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 .
Election/Restrictions
Applicant’s amendment submitted on 6/16/2026 is acknowledged. New claim 18 is added.
Applicant’s election without traverse of the required species, one purification step to be performed after step d) or step e) in the reply filed on 2/5/2026 is acknowledged. Applicant did not elect a particular species from the group “one or more purification steps performed after step d) or e)”, so the examiner hereby withdraws the election of species requirement, and each species will be examined.
Claims 1-18 are under examination on the merits.
Withdrawn Objections
The previous objections are hereby withdrawn, due to Applicant’s amendment submitted 6/16/2026:
Claim objections: claims 1 and 7 for minor informalities.
Withdrawn Rejections
The previous rejections are hereby withdrawn, due to Applicant’s amendment submitted 6/16/2026:
35 U.S.C. §112(b): claims 6, 8-10, and 17 for being indefinite.
35 U.S.C. §103: claims 1-7 and 9-16 under 35 U.S.C. 103 as being unpatentable over Fiedler (WO 2019133677 A1, published 7/4/2019) in view of Oh (WO 2019006390 A1, published 1/3/2019); and Claims 8 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Fiedler and Oh (supra), as applied to claims 1-7 and 9-16, in further view of Hanak, et al. (CA 3112824 A1, published 3/26/2020).
Claim Interpretation
Claims 8 and 17 refer to “about pH”. The specification indicates “the term pH of about 5 is understood to mean within a range of pH values from 4.0 to 6.0” (p. 12). The examiner is interpreting claim 17’s limitation to require a pH from 4 to 6.
New Objections
Claim Objections
Claim 8 is objected to because of the following informalities: the claim contains hyphens on lines 4-7. To put the claim in better form, it is recommended that they are changed to roman numerals or letters in parentheses. Appropriate correction is required.
New Rejections
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
(New Rejection) Claims 8 and 17 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 8 recites “a re-equilibration of the cation exchange chromatography material to about pH 3.5 ± 0.5” on lines 4-5. The specification does not define “about”, but indicates the term pH of about 5 is understood to mean within a range of pH values from 4.0 to 6.0” (p. 12). Based on the teachings of the specification and claim 8 requiring “about pH 3.5 ± 0.5”, the metes and bounds of the claim are unclear. A person of ordinary skill in the art would not know which pHs are claimed. The term “about” in claim 8 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 17 is also indefinite because it depends on claim 8 but does not resolve this lack of clarity.
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.
(New Rejection) Claims 1-7, 9-16, and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Fiedler (WO 2019133677 A1, published 7/4/2019) in view of Oh (WO 2019006390 A1, published 1/3/2019), Fabre (PGPub US 20100260798 A1, published 10/14/2010), Boudeffa (PGPub US 20170002332 A1, published 1/5/2017), and Liu (PGPub US 20100098725 A1, published 4/22/2010).
The claimed invention encompasses a method for reducing a contaminating DNA content of a preparation containing AAV capsids and contaminating DNA, comprising the steps of: a) performing an extraction of DNA with a solid phase bearing positive charges at its surface said solid phase is contacted with the preparation at a pH 7.0 ± 1.0, and a salt concentration of 10 mM to 200 mM yielding a first fraction, b) diafiltering the first fraction by a first tangential flow filtration to obtain a second fraction, c) treating the second fraction with DNase, d) diafiltering the DNase treated second fraction obtained by step c) by a second tangential flow filtration to a buffer with pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 20 mM to yield a third fraction, and e) optionally, concentrating the third fraction by tangential flow filtration before supplemental chromatography to yield a concentrated third fraction, as recited by a representative claim 1.
The Prior Art
Fiedler teaches methods of producing and purifying full adeno-associated virus (AAV) capsids from a concentrated AAV fraction comprising empty AAV capsids and full AAV capsids (Abstract; para. [0069]). Fiedler’s methods encompass embodiments wherein the solution containing the AAV particles undergoes ion exchange chromatography, such as anion exchange that can remove residual particle contaminants, acidic impurities, proteases, and/or host cell DNA, and may be conducted with membrane-based separation (para. [00162]). The anion exchange support can be PEI-based resins (para. [00162]).
