DETAILED ACTION
Final Rejection
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Withdrawal of Restriction/Election of Species
2. Restriction on election of species of non-ionic surfactant as applied as per prior office action of 09/13/2021 is withdrawn.
3. Restriction on election of species of transgene as applied as per prior office action of 09/13/2021 is withdrawn.
Information Disclosure Statement
4. The information disclosure statements (IDS) submitted on 01/18/2019, 05/05/2020, 07/10/2020, 03/10/2021, 12/19/2024 are acknowledged. The submission is in-compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Priority
5. This application is a 371 of PCT/US17/43291 07/21/2017 which claims benefit of 62/865,312 07/21/2016.
Status of claims
6. Claims 1-3, 5-7, 10-11, 13, 16, 19, 21-26, 28, 30-31, 34, 36, 40-43, 45, 47, 49, and 53-73 as amended and filed on 10/08/2025 are pending and are under examination in this office action.
7. Claims 1-3, 5-7, 10-11, 13, 16, 19, 21-26, 28, 30-31, 34, 36, 40-43, 45, 47, 49, and 53-73 are under examination.
Withdrawn Claim Objections
8. Withdrawn rejection of claims 1, 13 and 16 in view of amendment of the claimed filed on 10/08/2025.
Withdrawn Objection to Specification
9. The objection to the specification is withdrawn in view of the amendments to the claims 1, and 16 that recites vector genomes/ml and corresponds to the support in the specification.
Withdrawn Claim Rejections - 35 USC § 112
10. The rejection of claim 1 and dependent claims 2-3, 5-7, 10-11, 13, 15-16, 19, 21-23,24-26, 28, 30-31, 34, 36, 40-43, 45, 47, 49, and 53-73 under 35 U.S.C. 112(b) is withdrawn in view of the amendment to the claim 1.
Claim Rejections - 35 USC § 103
11. 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.
12. Claims 1-3, 16, 19, 21-26, 28, 30-31, 34, 36, 40-43, 45, 53-60, and 73 are rejected under 35 U.S.C. 103 as being unpatentable over Wright et al 2013 (US20130072548A1 published 03/21/2013), Tomono et al 2016 (Molecular Therapy, Methods & Clinical Development, 3, 15058), Florencio et al 2015 (Molecular Therapy-Methods & Clinical Development, 2, 15024), Pierce et al 2006 (US20060194313A1 published 08/31/2006), Bennicelli et al 2008 (Mol Ther., 2008, 16(3):458-65), Wright 2008 (Gene Therapy, (2008) 15, 840–848), Mehtali et al 2015 (US20150275186A1, published 10/01/2015), and Fabre et al 2010 (US 2010/0260798A1 published 10/14/2010).
The instant claim 1 is interpreted as directed to: a method of production of recombinant adeno-associated virus (rAAV) vector particles in cell culture and purification at high recovery or high titer, inter alia, by a process of purification involving harvesting cell culture produced rAAV, lysis of cell culture comprising rAAV, clarification of lysate, filtration of lysate, ion exchange column chromatography purification of the the clarified lysate to produce purified rAAV vector particles, cesium chloride gradient ultracentrifugation to separate bona fide rAAV vector particles from empty capsid AAV particles and other AAV vector related impurities; collecting bona fide rAAV vector particles (step (f)(1)); formulating bona fide rAAV vector particles in a buffer with a non-ionic surfactant(step (f)(2)); subjecting said bona fide rAAV vector to a buffer exchange by tangential flow filtration (TFF) to produce an AAV vector formulation (step (g); filtering said AAV vector formulation produced to produce a formulation of bona fide rAAV vector particles at high recovery or high titer, and wherein the collected bona fide rAAV vector particles of step (f)(1) are diluted to a concentration of less than about 5x1012 rAAV vector particles/ml prior to, substantially simultaneously or after step (f)(2) and prior to step (g).
The phrase “bona fide” is interpreted as "bona fide rAAV vector" refers to AAV vectors comprising a transgene of interest which are capable of infecting target cells (specification para [0029]), high rAAV vector recovery is interpreted as “70% or greater rAAV vector yield” from start to finish (specification para [0007]); rAAV vector high titer is interpreted as at least at “1E+12, 5E+12, 1E+13, or 5E+13 vector genomes/ml or rAAV vector particles/ml.
