Method for culturing adherent cells

The subject of the invention is a process for producing adherent cells, according to which adherent cells are introduced into a culture vessel which contains microcarriers in a culture medium, and several successive cell passages are performed in this same vessel, each time using all or part of the cell population of the preceding cell passage for carrying out the next cell passage. The invention also relates to the use of this process for the production of biological agents, in particular for preparing vaccines or drugs.

In the 1980s, the development of the technology of cell culture on microcarriers facilitated the large-scale production of adherent cells and consequently the production of biological agents. The production of adherent cells intended for the production of biological agents for pharmaceutical use must nevertheless observe a certain number of regulatory requirements, in particular; that of prohibiting the use of adherent cells beyond a certain number of "cell passages", since there is a risk of morphological and/or biological transformation of the cells. This is in particular the case for cells of the Vero line.

US 4,664,912 describes a process used on the industrial scale for producing a batch of adherent cells from a cell seed originating from a working cell bank. It is based on a succession of cell passages which each time take place in different bioreactors, the working volumes of which increase over the course of the successive cell passages. This makes it possible each time to increase the amount of microcarriers while at the same time maintaining an optimum concentration of microcarriers in the culture medium which is generally between 1 and 5 g/1. The cell biomass thus increases over the course of the successive cell passages until the desired industrial batch of cells is obtained. The transfer of the cells from one bioreactor to another bioreactor is carried out after having detached the adherent cells from the microcarriers by means of treatment with trypsin and then by blocking the action of the enzyme by introducing serum proteins or serum into the medium so as to preserve as much as possible the integrity of the cells. The cell suspension obtained is then transferred (in the presence or absence of the used microcarriers) into a larger bioreactor which contains a greater amount of new microcaxriers. However, this method of industrial production of adherent cells requires the use and the handling of a large amount of material, which has an effect on the production costs for the biological agents.

In order to reduce the production costs for adherent cells intended for the production of biological agents, EP 1060241 proposes a faster method of production which no longer requires the need to start again from a cell seed originating from a working bank each time it is desired to produce an industrial batch of cells. The method consists in transferring, after each cell passage, most of the cells (80 to 90% of the cell biomass) to one or more other bioreactors in order to continue the amplification of the cell biomass and to constitute an industrial production batch of cells, while the remaining 10 to 20% of the cells are maintained in order to keep a stock "of feeder cells" from which further batches of cells can be produced. This method nevertheless has the following drawbacks:

- the cell batches produced display a certain heterogeneity insofar as they do not all have the same number of cell passages;

- the maintaining of a stock of "feeder" cells in culture at the time of each transfer operation inevitably leads to an "aging" of the cells which is directly linked to the number of cell passages performed, and can therefore be used only for a limited period of time for the regulatory reasons already mentioned.

In order to dispense with the use of proteolytic enzymes, such as trypsin which is harmful to the integrity of the cells, Ohlson et al., in Cytotechnology (1994), vol. 14, pages 67-80, describes a technology for microcarrier to microcarrier transfer (bead to bead transfer) of adherent cells in the absence of any enzymatic treatment. The close contact between microcarriers covered with adherent cells and bare microcarriers promotes transfer of the cells onto the bare microcarriers from which the cells can be amplified. In order to increase cell growth, bare microcarriers are therefore added to a culture medium which contains microcarriers covered with adherent cells and intermittent stirring of the culture medium is carried out in order to promote contact between microcarriers. Nevertheless, the cell population that is produced is "desynchronized" since the cells are at various stages of the cell cycle, which can be a major drawback for producing biological agents.

There is still the need to optimize the methods of large-scale production of adherent cells and also the production of biological agents which derive there from, so as to reduce production costs.

To this effect, the subject of the present invention is:

A process for producing adherent cells, according to which:

a. adherent cells are introduced into a culture vessel which contains microcarriers in a culture medium:
b. the cells are amplified by performing successive cell passages in this same culture vessel, wherein each cell passage subsequent to the first cell passage is carried out:

i. using all or part of the cell population that was obtained during the preceding cell passage, after having subjected the cell population to an enzymatic treatment in order to detach the cells from the microcarriers; and ii. by introducing culture medium and an increasing amount of microcarriers; and

c. the cell population produced during the final cell passage is harvested after having optionally subjected said cell population to an enzymatic treatment in order to detach the cells from the microcarriers.

The subject of the present invention is also:

A process for producing a biological agent produced by adherent cells, according to which:

a. adherent cells are introduced into a culture vessel which contains
microcarriers in a culture medium;

b. the cells are amplified by performing several successive cell passages in
this same culture vessel, wherein each cell passage subsequent to the first
cell passage is carried out:

i. using all or part of the cell population that was obtained during the preceding cell passage, after having subjected said cell population to an enzymatic treatment in order to detach the cells from the microcarriers, and
ii. by introducing culture medium and an increasing amount of microcarriers;

c. the cell population produced during the final cell passage is treated in such a way that it produces the biological agent, said treatment being carried out in the same culture vessel as that which was used to amplify the cells; and

d. the biological agent is harvested.

According to one aspect of the process for producing the biological agent, the biological agent is an infectious agent and the treatment of the cell population is performed by infecting the cell population, produced during the final cell passage, with said infectious agent in an infection medium.

According to one particular aspect, the infectious agent is the rabies virus and the infection medium is a viral infection medium free of any product of animal origin.

Generally, the number of cell passages performed in the same culture vessel is 2,3 or 4.
The concentration of microcarriers in the culture medium during the first cell passage is generally < 1 g/1, and preferably < 0.5 g/1.

Very preferably, the enzymatic treatment employs a solution containing a proteolytic enzyme, such as trypsin.

According to another aspect of the process according to the invention, each cell passage subsequent to the first cell passage is carried out while increasing the volume of the culture medium.

Preferably, the first cell passage is performed in a culture medium volume which is between 1/5 and half the working volume of the culture vessel.

In another embodiment of the process according to the invention, the culture medium is free of serum of animal origin.

Preferably, the culture medium is free of product of animal origin.

According to another aspect, the protein concentration in the culture medium is <15mg/l.

According to yet another aspect, the culture medium also contains a cell protection agent.
Preferably, the cell protection agent is a polyvinylpyrrolidone or a poloxamer.

According to yet another embodiment of the process according to the invention, the culture vessel is a bioreactor which has a working volume of between 3 and 3000 liters, preferably between 20 and 1000 liters, and particularly preferably between 20 and 500 liters.

In yet another embodiment, the culture vessel is a single-use bioreactor.

In one particular aspect of the process according to the invention, the adherent cells are Vero cells.

In general, the cell population which is harvested contains at least 60 times the amount of cells that were initially introduced into the culture vessel.

In another aspect, the invention relates to a process for producing adherent cells, according to which:

a. a stock of adherent cells is thawed, then
b. the thawed adherent cells are subjected to one of the embodiments of the process of the invention.

In yet another aspect, the invention relates to a process for producing adherent cells, according to which, after having produced adherent cells in a first culture vessel according to a process of the invention:

a. the cell population harvested after having subjected said cell population to an enzymatic treatment in order to detach the cells from the microcarriers is transferred into a second culture vessel which has a larger working volume and which contains a culture medium containing a larger amount of microcarriers than the amount of microcarriers that was present during
the final cell passage performed in the first culture vessel, and
b. one of the embodiments of the process of the invention is implemented in this second vessel.

In one particular aspect, one of the embodiments of the process according to the invention is repeated in a third culture vessel which has a larger working volume than the working volume of the second culture vessel.

The invention also relates to the use of the cells which have been produced according to the process of the invention, for the production of biological agents.

Finally, it relates to a process for producing adherent cells in a culture vessel which contains microcarriers in a culture medium, according to which the amount of cells produced is increased by a factor > 60 by carrying out successive cell passages in one and the same culture vessel.

Detailed description of the invention

The invention relates to a process for producing adherent cells, according to which, in order to amplify cells and to form industrial batches of cells, several successive cell passages are performed in one and the same cell culture vessel. By virtue of this process, the nxmiber of culture vessels to be used is reduced and the batches of cells produced are homogeneous since they all have the same number of cell passages. This process also serves for the production of biological agents.

