MXPA99007121A - Methods for cultivating cells and propagating viruses - Google Patents

Abstract

This invention relates to methods for cultivating cells, and in particular to methods for propagating viruses, and more particularly still, to methods for propagating recombinant viruses for gene therapy.

Description

METHODS FOR CULTIVATING CELLS AND PROPAGATING VIRUSES BACKGROUND OF THE INVENTION Many established cell lines are available for various purposes in biotechnology. Some cell lines can be grown as suspensions of individual cells, 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 cells to grow, since many cell lines will form only one single cell layer on the surface. In addition, some cell lines may tend to grow in bulk or aggregates in the absence of a support, which is an inconvenient result when they are required as suspensions of individual cells, but more especially when the cells will be infected with a virus, or transformed with a recombinant ve, since the virus or ve may not have access to cells within the mass or aggregate. In this way, there may be severe problems in expanding the growth of a cell line in proportion, in particular to provide efficient surface areas for the cells to grow and / or to prevent their stacking. The technology of microcarriers has been used to maintain cells in culture. For example, Foresten et al (Biotech, Bioenq.40: 1039-1044 (1992)) described the extended subculture of fibroblasts in series. human diploids on microcarriers using a complement in the medium that reduced the need for serum by cultured cells. In addition, Ohison et al. (Cvtotechnoloqy 14: 67-80 (1994)) described the transfer, drop by drop, of Chinese hamster ovary cells using macroporous gelatin microvehicles. Finally, Hu et al (Biotech, Bioenq 27: 1466-1476 (1985)) described the serial propagation of mammalian cells on microcarriers using a trypsinization technique and pH for selection. However, in view of the problems outlined above, there is a need for improvements in methods for growing cell lines, in methods for producing viruses for clinical uses and in methods for expanding the production of viruses for commercialization on a larger scale, especially recombinant viruses for gene therapy. The present invention satisfies these needs and more.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention is a method for growing cells, comprising: (a) culturing the cells on a first batch of microcarriers until the cells are substantially confluent; (b) separating the cells from the microcarriers without removing them from the suspension; (c) adding a second batch of microcarriers; Y (d) then culturing the cells. Another aspect of the invention is a method for separating cells from a first batch of microcarriers, comprising the following steps: (a) washing the microcarriers and the adhered cells to remove the soluble materials; (b) contacting the microcarriers and the washed cells with a chelating agent; (c) removing the chelating agent; (d) trypsinizing the cells for a short period to separate them from the microcarriers; and (e) neutralizing trypsin by the addition of protein, wherein steps (a) - (e) are carried out in an individual culture vessel. A further aspect of the invention is a method for separating cells from microcarriers on which they have been grown, but from which they have been separated, which comprises introducing an aqueous suspension of cells and microcarriers through an inlet into a device. separation, the device comprising: (a) an input; (b) a column; (c) an outlet for collecting the cells and the aqueous solution; and (d) a mesh screen; wherein the microvehchules are retained in suspension by an upward flow in the separation device, and are retained in said device by a mesh screen, and wherein the cells and the aqueous solution are collected through the outlet. A further aspect of the invention is a system for separating cells from the microcarriers on which the cells have been cultured, the system comprising: (a) a biorea in which the cells were grown 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 collecting the 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 said device by a mesh screen, and wherein the cells and the 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.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a side view of a system in accordance with the present invention used to separate media containing free cells and viruses from microcarriers. Figure 2 is a schematic view of a separation device according to the present invention used to separate media containing free cells and microcarrier viruses.