Regarding Claim 1: Wright et al 2013 (US20130072548A1) partially teaches instant claim 1 directed to a method for bona fide recombinant AAV (rAAV) viral vector purification for use in gene therapy by disclosing (See, US20130072548A1, title, abstract, para [0010], claim 1): a method for purifying bona fide AAV vector particles comprising a transgene encoding a therapeutic protein or fragment thereof from an AAV preparation comprising AAV vector particles, empty capsids and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids, said method comprising:
The instant claim 1 directed to a method for producing recombinant adeno-associated virus (rAAV) vector particles at high recovery or high titer, said method comprising the steps are taught as follow:
(a) harvesting cells and/or cell culture supernatant comprising rAAV vector particles is taught by Wright et al 2013 claim 1 step a)
(b) lysing said harvest produced in step (a) to produce a lysate is taught by Wright et al 2013 claim 1 step c)
(c) filtering said lysate produced in step (b) to produce a clarified lysate is taught by Wright et al 2013 claim 1 step d)
(d) subjecting said clarified lysate produced in step (c) to ion exchange column chromatography to produce a column eluate comprised of purified rAAV vector particles is taught by Wright et al 2013 claim 1 steps b) and e)
(e) mixing said column eluate produced in step (d) with cesium chloride to produce a mixture, and subjecting said mixture to gradient ultracentrifugation to substantially separate bona fide rAAV vector particles from empty capsid AAV particles and other AAV vector related impurities is taught by Wright et al 2013 claim 1 steps f) and g)
(f)(1) collecting said bonafide rAAV vector particles separated in step (e); (step ( e) and (f)(1) teaches instant claim 30).
(f)(2) formulating said collected bonafde rAAV vector particles in a buffer with a non-ionic surfactant.
(g) subjecting said bonafide rAAV vector particles in step (f)(2) to a buffer exchange by tangential flow filtration to produce an AAV vector formulation;
Wright et al 2013 (US20130072548A1) does not teach instant claim 1 step (f)(2) formulating said collected bona fide rAAV vector particles in a buffer with a non-ionic surfactant and step (g) subjecting said bona fide rAAV vector particles in step (f)(2) to a buffer exchange by tangential flow filtration (TFF) to produce an AAV vector formulation. Wright 2013 does not explicitly teach formulating the rAAV vector particles with non-ionic surfactant prior to the tangential flow filtration of claim 1 step (h).
Florencio et al 2015 teaches a simple downstream process based on a non-ionic surfactant that has a hydrophilic polyethylene oxide and an aromatic hydrophobic group, Triton-X-100 (instant claim 72 limitation) treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors. Florencio et al teaches a new rAAV vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture with medium at the time of harvest. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies (See, abstract, fig 1, fig 2, page 4 col 2, discussion section, para 1, entire article). Thus, Florencio et al 2015 teaches added limitation of instant claim 45, wherein the method produces rAAV vector particles having a higher titer than rAAV vector particles produced where a nonionic surfactant is added to the AAV vector formulation after buffer exchange by tangential flow filtration of step (g). Based on the teachings of Florencio et al 2015 a non-ionic surfactant is added to the medium at the time of cell harvest and lysis and the non-ionic surfactant is comprised in the AAV vector preparation prior and through buffer exchange by TFF (Florencio et al 2015 teaches added limitation instant claim 45). The instant claim 45 is directed to functional result. Given the method, including the steps and ingredients used, has been met, the functional results for instant claim 45 must also be met.
Tomono et al 2016, Pierce et al 2006 (US20060194313A1) and Mehtali et al 2015 (US20150275186A1) teaches the above recited instant claim 1 steps (f)(2) and (g) as recited below.
Tomono et al 2016 teaches ultracentrifugation-free chromatography including culture supernatant ultrafiltration tangential flow filtration (TFF) for large-scale purification of recombinant rAAV serotype 1 for gene therapy. Tomono et al 2016 teaches a NaCl salt buffer comprising 0.01% Pluronic F-68 (non-ionic surfactant) and further purification of TFF filtered culture medium and ammonium sulfate concentrated rAAV on dual-ion exchange columns to further remove impurities from rAAV and to separate empty AAV particles from rAAV particles (See, Tomono et al 2016, page 2, abstract, figure 3, page 7, col 1, para 3). Thus, Tomono et al 2016 teaches comprising 0.01% Pluronic F-68 (non-ionic surfactant) in a buffer prior to separation of empty AAV from bona fide rAAV particles.
