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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 November 13, 2025 has been entered.
Information Disclosure Statement
The Information Disclosure Statement (IDS) filed on 11/20/2025 has been considered by the Examiner inasmuch as foreign documents have been submitted into the file wrapper in English.
Claim Status
Claims 2 and 22 are canceled. Thus, claims 1, 3-21 and 23-24 as amended are examined on the merits herein.
Claim Interpretation
Claim 1, Claim 23 and Claim 24 recite “isolating and concentrating nucleic acids of target sizes”, see claim 1, line 1; claim 23, lines 1-2 and claim 24, line 1. The Examiner respectfully notes that the specification does not explicitly define the phrase “target sizes” when referring to the nucleic acids that are isolated and concentrated as required in claim 1, claim 23 or claim 24 as discussed above.
The Examiner notes the specification exemplifies the analyte is a nucleic acid fragment below a selected size, e.g. fewer than 10,000 bp etc., see paragraph [0006], pg. 3, lines 4-7.
The Examiner also notes the specification discloses “target size cut-offs may be selected through appropriate selection and specific ordering of ATPS phase forming and fractionating components, and centrifugation, mixing and incubation steps, see pg. paragraph [0006], pg. 3, lines 9-11.
However, the Examiner did not find anything within the specification specifically excluding isolation and concentration of any target size ranges of nucleic acids.
Therefore, the Examiner reasonably interprets the limitation “nucleic acids of target sizes” to include any sized nucleic acid, and thus the isolation and concentration of nucleic acids of target sizes as required in claim 1, claim 23 and claim 24 includes the recitation of isolation and concentration of any sized nucleic acid.
Furthermore, in view of the interpretation above, the Examiner reasonably interprets that the recitation of isolating and concentrating nucleic acids of target sizes wherein the first ATPS as recited in claim 1, lines 3-7, the partitioning of the target nucleic acid fragments below a target size, see claim 1, lines 5-6; the second ATPS as recited in claim 1, lines 8-12; and the partitioning and concentrating of the target nucleic acid fragments into the third phase solution as required in claim 1, lines 10-11, includes any sized nucleic acids, and thus the Examiner reasonably interprets the second phase solution, as required in claim 1, line 6 and the recovered third phase solution, as required in claim 1, line 13, includes various nucleic acids of any size as discussed above.
Response to Arguments
The Examiner notes the rejection of claims 1, 3-21 and 23-24 under 35 U.S.C. 103 has been modified as explained in further detail below.
Applicant argues:
(A) The cited references do not disclose the following features of amended independent claim 1: (i) the combination of a first polymer-salt ATPS and a second polymer-salt ATPS; (ii) in the second ATPS, contaminants partition to the fourth phase solution; and (iii) the first phase forming polymer has a higher molecular weight than the second phase forming polymer, see Applicant’s remarks, pg. 8 of 12, Independent claim 1, paragraph 1.
(B) The PEG300 systems in Ribeiro are different because they result in both proteins and plasmid DNA concentrating in the same phase; see Applicant’s arguments, pg. 9 of 12, paragraph 2.
(C) Ribeiro only describes single ATPS systems where plasmids consistently partition to one phase or the other depending on PEG molecular weight, but never teaches or suggests a sequential system where the partitioning direction reverses between steps, see Applicant’s remarks, pg. 9 of 12, paragraph 3.
(D) The method recited in amended claim 1 represents a coordinated, opposite-direction partitioning behavior that is not taught by any of the cited references, see Applicant’s remarks, pg. 9 of 12, paragraph 3.
(E) The method of amended claim 1 extracts with a first ATPS and then mixes it with phase forming components with different chemical properties than the phase forming components of the first ATPS to form a second ATPS which partitions the target nucleic acids into a phase opposite of the phase they partitioned into within the first ATPS, which results in phase-switching behavior not taught or suggested by Ribeiro. See Applications remarks, pg. 9 of 12, last paragraph.
With respect to Applicant’s arguments (A)-(E), the Examiner notes the new 103 rejections below incorporate the combination of Frerix and Ribeiro to teach independent claim 1.
The Examiner notes Frefix teaches a process comprising a capture step and a polishing step of plasmid DNA (pDNA) by exploiting the ability of aqueous two-phases systems to differentiation between different forms of DNA.
The Examiner notes Frerix teaches during the ATPS capture and polishing processes each process uses potassium phosphate buffers (e.g. the salt) and PEG solutions (e.g. the polymer) to form the ATPS.
