Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Reexamination Certificate
2000-03-29
2001-12-04
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S235100, C435S320100
Reexamination Certificate
active
06326183
ABSTRACT:
BACKGROUND OF THE INVENTION
Baculovirus are a diverse group of viruses having a host range which is restricted to insects. They are large viruses having a lipid envelope and containing a genome of double-stranded circular DNA. Recombinant baculovirus (rBV) vectors, primarily derived from the baculovirus
Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV), are commonly used in industry and academia for the high-level production of heterologous proteins in insect cells. This expression system involves the infection of cultured insect cells with rBV into which the gene encoding the protein to be expressed has been inserted. See O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992)
Baculovirus Expression Vectors: A Laboratory Manual
. (Freeman, New York) for review of the baculovirus expression system.
Another potential use of rBV has recently appeared. It is demonstrated that rBV can transfer genetic material into mammalian cells, with a preference for hepatocytes. As long as a gene of interest is preceded by a promoter that is active in mammalian cells, the gene will be efficiently expressed in the target mammalian cells. See Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P., and Strauss, M. (1995), “Efficient gene transfer into human hepatocytes by baculovirus vectors”,
Proc. Natl. Acad. Sci. USA
92, 10099-10103; Boyce, F. M. and Bucher, N. L. R. (1996) “Baculovirus-mediated gene transfer into mammalian cells”,
Proc. Natl. Acad. Sci. USA
93, 2348-2352, incorporated herein by reference. This previously unobserved characteristic makes rBV potentially useful in human gene therapy. Recombinant baculovirus has several potential advantages for gene therapy. These include:
1. a very large DNA insert capacity
2. A fairly high viral titer
3. absence of a pre-existing immune response in humans
4. lack of replication in mammals
5. lack of toxicity in mammals
6. lack of expression of viral genes in mammalian cells due to the insect-specificity of the baculovirus transcriptional promoters, and, therefore, a potential absence of a cytotoxic T lymphocyte response directed against these viral proteins
Nevertheless, there exists a need for a method to concentrate rBV to a very high titer. This will be required for in vivo gene therapy applications of high doses of rBV. This virus must be at high titer and must be present in a physiological buffer. Also, a purification/concentration step is critical in large scale in vitro production of proteins in insect cells. In order to produce proteins at very high levels, the insect cells must be infected with rBV at a high multiplicity of infection (moi). If the rBV has not been previously concentrated, but is rather simply the conditioned medium from the insect cells producing the virus, the volume of the inoculum would be a significant proportion of the total bioreactor volume. This may not be desirable, nor in some cases even possible, in large-scale manufacturing.
Viruses are conventionally concentrated to high titer by a combination of pelleting the virus through use of an ultracentrifuge and by banding the virus in, for instance, a sucrose gradient. See, for example, Sandig, V., Hofmann, C., Steinert, S., Jennings, G., Schiag, P., and Strauss, M. (1996) “Gene transfer into hepatocytes and human liver tissue by baculovirus vectors”,
Human Gene Therapy
7: 1937-1945 and Bowles, N. E., Eisensmith, R. C., Mohuiddin, R., Pyron, M. And Woo, S. L. C. (1996) “A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo”,
Human Gene Therapy
7: 1735-1742. Unfortunately, rBV preparations made in this manner tend to be badly aggregated (See Example 1). Therefore, a concentration step is needed that avoids this problem, but such a step has not heretofore been available.
SUMMARY OF THE INVENTION
In accordance with this invention, there is provided a process for producing chromatographically concentrated baculovirus, which process comprises contacting a preparation containing a baculovirus with an ion exchange chromatographic resin and eluting the bound baculovirus from the resin.
Briefly stated, the present invention provides methods to separate lipid envelope viruses, e.g., baculoviruses, from preparations. In one aspect, the present invention provides methods for the separating a baculovirus from contaminating substances comprising the steps of: (a) contacting a preparation containing a baculovirus with an ion exchange chromatographic resin, so that the virus binds to the resin and (b) eluting the bound baculovirus from the resin in a volume that is smaller than that of the original starting volume. The method may further include the step of separating from the resin, prior to the elution step, that portion of the preparation which is not bound to the resin.
In another method, the resin is generated in buffer and, after the resin is contacted with the baculovirus but before the elution step, the resin is washed with a buffer having properties identical to the buffer used to generate the resin. Preferred resins include those comprising derivatized agarose in which the derivative is at least one aryl sulfate group or a sulfopropyl group. Additional resins include those selected from the group consisting of: (i) a mixed resin column of silica derivatized with mixed cationic and anionic groups; (ii) a silica resin comprising carboxyl and sulfate functional groups; (iii) a cellulose resin with a carboxymethyl functional groups; (iv) an acrylamide resin with carboxymethyl functional groups; (v) a ceramic resin with sulfate functional groups and (vi) a sepharose resin with carboxymethyl functional groups.
A further aspect of the invention is a chromatographically concentrated preparation of baculovirus having the following properties: (a) concentrated at least five fold from an original preparation; (b) having less than about 50% soluble protein, exclusive of viral protein, and (c) in contact with a physiological buffer. Another chromatographically concentrated preparation of baculovirus has the property of lacking significant aggregation when tested by centrifugation at about 4000 g for about 6 minutes.
The method presented is an easy and rapid single step concentration using column chromatography. This protocol has advantages over other methods in that no ultracentrifugation is required, the virus does not appear to aggregate appreciably during the concentration and the method can be easily scaled up to a very large size.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based, in part, on the discovery that protein purification schemes using column chromatography can be applied to the purification and concentration of baculovirus. In particular, we are now able to generate “chromatographically concentrated baculovirus” using ion exchange chromatographic procedures, defined as baculovirus that has been concentrated by a factor of at least five, preferably ten to thirty, most preferably about fifty to one hundred fold, over a titer of baculovirus prior to the chromatographic procedure. Moreover, a chromatographically concentrated” preparation has a protein concentration, exclusive of viral protein, having a value reduced to less than 50% of its original protein concentration. A further property of a chromatographically concentrated preparation is that at least fifty (50%) percent of the baculovirus is in a non-aggregated state as compared to the baculovirus preparation prior to the chromatographic procedure. The term “non-aggregated state” refers to the ability of the baculovirus in the chromatographically concentrated preparation not to form pellets under low speed (e.g., no greater than about 4000 g) centrifugation. That is, that portion of the viral preparation remaining in the supernatant after low speed centrifugation is the “non-aggregated” portion.
The term “ion exchange chromatography resin” refers to a matrix whose components carry functional groups that have either positive or negative charges (anion-exchangers and cation-exc
Biogen Inc.
Mosher Mary E.
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