WO1998050522A1

WO1998050522A1 - Method and apparatus for high volume production of viral particles

Info

Publication number: WO1998050522A1
Application number: PCT/US1998/008797
Authority: WO
Inventors: Van C. Taiariol; Girish J. Pendse; John Vernachio
Original assignee: Baxter International Inc.
Priority date: 1997-05-07
Filing date: 1998-04-29
Publication date: 1998-11-12
Also published as: AU7172598A

Abstract

The present invention provides a method of making large volumes of high numbers of functional viral particles by culturing virus-producing cells in an internally oxygenated bioreactor which provides an oxygen-containing gas to the culture. The present invention also provides an internally oxygenated bioreactor suitable for use in such a method.

Description

METHOD AND APPARATUS FOR HIGH VOLUME PRODUCTION OF VIRAL PARTICLES

BACKGROUND OF THE INVENTION

The present invention relates generally to cell culture and, more specifically, to the in vitro culturing of cells using an internally oxygenated bioreactor for the purpose of recovering viruses produced by the cells .

In vitro culture of cells is well known for a variety of purposes, such as studying cell-cell interactions, studying or assaying interactions between cells and various exogenous substances or fluids, and the like. Among the most important uses for in vitro culturing of cells is for the production of mass quantities of cells and for the recovery of useful products, such as viruses, produced by the cultured cells. The prior art is replete with methods and apparatus for the in vitro culturing of cells for the purposes of culturing the cells to a substantial density per se and/or for obtaining quantities of useful products secreted by them. Anchorage-dependent cells, those which require affixation to a surface in order to grow and survive, have been cultured in a variety of flasks and roller bottles where cells attach to the flask or bottle surfaces, on microcarriers suspended in culture medium, on the surfaces of hollow fibers, and in other similar systems. Anchorage-independent cells, those which are capable of being cultured unattached to a substrate, have been grown in a variety of suspension culture vessels, hollow fiber devices, and the like.

As the fields of genetic engineering and gene therapy have advanced, the need for methods capable of producing large volumes of high titer viral vector preparations has steadily increased. The production of sufficient numbers of functional viral vectors is one of the most important problems in making gene therapy practical. Preferably, such virus preparations would be made quickly and with a minimum of operator contact and monitoring. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION The present invention provides a method of producing viruses by culturing cells capable of producing viruses in an internally oxygenated bioreactor of this invention, as set forth below.

The present invention also provides an internally oxygenated bioreactor useful in culturing cells; and especially so for producing high numbers of viral particles as discussed below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of an internally oxygenated bioreactor.

FIG. 2 is a schematic diagram of an apparatus including an internally oxygenated bioreactor (I/O Bioreactor) used to culture cells to produce high titers of retrovirus vectors.

FIG. 3 is a diagram of the squeeze tube of FIG. 2. FIG. 4 is a map of plasmid pLXSN-T84.66γ . FIG. 5 is a map of plasmid pLXSN-N29γ, which was used to make plasmid pLXSN-T84.66γ of FIG 4.

FIG. 6 diagrams the glucose concentration in culture media within two internally oxygenated bioreactors (squares) and the perfusion rate of cell culture media into the same bioreactors (diamonds) during the production of Moloney murine leukemia retrovirus by retrovirus producer cells transformed with plasmid pLXSNT84.66γ#22 over a period of thirteen days . The dotted line indicates the day when serum-free media replaced media supplemented with 5% FBS. FIG. 7 diagrams the titers of Moloney murine leukemia virus produced by two bioreactors. These data were obtained from the bioreactors used in FIG. 6.

DETAILED DESCRIPTION OF THE INVENTION This invention provides a method for the production of high numbers of functional viral particles. Furthermore, the instant method provides for low residence time of the viral particles in a bioreactor. Low residence time in the bioreactor means that the viral particles so produced are only briefly subjected to the virus-destroying temperatures needed to culture the virus-producing cells. Furthermore, low residence time also results in a much shorter period needed to produce such high numbers .

Specifically, the method employs an internally oxygenated bioreactor containing cells capable of producing viral particles. A mixture of gasses containing at least oxygen and carbon dioxide are passed through the bioreactor. Cell culture medium appropriate for the production of viral particles is passed through the bioreactor at a flow-rate such that the average residence time of the viral particles in the bioreactor produced by the method is between about 0.5 and about 8.0 hours. During this process the bioreactor is maintained at a temperature appropriate for the production of viral particles, and the cell culture medium is collected after it has passed through the bioreactor.

More specifically, the bioreactor contains the cell culture spaces as discussed below. The culture spaces are inoculated with cells capable of producing viral particles. Alternatively, the bioreactor can be inoculated first by virus-free cells. These cells can be converted to virus- producing cells by transfection with the appropriate vector. In the following description, the term "virus" and "viral particles" are used interchangeably to denote a preparation which can contain infectious and non-infectious virus particles produced by a virus-producing cell. In addition, the term can include non- infectious virus-like particles, which are useful as vaccines or immunoreagents (see, for example, Lowy et al . , Proc. Natl. Acad. Sci. USA, 91:2436-2440 (1994), which is incorporated herein by reference) .

The instant method can be used for the production of high titers of viral particles from prokaryotic or eukaryotic cells, such as bacteria, plant cells, animal cells, mammalian cells, human cells, or insect cells. Preferred cells are those which are capable of long-term growth and subdivision in culture, such as chronically infected cells or virus packaging cells, which also continuously produce viral particles. However, all other types of cells which are capable of producing viruses are useful in the present method. The cells that produce viral particles can be anchorage-dependent, such as fibroblasts, or can be anchorage-independent, such as leukocytes. As discussed above, the cells can be capable of producing virus before or after being inoculated into the internally oxygenated bioreactor.