The Examiner notes the ATPS capture process of Frerix corresponds to the first polymer-salt ATPS to isolate and concentrate the target nucleic acid fragments from the contaminants, exemplifying the nucleic acid as supercoiled plasmid DNA (scpDNA) and the contaminant as proteins.
The Examiner notes Frerix teaches in these systems supercoiled plasmid DNA (scpDNA) exhibits a near quantitative partitioning in the salt-rich bottom phase; and therefore, the Examiner notes Frerix teaches the limitation “contaminants partition to the first phase”, required in claim 1, line 6-7; as evidenced by Frerix’s teaching the addition of the PEG solution within the ATPS capture process results in a rapid separation of plasmid DNA (pDNA) from endogenous proteins as discussed above.
The Examiner notes the ATPS capture step of Frerix uses potassium phosphate buffer and a PEG800 solution comprising PEG600 and PEG1000.
Although, Frerix does not teach the second phase ATPS to partition and concentrate the target nucleic acid fragments in the third phase solution and contaminants partition to the fourth phase solution.
However, in the same field of endeavor of using ATPS to isolate and concentrate nucleic acids, Ribeiro teaches isolation of plasmid DNA from cell lysates with various PEG/K2HPO4 aqueous two-phase systems (ATPS), including either PEG300, PEG600 or PEG1000.
Ribeiro teaches in Table 1, concentrations of plasmid, protein and gDNA in the lysate and after extraction with the ATPS where PEG600 contained no protein within the bottom phase of the tested lysate loads of 20%, 40% and 60% (w/w).
Ribeiro teaches recovery yield of plasmid DNA in the bottom phase when using PEG600 was 34% and was present in the bottom phase.
Ribeiro shows in Table 1 the recovery yield of plasmid DNA was 62% in the top phase of PEG300 with a lysate load of 20% (w/w); and during agarose gel analysis of plasmid and RNA partitioning of the PEG 300; ATPS partitions and concentrates the open circular form (oc) and the supercoiled (sc) form of plasmid and RNA in the top phase of the ATPS as compared to PEG600 which partitions the plasmid and RNA to the bottom phase.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have (a) substituted the PEG800-potassium phosphate ATPS of Frerix for the PEG600-potassium phosphate ATPS of Ribeiro; and (b) to have added an additional ATPS step comprising the PEG300-potassium phosphate ATPS as taught by Riberio above in between the ATPS capture step and ATPS polish step as taught by Frerix above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been particularly motivated to have (a) substituted the PEG800 of Frefrix for the PEG600 of Ribeiro in order to remove all protein from the sample by isolating plasmid DNA within the bottom phase of the PEG600-potassium phosphate ATPS as taught by Ribeiro above; and (b) to have added the additional ATPS step of using a PEG300-potassium phosphate ATPS within the process as taught by Frerix above as Ribeiro teaches an increased recovery yield of plasmid DNA with PEG300 when compared to PEG600 when the lysate load is 20% (w/w) as discussed above; and wherein the Examiner further notes the additional ATPS step as discussed above is particularly motivating in view of Ribeiro explicitly teaching product recovery costs become critical to the overall economics of modern biotechnology processes as discussed above.
Accordingly, one of ordinary skill in the art would have had a reasonable expectation of success to have made the substitution (a) and addition (b) as discussed above, because Frerix and Ribeiro are both drawn to processes of using PEG-potassium phosphate ATPS systems in isolating and recovering plasmid DNA as discussed above.
Thus, Applicant’s arguments (A)-(D) have been fully considered but are not found persuasive.
Applicant argues:
(F) Ribeiro even teaches away from the Examiner’s proposed combination, as the specific PEG molecular weights relied upon by the Examiner result in poor nucleic acid recovery performance as shown in Table 1 and Figure 3 of Ribeiro, see Applicant’s remarks, pg. 9 of 12, last paragraph of the page.
(G) In fact, Ribeiro explicitly taught that that “systems using PEG300 are not adequate for plasmid purification”; and therefore, the inadequate recovery and failure to separate nucleic acids from protein contaminants in PEG300 system will discourage one of ordinary skill in the art from pursuing the molecular weight combinations suggested by the Examiner, see Applicant’s remarks, pg. 10 of 12, paragraph 1.