Fiedler also contemplates that an acidic component, such as host cell DNA, is removed in some embodiments (para. 24]), and that in some embodiments, the DNA from any host cell used is removed by anion exchange and wash steps, and Benzonase and/or DNase is not used (para. [163]). Fiedler specifically contemplates contacting an AAV containing solution with a filter comprising positively charged groups effective to deplete acidic charged contaminants from the AAV containing solution (claim 79; see also claims 1-78).
Fiedler’s methods comprise any combination of steps disclosed therein, and may be combined with additional steps (para. [180]). Fiedler’s methods also encompass negative anion exchange chromatography on a fraction comprising AAV particles, by applying the fraction to a chromatography column or membrane under conditions that allow for the AAV to flow through the AEX chromatography column or membrane; the loading buffer may comprise about 100 mM to about 150 mM salt, and the pH of the loading buffer is about 8 to about 9 (para. [184]). In the negative chromatography step, unwanted components bind to the chromatography resin and the desired AAV does not bind to the chromatography resin, and advantages of this “non-binding mode” include relative ease of carrying out the procedure and in conducting subsequent assaying (para. [183]). Fiedler discloses that the method may comprise concentrating AAV fractions using ultra/diafiltration, such as tangential flow filtration (TFF) steps, and the ultra/diafiltration may be performed before, after, or before and after performing anion exchange chromatography (para. [185]). Additionally, Fiedler describes diafiltration with a 300 kDA cassette (para. [297]). Fiedler also discloses a method of purifying an adeno-associated virus (AAV) comprising: (a) loading an AAV containing solution onto an affinity resin targeted against AAV under conductions that allow binding between the AAV in the solution and the affinity resin; (b) at least two wash steps; and (c) eluting the AAV from the affinity resin (para. [0007])
Fiedler teaches that the AAV purified by its methods may be of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 serotype, AAV11, AAV12, AAV13, serotypes, or an AAV modified by genetic engineering or that is chemically modified (para. [0076]).
However, Fiedler does not specifically disclose treating with DNase.
Oh discloses purification, production, and manufacturing methods for recombinant AAV vector particles, wherein the methods include at least 2 column chromatography steps, which can include cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, and/or AAV affinity chromatography alone or in combination and in any order (Abstract; Fig. 1). Oh teaches treating with a nuclease, such as benzonase, to reduce contaminating nucleic acid (para. [0020]). Oh also teaches diafiltration buffer and equilibration buffers to be used for TFF that are 10 mM Na-P (sodium phosphate), pH 7.3, 180 mM NaCl (also referred to PBS180, 10 mM sodium phosphate with 180 mM NaCl, pH 7.3; pp. 42-43, printout pp. 44-45). Additionally, Oh discloses that alternating tangential flow (ATF) or TFF with either spiral-wound membrane module or flat plate module are alternatives to TFF with hollow fiber cartridge (p. 43, p. 45 printout, section 2.2). Oh also specifically discloses cation exchange chromatography followed by anion exchange chromatography (Fig. 1; paras. [0010-0015). Oh also discloses typical equilibration buffers and solutions for washes and elutions for cation exchange chromatography are at an appropriate pH of about pH 3 to about pH 8 (para. [0132]).
Oh further discloses that the method may comprise a concentrating step, (b), which is concentrating cells and/or cell culture supernatant comprising rAAV vector particles to produce a lysate and may be via ultrafiltration/diafiltration, such as by TFF (paras. [0010-0016]). Oh also discloses that the buffer, pH, and conductivity of physical cell lysis all depend on the first loading condition, in most cases the diafiltration buffer is the same or an equivalent buffer used for the equilibration in the first column, and for Benzonase treatment, both pH and conductivity should be within the range of benzonase working condition (e.g., about pH 6.5-8.5, conductivity >15 mS/cm), and 2 mM MgCl2 should be added for proper digestion (pp. 41-42, printout pp. 43-44; “Exemplary Physical lysis method”). Oh also discloses a chemical lysis method, wherein Triton X-100 or an equivalent non-ionic surfactant is used, and benzonase treatment can be simultaneously or sequentially with surfactant, with MgCl2 concentration of 1-5 mM (p. 41, printout p. 43; “Exemplary cell lysis and preparation prior to column purification”). Further, Oh discloses that its AAV vector purification and production methods are applicable to a wide variety of AAV serotypes/capsid variants, and may purify many different serotypes/pseudotypes of rAAV vectors (para. [0072]).