The viral particles produced by the cultured cells in the instant method can be any viral particle capable of being produced by cultured cells. Such viruses can be produced by the cells continuously, such as by budding in the case of retroviridae, or can be produced by lysing the host cell at the end of the infection cycle, such as in the case of picornaviridae or adenoviridae . The viral particle produced in the instant method can be of any origin, such as bacterial, insect, plant or animal.

The virus-producing cells can be cultured in any media appropriate for promoting either the growth of the cells or the production of viral particles, or both. Different cell types have different nutrient and environmental needs . Therefore, the particular culture media used in operating the bioreactor is dependent upon the type of cell being cultured.

Furthermore, some viruses require that the virus- producing host cells be actively dividing in order to produce viruses, as is the case for retrovirus. Adenovirus infection, however, does not require such an active division stage. Therefore, depending on the cell type and virus type, the culture media may include growth factors, nutrients such as glucose, and complex supplements, such as serum, in order for the cell culture in the bioreactor to produce viral particles. A wide variety of commercially available culture media exists which contain any or all of these factors, nutrients and supplements, and all of these media can be used with the appropriate cell types in the instant method.

The concentration and type of serum used as a supplement in the instant method is dependent upon the type of virus-producing cell being cultured. For example, human serum and mammalian serum, such as bovine serum or fetal bovine serum, can be used to support the growth of human cells. Depending on the cell growth rate desired, the concentration of serum can be increased or decreased. Normally, mammalian cell growth is sustained at serum concentrations between about 2 and about 10%. The type of media used to culture the cells can be selected on the basis of maximizing the amount of virus produced by the cultured cells.

The ultimate use of the viral particles produced by the instant method can also influence the type of serum used. For example, if the virus preparation is to be used in humans , then xenotypic animal serum should be avoided to prevent undesirable transfer of etiological agents and undesirable immunological reactions in the human recipient. Even when human serum is used to culture cells producing viruses for use in humans, care should be taken to determine that the serum is free of undesirable etiological agents, such as HIV or hepatitis type B virus. This problem can be avoided by using serum free media.

In order to maintain concentrations of nutrients and growth factors appropriate to sustain the growth of the cell culture, support the production of viral particles by the cell culture, fresh medium is introduced into the bioreactor and the resulting culture fluid (i.e., spent culture medium plus any viral particles) is removed. The average time which the culture media and viral particles remain in the bioreactor is termed the residence time.

In the instant method, the residence time utilized is between about 30 minutes and about 8 hours. Better conditions include a residence time of 30 minutes and 4 hours, with the optimal conditions being between 30 minutes and 2 hours. The residence time ranges set forth above are for the production of viral particles. Longer residence times may be necessary prior to this production time. Thus, lower culture media flow rates may first be used to allow the virus-producing cells to affix and grow to a suitable density with the bioreactor. After such density has been reached, the production of viral particles by the instant method may commence. In order to maintain an appropriate residence time, the cell culture medium and fluid are monitored for one or more parameters such as glucose concentration, the partial pressure of oxygen and carbon dioxide, and pH.

In addition to nutrients and growth factors, the cells used in the instant method require gasses in order to grow and produce viral particles. Oxygen and other essential gasses must be supplied to the bioreactor, as the need for these gasses by the virus-producing cells soon exceed the amount found in the culture medium. The bioreactor of the present invention uses a gas-permeable membrane envelope through which oxygen-containing gasses are passed in order to maintain appropriate concentrations of oxygen, and other gasses, such as carbon dioxide, in the culture medium. The amount of oxygen delivered to the culture medium can be modulated by adjusting the concentration of oxygen in the gas delivered to the bioreactor and the flow rate of the gas through the gas-permeable membrane envelope therein. Appropriate gas mixtures and flow rates can be determined by monitoring the partial pressure of oxygen in the cell culture media within the bioreactor. Low concentrations of oxygen indicate that the gas mixture should have more oxygen or that the gas flow rate should be increased. The concentration of other gasses, such as carbon dioxide, are also important for maintaining healthy cells. A useful gas mixture is air, or air supplemented with about 1% to about 10% carbon dioxide. The p0₂ in the culture fluid in the bioreactor can be between about 50 and 120 mmHg during production of virus and about 160 mmHg when cells are seeded into the bioreactor. The pC0₂ in the culture flow in the bioreactor can be between about 10 and 90 mmHg.

In addition, in the instant method the bioreactor should be maintained at appropriate temperature or temperatures to stimulate cell growth and viral particle production. Different types of cells require different temperatures to grow efficiently. Mammalian cells, for example, grow best at about 30°C to about 40°C, with 37 C usually being preferable. However, lower culture temperatures can also be used and can unexpectedly enhance the production of viruses from the cell culture. For example, maintaining the bioreactor at about 37°C during growth phase encourages the cell culture to reach high cell densities quickly. Subsequently, the temperature can be reduced to about 32°C, which reduces the cellular growth rate, but can encourage the enhanced recovery of viral particles. The aforementioned factors of cell type, virus type, culture medium, serum requirements, residence time, oxygen demand, and temperature can all be monitored and modulated in order to enhance cell growth and viral particle production.