(H) Furthermore, Ribeiro teaches away from using a second ATPS with a low MW polymer (e.g. PEG300) for second extraction after initial concentration with the first ATPS, since Ribeiro teaches that for PEG300 system an increase in the lysate load deteriorates the performance of the extraction, both in terms of yield and purification; and thus goes against the teaching of Ribeiro as one of skill in the art will be steered away from using a second ATPS as an additional step based on the “one-step separation” teaching of Ribeiro, let alone using a second ATPS with a low MW polymer such as PEG 300 for second extraction, see Applicant’s remarks, pg. 10 of 12, paragraph 2.
With respect to Applicant’s arguments (F)-(H), the Examiner reiterates their arguments above and further notes Ribeiro teaches within Table I as the lysate load decreases from 60% to 40% to 20% (w/w) within the PEG300 system the process results in both an increase in nucleic acid recovery yield and purity of the sample; which the Examiner notes neither of which are observed when using the PEG600 system as the ATPS as taught in Table I of Ribeiro.
Additionally, the Examiner respectfully reiterates that Ribeiro does explicitly teach within all tested lysate loads of 20%, 40% and 60% (w/w) the PEG600 system removes all protein from the sample; and wherein the Examiner also notes this teaching is not observed when using the PEG300 system as the ATPS as taught in Table I of Ribeiro as argued by Applicant above.
Consequently, the Examiner notes the two preceding paragraphs provide support and justification for why the Examiner stated it would have been particularly motivating to one of ordinary skill in the art to have combined the teachings of Frerix and Ribeiro as discussed above, because the motivation to combine the Frerix and Ribeiro references is due to Ribeiro’s explicit teaching that product recovery and safety are critical considerations to the overall economics of modern biotechnology processes as discussed above.
Furthermore, the Examiner notes amended claim 1 does not recite a limitation requiring (a) any level or percentage nucleic acid recovery required for the method; (b) any level or percentage of nucleic acid purity for the method; or (c) a specific lysate load of the fluid mixture recited in independent claim 1, line 2; independent claim 23, line 2 or independent claim 24, line 2.
Thus, Applicant’s arguments (F)-(H) have been fully considered but are not found persuasive.
Applicant argues:
(I) Matos does not cure this deficiency because Matos teaches a polymer-free phase in a downstream process is more attractive since it is often necessary to further purify the product by filtration or chromatographic separations, see Applicant’s remarks, pg. 10 of 12, paragraph 3.
Therefore, Matos directly teaches away from the feature of concentrating the target nucleic acids in the polymer-rich phase in the second ATPS in amended claim 1, see Applicant’s remarks, pg. 10 of 12, paragraph 3.
With respect to Applicant’s argument (I), the Examiner reiterates their arguments above and further notes Matos is only relied on for its teaching of a kit, required in independent claim 24; and therefore, Matos is not relied on for teaching any limitation within independent claim 1.
Thus, Applicant’s argument (I) has been fully considered but is not found persuasive.
(J) Lis does not teach or suggest using ATPS, let alone teach or suggest using sequential ATPS to concentrate nucleic acids of target sizes, see Applicant’s remarks, pg. 10 of 12, paragraph 4.
With respect to Applicant’s argument (J), the Examiner reiterates their arguments above and further notes Lis is only relied on for its teaching that the size of DNA molecule precipitated by PEG is dependent on the concentration of PEG as required in dependent claim 21; and therefore, Lis is not relied on for teaching any limitation within independent claim 1.
Thus, Applicant’s argument (J) has been fully considered but is not found persuasive.
Applicant further argues:
(K) Such features of the method of amended claim 1 achieve a surprisingly high nucleic acid recovery described in the specification in paragraph [0007] where the disclosed process accomplishes nucleic acid purification with little to no loss of nucleic acid (i.e., very high recovery), see Applications remarks, pg. 9 of 12, last paragraph.
(L) The surprisingly high recovery efficiency of target nucleic acids (e.g. DNA) in Example 4 of the specification (paragraphs [0088]-[0089]) demonstrates that the claimed method showed a significantly improved performance (close to 100% recovery) compared to the commercially available QIAamp MinElute ccfDNA Mini Kit (about 50% recovery, see Applicant’s remarks, pg. 10 of 12, last paragraph of the page – pg. 11 of 12, first paragraph of the page.
With respect to Applicant’s arguments (K)-(L), the Examiner reiterates their arguments above and further notes Example 4 of the specification recites specific reaction volumes; reaction inputs for the second ATPS; working concentrations of glycol polymer 6000, salt, and EDTA in Table 5, on pg. 26. The Examiner notes none of the reaction conditions within the ATPS system of Example 4 described above are recited within independent claims 1, 23 or 24.
