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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous office action has been withdrawn pursuant to 37 CFR 1.114.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 05 August 2025 has been entered.
The Request for Continued Examination filed has been considered. The following information has been made of record in the RCE filed for the instant application:
1. Claims 2, 12, 16, 18, and 26-28 have been canceled.
2. No new Claims have been added.
3. No Claims have been amended.
4. Response to rejections under 35 USC 102 and 103.
The following rejection has been withdrawn:
5. The rejection of Claims 26-27 under 35 U.S.C. 102(a)(1) as being anticipated by Medrano et al (Bioprocess Biosys Eng, 2017, 40, 463-471; cited in IDS filed 08/27/2021; of record and newly cited necessitated by amendment) has been rendered moot by cancelation.
6. The rejection of Claim(s) 26-26 under 35 U.S.C. 103 as being unpatentable over Medrano et al (Bioprocess Biosys Eng, 2017, 40, 463-471; cited in IDS filed 08/27/2021; of record) in view of Freitas et al (Separation and Purification Technology, 2009, 65, 95-104; newly cited necessitated by amendment) and further in view of Urthaler (EP 2088196 A1; cited in the search report filed 08/27/2021; of record) has been rendered moot by cancelation.
Claims 1, 3-11, 13-15, 17, and 19-25 are pending in the case. Claims 22-23 have been withdrawn from consideration. Claims 1, 3-11, 13-15, 17,19-21, and 24-25 are under prosecution in this Action.
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.
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.
Claim(s) 1, 3-11, 13-15, 17, 19-21, and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Medrano et al (Bioprocess Biosys Eng, 2017, 40, 463-471; cited in IDS filed 08/27/2021; of record) in view of Freitas et al (Separation and Purification Technology, 2009, 65, 95-104; of record) and further in view of Urthaler (EP 2088196 A1; cited in the search report filed 08/27/2021; of record).
Medrano et al teaches isolation/purification of plasmid DNA (pVAX1-NH36) by contacting it with an anion exchange column (Mustang Q). After the adsorption step, the column was washed with 0.6M NaCl in TE buffer which removed RNA. Elution of the bound material was performed using a saline gradient, wherein in the first gradient the molarity was increased from 0.600 to 0.725m NaCl in TE buffer, while the second gradient was from 0.725 to 1.100M NaCl in TE buffer (page 465, right col., part of the method of claim 1 regarding isolating/purifying plasmid DNA and elution with salt; limitation of claim 4, limitation of claim 6, part (ii)-sodium chloride; limitation of claim 7-use of anion exchange membrane). The purity level of the plasmid DNA is 94.1% (page 469, Table 2, membrane fraction B10, HPLC purity %; limitation of claim 3). The elution step included fractionation of plasmid DNA (page 466, right col. last para through page 467; as in claim 5). The method comprises separating solid components from the mixture from the pDNA from solid components via precipitation (page 465, left col. second full para; limitations of claims 8-9; precipitating and removing the solid from the liquid phase reads on two-phase separation as in claim 9). The column was washed with a wash buffer prior to elution (page 465, right col., see second para below subtitle: AEC membranes; limitation of claim 10(v)). After the elution step a purification step of the pDNA was also performed (page 466, left col., first para; as in claim 11).
The method of Medrano includes the steps recited in claim 13 (page 464, right col. para below subtitle-Host strain plasmid through page 465, left col, second para). The method involves removal of DNA wherein precipitation with CaCl2 is not used for its removal (as in claim 14). Fractions of the pDNA are collected from the elution stage (page 465, right col. last para, line 11; limitation of claim 17 (i)). The nucleic acid is a pDNA comprising bacterial DNA (page 464, right col., first para under subtitle-Host strain and plasmid; limitation of claim 19(ii)). The membrane used has an average pore diameter of 800nm, which is equal to 0.8m (limitation of claim 20 (i)).
In the method of Medrano solid components from the mixture comprising the biological molecules (pDNA) of interest is separated prior to contacting it with the anion exchange column (page 465, left col., para under sub title-Primary recovery). In this step, a buffer is added to the mixture (as in claim 21, part (i)).