Fabre teaches a method for purifying the rabies virus, the method comprising the steps of 0.45 µm clarified harvest, Fractogen EMD SO3- chromatography, concentration/diafiltration by ultrafiltration, adjustment of 2 mM MgCl2, benzonase treatment, and sucrose gradient ultracentrifugation (Abstract; Table IV).
Boudeffa teaches a process for purifying viruses, with the steps in order of benzonase, 0.45 µm clarification, concentration by TFF ultrafiltration/diafiltration, and then anion exchange chromatography (Fig. 1; abstract).
Liu teaches purification methods for purifying influenza viruses with high overall recovery of virus and result in levels of host DNA, host cell protein, and non-specific endonuclease (e.g., Benzonase), which are below the specifications required by regulatory agencies (Abstract). Liu’s method involves the step of on-column benzonase treatment, on a CS column which binds the virus (Fig. 10A; para. [0492]).
It would have been obvious to one of ordinary skill in the art to modify the methods of Fiedler to arrive at the claimed method steps of: a) performing an extraction of DNA with a solid phase bearing positive charges at its surface said solid phase is contacted with the preparation at a pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 200 mM yielding a first fraction, b) diafiltering the first fraction by a first tangential flow filtration to obtain a second fraction, c) treating the second fraction with DNase, d) diafiltering the DNase treated second fraction obtained by step c) by a second tangential flow filtration to a buffer with pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 20 mM to yield a third fraction, and e) optionally, concentrating the third fraction by tangential flow filtration before supplemental chromatography to yield a concentrated third fraction.
With respect to the claims’ requirements of: I) performing an extraction of DNA at a pH of 7.0 ± 1.0, and a salt concentration of 10 mM to 200 mM, and II) a second TFF to a buffer with pH of 7.0 ± 1.0 and a salt concentration of 10 mM to 20 mM (claim 1), these are clearly result effective variables because pH values will affect the result as it determines the amount of charge on the column and stability of the extract, and salt concentration affects the binding. This is also the case for other pHs or concentrations present in the claims. Similarly, the TFF membrane porosity with a molecular weight cutoff in the range of 10 kDa to 300 kDA (claim 5), 60 kDa to 300 kDa (claim 13), 100 kDa to 300 kDa (claim 14), 200 kDa to 300 kDa (claim 15), or 250 kDa to 300 kDa (claim 16) is a result effective variable because it determines which molecular species are removed or retained in the method, and thus would also be obvious to optimize. It has long been settled to be no more than routine experimentation for one of ordinary skill in the art to discover an optimum value of a result effective variable. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum of workable ranges by routine experimentation." Application of Aller, 220 F.2d 454, 456, 105 USPQ 233, 235-236 (C.C.P.A. 1955). "No invention is involved in discovering optimum ranges of a process by routine experimentation." Id. at 458, 105 USPQ at 236-237. The "discovery of an optimum value of a result effective variable in a known process is ordinarily within the skill of the art." Application of Boesch, 617 F.2d 272, 276, 205 USPQ 215, 218-219 (C.C.P.A. 1980). Since Applicant has not disclosed that the specific limitations recited in instant claims are for any particular purpose or solve any stated problem, and the prior art teaches that parameter magnitudes that are encompassed by instant claims, often vary according to the sample being analyzed and various matrices, solutions and parameters appear to work equally as well, absent unexpected results, it would have been obvious for one of ordinary skill to discover the optimum workable ranges of the methods disclosed by the prior art by normal optimization procedures known in the art.