The Examiner also notes Example 3 of the specification (paragraphs [0079]-[0082]) discloses the recovery of isolated and concentrated nucleic acids within exemplified first ATPS components (of the first ATPS) in Table 1 (see pg. 21) and second ATPS components (of the second ATPS) in Table 2 (see pg. 22).
The Examiner further notes in both Tables 1 and 2 the components recited within the first and second ATPS, respectively, recite a specific (w/w) percentage of non-phasic salt; a specific glycol polymer with a MW of 600 or a MW of 200; and specific volumes of sample, salt, and polymer within each ATPS. The Examiner notes none of these reaction conditions within the first and second ATPS components as discussed above are disclosed within independent claims 1, 23 or 24.
Moreover, the Examiner notes independent claims 1, 23 and 24 recite:
(i) a first ATPS comprising a first phase forming polymer in a first phase solution and a salt dissolved in a second phase solution; and
(ii) a second ATPS comprising a third phase forming polymer in a third phase solution and a salt dissolved in a fourth phase solution.
Accordingly, the Examiner notes independent claims 1, 23 and 24 do not recite any particular polymer or salt within either the first or second ATPS; nor do these claims recite concentrations of any of these components comprised within these solutions; nor do any of these claims recite volumes of any component within the first-phase through fourth-phase solutions or volumes of the first-phase through fourth-phase solutions themselves as components within the first or second ATPS respectively; and finally none of these claims recite any volumes of the fluid mixture comprising the nucleic acids and contaminants to be purified with the first and second ATPS as discussed above.
Consequently, the Examiner notes the claim scope of independent claims 1, 23 and 24 are broader than Example 4 within Applicant’s argument (L) as discussed above.
Furthermore, Ribeiro teaches the partitioning of nucleic acids in ATPS depends on many factors, such as the size and chemical properties of the macromolecule, the properties of the system components, and the ionic composition, see Ribeiro, pg. 377, left column, first full paragraph.
Therefore, in view of at least the foregoing reasons as discussed above, the Examiner reasonably interprets the Applicant’s argument of unexpected results within arguments (K)-(L) discussed above are not commensurate in scope with the claimed invention in either independent claims 1, 23 or 24.
Thus, Applicant’s arguments (K)-(L) have been fully considered but are not found persuasive.
(M) As demonstrated in Example 10 of the specification (paragraphs [00104]-[00106] and Table 11), Example 10 show the choices of molecular weight of polymer in the first and the second ATPS systems significantly affect the nucleic acid recovery; and that Example 10 demonstrates that the first polymer component having a higher molecular weight than the second polymer would significantly improve the yield and purity of the resulting nucleic acid product of the ATPS, see Applicant’s remarks, pg. 11 of 12, paragraph 1.
(N) Neither Ribeiro, Matos, nor Lis can reasonably predict the surprising technical effect achieved of the present invention, i.e. that in a sequential ATPS comprising first and second polymer-salt ATPS systems with the first polymer component (of the first ATPS) having a higher molecular weight than the second polymer component (of the second ATPS), where the yield and purity of the isolated target nucleic acid fragments would have been significantly improved as compared to the ATPS system taught by Ribeiro using PEG600 and PEG300 which resulted in poor recovery performance (<62% recovery), see Applicant’s remarks, pg. 11 of 12, paragraph 2.
With respect to Applicant’s arguments (M)-(N), the Examiner reiterates their arguments above and further notes Example 10 within the specification discloses the DNA concentration recovered as a result of the molecular weight of the first phase forming polymer in the first ATPS and the second phase forming polymer within the second ATPS within Table 11 (see pg. 34).
The Examiner notes the results within Example 10 are associated with the word “High” or “Low” for the first and second phase forming polymer within their respective ATPS within Table 11.
Additionally, the Examiner particularly notes within the specification the example embodiment within Table 11 describes the molecular weight difference from “Low” to “High” is within 200-2000Da (see pg. 33, paragraph [00105], last two lines of the paragraph).
Consequently, the Examiner notes the ranges of molecular weight difference between the first and second phase forming polymers within the first and second ATPS respectively described within Table 11 of Example 10 above are not recited within either independent claims 1, 23 or 24.
Moreover, the Examiner notes the molecular weight difference between the first ATPS comprising PEG600 and the second ATPS comprising PEG300 used within the combined method of Frerix and Ribeiro a discussed above is 300Da; and therefore, the combined method of Frerix and Ribeiro teach a molecular weight difference between the first and second phase forming polymers that is within the disclosed range of the “Low” to “High” designations used by Applicant within Table 11 of Example 10 as discussed above.