DETAILED DESCRIPTION OF THE INVENTION The present invention emphasizes the large-scale cultivation of cells for the propagation of viruses, especially recombinant viruses for gene therapy, vaccine production, etc. In particular, the present invention highlights three aspects of large-scale cultivation; the use of the transfer, from drop to drop, of adherent cells to sequentially expand the number of cells in culture, including the use of trypsin to dissociate cells from the microcarriers in the bireareactors, the use of the cell separation of the drops such as from a fluidized bed during harvest, and the use of microfiltration to disintegrate the cells in order to release viral particles. The term "virus", as used herein, includes not only naturally occurring viruses, but also recombinant viruses, viruses attenuated, vaccine strains, etc. Recombinant viruses include, but are not limited to, viral vectors comprising a heterologous gene. In some embodiments, some auxiliary function for the replication of the viruses is provided by the host cell, an auxiliary virus or an auxiliary plasmid. Representative vectors include, but are not limited to, those that will infect mammalian cells, especially human cells, and can be derived from viruses such as retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and avipox viruses. Adenoviral vectors are preferred. Adenoviral vectors types 2 and 5 are most preferred, with adenoviral vectors of type 5 being especially preferred. ACN53 is a recombinant type 5 adenovirus that codes for the wild type human tumor suppressor p53 protein and is described, for example, as in the published PCT international patent application WO 95/11984. As used herein, the term "confluent" indicates that the cells have formed a coherent monocellular layer on the surface (e.g., the microcarrier), so that virtually all of the available surface is used. For example, "confluent" has been defined (RI Freshney, Culture of Animal Cells - A Manual of Basic Techniques, Wiley-Liss, Inc. New York, NY, 1994, p.363) as the situation where "all cells they are in contact around their entire periphery with other cells and no available substrate is left uncovered. " For the purposes of the present invention, the term "substantially confluent" indicates that the cells are generally in contact on the surface, even when there may be interstices, so that more than about 70%, preferably more than about 90%, of the available surface is used. Here, "Available surface" means sufficient surface area to accommodate a cell. In this way, small interstices between adjacent cells that can not accommodate an additional cell, do not constitute an "available surface". The culture steps in the methods of the present invention can be carried out in a bioreactor or fermentor known in the art of about 1 to 5000 liters equipped with appropriate inputs to introduce the cells and microcarriers, sterile oxygen, various media for culture , etc; outputs to remove cells, microcarriers and means; and means for shaking the culture medium in the bioreactor, preferably a rotating filter (which also functions as an outlet for the media). Examples of means are described in the art; see, for example, Freshney, Culture of Animal Cells - A Manual of Basic Techniques, Wiley-Liss, Inc. New York, NY, 1994, pp. 82-100. The bioreactor will also have means for controlling the temperature, and preferably means for electronically monitoring and controlling the functions thereof. Examples of microcarriers on which the cells are allowed to grow are known in the art, and preferably are specially adapted for the purpose of growing cells. General reference is made to the Microcarrier Cell Culture - Principies &amp manual; Methods. published by Pharmacia. However, it should be noted that some lines of cells used in the present invention may not solidly adhere to the surface of the microcarriers; it is well within the ability of the skilled artisan to determine an appropriate combination of a cell line, virus (when applicable), microcarrier and culture conditions. The microcarrier preferably has a particle size in the range of about 100 to 250 microns, more preferably in the range of about 130 to 220 microns, and must be formed of a non-toxic material. The median of the sample size is preferably in these scales, so that these size scales are preferably those of at least 45% of the microcarrier sample. In a preferred embodiment, the microcarrier consists of substantially spherical microdroplets with an average particle size of about 150 to 200 microns, preferably 170 to 180 microns. The surface of the microcarrier can be treated to modify the adhesion of the cells, in particular to increase the adhesion of the same allowing their proliferation and dispersion; thus, the microcarriers can be coated, for example, with collagen. Preferably, the microcarriers are slightly denser than the culture medium, so that gentle agitation will keep them in suspension, while simple means such as sedimentation or centrifugation allow their separation. A density of 1.03 to 1045 g / ml is suitable when the microcarriers are balanced with a standard solution such as 0.9% NaCl (or with the culture medium). The present inventors have found that the Cytodex-3 microcarriers from Pharmacia in general they will satisfy these needs, although the particular requirements that apply to certain cell lines or viruses may require the selection of a particular Cytodex microcarrier. The cells can be those of any suitable host cell line that are capable of replicating themselves, and in particular to support the replication of the virus of interest. A particularly preferred cell line is the human embryonic kidney 293 cell line (catalog number ATCC CRL 1573). These cells do not adhere strongly to all microvehicules, and are preferably used with Pharmacia Cytodex-3 microcarriers, which are coated with collagen for better cell adhesion. The Cytodex-3 microcarriers have an average path size of approximately 175 microns, with 45% of the sample having a size of approximately 140 to 210 microns; the density of said microcarriers when equilibrated with 0.9% NaCl is 1.04 g / ml. The cells are preferably cultured on said microcarrier in a first step, and then they are loosened from it and transferred to additional microcarriers for a production step. Stirring can be conveniently effected not only by a paddle at the bottom of the bioreactor, but also by a rotary rotation filter, which preferably extends downward from the top of the bioreactor in the volume of the medium. The cells and microcarriers can be kept in suspension in the culture by rotating the rotary filter; the rotary filter can be equipped with holes thin that allow the removal of the medium without loss of cells. AND! medium can be removed and can be replaced simultaneously or alternatively; it is often convenient to remove a substantial fraction (eg, up to about 50%) of the medium, and then replenish it with the appropriate replacement medium while the medium is still removed, for example, by the rotary filter. Typically, the cells are enlarged in proportion from a master vessel of the useful cell bank, through various sizes of T-flasks and, preferably, finally to the bioreactors. A preferred flask is the CELL FACTORY ™ tissue culture flask (CF; NUNC), a specially designed long flask that conveniently has several internal compartments that provide a large surface area to which cells can adhere or fix, and on which they can grow. After cultivation until they are substantially confluent, the cells can be loosed by trypsinization, and are isolated. The trypsinization is carried out for a short period (preferably less than 5 minutes, preferably about 3 minutes), and the trypsin is then neutralized by the rapid addition of serum in the growth medium. If desired, the cells can be centrifuged, and the medium containing trypsin can be removed before adding the serum. The resulting cell suspension is then typically fed into a bioreactor is seeded for production (typically with a volume of 20 to 30 liters) for culture additional, and in some embodiments, to a larger production bioreactor (typically with a volume of 150 to 180 liters). The volume ratio of the second (larger) bioreactor: the seed bioreactor depends on the degree to which the cell line is propagated in the first bioreactor, but is typically 5: 1 to 10: 1, for example, on the scale from (6-8): 1. The cells are separated from the microcarriers by a trypsinization procedure performed in the culture vessel, preferably a bioreactor, while the microcarriers are suspended. The rotating filter is used to perform media exchanges to reduce serum and calcium levels in the medium, which increases the efficiency of the trypsinization while maintaining a constant volume in the bioreactor. The colonization steps, which could cause damage to the cells on the microcarriers, are avoided. The resulting suspension of cells / microcarriers can then be transferred to a production bioreactor, which is previously loaded with culture media and microcarriers. After transfer of the cell / microcarrier suspension from the seed bioreactor, the production bioreactor (eg, about 200 I) is operated, for example, at about 37 ° C and at a pH of about 7.3. An infusion of fresh medium during the propagation of the cells can then be carried out to maintain the concentration of lactate below approximately 1.0 g / l. Typically, the cells are allowed to grow on the microcarriers for about 4 to 7 days, until more than 50% of the microcarriers are completely confluent. Preferably, a virus infection procedure is then initiated. A 40 to 50 ml container of viral inoculum, typically containing approximately 1. 0x1013 total viral particles, is used to infect the production bioreactor. The virus is allowed to replicate in the production bioreactor for about 3 to 5 days until almost the time of maximum virus titer. Typically, more than 90% of the cells will have separated from the microcarriers due to the cytopathic effects of the virus. The final yield of recombinant adenovirus from the production bioreactor is typically about 8.5x10 9 viral particles / ml. This gives a total yield of viral particles of 1.4x1015 from each batch of 160 I. In other embodiments of the invention, the production bioreactor is inoculated with harvested cells by trypsinization, and is then used directly to inoculate the production bioreactor. Typically, 8 to 12 CELL FACTORY ™ tissue culture flasks are used to achieve the total density of inoculum sowing in the bioreactor from 0.6 to 1x105 cells / ml. The typical yield of the virus in this method varies from approximately 1.7 to 2.6x1010 viral particles / ml. Therefore, this particular method provides a total number of viral particles of approximately 3 to 4x1015 from each batch of 160 I.

In some embodiments of the invention, a process similar to the fluidized bed is used to harvest the bioreactor cells. Typically, the bioreactor is harvested after approximately 90% of the cells are separated from the microcarriers. Without being limited to any theory, the cytopathic effect of viral propagation in host cells seems to be the cause of cell separation. In other embodiments, uninfected cells can be separated from the microcarriers by the trypsinization method of the present invention. After the bioreactor is harvested, the broth contains cells, microcarriers and culture medium. The virus is present in the cells and in the medium. Therefore, all this material is preferably collected for processing. The specific gravity (density) of the microcarriers is similar to that of the cells. Preferably, the microcarriers remain freely suspended while the cells are separated from the drops, since the processing steps using sedimentation cause the cells to settle with the microcarriers, which results in recovery losses. In Figures 1 and 2 a preferred embodiment of a separation device is provided. In some embodiments of the invention, the separation device is provided as part of a system. An example of a system is shown in Figure 2. The system thus comprises a bioreactor 100 in which the cells are cultivated on microcarriers.; a flow path 102 from the bioreactor to the separation device 104, the separation device comprising a column 106, an outlet 108 for collecting the cells and the aqueous solution, and a 110 mesh screen. The microcarriers are retained in suspension by an upward flow in the separation device, and are retained in said device by the sieve mesh, and the cells and the aqueous solution are collected through the outlet. Also provided in the system is a pump 112, wherein the pump directs the flow of the aqueous solution from the bioreactor to the outlet.

