Method of producing adenoviral vector stocks

Details Method of producing adenoviral vector stocks Method of producing adenoviral vector stocks

2000-04-07

2001-01-02

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Virus or bacteriophage, except for viral vector or...

Utility Patent

active

06168941

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention pertains to the production of viral vector stocks.
BACKGROUND OF THE INVENTION
Modified viruses, also known as viral vectors, have proven convenient vector systems for investigative and therapeutic gene transfer applications, and adenoviral vector systems present several advantages for such uses. Due to these advantages, researchers have developed numerous gene therapy applications based upon adenoviral vectors. As such adenoviral vector based applications move through clinical trials and into approved medical applications, there will be an increasing need for efficient large-scale production of adenoviral vector stocks.
Systems for production of virus stocks in cell cultures are known in the art. Several references in the prior art teach general methods for the production of virus stocks in cell cultures (see, e.g., U.S. Pat. Nos. 4,055,466, 4,072,565, and 4,080,258). Due to the variations in stock production amongst the wide variety of viral vectors and cell types permissive for growth thereof, generalized methods have often proven unsuitable for large-scale production of viral vector stocks, particularly stocks of recombinant viral vectors.
More recently, researchers have studied the production of recombinant viral vector stocks by focussing on specific types of viral vectors. For example, systems drawn to production of recombinant baculovirus stocks in insect cells have been extensively researched (see, e.g., Kamen et al.,
Biotechnology and Bioengineering,
50(1), 36-48 (1996)). Other researchers have studied systems for the production of recombinant retroviruses (see, e.g., Le Doux et al.,
Biotechnology and Bioengineering,
63(6), 664-652 (1999)). The adsorption and replication rates of Vaccinia virus in batch cultures has also been previously studied (see Chillakuru et al.,
Biotechnol. Prog.,
7, 85-92 (1991)).
In comparison to the procedures available to produce baculovirus and other non-adenoviral vector stocks, relatively few systems for production of adenoviral vector stocks are known. U.S. Pat. No. 5,994,134 teaches the use of a microcarrier-based bioreactor process for producing viruses in cells at high densities, including adenoviruses, in serum-based medium. The limitations attendant serum-based mediums and microcarrier bioreactors limit the applicability of this method. Garnier et al.,
Cytotechnology,
15, 145-55 (1994), provides a method of producing high levels of recombinant proteins in 293 cells infected with recombinant adenoviral vectors by infecting the cells at various concentrations in combination with single or double medium replacements post infection. Furthering this research, C{circumflex over (o)}t{acute over (e)} et al.,
Biotechnology and Bioengineering,
59(5), 567-75 (1998), teaches production of recombinant proteins in adenoviral vector-infected 293 cells at cell densities of up to 1×10
7
cells/ml. However, neither Garnier et al. nor C{circumflex over (o)}t{acute over (e)} et al. addresses the production of adenoviral vector stocks.
Accordingly, there is a need for alternative methods of producing adenoviral vector stocks. The present invention provides such methods. This and other advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of producing an adenoviral vector stock. The steps of the method include providing a culture of cells permissive for growth of adenoviral vectors, wherein the cells are in a medium, culturing the culture under conditions to permit growth of the cells, perfusing fresh medium through the culture, in an amount of at least about two times the volume of medium in the culture, while the density of the cells in the medium is about 40-70% of the density of cells obtained in the medium when the growth of the culture is in the stationary phase, contacting the culture with adenoviral vectors under conditions permissive for the infection of the cells after the perfusion of fresh medium is substantially completed, culturing the infected cells to replicate the adenoviral vectors, and harvesting the infected cells to obtain an adenoviral vector stock.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method of producing an adenoviral vector stock. The steps of the method include (a) providing a culture of cells permissive for growth of adenoviral vectors, wherein the cells are in a medium, (b) culturing the culture under conditions to permit growth of the cells, (c) perfusing fresh medium through the culture for a period of about 1-6 hours, in an amount of at least about two times the volume of medium in the culture, while the density of the cells in the medium is about 40-70% of the density of cells obtained in the medium when the growth of the culture is in the stationary phase (also referred to as the “stationary phase density”), (d) contacting the culture with adenoviral vectors under conditions permissive for the infection of the cells after the perfusion of fresh medium in step (c) is substantially completed, (e) culturing the infected cells to replicate the adenoviral vectors, and (f) harvesting the infected cells to obtain an adenoviral vector stock.
The cells can be any suitable type of cells for producing an adenoviral vector stock. Thus, any cells that are capable of acting as adenoviral vector producing cells are suitable. Examples of suitable cells include cells of primary cell lines, such as human embryonic kidney (HEK), human embryonic lung (HEL), and human embryonic retinoblast (HER) cells, although other cells (e.g., HELA cells or cultured tissue cells) can be suitable. Preferably, the cells are, or are derived from, anchorage dependent cells, but which have the capacity to grow in suspension cultures. Preferred HEK cells are 293 cells, and cells from cell lines which are derived from 293 cells. Preferred HEL cells are A549 cells, and cells from cell lines derivative thereof.
Preferably, the cells are modified to contain DNA corresponding to, or derived from, some portion (e.g., one or more nucleotide sequences) of the adenoviral genome so as to be capable of complementing an adenoviral vector deficient in such sequences, particularly a replication-deficient adenoviral vector such as are generally described in U.S. Pat. No. 5,994,106 and International Patent Application WO 95/34671. More preferably, the cell is capable of such complementation of the adenoviral vector, while preventing homologous recombination between the adenoviral vector and complementing cell.
Preferably, the modified cells are of, or derived from, 293, A549 or modified HER cells. Modified HER cells, in particular offer several advantages over other cells capable of producing adenoviral vector stocks. Thus, it is desirable, for example, that such modified HER cells are highly transfectable, exhibit much lower frequencies of homologous recombination as 293 cells, and, when infected with adenoviral vectors, are associated with the appearance of plaques in monolayers of the cells in about 3-4 days (versus, for example, 6-10 days associated with 293 cells). Thus, such modified HER cells when infected with adenoviral vectors enable faster performance of plaque assays, than, for example, 293 cells. More desirably, the HER cells are associated with yields of adenoviral vectors at higher rates than in other cell types, more preferably at rates of about 2-3 times as high as those achieved with 293 cells infected with similar adenoviral vectors. Examples of suitable modified HER cells include 911 cells, described in Fallaux et al.,
Human Gene Therapy,
7, 215-222 (1996). Particularly preferred modified HER cells are PER.C6 cells (Introgene, Inc.), which are described in International Patent Application WO 97/00326.
The cells form a culture, which is maintained in a suitable medium. The culture of cells can be any suitable culture for the production of adenoviral vector stocks where fresh medium can be perfu

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