Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Insect cell – per se
Reexamination Certificate
1997-12-08
2001-04-03
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Insect cell, per se
C435S404000
Reexamination Certificate
active
06210966
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This application concerns a culture medium for insect cells.
2. Brief Description of the Art
The baculovirus expression system using insect cells has become an important tool for production of recombinant proteins for several reasons. First, the expression levels are often very high compared to those obtained in other animal cells, such as CHO-cells. Second, proteins produced using this system are often biologically active due to the insect cells' ability to perform post-translational modifications, folding and assembly of most proteins. Third, the time from cloning to production of the protein is short compared to the time needed to construct a stably transformed animal cell line. Fourth, cell death inevitably follows virus infection of insect cells. This can be an advantage over other expression systems because it may permit better expression of cytotoxic, regulatory or essential cellular genes.
Foreign proteins are produced during a lytic infection of insect cells with a recombinant virus. Baculoviruses contain a very late hyper-expressed gene, polyhedrin, which is not essential for viral replication. Placing a gene under control of the polyhedrin promoter allows the production of large quantities of a recombinant protein. The strategy for production of a protein involves three distinct stages:
(a) growing the insect cells from mid to late growth phase;
(b) infecting the cells with virus containing a gene encoding the protein of interest; and
(c) harvesting and purification of the protein product.
Insect cells are conventionally cultured in complex media containing inorganic salts and sometimes organic salts, amino acids, sugars, vitamins, trace elements, lipids and protein hydrolysates. For example one commercially available medium, called, IPL-41, is available from a number of suppliers. Apart from these components, serum or yeast extract has conventionally been added to provide undefined but essential nutrients. The amino acid glutamine has hitherto been assumed to be essential for cultured insect cells (Wang, M-Y et al Biotechnol. Prog. 1993, 9, 355-361; Kamen, A. A. et al Biotechnol. Bioeng. 1991, 38, 619-628;Wang, M-Y et al Biotechnol. Prog. 1993 in press), as it is for most cultured mammalian cell lines.
BRIEF SUMMARY OF THE INVENTION
We have surprisingly found that insect cells are capable of synthesising glutamine themselves. The ability of insect cells to grow without an external glutamine source has not been described in the literature before. We have now shown that insect cells are, indeed, able to grow without glutamine supplied to the medium and that insect cells are, in fact, capable of growing in a medium without glutamine, glutamate or aspartate.
According to one aspect of the invention there is provided a medium, for example for culturing insect cells for producing proteins or polypeptides using a baculovirus expression system, characterized in that the medium does not contain glutamine.
The medium may also optionally not contain glutamate and/or aspartate.
The medium of the invention can be used for growth of the cells for the production of recombinant proteins.
The medium of the invention has several advantages over traditional, glutamine-containing, insect cell culture media. First, glutamine is generally considered problematic in cell culture because of its instability. It decomposes spontaneously to pyrrolidone carboxylic acid and ammonia in aqueous solution. This instability is the major factor limiting the storage life of a cell culture medium. A medium without glutamine thus results in a markedly longer storage life. The medium of the invention is also less expensive and simpler to prepare. Further, cells growing in the medium of the invention exhibit a more efficient metabolism and a decreased secretion of by-products that may be inhibitory to growth.
Preferably, the medium comprises an ammonium salt or another ammonium ion source or at least one amino acid or another compound that can be converted to ammonium ions. An external ammonium ion source is then used as a nitrogen source for biosynthesis of amino acids. Preferably, the concentration of ammonium in the medium is up to 20 mM. However, during certain conditions, as for example, in substrate limited cultures, the cells may liberate ammonium themselves which in turn can be used for biosynthesis. Under these conditions addition of ammonium is not necessary for growth.
According to another aspect of the invention, there is provided a method of culturing insect cells, the method comprising growing the cells in a medium according to the invention.
According to a further aspect of the invention we provide a method of obtaining a polypeptide including transforming insect cells with recombinant baculovirus including a gene coding for said polypeptide, and growing the transferred cells in a culture medium that does not contain glutamine but which does contain an ammonium ion source.
The insect cells may for example be from the fall armyworm,
Spodoptera frugiperda.
REFERENCES:
patent: 2205572 (1974-05-01), None
Suzuki et al., Elucidation of Amidating Rxn Mech. by Frog amidating enzyme . . . Expressed in Insect Culture, EMBO, see abstract, 1990.*
Invertebrate Cell Culture Applications, 1982, Academic Press, Inc., New York; pp. 9 & 38.*
Biotechnology and Bioengineering Including: Symposium Biotechnology In Energy Production and Conservation., vol. 42, No. 6, May 9, 1993, New York, US., see pp. 697-707.*
Ohman et al., “Induction of a Metabolic Switch in Insect Cells by Substrate-Limited Fed Batch Cultures”, Appl Microbial Biotechnol. 73:1006-1013, pp. 1006-1013, 1995.
Häggström Lena
Öhman Lars
Garabedian Todd E.
Karo Bio AB
Naff David M.
Ware Deborah K.
Wiggin & Dana
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