In some embodiments, a microfilter 114 and an ultrafilter may be provided 116 as system components. In the embodiment shown in Figure 1, the separation device typically comprises a column 106, such as a chromatography column, having an inlet 114 through which an aqueous suspension of cells and microcarriers from a bioreactor 100 is introduced into the separation device 104; and at least one outlet 108 for collecting the cells and the aqueous solution; and a 110 mesh screen. The microcarriers are retained in suspension in the column by an upflow in the separation device, and are retained therein by a mesh screen, and wherein the cells and the aqueous solution are collected through the filter. the exit. The flow rate in the separation device is from about 1 to about 3 cm / min. Typically, an upward flow through the column is generated by pumping an aqueous solution, such as the cell suspension or a pH regulator, to through the entrance, where the entrance is located at the bottom of the device and the exit is located at the top of it. Figure 2 is an enlarged schematic view of a separation device 200, showing in more detail an exit assembly 210, a mesh screen assembly 212, an inlet 214, and a column 216 having an upper section 218 and a lower section 220. The lower section typically comprises about 20 to 50%, more preferably about 30% of the volume of the column, and contains the inlet. The lower section is preferably conical, with a preferred angle of about 15 to about 45 degrees. In this way, the fermentation broth of the bioreactor is pumped at the base of the column. The flow rate is regulated to provide sufficient upward flow to maintain suspended cells and viral particles in the medium, while allowing retention of the microcarriers within the separation device. Preferably, the flow rate is about 1 to 2 cm / min, since the cells have a specific gravity similar to that of the microcarriers. The purified broth containing cells and virus passes through the mesh screen on the upper end of the column similar to the fluidized bed, and is collected for microfiltration. For a device on the 200 I scale, the lower section of the column is preferably conical. The cone allows a gradual reduction in the linear velocity of the fermentation broth entering the cone. Speed Flow of the input line is reduced to achieve a reduced linear flow in a uniform distribution across the cross-sectional area at the upper end of the cone. The walls of the cone are at an angle that allows the droplets that settle on the walls to move down towards the entrance. In this way, these drops are resuspended to avoid trapping the cells between the sedimented drops. The angle of the conical walls is preferably approximately 30 degrees. Angles less than 15 degrees provide an exceptionally long cone, and angles greater than approximately 45 degrees may not effectively disperse the input power. The upper section of the column functions as an area in which the droplets settle at a rate greater than the linear flow rate of the fermentation medium. This section of the column is cylindrical in shape. Within this area a limit is formed, so that the microcarriers accumulate in the lower region of the column. A terminal plate assembly 222 (FIG. 2) of the column functions as a collection point for the purified fermentation media containing cells and viruses. This consists of a terminal plate 224 adapted with a mesh screen assembly. This screen, preferably mesh of approximately 50 to 120, more preferably mesh of approximately 100, functions as a second point for the removal of the microcarriers. The embodiment described above is the preferred embodiment used in the examples herein. The dimensions of the column and screen mesh can be varied based on the volume of the solution which will be processed, the concentration of drops, the particular microvehicle used and the formulation of the means (for example, specific gravity of the media). A preferred column consists of a custom made lower cone made of stainless steel that will be attached to two tubes of KS370 section from Pharmacia adapted with a KS370 end assembly, for which the automatic distribution screen was replaced with a stainless steel mesh (ss) (preferably mesh of about 50 to 120, more preferably mesh of about 100). After the cells are collected, they are preferably used to release the additional viral particles. Homogenization or freeze-thaw can be used to release the viral particles. In a preferred embodiment of the invention, microfiltration is used to simultaneously lyse the virus-containing cells and purge the broth from cell debris that would otherwise interfere with the purification of the viruses. For example, microfiltration can be carried out using a Prostak system (Millipore) with a hydrophilic or hydrophobic membrane of 0.65 microns, and at a shear rate of 7000 l / sec. The shear rate is generated by the flow of material retained through the tangential flow channels of the membrane. Therefore, the transverse flow is used not only to prevent the membrane from binding, but can also be used to create sufficient shear to lyse the cells. The pore size of the filter should be sufficient to allow the passage of the virus, while retaining the cell debris. Thus, typically the pore size scale is of approximately 0.2-0.65 views. The shear rate scale is typically from about 2000 to 10,000 l / sec, more preferably from about 7000 l / sec. Typically, BENZONASE ™ Endonuclease Cleaned Broth (American International Chemical, Inc.) is added to digest cellular nucleic acids, since viral particles can complex with said nucleic acids. In a preferred embodiment, ultrafiltration is used using a Pellicon system (Millipore) with a Pellicon I regenerated cellulose membrane, with a nominal molecular weight limit of one million to concentrate the virus. The ultrafiltration step accomplishes two functions: the virus is concentrated for purification, and the diafiltration is carried out to exchange the pH regulator, so that the virus suspension can be applied directly to a DEAE column. The eluate of the microfilter contains the released virus, and is preferably concentrated, for example, by ultrafiltration. Between each culture step, the cells can be loosened and separated from the microvehicle by means of trypsinization, for example, by treatment with trypsin. In the present invention, it is preferred to remove the serum used in the culture, since the whey proteins inhibit trypsin; the removal of the serum therefore allows a smaller amount of trypsin to be used. This is advantageous, since the addition of a larger amount can cause localized high concentrations of trypsin that could damage the cells. With respect to the next step, the ions Ca ++ are removed, since the removal of these ions from the cells tends to loosen them, and allows to use less trypsin. In this way, the loosening and separation of the cells, especially from the human embryonic kidney 293 cell line, can conveniently include the following steps: i) rapidly washing the cells to remove the serum and other soluble materials; I) removing Ca ++ from the washed cells by the addition of a chelating agent; iii) quickly remove the chelating agent; iv) quickly add trypsin; v) trypsinize the cells for a short period (preferably varying from about 3 minutes to about 15 minutes); vi) rapidly neutralize trypsin by adding protein. In step (i) above, the phrase "wash quickly" means, at a constant volume in the bioreactor, to perfuse a change in volume of medium at a rate of about 1 to 3 liters per minute, more preferably about 2 liters. by minutes. In step (iii) above, the phrase "rapidly removing the chelating agent" means, at a constant volume in the bioreactor, perfusing changes of 1.5 volumes of medium at a rate of approximately 1 to 3 liters per minute, more preferably about 2 liters per minute. In step (iv) above, the phrase "quickly add trypsin" means adding the appropriate volume of trypsin solution (typically a 2.5% solution) at a rate of about 1 to 3 liters per minute, more preferably about 2 liters per minute. minutes In step (vi) above, the phrase "Rapidly neutralizing trypsin by the addition of serum" means adding the appropriate volume of serum at a rate of about 1 to 3 liters per minute, more preferably about 2 liters per minute. If the serum is not removed in step (i), then the addition of the necessary large amounts of trypsin can lead to locally high concentrations thereof, which can actually damage or even destroy the cells, rather than simply loosen them. Removal of the serum in step (i) and Ca ++ in step (ii) reduces the amount of trypsin required in steps (iv) and (v). In order to avoid actually damaging or even destroying the cells, treatment with the chelating agent and trypsin should preferably be maintained for a short time (ie, long enough to separate the cells from the microcarriers, but preferably no longer) . Examples of preferred chelating agents include EDTA (ethylenediaminetetraacetic acid) and EGTA (ethylene-bis (oxyethylene-nitrile) -tetraacetic acid). The serum is removed by a medium exchange procedure; For example, the medium can be pumped through a filter rotary. Serum-free washing medium is added to replace the one that was pumped, and the mixture is stirred. Alternatively, the addition of serum-free washing medium can be continuous with the removal of media through the rotary filter. The procedure is repeated until the serum concentration has been reduced to a sufficiently low level, eg, less than about 1.0 to 0.2%, preferably about 0. 2%. The chelating agent, preferably EDTA, is added to the serum-free chelating agent, the mixture is stirred again, and the chelating agent is pumped. Alternatively, the addition of the chelating agent in the serum free medium can be continuous with the removal of the medium through the rotary filter. The trypsin is preferably used in step (v) to provide a concentration in the bioreactor of about 0.05 to 0.1%, and it is allowed to act on the cells for 5 to 10 minutes, for example, preferably a trypsin concentration of about 0.065% for approximately 8 minutes. The protein, typically in the form of bovine serum, is preferably added to the bioreactor at a final concentration of about 10 to 20% to inhibit trypsin. In this way, the addition of serum in step (vi) not only prepares the cells for subsequent culture, but also neutralizes the residual trypsin. The complete sequence of steps (i) - (vi) can be carried out in situ in the bioreactor; In some embodiments, the suspension of microcarriers and cells can be transferred to a larger bioreactor, in where other microcarriers are added for the next step of cultivation. The cells are allowed to adhere to the microcarriers, and are then cultured. Once they become substantially confluent again (for example, culture at about 37 ° C for 3 to 4 days), can be carried through the next stage, which may be for example harvesting, loosening for an additional further step, or inoculation with the virus. If the cells have been cultivated simply for harvest, then they can be harvested at this stage, for example, by repeating steps (i) through (vi) above. If they are required for an additional subsequent stage, then steps (i) - (vi) above can be repeated. If they have been grown for the propagation of a virus, the virus can now be inoculated into the medium. The examples herein serve to illustrate, but in no way limit, the present invention. The vectors and selected hosts and other materials, the concentration of reagents, temperatures and the values of other variables, are only to exemplify the application of the present invention, and should not be considered as limitations thereof.