Pierce et al 2006 (US20060194313A1) is in the art of recombinant expressed protein produced by a cells and their concentration and purification that involves addition of a non-ionic surfactant, e.g., Pluronic® F68, polyethylene glycol, or other non-ionic block copolymer surfactants, to the buffer used in a diafiltration step as part of a microfiltration process (e.g., tangential flow filtration; TFF) can reduce turbidity of the process fluid, a goal of procedures such as those described above is to maximize the amount of product, e.g., a secreted protein or peptide product, recovered from the cell culture or retentate, while simultaneously minimizing the amount of undesirable material in the cell culture and process stream. An increase in turbidity load in the cell culture or retentate is generally associated with an increase in the turbidity of the permeate, excessive turbidity in a solution in which cells are suspended, such as culture medium or a buffer or a mixture of both, is undesirable because turbidity indicates conditions that can cause filter blinding, blockage in the clarification train, the turbidity of the filtrate is typically reduced typically, the non-ionic surfactant is Pluronic F68 or polyvinyl alcohol (PVA), in some embodiments, the non-ionic surfactant is in a buffer, e.g., phosphate buffered saline (PBS). The concentration of the non-ionic surfactant in a buffer can be between 0.2 g/L (0.02% w/v) and 4 g/L, 0.2 and 3 g/L, 0.5 and 2.5 g/L, 0.5 and 2 g/L, 0.5 and 1.5 g/L. In some cases, the concentration of the non-ionic surfactant in the buffer is about 1 g/L (0.1% w/v) or 2.5 g/L. (See, abstract, para [0004], [0010], [0012], [0015], [0024], [0026]- [0028], claim 2, 8, 21, 23). Thus, Pierce et al 2006 (US20060194313A1) teaches addition of non-ionic surfactant is Pluronic® F68 to the cell culture medium comprising expressed recombinant protein prior to diafiltration step as part of a microfiltration process (e.g., tangential flow filtration; TFF). Pierce et al 2006 (US20060194313A1) teaches a non-ionic surfactant, e.g., Pluronic® F68, polyethylene glycol (See, para [0004]), non-ionic surfactants that can be used include polyethylene glycol (PEG), for example, PEG having a molecular weight of at least about 1000 (See, para [0013]),
Mehtali et al 2015 (US20150275186A1) is in the analogous art directed to a development and manufacturing, in particular industrial production of viral vectors and vaccines using cell cultures. Mehtali et al 2015 teaches that a non-ionic surfactant, such as polypropylene glycol (PLURONIC F-61, PLURONIC F-68, SYNPERONIC F-68, PLURONIC F-71 or PLURONIC F-108) can be added to the medium as a de-foaming agent preferably between about 0.05 g/L (0.005% w/v) and about 10 g/L, typically between about 0.1 (0.01% w/v) and about 5 g/L. The surfactant in cell culture medium may be decrease the size of the cell clumps (prevent to enlarge the size of the cell clumps) (See, para [0076]).
Step (h) of instant claim 1: filtering said AAV vector formulation produced in step (g) thereby producing a formulation of bonafide rAAV vector particles at high recovery or high titer, wherein the collected bona fide rAAV vector particles of step (f)(1) are diluted to a concentration of less than about 5x1012 rAAV vector particles/ml prior to, substantially simultaneously or after step (f)(2) and prior to step (g).
Wright et al 2013 teaches step (h) of the instant claim 1 step (h) by disclosing: formulating purified AAV particles with surfactant to provide an AAV particle formulation; filtering said formulation to remove any remaining impurities, wherein said bona fide AAV vector particles are present in said AAV product in an amount of at least 95% (See, Wright et al 2013 claim 1, step i) and h). The recited step also teaches instant claim 73 limitation.
Wright et al 2013 further teaches doses of AAV could be prepared: assuming a dose of 1×1011 AAV vector expressing a transgene to treat blindness administered to the eye; assuming a dose of 1×1012 AAV vectors expressing a transgene for Parkinson's Disease administered to CNS (teaches instant claim 28) (See, US20130072548A1, para [0070], [0074]). Therefore, Wright et al 2013 and Mehtali et al 2015 teaches an approach for dilution of rAAV to arrive at a desired dose concentration of AAV.
Fabre et al 2010 (US20100260798A1) is in the analogous art and teaches a method for purifying the Rabies virus comprising a single ion-exchange chromatography step, to prevent any aggregation phenomenon, a very small amount of a surfactant, which is preferably non-ionic, such as poloxamer 188 (instant claim 68 limitation) (Pluronic F 68) can be added, at a very low concentration, to the chromatographic eluate (See, abstract, para [0067]).
Bennicelli et al 2008 describes AAV2 vector recovery in the presence or absence of the non-ionic surfactant Pluronic F68 (PF-68) following vector dilution and passage through injection devices. See p. 459, col. 1. See Figure 1 and p. 459, col. 1 for the following recitations: “As shown in Figure 1a, stock AAV2.RPE65 vector diluted to a target concentration of 1 × 1011 vector genomes (vg)/ml either supplemented (+PF68) or not (–PF68) with 0.001% PF68 and then passed through device A, was recovered at 106.8% ± 9.3% and 50.9% ± 6.0%, respectively. The vector, similarly, diluted and passed through device B, resulted in an average recovery of 104.3% ± 7.0% when PF68 was present and 23.7% ± 4.8% in the absence of the surfactant. For device C, average vector recoveries were 94.1% ± 5.5% and 27.5% ± 7.4% in the presence or absence, respectively, of PF68. These results indicated that the surfactant was required to achieve consistent and quantitative recovery of the vector, diluted to concentrations relevant to animal models and prospective clinical studies.” Also see the Discussion, p. 463, col 2 to p. 464 for the following recitation: “Perhaps the most dramatic improvement with respect to delivery of the AAV was due to the addition of a low concentration of surfactant to the vehicle. Without surfactant, up to 75% of the vector was lost to inert surfaces, significantly reducing the dose of vector delivered. In this study, an approach was developed that allows consistent and predictable delivery of a given dose of vector” (See, Bennicelli et al 2008).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to the modify the prior art teachings of Wright et al 2013 (US20130072548A1) to incorporate a non-ionic surfactant, taught by Florencio et al or Pluronic F68, to a buffer used in a diafiltration step taught by Tomono et al 2016 and in a diafiltration step as part of a microfiltration process tangential flow filtration (TFF) in the method taught by Pierce et al 2006 (US20060194313A1). One of ordinary skill in the art would have been motivated to do so for the prevention of filter blinding/blockage in the clarification train through pore plugging and other mechanisms and the discovery that the addition of a non-ionic surfactant, e.g., Pluronic® F68, polyethylene glycol, or other non-ionic block copolymer surfactants, to the buffer used in a diafiltration step as part of a microfiltration process (e.g., tangential flow filtration; TFF) can reduce turbidity of the process fluid (See, Pierce et al 2006 (US20060194313A1), para [0004], [0012], [0027]) and for the advantage of achieving consistent and quantitative recovery of the vector by preventing aggregation and for removing cellular lysate impurities as taught by Tomono et al 2016, Mehtali et al 2015 teaches the concentration of non-ionic surfactant 0.005% to 0.01% w/v (See para [0076]). One of the ordinary skills would have been apprised of a reasonable expectation of success to arrive at the invention of claim 1 given the teachings in the art by Wright et al, Tomono et al, Florencio et al, Tomono et al, Pierce et al, Mehtali et al, Bennicelli et al, Fabre et al and Wright 2008 as recited supra.