Fiedler teaches removal of DNA from AAV preparations by anion exchange chromatography, where unwanted components bind to the chromatography resin and the desired AAV does not bind to the chromatography resin, and advantages of this “non-binding mode” include relative ease of carrying out the procedure and in conducting subsequent assaying. During a similar AAV processing method, Oh teaches the use of benzonase treatment to remove unwanted DNA. Fabre teaches benzonase treatment and sucrose gradient ultracentrifugation that occur after a column treatment, it would be obvious to add the diafiltration to before the sucrose, or substitute it for the sucrose gradient, because it is substituting one benzonase removal method for another, to arrive at predictable results. Liu teaches on-column benzonase treatment on a column that binds the virus. From these references, it is clear that benzonase and diafiltration steps may be performed at any place in a method of virus purification and contaminant removal, with predictable results.
Also, since Applicant has not shown the criticality of their step order, changing this order and that of the prior art is obvious, since for the reasons of record, the results would be expected. See Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.). Therefore, the order of Applicant’s steps, which provides expected results, is obvious.
It would be obvious to one of ordinary skill in the art to incorporate each of these similar procedures, which have art recognized equivalence for the same purpose. See MPEP §2144.06. Fiedler explicitly teaches that its methods may be performed in any order, and with additional steps. Notably, Oh discloses each form of chromatography claimed and says that any combination may be used in a method to purify AAV. Both Fiedler and Oh state that their methods are applicable to purification of a wide variety of AAV serotypes, including rAAV and AAV1-13 serotypes, as well as genetically engineered or chemically modified AAV. Oh discloses a non-limiting example of ultrafiltration/diafiltration is tangential flow filtration (TFF), for example, a hollow fiber membrane with a nominal pore size corresponding to a 100 kDA molecular weight cutoff, so that large amounts of AAV vector can be prepared when present in larger volumes of eluate. Thus, claim 5 would have been obvious before filing. The examiner is interpreting that to read on a single hollow fiber membrane. Further, it would have been obvious to one of ordinary skill in the art to select an appropriate filter for AAV, through routine optimization, see MPEP §2144.05, and Fiedler discloses diafiltration with a 300 kDA cassette.
Regarding claim 3’s limitation that TFF is performed with membranes selected from the group consisting of hollow-fiber membranes and flat membranes, Oh further discloses a non-limiting example of ultrafiltration/diafiltration is tangential flow filtration (TFF), for example, a hollow fiber membrane with a nominal pore size corresponding to a 100 kDA molecular weight cutoff, so that large amounts of AAV vector can be prepared when present in larger volumes of eluate (para. [0154]), thus use of hollow-fiber membranes for TFF would have been obvious to one of ordinary skill in the art
Regarding claim 4’s limitation, the first filtration is performed in a buffer which is formulated to facilitate DNA digestion by a DNase, the teachings of Oh render that obvious, because Oh discloses that TFF may be a concentrating step prior to cell lysis, that cell lysis may be performed simultaneously with benzonase treatment, and that in most cases the diafiltration buffer is the same or an equivalent buffer used for the equilibration in the first column, and for Benzonase treatment, both pH and conductivity should be within the range of benzonase working condition. Also, it would be routine and standard practice for one of ordinary skill in the art to ensure proper buffer conditions for optimal nuclease treatment or action of any enzyme.
With respect to claim 7 and 9-10, Oh discloses purification, production, and manufacturing methods for recombinant AAV vector particles, wherein the methods include at least 2 column chromatography steps, which can include cation exchange chromatography, anion exchange chromatography, size exclusion chromatography, and/or AAV affinity chromatography alone or in combination and in any order (Abstract; Fig. 1), therefore, performing cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography after step d) or step e) would have been obvious to one of ordinary skill in the art. With respect to claim 9, this claim modifies step (ii) of claim 7 which need not be chosen for the method of claim 7. Therefore, if affinity chromatography step was not selected to make claim 7 obvious claim 9 limitations are also met by the claim 7 obviousness argument above. With respect to claim 10, this claim modifies step (iii) of claim 7 which need not be chosen for the method of claim 7. Therefore, if anion exchange chromatography step was not selected to make claim 7 obvious claim 10 limitations are also met by claim 7 obvious argument above.
Regarding claim 11, which requires the hollow-fiber membranes are single hollow fiber membranes or membranes in bundles, that would be obvious to use in the obvious method because Oh teaches use of a hollow-fiber membrane, which the examiner is interpreting to be a single membrane.