Medrano does not expressly teach the use of the kosmotropic salts in the concentration range as in claim 1, does not teach that when contacting the mixture with the anion exchange material the density of the mixture is to be greater than 1.1kg/L on adding the buffer as in claim 21, part (i), and the limitations of claims 24-25.
Freitas et al teaches the use of ammonium sulfate, sodium citrate and potassium phosphate as kosmotropic salts in the purification of pDNA. The concentrations used 1.5M and 2M for potassium phosphate (Abstract; page 97, parts 2-3-2.4; page 102, Table 2, page 103, part 3.4 and part 4; kosmotropic salt and concentrations as in claims 1, 24 and 25). In view of this teaching, it would be obvious to one of ordinary skill in the art would use the kosmotropic salts and concentrations taught by Freitas in the method of Medrano for purifying pDNA as instantly claimed. One of ordinary skill in the art will also adjust the concentration of the kosmotropic salt as in claim 1 for the purpose of optimization of selective binding.
Urthaler teaches a method of isolating or purifying pDNA wherein a tulip shaped vessel is used for separating the solid components from the mixture comprising a biological molecule of interest prior to contacting with an anon exchange material that phase separates from the clear lysate, which is removed from the device through an outlet at the bottom (page 21, claims1-2 of Urthaler and Figures 13-14; limitation of claim 15).
Since the step taught by Medrano involves the use of two-phase separation wherein the solids are removed from the mixture, it would be obvious to one of ordinary skill in the art to add buffer to increase the density of the mixture to be greater than about 1.1kg/L so that the lighter solids will be on top and the liquid part will be at the bottom in view of the teachings of Urthaler (as in claim 21, part i). This would facilitate easy removal of the liquid part which contains the pDNA of interest to be drained out and thereby separating it from the solid.
One of ordinary skill in the art would be motivated to perform the step as claimed since Medrano teaches centrifuging for 30 minutes to remove the solids that are separated, which takes time. Draining the bottom-heavy liquid containing the biological molecule of interest is easy and faster than using centrifugation.
MPEP 2141 states, "The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit. The Court quoting In re Kahn, 441 F.3d 977, 988, 78 USPQ2d 1329, 1336 (Fed. Cir. 2006), stated that "[R]ejections on obviousness cannot be sustained by mere conclusatory statements; instead, there must be some articulated reasoning with some rational underpinning to support the legal conclusion of obviousness.'" KSR, 550 U.S. at, 82 USPQ2d at 1396. Exemplary rationales that may support a conclusion of obviousness include: (A) Combining prior art elements according to known methods to yield predictable results; (B) Simple substitution of one known element for another to obtain predictable results; (C) Use of known technique to improve similar devices (methods, or products) in the same way; (D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results; (E) " Obvious to try " choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success; (F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art; (G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention."
According to the rationale discussed in KSR above, the rationale in (G) above is seen to be applicable here since based on the prior art teachings, isolation and purification of a biological molecule of interest like plasmid DNA using anion exchange chromatography, kosmotropic salt, and salt solution for elution using the claimed method steps is known in the art.
Thus, the claimed invention as a whole would have been obvious to one of ordinary skill in the art before the effective filing date of the instant invention over the combined teachings of the prior art. Method improvement is the motivation. The artisan would also be motivated to use the claimed method (also taught by the prior art) since the method gives significantly increased yield of the desired product (Freitas). Medrano also teaches that a 21-fold global purification factor, and an 88% yield of pDNA is obtained (Abstract). Therefore, one of ordinary skill in the art would use the claimed method for isolating and purifying pDNA from a mixture in view of the combined teachings of the prior art.
Response to Applicant’s Remarks
Applicant has traversed the rejection of claims under 35 USC 103 arguing that the sequential use of kosmotropic and chaotropic salts in the claimed method results in a simplified, one step purification of the pDNA to a high degree, with very high yields that could not be obtained from the salt gradient anion exchange chromatography (AEX) purification method such as Medrano. Use of high salt concentration of at least 0.8M for the loading step of a pDNA sample allows selective binding of only the desired pDNA, while other components such as RNA or genomic DNA do not bind to the AEX material under such loading conditions.