EXPERIMENTAL EXAMPLES 1. REVIEW A. Preparation of cell inoculum Each batch of viral fermentation originates from a cell line propagated from a cell of the 293 Cell Workers Working Cell Bank (MWCB). Bioreactors are inoculated with 293 cells (catalog number of ATCC CRL 1573) maintained by propagation in T and CELL FACTORY ™ flasks using growth medium (medium 1), as illustrated in Table 1 below. Each transfer represents a passage. Typically, passage numbers 4 to 30 are used for the inoculation of a seeding bioreactor.

TABLE 1 Composition of the means 1 DMEM Powder (available from American Biorganics, Catalog No. D2807): preferably used to provide 4.5 g / L of glucose and 0.584 g / L of L-glutamine; without sodium bicarbonate and without HEPES. 2 Ca ++ free DMEM powder (available from American Biorganics, Catalog No. D2807403): preferably used to provide 4. 5 g / l of glucose and 0.584 g / l of L-glutamine; without sodium bicarbonate, without calcium chloride and without HEPES. 3 Bovine serum: available from Hyclone, Catalog No. 2151. 4 EDTA supply solution: 186.1 g / l of ETDA and 20 g / l of NaOH pellets. 5 Glutamine supply solution: 29.22 g / l. To prepare the transfer of cells from one flask to another during the expansion of the cells, the spent medium is poured, and the cells in the flask are then washed with pH regulated saline with phosphate (PBS). A solution of trypsin is added to the monolayer of cells on the surface of the flask, and the cells are exposed until they are separated from the surface. The action of trypsin is then neutralized mainly by the addition of growth medium (medium 1, table 1) containing serum; Complete neutralization is not necessary, since the residual trypsin will have low activity. The cells can be recovered by centrifugation, and resuspended in fresh growth media (mediol). Table 2 shows the typical volumes used.

TABLE 2 Typical volumes used in cell transfer B. Preparation of the virus inoculum Inocula of the virus can be prepared by infecting flasks for CELL FACTORY ™ mature tissue culture. In this procedure, 293 cells are first propagated from the T flasks to the CELL FACTORY ™ tissue culture flasks. When cultures of CELL FACTORY ™ tissue culture flasks are mature (typically 80 to 90% confluent), they are infected with an inoculum from the manufacturer's useful virus bank (MWVB). The infected CELL FACTORY ™ tissue culture flasks are incubated until the 293 cells separate from their supporting surface. The cells are harvested by centrifugation and fractured by multiple freeze-thaw cycles. After a subsequent centrifugation, the virus is recovered in the supernatant, and stored as aliquots at -20 ° C or less. This material is the "virus inoculum", which is used to infect bioreactors Optionally, the inoculum of the virus can also be derived from the harvesting of the bioreactor, which is sterilized with a filter (see "harvesting the production bioreactor" below).

C. Preparation and operation of the seed bioreactor Preferably, a seed bioreactor is used to prepare the inoculum of 293 cells for the production bioreactor. The seed bioreactor is steam sterilized and loaded with a batch of growth medium sterilized with filter (medium 1, table 1 above) for the free cell suspension procedure. However, for the microcarrier method, sterile dilated microcarrier droplets (Cytodex 3 or equivalent) are preferably added in this step. The seed bioreactor is inoculated with the 293 cells harvested from the CELL FACTORY ™ tissue culture flasks. The operating conditions are established as shown in table 3. The pH and dissolved oxygen (DO) are controlled by spraying CO2 and oxygen, respectively. Extra growth medium can be added to the bioreactor by perfusion. Cell growth is monitored by microscopic examination, and measuring lactate production and glucose consumption. Typically, when the density of cells in the suspension culture reaches 1 x 106 cells per milliliter in the seed bioreactor, it is ready to inoculate the production bioreactor. However, the inoculation requires additional steps to carry out the procedure with microcarriers. Typically, when the cells on the microcarriers are more than 50% confluent, the serum and calcium in the bioreactor medium are removed using the media described in table 1. Trypsin is then added rapidly, and when the cell separation reaches a typical level, serum is added to inactivate it. The contents of the seed bioreactor are now transferred to the production bioreactor. Optionally, 293 cells harvested from multiple flasks for CELL FACTORY ™ tissue culture can be used directly as inoculum for a production bioreactor. Typically, 8 to 12 of these crops are harvested and mixed to provide an inoculum.

TABLE 3 Operating conditions of the planting bioreactor D. Preparation and operation of the production bioreactor The viral production procedure is exemplified in a 200 liter production bioreactor using growth medium (medium 1, table 1). In the suspension culture method, the sterilized medium with filter is distributed in batches in the bioreactor. However, in the microcarrier method, the microcarriers are sterilized in situ in the production bioreactor, or are externally autoclaved and charged. These microcarriers are then conditioned in the growth medium (medium 1) before their inoculation with 293 cells.

The production bioreactor is inoculated with the 293 cells of the seed bioreactor. The operating conditions are as shown in table 4. The pH and dissolved oxygen (DO) are controlled by spraying CO2 and oxygen, respectively. Optionally, extra growth medium can be added to the bioreactor by perfusion. The growth of the cells is monitored by microscopic examination and measuring lactate production and glucose consumption. The cells are allowed to grow to approximately 1 x 106 cells per milliliter. The bioreactor is then inoculated with the virus. Preferably, a multiplicity of infection ratio (MOI) expressed as the total viral particles per cell of 50: 1 to 150: 1 is used. The viral titer is typically carried out using the CLAR Resource Q. The virus is allowed to spread until the viability of the cells decreases to approximately 10%. The type of virus and its action on the host cells can be determined if necessary to separate the host cells from the microcarriers and / or lyse the cells. Almost at the time of the maximum titer of the virus (often when the cells start to separate from the microcarrier and some of which can be lysed, so that the virus starts to escape), the incubation can be stopped and the cells and the virus You can harvest. In addition, in the microcarrier procedure, 80 to 90% of the cells, sometimes even more than 90%, can be separated from the microcarriers. Without being limited to any theory, the cytopathic effect of viral propagation in host cells seems to determine the separation of the cells. In this way, when adenovirus ACN53 is used with cells 293, the cells begin to separate from the microcarriers after culture with the virus for 3 or 4 days.

