DETAILED ACTION
Disposition of Claims
Claims 1-24 and 48-77 are pending.
Examiner’s Note
All paragraph numbers (¶) throughout this office action, unless otherwise noted, are from the US PGPub of this application US20250035532A1, Published 01/30/2025.
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Information Disclosure Statement
The information disclosure statements (IDS) submitted on 12/19/2024 and 11/05/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Notably, the disclosure statement filed lists a Search Report. The listing of the references cited in a Search Report itself is not considered to be an information disclosure statement (IDS) complying with 37 CFR 1.98. 37 CFR 1.98(a)(2) requires a legible copy of: (1) each foreign patent; (2) each publication or that portion which caused it to be listed; (3) for each cited pending U.S. application, the application specification including claims, and any drawing of the application, or that portion of the application which caused it to be listed including any claims directed to that portion, unless the cited pending U.S. application is stored in the Image File Wrapper (IFW) system; and (4) all other information, or that portion which caused it to be listed. In addition, each IDS must include a list of all patents, publications, applications, or other information submitted for consideration by the Office (see 37 CFR 1.98(a)(1) and (b)), and MPEP § 609.04(a), subsection I. states, "the list ... must be submitted on a separate paper." Therefore, the references cited in the Search Report have not been considered. Applicant is advised that the date of submission of any item of information or any missing element(s) will be the date of submission for purposes of determining compliance with the requirements based on the time of filing the IDS, including all "statement" requirements of 37 CFR 1.97(e). See MPEP § 609.05(a).
Note: If copies of the individual references cited on the Search Report are also cited separately on the IDS (and these references have not been lined-through) they have been considered.
Specification
The use of the terms POROS™; CAPTURESELECT™, and SEPHAROSE™, which are trade names or marks used in commerce, have been noted in this application. The terms 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 Objections
Claim 1 is objected to because of the following informalities: the definition of the abbreviation “AAV” is not provided. For clarity, it is requested that the first recitation of an abbreviation within a claim set be preceded by its full-length name (i.e. … adeno-associated virus (AAV)...).
Appropriate correction is required.
Claims 22 and 70 are objected to because of the following informalities: Claim 22 is drawn to the method of claim 1, further comprising quantifying empty capsids, partially filled capsids, and/or full capsids of said at least one AAV vector. However, it is not technically correct to say that “empty capsids” would be a “vector”, so the suggested wording that would be more appropriate would be along the lines of “quantifying empty, partially filled, and/or full AAV capsids in said sample”. Claim 70 is objected to for similar reasoning.
Appropriate correction is required.
Claim 66 is objected to because of the following informalities: the claim recites “wherein in said rotor” and it should be “wherein said rotor”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b); Second Paragraph
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.
Claim 1 and dependent claims 2-24 thereof are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is drawn to a method of characterizing at least one AAV vector in a crude sample or in a sample containing at least one impurity, comprising:
(a) subjecting a sample containing at least one AAV vector to affinity purification to produce an enriched sample;
(b) contacting said enriched sample to a salt solution to form a homogeneous solution; and
(c) subjecting said homogeneous solution to density gradient equilibrium analytical ultracentrifugation to characterize the at least one AAV vector. However, it is unclear whether “a sample containing at least one AAV vector” recited in part (a) is the “crude sample” or “sample containing at least one impurity” recited in the preamble. Step (a) introduces “a sample” without clear reference to either sample recited in the preamble through the use of the indefinite article “a”, and therefore it is unclear whether the sample subjected to affinity purification is required to be crude or contain at least one impurity. One suggestion would be to amend the claim along the lines of “(a) subjecting said crude sample or said sample containing at least one impurity, wherein the sample contains at least one AAV vector, to affinity purification to produce an enriched sample;” or “A method of characterizing at least one AAV vector in a sample that is crude or contains at least one impurity, comprising: (a) subjecting said sample to affinity purification…”
Since a skilled artisan would not be reasonably apprised as to the metes and bounds of the claimed invention, instant claim 1 is rejected on the grounds of being indefinite. Claims 2-24 are also rejected since they depend from claim 1, but do not remedy these deficiencies of claim 1
Claims 6 and 54 and dependent claims 7 and 55 thereof are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 6 recites “wherein said affinity ligand comprises an antigen-binding protein, an antibody, a variant thereof, or a fragment thereof.” However, it is unclear if “a variant thereof” and/or “a fragment thereof” modify only the antibody, only the antigen-binding protein, or both.
Claim 54 is rejected for similar reasoning.
For at least these reasons, claims 6 and 54 are rejected on the grounds of being indefinite. Claims 7 and 55 are rejected for depending upon claim 6 or 55 but for not clarifying the metes and bounds of claim 6 or 55.
Claims 7 and 55 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 7 contains the trademarks/trade names POROS™, CAPTURESELECT™, and SEPHAROSE™. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe components of affinity purification, namely resins, and, accordingly, the identifications/descriptions are indefinite.
Claim 55 is rejected for similar reasoning.
For at least these reasons, claims 7 and 55 are rejected on the grounds of being indefinite.
Claims 9 and 57 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 9 comprises the limitations of “wherein a wash buffer used in the wash step(s) comprises about 1×TBS, about 2×TBS, about 1×TBS with 20% ETOH, about 1×PBS, about 2×PBS, about 1×PBS with 18% EtOH, or combinations thereof.” The term “about” in claim 9 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While the specification defines “about” at ¶[0110], this definition does not provide clarity as to the metes and bounds as to what would be considered the upper and lower limits of “about” numerically.