This is evidenced by the results, specifically, Table 4 at paragraph 0115, which shows that RNA was removed and low levels of pDNA and linear pDNA remained, 4 and 3% respectively. Gradient elution allowed separation of RNA, oc pDNA and sc pDNA and Fig 7 confirmed that proteinaceous impurities were additionally removed in the AEV chromatography step.
Freitas is directed to the use of hydrophobic interaction chromatography (HIC). This method separates molecules based on hydrophobicity; an entirely different separation principle compared to anion exchange chromatography of the present claims which separates biomolecules based on their net charge. Freitas provides that in HIC, binding of a charged macromolecules is promoted by high concentrations of kosmotropic salts to enhance hydrophobic interactions by reducing solvation.
In contrast, kosmotropic salts like ammonium or sodium citrate are not commonly used in AEX chromatography due to their stabilizing effects on macromolecular structures, which can reduce elution efficiency unless specifically optimized. Therefore, the artisan would not look to Freitas’ disclosure directed to an entirely different process and would not have a reasonable expectation of success.
The binding behavior of various components in Freitas’ HIC separation is different from AEX chromatography of the present invention. Fig. 3 shows that the desired plasmid DNA is eluted first while impurities (in particular RNA) are eluted later. Further, the elution profiles of Fig. 4 additionally show that RNA is eluted after pDNA.
The method of Freitas involves binding of several components in the loading phase, with subsequent separation of pDNA from RNA through elution buffer gradients, resulting in a lower yield of pDNA compared to the claimed method. This can be seen from Table 2 of Freitas on page 102, which provides that the method resulted in pure pDNA at a mere 73.5% yield, which is much lower compared to the present application’s very high yield and purity levels. Freitas also admits that high sample loads quickly decreased the purity of the pDNA. For these reasons are not obvious over the prior art (Remarks-pages 7-11).
Applicant’s arguments have been considered but are not found to be persuasive. The method of Medrano in combination with that of Freitas would result in the method wherein kosmotropic and chaotropic salts are used sequentially and is also a one-step purification of pDNA. Medrano teaches most of the steps recited in the instant claims and does not teach the use of kosmotropic salts as claimed. The use of kosmotropic salts for the purification of pDNA is taught by Freitas.
Applicant argues that kosmotropic salts are not commonly used in AEX chromatography. This means that it may be used. Freitas teaches that both anion exchange and HIC have been used for purification of pDNA (page 95, right col. first full para). When both these chromatographic methods can be used for purifying pDNA then the kosmotropic salts used in the HIC method of Freitas can be used in the AEX method of Medrano. One of ordinary skill in the art will have a reasonable expectation of success in doing so.
The claimed method is drawn to purifying pDNA. It does not matter which component is eluted first and which is eluted last. As long as the method is amenable to separation and purification of the desired component (pDNA in the instant case) the artisan will use it. The yield of 73.5% reported by Freitas is a high yield. Applicant states that the yield of 73.5% reported by Medrano is much lower compared to yield in the present method but has not disclosed what the yield is in the present method. Perusal of the examples did not clarify as to how high the yield in the present method is compared to that of Medrano. If high sample loads decreased the purity of the pDNA according to Medrano, one of ordinary skill in the art can optimize the sample load for maximum yield and purity level. This is routine experimentation and can be recognized and performed by one of ordinary skill in the art.
The combined teachings of the prior art do render the instant claims obvious. The rejection is maintained.
Conclusion
1. Elected Claims 1, 3-11, 13-15, 17,19-21, and 24-25 are rejected.
2. Claims 22-23 have been withdrawn from consideration.
3. Claims 2, 12, 16, 18, and 26-28 have been canceled.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to GANAPATHY KRISHNAN whose telephone number is (571)272-0654. The examiner can normally be reached M-F 8.30am-5pm.
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/GANAPATHY KRISHNAN/Primary Examiner, Art Unit 1693