Thus, Applicant’s arguments (M)-(N) has been fully considered but are not found persuasive.
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.
(I) Claims 1, 3-20 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Frerix et al. (Published 25 August 2006, Biotechnology and Bioengineering, Vol. 96, Issue 1, pp. 57-66, PTO-892) in view of Ribeiro et al. (Published 15 March 2002, Biotechnology and Bioengineering, Vol. 78, Issue 4, pp. 376-384, PTO-892 mailed 03/11/2025).
Regarding claims 1, 3-20 and 23, Frerix teaches a process for the capture of plasmid DNA (pDNA) by exploiting the ability of aqueous two-phases systems to differentiation between different forms of DNA, see pg. 57, abstract.
Frerix teaches a hundred-gram biomass was dissolved with P1 resuspension buffer to reach 1,000 g of a 10% w/w solution (e.g. a fluid mixture, required in claim 1, line 2; and after complete dissolution; 1,000 g P2 lysis buffer was added and mixed; and after incubation of said mixture; a potassium phosphate buffer and 1,000 g PEG 800 solution were added (e.g. combining said fluid mixture and the first aqueous two-phase system, required in claim 1, lines 3-4. See pg. 60, left column, ATPS Capture, paragraph 1.
Frerix teaches the PEG 800 stock solutions were made of 28.5 g PEG 600, 46.5 g PEG 1000 (p.A.) and 25 g deionized water, see pg. 58, right column, polyethylene glycol and potassium phosphate, paragraph 1.
Frerix teaches the resulting bottom phases of the capture step were pooled, filtered, membrane-absorbed, and eluted in order to receive the desalted pDNA in the filtrate (e.g. extracting the second phase mixture, required in claim 1, line 8), see pg. 60, left column, ATPS Capture, paragraph 1.
Frerix teaches during the ATPS capture process the alkaline lysate is neutralized using potassium phosphate in order to ease and enable the immediate formation of an ATPS system after addition of the PEG solution. The latter procedure results in a rapid separation of pDNA from endogenous proteins, see pg. 61, left column, Neutralization, paragraph 1.
Frerix teaches in these systems supercoiled plasmid DNA (scpDNA) exhibits a near quantitative partitioning in the salt-rich bottom phase (e.g. such that target nucleic acid fragments below a target size partition to said second phase solution, required in claim1, lines 5-6), see pg. 57, abstract.
Consequently, based on the teachings of Frerix within the preceding two paragraphs, the Examiner respectfully notes Frerix also teaches the limitation “contaminants partition to the first phase”, required in claim 1, line 6-7; as evidenced by Frerix’s teaching that addition of the PEG solution within the ATPS capture process results in a rapid separation of pDNA from endogenous proteins as discussed above.
Frerix teaches 300 µL of the desalted plasmid preparation of the capture step and 300 µL NaOH solution were mixed and incubated for denaturing experiments; neutralization occurred by addition of potassium phosphate buffer; and then PEG 800 stock solution was added (e.g. the at least one size fractionating component selected from a polymer, required in claim 13, lines 4-5 and claim 17; and the molecular weight, required in claim 14), wherein said mixture was mixed via vortex and centrifuge; and the resulting bottom phase was recovered, (e.g. mixing the third phase solution with at least one size fractionating component, e.g. the polymer, in order to form a supernatant and a precipitated pellet and separating the supernatant from the precipitated pellet, see claim 13, pg. 5, lines 2-8), see pg. 59, left column, Polish Procedure, paragraphs 1-2.
Frerix teaches the polish operation to meet final purity demands is based on a system exploiting the combination of the denaturation of the nucleic acids present, specific renaturation of scpRNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as open circular plasmid DNA (ocpDNA) and genomic host DNA (gDNA), see pg. 57, abstract.
Frerix teaches this polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids, see pg. 57, abstract.
Frerix teaches the potassium phosphate buffer is comprised of dipotassium phosphate and monopotassium phosphate (e.g. dipotassium phosphate and monopotassium phosphate, required in claim 5, lines 2-3), see pg. 58, right column, polyethylene glycol and potassium phosphate, paragraph 1.