Additionally, Claim 8 permits different buffers used in different wash steps. With the use of “combinations thereof”, it is unclear if this means that in the first wash, 1xTBS is used while in the second wash, 1x PBS is used. Conversely, the “combination thereof” may refer to different combinations of components in the wash buffer (e.g. a wash buffer with 1x PBS and 1xTBS). Therefore, it is unclear as to what the “combinations thereof” modifies and how it affects the metes and bounds of the claim.
Claim 57 is rejected for similar reasoning.
For at least these reasons, claims 9 and 57 are rejected on the grounds of being indefinite.
Claims 12 and 60 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 12 is drawn to the method of claim 10, wherein said elution buffer comprises about 50 mM glycine and about 0.01% poloxamer 188 at a pH of about 3 or about 100 mM glycine and about 0.01% poloxamer 188 at a pH of about 2. Due to the punctuation in the claim, it is unclear if the alternate options are 1) about 50 mM glycine and 2) about 0.01% poloxamer 188 at pH of about 2 or about 3; OR 1) about 50 mM glycine, 2) about 0.01% poloxamer 188 at a pH of about 3, or 3) about 0.01% poloxamer 188 at a pH of about 2.
Furthermore, the term “about” in claim 12 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While the specification defines “about” at ¶[0110], this definition does not provide clarity as to the metes and bounds as to what would be considered the upper and lower limits of “about” numerically.
Claim 60 is rejected for similar reasoning.
For at least these reasons, claims 12 and 60 are rejected on the grounds of being indefinite.
Claims 14-15 and 63-64 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 14 is drawn to the method of claim 1, wherein a concentration of salt in said salt solution is about 1.30 g/mL to about 1.40 g/mL or about 1.32 g/mL to about 1.36 g/mL, while claim 15 depends upon claim 14 and limits to wherein said salt concentration is about 1.34 g/mL. The term “about” in claims 14-15 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While the specification defines “about” at ¶[0110], this definition does not provide clarity as to the metes and bounds as to what would be considered the upper and lower limits of “about” numerically.
Claims 63-64 are rejected for similar reasoning.
For at least these reasons, claims 14-15 and 63-64 are rejected on the grounds of being indefinite.
Claims 17 and 66 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.
Claims 17 and 66 provide for “wherein said rotor speed is about 40 k rpm”. The term “about” in claim 17 is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While the specification defines “about” at ¶[0110], this definition does not provide clarity as to the metes and bounds as to what would be considered the upper and lower limits of “about” numerically.
Claim 66 is rejected for similar reasoning.
For at least these reasons, claims 17 and 66 are rejected on the grounds of being indefinite.
Claims 18 and 68 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.
Claims 18 and 68 are drawn to the method of claim 1, wherein said at least one AAV vector comprises a serotype selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, variations thereof and combinations thereof. The claim does not define what constitutes a “variation” of any of AAV1-AAV12. Further, it is unclear whether “combinations thereof” refers to a sample containing separate AAV vectors of two or more serotypes, a single AAV vector having components or characteristics derived from more than one serotype, or some other combination. Therefore, the metes and bounds of the claimed AAV serotypes cannot be determined with reasonable certainty.
For at least these reasons, the metes and bounds of claims 18 and 68 are unclear.
Claims 19-20 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.
Claims 19 and 20 recite the limitation "said sample". There is insufficient antecedent basis for this limitation in the claim, as multiple samples are recited in claim 1. With the suggestions offered supra to amend claim 1, this should allow for proper antecedent basis of “said sample”, but as the claims are currently drafted, it is unclear which sample is being referenced.
For at least these reasons, claims 19 and 20 are rejected on the grounds of being indefinite.
Claims 21 and 69 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 21 is drawn to the method of claim 1, wherein a concentration of said at least one AAV vector in said sample is from 1×1010 to 1×1012 cp/mL. However, it is unclear if the “1010” and “1012” is meant to be an exponential (e.g. “10^10” or “1010”) or if said concentration is meant to be multiplied by that exact number. Claim 69 is rejected for similar reasoning. For the purpose of prior art, the claim will be interpreted as reading on an exponential, but the claims must be amended for further clarification.
For at least these reasons, claims 21 and 69 are rejected on the grounds of being indefinite.
Claims 24 and 72 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 24 is drawn to the method of claim 1, further comprising detecting and/or quantifying high molecular weight species. However, it is unclear what the “high molecular weight species” encompass. The claim does not specify whether the high molecular weight species are high molecular weight species of the recited AAV vector, such as aggregated AAV capsids, or other high molecular weight species present in the sample. Accordingly, the scope of the material being detected and/or quantified cannot be determined with reasonable certainty.
Claim 72 is rejected for similar reasoning.
For at least these reasons, claims 24 and 72 are rejected on the grounds of being indefinite.
Claim 48 and dependent claims 49-74 thereof are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 48 is drawn to a method for characterizing a viral vector, comprising:
(a) subjecting cells that recombinantly produce a viral vector to lysis to form a lysed sample;
(b) subjecting said lysed sample to filtration to form a harvest pool;
(c) subjecting said harvest pool to affinity purification to form an enriched viral vector sample; and
(d) subjecting said enriched viral vector sample to density gradient equilibrium analytical ultracentrifugation to characterize said viral vector. However, the “a viral vector” in part (a) does not have clear antecedent basis as it uses the indefinite article “a” instead of the definite article “the” or “said” to refer back to the viral vector recited in the preamble. Therefore, it is unclear if the viral vector of part (a) is the same viral vector that is characterized or a different viral vector.
Since a skilled artisan would not be reasonably apprised as to the metes and bounds of the claimed invention, instant Claim 48 is rejected on the grounds of being indefinite. Claims 49-74 are also rejected since they depend from claim 48, but do not remedy these deficiencies of claim 48.
