US6146874A
Expired / Lapsed
US
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Method of preparing recombinant adeno-associated virus compositions
Assignee: University of Florida Research Foundation Inc
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Filing: 1999-05-27
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Granted: 2000-11-14
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Expires: 2019-05-27
Abstract
Abstract Disclosed are methods for the isolation and purification of high-titer recombinant adeno-associated virus (rAAV) compositions. Also disclosed are methods for reducing or eliminating the concentration of helper adenovirus in rAAV samples. Methods are disclosed that provide highly-purified rAAV stocks having titers up to about 1013 particles/ml at particle-to-infectivity ratios of less than 100 in processes that are accomplished about 24 hours or less.
Inventors
- 👤 Sergei Zolotukhin
- 👤 Barry J. Byrne
- 👤 Nicholas Muzyczka
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-02 20:03
CPC Classifications
C; C12; C12N; C12N7/00; C12N15/00; C12N15/09; C12N15/63; C12N15/79; C12N15/85; C12N15/86; C12N2750/00; C12N2750/00011; C12N2750/14011; C12N2750/14111; C12N2750/14141; C12N2750/14143; C12N2750/14151; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S435/8215; Y10S435/948; Y10S530/00; Y10S530/82; Y10S530/826
Claims Analysis
Total Claims: 35
Claim 1
Independent Claim
29 words
A method of isolating a recombinant adeno-associated virus, comprising applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting said recombinant adeno-associated virus from said gradient.
Claim 2
Independent Claim
29 words
A method of isolating a recombinant adeno-associated virus, comprising applying a sample containing recombinant adeno-associated virus to an iodixanol gradient, and collecting said recombinant adeno-associated virus from said gradient.
Claim 3
Dependent Claim
13 words
The method of claim 1, wherein said iodixanol gradient is a discontinuous gradient.
Claim 4
Dependent Claim
31 words
The method of claim 2, wherein said iodixanol gradient comprises an about 15% iodixanol step, an about 25% iodixanol step, an about 40% iodixanol step, and an about 60% iodixanol step.
Claim 5
Dependent Claim
17 words
The method of claim 3, wherein said recombinant adeno-associated virus is collected from said 40% iodixanol step.
Claim 6
Dependent Claim
16 words
The method of claim 3, wherein said 15% iodixanol step further comprises about 1 M NaCl.
Claim 7
Dependent Claim
17 words
The method of claim 1, wherein said iodixanol gradient is subjected to centrifugation after applying said sample.
Claim 8
Dependent Claim
42 words
The method of claim 1, further comprising contacting said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix, removing non-bound species from said matrix, and eluting said virus from said matrix.
Claim 9
Dependent Claim
18 words
The method of claim 7, wherein said matrix comprises heparin agarose type I or heparin agarose type II-S.
Claim 10
Dependent Claim
14 words
The method of claim 7, wherein said matrix is comprised within an HPLC column.
Claim 11
Dependent Claim
21 words
The method of claim 7, wherein said virus is eluted from said matrix with a solution comprising about 1 M NaCl.
Claim 12
Dependent Claim
37 words
The method of claim 1, further comprising contacting said recombinant adeno-associated virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix, and collecting non-interacting virus from said hydrophobic matrix.
Claim 13
Dependent Claim
12 words
The method of claim 11, wherein said hydrophobic matrix comprises phenyl groups.
Claim 14
Dependent Claim
11 words
The method of claim 12, wherein said hydrophobic matrix is phenyl-sepharose.
Claim 15
Dependent Claim
28 words
The method of claim 1, further comprising applying said recombinant adeno-associated virus to a cesium chloride equilibrium density gradient, and collecting said recombinant adeno-associated virus from said gradient.
Claim 16
Dependent Claim
46 words
The method of claim 1, further comprising contacting said recombinant adeno-associated virus with at least a first ion exchange chromatography medium, under conditions effective to permit interaction of said virus with said medium, removing non-interacting species from said medium, and eluting said virus from said medium.
