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 .
Response to Amendments
Applicant's amendments to claims 1, 3, 6 and 19, filed 9/25/2025, have been entered. Claims 2 and 18 have been canceled. Claims 1, 3, 6, 12, 16, 19-22, 25-28, 31, 36-40, 42, 45, 49-52, and 54 remain pending and are being considered on their merits. Any rejections of record not particularly addressed below are withdrawn in light of the claim amendments and applicant’s comments.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1, 3, 6, 12, 16, 19-22, 37-39, 42, 45, 49, 52 and 54 remain rejected under 35 U.S.C. 103 as being unpatentable over Akshita (2017, Doctoral thesis, Nanyang Technological University, Singapore) as evidenced by Microfluidics (Application Note) and in view of Widmaier et al (U.S.PGPUB 20160222174).
Akshita teaches suckerin proteins share sequence homology to silk proteins, and specifically that the block copolymer primary sequences of the suckerin family are similar to the repetitive spider silk sequences (see abstract and sections 2.5 & 5.2.2).
Regarding claims 1, 6, 12, 16, 37 and 42, Akshita teaches a method for isolating a recombinant suckerin protein from a host cell that is a culture of E. coli expressing the recombinant protein, said method comprising providing a cell pellet (reads on an insoluble mass) comprising a recombinant suckerin protein, adding the cell pellet (reads on an insoluble mass) to an aqueous solution comprising a solvent, applying a shear force via microfluidization with a M110P Microfluidizer at 25,000 psi for up to six passes to the aqueous solution comprising the insoluble mass thereby solubilizing the recombinant suckerin protein in the aqueous solution, and isolating the recombinant protein from the solution (see section 3.2). Regarding claim 3, is cited solely as evidence that the M110P Microfluidizer inherently can produce high shear rates within the range from 6*10-6 s-1 to 10*106 s-1.
Regarding claims 1, 19 and 21-22 Akshita teaches including the chaotropic agent urea helps with the removal of contaminants such as bacterial membrane proteins that are non-specifically adsorbed onto the hydrophobic inclusion bodies during processing, and Akshita teaches including both 2 M and 8 M urea (reads on about 10 M) (see sections3.2 and 4.2.2.2).
Akshita does not teach that the recombinant protein is a recombinant spider silk protein (claims 1, 38-39, 45, 49, 52 and 54), or the percent of the insoluble mass that is added to the aqueous solution (claim 20).
Regarding claims 1, 49, 52 and 54, Widmaier is drawn to methods and compositions directed to synthetic block copolymer proteins, specifically spider silk protein, and expression of recombinant spider silk protein constructs in E. coli for their production, and purification of proteins spider silk proteins (see paragraphs 3-12 and 65-71). Regarding claims 1, 38-39, 45, 52 and 54, Widmaier teaches that said proteins have crystalline and amorphous regions, and Widmaier teaches the recombinant spider silk protein comprising SEQ ID NO 1249 is specifically useful (see paragraphs 4 & 61 and SEQ ID NO 1249); SEQ ID NO 1249 is identical to SEQ ID NO 23 of the instant specification, and the protein produced by this sequence reads on a “full-length recombinant” spider protein since the entire recombinant sequence is expressed. Regarding claim 49, Widmaier teaches the spider silk protein can be purified, and that it is useful for the commercial production of silk-like fibers (see abstract and paragraphs 88-90); reads on a purity of any level.
It would have been obvious to combine Akshita and Widmaier to purify Widmaier’s spider silk protein in Akshita’s method. A person of ordinary skill in the art would have had a reasonable expectation of success in purifying Widmaier’s spider silk protein in Akshita’s method because Akshita teaches the method is useful for purifying proteins that share sequence homology to silk proteins, and specifically that the repetitive spider silk protein sequences, and therefore the structure and properties of the proteins are similar. The skilled artisan would have been motivated to purify Widmaier’s spider silk protein in Akshita’s method because recombinant spider silk protein comprising SEQ ID NO 1249 is specifically useful for the commercial production of silk-like fibers.
Regarding the percent of the insoluble mass that is added to the aqueous solution, this amount is result effective as Akshita teaches that the aqueous solutions are used to remove contaminants such as bacterial membrane proteins, and therefore it is obvious to adjust the amount of solutioned added to achieve the desired level of purification of the target protein from said contaminates.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill at the time the invention was made.
Claims 25-28, 31, 36, 40 and 50-51 remain rejected under 35 U.S.C. 103 as being unpatentable over Akshita as evidenced by Microfluidics and in view of Widmaier as applied to claims 1, 3, 6, 12, 16, 19-22, 37-39, 42, 45, 49, 52 and 54 above, and further in view of Klimov et al (U.S.PGPUB 20120085262) and Will et al (U.S.PGPUB 20170327815).
The teachings of Akshita and Widmaier are discussed and relied upon above. Additionally, regarding claim 36, Akshita teaches the purification method includes multiple incubation steps with the chaotropic agent urea, and centrifugation steps to re-pellet the mass in the solution (see sections3.2 and 4.2.2.2).
Akshita and Widmaier do not teach that the chaotropic reagents are including guanidine hydrochloride (GdnHCL) or guanidinium thiocyanate (GdmSCN) (claims 25-26), the molarity of the chaotropic reagents (claims 25-27, 36 and 50-51), or the temperature and length of incubation time (claims 28 and 31).
