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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Sep. 7, 2024. Claims 1-13 are pending and currently examined.
Specification
The use of the terms, such as the Eshmuno® CMX column (see [0044]), the AKTA™ system (see [0044]), and Natrix® CH membrane device (see [0223]), etc., which are trade names or marks used in commerce, have been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objection
Claims 2, 10, 11 and 13 are objected to for the following abnormalities:
1. These claims recite “Method according to claim……” which should be changed to “The method according to claim……”.
2. These claims recite “whereby”, which should be changed to “wherein”.
3. Claims 2, 11 and 13 lack punctuation, as well as a conjunction phrase connecting the steps.
4. Claim 10 recites the term of “HCPs”. This term is not immediately apparent for what it refers to. It appears to be an abbreviation and must be spelled out.
Applicant is required to make proper corrections.
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.
Claims 1-13 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention. This rejection has the following grounds.
A. Claim 1 recites “A method for purifying adeno-associated virus (AAV) particles by contacting a sample comprising said AAV particles with a mixed mode cation exchange chromatography matrix.” This claim lacks essential steps. See MPEP § 2172.01. First, by reciting the limitation “by contacting a sample comprising said AAV particles with a mixed mode cation exchange chromatography matrix”, it is not clear if the claimed method requires the act of “contacting” as an active step. Applicant may consider using the wording “A method for purifying adeno-associated virus (AAV) particles, comprising contacting a sample comprising said AAV particles with a mixed mode cation exchange chromatography matrix.” Secondly, the claim only specifies a contacting step, omitting an essential step that is required to produce purified AAV particles.
B. Claim 2 depends from itself. Additionally, claim 2 lacks a conjunction phrase to link the recited steps. Therefore, it is not clear what its metes and bounds are. To facilitate examination, claim 2 is considered as depending from claim 1, and the recited steps are linked by “and”.
C. Claims 2-10 and 12-13 recite “whereby” which renders to claims indefinite because it is not clear how the limitations following this word further limit the claims. If it is intended that the limitations are required by the claims, Applicant may consider using word “wherein”.
D. Claims 3, 9, and 10 depend from claim 1 and recite “step c)” and/or “step a)”. However, claim 1 does not specify “step c)” and “step a)”. Therefore, it is not clear what metes and bounds of these claims are.
E. Claim 6 recites “whereby is a hydrogel membrane” which renders the claim indefinite. To facilitate examination the limitation is considered as if it were “wherein the chromatography matrix is a hydrogel membrane”.
F. Claims 11 and 13 lack conjunction phrase to link the recited steps. To facilitate examination, claims 11 and 13 are considered as if the recited steps were linked by “and”.
G. Claim 10 recites the term “HCPs”. The specification does not define it, and, thus it is not clear what it refers to. To facilitate examination, the term “HCPs” is interpreted as abbreviation for “host cell proteins”.
H. Claim 13 recites “e.g. RNase and/or DNase” which renders the claim indefinite because it is not clear if this limitation is required or just exemplary.
It is noted that any interpretation of the claims set forth above does not relieve Applicant of the responsibility of responding to this rejection. If the actual interpretation of the claims is different than that posited by the Examiner, additional rejections and art may be readily applied in a subsequent final Office action.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 2, 5, 8, 11 and 13 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by O'Riordan et al. (J Gene Med 2000; 2: 444-454), as evidenced by Moleirinho et al. (Expert Opinion on Biological Therapy, 2020, 20:5, 451-465).
These claims are directed to a method for purifying adeno-associated virus (AAV) particles by contacting a sample comprising said AAV particles with a mixed mode cation exchange chromatography matrix.
Clarification and interpretation of the claims are discussed in the 112b rejection above.
O'Riordan teaches that traditionally AAV has been purified from cell lysates using CsCl gradients, and this approach however is not likely to be useful in large-scale manufacturing; O'Riordan teaches that the preferred method for the purification of rAAVbgal includes treatment of virally infected cell lysates with both trypsin and nuclease followed by ion exchange chromatography using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine sulphate affinity chromatography. See Abstract.