TABLE 4 Operating conditions of the production bioreactor E. Harvesting the production bioreactor 1. Separation of cells from the microcarrier During harvest, the contents of the bioreactor have to be handled differently, depending on whether the procedure uses free suspension or a microcarrier. In the procedure with microcarriers, a fluidized bed column is preferably used to separate the microcarriers from the cells and the supernatant. An upward flow velocity is maintained in order to retain the microcarriers, while the cells and the supernatant pass through. The fluidized bed is washed with washing medium or pH regulator to recover most residual cells and viruses, and the washes are combined with the cells and the supernatant as an eluate. The fluidized bed operation is not required for the free cell suspension procedure. The eluate containing cells and the virus is further processed, since the cells are used by high shear to release the virus, and the eluate is then purified by cross-flow microfiltration. Typically, Durapore (Millipore) 0.65 μm membranes or equivalent membranes are used. Towards the end of the microfiltration, the retained material is washed with the pH regulator for washing to recover residual virus in the permeate. After microfiltration, the permeate can optionally be treated with a nuclease such as the BENZONASE ™ endonuclease. The permeate material of the microfiltration is concentrated by ultrafiltration (with a molecular weight limit of typically 1 million), and an exchange of the pH regulator is carried out using the pH regulator for washing. The concentrated and diafiltered retained material containing the virus is then passed through a final filter. The resulting filtrate is stored as "viral concentrate" in a freezer at -20 ° C or less. 2. Composition and preparation of the media Table 1 above indicates the means used in the preparation of the viral inoculum and in the fermentation process. All these culture media are prepared by first dissolving the dry DMEM powder and other reagents in purified water. After dissolving the dry powders, these media are adjusted to pH 7.2-7.6 with hydrochloric acid. The media is then sterilized by passing them through a 0.2 μm filter into an appropriate storage container. These sterile media are refrigerated below 10 ° C, and discarded one month after their preparation. Table 5 includes several pH regulators used in the process.

TABLE 5 pH regulators used in fermentation and harvesting Table 6 summarizes the controls of the procedure during the fermentation and harvesting procedures.

TABLE 6 Procedural controls TABLE 6 (CONTINUED) verification of contaminants sterility of the sample Observation bioreactor Growth and microscopic production, cellular morphology measurement of the normal ones on the microcarrier concentrations, less lactate and glucose, of 1.2 g / l of lactate cell count of the former of the infection, the supernatant, most of the cells verifying the separation from the sterility of the microcarriers in the sample harvests, without contaminants. Fluidized bed Visual observation The fluidized bed volume of the fluidized bed will be less pressure than the total feed volume and column, and the feed flow velocity pressure should feed. be less than the leakage point of the column assembly Microfiltration Observation and control Flow rate of the feed flow adjusted feeding, flow to achieve high permeate material, shear stress without pressure to exceed a feed pressure, transmembrane of 0.5 transmembrane and barias. The material temperature retained, must not exceed 37 ° C. temperature of the material retained. Ultrafiltration Observation and control The pressure of the supply pressure must not feed and exceed 1 bar. The temperature of the temperature should not material held exceed 37 ° C. Final filtration Observation and control The pressure of the feed pressure must not feed exceed 2 bar II. Drop transfer exhausts from the seed bioreactor to the production bioreactor A. Preparation of the 293 cell nodule for the seed bioreactor 1. Progressive increase of the vessel to the T75 flask A frozen container of the 293 cell line containing a total of 2 x 107 cells in a 37 ° water bath is thawed. C. The cells were washed with 10 ml of medium 1. The washed cells were resuspended in a total volume of 30 ml of medium 1, and placed in a 75 cm2 tissue culture flask (T75). The culture was placed in an incubator at 37 ° C with an atmosphere of CO2 at 5% and a humidity level of 100%. This was passage 1 of the crop. 2. Progressive increase of the T75 flask to the T500 flask using trypsinization The T75 culture reached a confluence level of 90% in three days. At this time, the T75 culture was trypsinized in the following manner. The 30 ml of the supernatant medium was removed from the flask. A volume of 10 ml of CMF-PBS (saline regulated in its pH with Dulbecco's phosphate without calcium chloride and without magnesium chloride) was used to wash the surface of the culture. The CMF-PBS of the supernatant was removed from the flask. 2 ml of TE solution (crude trypsin at 0.05% with EDTA-4Na at 0.53 mM) were added to the flask. The flask moved, so that the solution covered the total area of the crop. The cells were separated from the surface of the flask within five minutes. 10 ml of medium 1 was added to the flask immediately after the cells were separated from the surface. The cell suspension was centrifuged at 1000 rpm for ten minutes at room temperature by pausing. The supernatant was removed. The cells were resuspended in 5 ml of medium 1. The cell suspension was transferred to a sterile tube with 200 ml of medium 1. The 200 ml of cell suspension was placed in a 500 cm2 tissue culture flask (T500). . The liquid in the T500 flask was allowed to equilibrate between the chambers before being placed horizontally in the incubator. The culture was placed in an incubator at 37 ° C with an atmosphere of CO2 at 5% and a humidity level of 100%. This was passage 2 of the crop. 3. Progressive increase and passage of the T500 culture using trypsinization The T500 culture reached a confluence level of 90% in four days. On the fourth day, the T500 culture was trypsinized and expanded in proportion as follows. Discarded the supernatant medium. The culture surface was washed with 25 ml of CMF-PBS. A volume of 25 ml of TE was added to the flask. The flask was moved, so that the TE solution covered all three layers of the crop surface. The cells were separated from the surface of the flask within five minutes. After the cells were separated, 50 ml of medium 1 was added to the flask. All surfaces they came in contact with the medium when moving the flask. The resulting cell suspension was poured into a 200 ml conical centrifuge tube. The cells were pelleted by centrifugation at 1000 rpm for ten minutes at room temperature by pausing. The supernatant was discarded. The cells were resuspended in 5 to 15 ml of medium 1. The cell suspension was placed in 800 ml (200 ml per flask T 500 new) of medium 1. The cell suspension was mixed. A volume of 200 ml of the cell suspension was added to each of the four T500 flasks. The liquid level in each flask was allowed to equilibrate between the chambers before being placed horizontally in the incubator. The separation ratio for this passage was 1: 4. This was passage 3. The culture was thus passed through passages 4 to 13. In passage 13, four T500 cultures were trypsinized in the manner described above. The cell suspensions were mixed and placed in a tube containing 1.5 liters of medium 1. This cell suspension was added to a 6000 cm2 CELL FACTORY ™ (CF) tissue culture flask. The liquid level in the CF was allowed to equilibrate between the chambers before being placed horizontally in the incubator. 4. Progressive increase and passage of cultures of the CELL FACTORY ™ tissue culture flasks The CF culture reached a confluence level of 80% in days. The trypsinization was carried out in the following manner for passage 15. The 1.5 liters of medium 1 were drained from the CF culture. The Crop surfaces were washed with 500 (+/- 100) ml of CMF-PBS.

After washing, 250 (+/- 50) ml of the TE solution was added to the CF culture. The CF was moved, so that the TE solution covered each of the surfaces. After the cells were removed from the surface, 500 ml of medium 1 was added to the CF. The CF was moved, so that the medium 1 came into contact with each of the surfaces. The resulting cell suspension was aliquoted into four 250 ml conical centrifuge tubes. The cells were transformed into pellets by centrifugation at 1000 rpm for ten minutes by pausing. The supernatant medium was discarded from each centrifuge tube. In each centrifuge tube, the cells were resuspended in 5 ml of medium 1. The cell suspensions were mixed in a centrifuge tube. The remaining three centrifuge tubes were washed with another 5 to 10 ml of medium 1, which was added to the overall cell suspension. This cell suspension was also separated between 6 tubes containing 1.5 liters of medium 1. Each of the six 1.5-liter cell suspensions was added to a CF. The liquid level of each CF was allowed to equilibrate between the chambers, before the CF was placed horizontally in the incubator. The crop was passed in the same way through passage 16. The data of the passage are given in table 7.