For the purpose of the present invention, a "cell passage" begins at the time a suspension of adherent cells is brought into contact with microcarriers in a culture medium and usually ends at the time the adherent cells are released from their microcarriers by enzymatic treatment and are again in the form of a suspension in the culture medium. A cell passage usually comprises the following phases:

- a microcarrier colonization phase, which corresponds to the period of time during which the cells which have been brought into contact with the microcarriers in a cvilture medium adhere to the microcarriers;

- a phase of amplification of the cells adherent to the microcarriers, which corresponds to the period of time during which the cells multiply on the microcarriers until the available surface of the colonized microcarriers is more than 70%, and preferably more than 80%, covered by the cells. When the cells have covered more than 70% of the available surface of the colonized microcarriers, it is considered that the adherent cells are "substantially confluent" or have reached the "stage of confluence"; and

- a phase of detachment of the substantially confluent cells from the microcarriers by means of an enzymatic treatment such that a maximum of cells are detached from their support (in general more than 80% and preferably more than 90%) in a short space of time (in general in less than 30 minutes and often in a period of time of less than 20 minutes). The cell population is then essentially in the form of a suspension of cells released from their microcarriers (or detached from their microcarriers).

In the case of the present invention, depending on the use of the adherent cells which are produced, the final cell passage performed in the culture vessel comprises or does not comprise a detachment phase.

In the context of the present invention, the successive cell passages are carried out in one and the same culture vessel using all or part of the cell population obtained during the preceding cell passage for carrying out the next cell passage. At least 80% of the cell population obtained during the preceding cell passage is normally used to carry out the next cell passage. Preferably, in order to produce a maximum amount of cells, the successive cell passages are performed by each time using the entire cell population obtained during the preceding cell passage to carry out the next cell passage. Although, at the end of each cell passage, the cells are released (detached) from their microcarriers by means of an enzymatic treatment, there is no transfer, that is recommended in the prior art, of the cell biomass into one or more other cell culture vessels to continue the cell amplification. The amplification of the cell biomass is carried out herein in one and the same culture vessel. This process is very advantageous since equivalent amounts of cells are produced in the same periods of time, without having to use and to handle several culture vessels, which reduces the space required for producing industrial amounts of cells and consequently substantially reduces production costs. Surprisingly, although the amplification of the cell biomass requires an enzymatic treatment at each cell passage, the amount of cells that is produced at the end of the implementation of the process according to the invention is significantly greater than that which is obtained using the conventional "microcarrier to microcarrier transfer" technology (of example 2).

Each new cell passage which corresponds to a cell passage subsequent to the first cell passage is performed in the same culture vessel. In order to start up a passage subsequent to the first cell passage, culture mediimi and a larger amoimt of microcarriers than the amount of microcarriers that was introduced during the preceding cell passage are introduced so as to increase the available cell support surface. It is understood that the term "new cell passage" or "passage subsequent to the first cell passage" denotes a cell passage following a cell passage that was carried out in the culture vessel. It is also understood that the introduction or the addition of microcarriers into the culture vessel corresponds to the introduction of bare microcarriers. Preferably, unused microcarriers are used in order to facilitate the adhesion of the adherent cells. Even though usually the volume of culture medium is concomitantly increased at each passage subsequent to the first cell passage, the increase in the amount of microcarriers is generally proportionally greater than the increase in volume of medium, which generally results in a gradual increase in the microcarrier concentration in the culture medixmi during the successive cell passages. During the final cell passage, when, for example, dextran microbeads sold under the name cytodex (Cytodex 1, 2 or 3) are used as microcarriers, the microcarrier concentration in the culture medium is generally between 1 and 7 g/1, but can reach 10 to 15 g/1. The process of the invention according to which the cell biomass is increased by successive cell passages in one and the same culture vessel is called an "all-in-one process" (see figure lb).

Usually 2 cell passages, 3 cell passages or 4 cell passages are performed in the:Same culture vessel. Depending on the subsequent use that is made thereof, the cell population that is harvested at the end of the implementation of the process according to the invention is either in the form of a suspension of cells released from their microcarrier (in this case, the final cell passage is performed with the cell detachment step being included) or in the form of a suspension of cells adherent to the microcarriers (in this case, the final cell passage is performed with the cell detachment step being omitted). When the process for producing adherent cells comprises two successive cell passages carried out in one and the same culture vessel, the process according to the invention amounts to implementing the following steps:

a. culture medium, microcarriers and adherent cells are introduced into a
culture vessel,
b. the cells are subjected to culture conditions which allow them to adhere
and to proliferate on the microcarriers,
c. the cells are detached from the microcarriers by means of an enzymatic
treatment and a part of the cells is optionally removed from the culture
vessel,
d. culture medium and microcarriers are again introduced, such that the
amount of microcarriers that is introduced is larger than the amount of
microcarriers that was previously introduced,
e. the cells are again subjected to culture conditions which allow them to
adhere to and proliferate on the microcarriers, and
f. the cell population obtained is harvested after having optionally detached
the cells from their microcarriers by means of an enzymatic treatment,
steps a) to e) being carried out in one and the same culture vessel.

Steps a) to c) correspond to the first cell passage and steps d) to f) correspond to the second cell passage which ends with the harvesting of the cells. When there are more than two successive cell passages carried out in the same vessel, this amounts to repeating again steps c), d) and e) after step e) in the same culture vessel, before implementing step f) of harvesting the cells. Usually, steps c), d) and e) are repeated once after step e), which corresponds to carrying out three successive cell passages, or steps c), d) and e) are repeated twice after step e), which corresponds to carrying out 4 successive cell passages. Preferably, step c (which corresponds to the detachment of the cells from their microcarriers) is implemented when the cells are substantially confluent. Usually, the volume of culture medium is also increased each time microcarriers are added (step d).

When the population of cells that is harvested at the end of the implementation of the process according to the invention is iised to form a stock of cells, the final cell passage generally comprises a step of detaching the cells by means of an enzymatic treatment that is generally performed in the same culture vessel. The cell population is then essentially in the form of a suspension of cells released from their microcarriers.

When the population of cells is used for the production of a biological agent, the final cell passage is often performed without including a cell detachment step. The population of cells produced, in the form of a suspension of cells adherent to the microcarriers, is then treated directly in the same culture vessel so that it produces the biological agent of interest. The term "biological agent" is intended to mean any substance or organism which can be produced by the adherent cells. These are in particular viruses or proteins (antibodies, antigens, enzymes, etc.).
When the process for producing a biological agent produced by adherent cells comprises two successive cell passages carried out in the same culture vessel, the process according to the invention therefore amounts to implementing the following steps:

a. culture medium, microcarriers and adherent cells are introduced into a culture vessel,
b. the cells are subjected to culture conditions which allow them to adhere to and proliferate on the microcarriers.
c. the cells are detached jfrom the microcarriers by means of an enzymatic treatment and a part of the cells is optionally removed from the culture vessel,
d. culture medium and microcarriers are again introduced, such that the amount of microcarriers newly introduced is larger than the amount of microcarriers that was previously introduced,
e. the cells are again subjected to culture conditions which allow them to
adhere to and proliferate on the microcarriers,
f. the cell population obtained is treated in such a way that it produces the biological agent, and
g. the biological agent is harvested, steps a) to f) being carried out in one and the same culture vessel.

When there are more than two successive cell passages carried out in the same vessel, this amoimts to repeating again steps c), d) and e) after step e) in the same culture vessel, before implementing step f) of producing the biological agent. Usually, steps c), d) and e) are repeated once after step e), which corresponds to carrying out three successive cell passages, or steps c), d) and e) are repeated twice after step e), which corresponds to carrying out 4 successive cell passages. Preferably, step c (which corresponds to the detachment of the cells from their microcarriers) is implemented when the cells are substantially confluent. Usually, the culture volimie is also increased each time microcarriers are added (step d).

When it is a question of producing a recombinant protein, such as, for example, a cytokine, an antibody or a vaccine protein, the cell suspension is placed under culture conditions which promote the production of this protein using suitable production media. By way of example, mention is made of the media described in EP 0354129 for the production of recombinant proteins by CHO and Vero cells. When the biological agent is an infectious agent, the suspension of cells adherent to the microcarriers is infected by introducing the infectious agent (bacteria, viruses, parasites, etc.) into the culture vessel after having generally replaced the culture medium with an infection medium. The infectious biological agent can in particular be a recombinant virus (recombinant poxviruses, recombinant adenoviruses) or viruses such as, for example, the rabies virus, influenza virus, poliovirus, etc. The biological agent is usually harvested by removing the culture supernatant in one or more stages - see example 7. When the biological agent is instead intracellular, as in the case of non-lytic viruses, it is often advantageous to harvest the supernatant and the cells, which are subsequently treated with lytic agents.