Claims 40-43: The added limitations of instant claims 40-43 are taught by Wright et al 2013 (US20130072548A1) by disclosing the eluate so generated is then added to an isopynic gradient and subjected to ultracentrifugation, the layer containing the viral particles is harvested and subjected to buffer exchange by Tangential Flow Filtration. The purified AAV particles are then formulated with surfactant and the resulting formulation filtered to remove any remaining impurities there by producing a highly purified AAV product, wherein said bona fide AAV vector particles are present in said AAV product in an amount of at least 95%, preferably at greater than 98% (See, para [0010], [0016], [0018], [0020], [0053], [0063], [0069], [0070], [0074]), adding nuclease to the lysate is taught by disclosing, …incubated in a neutral buffered salt solution containing a nuclease (Benozonase) at a concentration of 100 Units/mL to digest and remove nucleic acid impurities, and further washed with a neutral buffered saline solution to remove residual nuclease (See, para [0074]).
The non-ionic surfactant concentration taught my Mehtali et al 2015 and Pierce et al 2006 were sufficient to prevent aggregation of protein or virus in a buffer that had the desired properties (prevention of protein or virus or cell aggregates). Therefore, determining or adjusting concentration of a non-ionic surfactant or buffer salt for diafiltration/microfiltration TFF process based on requirement for optimal performance is a routine laboratory approach. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382.
Furthermore, according to section 2144.05 of the MPEP, 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.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages”).
Claims 16, 19, 31, 34, and 36: The instant claims 16, 19, 31, 34, and 36 (dependent on base claim 1) has added limitations, wherein the yield of said bona fide rAAV vector particles in step (h) is at least about 5x1012 bona fide rAAV vector particles/ml (claim 16); wherein the recovery of said bona fide rAAV vector particles in step (h) is at least about 60% of the total rAAV vector particles of step (a) (claim 19); wherein purity of said bona fide rAAV vector particles in said filtrate of step (h) is at least about 80% (claim 31); wherein said empty capsid particles are present in said filtrate of step (h) in an amount of 10% or less (claim 34); wherein said filtrate of step (h) has no greater than about 10% aggregated rAAV particles (claim 36). The instant claims 16, 19, 31, 34, and 36 are directed to functional results. Given the method, including the steps and ingredients used, has been met, the functional results must also be met.
Claims 2-3: Wright et al 2013 (US20130072548A1) further teaches instant claim 2 and claim 3 added limitations, wherein said buffer of step (f)(2) comprises sodium chloride, and/or sodium phosphate (instant claim 2 limitation); wherein said buffer of step (f)(2) comprises sodium chloride from a concentration of about 5mM to a concentration of about 500 mM (instant claim 2 limitation) by disclosing finally AAV particles were eluted using a neutral buffered saline solution at a salt concentration of approximately 400 mM NaCl, providing sufficiently elevated ionic strength to disrupt binding of AAV particles from the chromatography resin (See, Example 2, para [0074]); AAV particles are eluted using a buffer of appropriate ionic strength. Suitable buffers include e.g., 10-50 mM sodium phosphate, preferably 15-40, such as 15, 20, 25, 30, 35, 40, etc. mM sodium phosphate containing salt, such as NaCl or KCl, at a concentration of e.g, 100-700 mM, such as 200-400 mM, e.g., 200, 300, 325, 350, 370, 380, 400, etc., or any concentration within these ranges (See, para [0056]; also see Sodium chloride salt concentrations used in AAV chromatography (See, para [0057], [0061]). This Wright et al 2013 teaches the claimed sodium chloride, and /or sodium phosphate buffer and sodium chloride concentration range from 5 mM to 500 mM.