Claim 53 and dependent claims 54-57 thereof are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 53 recites “wherein said affinity purification comprises contacting said sample to a resin conjugated to an affinity ligand.” However, claim 48, upon which claim 53 depends, recites multiple samples throughout the claim, so it is unclear as to which sample claim 53 is referencing.
Since a skilled artisan would not be reasonably apprised as to the metes and bounds of the claimed invention, instant Claim 53 is rejected on the grounds of being indefinite. Claims 54-57 are also rejected since they depend from claim 53, but do not remedy these deficiencies of claim 53.
Claims 69-70 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.
Claims 69 and 70 recite “said at least one viral vector” in the claim, but claim 48 recites “a viral vector” in the preamble and part (a), so the antecedent basis of “at least one viral vector” is unclear because the numerical value of “vector” fails to agree with the two different vectors recited in claim 48, and as set forth supra there are two different viral vectors recited (preamble and part (a)) so it is unclear which vector is being referenced.
For at least these reasons, claims 69-70 are rejected on the grounds of being indefinite.
Claim 75 is 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.
The “a viral vector” in part (a) of claim 75 does not have clear antecedent basis as it uses the indefinite article “a” instead of the definite article “the” or “said” to refer back to the viral vector recited in the preamble. Therefore, it is unclear if the viral vector of part (a) is the same viral vector that is characterized or a different viral vector, and it is unclear if the “said viral vector” in part (b) refers back to the viral vector of part (a) or the preamble.
For at least these reasons, the metes and bounds of the claim are unclear.
Claim 76 and dependent claim 77 thereof 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.
The “a viral vector” in part (a) of claim 76 does not have clear antecedent basis as it uses the indefinite article “a” instead of the definite article “the” or “said” to refer back to the viral vector recited in the preamble. Therefore, it is unclear if the viral vector of part (a) is the same viral vector that is characterized or a different viral vector, and it is unclear if the “said viral vector” in part (b) refers back to the viral vector of part (a) or the preamble.
For at least these reasons, the metes and bounds of the claim are unclear. Claim 77 is rejected for depending upon claim 76, but not clarifying the metes and bounds of claim 76.
Claim 77 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 77 is indefinite because it is unclear what is meant by the “empty/full ratio of said viral vector” and the “empty/partially filled/full ratio of said viral vector.” The specification defines a viral vector as a recombinant virus comprising a nucleic acid to be delivered into a host cell, whereas an empty capsid lacks packaged transgene(s) (¶[0006][0127]). It is therefore unclear whether the claim refers to ratios of empty, partially filled, and full viral capsids within a preparation of the recited viral vector, or to some other ratio attributable to the viral vector itself. One suggestion would be to amend the claim along the lines of the following: “…wherein said critical quality attributes include a ratio of empty viral capsids to full viral capsids in said enriched sample, a ratio of empty, partially filled, and full viral capsids in said enriched sample…”
For at least these reasons, the metes and bounds of claim 77 are unclear.
Claim Interpretation
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art.
NB: “Small-scale” is defined by the specification at ¶[0134] as experimental volumes ranging from 1 uL to 10 mL.
“Medium throughput” is defined by the specification at ¶[0135] as processing 5 to 50 or more samples at one time.
Claim 1 is drawn to a method of characterizing at least one AAV vector in a crude sample or in a sample containing at least one impurity, comprising:
(a) subjecting a sample containing at least one AAV vector to affinity purification to produce an enriched sample;
(b) contacting said enriched sample to a salt solution to form a homogeneous solution; and
(c) subjecting said homogeneous solution to density gradient equilibrium analytical ultracentrifugation to characterize the at least one AAV vector.
Further limitations on the method of claim 1 are wherein said affinity purification comprises affinity chromatography (claim 2); wherein the affinity purification is a small-scale affinity purification (claim 3); wherein the affinity purification occurs in a medium throughput manner (claim 4); wherein said affinity purification comprises contacting said sample to a resin conjugated to an affinity ligand (claim 5), wherein said affinity ligand comprises an antigen-binding protein, an antibody, a variant thereof, or a fragment thereof (claim 6), wherein said resin is POROS™ CaptureSelect™ AAVX Affinity Resin, POROS™ CaptureSelect™ AAV8 Affinity Resin, POROS™ CaptureSelect™ AAV9 Affinity Resin, AVB Sepharose™, or a combination thereof (claim 7); further comprising subjecting said resin to at least one wash step, at least two wash steps, at least three wash steps, or at least four wash steps (claim 8), wherein a wash buffer used in the wash step(s) comprises about 1×TBS, about 2×TBS, about 1×TBS with 20% ETOH, about 1×PBS, about 2×PBS, about 1×PBS with 18% EtOH, or combinations thereof (claim 9); wherein said affinity purification comprises an elution step (claim 10), wherein said elution step includes an elution buffer, optionally wherein said elution buffer comprises glycine, poloxamer 188, or a combination thereof (claim 11), wherein said elution buffer comprises about 50 mM glycine and about 0.01% poloxamer 188 at a pH of about 3 or about 100 mM glycine and about 0.01% poloxamer 188 at a pH of about 2 (claim 12); wherein said salt solution includes cesium chloride, cesium sulfate, cesium bromide, cesium formate, cesium acetate, cesium iodide, cesium selenite, potassium bromide, rubidium bromide, rubidium chloride, cesium TFA, sodium iothalamate, or a combination thereof (claim 13); wherein a concentration of salt in said salt solution is about 1.30 g/mL to about 1.40 g/mL or about 1.32 g/mL to about 1.36 g/mL (claim 14), wherein said salt concentration is about 1.34 g/mL (claim 15); wherein a rotor speed of the analytical ultracentrifuge is from 10 k to 60 k rpm (claim 16), wherein said rotor speed is about 40 k rpm (claim 17); wherein said at least one AAV vector comprises a serotype selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, variations thereof and combinations thereof (claim 18); wherein said sample is a crude sample (claim 19); wherein said sample comprises a harvest pool or a tangential flow filtration pool (claim 20); wherein a concentration of said at least one AAV vector in said sample is from 1×10^10 to 1×10^12 cp/mL (claim 21); further comprising quantifying empty capsids, partially filled capsids, and/or full capsids of said at least one AAV vector (claim 22); further comprising quantifying a ratio of absorbance at 260 nm to absorbance at 280 nm wavelengths of AAV vector species separated by density gradient equilibrium analytical ultracentrifugation (claim 23); and further comprising detecting and/or quantifying high molecular weight species (claim 24).