TABLE 7 Data of the passage . Preparation of inoculum cell cultures flasks for tissue culture CELL FACTORY ™ to seed bioreactor cultures CF reached a level of confluence of 80% in days. Four of the six CF cultures were used to inoculate the seed bioreactor in the following manner. The 1.5 liters of medium 1 were drained from the CF culture. The culture surfaces were washed with 500 (+/- 100) ml of CMF-PBS. After washing, 250 (+/- 50) ml of the TE solution was added to the CF culture. The CF was moved, so the solution of I will cover each one of the surfaces. Immediately after the cells were separated from the surface, 500 ml of medium 1 was added to the CF.

The CF was moved, so that the medium 1 came into contact with each of the surfaces. The resulting cell suspension was aliquoted into four 250 ml conical centrifuge tubes. The cells were pelleted by centrifugation at 1000 rpm for ten minutes at room temperature by pausing. The supernatant medium was discarded from each centrifuge tube. In each centrifuge tube, the cells were resuspended in 5 ml of medium 1. The cell suspensions were mixed in a centrifuge tube. The remaining three centrifuge tubes were washed with another 5 to 10 ml of medium 1, which was added to the overall cell suspension. The cell suspensions of each of the four centrifuge tubes were then combined, yielding a total volume of 50 to 100 ml. An additional volume of medium 1 was added to bring the total volume to 1000 ml. This was the inoculum of cells. The total number of cells in the cell inoculum was 2.88 x 109 total cells and 2.84 x 109 viable cells. The 1000 ml of the cell inoculum was transferred to a sterile Erlenmeyer flask, and inoculated in the 30 liter bioreactor containing a total volume of 18 liters of medium 1 with 66 g of Cytodex 3 microcarriers.

B. Seed bioreactor 1. Preparation of Cvtodex 3 microcarriers for the 30 liter seed bioreactor. A batch of 66 g of Cytodex 3 microcarriers was prepared as follows. The 66 g of Cytodex 3 microcarriers were placed in a five liter glass Erlenmeyer flask. Two liters of CMF-PBS were added with 0.2 ml of Tween 80. The microcarriers were allowed to expand at room temperature for five hours thirty minutes. After this period of dilatation, the CMF-PBS of the supernatant was decanted from the flask, leaving the suspension of microcarriers Cytodex 3. The suspension of microcarriers Cytodex 3 was washed with two liters of CMF-PBS, and then resuspended in CMF-PBS up to a total volume of two liters. The Cytodex 3 batch was autoclaved in the five liter flask at 121 ° C for 3.5 hours over a liquid cycle. The batch of sterilized Cytodex 3 was used the next day for the 30 I seed bioreactor. On the day of the addition of Cytodex 3 to the thirty liter bioreactor, the following actions were carried out. The CMF-PBS was decanted from the supernatant of the 5 liter flask. The suspension of microcarriers was washed with two liters of medium 1. After washing, medium 1 was added to the flask to a final volume of 2 liters. 2. Preparation of 30-liter seed bioreactor The 30-l seed bioreactor containing a rotating filter was washed and sanitized with steam. The bioreactor was sterilized for 50 minutes at 121 ° C. One day before inoculation of 293 cells, the 30-liter seed bioreactor was filled with 18 liters of medium 1. The two liters containing 66 grams of the Cytodex 3 microcarriers with medium solution 1 were added to the 30 I bioreactor. The operating conditions of the 30 I seed bioreactor are given in Table 8.

TABLE 8 Operating conditions of the 30 I sowing bioreactor 3. Cultivation of 293 cells in the 30 I seed bioreactor 293 cells were propagated on the microcarriers Cytodex 3 for 5 days. The actual operating conditions in the 30 I sowing bioreactor during this period are given in Table 9.

TABLE 9 Operating conditions in the 30 I seed bioreactor On the fifth day of culture, 54% of the microcarrier population contained more than 50% cells / microcarrier, 38% contained 1 to 25 cells, 8% did not contain cells, and 8% were in aggregates of 2 microcarriers, according to was determined by examining a sample under the microscope at a magnification of 100X. The results are given in table 10.

TABLE 10 Results of the microscopic examination on the fourth day of cell culture 293 on microcarriers Cytodex in the bioreactor of 30! B. Dropwise transfer procedure 1. Preparation of Cvtodex 3 microcarriers for the production bioreactor A batch of 420 grams of Cytodex 3 microcarriers was prepared in the following manner. The 420 grams of Cytodex 3 microcarriers were placed in a 50 I jug. A volume of 21.5 I of CMF-PBS was added with 2.0 ml of Tween 80. The microcarriers were allowed to expand at room temperature for 17 hours. After this period of dilatation, the CMF-PBS of the supernatant was removed from the jug, leaving the suspension of microcarriers Cytodex 3. The suspension of microcarriers Cytodex 3 was washed with 25 liters of CMF-PBS, and then resuspended in CMF-PBS up to a total volume of 20 liters. The 200 I bioreactor containing a rotating filter was washed and sanitized with steam. The suspension of Cytodex 3 20 I microcarriers was transferred to the bioreactor. 5 liters of CMF-PBS were used to wash the 50 I jug, and transferred to the bioreactor. The bioreactor was sterilized for 50 minutes at 123 ° C. The bioreactor was maintained at 4 ° C overnight. The next day, 120 I of medium 1 was added to the 200 I bioreactor. The microcarrier solution was stirred at 90 rpm for 10 minutes in the bioreactor. The volume was reduced to 55 I by separating the liquid through the rotary filter. Another 110 I of medium 1 was added to the 200 I bioreactor. The microcarrier solution was stirred at 90 rpm for 10 minutes in the bioreactor. The volume was reduced to 55 l by separating the liquid through the rotary filter. Medium 1 was added to the bioreactor to bring the volume up to 125 I. The operating conditions of the bioreactor were determined in accordance with Table 11.

TABLE 11 Operating conditions of the bioreactor The 200 I production bioreactor was ready to receive the inoculum from the seed bioreactor. 2. Tripsinization of the seed bioreactor culture On the fifth day of culture of the 293 cells on the Cytodex 3 microcarriers, the dropwise transfer procedure was carried out. The serum and calcium levels of the medium in the culture were reduced by perfusing 22 liters of medium 2 at a rate of 2 I per minute using the rotary filter with a constant volume in the 20 I bioreactor. perfusion was continued with 22 I of medium 3 at an infusion rate of 2 I per minute with a constant volume in the 20 I bioreactor. This further reduced serum and calcium levels. Medium 3 contained disodium ethylenediaminetetraacetate dihydrate (EDTA), which chelates divalent cations such as magnesium and calcium. A third round of perfusion was carried out using 33 liters of medium 2, which was designed to further reduce serum and calcium levels, and to reduce the concentration of EDTA in the medium. At this point, the medium was isolated through the rotary filter to reduce the total culture volume to 15.5 I. A volume of 480 ml of a 2.5% trypsin solution was added to the bioreactor in one minute. By microscopic observation, 8 minutes after the addition of the trypsin solution, 90% of the cells had been separated from the microcarriers. At this point, 4 I of serum was added to the bioreactor in 2 and a half minutes to inhibit the action of trypsin and protect the cells from shear during the transfer process to the production bioreactor. The trypsinized cells and the microcarriers were transferred to the production bioreactor by pressure. The transfer was achieved in eight minutes. Immediately after the transfer, 5 l of medium 1 was added to the seed bioreactor as a jet, and transferred to the production bioreactor by pressure. The operating conditions of the sowing bioreactor during the dropwise transfer procedure are given in Table 12.