Wright 2008 reviews manufacturing and characterizing AAV-based vectors for use in clinical studies; see whole document, including title. See p. 844, col. 1 for teaching that the release of vector can be enhanced by elevated ionic strength (e.g. 300 mM NaCl), likely by inhibiting ionic bond formation between vector particles and cell debris. See p. 845, col. 1 for teaching that nonspecific adsorption of vectors to various surfaces, including plastics, glass, metal, etc., may occur and the inclusion of a surfactant has been reported to prevent vector losses.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the claimed sodium chloride, and /or sodium phosphate buffer and sodium chloride concentration range from 5 mM to 500 mM buffer in step (f)(2) the TFF step in the method taught by Wright et al 2013 (US20130072548A1) to arrive at the inventions of claims 2-3. One would have been motivated to do so because Wright 2008 teaches that elevated ionic strength enhances the release of vector by inhibiting ionic bond formation between vector particles and debris. There would have been a reasonable expectation of success given the underlying materials and methods are widely known and commonly used as evidenced by the prior art that high concentrations of salt inhibit the binding of vector particles and debris, enhancing vector release. The invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time of the invention. One of the ordinary skills would have been apprised of a reasonable expectation of success to arrive at the invention of claims 2-3 given the combined teachings in the art by Wright et al 2013, Pierce et al 2006, Mehtali et al 2015 and Wright 2008 as recited supra.
Determining or adjusting concentration of a salt in buffer for diafiltration/microfiltration TFF process based on requirement for optimal performance is a routine laboratory approach. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382. Furthermore, according to section 2144.05 of the MPEP, 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.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages”).
Claims 53-60: The instant claims 53-60 (dependent on base claim 1) presented incorporating the added limitations are, wherein said harvest of step (a) is concentrated before step (b) to produce a concentrated harvest (limitation of claim 53); wherein said concentration is achieved by tangential flow filtration (limitation of claim 54); wherein said lysing of said harvest of step (b) is by microfluidization (limitation of claim 55); wherein the method comprises lysing said concentrated harvest to produce a lysate (limitation of claim 56); wherein the lysing of the concentrated harvest is by microfluidization (limitation of claim 57); wherein said column eluate of step (d) is concentrated to produce a concentrated column eluate (limitation of claim 58); wherein said concentration is achieved by tangential flow filtration (limitation of claim 59); wherein said concentrated column eluate produced is mixed with cesium chloride to produce a mixture (limitation of claim 60).
The recited added limitations of instant claim 53-60 are obvious variations of the steps taught by the combined teachings of Wright et al 2013 and additional prior arts as applied to the instant claim 1.
13. Claims 5-7, 10-11, 13, 67-72 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Wright et al 2013 (US20130072548A1 published 03/21/2013), Tomono et al 2016 (Molecular Therapy, Methods & Clinical Development, 3, 15058), Florencio et al 2015 (Molecular Therapy-Methods & Clinical Development, 2, 15024), Pierce et al 2006 (US20060194313A1 published 08/31/2006), Bennicelli et al 2008 (Mol Ther., 2008, 16(3):458-65), Wright 2008 (Gene Therapy, (2008) 15, 840–848), Mehtali et al 2015 (US20150275186A1, published 10/01/2015), and Fabre et al 2010 (US 2010/0260798A1 published 10/14/2010) as applied to claims 1 above and further in view of Chan et al 2016 (AU2014333884A1, published 05/12/2016) and Mertens et al 2015 (Soft Matter, 2015, 11(44):8621-31).
Claims 5-7, 10-11, 13 and 67-72: The instant claims 5-7, 10-11, 13 and 67-72 (dependent on claim 1 or 7) are directed to modify the instant claim 1 with limitations on non-ionic surfactants of step (f)(2) of bona fide recombinant AAV purification, inter alia, comprising polyalkylene glycol (instant claim 5 limitations); a block co-polymer comprising at least one polyethylene glycol block and at least one polypropylene glycol block (instant claim 6 limitation); an alkyl polyglycoside, cetostearyl alcohol, cetyl alcohol, cocamide DEA, cocamide MEA, decyl glucoside, ethoxylate, octylphenoxypolyethoxyethanol, polyethylene glycol monoisohexadecyl ether, glucosides, maltosides, monolaurin, octylphenoxypolyethoxyethanol, nonoxynols octaethylene glycol monododecyl ether, N-octyl beta-D-thioglucopyranosides, octyl glucosides, oleyl alcohol, PEG-10 sunflower glycerides, pentaethylene glycol monododecyl ether, poloxamers-, polyglycerol polyricinoleate, sorbitans, stearyl alcohol, 2- [4-(2,4,4-trimethylpentan-2-yl)phenoxy] ethanol, and polysorbate 80 (instant claim 7 limitations); cetomacrogol 1000, polysorbates; polyglycerol polyricinoleate, octadecanoic acid [2-[(2R,3S,4R)-3,4-dihydroxy-2-tetrahydrofuranyl]-2-hydroxyethyl] ester, octadecanoic acid [(2R,3S,4R)-2-[1,2-bis(1-oxooctadecoxy)ethyl]-4-hydroxy-3- tetrahydrofuranyl] ester; C8 to C22 long chain alcohols; substituted or unsubtituted octylphenol in which the substituents can include a polyethoxyethanol group or any other substituent that will form a non-ionic surfactant with octylphenol; polyethylene glycol monoisohexadecyl ether; dodecanoic acid 2,3-dihydroxypropyl ester; glucosides may include lauryl glucoside, octylglucoside and decyl glucoside; fatty acid amides; and nonionic surfactants that have a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophilic group (instant claim 10 limitations); a polyethylene glycol-block-polypropylene glycol-block-polyethylene glycol or a triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) (instant claim 11 limitations); wherein said non-ionic surfactant of step (f)(2) has a concentration of about 0.0001% to a concentration of about 0.1% (instant claim13 limitation).