Claim 48 is drawn to a method for characterizing a viral vector, comprising:
(a) subjecting cells that recombinantly produce a viral vector to lysis to form a lysed sample;
(b) subjecting said lysed sample to filtration to form a harvest pool;
(c) subjecting said harvest pool to affinity purification to form an enriched viral vector sample; and
(d) subjecting said enriched viral vector sample to density gradient equilibrium analytical ultracentrifugation to characterize said viral vector.
Further limitations on the method of claim 48 are wherein the method is further comprising subjecting said harvest pool to tangential flow filtration prior to step (c)(claim 49); wherein the affinity purification is a small-scale affinity purification (claim 50); wherein the affinity purification occurs in a medium throughput manner (claim 51); wherein said affinity purification comprises affinity chromatography (claim 52), further comprising detecting and/or quantifying high molecular weight species (claim 72); wherein said affinity purification comprises contacting said sample to a resin conjugated to an affinity ligand (claim 53), wherein said affinity ligand comprises an antigen-binding protein, an antibody, a variant thereof, or a fragment thereof (claim 54), wherein said resin is POROS™ CaptureSelect™ AAVX Affinity Resin, POROS™ CaptureSelect™ AAV8 Affinity Resin, POROS™ CaptureSelect™ AAV9 Affinity Resin, AVB Sepharose™, or a combination thereof (claim 55), further comprising subjecting said resin to at least one wash step, at least two wash steps, at least three wash steps, or at least four wash steps (claim 56), wherein a wash buffer used in the wash step(s) comprises about 1×TBS, about 2×TBS, about 1×TBS with 20% ETOH, about 1×PBS, about 2×PBS, about 1×PBS with 18% EtOH, or combinations thereof (claim 57); wherein said affinity purification comprises an elution step (claim 58), wherein said elution step includes an elution buffer, optionally wherein said elution buffer comprises glycine, poloxamer 188, or a combination thereof (claim 59), wherein said elution buffer comprises about 50 mM glycine and about 0.01% poloxamer 188 at a pH of about 3 or about 100 mM glycine and about 0.01% poloxamer 188 at a pH of about 2 (claim 60); further comprising contacting said enriched viral vector sample to a salt solution to form a homogeneous mixture prior to step (d)(claim 61), wherein said salt solution includes cesium chloride, cesium sulfate, cesium bromide, cesium formate, cesium acetate, cesium iodide, cesium selenite, potassium bromide, rubidium bromide, rubidium chloride, cesium TFA, sodium iothalamate, or a combination thereof (claim 62), wherein a concentration of salt in said salt solution is about 1.30 g/mL to about 1.40 g/mL or about 1.32 g/mL to about 1.36 g/mL (claim 63), wherein said salt concentration is about 1.34 g/mL (claim 64); wherein a rotor speed of the analytical ultracentrifuge is from 10 k to 60 k rpm (claim 65), wherein said rotor speed is about 40 k rpm (claim 66); wherein said viral vector comprises an AAV vector (claim 67), wherein said AAV vector comprises a serotype selected from a group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, variations thereof and combinations thereof (claim 68); wherein a concentration of said at least one viral vector in said sample is from 1×10^10 to 1×10^12 cp/mL (claim 69); further comprising quantifying empty capsids, partially filled capsids, and/or full capsids of said at least one viral vector (claim 70); further comprising quantifying a ratio of absorbance at 260 nm to absorbance at 280 nm wavelengths of viral vector species separated by density gradient equilibrium analytical ultracentrifugation (claim 71); wherein said cells are HEK293, HeLa, or Chinese Hamster Ovary (CHO) cells (claim 73), and wherein said cells are HEK293 cells (claim 74).
Claim 75 is drawn to a method for quantifying the capsid content of a viral vector, comprising:
(a) subjecting a sample including a viral vector to affinity purification to form an enriched sample; and
(b) subjecting said enriched sample to density gradient equilibrium analytical ultracentrifugation to quantify the capsid content of said viral vector.
Claim 76 is drawn to a method for characterizing critical quality attributes of a viral vector, comprising:
(a) subjecting a sample including a viral vector to affinity purification to form an enriched sample; and
(b) subjecting said enriched sample to density gradient equilibrium analytical ultracentrifugation to characterize critical quality attributes of said viral vector.
Further limitations on the method of claim 76 are wherein said critical quality attributes include the empty/full ratio of said viral vector, the empty/partially filled/full ratio of said viral vector, low molecular weight species of said viral vector, and/or high molecular weight species of said viral vector (claim 77).
Claim Rejections - 35 USC § 112(a); First Paragraph
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 18 and 68 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The written description requirement is separate and distinct from the enablement requirement. To satisfy the written description requirement, the specification must reasonably convey to one skilled in the relevant art that the inventor had possession of the claimed invention as of the filing date. Possession may be shown by a description of the complete structure of the claimed invention, a representative number of species falling within the scope of a claimed genus, or relevant identifying characteristics sufficient to show that the inventor had possession of the claimed subject matter.