O'Riordan teaches that cell lysates from 293 cells infected with adeno-associated virus (in the presence of Ad5ts149) were chromatographed on a hydroxyapatite resin (40 ml) which was preequilibrated with 10 mM sodium phosphate pH 6.4, containing 10 mM NaCl, 0.1% Tween-80, 10% glycerol and 2 mM MgCl2. rAAVbgal was applied to the resin in the same buffer. Bound proteins were eluted from the resin using a linear salt gradient (120 ml) of 10-400 mM sodium phosphate at pH 6.4. Fractions collected from the resin were assayed for rAAVbgal proteins by immunoblotting using an antibody against the three capsid proteins of rAAVbgal, VP1, VP2 and VP3 (Catalog 03- 65158, American Research Products, Belmont, MA, USA). See page 446, left column, para 2.
O'Riordan teaches that a DEAE Macroprep resin (BioRad) (5 ml) was equilibrated with 10 mM sodium phosphate buffer containing 50 mM NaCl, 10% glycerol, pH 7.5. rAAVbgal-containing fractions eluted from the hydroxyapatite column were pooled and dialyzed into the same buffer used for equilibration of the DEAE resin. A linear gradient (50 mM±1 M NaCl in 10 mM sodium phosphate, pH 7.5, 10% glycerol, 0.05% Tween-80) was applied to the resin at a flow rate of 5 ml/min, the volume of the gradient was 50 ml. Bound proteins were eluted from the resin and collected in 2.5-ml fractions. Each fraction was assayed for rAAVbgal proteins and contaminating adenoviral proteins (Coomassie blue staining and immunoblotting). See page 446, left column, para 3.
O'Riordan teaches that Cellufine sulphate resin (3 ml column volume) was equilibrated with phosphate buffer saline (PBS) containing 10% glycerol. Fractions eluted from the DEAE resin containing both rAAVbgal proteins and DNA were pooled and applied to the resin at a flow rate of 4 ml/min. The resin was washed with 250 mM NaCl and a linear salt gradient (0.25-1 M NaCl in PBS/10% glycerol) was applied. The volume of the gradient was 10 ml. The eluted fraction and the flow through were analyzed for both rAAVbgal and Ad5ts149 proteins (immunoblotting) and infectivity (titer analysis). The final fraction was also assayed for rAAVbgal DNA by slot blot analysis. See page 446, left column, para 4.
Accordingly, O'Riordan teaches a method for purifying AAV particles comprising contacting a cell lysate sample containing AAV particles with a hydroxyapatite chromatography, followed by washing, elution and dialysis, and two succeeding different chromatography with DEAE Macroprep resin and Cellufine sulphate resin, respectively. O'Riordan is silent on if any of the three chromatography resins (matrices) is a mixed mode cation exchange chromatography matrix.
Moleirinho teaches that mixed-mode chromatography (MMC) is based on a combination of different binding mechanisms such as, for example, ligands that combine electrostatic and hydrophobic interactions; that Hydroxyapatite is one of the examples of MMC where the resins bind both positively charged functional groups and negatively charged phosphate groups, which have been used for the purification of adenovirus, dengue virus and retrovirus. See page 456, right column, para 3. According to Moleirinho, hydroxyapatite chromatography matrix is a mixed mode cation exchange chromatography matrix.
Therefore, O'Riordan teaches each and every aspect of claims 1, 2, 5, 8, 11 and 13.
Claims 1, 2, 5, 8, 11 and 13 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Qu et al. (Journal of Chromatography B, 990 (2015) 15–22), as evidenced by Moleirinho et al. (Expert Opinion on Biological Therapy, 2020, 20:5, 451-465).
Qu teaches that ceramic hydroxyapatite (CHT) chromatography resin with a polyethylene glycol (PEG) modulation has recently been used for rAAV1 and rAAV9 vectors. This study reports the use of CHT chromatography modulated by calcium ions instead of PEG for rAAV9 purification. Calcium-ion-containing buffers effectively improve the inclusion of CHT as a capture resin, the resin-binding capacity and the yield. A previously developed purification strategy consists of CHT followed by ANX anion exchange chromatography. The vector yield of this approach is approximately 70%, and a software analysis indicated a vector purity exceeding 98%. The residual host cell (HEK293) protein contents are 24.75 ± 2.32 ng and 67.21 ± 2.10 ng, and the Benzonase residue contents are 1.55 ± 0.10 pg and 1.95 ± 0.16 ng per 1013 vector genome copies (G.C.) separated by CHT/ANX and CsCl. See Abstract.