The culture fluid produced in the instant method can be treated to prevent the loss of virus titer. For example, many viruses are sensitive to the temperatures used to culture the cells which produce them. In such cases, the culture fluid can be continuously stored at a lower temperature, such as between about 0°C and 24°C, and preferably about 2°C to about 8°C. This temperature range is also useful in light of the fact that some viruses, such as retroviruses and herpesviruses, are labile at room temperature and lose infectivity when frozen. In such cases, the culture fluid can flow by way of tubing from the bioreactor to a container stored under controlled storage conditions, such as at between about OS to about 4£, and preferably, about 4C. Because each viral particle has its own temperature sensitivity profile, the ultimate choice of collection temperature depends on the particular viral particle being produced. Furthermore, the collection conditions can also be further modified to optimize the storage of the particles. For example, soluble protein can stabilize certain viral particles. Therefore, for such particles, the culture fluid can be supplemented with protein, such as albumin, in order to maintain the high titers produced in the bioreactor. Finally, the culture fluid can be continuously or intermittently subjected to physical or immunochemical procedures for isolating the viral particles.

The internally oxygenated bioreactor used in the instant method comprises a generally concentric, generally annular length of at least one gas-permeable, liquid impermeable membrane envelope 10. The gas-permeable envelope is an edge-sealed composite of a spacer layer sandwiched between a liquid impermeable base layer and a liquid- impermeable top layer, with at least one of the base and top layers being permeable to gasses such as oxygen and carbon dioxide. The gas-permeable membrane envelope is also in communication with one or more gas inlet means for providing an oxygen-containing gas to the enclosed spaces and gas outlet means for removing gas from these spaces. The area between adjacent lengths of the generally concentric annular lengths of the gas-permeable membrane envelope define narrow annular culturing spaces 30 along the lengths for growth of virus-producing cells in contact with culture medium. The annular culturing spaces 30 further contain a cell culture matrix 20. The bioreactor has a liquid inlet face at one end of its length and a liquid outlet face of the opposite end of its length 17 by which cell culture medium can directly be introduced to and withdrawn from the culture spaces. Furthermore, the bioreactor is encased in a fluid-tight housing. The housing has access means for accessing the gas inlet and gas outlet means of the gas-permeable membrane envelope, the liquid inlet means for introducing a flow of culture medium to the liquid inlet face 61 of the bioreactor and liquid outlet means for removing culture liquid from the liquid outlet face 63 of the bioreactor. In addition, the housing may have means for introducing probes measuring the concentration of, for example, the pH, p0₂, pC0₂, or concentration of glucose within the bioreactor. More specifically, and as illustrated in FIG. 1, the liquid-impermeable, gas-permeable membrane envelope 10 comprises a spacer element 16 interleaved between a top layer 12 and bottom or base layer 14. Top layer 12 and base layer 14 are formed of liquid- impermeable material and at least one of the layers also is gas-permeable . Suitable liquid- impermeable, gas-permeable materials for these layers are silicone rubbers, or materials such as polyethylene, polypropylene, or polytetrafluoroethylene. Fabrics or meshes such as fiberglass, polyester, or nylon can form a base structure upon which gas-permeable materials may be placed. For example, polyester mesh can be sprayed with medical grade silicon rubber to form gas-permeable materials. Spacer element 16, employed to prevent collapse of the gas-permeable membrane envelope 10, can be made of any suitable inert material, such as polypropylene, fiberglass, nylon or other polymeric plastics, typically in a porous screen or mesh-like configuration .

A gas inlet port 18 and a gas outlet port 19, constructed, for example, of silicone rubber tubing, are arranged along lengths of the sealed membrane envelope. A rigid flexible means, such as a spring, can be placed inside the tubing where it intersects with the membrane envelope . The spring can extend into the membrane envelope and outside the membrane envelope so long as it remains within the tubing. The spring is of such a diameter and composition to prevent the tubing from crumpling when being bent or compressed.

Depending upon the longer dimension of the pre-wound envelope 10, more than one set of gas inlet and outlet ports (18, 19) can be provided. The composite of spacer 16 and top and base layers 12 and 14 is sealed about its edges in any appropriate manner to provide an enclosed space within the membrane envelope for introducing a oxygen-containing gas which can exit only by diffusion across an gas-permeable layer or through a gas outlet port

19. In order to form an envelope with an internal space, one of the top layer 12 and bottom layer 14 can be slightly wider than the other.

The bioreactor can be formed, for example, by winding or rolling the membrane envelope about itself, preferably making the first wind about a centrally-disposed supporting core element 26 having a length substantially the same as the length 17 of the membrane envelope. The winding can start with an extended portion of at least one of the gas- permeable membranes (12, 14) . One of the membranes (12, 14) can be longer than the other to serve as a leader 52 to facilitate the beginning of the winding or rolling process. Another extended portion of at least one gas permeable membrane (12, 14) can serve as a tail 49 to facilitate the completion of the winding or rolling process. In this form, the cell culture spaces 30 defined by adjacent lengths of winds of the membrane envelope are bounded solely by the outer surfaces of the liquid-impermeable and gas-permeable layers (12 and 14) of the membrane envelope. The culture spaces 30 of the internally oxygenated bioreactor are provided with a separate independent cell culture matrix 20 on which the virus-producing cells can affix and grow. As illustrated in FIG. 1, the cell culture matrix 20 consists of a composite laminate of sheets 22 which, for example, may be from about 100 to 300 microns in thickness. These sheets can be sandwiched about an optional second spacer 24, which can be a plastic material such as polypropylene, fiberglass, or nylon sheet. The cell culture matrix has dimensions that generally coincide with the length and width of the membrane envelope 10, and is laid over the membrane envelope 10 before the winding operation. The width of the membrane envelope 10 corresponds approximately to the length 17 of the bioreactor 51. The layered arrangement of the membrane envelope 10 and the cell culture matrix 20 is then wound about itself, beginning with a first wind about supporting core element 26, to form an internally oxygenated bioreactor of predetermined length having a generally spiral or "jelly-roll" cross-section. As seen in FIG. 1, the bioreactor consists of spiral winds of membrane envelope 10 in which narrow annular cell culture spaces 30 are defined by the areas between adjacent winds. As mentioned above, by reason of the lamination/winding process, these cell culture spaces contain a cell culture matrix 20 on which cells can affix to or be substantially immobilized on and grow. The supporting core element 26 has as its essential function the provision of longitudinal structural support to the membrane envelope and cell culture matrices when used. The supporting core element can be formed of any suitable inert material, such as plastic, having a sufficient degree of rigidity. Materials such as polycarbonates or fluorocarbon polymers such as polytetrafluoroethylene are suitable. For this purpose, the supporting core element can be in the form of a solid rod-, spool-, or cylinder-like element having a cross-sectional diameter appropriately large enough to provide support and to reduce circumferential differences between the two sides of the membrane envelope. Alternatively, the supporting core element 26 can be a hollow rod, spool, or cylinder capped at both ends to prevent the flow of fluid therethrough.

