Method for the production and purification of adenoviral vectors
DCFirst Claim
1. A method for producing a purified adenovirus composition comprising:
- a) growing host cells in a media;
b) providing nutrients to said host cells by perfusion or through a fed-batch process;
c) infecting said host cells with an adenovirus;
d) lysing said host cells to provide a cell lysate comprising adenovirus, wherein said lysis is achieved through autolysis of infected cells; and
e) purifying adenovirus from said lysate to provide a purified adenovirus composition.
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Abstract
The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production witrh cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.
175 Citations
89 Claims
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1. A method for producing a purified adenovirus composition comprising:
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a) growing host cells in a media;
b) providing nutrients to said host cells by perfusion or through a fed-batch process;
c) infecting said host cells with an adenovirus;
d) lysing said host cells to provide a cell lysate comprising adenovirus, wherein said lysis is achieved through autolysis of infected cells; and
e) purifying adenovirus from said lysate to provide a purified adenovirus composition. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 88, 89)
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30. A method for producing an adenovirus composition comprising:
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a) growing host cells in a media comprising glucose;
b) providing nutrients to said cells by perfusion or a fed-batch process at a rate to provide a glucose concentration of less than 2.0 g/L;
c) infecting said host cells with an adenovirus; and
d) harvesting and lysing said host cells to produce a lysate comprising said adenovirus composition. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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61. A method for preparing a purified adenovirus composition comprising:
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a) growing host cells;
b) providing nutrients to said host cells by perfusion or through a fed-batch process;
c) infecting said host cells with an adenovirus;
d) lysing said host cells using a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication to produce a crude lysate composition comprising adenovirus; and
e) purifying adenovirus from said lysate by a process that includes one or more chromatography steps without the use of cesium chloride density gradient centrifugation, to provide a purified adenovirus composition. - View Dependent Claims (62, 63, 64, 65, 66, 67, 68, 69, 70)
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71. A method for preparing a purified adenovirus composition comprising:
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a) growing host cells in a serum-free media;
b) infecting said host cells with an adenovirus;
c) harvesting and lysing said host cells to provide a lysate comprising adenovirus; and
d) purifying adenovirus from said lysate by a process that includes a chromatography step without the use of cesium chloride density gradient centrifugation, wherein said chromatography step involves a single chromatography step, to provide a purified adenovirus composition wherein the recovery of purified adenovirus after the chromatography step is 70% ±
10% of the starting PFU.- View Dependent Claims (72, 73, 74, 75, 76, 77)
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78. A method for preparing a purified adenovirus composition comprising:
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a) growing host cells in a media;
b) infecting said host cells with an adenovirus;
c) harvesting and lysing said host cells to provide a lysate comprising adenovirus;
d) treating the lysate with a nuclease to reduce the concentration of contaminating nucleic acid; and
e) purifying adenovirus from said lysate by a process that includes a chromatography step without the use of cesium chloride density gradient centrifugation, wherein said chromatography step involves a single chromatography step, to provide a purified adenovirus composition wherein the recovery of purified adenovirus after the chromatography step is 70% ±
10% of the starting PFU.- View Dependent Claims (79, 80, 81, 82, 83, 84, 85)
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86. A method for preparing a purified adenovirus composition comprising:
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a) growing host cells;
b) providing nutrients to said host cells by perfusion or through a fed-batch process;
c) infecting said host cells with an adenovirus;
d) lysing said host cells using a process that includes hypotonic solution, hypertonic solution, impinging jet, microfluidization, solid shear, detergent, liquid shear, high pressure extrusion, autolysis or sonication to produce a crude lysate composition comprising adenovirus; and
e) purifying adenovirus from said lysate by a process that includes a chromatography step without the use of cesium chloride density gradient centrifugation, wherein said chromatography step involves a single chromatography step, to provide a purified adenovirus composition. - View Dependent Claims (87)
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Specification