ZA200408366B - Improved viral purification method - Google Patents
Improved viral purification method Download PDFInfo
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- ZA200408366B ZA200408366B ZA200408366A ZA200408366A ZA200408366B ZA 200408366 B ZA200408366 B ZA 200408366B ZA 200408366 A ZA200408366 A ZA 200408366A ZA 200408366 A ZA200408366 A ZA 200408366A ZA 200408366 B ZA200408366 B ZA 200408366B
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- reovirus
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- 238000000034 method Methods 0.000 title claims description 25
- 238000000746 purification Methods 0.000 title description 4
- 230000003612 virological effect Effects 0.000 title description 3
- 241000702263 Reovirus sp. Species 0.000 claims description 27
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- 238000001914 filtration Methods 0.000 claims description 7
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 230000002458 infectious effect Effects 0.000 claims description 4
- 239000003599 detergent Substances 0.000 claims description 2
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- 239000000725 suspension Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims 4
- 239000000706 filtrate Substances 0.000 claims 3
- 102000004190 Enzymes Human genes 0.000 claims 2
- 108090000790 Enzymes Proteins 0.000 claims 2
- 239000013592 cell lysate Substances 0.000 claims 2
- 238000011026 diafiltration Methods 0.000 claims 2
- 238000004255 ion exchange chromatography Methods 0.000 claims 2
- 238000001542 size-exclusion chromatography Methods 0.000 claims 2
- HNLXNOZHXNSSPN-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCOCCOCCOCCO)C=C1 HNLXNOZHXNSSPN-UHFFFAOYSA-N 0.000 claims 1
- 238000005571 anion exchange chromatography Methods 0.000 claims 1
- 210000003734 kidney Anatomy 0.000 claims 1
- 210000001161 mammalian embryo Anatomy 0.000 claims 1
- 244000309711 non-enveloped viruses Species 0.000 claims 1
- 229940098514 octoxynol-9 Drugs 0.000 claims 1
- 229920002114 octoxynol-9 Polymers 0.000 claims 1
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- 238000002560 therapeutic procedure Methods 0.000 description 7
- 208000008104 Reoviridae Infections Diseases 0.000 description 6
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- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
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- 101150040459 RAS gene Proteins 0.000 description 1
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- 230000003213 activating effect Effects 0.000 description 1
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- 210000004899 c-terminal region Anatomy 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
} IMPROVED VIRAL PURIFICATION METHODS . RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Applications Serial
Number 60/377,273, filed April 30, 2002; and Serial Number 60/443,176, filed January 29,2003. The entire disclosure of these prior applications is hereby incorporated by reference.
This invention relates to a method of extracting virus from a cell culture. In particular, the method is useful for extracting infectious virus which is suitable for clinical administration to mammals, including human.
U.S. Patent Application Publication No. 20020037576, published March 28, 2002.
W099/08692A1, published February 25, 1999.
Japanese Patent 63044532A, published February 25, 1988.
Berry et al., Biotechnology and Bioengineering, "Production of Reovirus : Type-1 and Type-3 from Vero Celis Grown cn Solid and Macroporous Microcarriers”,
Biotechnology and Bioengineering 62: 12-19 (1999).
Bos, J.L.,"Ras Oncogenes in Human Cancer: A Review", Canc. Res. 49(17): - 4682-4689 (1989).
Chandron and Nibert, "Protease cleavage of reovirus capsid protein mul and . mulC is blocked by alkyl sulfate detergents, yielding a new type of infectious subvirion particle", J. of Virology 72(1):467-75 (1998).
Coffey, M.C., et al., “Reovirus therapy of tumors with activated Ras pathway”, Science 282: 1332-1334 (1998).
Davis, et al., Microbiology, Lippincott, Philadelphia (1990).
Drastini, Y. et al., "Comparison of eight different procedures for harvesting avian reoviruses grown in Vero cells", J. Virological Methods 39: 269-278 (1992).
Duncan et ai., "Conformational and functional analysis of the C-terminal globular head of the reovirus ceil attachment protein”, Virology 182(2):810-9 (1991).