The cell culture matrix 20 in FIG. 1 can be in the form of one or more sheets of any suitable inert material, such as cellulosic materials, woven fiberglass, nylon, polyester, plastics and the like. The cell culture matrix can be in the form of a flat sheet, such as a thin plastic flexible sheet, but also can be in a form to provide additional surface area for cell growth, such as being corrugated, pleated, or convoluted. Also, additional spacing between adjacent membrane envelope layers may be used to enlarge the cell culture space to more easily accommodate the flow of culture medium. Such spacing may be provided using spacer elements such as those used in the membrane envelope 10. The thickness of sheet-like cell culture matrix should be on the order of less than about 300 microns thick, and more preferably less than about 200 microns thick, owing to the fact that diffusional limitations are encountered in cell multilayers above about these thicknesses . The cell culture matrix 20 normally may be present in all the narrow annular culture spaces of the bioreactor and throughout the full length of each such space, as will be the case when the cell culture matrix is a sheet material layered over the membrane envelope 10 by a spiral winding or rolling operation. However, it is not strictly necessary for the cell culture matrix to be so arranged, and the present method contemplates the presence of a cell culture matrix in less than all the cell culture spaces and/or along only a portion of the length or width of such spaces .

The bioreactor has at each end an inlet or outlet face through which culture medium can be introduced into the cell culture spaces and from which culture fluid can be removed from the cell culture spaces. The bioreactor can contain one or more sets of accessible gas inlet and gas outlet ports (18, 19) communicating with the interior of the membrane envelope 10.

The bioreactor can be enclosed in any suitable fluid-tight housing 50, such as polycarbonate or silicone rubber, having liquid inlet and outlet ports and ports through which the gas inlet and gas outlet ports 18 and 19 of the membrane envelope may be accessed. The housing can also be provided with at least one port through which cells can be introduced into the bioreactor so as to occupy areas in the cell culture spaces whether provided with a separate cell substrate element or not. Alternatively, the cells can be introduced into the cell culture spaces 30 by including them in the initial charges of culture media introduced into the cell culture spaces 30.

The bioreactor can be formed from a single given integral length of membrane envelope 10 having an appropriate number of gas inlet and gas outlet ports (18, 19) . Alternatively, the bioreactor can be constructed from more than one such membrane envelope, with a first being spirally wound for the number of winds possible, and a next membrane envelope being wound from the terminus of the last wind of the first membrane envelope, and so on. In bioreactors made in this way, each membrane envelope will have its own independent gas inlet and gas outlet ports. All such ports can be attached to a manifold so as to be in communication with a single gas inlet source and gas outlet source or, alternatively, can be in communication with a separate, captive gas source and gas outlet.

Also, a plurality of lengths of membrane envelopes can be used to form the bioreactor, each such length constituting a separate single wind over which the next length is wound or wrapped. Interleaved between one and preferably all of the lengths of membrane are the cell culture matrix. In this manner, the individual bioreactors assume a cylindrical configuration in which there are a series of generally concentric, generally annular spaces between these concentric annular lengths which form cell culture spaces. However, these cell culture spaces are not in fluid communication with other cell culture spaces, and each membrane envelope length is required to have its own captive gas inlet and gas outlet ports for introducing and withdrawing an oxygen-containing gas into the interior of each envelope .

The bioreactor can be constructed of any suitable cross-sectional diameter and any suitable length from inlet face to outlet face. Several of the bioreactors can be abutted, attached or unattached, in fluid and/or gas communication in series throughout the length of a single fluid-tight housing 50. In the instant method, the bioreactor 51 preferably is pre-sterilized using any appropriate means and then can be arranged, either vertically or horizontally or in any other desired position on any suitable support member, although a vertical arrangement is preferred. As shown in FIG.2, a liquid inlet port 52 is connected by suitable sterile connections to a source of appropriate cell culture media and a liquid outlet port 54 is similarly connected to an appropriate vessel or line through which culture fluid can be withdrawn, collected and processed. Gas inlet and gas outlet ports (18, 19) of the membrane envelope 10 are connected, respectively, to a source of oxygen-containing gas and to a line for drawing off gas. Although gasses can be provided under positive and negative pressure, positive pressure is preferable because negative pressure can cause condensation to form on the inner surface of the membrane envelope 10, which can prevent the efficient exchange of gasses across the membrane envelope 10 to the culture space 30. Where the membrane envelope 10 is provided with a number of such gas inlets and/or outlets, these can be attached to a manifold, within or outside the housing, into a single inlet or outlet line.

