Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Recovery or purification
Utility Patent
1998-01-28
2001-01-02
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Recovery or purification
C435S041000, C435S325000, C435S366000, C435S378000, C435S380000, C435S403000, C435S291300, C435S306100
Utility Patent
active
06168944
ABSTRACT:
BACKGROUND OF THE INVENTION
Many established cell lines are available for a variety of purposes in biotechnology. Some cell lines can be cultivated as single-cell suspensions, but other cell lines do not grow well without a support. The growth of a cell line that requires support is often limited by the surface area available for the cells to grow on, since many cell lines will form only a monocellular layer on the surface. In addition, some cell lines may tend to grow in clumps or aggregates in the absence of a support, which is an undesirable result when they are needed as single-cell suspensions, but more especially when the cells are to be infected with a virus or transformed with a recombinant vector, since the virus or vector may not gain access to the cells within the clump or aggregate. Thus, there can be severe problems in scaling up the cultivation of a cell line, in particular in providing enough surface area for the cells to grow on and/or avoiding clumping of the cells.
Microcarrier technology has been used to cultivate cells in culture. For example, Forestell et al. (
Biotech. Bioeng.
40: 1039-1044 (1992)) disclosed extended serial subculture of human diploid fibroblasts on microcarriers using a medium supplement that reduced the need of the cultured cells for serum. Furthermore, Ohlson et al. (
Cytotechnology
14:67-80 (1994)) disclosed the bead to bead transfer of Chinese hamster ovary cells using macroporous gelatin microcarriers. Finally, Hu et al. (
Biotech. Bioeng.
27: 1466-1476 (1985)) disclosed the serial propagation of mammalian cells on microcarriers using a selection pH trypsinization technique.
However, in view of the problems noted above, there is a need for improvements in methods of cultivating cell lines, in methods of producing viruses for clinical uses, and, in methods of scaling up the protection of viruses for larger-scale commercialization, especially recombinant viruses for gene therapy. The Instant invention meets these needs and more.
SUMMARY OF THE INVENTION
One aspect of the invention is a method for producing a recombinant virus for gene therapy, the method comprising
(a) cultivating the cells on a first batch of microcarriers until the cells are substantially confluent;
(b) transferring the cells from the microcarrriers by detaching the cells from the microcarriers by adding trypsin without removing the microcarriers from suspension, and adding a second batch of microcarriers;
(d) infecting the cells with a recombinant virus;
(e) separating cells of (d) from microcarriers on which they have been cultivated but from which they have become detached, comprising introducing an aqueous suspension of cells and microcarriers through an inlet into a separation device, the device comprising:
(i) an inlet;
(ii) a column;
(iii) an outlet for the collection of cells and the aqueous solution; and
(iv) a mesh screen;
wherein the microcarriers are retained in suspension by an upward flow in the separation device and are retained in the separation device by a mesh screen, and wherein the cells and aqueous solution are collected through the outlet; and
(f) liberating virus from collected cells of (e), which comprises mechanically shearing the cells by means of exposing the cells to a shear rate, wherein the shear rate for mechanically shearing the cells is generated by collecting the cells in a microfilter and rapidly recirculating the aqueous medium through the microfilter.
A further aspect of the invention is a method for the separation of cells from microcarriers on which they have been cultivated but from which they have become detached, wherein the cells have been infected with a recombinant virus for gene therapy, comprising introducing an aqueous suspension of infected cells and microcarriers through an inlet into a separation device, the device comprising:
(a) an inlet;
(b) a column;
(c) an outlet for the collection of cells and the aqueous solution; and
(d) a mesh screen;
wherein the microcarriers are retained in suspension by an upward flow in the separation device and are retained in the separation device by a mesh screen, and wherein the cells and aqueous solution are collected through the outlet.
A further aspect of the invention is a method for the liberation of a recombinant virus for gene therapy from cultivated cells in an aqueous medium, which comprises mechanically shearing the cells by means of exposing the cells to a shear rate, wherein the shear rate for mechanically shearing the cells is generated by collecting the cells in a microfilter and rapidly recirculating the aqueous medium through the microfilter.
A further aspect of the invention is a system for separating cells infected with a recombinant virus for gene therapy from microcarriers on which the cells have been cultivated, the system comprising:
(a) a bioreactor in which the infected cells were cultivated on the microcarriers;
(b) a flow path from the bioreactor to a separation device;
(c) a separation device comprising
(i) an inlet;
(ii) a column;
(iii) an outlet for the collection of cells and the aqueous solution; and
(iv) a mesh screen;
wherein the microcarriers are retained in suspension by an upward flow in the separation device and are retained in the separation device by a mesh screen, and the cells and aqueous solution are collected through the outlet; and
(d) a pump, wherein the pump directs the flow of the aqueous solution from the bioreactor to the outlet.
REFERENCES:
patent: 5707868 (1998-01-01), Boulay et al.
patent: 5846829 (1998-12-01), Worden et al.
patent: WO 86/01531 (1986-03-01), None
patent: WO 95/11984 (1995-05-01), None
patent: WO 95/16772 (1995-06-01), None
patent: WO 95/24468 (1995-09-01), None
patent: WO 96/22378 (1996-07-01), None
Forestell, et al. Biotech. Bioeng 40: 1039-1044 (1992).
Ohlsen, et al. Cytotechnology 14: 67-80 (1994).
Hu, et al. Biotech Bioeng. 27: 1466-1476.
Huyghe, et al. Human Gene Therapy 6:1403-1416 (1995).
Billig et al., vol. 55, 1984, pp. 67-75.
Chemical Abstract 105:41214, Vretblad et al.
Maiorella et al., Biotech. Bioeng vol. 37, pp. 121-126 (1991).
Batandolo Serge
Condon Russell G. G.
Connelly Nancy V.
Frei Andreas
Glowacki Edward
Gould James M.
Schering Corporation
Stucker Jeffrey
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