Regarding claims 25-27, 36, 40 and 50-51, Klimov teaches methods of making of aqueous protein dispersion of spider silk proteins, and that solubility of such spinnable hydrophobic proteins can be greatly increased by means of known chaotropic reagents, including guanidine hydrochloride (GdnHCL) (reads on “guanidine chloride”) and guanidinium thiocyanate (GdmSCN), with the highest experimentally observed solubility in 10 M GdmSCN solution is about 38% (see paragraphs 10, 27 & 33 and Example 1); reads on the protein having a lower solubility threshold in a non-chaotropic solvent. Regarding claim 28, Klimov teaches that solubilizing by incubating the spider silk protein and in the solution at 20-25 degrees Celsius (see Example 1).
Regarding claims 25-27, 36 and 50-51, Will also teaches solubilizing proteins including silk proteins in chaotropic solutions, and Will teaches the chaotrope concentration in solution is 2 M or higher, e.g., about 3-8 M, 3-6 M, 3.5-5.5 M, 3.5-5 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 8 M (e.g., up to limit of solubility), and that appropriate chaotropes include guanidine thiocyanate (GdmSCN), guanidine hydrochloride (GdnHCL) (reads on “guanidine chloride”), and urea (see paragraphs 6, 8, 11, 33 and 48-49). Regarding claim 31, Will teaches solubilizing the protein composition by incubating it with the chaotrope solution for 10-180 minutes (see paragraph 9).
It would have been obvious to combine Akshita and Widmaier with Klimov and Will to purify Widmaier’s spider silk protein in Akshita’s method using any of the known chaotropic reagents GdnHCL, GdmSCN, or urea, and molarities ranging from 2 to 10 M, and incubating the composition for 10-180 minutes at 20-25 degrees Celsius. A person of ordinary skill in the art would have had a reasonable expectation of success in purifying Widmaier’s spider silk protein in Akshita’s method using any of the known chaotropic reagents GdnHCL, GdmSCN, or urea, and molarities ranging from 2 to 10 M, and incubating the composition for 10-180 minutes at 20-25 degrees Celsius because Akshita teaches that chaotropic reagents should be used in the method, while both Klimov and Will establish that different known chaotropic reagents can be used to purify silk proteins and provide examples of incubation conditions. The skilled artisan would have been motivated to purify Widmaier’s spider silk protein in Akshita’s method using any of the known chaotropic reagents GdnHCL, GdmSCN, or urea, and molarities ranging from 2 to 10 M, and incubating the composition for 10-180 minutes at 20-25 degrees Celsius because Akshita teaches that chaotropic reagents should be used in the method, while both Klimov and Will establish that different known chaotropic reagents can be used to purify silk proteins and provide examples of incubation conditions. Additionally, the choice of chaotropic reagent, molarity, and incubation conditions are result effective as Akshita teaches that the aqueous solutions are used to remove contaminants such as bacterial membrane proteins, and therefore it is obvious to adjust the amount of solutioned added to achieve the desired level of purification of the target protein from said contaminates.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill at the time the invention was made.
Response to Arguments
Applicant's arguments filed 9/25/2025 have been fully considered but they are not persuasive.
Applicant alleges microfluidization was not sufficient to solubilize proteins of Akshita, noting that following the microfluidization to lyse cells, Akshita performed subsequent steps to ultimately isolate and solubilize target suckerin proteins. However, the claimed method is not limited to no additional steps being performed after the microfluidization as the method uses the open claim language “comprising”. Indeed, claims 36-37 and 50-51 all specifically limit to additional steps following the microfluidization. Therefore, as the claimed method does not require no additional steps following the microfluidization step, this argument is not persuasive.
Applicant highlights that Akshita does not exemplify performing microfluidization in the presence of a chaotropic agent as claimed. However, the rejection above is an obviousness type of rejection, and therefore does not allege that Akshita exemplifies all of the claimed features. Rather, the rejection specifically highlights that for the protein isolation, Akshita teaches (1) applying a shear force via microfluidization to an aqueous solution comprising an insoluble mass comprising the protein, and (2) inclusion of the chaotropic agent urea helps with the removal of contaminants that are non-specifically adsorbed onto the hydrophobic inclusion bodies during processing. Therefore, these combined teachings of Akshita render it obvious to include Akshita’s chaotropic agent urea during Akshita’s processing steps since Akshita specifically teaches there are beneficial effects to including it, and this argument is not persuasive.
Applicant alleges that none of the secondary references remedy the alleged deficiencies in Akshita. However, as applicant’s arguments alleging deficiencies in Akshita were not persuasive, this argument is not persuasive.
Applicant alleges present application demonstrates a benefit to protein solubilization by including a chaotropic agent with a step of applying shear force via microfluidization, pointing to the results in Table 1. Applicant notes that their results show that (1) all conditions that included a step of microfluidization resulted in better isolation of the protein, and (2) that the conditions that include a chaotropic agent also yielded better results of isolation of the protein. Applicant concludes that this demonstrates a benefit to including a chaotropic agent with microfluidization for the protein isolation. However, as stated in the above rejection, both the use of microfluidization and a chaotropic agent are specifically taught to be beneficial for protein isolation by the primary reference Akshita. Indeed, in the portion of Akshita cited in the rejection, Akshita specifically states “[m]icrofluidization has been successfully used for suckerin protein extraction with higher yields” and Akshita outlines the beneficial roles of chaotropic agents for protein isolation (see section 4.2.2.2, which is cited in the above rejection). Therefore, applicant’s observed benefits are specifically predicted by the teachings of Akshita, and applicant’s position is not persuasive.
Conclusion
No claims are free of the art. No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/S.A.M/Examiner, Art Unit 1653