The combined teachings of Wright et al 2013 (US20130072548A1) and the additional prior arts as applied to claim 1 supra teaches instant claim 1.
Wright et al 2013 further teaches use of surfactant in purified AAV particles formulation for filtration to remove any remaining impurities there by producing a highly purified AAV product; and in buffer used to wash the column after the clarified AAV cell lysate was applied to the resin in neutral buffered saline (salt concentration of approximately 200 mM NaCl) (i.e. the resin subjected to solutions that remove impurities, but not bound AAV particles) with a neutral buffered saline solution containing 5 mM sarkosyl (a surfactant) (See para [0010], [0074], [0075], claim 1). Sarkosyl is an ionic/anionic surfactant.
Wright et al 2013 does not teaches non-ionic surfactant in a bona fide recombinant AAV purification/preparation method.
Tomono et al 2016, as recited supra, inter alia, teaches NaCl salt buffer comprising 0.01% Pluronic F-68 (non-ionic surfactant) use for dual io-exchange for removal of impurities from rAAV preparation and to separate empty AAV particles from rAAV particles (See, Tomono et al 2016, page 2, abstract). Thus, Tomono et al 2016 teaches comprising 0.01% Pluronic F-68 (non-ionic surfactant) in a buffer prior to separation of empty AAV from bona fide rAAV particles.
Florencio et al 2015 teaches a simple downstream process based on a non-ionic surfactant that has a hydrophilic polyethylene oxide and an aromatic hydrophobic group, Triton-X-100 (instant claim 72 limitation) treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors (See, abstract, fig 1b, page 4 col 2, discussion section, para 1).
Pierce et al 2006 (US20060194313A1) teaches a non-ionic surfactant (e.g., a non-ionic block copolymer surfactant such as Pluronic F-68 or polyvinyl alcohol (PVA), polyethylene glycol (PEG), (See, para [0004], [0006]-[0007], [0013]).
The combined teachings of Tomono et al 2016, Pierce et al 2006 (US20060194313A1) and Mehtali et al 2015 (US20150275186A1) teaches the added limitation of instant claim 13, wherein said buffer of step (f)2 contains about 0.0001% to about 0.1% of a non-ionic surfactant. Tomono et al 2016, as recited supra, teaches a NaCl salt buffer comprising 0.01% Pluronic F-68 (non-ionic surfactant) for purification of bona fide rAAV particles. Pierce et al 2006 (US20060194313A1) is in the art of recombinant expressed protein, as recited supra, teaches non-ionic surfactant is Pluronic F-68 teaches concentration between 0.2 g/L (0.02% w/v) and 1 g/L (0.1% w/v) (See, abstract, para [0004], [0010], [0012], [0015], [0024], [0026]- [0028], claim 2, 8, 21, 23). Mehtali et al 2015 (US20150275186A1) is in the analogous virus purification art directed to industrial production of viral vectors and teaches that a non-ionic surfactant, PLURONIC F-68, can be added to the medium as a de-foaming agent preferably between about 0.05 g/L (0.005% w/v) and about 10 g/L, typically between about 0.1 (0.01% w/v) and about 5 g/L. The surfactant in cell culture medium may be decrease the size of the cell clumps (prevent to enlarge the size of the cell clumps) (See, para [0076]).