The media used for the production of biological agents, in particular the infection media which are used for the production of viruses such as the rabies virus, can be advantageously free of serum of animal origin, of protein of animal origin, or even of any product of animal origin.
The microcarriers suitable for the subject of the invention are usually in the form of microbeads, which are preferably nonporous so as to facilitate the action of the enzymes. They have a diameter generally between 90 and 250 m. Their density is slightly higher than that of the culture medium so as to facilitate their recovery by simple settling out, but at the same time it should not be too high to obtain complete resuspension of the microbeads once the medium is subjected to moderate stirring. Under standard culture conditions, the density of the microcarriers is usually between 1.020 and 1.050 g/ml. The smface of the microbeads is chosen so as to facilitate the adherence of the cells. The matrix of the microcarriers is preferably non-rigid so as to provide better preservation of the cells when collisions occtir between microbeads. The mean available surface for adhesion of the cells is usually between 4000 and 5000cm/g of microbeads. These characteristics are foimd in particular for microbeads with a crosslinked dextran matrix, sold imder the name cytodex (cytodex 1, cytodex 2, cytodex 3), but can also be found for other microbeads, the matrix of which is based on crosslinked polystyrene (Biosilon, Solohill) or for glass microbeads (Sigma Aldrich).

In the context of the present invention, the microcarrier concentration during the first cell passage, in particular when cytodex' microbeads such as cytodex" 1 microbeads are used as microcarriers, is generally reduced to a concentration <1 g/1, whereas, in the prior art, the microcarriers are used at a concentration of between 1 and 5 g/1. It is usually <0.5 g/1; more specifically, it is between 0.1 and 0.4 g/1 and more particularly it is between 0.1 and 0.3 g/1. This concentration corresponds in fact to the initial concentration of microcarriers in the culture medium after the introduction of the cells into the culture vessel. It is therefore < 1 g/1, preferably <0.5 g/1; it is in particular between 0.1 and 0.4 g/1 and more specifically it is between 0.1 and 0.3 g/1.

The initial amount of cells which is introduced into the culture vessel is chosen such that more than 80% of the microcarriers are colonized by the cells. In order to obtain this degree of colonization, an initial amount of cells which is at least 5 to 10 times greater than the amount of microcarriers that is present in the culture medium is conventionally introduced into the culture vessel. For example, in the case of a Vero cell production, the initial amount of cells that is introduced into the culture vessel is generally between 5 x lO'' and 5 x 10'* cells/cm of cytodex microbeads, which represents approximately between 5 cells and 50 cells per microbead. In fact, since the microcarrier concentration in the culture medium during the first cell passage is lower than that which is conventionally used in the prior art, it consequently follows that the initial cell concentration is also lower.

At the end of each cell passage, the cells are detached fi"om the microcarriers in a short period of time (in general less than 30 minutes and preferably less than 15 minutes) by treating the cells with an enzyme solution which has a proteolytic activity (protease). In the context of the invention, the cells are usually detached from the microcarriers in the culture vessel that is used to perform the successive cell passages, which means that all the cell culture phases and all the treatments which are performed on the cells during the successive passages are carried out in one and the same culture vessel. It is optionally possible to detach the cells from the microcarriers after having transferred them into a secondary vessel where the enzymatic treatment takes place, and then to reintroduce the cell suspension obtained into the single culture vessel where the successive cell passages take place. This method is not advantageous because it leads to a loss of cells during the transfer operations.

The proteolytic enzyme solution usually contains a serine protease such as trypsin, pronase® or dispase®. Papain, ficin or coUagenase can also be used when the microcarriers are cytodex 3 microbeads. A trypsin solution is commonly used to detach the adherent cells from cytodex' microbeads. Preferably, the protease is of nonanimal origin, which indicates that it has been produced using a process which does not use material of animal origin. It is produced, for example, using a plant material, by chemical synthesis, or by genetic recombination using bacteria, yeast, fungi or plants. An en2yme solution free of any product of animal origin, sold by Invitrogen under the trade name TrypLE™ Select or TrypLE™ Express, can for example be used. This protease, the protein sequence of which is described in WO 94/25583, is produced by fermentation of the Fusarium oxysporum DSM 2672 strain or produced by genetic recombination. It has an enzymatic activity similar to trypsin. In order to facilitate the detachment of the cells, a chelating agent which binds calcium ions, such as, for example, EDTA, EGTA or citrate, can be added to the enzyme solution or, optionally, the adherent cells can be treated with a chelating agent before performing the enzymatic treatment. The concentration of protease and, optionally, of chelating agent in the medium and also the temperature at which the enzymatic treatment of the cells is carried out (usually between 20 and 38°C) are set such that more than 80% of the cells are detached from their support in a short period of time (<30 minutes). Prior to the actual enzymatic treatment, at least half the volume of culture medium is generally removed. Approximately 2/3 of the volume of culture medium is iisually removed. The proteolytic activity is then neutralized by adding to the medium an inhibitor generally of peptide or protein origin which neutralizes the action of the proteases. Preferably, the composition of the inhibitor is free of any contaminant of animal origin. This inhibitor is, for example, recombinant aprotinin, or extracts or purified fractions containing a trypsin inhibitor originating from soybeans or from lima bean (Worthington Biochemical). The mediimi is generally kept stirring throughout the phase of detachment of the cells from the microcarriers, except during the period when culture medium is removed.

The cell suspension obtained is generally quantified using conventional counting systems which can also determine the cell viability. Although a part of the cell population can be removed from the culture vessel when there has been too much cell growth, the entire cell population is often used to initiate a cell passage subsequent to the first cell passage which takes place in the same vessel. In order to increase the cell biomass during the successive passages, it is necessary to introduce, into the culture vessel at the beginning of each passage subsequent to the first cell passage (which begins after the cell detachment step using enzymatic treatment), a larger amount of microcarriers than the amount of microcarriers that was previously introduced. If the same volume of culture medium is maintained during the successive cell passages, this amounts to increasing the microcarrier concentration at each cell passage subsequent to the first cell passage. On the other hand, the same microcarrier concentration can be maintained during the successive cell passages if the culture medium volume is increased in the same proportion at each new cell passage. Very preferably, at the start-up of each cell passage subsequent to the first cell passage, both the culture medium volume and the microcarrier concentration are increased in the culture vessel in order to obtain maximirai amplification of the cells. By way of indication, at each new cell passage, the cells are cultured in a culture medium volume which is between 1.2 and 3 times larger than the volume in which the cells are contained during the preceding passage. Likewise, at each new cell passage, the microcarrier concentration in the culture medivim is between 2 and 10 times higher than that which existed during the preceding cell passage. In the context of the present invention, it is not generally usefial to remove the used microcarriers at the end of each cell passage (i.e. at the end of the cell detachment step). Even though said microcarriers can be recolonized by the cells, the amount of used microcarriers originating from the preceding cell passages is not generally taken into account in calculating the amount of microcarriers to be introduced at the beginning of each new cell passage. The phase of adhesion of the cells to the microcarriers generally lasts between 1 and 10 hours depending on the cell type. After the adhesion phase, it may be advantageous to remove all or part of the culture medium after having allowed the microcarriers to settle out, and to replace it with new medium in order to accelerate the proliferation of the cells adherent to the microcarriers.

The culture media suitable for the subject of the invention can be conventional cell culture media supplemented with serum of animal origin. Advantageously, the culture media contain neither serum nor serum protein. The culture media can in particular be fi-ee of any protein of animal origin or even of any product of animal origin. The term "protein or product of animal origin" is intended to mean a protein or a product of which the production process comprises at least one step in which a material originating from animals or from humans is used. Particularly advantageously, the media used for culturing the cells can be free of any protein or can contain very small amounts of proteins in the form of recombinant proteins or proteins extracted from plants (soy, rice, etc.) or from yeasts. They most commonly contain low-molecular-weight proteins (< 10 KD) (also called polypeptides) at very low concentrations. The total protein concentration in these culture media is generally < 15 mg/1 measured by the Bradford method. This is the case in particular for the VP SFM medium sold by InVitrogen, which is suitable for the process according to the invention, in particular for culturing Vero cells. Mention is also made of the media Opti Pro' serum-free (InVitrogen), Episerf (InVitrogen), Ex-cell® MDCK (Sigma-Aldrich), Ex-Cell™ Vero (SAFC biosciences) MP-BHK® serum free (MP Biomedicals), SFC-10 BHK express serum free (Promo cell), SFC-20 BHK express protein free (Promo cell), HyQ PF Vero (Hyclone ref SH30352.02), Hyclone SFM4 Megavir, MDSS2 medium (Axcell biotechnology), Iscove's modified DMEM medium (Hyclone), Ham's nutritive media (Ham -FIO, Ham-F12), Leibovitz L-15 medium (Hyclone), Pro Vero medium (Lonza) and Power MDCK medium (Lonza) which are free of any product of animal origin and which contain few or no proteins.