Regarding claim 12, which requires the flat membranes are single membranes or membranes that are stacked or rolled into cylinders, that would be obvious because to use in the obvious method because Oh teaches that TFF with either spiral-wound membrane module or flat plate module are alternatives to TFF with hollow fiber cartridge.
Regarding claim 18, which requires the third fraction has a concentration factor of 10-20 with respect to the preparation, that would be obvious, because the method is rendered obvious by the cited references, and so whatever results would naturally flow from the obvious method. Additionally, the third fraction’s concentration factor could be optimized by routine experimentation, for example, by eluting with a lower volume to generate a higher concentration factor. The determination of whether each of these clauses is a limitation in a claim depends on the specific facts of the case. See, e.g., Griffin v. Bertina, 283 F.3d 1029, 1034, 62 USPQ2d 1431 (Fed. Cir. 2002)(finding that a “wherein” clause limited a process claim where the clause gave “meaning and purpose to the manipulative steps”). In Hoffer v. Microsoft Corp., 405 F.3d 1326, 1329, 74 USPQ2d 1481, 1483 (Fed. Cir. 2005), the court held that when a “‘whereby’ clause states a condition that is material to patentability, it cannot be ignored in order to change the substance of the invention.” Id. However, the court noted (quoting Minton v. Nat’l Ass’n of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003)) that a “‘whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.’” Id.
In applying the broadest reasonable interpretation and absent guidance in the specification, the examiner is interpreting “salt”, to mean any salt, not to be limited to NaCl. Sodium phosphate, disclosed to be a diafiltration buffer, reads on the claim limitation “salt”.
One of ordinary skill in the art would have been motivated to remove unwanted DNA impurities from AAV preparations. There would be a reasonable expectation of success because each of the methods of DNA removal were known in the art, and the cited references demonstrate that the method steps may be performed in different orders. Therefore, claims 1-7, 9-16, and 18 were prima facie obvious before the priority date of the instant invention.
(New Rejection) Claims 1-18 are rejected under 35 U.S.C. 103 as being unpatentable over Fiedler (WO 2019133677 A1, published 7/4/2019) in view of Oh (WO 2019006390 A1, published 1/3/2019), Fabre (PGPub US 20100260798 A1, published 10/14/2010), Boudeffa (PGPub US 20170002332 A1, published 1/5/2017), Liu (PGPub US 20100098725 A1, published 4/22/2010), as applied to claims 1-7, 9-16, and 18, in further view of Hanak, et al. (CA 3112824 A1, published 3/26/2020).
The teachings of Fiedler, Oh, Fabre, Boudeffa, and Liu are described above.
However, Fiedler, Oh, Fabre, Boudeffa, and Liu do not teach cation exchange chromatography elution that is performed with a salt gradient.
Hanak teaches a method of purifying a recombinant AAV (rAAV) particle from a mammalian host cell culture, comprising the steps of: (b) purifying a HIC eluate through cation exchange chromatography (CEX) to produce a CEX eluate comprising a plurality of rAAV particles; (c) isolating a plurality of full rAAV particles from the CEX eluate of (b) by anion exchange (AEX) chromatography to produce an AEX eluate comprising a purified and enriched plurality of full rAAV particles; and (d) diafiltering and concentrating the AEX eluate into a formulation buffer by TFF to produce a final composition (claim 1). Hanak specifically discloses selecting a fraction from a CEX chromatogram containing rAAV particles to produce the CEX eluate, wherein the rAAV particles are eluted using a step gradient, which may be a pH gradient, salt gradient, or both (claims 57, 70 and 71).
Regarding claims 8 and 17, it would have been obvious to one of ordinary skill in the art to modify the methods rendered obvious by Fiedler, Oh, Fabre, Boudeffa, and Liu to incorporate a salt gradient elution during CEX, as disclosed by Hanak. Oh also discloses typical equilibration buffers and solutions for washes and elutions for cation exchange chromatography are at an appropriate pH of about pH 3 to about pH 8, and Hanak teaches that the plurality of rAAV particles may be eluted using a salt gradient during CEX. One of ordinary skill in the art would have been motivated to elute rAAV particles. There would be a reasonable expectation of success because the individual parameters of elution are disclosed by Oh and Hanak. Therefore, claims 1-18 were prima facie obvious before the priority date of the instant invention.