Although, Frerix does not teach (a) such that the target nucleic acid fragments partition to and concentrate in the third phase solution and contaminants partition to the fourth phase solution, required in claim 1, lines 10-12; (b) the molar concentration and mass concentration required in claims 3-4 and 15-16; and (c) the excluded volume, hydrophilic/hydrophobic and electrostatic interactions, required in claims 7-12.
However, in the same field of endeavor of isolating plasmid DNA, with respect to limitation (a) Ribeiro teaches plasmid DNA used for non-viral therapeutic transfer or nucleic acid vaccination has to be highly purified and free from contaminating components such as bacterial proteins, toxins, genomic DNA or RNA. Additionally, Ribeiro teaches product recovery costs become critical to the overall economics of modern biotechnology processes and the need to have a process complying with guidelines issued by regulatory agencies has increased the interest in developing alternative methods for the downstream processing of plasmids. See pg. 376, right column, paragraph 1.
Ribeiro teaches isolation of plasmid DNA from cell lysates, see pg. 376, title; where PEG/K2HPO4 aqueous two-phase systems (ATPS) were prepared by combining water, PEG, salt and lysate and vortex mixing, wherein the mixture was centrifuged for 20 s at 2000g to facilitate phase separation and the top and bottom phases were carefully isolated; wherein different PEG molecular weights (MW) were chosen, for example:
PEG 300 (20/20% w/w) (e.g. the second phase polymer, required in claim 1, line 9 and claim 18);
PEG 600 (20/20% w/w) (e.g. the first phase polymer, required in claim 1, line 4 and claim 18) and;
PEG 1000 (15/13% w/w), see pg. 377, right column, aqueous two-phase systems, paragraph 1.
The Examiner respectfully notes K2HPO4 as taught by Ribeiro above is dipotassium phosphate, the salt required within instant claim 6; and is consistent with dipotassium phosphate contained within the potassium phosphate buffer taught by Frerix above.
Ribeiro shows in Table 1, concentrations of plasmid, protein and gDNA in the lysate and after extraction with ATPS, where PEG600 contained no protein within the bottom phase of the lysate loads 20%, 40% and 60% (w/w) tested, see pg. 382, Table 1.
Ribeiro teaches recovery yield of plasmid DNA in the bottom phase when using PEG 600 was 34%, see pg. 382, Table 1.
Ribeiro teaches extraction of the plasmid in the lysate in the bottom phase, see pg. 381, plasmid quantification, paragraph 1.
Ribeiro teaches the recovery yields of plasmid DNA were found to be proportional to the plasmid concentration in the lysate, see pg. 376, abstract, paragraph 3.
Ribeiro shows in Table 1 the recovery yield of plasmid DNA was 62% in the top phase of PEG300 with a lysate load of 20% (w/w), see pg. pg. 382, Table 1; and during agarose gel analysis of plasmid and RNA partitioning of the PEG 300; ATPS partitions and concentrates the open circular form (oc) and the supercoiled (sc) form of plasmid and RNA in the top phase of the ATPS as compared to PEG 600 which partitions the plasmid and RNA to the bottom phase (e.g. the target nucleic acid fragments partition to and concentrate in the third phase solution and contaminants partition to the fourth phase solution, required in claim 1, lines 10-12), see pg. 379, Figure 1 and pg. 380, left column, paragraph 1.
Ribeiro teaches extraction of the plasmid in the lysate in the top phase (e.g. recovering the concentrated target nucleic acid fragments from the third phase solution, required in claim 1, line 13), see pg. 381, plasmid quantification, paragraph 1.
The Examiner respectfully notes in the first phase extraction wherein the plasmid was partitioned to the bottom phase, PEG 600 was used, and in the second extraction wherein the plasmid was partitioned to the top phase, PEG 300 was used. Thus, the Examiner respectfully notes the use of PEG 600 as a first phase forming polymer and PEG 300 as the second phase forming polymer corresponds to the limitation “wherein the first phase forming polymer has a higher molecular weight than the second phase forming polymer”, required in claim 1, lines 14-15.
Ribeiro teaches the simplicity, the rapidity, and the ease of scale up makes ATPS an attractive method for plasmid isolation and purification from cell lysates, see pg. 383, left column, last paragraph of the column.
Ribeiro teaches within Figure 1 the top phase of the PEG300/K2HPO4 ATPS with 40% (w/w) of lysate where the size fractionating of sc plasmid with a size between 6.56-4.36 kb (6,560-4,360 bp) was observed (e.g. the nucleic acid target size, required in claim 20), see pg. 379, Figure 1.
