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 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 1-17 are under examination on the merits.
Information Disclosure Statement
The Information Disclosure Statements (IDSs) submitted on 12/2/2022 and 4/3/2024 are in compliance with 37 CFR 1.97. Accordingly, the IDSs are being considered by the examiner.
Claim Objections
Claims 1 and 7 are objected to because of the following informalities: “Performing” on line 3, “Diafiltering” on lines 6 and 9, “Treating” on line 8, and “Optionally” and “Concentrating” on line 12 of claim 1, and “A”, “An”, and “An” on lines 3-5 of claim 7 should not be capitalized. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claims 6, 8-10, 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.
Regarding claim 6, it recites “the preparation [] contains AAV particles selected from the group consisting of [..] recombinant serotypes with features of more than one serotype” on lines 3-4. Claim 6 is indefinite because the metes and bounds of the limitation “features of more than one serotype” are unclear. The specification does not define features, and is not clear what kind of features the claim refers to. Examples of such a feature could be chemical, physical, and structural characteristics, genes encoded, tropism, etc.
Regarding claims 8-10, the claims recite “the fraction of step d) or e)”, however, claim 1, from which claims 8-10 depend on through claim 7, mentions two fractions “a second fraction” and “a third fraction” on lines 9 and 11, respectively. It is unclear which fraction claims 8-10 are referring to.
Regarding claim 17, the claim recites “[t]he method of claim 8, wherein the pH is about 5” on line 1. However, claim 8 recites two distinct pHs, the pH when the fraction is loaded on a cation exchanger chromatography material (pH 4 to 6), and a re-equilibration of the cation exchange chromatography material (pH 3.5 ± 0.5). It is not clear which pH claim 17 is referring to.
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.
Claims 1-7 and 9-16 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).
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, 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 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]). 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]).
It would have been obvious to one of ordinary skill in the art to modify the methods of Fiedler to incorporate DNase treatment steps based on the teachings of Oh. 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 ofbenzonase treatment to remove unwanted DNA. 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. The examiner is reading 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 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 for one of ordinary skill in the art to ensure proper buffer conditions for optimal nuclease treatment.
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, the cited references say that each o. Therefore, claims 1-7 and 9-16 were prima facie obvious before the priority date of the instant invention.
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).
The Prior Art
The teachings of Fiedler and Oh are described above. However, they 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).
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Fiedler and Oh to incorporate a salt gradient elution during CEX, as disclosed by Hanak. Oh teaches that 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 8 and 17 were prima facie obvious before the priority date of the instant invention.
Conclusion
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
/BENJAMIN P BLUMEL/Primary Examiner, Art Unit 1671