Cells can be inoculated into the bioreactor either through one or more inoculum ports 56 generally arranged so as to be in proximity to the open inlet face of the spirally wound element 10, 20, 26, or by means of liquid flowed through the culture spaces 30. Culture media is introduced into the bioreactor through liquid inlet port 52 and can, if desired, pass through a distributor means, such as a perforated plate, before contacting the inlet face of the bioreactor to promote relatively even distribution of culture media within the cell culture space 30. Also, filtering means can be used to remove debris or contamination from the culture media prior to being introduced into the bioreactor unit .

Initial flow rates and nutrient conditions are chosen to permit cells to become affixed to or associated with the cell culture matrix 20 which has been provided in the cell culture spaces 30, and thereafter can be optimized and controlled to effect desired flow rates and growth conditions. Culture media can be introduced into the cell culture spaces using either positive or negative pressure, with positive pressure being preferred. Culture fluid, the spent culture medium together with any cell products, viruses, wastes, and the like, can be withdrawn from the opposite liquid outlet end 54 of the unit.

During the instant method, the flow of inlet culture medium and inlet oxygen-containing gas may be continuous, pulsed, or intermittent. Preferably, culture media can, at least periodically, be fed to the bioreactor from the normally outlet end to promote a relatively uniform nutrient environment in the culture spaces 30 across the length of the bioreactor. The bioreactor can, if desired, be rotated about its core axis or subjected to any other gentle motion to promote a uniform culturing environment throughout its length and in all spaces between adjacent winds or lengths of the membrane envelope 30. Furthermore, a portion of the cell culture fluid can be continuously recirculated from the medium outlet port to the input port to promote a uniform culturing environment.

The instant invention is also directed to a bioreactor as described above for the instant method, except for the fact that the cell culture spaces 30 contain only a cell culture matrix 20 consisting of one or more thin plastic sheets. In other words, no spacer element is present in the cell culture space 30.

Thus, the instant internally oxygenated bioreactor comprises a generally concentric, generally annular length of at least one gas-permeable, liquid impermeable membrane envelope. The gas-permeable envelope is an edge-sealed composite of a spacer layer sandwiched between a liquid impermeable base layer and a liquid-impermeable top layer, with at least one of the base and top layers being permeable to gasses such as oxygen and carbon dioxide. The gas-permeable membrane envelope is also in communication with one or more gas inlet means for providing an oxygen- containing gas to the enclosed spaces and gas outlet means for removing gas from these spaces. The area between adjacent lengths of the generally concentric annular lengths of the gas-permeable membrane envelope define narrow annular culturing spaces along the lengths for growth of virus-producing cells in contact with culture medium. The annular culturing spaces further consist essentially of a cell culture matrix. The bioreactor has a liquid inlet face at one end of its length and a liquid outlet face of the opposite end of its length by which cell culture medium can directly be introduced to and withdrawn from the culture spaces. Furthermore, the bioreactor is encased in a fluid-tight housing. The housing has access means for accessing the gas inlet and gas outlet means of the gas-permeable membrane envelope, the liquid inlet means for introducing a flow of culture medium to the liquid inlet face of the bioreactor and liquid outlet means for removing culture liquid from the liquid outlet face of the bioreactor.

An optimum configuration for such a bioreactor contains a cell matrix comprised of from one to three, and especially three, such thin plastic sheets. The plastic sheets can be solid or porous, and preferably are made of fiberglass or nylon fabric.

The bioreactor can be used to culture virus-producing cells as described above for the instant method or for culturing any other variety of mammalian or non-mammalian cells using standard cell culture conditions. An optimal embodiment of the bioreactor of this invention is described below in Example II.

The following Examples are intended to illustrate but not limit the present invention. EXAMPLE I

PREPARATION OF LXSN84.66γ#22 PRODUCER CELLS This example describes the preparation of LXSN84.66γ#22 producer cells by transfecting the plasmid pLXSNT84.66γ into the packaging cell line PA317 (ATCC Accession No: CRL-9078) . These cells are neomycin resistant and produce replication defective moloney murine leukemia retrovirus.

Cell line PA317 was derived from the murine fibroblast cell line NIH/3T3 TK^" as described by Miller and Buttimore (Mol. and Cell. Biol., 6:2895-2902 (1986), which is incorporated herein by reference) . The PA317 cell line is a contact inhibited adherent cell line. This trait was retained in the retrovirus producing clone LXSN84.66γ#22. Plasmid pLXSNT84.66γ (see FIG. 4) was made using plasmid pLXSN-N29γ (see FIG. 5) as the starting vector.

The plasmid pLXSN-N29γ was derived from the plasmid pLXSN, which was originally described by Miller and Rosman

(BioTechniques, 7:980-990 (1989), which is incorporated herein by reference) . Plasmid pLXSN-N29γ was linearized with the restriction endonucleases EcoR I and Xho I and then blunt ended. The linearized plasmid was digested with Xho I to excise the N29 SCA region. The linearized plasmid without the N29 SCA region was isolated using well known methods (see Sambrook et al . , Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989, which is incorporated herein by reference) . A chimeric DNA molecule encoding regions of the V_H and V_L genes for monoclonal antibody T8466 and the Fee receptor γ chain obtained from pLXSN-N29γ

("γ-chain"), which is highly homologous to the zeta-chain of the T-cell receptor.