Ribeiro teaches the production of plasmid DNA includes primary isolation and purification steps where techniques most commonly used for purification have been based on chromatographic methods; and wherein complexity of the feedstock; a precipitation step with appropriate agents is usually necessary (e.g. precipitating the target nucleic acid fragments from the supernatant, required in claim 13, line 10), see pg. 377, left column, second full paragraph.
It would have been prima facie obvious to one of ordinary skill in the art at the invention’s effective filing date to have (a) substituted the PEG800-potassium phosphate ATPS of Frerix for the PEG600-potassium phosphate ATPS of Ribeiro; and (b) to have added an additional ATPS step comprising the PEG300-potassium phosphate ATPS as taught by Riberio above in between the ATPS capture step and ATPS polish step as taught by Frerix above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been particularly motivated to have (a) substituted the PEG800 of Frefrix for the PEG600 of Ribeiro in order to remove all protein within the bottom phase of PEG600 when isolating plasmid DNA as taught by Ribeiro above; and (b) to have added the additional ATPS step using PEG300 within the process taught by Frerix above; as Ribeiro teaches an increased recovery yield of plasmid DNA with PEG300 when compared to PEG600 when the lysate load is 20% (w/w) as discussed above; and particularly in view of Ribeiro explicitly teaching product recovery costs become critical to the overall economics of modern biotechnology processes as discussed above.
One of ordinary skill in the art would have had a reasonable expectation of success to have made the substitution and addition as discussed above, because Frerix and Ribeiro are both drawn to processes of using PEG-potassium phosphate ATPS systems in isolating and recovering plasmid DNA as discussed above.
With respect to limitation (b) the Examiner respectfully notes since the combination of Frerix and Ribeiro teach the claimed first ATPS and second ATPS required in claim 1 and different concentrations of PEG within the ATPS, for example 75% (w/w) of PEG 800 which comprised PEG 600 and PEG 1000 as taught by Frerix above; and 20% (w/w) of PEG 600 and 15% (w/w) of PEG 1000 as taught Ribeiro above; then using Frerix and Ribeiro as a starting point to vary the PEG solutions through routine experimentation and optimization would allow the artisan to arrive at the molar concentrations required in limitation (b) as obvious to one of ordinary skill in the art as Frerix and Ribeiro demonstrate varying the molar concentration and mass concentration is a known consideration in the prior art.
In addition, the Examiner respectfully notes MPEP 2144.05(II)(A) states “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.".
With respect to limitation (c), the Examiner reasonably interprets this limitation to be a physical consequence of using (1) the first ATPS to partition out the contaminants in the first phase solution and (2) the second ATPS to partition out and concentrate the target nucleic acid fragments in the third phase solution. Since the combination of Frerix and Ribeiro teach the structural limitations required to remove all protein and isolate plasmid DNA in the salt-rich bottom phase as demonstrated with the PEG600/K2HPO4 ATPS; and wherein a PEG300/K2HPO4 ATPS is used to partition out and concentrate plasmid DNA with increased plasmid DNA recovery yield when compared to PEG600 in the top phase as discussed above; the excluded volume interactions, required in claims 7-8; hydrophilic/hydrophobic interactions, required in claims 9-10; and electrostatic interactions, required in claims 11-12 will be met by the combined teachings of Frerix and Ribeiro as discussed above as this combination teaches the fractioning of the first ATPS as required in claim 1, lines 5-7 and the second ATPS, as required in claim 1, lines 10-11.
Accordingly, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have incorporated limitations (a)-(c) as discussed above into the method as taught by Frerix above using the teachings of Ribeiro above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to use the teachings of Ribeiro above in order to improve the recovery yield of the supercoiled plasmid DNA as taught by the method of Frerix above. One of ordinary skill in the art would have had a reasonable expectation of success to have incorporated the limitations (a)-(c) as discussed above into the method of Frerix above, because Frerix and Ribeiro are both drawn to processes of using PEG-potassium phosphate ATPS systems in isolating and recovering plasmid DNA as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(II) Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Frerix et al. (Published 25 August 2006, Biotechnology and Bioengineering, Vol. 96, Issue 1, pp. 57-66, PTO-892) and Ribeiro et al. (Published 15 March 2002, Biotechnology and Bioengineering, Vol. 78, Issue 4, pp. 376-384, PTO-892 mailed 03/11/2025) as applied to claims 1, 3-20 and 23 above, and further in view of Lis et al. (Published 1 March 1975, Nucleic Acids Research, Vol. 2, Issue 3, pp. 383-390, PTO-892 mailed 03/11/2025).
