Method for purifying virus

A virus and anion technology, applied in the field of virus purification, can solve the problems of difficult to scale-up industrial-scale production, time-consuming, expensive and other problems

Inactive Publication Date: 2006-08-09
ONYX PHARMA INC
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the method has proven effective for use as a research tool, it is too expensive, time-consuming and not easily scaled up for industrial-scale production

Method used

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  • Method for purifying virus
  • Method for purifying virus
  • Method for purifying virus

Examples

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preparation example Construction

[0066] Preparation of cell lysates

[0067] Virus can be purified from cell lysates prepared from a number of sources mentioned above, including cell lines, tissues, body fluids, organs, and the like. Viruses are often purified from cell lysate preparations prepared from virus-infected cells, where the cells have been cultured using cell culture methods. For example, adenovirus can be isolated from virus-infected cells such as 293 cells, HeLa cells, and the like. Cells can be infected at a high multiplicity of infection (MOI) to optimize yield.

[0068] Virus-containing cell lysates can be prepared by any method suitable for the release of virus from infected cells. Virus can be released from infected cells using techniques known in the art or by autolysis. Preferred methods of lysing virus-infected cells include the use of hypotonic solutions, hypertonic solutions, sonication, pressure, or detergents. A preferred technique is the use of detergents, more preferably a combi...

Embodiment 1

[0086] Purification of Adenovirus

[0087] figure 1 A general purification scheme is shown. Viruses were monitored at certain steps of the purification process described in this example. The method involves measuring virus concentrations by AEX-HPLC similar to that described in Huyghe, et al, Human Gene Therapy 6: 1403-1416 (November 1995). In summary, a 1 ml Resource-Q column was equilibrated with a buffer containing 300 mM NaCl and 20 mM phosphate buffer pH 7.5. Virus was eluted by running a linear gradient of 300 mM → 600 mM NaCl and monitoring the UV absorbance at 260 and 300 nm. The areas of the virus peaks were integrated and compared to a cesium chloride purified reference standard.

[0088] Preparation of cell lysates

[0089] Hela-S3 cells, obtainable from American Type Culture Collection, Accession No. ATCC CCL-2.2, were infected with adenovirus Onyx 411 as described by Johnson, et al., Cancer Cell. 2002 May;1(4):325-37 . 70 liters of cell culture harvest was ...

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Abstract

A process for the purification of viruses from a cell lysate preparation is described, consisting of preferably two successive chromatographic steps; the first a clarification step utilizing size exclusion chromatography, and the second, a virus capture and release step using anion exchange chromatography, which successive chromatographic steps have the advantage of purifying virus, and avoiding chromatography buffer conductivity adjustments.

Description

field of invention [0001] The invention described herein is in the field of virus purification. Background of the invention [0002] There has been a resurgence in the use of viruses to treat cancer (Lancet Oncology Vol. 3, January 2002, page 17). Probably the most studied virus is the adenovirus, and most of the recent work has been done by McCormick and colleagues. The adenovirus they used had a deletion of the E1B gene, which encodes a 55kd protein that can bind to the tumor suppressor gene p53 and inhibit its function (Science, 1996; vol.274: page 373). The virus targets tumors that lack p53 function and has entered human clinical trials. Another virus that has been genetically engineered to treat cancer is the herpes simplex virus. Different mutants have been constructed and tested, including mutants with mutations in the ICP34.5 and ICP6 genes. The former encodes so-called neurovirulence factors, while the latter encodes the large subunit of ribonucleotide reductas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02A61K
CPCC12N7/00C12N2710/10351
Inventor P·克斯特尔K·亚纳吉马奇C·奥尔森
Owner ONYX PHARMA INC
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