Claim 17
Dependent Claim
12 words
The method of claim 1, wherein said sample further comprises a virus.
Claim 18
Dependent Claim
12 words
The method of claim 16, wherein said sample further comprises an adenovirus.
Claim 19
Dependent Claim
17 words
The method of claim 1, wherein said sample further comprises at least a first polypeptide or protein.
Claim 20
Dependent Claim
17 words
The method of claim 1, wherein said sample further comprises a cell extract or a growth medium.
Claim 21
Independent Claim
85 words
A method of isolating a recombinant adeno-associated virus, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; and e) eluting said virus from said matrix.
Claim 22
Independent Claim
85 words
A method of isolating a recombinant adeno-associated virus, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; and e) eluting said virus from said matrix.
Claim 23
Independent Claim
116 words
A method of isolating a recombinant adeno-associated virus, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; e) eluting said virus from said matrix; f) contacting the eluted virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix; and g) collecting the non-interacting virus from said hydrophobic matrix.
Claim 24
Independent Claim
116 words
A method of isolating a recombinant adeno-associated virus, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; e) eluting said virus from said matrix; f) contacting the eluted virus with a hydrophobic matrix, under conditions effective to permit interaction of hydrophobic species with said hydrophobic matrix; and g) collecting the non-interacting virus from said hydrophobic matrix.
Claim 25
Independent Claim
45 words
A method for reducing or eliminating adenovirus from a recombinant adeno-associated virus composition contaminated with adenovirus, comprising applying a sample containing recombinant adeno-associated virus and adenovirus to an iodixanol gradient, and collecting from said gradient at least a first fraction comprising said recombinant adeno-associated virus.
Claim 26
Independent Claim
45 words
A method for reducing or eliminating adenovirus from a recombinant adeno-associated virus composition contaminated with adenovirus, comprising applying a sample containing recombinant adeno-associated virus and adenovirus to an iodixanol gradient, and collecting from said gradient at least a first fraction comprising said recombinant adeno-associated virus.
Claim 27
Independent Claim
96 words
A method of producing a recombinant adeno-associated virus having a particle-to-infectivity ratio of less than about 100 to 1, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; and e) eluting said virus from said matrix.
Claim 28
Independent Claim
96 words
A method of producing a recombinant adeno-associated virus having a particle-to-infectivity ratio of less than about 100 to 1, comprising the steps of: a) centrifuging a sample containing recombinant adeno-associated virus through an iodixanol gradient; b) collecting from said iodixanol gradient at least a first fraction comprising said recombinant adeno-associated virus; c) contacting said at least a first fraction comprising said recombinant adeno-associated virus with a matrix comprising heparin, under conditions effective to permit binding of said virus to said matrix; d) removing non-bound species from said matrix; and e) eluting said virus from said matrix.
Claim 29
Dependent Claim
19 words
A kit comprising, in a suitable container, iodixanol, a matrix comprising heparin and instructions for isolating recombinant adeno-associated virus.
Claim 30
Dependent Claim
19 words
A kit comprising, in a suitable container, iodixanol, a matrix comprising heparin and instructions for isolating recombinant adeno-associated virus.
Claim 31
Dependent Claim
14 words
The kit of claim 24, wherein said iodixanol is formulated as an iodixanol gradient.
Claim 32
Dependent Claim
18 words
The kit of claim 24, wherein said matrix comprises heparin agarose type I or heparin agarose type II-S.
Claim 33
Dependent Claim
10 words
The kit of claim 24, further comprising a hydrophobic matrix.
Claim 34
Dependent Claim
12 words
The kit of claim 27, wherein said hydrophobic matrix comprises phenyl groups.
Claim 35
Dependent Claim
11 words
The kit of claim 28, wherein said hydrophobic matrix is phenyl-sepharose.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2003-11-14 | 2004-05-15 | 2004-11-14 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2007-11-14 | 2008-05-15 | 2008-11-14 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2011-11-14 | 2012-05-15 | 2012-11-14 | $7700.00 | Window Closed / Lapsed |