When the culture medium is free of animal serum, of serum protein or has a total protem concentration < 15 mg/1 (Bradford), a cell protection agent, which protects the cells against the shear forces that are exerted when the medium is stirred, is generally added. The cell protection agents most commonly used generally have surfactant properties. They are in particular vinyl alcohol polymers, also known as polyvinyl alcohols (PVAs), ethylene glycol polymers, also known as polyethylene glycols (PEGs), l-vinyl-2-pyrroHdone polymers, also known as polyvinylpyrrolidone (PVP), or poloxamers which are "block copolymers" of ethylene oxide and of propylene oxide having the chemical formula HO(C2H40)a(C3H60)b(C2H40)aH according to which a denotes the number of ethylene oxide units and b denotes the number of propylene oxide units. These cell protection agents are generally used in a concentration range of from 0.001% to 2% (w/v) in the culture medium. Among the cell protection agents that are particularly preferred, mention may be made of poloxamer 188 and PVP. Poloxamer 188 has an average molecular weight of approximately 8400 daltons and is used in the culture medium at a concentration usually between 0.05 and 0.2% (w/v). PVP is also recommended since it stimulates cell growth, as is described in WO 01/40443. PVP is generally used in an average molecular weight range of between 20 KDa and 360 KDa, preferably in an average molecular weight range of between 20 KDa and 40 KDa, at a concentration in the culture medium which is generally between 0.01% and 2% (w/v) and preferably at a concentration between 0.05% and 0.5% (w/v). PVP can also be characterized no longer by means of its molecular weight only, but according to its K value which takes into account the average molecular weight of a PVP and also the variations in molecular weight on either side of the average value. For the calculation of the K value, reference is made to the equation as defined in the article Cryobiology, 8,453-464 (1971): the K value is calculated on the basis of the relative viscosity of a 1% solution of PVP according to the formula:

Log ri rel/C= 75KoV (1+1.5 KQC) + Ko
K= 1000 Ko

C represents the PVP concentration in grams per 100 ml of medium.
tj rel is the viscosity of the solution compared with that of the solvent. A PVP suitable for the subject of the invention has a K value which is generally between 18 and 60, preferably between 26 and 35. By way of indication, for producing a stock of Vero cells according to the process of the invention, a culture medium based on VPSFM sold by Invitrogen containing as cell protection agent a PVP having a K value of about 30 at a concentration of 0.1 % (w/v) or poloxamer 188 at a concentration of 0.1% (w/v), can be used as culture medium free of any product of animal origin and having a very low protein content (< 15 mg/1 by the Bradford method).

Usually, the composition of the culture medium is the same during the successive cell passages, but, as required, it may prove to be useful to provide nutritive supplements such as glucose and/or glutamine. During the successive cell passages, it may also be useful, during the cell proliferation phases, to renew all or part of the culture medium as required by the cells. This is evaluated by means of conventional testing methods at the disposal of those skilled in the art, such as the measurement of glucose, glutamine, lactate, ammonium ion levels. In the context of the present invention, the culture medium is generally continually stirred with a strength that is just sufficient to continually keep the microcarriers in suspension in the culture medium, except when all or part of the culture medium is removed.

The volume of the culture medium during the first cell passage usually represents between half and 1/5 of the working volume of the culture vessel. In the case of culture vessels with a large capacity (bioreactors of more than 100 liters), the particular configuration of which enables the culturing of cells adherent to microcarriers in a small volume (such as, for example, a bioreactor equipped with a conical-bottomed sedimentation zone), the volume of the culture medium may be smaller (between 1/6 and 1/10 of the working volume of the bioreactor) or even smaller, and represent only 1/20 of the working volume of the bioreactor. As previously mdicated, the culture mediirai volumes are generally increased over the course of the successive cell passages, the final cell passage often being carried out in the presence of a culture medium volume which corresponds to at least 70% of the working volume of the vessel.

The culture vessel is equipped with a stirring system (mechanical, by means of a current of air, etc.) for maintaining the microcarriers in suspension in the cell culture medium, and has means for renewing the media according to the needs of the culture and/or means for testing and regulating the temperature, the pH, the oxygen pressure, the gassing optionally with nitrogen or with air, and the metabolites or nutrients (lactates, glucose, glutamine, ammonium ions, etc.). These devices are well known to those skilled in the art who know how to use them according to the size and the configuration of the vessel used. By way of example, the culture vessel according to the invention may be in the form of spinners or of a bioreactor. When the working volume of the vessel is > 2 liters, use is normally made of a bioreactor which may be conventionally in the form of a reusable glass tank or metal tank or, when the bioreactors are single-use bioreactors, of vessels in the form of single-use bags sold in particular by P. Guerin under the name Nucleo PG-ATMI™. Use may also be made, by way of example, of the Biowave system (Wave Bioreactor sold by General Electrics, the STR single-use Bioreactor™ system (Sartorius), the SUB™ system (Hyclone), or the cell ready system (Millipore). In the context of the process according to the invention, the main objective of which is to produce cell batches on an industrial scale, a bioreactor of which the working volume is between 3 liters and 1000 liters is used, but more generally, a bioreactor of which the working volume is between 20 liters and 500 liters is used.

For the purpose of the invention, the "adherent cells" are cells estabUshed as lines or cells resulting directly from the extraction of animal or human, healthy or tumor tissues, which, under the culture conditions used, need a solid support in order to multiply and develop normally. They usually form a single-cell layer on their support owing to the contact inhibition phenomenon. Cells which, under the culture conditions used, do not need a solid support in order to multiply and which can grow in suspension in the culture medium are in fact therefore excluded. The adherent-cell lines can be derived from primary cultures of healthy or tumor cells, but also can be obtained by transformation of cells using immortalizing agents, as is the case for the PER.C6 line.
As an example of adherent-cell lines suitable for the subject of the invention, mention is made of murine cell lines such as the 3 T3, NTCT or WEHI line, hamster cell lines such as the BHK line (in particular the BHK21 line) or the CHO line, canine cell lines such as the MDCK line, porcine cell lines such as the PK15 line, bovine cell lines such as the MDBK line, simian cell lines such as the Vero, LLC-MK2, FRHL2 or MAI04 line, and human cell lines such as the MRC5, 293, PER.C6, Hela, ECV or A 431 line. These adherent-cell lines may also be in the form of lines transfected with a recombinant vector (plasmid, virus, etc.) when they are intended for the production of recombinant proteins.

By virtue of the process according to the invention, the population of adherent cells is increased by a factor of at least 40, preferably of at least 60 and particularly preferably of at least 100 by carrying out successive cell passages in one and the same culture vessel. This can be done because the process according to the invention makes it possible to increase by between 5 and 40 times, preferably between 10 and 30 times, the surface area of the cell support during the successive cell passages which are carried out in this single vessel. In the prior art methods, a cell amplification of this order is observed using at least two vessels, but more generally three culture vessels of different sizes (see example 5). The process according to the invention is very advantageous since the same industrial amounts of cells as those which are obtained with the prior art methods are also produced in the same time periods, while at the same time reducing the costs related to the use and maintenance of culture vessels and the space necessary for producing these cell batches.

The process for producing adherent cells according to the invention can advantageously be carried out by directly introducing into the culture vessel cells which have just been thawed, without recourse to a conventionally recommended adaptation period, during which one or more "adaptation" cell passages are performed in order to "adapt" the cells to more difficult culture conditions, such as culturing in the presence of cytodex microbeads at a concentration < 0.5 g/1 in the culture medium, and/or culturing in media which do not contain serum or which contain very little protein (< 15 mg/1). In the case in point, a stock of adherent cells is thawed according to procedures well known to those skilled in the art, and then the suspension of thawed cells is introduced directly into the culture vessel which contains microcarriers in a culture medium so as to carry out the process as described in the invention. As previously indicated, in particular when cytodex™ microbeads are used as microcarriers, the microcarrier concentration in the culture medium dviring the first cell passage is generally reduced to a concentration <0.5 g/1; it is generally between 0.1 and 0.4 g/1 and more specifically it is between 0.1 and 0.3 g/1. The culture medium also does not need to contain serum or serum proteins. The ctilture medium can even be completely fi-ee of proteins or have a very low total protein content (<15 mg/1). The stock of fi'ozen cells can originate from a vial (in this case, the amount of cells is generally relatively low, 10 to 5x10 cells) or advantageously originate from a bag which contains up to 100 times more cells. The large-scale cell production process is in particular accelerated since the content of thawed bags can be seeded directly into a culture vessel of large capacity.

When the stock of adherent cells which has been produced using the process of the invention is not sufficient, the cell biomass which has been obtained from one and the same cell culture vessel can be further increased by transferring the cell population:

o either into one or more culture vessels which can be used to carry out successive cell passages conventionally, i.e. by transferring, after each cell passage, the cell biomass obtained into another larger culture vessel; or more advantageously, o into a second culture vessel which has a much larger working volume (generally at least 10 times larger, most commonly between 10 and 50 times larger than the first vessel) and again applying the process according to the invention to the cells which have been transferred into this second vessel. By carrying out the process in this way, the number of culture vessels to be used in order to produce industrial batches of cells, and also the space required, are even more significantly reduced.