This chimeric DNA molecule was made by obtaining the mRNA for the V_H, V_L, and γ-chain molecules from the appropriate cell line and making cDNA from the mRNA using well-known methods (Sambrook et al . , supra , 1989) . The V_L cDNA was linked in-frame to the V_H cDNA by a Genex 212 linker using spice overlap extension PCR. The V_H cDNA was linked in- frame to the γ-chain cDNA by blunt end ligation. A SnaB I restriction site was added upstream of the V_L coding region and a Xho I restriction site added downstream of the γ-chain coding region. The Xho I restriction site was made partially single stranded to allow directed cloning. This chimeric DNA molecule was inserted into the linearized pLXSN-N29γ without the N29 SCA region and ligated to make plasmid pLXSNT84.66γ .

The plasmid pLXSNT84.66γ was transfected into HAT- and HT-selected PA317 cells using LIPOFECTAMINE (BRL/Gibco Life Sciences, Gathersberg MD) or DOTAP (Boehringer Mannheim, Indianapolis, IN) following the manufacture's instructions. Because cells transfected with the plasmid pLXSNT84.66γ are resistant to neomycin, stably transfected clones were selected in DMEM with 10% FCS supplemented with 0.5 mg/ml of active G418 (BRL/Gibco Life Sciences) until resistant colonies were visible.

Individual resistant colonies were harvested and inoculated into 24-well tissue culture plates (Corning Glass Works, Corning NY) and cultured in DMEM with 10% FCS supplemented with 0.5 mg/ml of active G418. Stable clones were expanded into 6 -well tissue culture plates (Corning) ,

2 and subsequently into 150 cm T-Flasks (Corning) , in DMEM with 10% FCS supplemented with 0.5 mg/ml of active G418. Cell-free supernatants from the T-Flask cultures were titered for Moloney murine leukemia virus on NIH/3T3 cells following the method of Cepko (_.In Current Protocols in Molecular Biology, Suppl. 17, pp. 9.11.5-9.11.12, Wiley- Interscience, NY (1992) , which is incorporated herein by reference). Clone #22, designated LXSN84.66γ#22 , was found to produce titers of 10 to 10 neomycin-resistant CFU/ml of culture supernatant . This producer clone was selected for use in the bioreactor. Samples of the LXSN84.66γ#22 cultures were found to be free of contamination by conventional methods.

EX7AMPLE II PRODUCTION OF VIRAL PARTICLES USING AN INTERNALLY OXYGENATED BIOREACTOR

This Example provides methods of obtaining large volumes of high titer retroviral preparations by culturing retrovirus producing cells in a culture unit. An internally oxygenated bioreactor (hereafter "bioreactor") was constructed using a liquid-impermeable, gas-permeable membrane envelope consisting of two lengths of a polyester mesh reinforced silicone rubber membrane about 0.2 mm thick. The silicone rubber membrane was made by spraying medical grade silicone rubber onto polyester mesh (Style 12525, Pomona Textile, Pomona, CA) . The membrane envelope 10 was made by centering a spacer element 16 made of polypropylene mesh (Vexar^®, Dupont Canada Inc., Whitby, Ontario, Canada) (60 inches in length and 10 1/4 inches in width) on top of the bottom layer 14 (65 inches in length and 11 inches in width) so that the spacer element 16 did not cover about 5 about inches of the length of bottom membrane 14. The top layer 12 (72 inches in length and 11 inches in width) was placed on top of the polypropylene mesh and bottom layer 14 so that the spacer element 16 was sandwiched between the top and bottom layer (12, 14) and about 10 V_ inches of the top layer were not contacting the spacer element 16 or the bottom layer 14. Two gas inlet tubes 18 comprised of about 1.5 inches of TEFLON tubing having an outside diameter of about 3/8 of an inch (Cole-Palmer Instrument Co, Vernon Hills, IL) were placed about 2 and about 57 inches along one length of the spacer element between the top and bottom layers (12, 14) so that about 0.5 inch protruded from the unsealed membrane envelope. A single gas outlet tube 19 was similarly placed about 30 inches along the other length of the spacer element 16. The TEFLON tubes were fitted internally with springs having an outside diameter corresponding to the inside diameter of the TEFLON tube for their entire length.

Within the membrane envelope 10, the TEFLON tube was split, one half the tube with the spring was placed on one side of the spacing element 16, and the remaining half the tube placed on the other. The TEFLON tubing extending from the membrane envelope was fitted with silicone rubber tubing having an outside diameter of about 3/8 of an inch. The silicone rubber tubing connected the membrane envelope to the gas inlet and gas outlet ports.

The membrane envelope 10 was formed by sealing the top and bottom layers (12, 14) together with silicone rubber cement at their overlapping portions surrounding the spacer element 16. In order to form a membrane envelope 10 with an internal volume, the bottom layer 14 was slightly wider (about % inch) than the top layer 12 and the edges of the top and bottom layers were joined flush. The top layer 12 of the membrane envelope 10 was covered with a cell culture matrix consisting of three layers of 0.26 mm thickness nylon fabric (Style 12619, Pomona Textile, Pomona, CA) (9.5 inches in width and 60 inches in length) . Starting with the 5.5 inches ("leader") of the bottom layer 14 that extended past the membrane envelope 10. The leader was glued to a hollow spool-shaped core 26 made of polycarbonate having a length of about 12.5 inches at a diameter of about 1 inch. The cell culture matrix was wound around the polycarbonate core 26 at a drag of about seven pounds. Each end of the spool was fitted a circular endcap about 3 inches in diameter. Both endcaps were fitted with one port for culture media input or output (52, 54) and one port for gas input or output (18, 19) , as the case may be. The gas ports of each endcap were connected to either of the two gas inlet tubes 18 or the gas outlet tube of the membrane envelope 10. The endcaps may optionally be fitted with sampling ports and a temperature probe port.