Chan et al 2016 (AU2014333884A1) teaches filtration of a virus, influenza virus, and virus-like particles (VLPs) using a membrane filter pre-treated with a surfactant, and a higher yield of recovery of the biological product (a virus or VLP) in the filtrate is achieved as compared to a non-pretreated filter control. The surfactant is preferably a non-ionic surfactant. The surfactant is preferably a hydrophilic surfactant. Preferred surfactants of the invention have a HLB (hydrophile/lipophile balance) of at least 10, preferably at least 15, and more preferably at least 16. The surfactant may be selected from Cetomacrogol 1000, Cetostearyl alcohol (instant claim 70 limitation), Cetyl alcohol, Cocamide DEA, Cocamide MEA (instant claim 71 limitation), Decyl glucoside, IGEPAL CA-630, Isoceteth-20, Lauryl glucoside, Monolaurin, Narrow range ethoxylate, Nonidet P-40, Nonoxynol-9, Nonoxynols, NP-40 (instant claim 67 limitation), Octaethylene glycol monododecyl ether, Octyl glucoside, Oleyl alcohol, Pentaethylene glycol monododecyl ether, Poloxamer, Poloxamer 407, Polyglycerol polyricinoleate, Polysorbate, Sorbitan monostearate, Sorbitan tristearate, Stearyl alcohol, and Triton X-100. In one aspect, the surfactants chosen should reduce hydrophobic interactions and …….. yet does not split or disrupt the proteins or its immunogenicity. It is particularly preferred that the surfactant is a polysorbate. Possible polysorbates are polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80 (instant claim 69 limitation). A particularly preferred form of polysorbate is polysorbate 80, which is also known as Tween 80 (See, para [39]-[56]). Chan et al 2016 (AU2014333884A1) also teaches a few non-ionic surfactants of claims 5-7, 10-11 as claimed and recited in claims above.
Mertens et al (2016) low to moderate amounts of mild non-ionic surfactants such as Tween 20 are even likely to enhance virus dispersivity, while potentially increasing their infectivity to some extent (See, page 10, para 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to the modify the prior art teachings of Wright et al 2013 (US20130072548A1) to incorporate an appropriately compatible non-ionic surfactant taught by Pierce et al, Florencio et al, Chan et al, Tomono et al, Mertens et al including Pluronic F-68 to a buffer used in a diafiltration step as part of a microfiltration process tangential flow filtration (TFF) in the method taught by Pierce et al 2006 (US20060194313A1). One of ordinary skill in the art would have been motivated to do so for the prevention of filter blinding/blockage in the clarification train through pore plugging and other mechanisms and the discovery that the addition of a non-ionic surfactant, e.g., Pluronic® F68, polyethylene glycol, or other non-ionic block copolymer surfactants, to the buffer used in a diafiltration step as part of a microfiltration process (e.g., tangential flow filtration; TFF) can reduce turbidity of the process fluid (See, Pierce et al 2006 (US20060194313A1), para [0004], [0012], [0027]) and for the advantage of achieving consistent and quantitative recovery of the vector by preventing aggregation. Mehtali et al 2015 teaches the concentration of non-ionic surfactant 0.005% to 0.01% w/v (See para [0076]). Further, Wright (2008) teaches that the inclusion of a surfactant prevents vector losses nonspecific adsorption of vectors to plastics, glass, metal and other surfaces during storage and handling of vector may occur. Inclusion of a surfactant in the drug product has been reported to prevent vector losses. One of the ordinary skills would have been apprised of a reasonable expectation of success to arrive at the invention of claim 1 given the teachings in the art by Wright et al 2013, Pierce et al 2006, Mehtali et al 2015 and Wright 2008 as recited supra.
The non-ionic surfactant concentration taught my Mehtali et al 2015 and Pierce et al 2006 were sufficient to prevent aggregation of protein or virus in a buffer that had the desired properties [prevention of protein or virus or cell aggregates]. Therefore, determining or adjusting concentration of a non-ionic surfactant or buffer salt for diafiltration/microfiltration TFF process based on requirement for optimal performance is a routine laboratory approach. See In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997); In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955); Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382.
Furthermore, according to section 2144.05 of the MPEP, 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.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages”).
Claims 21-26 and 28: The combined teachings of Wright et al 2013 (US20130072548A1) and the additional prior arts as applied to claim 1 supra teaches instant claim 1.
Wright et al 2013 further teaches added limitations of instant claims 21-26 and 28 (dependent on claim 1);
Claim 21: The added limitation of instant claim 21 limitation, wherein said bona fide rAAV vector particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 are taught by Wright et al 2013 (See, US20130072548A1, claim 6, para [0012], [0023], [0054], [0069]).
Claim 22: The added limitation of instant claim 22 limitation, wherein said bona fide rAAV particles comprise a transgene that encodes an inhibitory nucleic acid; and
Claim 23: The instant claim 23 limitation, wherein said inhibitory nucleic acid is selected from the group consisting of siRNA, an antisense molecule, miRNA, ribozyme and shRNA, are disclosed by Wright et al 2013 as said transgene encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, and a miRNA a ribozyme and a shRNA (See, US20130072548A1, claim 8, para [0014], [0046]).
Claim 24: The added limitations of instant claims 24, wherein said bonafide rAAV particles comprise a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGF3, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase are disclosed and taught by Wright et al 2013 (See, US20130072548A1, claim 9, para [0046]).
Claim 25: The added limitation of instant claims 25, wherein said bonafide rAAV particles comprise a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors a and 3, interferons a,3, and y, stem cell factor, flk-2/flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules are disclosed and taught by Wright et al 2013 (See, US20130072548A1, claim 10, para [0047]).