Claims 18 and 68 recite an AAV vector comprising a serotype selected from AAV1 through AAV12, along with “variations thereof and combinations thereof.”
The specification describes AAV1 through AAV12 and likewise states that the AAV vector may comprise “variations thereof and combinations thereof” (¶[0022][0042][0063]). The specification further describes an AAV capsid as the protein shell of the virus and provides general discussion of recombinant AAV vectors (¶[0125-0128]).
However, the specification does not describe what structural features constitute a “ variation” of any of AAV1 through AAV12 or what AAV structures are encompassed by a “combination” of those serotypes. The disclosures does not identify a degree of sequence variation, particular capsid substitutions, regions that may be altered, or other structural characteristics sufficient to identify the AAV variants falling within the claimed scope. Similarly, the disclosure does not identify whether a claimed “combination” is a chimeric or mosaic capsid, another recombinant arrangement containing components from different AAV serotypes, or some other AAV structure.
The experimental disclosure does not provide representative species across this broader scope. Example 1 evaluates purified AAV8 and AAV5 vectors using DGE-AUC (¶[0139-0142]). Example 6 describes affinity purification and DGE-AUC characterization of AAV5, and Example 7 describes purification and characterization of an AAV5-CAG-GFP sample from a cell lysate (¶[0156-0157]). These disclosed AAV5 and AAV8 embodiments do not provide representative examples of the broadly recited variations or combinations of AAV1 through AAV12
The specification therefore does not describe a sufficient number of species representative of the claimed scope or identify structural features common to the claimed group which would allow one skilled in the art to recognize which additional AAVs constitute the recited “variations thereof and combinations thereof.” Instead, one skilled in the art would be required to determine what modifications or combinations of the listed AAV serotypes are intended to fall within the claimed genus.
Accordingly, the disclosure does not reasonably convey to one skilled in the art that the inventor had possession of the full scope of the AAV “variations thereof and combinations thereof” recited in claims 18 and 68 at the time the application was filed.
Claims 48-66 and 69-77 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for characterization of AAV vectors, including the disclosed AAV5 and AAV8 embodiments, does not reasonably provide enablement for characterization of viral vectors generally. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims.
The legal considerations that govern enablement determinations pertaining to undue experimentation have been set forth in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). The factors to be considered include: (1) the breadth of the claims; (2) the nature of the invention; (3) the state of the prior art; (4) the level of one of ordinary skill; (5) the level of predictability in the art; (6) the amount of direction provided by the inventor; (7) the existence of working examples; and (8) the quantity of experimentation needed to make or use the invention based on the content of the disclosure. The factors are considered as a whole in determining whether any necessary experimentation would have been undue.
Nature of the invention and breadth of the claims. The claimed invention is directed to methods in which a viral vector is affinity purified and subsequently subjected to density gradient equilibrium ultracentrifugation (DGE-AUC) for characterization of the viral vector, including quantification of capsid content or characterization of critical quality attributes. Claim 48 additionally required production of the viral vector in cells, lysis of the cells, and filtration before the affinity purification step. Claims 75-77 broadly recite affinity purification followed by DGE-AUC without limiting the viral vector to AAV. The breadth of “viral vector” is any vector which has been engineered to deliver a therapeutic payload to a host, cell, or system.
The specification describes the method of claim 48 in ¶[0048], and the broader methods for quantifying capsid content and characterizing critical quality attributes of a viral vector in ¶[0070-0072]. The specification states that a “viral particle” includes a viral capsid and an encapsulated viral genome and expressly contemplates application of the disclosed methods to viruses from multiple viral families, including Adenoviridae, Retroviridae, Baculoviridae, and Herpesviridae (¶[0128]). The specification further provides general descriptions of DGE-AUC and affinity purification (¶[0130-0133]).
However, the claims are not limited to the disclosed AAV embodiments. Claims 48-66 and 69-77 encompass substantially different viral vectors having different particle structures, surface features, densities, production systems, and purification requirements. The expressly contemplated scope includes both AAV and materially different non-AAV viral vectors (¶[0128]). The breadth is material to both principal steps of the claimed methods. Affinity purification requires an affinity ligand and purification conditions capable of capturing the particular viral vector without unacceptable loss or damage. DGE-AUC further requires gradient conditions capable of providing useful separation of the viral particles or particle species being characterized. The specification does not provide a common affinity interaction or a common set of DGE-AUC conditions that apply across the full viral vector scope. The claimed scope therefore extends beyond the embodiments described in the specification.
State of the prior art and predictability of the art. At the time the application was filed, it was known that viral vector purification conditions were dependent on the particular vector being processed. Rieser et. al. (Rieser R, et. al. Pharmaceutics. 2021 May 18;13(5):748.) expressly reports that “for the used affinity chromatography (AC), a serotype dependence was observed.” Rieser further explains that chromatography resins and conditions were often tailored to individual AAV serotypes and reports that engineered AAV capsids presented additional problems for affinity purification. Therefore, material variation in affinity purification was known even with the considerably narrower AAV genus.
Wolf et. al. (Wolf MW, et. al. Expert Rev Vaccines. 2011 Oct;10(10):1451-75.) describes different downstream approaches for adenovirus (AdV), AAV, and retroviral vectors rather than a common viral vector purification procedure. With respect to separation of gene therapy vector particles, the authors state that application and optimization of such methods must be considered “on a by-case basis.” This teaching is consistent with a skilled artisan expecting vector-specific process development rather than extrapolating a purification protocol established for one viral system to viral vectors generally.