In order to assess the economic advantage of the implementation of the process according to the invention in the context of an industrial-scale production of adherent cells, reference may be made to the conventional scheme for the industrial production of Vero cells intended for the production of poliovirus, as described in Reviews of Infectious Diseases, vol 6, supplement 2, S341-S344 (1984). The conventional scheme comprises five successive cell passages, the fu-st being carried out in a 1-liter bioreactor, the second in a 5-liter bioreactor, the third in a 20-liter bioreactor, the fourth in a 150-liter bioreactor and, finally, the fifth in a 1000-liter bioreactor. By virtue of the process according to the invention, it is possible to perform the first three cell passages in a single 20-liter bioreactor, and then to perform the last two passages conventionally by transferring the cells into a 150-liter bioreactor and then into a 1000-liter bioreactor. It is also possible to repeat the process according to the invention twice performing the first three cell passages in a single 20-liter bioreactor and then transferring the cells obtained directly into a single 500-liter or 1000-liter bioreactor in which the last two cell passages are performed. In both cases, an amovmt of cells which is of the same order as that which is obtained when the conventional scheme is applied is produced in the same time periods, but in the fu-st case, a saving of 2 bioreactors (1 liter and 5 liters) is made and, in the second case, a saving of 3 bioreactors (1 liter, 5 liters and 150 liters) is made (cf. example 5).

A process for producing adherent cells that is particularly advantageous from the economic standpoint consists in repeating the process according to the invention in two culture vessels of very different size. To this effect, the subject of the invention is therefore: A process for producing adherent cells, according to which:

a. adherent cells are introduced into a first culture vessel which contains microcarriers in a culture medium;

b. the cells are amplified by performing several successive cell passages in this first culture vessel, wherein each cell passage subsequent to the first cell passage is carried out, each time using all or part of the cell population that was obtained during the preceding cell passage and the cells of which were detached from the microcarriers by means of an enzymatic treatment, and each time adding culture medium and an increasing amount of microcarriers;

c. the cell population obtained during the final cell passage carried out in this first culture vessel is harvested after having detached the cells from the microcarriers by means of an enzymatic treatment;

d. the cell population harvested is transferred into a second culture vessel which has a larger working volvime and which contains a culture medium containing a larger amoimt of microcarriers than the amount of microcarriers that was present during the final cell passage performed in the first culture vessel;

e. the cells are amplified by performing several successive cell passages in this second culture vessel wherein each cell passage subsequent to the first cell passage is carried out, each time using all or part of the cell population that was obtained during the preceding cell passage and the cells of which were detached from the microcarriers by means of an enzymatic treatment, and each time adding culture medium and an increasing amount of microcarriers;

f. the cell population obtained during the final cell passage carried out m this second culture vessel is harvested after having optionally detached the cells from the microcarriers by means of an enzymatic treatment, and, optionally.

g. steps d to f are repeated again in a third culture vessel having an even larger working volume. In general, the working volume of the second culture vessel is 20 to 50 times larger than that of the first culture vessel.

Advantageously, the adherent cells which are introduced in step a) of the process originate from a stock of frozen cells, which were thawed just before introduction into the first culture vessel.

The repetition of the process according to the invention can also be implemented for producing a biological agent. To this effect, the subject of the invention is therefore:

A process for producing a biological agent produced by cells adherent to microcarriers, according to which:

a. adherent cells are introduced into a first culture vessel which contains microcarriers in a culture medium;

b. the cells are amplified by performing several successive cell passages in this first culture vessel, wherein each cell passage subsequent to the first cell passage is carried out, each time using all or part of the cell population than was produced during the preceding cell passage and the cells of which were detached from the microcarriers by means of an enzymatic treatment, and each time adding culture medium and an increasing amount of microcarriers;

c. the cell population obtained during the final cell passage carried out in this first culture vessel is harvested after having detached the cells from the microcarriers by means of an enzymatic treatment;

d. the cell population harvested is transferred into a second culture vessel which has a larger working volume and which contains a culture medium containing a larger amount of microcarriers than the amount of microcarriers that was present during the final cell passage performed in the first culture vessel;

e. the cells are amplified by performing several successive cell passages in this second culture vessel wherein each new cell passage is carried out, each time using all or part of the cell population that was produced during the preceding cell passage and the cells of which were detached from the microcarriers by means of an en2ymatic treatment, and each time adding cixlture medium and an increasing amount of microcarriers; f the cell population produced during the final cell passage carried out in the second culture vessel is treated such that it produces the biological agent, said step f) being carried out in the same culture vessel as that which was used to carry out steps d) and e); and g. the biological agent is harvested.

Optionally, steps d) and e) can be repeated in a third culture vessel before treating the
cell population such that it produces the biological agent.

As previously indicated, the biological agent which is produced can be, for example, a recombinant protein or a virus such as the rabies virus.

The subject of the invention is also the use of the cells which have been produced by means of one of the processes according to the invention, for the production of biological agents.
Finally, the invention relates to a process for producing adherent cells in a culture medium which contains microcarriers, according to which successive cell passages are carried out in one and the same culture vessel in order to increase the cell population by a factor > 40, preferably by a factor > 60 and particularly preferably by a factor > 120. The culture vessel used is preferably a bioreactor which has a working volume of at least 20 liters.

The culture medium, as has been indicated in the context of the invention, can be a conventional medium supplemented with serum, but preferably the medivim is free of serum of animal origin. Particularly preferably, a medium free of any product of animal origin and the protein concentration of which is < 15 mgA is used.

Figure 1 represents two processes for amplifying cells adherent to microcarriers by means of successive cell passages: a) according to the conventional process, the successive cell passages are carried out in different culture vessels having increasing working volumes, and b) according to the process according to the invention (all-in-one process), the successive cell passages are carried out in one and the same culture vessel. The cells are in frozen form at stage 0. Step 01 corresponds to the transfer of the thawed cells into a bioreactor. Step 1 corresponds to the first cell passage. Step 1-2 corresponds to the transfer of the cell population obtained at the end of the first cell passage after treatment with a proteolytic enzyme in order to detach the cells from the microcaniers, either, in the case of process a), into a second bioreactor which has a larger working volume, or, in the case of process b), into the same culture vessel. Step 2 corresponds to the second cell passage. In the case of process b), the second cell passage is generally carried out in a larger culture medium volume and in the presence of a higher microcarrier concentration. Step 2-3 corresponds to the transfer of the cell population obtained at the end of the second cell passage after treatment with a proteolytic enzyme in order to detach the cells from the microcaniers, either, in the case of process a), into a third bioreactor which has a larger working volume, or, in the case of process b), into the same culture vessel. Step 3 corresponds to the third cell passage. In the case of process b), the third cell passage is generally carried out in a larger culture medium volume and in the presence of a higher microcarrier concentration than during the second cell passage.

Figure 2 represents the appearance of the microbeads under a microscope (magnification X20) after a period of 8 days of Vero-cell culture implementing a) the all-in-one process or b) the bead-to-bead transfer process (cf example 2 for the operating conditions).

The present invention will be understood more clearly in the light of the following examples which serve to illustrate the invention without, however, limiting the content thereof

Example 1: Amplification of Vero cells bv carrying out 3 successive cell passages in one and the same 2-liter bioreactor

In this example, the role of various parameters, such as the initial microcarrier concentration, the presence or the absence of serum in the culture medium and the nature of the cell protection agent, on the cell growth was studied.

1.1) Material used

Bioreactor:

The experiments were carried out in single-use bioreactors with a capacity of 2.4 liters, sold by Millipore under the name Cellready (Mobius). They are equipped with a pH probe, a pOa probe and a temperature probe and a marine propeller stirring paddle.

Microcarriers:

Cytodex 1 micrpbeads supplied by GE Healthcare were used. The microbeads were hydrated for 24 hours in a phosphate buffer (IxC PBS, pH~7.4) after having removed the amount necessary to carry out each cell passage. They were then rinsed 3 times in the same buffer and then sterilized by autoclaving. Just before introduction into the bioreactor, the sterilization buffer was replaced with an equivalent volume of culture medium after settling out the microbeads. 1 g of microbeads represents an adhesion surface of approximately 4400 cm.

Culture media tested

VPSFM/K30: VPSFM medium (Invitrogen), serum free and free of product of animal origin, supplemented with 0.1% w/v of polyvinylpyrrolidone (PVP) K30 supplied by ISP.

VPSFM/F68: VPSFM medium (Invitrogen), serum free and free of product of animal
origin, supplemented with 0.1% w/v of poloxamer 188 supplied by BASF.

VPSFM/K30/SVF: VPSFM/K30 medium supplemented with 4% decomplemented fetal calf serum.