TABLE 12 Operating conditions of the sowing bioreactor during the drop-by-drop transfer procedure C. Culturing 293 cells in the 200 I production bioreactor before infection 293 cells were propagated on the Cytodex 3 microcarriers for six days. The actual operating conditions in the 200 I production bioreactor during this period are given in Table 13.

TABLE 13 Operating conditions in the 200 I roduction bioreactor A total volume of 115 liters of medium 1 was perfused from days 4 to 6. The regimens were as follows: 24 liters were perfused in one hour on day four, 40 liters were perfused in one hour on day five, and perfused 50 liters in one hour on day six. The rate of oxygen uptake measured as the decrease in dissolved oxygen level (% air saturation,% OD) per minute (decrease in DO % / min) reached 1.65% per minute on day six. The results of the microscopic examination on the sixth day of cell culture 293 on microvehicles Cytodex 3 in the 200 I bioreactor are given in Table 14.

TABLE 14 Results of the microscopic examination on the sixth day of 293 cell culture on Cvtodex 3 microcarriers in the 200 I bioreactor Percentage of total microcarrier population > 50 cells 10-50 cells per 1-10 cells per microvehicle microcarrier microcarrier empty bodies microcarrier 31% 23% 25% 21% D. Infection of 293 cells in the 200 I production bioreactor The bioreactor culture was inoculated with virus on day 6. The viral inoculum had been stored frozen at -80 ° C. A volume of 45 ml of the viral inoculum, 2-2, was thawed in a water bath of 20 to 25 ° C. The total amount of virus added to the tank was 1.1 x 1013 viral particles, measured by the CLAR Resource Q test. The viral inoculum was mixed and placed in a tube with one liter of medium 4 (Eagle's medium modified from Dulbecco 293-1 -R07 with L-glutamine and sodium bicarbonate (3.7 g / l)). The viral solution was filtered through a Maxi Gelman culture dish in a sterile five liter addition flask. The viral suspension was added to the 200I production bioreactor with sterile connections made by the tubing welder. The operating conditions of the production bioreactor after infection are given in Table 15.

TABLE 15 Operating conditions of the production bioreactor after infection Three days after infection, 89% of the microcarriers had no adhered cells, and the oxygen uptake rate measured was 0.53% per minute. The total concentration of infected cells present in the broth of the supernatant was 1.0 x 106 cells / ml. The total volume in the bioreactor was 162 liters. The bioreactor was harvested on this occasion.

E. Recovery operations A volume of 400 liters of pH buffer for harvest recovery was prepared and filtered through a Pall Ultipor N66 (0.2 micron pore size), and was aliquoted into sterile containers of the following way. Three aliquots of 210 liters, 130 liters and 50 liters were prepared. The volume of 210 liters was used for the washing operation of the bioreactor and the fluidized bed column. The aliquot of 130 liters was used during the microfiltration. The aliquot of 50 liters was used during the ultrafiltration process.

F. Separation of the cells from the microcarriers using the fluidized bed column The fluidized bed was sanitized using a caustic solution (0.1 N sodium hydroxide). A T-shaped fitting was connected to the bioreactor harvest hole. On one side of the T-shaped fitting a sanitary hose (inner diameter of 15.9 mm) and a valve to the fluidized bed column were connected. A peristaltic pump was placed on this line (Watson Marlow, model 604 S). The second side of the T-shaped fitting was connected to a sanitary hose (inner diameter of 15.9 mm) and valve, leading to the pH regulator tank. The output of the fluidized bed column, through which the broth containing the cells and the virus was passed, was connected to a tank used as the recirculation vessel for microfiltration. The bioreactor broth was passed through the fluidized bed column at an objective flow rate of 2 to 3 liters per minute. The flow rate was controlled with the peristaltic pump. Stirring was maintained in the bioreactor. When the volume of the bioreactor was lower than 100 liters, the rotary filter went out. When the volume of the bioreactor was less than 30 liters, the agitator was turned off. After the contents of the bioreactor were processed through the fluidized bed column, the bioreactor was washed with 90 liters of pH buffer for crop recovery. This washing material was processed through the fluidized bed column. At the end of the procedure, the microcarriers remained in the fluidized bed column and were discarded. The data is given in table 16.

TABLE 16 Data of a fluidized bed column operation G. Microfiltration of microcarrier depurated broth - lysis of infected cells and treatment with endonuclease BENZONASE ™ The starting material for the microfiltration process was the broth from the fluidized bed column that was purified from the microcarriers and that contained cells and viruses . During the microfiltration step, the cells were used due to the shear rate used, the broth was cleared from debris larger than 0.65 microns, and the residual nucleic acids from the cells used were digested by BENZONASE ™ endonuclease (eg, 0.5 units of million per lot of 200 I), an enzyme preparation. The microfiltration unit was a Prostak system (Millipore). It contained a Durapore hydrophilic membrane of 0.65 micron pore size (catalog number SK2P446EO) with a surface area of 5,022 m2.

The feed and retentate lines of the Prostak filter unit were connected to the microfiltration recirculation vessel containing the purified microcarrier broth of the fluidized bed column. A line used to feed the harvest recovery pH regulator to the microfiltration recirculation vessel was connected. The line of permeate material from the Prostak unit was connected to the recirculation vessel for ultrafiltration. The temperature of the broth was maintained in the range of 25-35 ° C. When the broth feed in the Prostak unit was reduced to a volume of 10 to 30 liters in the microfiltration recirculation vessel, 50 liters of the crop recovery pH regulator was added to the vessel, and microfiltration was continued. This step was repeated once. The microfiltration was continued until the volume in the recirculation vessel for microfiltration was reduced from 10 to 30 liters. On this occasion, 0.5 units of one million BENZONASE endonuclease were added to the purified broth in the ultrafiltration recirculation vessel. The contents of the vessel were mixed well, and the broth was maintained for two hours before the ultrafiltration was initiated. The data is given in table 17.

TABLE 17 Data of a microfiltration operation H. Ultrafiltration of the broth to concentrate the virus with diafiltration to carry out the buffer exchange The starting material for the ultrafiltration process in the recirculation vessel for ultrafiltration was the purified broth treated with endonuclease BENZONASE ™ from the material permeated by microfiltration. The ultrafiltration unit was a Pellicon system (Millipore). It contained a Pellicon II regenerated cellulose membrane with a nominal molecular weight limit of one million (catalog number P2C01 MC05) with a surface area of 5,022 m2. The feed and retentate lines of the Pellicon unit were connected to the ultrafiltration recirculation vessel. The line of material permeated by ultrafiltration was connected to a waste container. A vessel containing the harvest recovery pH buffer was connected (Tris base at 50 mM, sodium chloride at 150 mM, magnesium chloride hexahydrate at 2 mM and sucrose at 2%) to the recirculation vessel for ultrafiltration. When the volume of material retained by nitration reached 5 to 10 liters, 15 liters of the harvest recovery pH regulator was added, and the ultrafiltration was continued. This step was repeated once. The ultrafiltration was continued, until the volume of material retained was less than 5 to 10 liters. The retained material from the ultrafiltration contained the concentrated virus. The retained material was collected from the Pelllcon unit. A 3 to 6 liter jet of the crop recovery pH regulator was used to collect all the material from the Pellicon unit. This material applied to the jet was added to the broth of the retained material of the ultrafilter. This was filtered through a Millipore filter, Durapore, pore size 0.45 microns (catalog number CVHL71 PP3) in a sterile bag. The material in the sterile bag was stored frozen at -80 ° C. The data is given in table 18.