Jiang et. al. (Jiang C, et. al. J Virol. 2004 Sep;78(17):8994-9006.) illustrates the problem for an expressly contemplated non-AAV vector. Jiang states that “HSV-1 is an enveloped virus that requires the development of gentle purification methods” and describes an affinity method in which the HSV-1 vector itself was engineered to display a cobalt-binding peptide. The reference further explains that gentle purification preferably avoided centrifugation, demonstrating that an affinity purification and centrifugation scheme suitable for AAV cannot simply be presumed suitable for an HSV viral vector encompassed by the present claims.
The art was not sufficiently predictable to support extrapolation from the limited AAV embodiments in the specification to the more broadly claimed scope of any viral vector purification. Accordingly, the results obtained using AAV5 and AAV8 in the specification would not have reasonably established that the broader claimed scope could be practiced without further experimentation.
Level of skill in the art. One skilled in the art would have been familiar with viral vector production, chromatography, density-gradient centrifugation, and analytical centrifugation. Such a person also would have been capable of screening affinity ligands, modifying buffer conditions, changing gradient composition and/or density, and testing configuration parameters. However, the existence of known methods for preparing and testing candidate embodiments does not establish that one skilled in the art would have known, without further experimentation, which additional affinity ligands, binding and elution conditions, gradient-forming materials, gradient densities, and centrifugation conditions would satisfy the claimed limitations of characterizing the different viral vectors.
Working examples. The specification provides seven numbered working examples directed to purification and characterization of AAV systems, namely AAV5 and AAV8. Example 1 evaluates DGE-AUC using purified AAV8 and AAV5 and used different gradient salts for the two serotypes (¶[0139-0142]). Examples 2-5 develop and validate the affinity purification/DGE-AUC approach using AAV process samples, while Examples 6 and 7 specifically characterize AAV5, including an AAV5-CAG-GFP sample produced in HEK293 cells (¶[0143-0157]). The working data also demonstrate that the analytical conditions were not interchangeable even among the tested AAV samples. For AAV8, Example 1 used a cesium chloride salt solution, whereas AAV5 was analyzed using cesium sulfate (¶[0141-0142]). During optimization, the specification reports that cesium chloride did not sufficiently separate partially filled and full AAV5 capsids, cesium bromide produced substantial noise, and cesium sulfate provided the best separation (¶[0145-0149]).
The specification does not provide working examples directed to non-AAV viral vectors, such as retroviral, lentiviral, baculoviral, herpesviral, or any other materially different viral vector family expressly contemplated by ¶[0128],. The disclosed examples therefore do not establish enablement across the full scope of the claims with respect to affinity purification followed by DGE-AUC, and do not establish this scope can be practiced without undue experimentation.
Guidance in the specification. The specification provides substantial guidance regarding AAV5 and AAV8. For example, ¶[0133] identifies POROS™CAPTURESELECT™ AAVX, AAV8, and AAV9 affinity resins and AVB SEPHAROSE™, while ¶[0139-0158] provide AAV-specific affinity purification and DGE-AUC experiments. The specification also describes adjustment of salt density, rotor speed, and gradient-forming salt for AAV samples (¶[0145-0150]).
However, the specification does not provide sufficient guidance regarding non-AAV viral vectors encompassed by the claims. In particular, the specification does not explain affinity ligands that may be used for said vectors and binding/elution conditions that will capture the various non-AAV vectors encompassed by the claims and ¶[0128], nor does it provide DGE-AUC conditions that will resolve the relevant viral vector species after such affinity purification. In particular, the specification does not explain how the AAV procedures should be modified for vectors having materially different particle structures or surface chemistry, or how appropriate gradient conditions would be selected to characterize the capsid content and critical quality attributes recited in claims 75-77.
Quantity of experimentation necessary. To practice the full scope of the claims, one skilled in the art would need to select or develop an affinity capture system for additional viral vector types and determine conditions that provide sufficient recovery while preserving the material to be characterized. The skilled artisan would ten need to establish suitable DGE-AUC conditions, including gradient composition and density and appropriate centrifugation parameters, and determine whether the resulting profiles permit the recited characterization. Such experimentation would not merely involve the routine application of known methods to embodiments reasonably expected to work, or applying the disclosed AAV methods to the non-AAV viral vectors. Instead, one skilled in the art would need to prepare and test additional embodiments to determine whether they satisfy the ability to separate non-AAV vectors from all impurities, such as cellular debris and non-viable vector species. Although the individual methods used to prepare and test candidate embodiments may have been known in the art, the relevant inquiry is not whether one skilled in the art could perform the required assays. The relevant inquiry is whether the specification provides sufficient guidance to identify and practice the embodiments falling within the full scope of the claims without undue experimentation. Here, one skilled in the art would need to prepare and test additional affinity purification and DGE-AUC conditions to determine which embodiments satisfy the ability to characterize the breadth of viral vectors encompassed by the claims.
Amgen. The Supreme Court has explained that a specification need not describe with particularity how to make and use every embodiment within a claimed class. However, the disclosure must enable one skilled in the art to make and use the full scope of the claimed invention. A reasonable amount of experimentation may be permissible depending on the nature of the invention and the underlying art. Amgen Inc. v. Sanofi, 598 U.S. 594, 610-13 (2023).
In the instantly claimed invention, the specification describes detailed examples for AAV5 and AAV8, but the claims also encompass other AAV species and non-AAV viral vectors. The specification identifies additional viral families to which the inventors contemplate applying the method (¶[0128]), but does not provide a general technical principle or sufficient working guidance that would allow one skilled in the art to carry the AAV affinity purification/DGE-AUC method across those materially different viral vector families without determining suitable conditions by further experimentation. The specification does not identify a general quality or provide sufficient guidance that would allow one skilled in the art to practice that broader scope without undue experimentation.