Frerix and Ribeiro address claims 1, 3-20 and 23 as written above.
Although, Frerix and Riberio do not teach the target size is selected within a 50 bp range below 1000 bp, required in claim 21.
However, in the same field of endeavor of isolating DNA with PEG, Lis teaches a method for fractionation of DNA according to size. The method is based on the fact that the size of DNA molecule precipitated by PEG is dependent on the concentration of PEG; and this degree of resolution has only been demonstrated in the range of 100-2000 base pairs. See pg. 383, Introduction, paragraph 2.
Therefore, the Examiner respectfully notes Frerix and Ribeiro teach the claimed first ATPS and second ATPS required in claim 1; in addition, the Examiner notes the teachings of Lis above disclose the fractionating of DNA according to size is dependent on the concentration of PEG and a degree of resolution as low as 100 base pairs was demonstrated as discussed above. Therefore, the Examiner notes using Lis as a starting point to vary the PEG solutions of Frerix and Ribeiro above through routine experimentation and optimization would allow the artisan to arrive at the target size selection of within a 50 bp range below 1000 bp required within instant claim 21 as obvious to one of ordinary skill in the art and within the scope of the artisan as Lis already demonstrated a degree of resolution of 100 bp via fractionating DNA according to size is dependent on the concentration of PEG as discussed above.
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have included the teachings of Lis into the combined method of Frerix and Ribeiro as discussed above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to include the teachings of Lis within the combined teachings of Frerix and Ribeiro above in order to develop alternative methods for the downstream processing of plasmids where product recovery costs become critical to the overall economics of modern biotechnology processes and in view of these processes complying with the guidelines issued by regulatory agencies as taught by Ribeiro above. One of ordinary skill in the art would have had a reasonably expectation of success to have incorporated the teachings of Lis into the combined method of Frerix and Ribeiro as discussed above, because Frerix, Ribeiro and Lis are all drawn to using PEG solutions to isolate DNA as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
(III) Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Frerix et al. (Published 25 August 2006, Biotechnology and Bioengineering, Vol. 96, Issue 1, pp. 57-66, PTO-892) and Ribeiro et al. (Published 15 March 2002, Biotechnology and Bioengineering, Vol. 78, Issue 4, pp. 376-384, PTO-892 mailed 03/11/2025) as applied to claims 1, 3-20 and 23 above, and further in view of Matos et al. (Published 10 February 2014, Separation and Purification Technology, Vol. 122, pp. 144-148, IDS filed 09/06/2022).
Frerix and Ribeiro address claims 1, 3-20 and 23 as written above. Although, Frerix and Riberio do not teach a kit, required in claim 24.
However, in the same field of endeavor of nucleic acid isolation using two phase systems, Matos teaches isolation of PCR fragments using aqueous two-phase systems, see pg. 144, title; specifically, plasmid DNA, see pg. 146, right column, paragraph 1; and that commercial kits allow the purification of the amplified products, see pg. 145, left column, paragraph 2.
The Examiner reasonably interprets within the commercial kits for purification of amplified products, such as plasmid DNA as taught by both Frerix and Ribeiro above, will include containers for the ATPS solutions thus separating them before use within the purification process. Therefore, the Examiner reasonably interprets the recitation of a kit as taught by Matos above when in combination with the teachings of Frerix and Ribeiro above corresponds to the kit recited in claim 24.
It would have been prima facie obvious to one of ordinary skill in the art before the invention was filed to have included the teachings of Matos into the combined method of Frerix and Ribeiro as discussed above as within the scope of the artisan as combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to include the teachings of Matos in order to develop a kit which provides an alternative method for the downstream processing of plasmids where product recovery costs become critical to the overall economics of modern biotechnology processes and in view of these processes complying with the guidelines issued by regulatory agencies as taught by Ribeiro above.
One of ordinary skill in the art would have had a reasonably expectation of success to have incorporated the teachings of Matos into the combined method of Frerix and Ribeiro as discussed above, because Frerix, Ribeiro and Matos are all drawn to using PEG-salt ATPS systems in isolating plasmid DNA as discussed above.
Thus, the claimed invention as a whole would have been prima facie obvious over the combined teachings of the prior art.
Conclusion
No claims are allowed in this action.
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/JARET J CREWS/Examiner, Art Unit 1691
/RENEE CLAYTOR/Supervisory Patent Examiner, Art Unit 1691
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