Qu teaches that mixed-mode ceramic hydroxyapatite resins (CHT type I 40 mm; Bio–Rad Laboratories, Inc. Hercules, CA, USA) were selected to capture rAAV9. Small-scale experiments were performed using an AKTA explorer FPLC system equipped with 2-ml resins packed in a 1-cm-diameter column running at a flow rate of 2 ml/min. Two experiments were conducted. In the first study, the cell lysate was mixed with CaCl2(30 ppm). A CHT chromatography column was pre-equilibrated with 5 column volumes (CV) of equilibration buffer (5 mM PB buffer containing Na2HPO4and NaH2PO4, 50 mMNaCl, 30 ppm Ca2+, pH 7.0), and 10 ml of cell lysate was then loaded after washing the column with 10 CV of equilibration buffer. A step gradient eluted the vectors with PB buffer (20 mM PB, 200 mM NaCl,pH 7.0). All of the solution was collected in a series of fractions and analyzed with qPCR or silver staining and a BCA assay. See page 16, right column, para 2.
Teachings of Moleirinho is set forth in the rejection above. It teaches that hydroxyapatite chromatography matrix is a mixed mode cation exchange chromatography matrix.
Accordingly, Qu teaches a method for purifying AAV particles comprising contacting a cell lysate sample containing AAV particles with a hydroxyapatite chromatography matrix (which is a mixed mode cation exchange chromatography matrix), combined with washing, elution, as well as other separation approaches, such as anion exchange chromatography (e.g. ANX).
Therefore, Qu teaches each and every aspect of claims 1, 2, 5, 8, 11 and 13.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102 of this title, 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.
Claims 1-13 are rejected under 35 U.S.C. 103 as being unpatentable over O'Riordan et al. (J Gene Med 2000; 2: 444-454), Qu et al. (Journal of Chromatography B, 990 (2015) 15–22), and Moleirinho et al. (Expert Opinion on Biological Therapy, 2020, 20:5, 451-465), in view of Schmidt et al. (J. Sep. Sci. 2014, 37, 5–13), Hou et al. (Biotechnol. Prog., 31:974–982, 2015), Henderson et al. (US 2022/0280633 A1, published on Sep. 8, 2022; PCT filed on Jul. 30, 2020).
Claim 1, 2, 5, 8, 11 and 13 are described and addressed in the 102 rejections above.
Claim 3 specifies that the elution buffer has a pH higher than the pH of the sample buffer. Claim 4 specifies that the method includes recovering AAV particles which flow through the chromatography matrix without binding to it. Claim 6 specifies that the chromatography matrix is a hydrogel membrane. Claim 7 specifies that the sample comprises empty and full AAV capsids and the empty AAV particles are partially or fully eluted prior to the full AAV particles. Claim 9 specifies that elution in step is performed with a linear pH gradient from a pH between 4 and 6.5 to a pH between 9 and 11 while conductivity is kept at a constant level. Claim 10 specifies that in one fraction of AAVs eluted in step c) HCPs are reduced with a log reduction of 2 log or more compared to the sample applied in step a) and the percentage of full AAVs is above 90%. Claim 12 specifies that lysis of the cells is done with a detergent selected from the group of alkyldimethylamine oxides and optionally sodium chloride.
Relevance of O'Riordan, Qu and Moleirinho are set forth supra.
For claims 3 and 9, O'Riordan, Qu and Moleirinho teach methods using buffers with fixed pH and salt gradients for elution.
Schmidt teaches studies on modeling of salt and pH gradient elution in ion-exchange chromatography. According to Schmidt, the separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed. See Abstract.
According to the teachings of Schmidt, chromatography elution with either salt gradient and pH gradient are known and practiced in the art at the time of invention for protein purification. It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to test different elution approaches known in the art, including pH gradient, to find an optimized elution condition. Additionally, such a combination, or a substitution of one element for another known in the field to have the same function for the same purpose, is evidence that the claimed invention may be found obvious. See MPEP 2144.06. Therefore, the instant invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the references, especially in the absence of evidence to the contrary.