Conclusion. For the reasons discussed above, the specification does not enable one skilled in the art to use the full scope of the invention recited in the claims without undue experimentation.
Claim Rejections - 35 USC § 102
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 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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 48, 50, 52-53, 58-59, 61-66, and 69-77 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yang et. al. (Yang X, et. al. J Pharm Sci. 2008 Feb;97(2):746-63.; CITED ART OF RECORD; hereafter “Yang.)
The Prior Art
Yang teaches adenoviruses (rAd), widely used as vectors for gene therapy, are generally purified by column chromatography and frequently contain empty capsids and other aberrant forms of virus particles, and to determine particle heterogeneity they utilized analytical ultracentrifugation (AUC) in CsCl density gradients (entire document; see abstract.) Yang teaches column purification to generate enriched samples of rAd (“Experimental Procedures: rAd Vector Production and Purification: Vector 1-3; pp. 747-8). Yang teaches subjecting said enriched rAd vector sample to density gradient equilibrium analytical ultracentrifugation (DGE-AUC)(pp. 748-749; “Analytical Ultracentrifugation on CsCl Density Gradients, Purification of Different rAd Particle Forms, Quantification of Empty Capsids by AUC”; instant claims 75-76). The AUC profile differences of various vectors and preparations allowed Yang to determine critical quality attributes of said viral vector, such as the amount of full vector to the amount of empty vector in a composition (p. 751, “AUC profile Differences of Various Vectors and Preparations”, instant claim 77).
Yang teaches that with Vector 1, the vector was grown in HEK293 cells engineered to express the type 5 rAd vector, and that said cells were lysed and cleared from cellular debris to form a pool, which was then subjected to anion exchange on DEAE-Fractogel and by gel filtration on Superdex 200 to form an enriched viral vector sample (“Vector 1” p. 747). Yang also teaches vector 3 was isolated in a similar way, but additionally “an immobilized zinc affinity chromatography (IZAC) step [was] inserted between the anion exchange and gel filtration purification steps.”(“Vector 3”, p. 748) The vectors were then subjected to DGE-AUC to characterize said vectors (pp. 749-750, “Differing Density Forms of rAd Particles in CsCl Are Observed by AUC”; instant claims 48, 50, 52-53, 58, 73-74.) Yang teaches with Vector 3, the elution was performed with 250 mM glycine in 450 mM NaCl (p. 748, “Vector 3”; instant claims 58-59, 61). The vectors were diluted to 8x10^11 vp/mL and were mixed with 456 mg of CsCl per mL of adenovirus solutions, and was selected to give the solution density of approximately 1.32 g/mL (p. 749, left col., Instant claims 61-64, 69). The AUC was performed at about 40k rpm (p. 749, left col., instant claims 65-66.) While Yang teaches A280 was used to monitor the DNA/protein content from the A260/A280 ratio, A320 was also employed for the more quantitative analysis of the empty capsids and other forms of virus particles because A320 results from the light scattering of particles and is less dependent upon the DNA or protein contents of virus particles (p. 749, left col., instant claims 70-71). Yang teaches analysis of empty/full viral particles as well as viral aggregates (p. 756, rt. Col., ¶3; instant claim 72).
For at least these reasons, Yang teaches the limitations of instant claims 48, 50, 52-53, 58-59, 61-66, and 69-77, and anticipates the invention encompassed by said claims.
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 1-24, 49, 51, 54-57, 60, and 67-68 are rejected under 35 U.S.C. 103 as being unpatentable over Yang as applied to claims 48, 50, 52-53, 58-59, 61-66, and 69-77 above, and further in view of Florea et. al. (US20230303983A1, Priority 03/28/2022, Pub. 09/28/2023; hereafter “Florea”) and Intas et. al. (WO2022043926A1, Priority 08/31/2020, Pub. 03/03/2022; CITED ART OF RECORD; hereafter “Intas”.)
The Prior Art
The teachings of Yang have been set forth supra. While Yang teaches the use of affinity purification methods to use in the methods to isolate and characterize viral vectors, Yang teaches IZAC and does not teach the use of an affinity purification method that utilizes a protein ligand, such as an antigen binding protein or antibody. While Yang teaches the majority of the analytical method with respect to rAd vectors, Yang is silent as to specific steps in the method, such as subjecting said harvest pool to tangential flow filtration (TFF), or applying such a purification method to adeno-associated virus, such as AAV1-AAV12. However, as rAd vectors are often employed as “helper” viruses to produce AAV, and TFF was a common method to isolate and purify virus from impurities, and the use of affinity purification that utilizes a protein ligand was common in the art, the application of the methods of Yang to these conditions would be obvious to a skilled artisan, especially in light of the teachings of Florea.
Florea teaches optimized methods of high efficiency purification of adeno-associated virus (AAV) particles, comprising a step of binding one or more AAV particles with a volume of chromatography resin medium comprising at least one ligand possessing a pan-AAV affinity (entire document; see abstract.) Florea teaches the separation methods can be used to quantify empty versus full AAV capsids (¶[0047][0079-0082]). Florea teaches that affinity chromatography can be used to separate the AAV, and teaches small scale separation using AAV resins such as POROS AAVX (Example starting at ¶[0122]; instant claims 3, 50). Florea teaches the resins can be used at larger scales (¶[0123]; instant claims 4, 51), and that the resins can be linked to antibodies that bind to the AAV (reference claim 14; ¶[0007]; instant claims 5-7, 53-55). Florea teaches the method may comprise one or more wash steps (reference claim 24; instant claims 8, 56) in any of tris-buffered saline (TBS), ethanol, guanidine HCL, phosphoric acid, glycine, Tris NaOH, water, a non-ionic surfactant, and NaCl (reference claim 24; instant claims 9, 57). Florea notes the binding capacity of the resins can be up to 1x10^14 vg/mL of the AAV, that aggregates can be measured and avoided through certain steps (method at ¶[0164]) and fractions of viral purifications eluted from the resin were measured at 280 nm (¶[0141]).