Claim 26: The added limitation of instant claims 26, wherein said bonafide rAAV vector particles comprise a transgene encoding a protein useful for correction of in born errors of metabolism selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha- 1 antitrypsin, glucose-6- phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta- synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence are disclosed and taught by Wright et al 2013 (See, US20130072548A1, claim 11, para [0048]).
Claim 28: The added limitation of instant 28, wherein said bonafide rAAV vector particles comprise a transgene encoding a protein useful for correction of neurodegenerative diseases.
Wright et al 2013 further teaches AAV vector expressing a transgene to treat blindness administered to the eye, a dose of 1×1012 AAV vectors expressing a transgene for Parkinson's Disease administered to CNS thus renders obvious instant claim 28 (See, US20130072548A1, para [0070], [0074]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to the modify the combined prior art teachings of Wright et al 2013 (US20130072548A1) and additional recited prior arts as applied to claim 1 and incorporate further teachings of Wright et al 2013 on different serotypes of AAV to construct rAAV vectors for delivery of different transgenes encoding functional therapeutic molecules (protein molecules, hormones or cytokines) directed for treatment of different diseases or immunomodulation or encoding therapeutic RNA molecules (siRNA/shRNA/miRNA, ribozymes) for silencing of undesired target gene transcripts/translation. One of ordinary skill in the art would have been motivated to do develop rAAV gene therapy vectors for treatment of diseases and for commercial success. One of the ordinary skills would have been apprised of a reasonable expectation of success to arrive at the claimed inventions given the combined prior art teachings as applied to the claims as recited supra and rAAV gene therapy art.
14. Claims 47, 49, and 61-66 are rejected under 35 U.S.C. 103 as being unpatentable over combined teachings of Wright et al 2013 (US20130072548A1 published 03/21/2013), Tomono et al 2016 (Molecular Therapy, Methods & Clinical Development, 3, 15058), Florencio et al 2015 (Molecular Therapy-Methods & Clinical Development, 2, 15024), Pierce et al 2006 (US20060194313A1 published 08/31/2006), Bennicelli et al 2008 (Mol Ther., 2008, 16(3):458-65), Wright 2008 (Gene Therapy, (2008) 15, 840–848), Mehtali et al 2015 (US20150275186A1, published 10/01/2015), and Fabre et al 2010 (US 2010/0260798A1 published 10/14/2010) as applied to claims 1 above and further in view of Wilson et al 2004 (Human Gene Therapy, 13:1921–1934), Wolff et al 2011 (Expert Rev. Vaccines 10(10), 1451–1475), Doria et al 2014 (Human Gene Therapy Methods, 24:392–398).
Claim 47, 49, 61-66: The instant claims 47, 49, 61-66 (dependent on claim 1) recites added limitations: wherein the tangential flow filtration of step (g) is carried out at a trans-membrane pressure of between about 2-15 psig (claim 47); wherein the tangential flow filtration of step (g) is carried out at a shear rate greater than about 3000 sec-1 (claim 49); wherein the tangential flow filtration is carried out at a trans-membrane pressure of between 2-15 psig (claim 61); wherein the tangential flow filtration is carried out at a trans-membrane pressure of between 4-12 psig (claim 62); wherein the tangential flow filtration is carried out at a shear rate greater than 3000 sec-1 (claim 63); wherein the tangential flow filtration is carried out at a shear rate greater than 5000 sec-1 (claim 64); wherein the tangential flow filtration is carried out at a shear rate between 5000-15,000 sec-1 (claim 65); wherein the tangential flow filtration is carried out at a shear rate between 8000-12,000 sec-1 (claim 66).
The combined teachings of Wright et al 2013 (US20130072548A1) and the additional prior arts as applied to claim 1 supra teaches instant claim 1. However, does not explicitly teach the tangential flow filtration (TFF) shear force and trans-membrane pressure parameters.
Wilson et al 2004 teaches scalable method for the purification of recombinant AAV from cell culture, for gene therapy, comprising tangential flow filtration (TFF) for ultrafiltration and diafiltration process and teaches all TFF steps were operated at a trans-membrane pressure of 10–15 psi (See, abstract and methods, entire article).
Doria et al 2014 teaches a simple method comprising TFF of the medium containing AAV2/8 vectors to purify research-grade vectors for use in animal models. The transmembrane pressure of 10–12 psi maintained throughout the procedure (See, abstract and methods, entire article).
Wolff et al 2011 reviewed downstream processing of cell culture-derived virus particles. Wolff et al teaches- important for concentration of active virus particles is a gentle processing for which TMP and retentate flux have to be optimized for low wall shear rates during filtration. The key parameters for an ultrafiltration process are the transmembrane pressure (TMP), feed (retentate) and permeate flux as well as the nominal molecular weight limit, also sometimes referred to as molecular weight cutoff.
It would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to the modify the prior art teachings of Wright et al 2013 (US20130072548A1) to alter different parameters in the method described by the combined teachings,