Cells:

Vero cells originating from a bank of cells stored in frozen form at 50 x 10 cells/ml in serum-free medium containing 10% dimethyl sulfoxide in tubes for freezing cells (Nunc tube ref: 430663, 5ml).

1.2) Operating protocol used to assess the parameters regarding initial microcarrier
concentration and cell protection agent in the "all-in-one process".

The same operating protocol was used to study these two parameters.

Concentrations of 0.1 g/1 and 0.3 g/1 were tested to assess the effect of a very low microcarrier concentration during the first cell passage on Vero cell growth.

66 X 10 cells in 2 liters of VPSFM/K30 medium containing 0.6 g of microcarriers were introduced into bioreactor 1 (bio 1) (which is equivalent to an amount of 25 000 cells/cm of adhesion surface and represents an initial microbead concentration of 0.3 g/1) after having adjusted the bioreactor regulation parameters such as the pH at 7.2-7.5, the temperature at 37°C and the p02 at =<25%.

22 X 10 cells in 2 liters of VPSFM/K30 medium containing 0.2 g of microcarriers were introduced into bioreactor 2 (bio 2) which is equivalent to an amoimt of 25 000 cells/cm of adhesion surface and represents an initial microbead concentration of 0.1 g/1).
Throughout the duration of the cell culture, the culture mediimi was subjected to continual stirring, except during the microbead settling-out phases, which are used to renew the culture medium or to reduce the culture medium volume. At day D3, the cells were treated with trypsin according to the following protocol: After settling out the microbeads, ~ 300 ml of VPSFM/K30 culture medium were left in the bioreactor and then == 300 ml of a 0.025M sodium citrate solution containing 600 mg of recombinant trypsin (ref.: Roche 04618734) in phosphate buffer without calcium and magnesium were added. The medium was maintained under moderate stirring. After having verified that the cells had indeed been detached, by means of a test sample (in general, this detachment took place in a period of between 15 and 30 minutes), the action of the trypsin was stopped by adding ~ 300 ml of a solution of VPSFM/K30 containing 1 mg/ml of trypsin inhibitor (ref: Sigma T6522). After cell counting, a part of the cell suspension was removed to adjust the remaining amount of cells such that there were approximately 25 x 10 cells/cm of adhesion surface after having introduced either 2.4 g of microcarriers (bio 1), or 0.6 g of microcarriers (bio 2) in a total volume of culture medium of 2 liters to carry out the second cell passage. The regulation parameters were then again adjusted as during the first cell passage. After 4 to 6 hours, the medium was replaced with new culture medium, and then was optionally renewed a second time after 24 to 48 hours of culture. At D7, the cells were again trypsinized according to the same protocol as that used at D3. After cell counting, a part of the cell suspension was also removed to adjust the remaining amount of cells such that there were == 25 x 10 cells/cm of adhesion surface after introduction of either 6 g of microcarriers (bio 1) or 2.4 g of microcarriers (bio 2) in a total volume of culture medium of 2 liters to carry out the third cell passage. The bioreactor regulation parameters were then again adjusted. After 4 to 6 hours, the mediimi was replaced with new culture medium, which was then optionally renewed a second time after 24 to 48 hours of culture. At DIO, the cells were harvested in order to assess the degree of cell amplification according to the initial microcarrier concentration in the culture vessel.
In order to assess the role of cell protection agents, poloxamer 188 and polyvinylpyrrolidone (PVP) K30 were tested using the same operating protocol and with an initial microcarrier concentration of 0.3 g/1.

1.3) Operating protocol used to assess the role of serum in the "all-in-one" process.

The same operating protocol as that described in section 1.2 was used with the following features:
- VPSFM/K30/SVF medium was tested,
- the initial microcarrier concentration was 0.3 g/1,
- the trypsin treatments were carried out on days D5 and D8. Before each trypsin treatment, the suspension of microbeads was "rinsed" 3 times with 600 ml of a rinse buffer (phosphate buffer (IxC PBS)) by means of a succession of steps comprising rinsing, settling out and removal of the rinse buffer, in order to remove the serum.

1.4) Results

To assess the role of the various parameters studied on cell growth, the cell concentration was measured at regular intervals using the Nucleocounter (Chemometec®) counting system and the number of cumulative cell generations was calculated. Each time, two samples of the microbead suspension were taken. The values indicated represent the mean values of the two samples taken for each time point analyzed.

The amounts of cells and the number of cumulative cell generations observed during the cultunng are given in the 3 tables below according to each of the parameters studied.

1.4.1) "Initial microcarrier concentration" parameter
*: before trypsin treatment
**: after trypsin treatment and cell concentration adjustment to 25 x 10 cells/cm of
adhesion surface.
***: For the calculation of the number of cumulative generations, the possible cell
concentration adjustments that were made during the successive cell passages were
taken into account and the general formula below was used:
logio {fmal [cell]/initial [cell]}
Number of generations =
Logio 2 final [cell]: corresponds to the cell concentration on the day under consideration
initial [cell]: corresponds to the initial cell concentration at DO

Even though the amount of cells produced after 3 successive cell passages is smaller in the bioreactor wherein the initial microcarrier concentration is 0.1 g/1, the cell population doubling time is, on the other hand, slightly faster in this bioreactor because the number of cumulative cell generations observed is higher throughout the duration of the test.

Surprisingly, a very low microcarrier concentration during the first cell passage (~ 0.1 g/1) has no negative impact on the cell growth. Conversely, the cells have a tendency to divide even more actively than when they are in a bioreactor which contains a higher microcarrier concentration (0.3 g/1).

1.4.2) "cell protection agent" parameter

*: before trypsin treatment
**: after trypsin treatment and cell concentration adjustment to 25 x 10 cells/cm of adhesion surface

The results show that the addition of poloxamer 188 or of PVP(K30) to a serum-flee culture medium has an overall equivalent effect on cell amplification carried out according to the all-in-one process.

1.4.3) "serum" parameter

*: before trypsin treatment * *: after trypsin treatment

These results show that the "all-in-one" process is also applicable to cells adherent to microcarriers cultured in a culture medium containing serum.

Example 2: Comparison of the Vero cell productions obtained either by using the process according to the invention ("all-in-one process") or by using the process by simple addition of microcarriers ("bead-to-bead transfer" technique)

Cell production was compared after 2 successive cell passages carried out according to the "all-in-one process" or using the "bead-to-bead transfer" technique.

The studies were carried out in glass bioreactors with a capacity of 4 liters, sold by Sartorius under the name Quattro. They are equipped with pH, p02 and temperature probes and a stirring paddle.

2.1) operating protocol used for implementing the "all-in-one process"

For the "all-in-one process", the same principle as the one described in section 1.2 was applied with the following features;

- The culture medium used was VPSFM/K30 medium.

- The volume of the culture medium remained constant during the 2 successive cell passages and was 4 liters.

- The first cell passage was carried out using a microbead concentration of 0.3 g/1 and by introducing an amount of cells such that there were 50 000 cells/cm of adhesion surface.

- The culture medium was renewed at D3.

- At D4, the cells were treated with trypsin according to the same procedures as those described in section 1.2. After trypsinization and counting of the cells, 2/3 of the cell suspension volume was removed so as to adjust the remaining amount of cells 25 000 cells/cm of adhesion surface after introduction of 4.8 g of microbeads (i.e. a concentration of 1.2 g/1) in a total volume of culture medium of 4 liters to carry out the second cell passage.

- The culture medium was renewed twice: a first time 4 to 6 hours after introduction of the microbeads and then a second time at D6.

- At D8, the cells were harvested and counted in order to assess the degree of cell amplification. An aliquot of the microbead suspension was also analyzed under a microscope in order to assess the degree of covering of the microbeads by the cells.

2.2) operating protocol used to implement the "bead-to-bead transfer" process

The same operating protocol as the one described in section 2.1 was appHed, except that the cells were not treated with trypsin at D4. At day D4, 2/3 of the microbead suspension volume was removed in such a way as to remove the same proportion of cells and of microbeads so as to be under the same culture conditions as those used in the "all-in-one" process. A microbead suspension containing 4.8 g of cytodex 1 (i.e. a concentration of 1.2 g/1) in a total volimie of cell culture medium of 4 liters was then introduced. The operating protocol is then the same as the one described in section 2.1.

2.3) Results

In order to compare cell growth during the two processes tested, the cell concentration was measured at regvilar intervals according to the same procedures as those described in section 1.4.
The amounts of cells and the number of cumulative cell generations observed during the culture are given in the table below.