Fields, B.N. et al., Fundamental Virology, 3rd Edition, Lippincott-Raven (1996).
Mah et al., "The N-terminal quarter of reovirus cell attachment protein sigma 1 possesses intrinsic virion-anchoring function”, Virology 179(1):95-103 (1990).
McRae, M.A. and Joklik, W.K., “The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3", Virology, 89:578- 593 (1979). . 20 Nibert et al., “Reovirus and their replication”, in Fields et al., Fundamental
Virology, 3rd Edition, Lippincott-Raven (1996).
Remington's Pharmaceutical Sciences, Mack Publishing Company, : Philadelphia PA 19" ed. (1995).
Smith, R.E., et al., “Polypeptide components of virions, top component and . cores of reovirus type 3", Virology, 39:791-800 (1969). :
Strong, J.E. and P.W. Lee, “The v-erbV oncogene confers enhanced cellular susceptibility to reovirus infection”, J. Virol. 70: 612-616 (1996).
Strong, J.E., et al., "Evidence that the Epidermal Growth Factor Receptor on
Host Cells Confers Reovirus Infection Efficiency”, Virology 197(1): 405-411 (1993).
Strong, J.E., et al., “The molecular basis of viral oncolysis: usurpation of the
Ras signaling pathway by reovirus”, EMBO J. 17: 3351-3362 (1998).
Taber et al., “The selection: of virus-resistant Chinese hamster ovary cells”,
Cell 8: 526-533 (1576.
Turner and Duncan, "Site directed mutagenesis of the C-terminal portion of reovirus protein sigmal: evidence for a conformation-dependent receptor binding domain", Virology 186(1):219-27 (1992).
All of the publications, patents and patent applications cited above or elsewhere in this application are herein incorporated by reference in their entirety to the same extent as if the disclosure of each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.
] Due to the vast number of diseases caused by viruses, virology has been an intensively studied field. There has always been the demand to produce viruses } efficiently in order to isolate and purify viral proteins, to generate vaccines, Or to provide infectious viruses for laboratory studies. Recently, the new development of virus therapy has further necessitated the need for efficient production of infectious viruses.
Reovirus therapy is an example of virus therapy. Reovirus is a double- stranded RNA virus capable of binding to a multitude of cells. However, most cells are not susceptible to reovirus infection and binding of reovirus to its cellular receptor results in no viral replication or virus particle production in these cells. This is probably the reason why reovirus is not known to be associated with any particular disease.
It was discovered recently that cells sansformed with the ras oncogene become susceptible to reovirus infection, while their untransformed counterparts are not (Strong et al., 1998). For example, when reovirus-resistant NIH 3T3 cells were transformed with activated Ras or Sos, a protein which activates Ras, reovirus infection was enhanced. Similarly, mouse fibroblasts that are resistant to reovirus infection became susceptible after transfection with the EGF receptor gene or the v-erbB oncogene, both of which activate the ras pathway (Strong et al., 1993; Strong et al., 1996). Thus, } reovirus can selectively infect and replicate in cells with an activated Ras pathway.
The ras oncogene accounts for a large percentage of mammalian tumors. } Activating mutations of the ras gene itself occur in about 30% of all human tumors (Bos, 1989), primarily in pancreatic (9C%), sporadic colorectal (50%) and lung (40%) . 25 carcinomas, as well as myeloid leukemia (30%). Activation of factors upstream or downstream of ras in the ras pathway is also associated with tumor. For example, overexpression of HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is common in breast cancer (25-30%), and overexpression of platelet-derived growth factor (PDGF) receptor or EGF receptor is prevalent in gliomas and glioblastomas (40- ] 50%). EGF receptor and PDGF receptor are both known to activate ras upon binding to their respective ligand, and v-erbB encodes a constitutively activated receptor lacking } S the extracellular domain.