TABLE 18 Data of an ultrafiltration operation I. General Comments All cultures of 293 cells in the T75, T500 and CELL FACTORY ™ tissue culture flasks were maintained in medium 1 in an incubator at 37 ° C, 100% humidity and an atmosphere of C02 at 5% . All exposed operations were carried out aseptically under a biosecurity cover (laminar flow). The saturations and additions of medium were carried out through an in-line filter Ultipor N66 PALL of 0.2 micron pore size installed on the bioreactor's feeding orifice, which was sterilized with steam for 30 minutes at 121 ° C. All other additions to the bioreactors were carried out using a sterile Erlenmeyer flask with PHARMED ™ tubing that was aseptically connected between the bioreactor and the addition flask by a tubing welder. All pH regulators used in the recovery procedures were filtered through a 0.2 micron Pore Size Ultipor N66 PALL in-line filter (SLK7002NFP) installed over a hole in the receiving container. Note that for microfiltration operations, a hydrophilic or hydrophobic membrane can be used. All publications and patent applications cited herein are incorporated by reference in their entirety as if each patent application or individual publication was specifically and individually indicated to be incorporated herein by reference. The modifications and variations of this invention will be apparent to those skilled in the art. The specific modalities described herein are offered by way of example only, so it should not be considered that the invention is limited thereto.

Claims (38)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for growing cells, characterized in that it comprises: (a) culturing the cells on a first batch of microcarriers until the cells are substantially confluent; (b) separating the cells from the microcarriers without removing them from the suspension; (c) adding a second batch of microcarriers; and (d) then culturing the cells.

2. The method according to claim 1, further characterized in that. the step for separating the cells from the first batch of microcarriers comprises the following steps: (a) washing the microcarriers and the adhered cells to remove the soluble materials; (b) contacting the microcarriers and the washed cells with a chelating agent; (c) removing the chelating agent; (d) trypsinizing the cells for a short period to separate them from the microcarriers; and (e) neutralizing trypsin by the addition of protein, wherein steps (a) - (e) are carried out in an individual culture vessel.

3. The method according to claim 2, further characterized in that the chelating agent is EDTA.

4. The method according to claim 2, further characterized in that the trypsin is used in step (d) to a concentration of about 0.05% to about 0.1% for 5 a 10 minutes.

5. The method according to claim 1, further characterized in that a virus capable of infecting the cells is added during step (d).

6. The method according to claim 5, further characterized in that the virus is added when the cells become substantially confluent on the microcarriers.

7. The method according to claim 5, further characterized in that the virus comprises an adenoviral vector.

8. The method according to claim 7, further characterized in that the virus is ACN53.

9. The method according to claim 1, further characterized in that the cells are 293 cells.

10. The method according to claim 9, further characterized in that the cells are incubated for 3 to 4 days.

11. A method for separating microcarrier cells on which they have been cultured, but from which they have been separated, characterized in that it comprises introducing an aqueous suspension of cells and microcarriers through an inlet in a separation device , the device comprising: (a) an entry; (b) a column; (c) an outlet for collecting the cells and the aqueous solution; and (d) a mesh screen; where the microcarriers are held in suspension by an upward flow in the separation device, and are retained in said device by a mesh screen, and wherein the cells and the aqueous solution are collected through the outlet.

12. The method according to claim 11, further characterized in that the aqueous suspension of cells and microcarriers further comprises viruses propagated in the cells.

13. The method according to claim 11, further characterized in that the cells are 293 cells.

14. The method according to claim 12, further characterized in that the virus is an adenovirus.

15. The method according to claim 11, further characterized in that the flow rate in the separation device is from about 1 to about 3 cm per minute.

16. The method according to claim 11, further characterized in that the collected cells and aqueous medium are subjected to microfiltration.

17. The method according to claim 16, further characterized in that the microfiltration comprises a shear rate of approximately 2000-10,000 l / sec.

18. The method according to claim 11, further characterized in that the upward flow is generated by pumping the aqueous solution through the inlet, where the inlet is located at the bottom of the device, and the outlet is located in the superior of it.

19. - The method according to claim 11, further characterized in that the column comprises an upper section and a lower section, the lower section comprising approximately 20 to 50% of the volume of the column, and containing the input.

20. The method according to claim 19, further characterized in that the lower section is conical.

21. The method according to claim 20, further characterized in that the angle of the lower section is from about 15 to about 45 degrees.

22. A method for the release of a cellular constituent from cells cultured in an aqueous medium, characterized in that it comprises mechanically shearing the cells exposing them at a shear rate, where the shear rate for shear Mechanically subjecting the cells to shear stress is generated by collecting the cells in a microfilter, and rapidly recirculating the aqueous medium therethrough.

23. The method according to claim 22, further characterized in that the shear rate is about 2000-10,000 l / sec.

24. The method according to claim 22, further characterized in that the cell debris is collected by the microfilter, and the desired cell constituents are passed therethrough.

25. - The method according to claim 22, further characterized in that the cellular constituent is a virus.

26. The method according to claim 25, further characterized in that the virus in an adenovirus.

27. The method according to claim 26, further characterized in that the adenovirus has a heterologous gene.

28.- A system for separating cells from microvehicles on which the cells have been cultured, the system comprising: (a) a bioreactor in which the cells were grown 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; (ii) an outlet for collecting the 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 said device by a mesh screen, and wherein the cells and the 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.

29. The system according to claim 28, further characterized in that it comprises a microfilter, wherein the cells and the aqueous solution of step (c) are subjected to microfiltration.

30. - The system according to claim 29, further characterized in that it comprises an ultrafilter, wherein the product of claim 46 is subjected to ultrafiltration.

31. The system according to claim 28, further characterized in that the cells are 293 cells.

The system according to claim 28, further characterized in that the upward flow comprises pumping the aqueous solution through the inlet, where the entrance is located at the bottom of the device, and the exit is located at the top of it.

33. The system according to claim 28, further characterized in that the column comprises an upper section and a lower section, the lower section comprising 20 to 50% of the volume of the column and containing the inlet.

34. The system according to claim 33, further characterized in that the lower section is conical.

35.- The system according to claim 34, further characterized in that the angle of the lower section is from about 15 to about 45 degrees.

36.- A method for producing a recombinant virus for gene therapy, the method comprising: (a) culturing the cells on a first batch of microcarriers until the cells are substantially confluent; (b) transfer the cells of the microcarriers by separating them from the same by the addition of trypsin without removing the microcarriers from the suspension, and add a second batch of microcarriers; (c) infecting the cells with a recombinant virus; (d) separating the cells from step (c) of the microcarriers on which they have been cultured but from which they have been separated, comprising introducing an aqueous suspension of cells and microcarriers through an inlet in a separation device, the device comprising: (i) an entry; (I) a column; (iii) an outlet for collecting the 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 said device by a mesh screen, and wherein the cells and the aqueous solution are collected through the outlet; and (e) releasing the virus from the cells harvested from step (d), which comprises mechanically shearing the cells by exposing them at a shear rate, where the shear rate to mechanically subject the cells Shear stress is generated by collecting the cells in a microfilter, and rapidly recirculating the aqueous medium therethrough.

37. The method according to claim 36, further characterized in that the virus comprises an adenoviral vector.

38. The method according to claim 37, further characterized in that the virus is ACN53. 39.- The method according to claim 36, further characterized in that the cells are 293 cells.