Regarding claim 6, O'Riordan, Qu and Moleirinho teach using chronography columns which are monolith, and are silent on a hydrogel membrane.
Hou teaches a study on use of hydrogel membrane chromatography for monoclonal antibody purification. Hou teaches that membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. See abstract.
Accordingly, teachings of Hou indicate that hydrogel membrane is known in the art at the time of invention in the separation of proteins via membrane chromatography, and that such hydrogel membranes may be made from ion exchange resins.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to combine the teachings of O'Riordan, Qu and Hou to arrive at the invention as claimed. One would have been motivated to do so to evaluate the hydrogel membrane chromatography disclosed in Hou in the purification of AAV particles, in place of column chromatography.
Regarding claim 7, O'Riordan, Qu and Moleirinho are silent on if empty AAV particles are partially or fully eluted prior to the full AAV particles. However, O'Riordan and Qu teach or suggest that the samples to be purified for AAV particles contain both full and empty particles and that full particles which include the genome nucleic acid molecules are desired. One of skill in the art would have found it obvious to modify the elution strategy to arrive at a desired elution effect, such as which component is eluted earlier and which later.
Additionally, taken the teachings of O'Riordan, Qu and Moleirinho that various different elution conditions, it is reasonable to speculate that at one of the conditions would result in elution of empty AAV particles partially or fully prior to the full AAV particles. The Office does not have the facilities and resources to provide the factual evidence needed in order to establish that the elution conditions of O'Riordan and Qu does not elute empty AAV particles prior to full particles. In the absence of evidence to the contrary, the burden is on the applicant to prove that the claimed elution effect are different from those taught by the prior art and to establish patentable differences. See In re Best 562F.2d 1252, 195 USPQ 430 (CCPA 1977) and Ex parte Gray 10 USPQ 2d 1922 (PTO Bd. Pat. App. & Int. 1989).
Regarding claim 10, O'Riordan, Qu and Moleirinho are silent on if there is “one fraction” of AAVs eluted in the process contains the HCPs (host cell proteins) and full AAVs at the claimed amount and purity as claimed. One of skill would have found it obvious to fine-tune the chromatography process by adjusting various conditions, such as buffers, pH, salts, washing conditions, elution conditions, etc., to arrive at the effect as claimed through routine experimental optimization. It is noted that the claim is highly general in the claimed method. Thus, the claimed effect is considered as intended results that can be achieved by routine experimental optimization.
Regarding claim 12, O'Riordan, Qu and Moleirinho are silent on lysis of the cells with a detergent selected from the group of alkyldimethylamine oxides.
Henderson teaches an invention relating to virus-like particles such as synthetic enveloped VLPs or synthetic membrane VLPs and method of using and making them. See Abstract. It teaches that a lysis buffer that used in the invention was made up of 20 mM phosphate buffer (pH 7.4), 150 mM NaCl, and 2 mM MgC12 (to support Benzonase activity) and 2% LDAO detergent (n-Dodecyl-N,N-Dimethylamine-N-Oxide, Anatrace). See [0315]. It further teaches that lysis of VrS01 bearing cell pellets was then performed in 50 mM HEPES buffer (pH 7.5), 500 mM NaCl, 2 mM MgCl2 (to support Benzonase activity) and 2% LDAO detergent (n-DodecylN, N-Dimethylamine-N-Oxide, Anatrace). See [0401]. Here, the LDAO detergent (n-Dodecyl-N,N-Dimethylamine-N-Oxide, Anatrace) is a member of the group of alkyldimethylamine oxides.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the current invention to introduce the LDAO detergent used in Henderson into the studies of O'Riordan and Qu, who use different detergents in cell lysis. One would have been motivated to do so to compare the results of lysing cells using different detergents that are known to be functional in cell lysis.
Relevant Prior Art References Not Used in the Rejections
Gagnon et al. (Journal of Chromatography A 1649 (2021) 462210; submitted in IDS filed on Sep. 17, 2024).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL ALLEN, on (571) 270-3497, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/
Primary Examiner, Art Unit 1671
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