Response to Arguments
Applicant presents the following arguments:
The presently claimed invention is directed to a method for reducing contaminating DNA in a preparation of AAV capsids. The method comprises performing an extraction of DNA with a solid phase bearing positive charges at its surface to yield a first fraction. The first fraction, which has a reduced content of DNA, is diafiltered by a first tangential flow filtration (TFF) and the obtained fraction (the "second fraction") is treated with DNase. The DNase treated second fraction is diafiltered by a second TFF. Thus, in the method of claim 1, treatment with DNase is performed after contacting the preparation of AAV capsids with the solid phase. The cited references do not teach or suggest the method according to instant claim 1.
Significantly, in contrast to the instantly claimed method, Fiedler teaches that "The anion exchanger and wash steps described [in the reference] can be effective to remove contaminating] DNA such that there is no need to treat with Benzonase or DNase." (See paragraph [00163] of Fiedler.) The Examiner has recognized that Fiedler does not specifically disclose treating with DNase, although Fiedler does disclose DNase in the context of an assay in Example 9.
The Examiner cited Oh for remedying this deficiency of Fiedler. Oh is directed to methods for purification and production of recombinant AAV vector particles which include at least 2 column chromatography steps. See, for example, the embodiments disclosed by Oh in the Summary (paragraphs [0011] to [0015]). These embodiments are multi-step methods. The Examiner cited Oh at paragraph [0020] for teaching that "step (d)" can comprise treating with a nuclease, such as benzonase, to reduce contaminating nucleic acids. However and significantly, the methods disclosed by Oh do not teach or suggest performing a treatment with DNase after an AAV sample has been subjected to an anionic chromatography step. Rather, in each embodiment of Oh, the step (d), which can be treatment with a nuclease, is performed before the first column chromatography step, step (f). In particular, the benzonase is used by Oh in the lysis step or following the lysis step where cells are processed to form a lysate from which the AAV particles can be purified. Oh does not teach use of nuclease after lysing and filtering the lysate in preparation for further purification. The combined teaching of Fiedler and Oh do not teach or suggest the method of claim 1, in which treating with DNase is performed after extraction of DNA with a solid phase bearing positive charges at its surface.
The present application describes on page 1, lines 21-27, that in prior-art processes the amount of DNA, despite the treatment with DNase, causes problems during tangential flow filtration and that concentrating can only be achieved by a factor of two. Using the method of the present invention allows for the effective use of TFF and permits concentrating the preparation by a factor of 10 to 20, see Summary of the invention.
In summary, the method of claim 1 is a novel method that provides surprising enhancements for purification of AAV, especially with respect to reducing DNA contamination. The method of claim 1 would not have been obvious to one of ordinary skill in the art from a reading of Fiedler in view of Oh.
Thus, since Fiedler and Oh fail to teach or suggest all of the features of independent claim 1, a prima facie case of obviousness has not been established.
The deficiencies of Fiedler and Oh with respect to claim 1 are explained above. With respect to dependent claims 8 and 17, the Examiner recognized that Fiedler and Oh do not teach cation exchange chromatography elution that is performed with a salt gradient. The Examiner further cited Hanak for teaching this feature. However, the disclosure of Hanak does not make up for the deficiencies of Fiedler and Oh with respect to independent claim 1.
Thus, since the combined teachings of Fiedler, Oh, and Hanak fail to teach or suggest all of the features of independent claim 1, a prima facie case of obviousness has not been established for dependent claims 8 and 17.
Applicant’s arguments are not entirely persuasive:
The examiner agrees that a prima facie case of obviousness was not established in the now withdrawn rejection, however, each and every limitation is rendered obvious in the new rejection under 35 U.S.C. §103.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JEFFREY MARK SIFFORD whose telephone number is (571)272-7289. The examiner can normally be reached 8:30 a.m. - 5:30 p.m. ET with alternating Fridays off.
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/JEFFREY MARK SIFFORD/Examiner, Art Unit 1671
/Michael Allen/Supervisory Patent Examiner, Art Unit 1671