Since a large number of human tumors are accounted for by genetic alteration of the proto-oncogene ras or a high Ras activity, reovirus therapy is a new, promising therapy for such conditions (Coffey et al., 1998). Reovirus therapy is highly selective for Ras-associated tumor cells and leaves normal cells uninfected. This therapy has wide applications and can be used in both human and non-human animals.
In order to produce reovirus suitable for clinical administration, fast and efficient methods of producing reovirus in cultured cells are needed. Moreover, the traditional method of purifying viruses from cultured cells is tedious and time consuming, rendering the cost of virus production toc high. Therefore, an improved method for virus purification is also needed.
The present invention relates to an improved method of extracting and purifying viruses from cell culture that can be applied to both small and large scale virus production. The method involves a simple extraction step in which a detergent is - directly added to the cell culture. Thereafter, cell debris can be removed from the extraction mixture by, for example, filtration or centrifugation. The resulting virus suspension can be further concentrated and/or enriched by chromatographic methods.
The virus prepared according to the present invention can be used for any purpose, including purification of viral proteins, vaccination, infection of host cells and clinical . 25 administration.
Claims (16)
1. A method of producing virus from a culture of cells, comprising the steps of: (a) providing a culture of cells which has been infected by the virus; Md) extracting the virus from the cells by adding a detergent to the culture of cells and incubating for a period of time to result in a cell lysate; (c) removing cell debris by step-wise filtration comprising: (1) filtering through a prefilter having a pore size of 5S uM or 8 uM, and (i1) filtering after step (i) through a combination filter having pore sizes of 3 uM and 0.8 uM; (d) purifying the virus by a combination of ion exchange and size exclusion chromatography; and (e) collecting the virus.
2. The method of claim 1 further comprising treating the cell lysate with a DNA- cleaving enzyme.
3. The method of claim 1 further comprising concentrating the filtrate.
4. The method of claim 3 wherein the filtrate is concentrated by diafiltration.
~ 5. The method of claim 1 wherein the virus is a non-enveloped virus.
6. The method of claim 1 wherein the virus is a reovirus.
7. The method of claim 6 wherein the reovirus is a mammalian reovirus.
8. The method of claim 7 wherein the mammalian reovirus is a human reovirus.
9. The method of claim 8 wherein the human reovirus is a serotype 3 virus. -31- AMENDED SHEET: 21 FEBRUARY 2006 —
10. The method of claim 9 wherein the serotype 3 reovirus is the Dearing strain.
11. The method of claim 6 wherein the reovirus is a recombinant reovirus.
12. The method of claim 1 wherein the cells are human embryo kidney 293 (HEK 293) cells.
13. The method of claim 12 wherein the HEK 293 cells are grown in suspension.
14. The method of claim 1 further comprising purifying the virus by anion exchange chromatography.
15. A method of producing infectious reovirus, comprising: (a) providing a culture of HEK 293 cells which has been infected by reovirus; (b) extracting the virus from the cells by adding octoxynol-9 to 10 to the culture of HEK 293 cells and incubating at about 25°C to about 37°C; (c) treating the mixture from step (b) with a DNA-cieaving enzyme; (d) removing cell debris by step-wise filtration comprising: (1) filtering through a prefiiter having a pore size of 5 pM or 8 uM, and (11) filtering after step (i) through a combination filter having pore sizes of 3 uM and 0.8 uM; - (e) concentrating the filtrate by ultrafiltration or diafiltration; i ® purifying the reovirus by a combination of ion exchange and size exclusion chromatography; and (g) collecting the reovirus.
16. A method according to claim 1, substantially as herein described with reference to the illustrative Examples. -32- AMENDED SHEET: 21 FEBRUARY 2006
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US37727302P | 2002-04-30 | 2002-04-30 |
Publications (1)
Publication Number | Publication Date |
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ZA200408366B true ZA200408366B (en) | 2006-07-26 |
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Application Number | Title | Priority Date | Filing Date |
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ZA200408366A ZA200408366B (en) | 2002-04-30 | 2004-10-15 | Improved viral purification method |
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ZA (1) | ZA200408366B (en) |
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2004
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