*:bei ore cell concen Ltration adjustment
**: after cell concentration adjustment and optionally trypsin treatment (in the case of the "all-in-one" process)

During the first cell passage (DO to D3), the cell growth is similar in the two bioreactors. On the other hand, during the second cell passage (D3 to D8), the cell amplification is much weaker in the bioreactor wherein the "bead-to-bead transfer" technology was carried out. At D8, the amount of cells harvested is approximately half that which was harvested in the bioreactor wherein the "all-in-one" process was carried out. The results regarding the change in the number of cumulative cell generations are along the same lines. At D8, the number of cumulative cell generations is > 6 for the cells that were amplified according to the all-in-one process. It is 5 for the cells that were amplified according to the "bead-to-bead transfer" technology. The cells that were amplified according to the "all-in-one" process therefore multiply more actively. These results are surprising insofar as the "all-in-one" process requires the use of trypsin, known to be detrimental to cell integrity, m order to carry out the successive cell passages on the cells. These results were confirmed in 2 independent experiments carried out under the same operating conditions. The analysis of variance on the number of cumulative cell generations at D8 shows a significant difference (p=0.0137).

The microscopy analysis of the microbeads at D8 shows that the vast majority of the microbeads are covered with cells when the all-in-one process is carried out (see figure 2a). On the other hand, only a portion of the microbeads are covered with cells when the "bead-to-bead transfer" technology is carried out. Continuous stirring of the medium or intermittent stirring of the medium (by repeating, for example, for 2 hours cycles comprising stirring for 5 minutes followed by a resting period of 20 minutes) after the addition of the cytodex 1 microbeads at D4 does not substantially modify the results (see figure 2b).

Example 3: Amplification of Vero cells by carrying out 2 successive cell passages in one and the same 20-liter bioreactor

3.1) Material used

Bioreactor:

A 20-liter bioreactor in the form of a single-use bag sold by ATM! under the name Nucleo-20 was used. The pH, p02 and temperature probes, after having been calibrated and then sterilized by autoclaving, by virtue of the probe holder bags, were installed on the bag according to the standard ATMI protocol: the Kleenpack® connections located, on the one hand, on the bag and, on the other hand, on the probe holder, were cormected and then the probe was introduced into the bioreactor through the connection thus created.

Microcarriers: cytodex 1 microbeads supplied by GE Healthcare (cf section 1.1).
Culture medium: VPSFM/K30 medium (cf. section 1.1).

Cells: Vero cells (cf. section 1.1).

3.2) Operating protocol

Six liters of VPSFM/K30 culture medium were introduced into the Nucleo-20 and then 1 liter of a microbead suspension containing 4 g of cytodex 1 (which represents an initial microbead concentration of 0.5 g/1 after the addition of the cells) was added. After having adjusted the regulation parameters inside the Nucleo-20, such as the temperature at 37°C, the pH at 7.2-7.4 and the p02 at =25%, and subjected the medium to moderate stirring so as to resuspend the beads in the culture medium, 500 X 10 cells, after having been thawed and taken up in 1 liter of VPSFM/K30 culture medium, were introduced into the Nucleo. Throughout the duration of the operating protocol, the culture medium was subjected to continuous stirring, except during the microbead settling-out phases which take place in order to renew the culture medium or to reduce the culture medium volume.

At day D2 (2 days after placing in culture), the medium was replaced with new VPSFM/K30 medium.

At day D5, the cells were trypsinized according to the following protocol: The stirring, the pH regulation and the pOa regulation were stopped. Only the temperature regulation was maintained. After settling out of the microbeads, ~ 3 liters of VPSFM/K30 culture medium were left in the bioreactor and then ~ 3 liters of a 0.025M sodium citrate solution containing 600 mg of recombinant trypsin (ref: Roche 04618734) in phosphate buffer without calcium and magnesium were added. The medium was then again stirred moderately. After having verified that the cells had indeed detached, by taking a test sample (in general, this detachment takes place in a period of between 15 and 30 minutes), the action of the trypsin was stopped by adding == 3 liters of a solution of VPSFM/K30 containing 1 mg/ml of trypsin inhibitor (ref: Sigma T6522). The microbead suspension containing 28 g of cytodex 1 was then added, which represents a microbead concentration of approximately 1.4 g/1 in the medium after having adjusted the culture medium volume to 20 liters with the VPSFM/K30 culture medium. The regulation parameters inside the Nucleo-20 were then again adjusted as during the first cell passage. After 4 to 6 hours following the introduction of the microbeads, the medium was replaced with new culture medium. A second replacement of the culture medium with new culture medium was carried out at D7. At D9, the cells were substantially confluent. They were then trypsinized according to the same protocol as that used at D5. The level of cell amplification obtained after 2 cell passages were formed in the same bioreactor was determined from the cell suspension obtained.

3.3) Results

In order to measure the level of cell amplification obtained, the cell concentration was measured at regular intervals according to the same procedures as those described in section 1.4.

The cell amounts and concentrations observed during the culture are represented in table I below:

*: before trypsin treatment
**: after trypsin treatment and adhesion of the cells
***: represents the initial amount of viable cells seeded

After two successive cell passages carried out in the same 20-liter bioreactor, the cell population increased by a factor of 68 after 9 days of culture, whereas the surface of the cell support was increased by a factor of 7.

Example 4: Amplification of Vero cells by carrying out 3 successive passages in one and the same 20-liter bioreactor

4.1) Material used
The material which was used is identical to the material described in the procedure of example 3.

4.2) Operating protocol
The cell passages were carried out according to the same protocol as that described in section 3.2, with the following variations:

- the first passage was carried out by introducing 250 x 10 cells into 8 liters of VPSFM/K30 culture medium containing 2 g of cytodex 1 microbeads (which represents a microbead concentration of 0.25 g/1);

- at D5, after the first trypsin treatment, to carry out the second cell passage, a microbead suspension containing 14 g of cytodex 1 was added, which represents a microbead concentration of approximately 1.07 g/1 after having adjusted the culture medium volume to 13 liters with the VPSFM/K30 culture medium;

- at D9, after the second trypsin treatment, to carry out the third cell passage, a microbead suspension containing 60 g of cytodex 1 was added, which represents a microbead concentration of approximately 3 g/1 after having adjusted the culture medium volume to 20 liters with the VPSFM/K30 culture medium.

At D12, the cells were substantially confluent. They were then trypsinized according to the same protocol as that used at D5.

The level of cell amplification obtained after 3 cell passages performed in the same bioreactor was measured using the final cell suspension obtained.

4.3) Results

The cell amounts and concentrations observed during the culture are represented in the table below:

*: before trypsin treatment
**: after trypsin treatment and adhesion of the cells

After three successive cell passages of Vero cells carried out in the same 20-liter bioreactor, the cell population increased by a factor of 126 after 12 days of culture, whereas the surface of the cell support increased by a factor of 30.

Example 5: Comparison of the production of Vero cells using either the process according to the invention (the successive cell passages are carried out in one and the same bioreactor) or the conventional cell expansion process (the successive cell passages are each time carried out in different bioreactors of greater size V

5.1) Operating protocol

The production of Vero cells was studied and compared by performing either 3 successive cell passages in a single 20-liter bioreactor according to the protocol described in example 4 and using an initial amount of 250 x 10 cells originating directly from a bank of frozen cells, or 3 successive cell passages in different stainless steel bioreactors, the first in a 2-liter bioreactor, the second in a 7-liter bioreactor and the third in a 28-liter bioreactor. The experimental conditions of the conventional process which were used are the following:

The Vero cells after thawing were first adapted to their culture conditions by performing an initial passage in Cell Factories (CFIO) by introducing 40 x 10 cells per cm of adhesion surface into 2 liters of VPSFM/K30 culture medium. After approximately 5 days of culture, the cell population obtained was harvested after a trypsinization step. The cell population harvested is tised to seed a bioreactor with a working volume of 2 liters containing a suspension of 2 g of cytodex 1 microbeads in 2 liters of VPSFM/K30 culture medium (concentration 1 g/1). After having tested and adjusted the regulation parameters, set at a temperature of 37°C, the pH at 7.2-7.4 and the p02 at -25%, and subjected the medium to moderate stirring, the 2-liter bioreactor was seeded with, on average, 220 x 10 cells. At D3, the culture medium was replaced with new culture medium. At D4, the substantially confluent cells were trypsinized and then transferred with the used microcarriers into a 7-liter bioreactor containing 7 liters of VPSFM/K30 culture medium to which 14 g of cytodex 1 microbeads were added beforehand, which represents a microbead concentration of approximately 2 g/1. At D6, the culture medium was replaced with new culture medium. At D8, the substantially confluent cells were trypsinized and then transferred in the same way into a 28-liter bioreactor containing 28 liters of VPSFM/K30 culture medium to which 70 g of cytodex 1 microbeads were added beforehand, which represents a microbead concentration of approximately 2.5 g/1. At DIO, the culture medium was replaced with new culture medium. At Dll, the substantially confluent cells were trypsinized and then harvested and counted.