Florea teaches that tangential flow filtration (TFF) is a step that can be used to concentrate viral vectors (¶[0138]; instant claims 20, 49). Florea teaches the method can be used to purify a variety of AAV, such as AAV1, AAV2, AAV2-7m8, AAV-HSPG, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, Rh10, Rh61, Rh8, Rh32.33, VR-865, PHP.B and Anc80 (¶[0104]; instant claims 1, 18, 67-68). Florea teaches the use of non-ionic surfactants in the steps of the method, such as PLURONIC F-68 (trade name for poloxamer 188) and further washing and elution in the additional presence of glycine and a non-ionic surfactant at an elution pH of about 2.0-2.5, wherein the elution buffer is about 0.2M glycine and 0.01 v/v % PLURONIC F68 (¶[0014][0034][0042]). Optimization of these wash and elution buffers is routine in the art, and since Yang teaches a higher concentration of glycine for rAd, it would be obvious to optimize this glycine/pluronic composition for the final elution while maintaining a low pH in order to change the electrical charge of the resins and any other electrostatic bonds holding onto the virus in order to release it from the solid support/resin. The non-ionic surfactants also further release the virions from any hydrophobic interactions while preventing aggregation of the virions and stabilize the viral particles; glycine also is an excellent low pH buffer and helps to further stabilize the virions. Therefore, optimizing the AAV elution buffer of poloxamer 188 and glycine at a pH of about 2-3 would be obvious given the teachings of Yang and Florea, rendering the limitations of instant claims 10-12 and 60 obvious to a skilled artisan.
Intas teaches a method for purification of adeno-associated virus (AAV) vector comprising a transgene encoding Factor IX (FIX), wherein said method comprising steps of in-vitro cell lysis and clarification, affinity chromatography and density gradient centrifugation (entire document; see reference claims 7, 10). Intas teaches wherein affinity chromatography comprises equilibration with 20 mM Tris buffer containing 150 mM NaCl at pH 7.5, washing the column with high salt wash buffer (20 mM Tris, 500 mM NaCl, pH 7.0) and low pH wash buffer (100 mM Sodium acetate pH 5.0 and elution with low pH buffer (100 mM Glycine, pH 2.7)(reference claim 8). Intas teaches wherein density gradient ultracentrifugation is performed with iodixanol gradient (reference claim 9). Intas teaches such AAV serotypes as AAV8 may be purified using this method (p. 3, ¶2).
Given the teachings of Yang, a skilled artisan would be apprised as to the use of DGE-AUC to separate rAd and analyze full versus empty virions, and given the related nature of rAd to AAV, it would be obvious to apply these methods to AAV to either separate rAd out from AAV compositions as impurities and to apply the DGE-AUC to quantify the empty/full virion ratio of AAV in the composition, especially give the teachings of Florea and Intas. Given the teachings of Yang, a skilled artisan would be apprised as to methods of purifying and qualifying the resulting vectors from cell culture, namely rAd vectors. Given that rAd was commonly used as a helper virus to propagate adeno-associated virus (AAV), a skilled artisan would find it obvious to quantitate the amount of rAd and/or AAV in an AAV cell culture, or to have these analyses performed simultaneously to determine if the helper rAd virus was present or persistent in the AAV cell culture so as to not elicit an undesirable response if said AAV was to be used in vivo. Given the teachings of Intas and Florea, it would be obvious to provide extra purification steps, such as TFF, to the purification methods of the viral vectors. Therefore, arriving at the limitations of instant claims 1-24, 49, 51, 54-57, 60, and 67-68 would be obvious to a skilled artisan, given the teachings of Yang, Florea, and Intas.
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Yang in order to purify AAV using DGE-AUC and affinity exchange methods, thereby providing purified compositions of AAV virions that can be further analyzed for genomic compositions. One would have been motivated to do so, given the suggestion by Yang that the methods can determine empty vs. full virions, and given the suggestion by Florea and Intas that determination of full/empty AAV capsids was an important step in therapeutic applications of AAV. There would have been a reasonable expectation of success, given the knowledge that further affinity resins for AAV were known in the art, as taught by Intas and Florea, and also given the knowledge that specific wash and elution buffer reagents were known and suggested in the art as routine for AAV, as taught by Florea and Intas. Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure and is listed below.
US20180180525A1. Teaches AAV characterization by AUC, affinity-purified AAV samples, empty/full capsid analysis, and analytical density gradient equilibrium concepts. Does not teach the specific claimed steps in the specific order, but provides each step and shows the non-obvious nature of the DGE-AUC method of purification of AAV. Not utilized as rejection would be redundant to those set forth supra.
US20230183658A1. Teaches purification of AAV from contaminant-containing mixtures and expressly evaluates empty/full capsid content by AUC. Not utilized as rejection would be redundant to those set forth supra.
US20220267796A1. Teaches AAV affinity purification using POROS™ CAPTURESELECT™ AAVX/AAV8/AAV9 resins and expressly uses AUC to determine relative amounts of empty/full capsids. Not utilized as rejection would be redundant to those set forth supra.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RACHEL B GILL whose telephone number is (571)272-3129. The examiner can normally be reached on M to F 8:00 AM to 5:00 PM Eastern.
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/RACHEL B GILL/
Primary Examiner, Art Unit 1671
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