A silicone rubber sleeve about 1/8 of an inch in thickness was vacuum-wrapped over the wound composite.

Each endcap was sealed with a silicone rubber cap made secure to each endcap with braided umbilical tape. Two layers of filament tape were wound around the silicone sleeve and silicone caps. The filament tape was covered with a single layer foam cosmetic, which was covered with a shrink tubing outer cover. The various ports were fitted with appropriate connecting tubing. The gas input and output tubes (18, 19) were each fitted with a 0.2 μm ACRODISK filter 58 (Gelman Sciences, Inc., Ann Arbor, MI). The bioreactor 51 and connecting tubing and filters were sterilized using ionizing radiation.

As shown in FIG. 2, the sterilized bioreactor was placed in a vertical position in a variable temperature incubator at 37°C. All tubing in FIG. 2 is Class 6 medical grade silicone tubing except for the tubing loop connected to the recirculation peristaltic pump 66, which tubing is neoprene (0.25 inch inner diameter, 1/16 inch wall thickness, Phar-Med^®, Norton Performance Plastics, Wayne, New Jersey) . The gas input line 18 was fitted with a source of compressed gas, and the gas output line 19 was fitted with a water trap 60 (Bubble Trap, #B-100, American Omni Medical Corp., Costa Mesa, CA) between the bioreactor 51 and the filter 58. The trap 60 was used to collect condensate to prevent the filter from becoming wet. The condensate trap was periodically drained using the condensate drain 62.

A recirculating loop 64 was formed by connecting the culture media input port 52 and the culture fluid outlet port 54 of the bioreactor 51 with neoprene tubing. The recirculating loop was fitted with a peristaltic pump 66 to allow recirculation of culture media and culture fluid during operation of the bioreactor. Another peristaltic pump 68 supplied culture media from a medium container stored at about 4°C to both the bioreactor and the recirculating loop using medical grade silicon tubing. A seeding port 56 and sample ports 72 were also connected to the bioreactor recirculating loop 64 to allow cells to be seeded into the bioreactor and samples to be taken from it, respectively. The sample ports used are described in Oakley, U.S. Patent No.: 4,999,307, issued March 12, 1991 (which is incorporated herein by reference) , although similar types of sample ports can be used. Tubing also connected the recirculating loop to a product container 74 used to store culture fluid exiting from the bioreactor 51. The product container was stored at about 4 C. Between the bioreactor 51 and the product container 74, a squeeze tube 76 was placed in line using union connectors. The squeeze tube 76 prevented the flow of culture fluid from the product container 74 back into the bioreactor. As shown in FIG. 3, the squeeze tube 76 consisted of an inner 80 and a shorter outer silicone tube 82, wherein the outer tube was sealed to the inner tube. The outer tube was fitted with an air input line 84 and a plugged air output line 86. In operation, about 5 p.s.i. of air pressure was provided to the outer tube 82 through the air input line 84, which compresses the inner tube 80. Once the pressure of the culture fluid in the inner tubing 80 exceeded 5 p.s.i., then the culture fluid could flow to the product container 74.

The sterilized and fitted bioreactor was aseptically filled with AIM V Serum Free Media (Biowhittaker, Walkersville, MD) supplemented with 5% Fetal Bovine Serum

(FBS) (Biowhittaker) and perfused through the bioreactor at an initial rate of about 75 ml per day. The primed bioreactor was maintained at 37 C, at a pH of 6.6 to 7.5 pH

(7.0 was optimal) . Air containing 10 to 15% C0₂ was provided into the membrane envelope 10 through the gas input line 18 at a flow rate of about 100 to 200 ml/min. The p0₂ in the culture fluid in the bioreactor ranged between about 160 mmHg at the start of the culture to between about 50 and 120 mmHg during production of retrovirus. During the retrovirus production phase, the p0₂ in the culture fluid in the bioreactor was about 100 mmHg. The pC0₂ in the culture fluid in the bioreactor was between about 10 and 90 mmHg, and averaged about 45 mmHg. The peristaltic recirculation pump was operating at a rate of approximately 670 ml/hour. The culture fluid was aseptically collected in the product container 74 and , o maintained at about 4 C. One day after priming, the two bioreactors 51 were inoculated with 1 x 10 viable LXSN84γ#22 packaging cells (Baxter Biotech, Gene Therapy Unit, Santa Ana CA, from Example I above) . The bioreactors inoculated with the LXSN84.66γ#22 cells were perfused with AIM V media with 5% FBS at an initial rate of 75 ml per day. The media was recirculated at 11.2 mL/min with a peristaltic pump 66 always in the same direction of flow. The resulting culture fluid was continuously collected at between 2° and 8°C and stored at - 70°C. After eight days of culturing, it was observed that the glucose uptake rate had reached a plateau, such that an increase in the cell culture medium rate would not result in any increased glucose uptake by the cultured cells . In other words, the cell growth rate had been maximized with the serum-containing AIM V media. At this point, production of the viral vectors for future use was begun.

The perfusion media was changed to AIM V Serum Free Media

(Biowhittaker) and the temperature was reduced to 32°C. The culture fluid produced by the cell culture in the bioreactor on the second day after the switch to AIM V Serum Free Media was not intermingled with that resulting from the use of AIM-V media supplemented with serum or the culture fluid collected on the first day that the serum- free media was used. FIG. 6 shows the perfusion rate of culture media into the bioreactor and the concentration of glucose in the culture fluid over time.

