first extracting keratin to obtain keratin protein.
Regarding Applicants’ remarks that the distinguishing feature of claim 1 is the ordered step-down denaturing-agent dialysis of steps (i) and (i-i) that produces keratin for self-assembly into a filament network, the remarks are not persuasive. As already noted, Applicants’ step-down denaturing-agent dialysis is essentially performing stepwise dialysis, which is recognized as an alternative to single step dialysis and for achieving the same purpose of removing unwanted chemicals or small substances from the protein solution.
Regarding Applicants’ remarks that Bowden et al. fail to disclose a keratin filament network, the remarks are not persuasive. The deficiency of Bowden et al. to not teach a keratin filament network is remedied by the teachings of Shuai noted above.
Applicants assert that there is no reasonable expectation of success.
Applicants’ remarks are not persuasive. Applicants’ step-down denaturing-agent dialysis is essentially performing stepwise dialysis, which is recognized as an alternative to single step dialysis and for achieving the same purpose of removing unwanted chemicals or small substances from the protein solution. Therefore, one of ordinary skill would have a reasonable expectation of success that stepwise dialysis of the keratin in acidic dialysis solution of Bowden et al. would still yield a keratin protein for further utilization, including for forming a keratin-gel filament network as disclosed in Shuai since stepwise dialysis, like one-step dialysis, removes unwanted chemicals and solutes to generate a keratin protein solution.
Applicants assert that the claimed “keratin filament network” is structurally distinct from the prior art disclosures. Applicants assert that the specification at page 3 lines 15-18 discloses that as used herein, “keratin filament network” refers to a network of assembled KIFPs. The network may be made up of interconnected KIFPs, etc. Applicants assert that this is fundamentally distinct and different from what Shuai teaches/discloses. Applicants assert that Shuai is directed to forming a hydrogel.
Applicants’ remarks are not persuasive. Instant claim 1 recites that the keratin filament network is formed by mixing the purified keratin (obtained from the dialysis of step i) with a salt in acidic buffer and drying the solution to form the keratin filament network (steps ii and iii).
Shuai discloses the method for forming a keratin-based gel network comprises adding a salt CaCl2 or reducing pH to about 4 using citrate buffer/PBS to a soluble keratin solution, obtained from dialysis (at least p. 61, 66-70, 91) and lyophilizing keratin gels (at least p. 68-69). Therefore, Shuai discloses the same steps recited in the instant claims 1(ii) and 1(iii) for forming a keratin filament network and therefore, Shuai can be deemed to disclose a keratin-based gel network. It is not clear how a keratin gel disclosed in Shuai would not be considered a “keratin filament network” since it comprises keratin proteins in a network which are the same proteins recited in the instant claim.
In this instance, the conditions for extracting and dialyzing keratin proteins and forming a keratin network are known and disclosed in the teachings of the prior art noted above. It was further disclosed that keratin extracted with a denaturant can be dialyzed against an acidic buffer solution, where it is known that dialysis can be performed in a stepwise manner comprising repeating the dialysis step with decreasing concentrations of the denaturing agent (urea), to obtain a keratin protein, that can be further induced to from a keratin-gel filament network.
Regarding Applicants’ remarks on the dependent claims, the remarks are not persuasive for the reasons noted above and in the 103 rejection.
See also the reasons previously noted for Shuai and Bowden et al. on at least p. 7-13 of the April 16, 2026, final office action.
For at least these reasons, the 103 rejection is maintained.
No claim is allowed.
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/Marsha Tsay/Primary Examiner, Art Unit 1656