Samples of the cell culture in the bioreactor were taken daily and supernatants were obtained by centrifugation. The glucose in these supernatants was measured using a LIFESCAN ONE TOUCH II kit (Johnson & Johnson, Milipitas, CA) . The media perfusion rate was adjusted to maintain a residual glucose concentration of more than 1.5 g/L in the supernatants. The media perfusion rate did not exceed 3L per day, which represents about nineteen 160 ml bioreactor volumes per day, or an average residence time of about 77 minutes.

The concentration of viruses in the supernatant was determined as colony forming units per milliliter (CFU/ml) using NIH3T3 murine fibroblasts or HT1080 human fibrosarcoma cells grown in 100 mm dishes as target cells following the method of Cepko (supra, 1992) . Briefly, retroviral supernatants were serially diluted in 5 ml of growth media containing 8 μg/ml of polybrene . The 5 ml samples were applied to the target cells and the cultures were incubated for three hours, after which an additional 15 ml of growth media were added per dish. The transduced cells were cultured forty-eight hours at 37°C in 7% C0₂ before being split 1:20 into selection media (growth media supplemented with 0.5 mg/ml active G418) . The transduced cells were cultured under selection conditions until resistant colonies were visible. As shown in FIG. 7, the supernatants had titers which ranged from 6.92 x 10 to 6.21 x 10⁵ CFU/ml. Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

Claims

We claim :

1. A method for the production of viral particles, comprising:

a) providing an internally oxygenated bioreactor which contains cells capable of producing viral particles;

b) passing a mixture of gasses containing at least oxygen and carbon dioxide through the bioreactor;

c) passing cell culture medium appropriate for the production of viral particles through the bioreactor such that the average residence time in the bioreactor of the viral particles is between about 0.5 and about 8.0 hours;

d) maintaining the temperature of the bioreactor at a temperature appropriate for the production of viral particles; and

e) collecting the cell culture fluid after it has passed through the bioreactor.

2. The method of claim 1, wherein the cells in step a) are mammalian cells.

3. The method of claim 1, wherein the cells in step a) are attachment-dependent mammalian cells.

4. The method of claim 1, wherein the mammalian cells in step a) are virus packaging cells.

5. The method of claim 1, wherein the viral particles in step a) are human viral particles.

6. The method of claim 1, wherein the viral particles in step a) are human retrovirus particles.

7. The method of claim 1, wherein the viral particles in step a) are human adenovirus particles.

8. The method of claim 1, wherein the residence time of the viral particles in step c) is between about 30 minutes and about 4 hours.

9. The method of claim 1, wherein the residence time of the viral particles of step c) is between about 30 minutes and about 120 minutes.

10. The method of claim 1, wherein the residence time of the viral particles in step c) is about 1 hour.

11. The method of claim 1, wherein the cell culture fluid in step c) is supplemented with mammalian serum.

12. The method of claim 1, wherein the cell culture fluid in step c) is supplemented with human serum.

13. The method of claim 1, wherein culture media in step c) is initially supplemented with serum, and subsequently is serum-free medium.

14. The method of claim 1, wherein the temperature of the cell culture fluid in step d) is between about 30┬░C and 40 ┬░C.

15. The method of claim 1, wherein the temperature of the cell culture medium in step d) is about

37┬░C.

16. The method of claim 1, wherein the temperature of the cell culture medium in step d) is about 32┬░C.

17. The method of claim 1, wherein the cell culture medium in step e) is collected in a container maintained at about 0┬░C to about 24┬░C.

18. The method of claim 1, wherein the cell culture medium in step e) is collected in a container maintained at about 2┬░C to about 8┬░C.

19. The method of claim 1, wherein the cell culture medium in step e) is collected in a container maintained at about 4┬░C.

20. The method of claim 1, which further comprises the additional step of recirculating through the bioreactor a portion of the cell culture media after it has passed through the bioreactor.

21. An internally oxygenated bioreactor comprising a generally concentric, generally annular length of at least one gas-permeable, liquid impermeable membrane envelope; a) the gas-permeable envelope comprising an edge- sealed composite of a spacer layer sandwiched between a liquid impermeable base layer and a liquid-impermeable top layer, at least one of the base and top layers being permeable to oxygen and carbon dioxide;

b) the gas-permeable membrane envelope having in communication therewith one or more gas inlet means for providing an oxygen-containing gas to the enclosed spaces thereof and gas outlet means for removing gas from the enclosed spaces thereof, and wherein the area between adjacent lengths of the generally concentric annular lengths of the gas -permeable membrane envelope define narrow annular culturing spaces along the lengths for growth of cells in contact with culture medium, wherein the annular culturing spaces consist essentially of a cell culture matrix;

c) the bioreactor having a liquid inlet face at one end of the length thereof and a liquid outlet face at the opposite end of the length thereof by which cell culture medium can directly or indirectly be introduced to and withdrawn from the culture space; and

d) the bioreactor being encased in a fluid-tight housing, the housing containing envelope access means for accessing the gas inlet and gas outlet means of the gas -permeable membrane envelope, liquid inlet means for introducing a flow of culture medium to the liquid inlet face of the bioreactor, and liquid outlet means for removing culture liquid from the liquid outlet face of the bioreactor .

22. The method of claim 21, wherein the cell culture matrix comprises at least one layer of mesh.

23. The method of claim 21, wherein the cell culture matrix comprises at least one layer of fiberglass fabric .

24. The method of claim 21, wherein the cell culture matrix comprises at least one layer of nylon fabric .

25. The method of claim 21, wherein the cell culture matrix comprises three layers of nylon fabric .