5.2) Results

In order to monitor the cell growth during the two processes tested, the cell concentration was measured at regular intervals according to the same procedures as those described in section 1.4.
The amounts of cells and the levels of amplification observed during the culture are given in the table below:

Day "all-in-one" process 1 single 20-liter bioreactor Conventional process 3 bioreactors (4, 7 and 28 liters)

*: the amounts expressed are the average values obtained on 8 different experiments
which were carried out.

**: the amounts expressed are the average values obtained on 3 different experiments which were carried out.

Using the "all-in-one" process, more than 29 billion cells are obtained on average after 12 days of culture after having introduced on average 253 million directly thawed cells into a 20-liter bioreactor, i.e. an average level of cell amplification of 115. Using the conventional process, 32 billion cells are obtained on average after 11 days of culture after having initially introduced on average 220 million into a 2-liter bioreactor, i.e. an average level of cell amplification of 145. The available cell support surface was increased by a factor of 30 in the two processes, but required the use of 3 bioreactors in the case of the conventional process. The amount of cells obtained with the conventional process is slightly higher. This comes from the fact that the cells that were used to implement the "all-in-one" process and the conventional process were not in the same physiological conditions. The cells that were used to seed the 20-liter bioreactor in the case of the "all-in-one" process had just been thawed, whereas the cells used to seed the 2-liter bioreactor were much more vigorous since they had been cultured beforehand in a Cell Factory. The results at Dl show this very clearly; in the all-in-one process, the decrease of about 40% in the number of cells at Dl is a result of the conventional "post-thaw lag" phenomenon. During the same period of time, the cells which had been cultured beforehand in a Cell Factory multiplied in the conventional process (the cell population doubled). Despite the initial culture conditions that are clearly unfavorable in the "all-in-one" process, it is noted that, at the end of the culture, there is in the end very little difference between the amounts of cells harvested in the two processes. It is concluded that, if the initial culture conditions had been the same in the two processes tested, the same amounts of cells and the same levels of cell amplification would have been obtained. The "all-in-one" process is therefore very advantageous compared with the conventional process since the same amount of cells is produced in the same period of time, with economy of means.
Example 6: Amplification of Vero cells bv carrying out 3 successive cell passages in one and the same 200-liter bioreactor

6.1) Material used

With the exception of the bioreactor, which, in the present case, is a 200-liter single-use bag sold by ATMI under the name Nucleo-200, the material which was used is the same as that which is described in example 3.

6.2) Operating protocol

The operating protocol used is similar to that which is described in example 3, with the following modifications:

The Vero cells, after having been thawed, were placed in culture beforehand in 2 Cell Factories (CFIO) in a proportion of 40 x 10 cells per cm of adhesion surface and 2 liters of culture medium per CFIO. After 5 days of culture, the cell population was harvested after a trypsin treatment step and was subsequently used to seed the Nucleo-200.

The first cell passage in the Nucleo-200 was carried out by introducing 2.2 x 10 cells into 50 liters of VPSFM/K30 culture medium containing 25 g of cytodex 1 microbeads (which represents a microbead concentration of 0.5 g/1). At day D3, the culture medium was replaced with new culture medium. At day D4, the cells were trypsinized according to the protocol of example 3, leaving ~ 20 liters of medium in the Nucleo, and then adding ~ 20 liters of a 0.025M sodium citrate solution containing 3000 mg of recombinant trypsin in a phosphate buffer containing neither calcium nor magnesium. After detachment of the cells, the action of the trypsin was stopped by adding 20 liters of a solution of VPSFM/K30 containing 1 mg/ml of trypsin inhibitor.
The second cell passage was carried out in the same Nucleo-200 by adding to the entire cell population obtained a suspension of microbeads containing 130 g of cytodex 1, which represents a microbead concentration of approximately 1 g/1 after having adjusted the total volume of the medium to 130 liters with the VPSFM/K30 culture medium. The culture medium was renewed twice: the first time just after adhesion of the cells to the microbeads, the second time at D6. At day D7, the cells were again trypsinized according to the same protocol that was used at D4. The third cell passage in the same Nucleo-200 was carried out by adjusting the cell population such that there was a concentration of 20 000 cells/cm of adhesion surface after introducing 450 g of cytodex 1 microbeads in a total volume of medium adjusted to 180 liters with the VPSFM/K30 culture medium. Similarly, the culture medium was renewed twice: just after adhesion of the cells to the microbeads, and then at DIO. The cell population obtained after the 3 successive cell passages performed in the same bioreactor was then quantified. The level of cell amplification can thus be measured.

6.3) Results

The cell amounts and concentrations observed during the culture are represented in the table below:

Day of culture Culture volume Cell concentration in the medium

*: before trypsin treatment
**: after trypsin treatment and adhesion of the cells
***: after trypsin treatment and adjustment of the cell population to a concentration of 20 000 cells /cm of adhesion surface

After three successive cell passages of Vero cells carried out in the same 200-liter bioreactor, the cell population increased by a factor of 140 after 11 days of culture, whereas the cell support surface was increased by a factor of 18. Tests for amplification of Vero cells according to the "all-in-one process" were also carried out by performing in particular two successive cell passages in a single 500-liter single-use bioreactor sold by ATMI under the name Nucleo-500. The levels of cell amplification obtained were similar to those that are obtained with the 200-liter bioreactors, which clearly shows that the process of the invention is suitable on a very large scale.
Example 7: Production of the rabies virus from a batch of cells that was obtained by carrying out 3 successive cell passages in one and the same 200-liter bioreactor.

The batch of cells was produced using the same protocol as that which was described in example 6.
At day D11, the culture medium was replaced with a VPSFM-based viral infection medium and then the cells were infected with the Pitman Moore strain of the rabies virus originating from the Wistar institute, at a multiplicity of infection of 0.01. The same regulation parameters regarding temperature, pH, p02 and moderate stirring of the medium, used for the cell culture were retained and adjusted for virus production. The viral infection medium was renewed at day D3, after viral infection, and then the culture supematants were harvested in order to measure the infectious titers at days D7, DIO and D14 after viral infection. After each viral harvest, new viral infection medium was again added. The infectious titers in the culture supematants were measured by means of a conventional immunofluorescence test on BHK21 cells. A series of dilutions was prepared for each of the culture supematants tested and then each dilution was distributed into 10 wells of a 96-well microplate. Two series of tests were carried out in parallel. A suspension of BHK21 cells was then added to each of the wells. The cells were mcubated for 48 hours at 37°C imder 5% CO2. After 48 hours, the wells were covered with a cell layer which was then fixed with acetone. After having removed the acetone and dried the microplates, 50 l of a l/70th dilution of a monoclonal antibody directed against the rabies virus (FDI

Future bio Diagnostics-Ref 800092) were added. After incubation for one horn-followed by several rinses, the microplates were analyzed under a fluorescence microscope. A well is considered to be positive once specific fluorescence is observed in at least one cell. BHK21 cells cultured in the absence of rabies virus were used as a negative control, and BHK21 cells cultured in the presence of a reference rabies virus strain were used as a positive control. The infectious titers of rabies viruses contained in the culture supematants tested were determined according to the Spearman-Karber method and expressed in logio cell culture infectious dose 50% (CCID 50) units. The infectious titers obtained in the culture supematants that were observed were about 7.0 logio CCID 50.

CLAIMS

1. A process for producing adherent cells, according to which:

a. adherent cells are introduced into a culture vessel which contains microcarriers in a culture medium;

b. the cells are amplified by performing several successive cell passages in this same culture vessel, wherein each cell passage subsequent to the first cell passage is carried out:

i. using all or part of the cell population that was obtained during the preceding cell passage, after having subjected the cell population to an enzymatic treatment in order to detach the cells from the microcarriers, and

ii. by introducing culture medium and an increasing amount of microcarriers; and

c. the cell population produced during the final cell passage is harvested after having optionally subjected the cell population to an enzymatic treatment in order to detach the cells from the microcarriers.

2. A process for producing a biological agent produced by adherent cells, according to which:

a. adherent cells are introduced into a culture vessel which contains
microcarriers in a culture medium;

b. the cells are amplified by performing several successive cell passages in this same culture vessel, wherein each cell passage subsequent to the first cell passage is carried out:

i. using all or part of the cell population that was obtained during the preceding cell passage, after having subjected the cell population to an enzymatic treatment in order to detach the cells from the microcarriers, and

ii. by introducing culture medium and an increasing amount of microcarriers;

c. the cell population produced during the final cell passage is treated such that it produces the biological agent, said treatment being carried out in the same culture vessel as that which was used to amplify the cells; and

d. the biological agent is harvested.

3. The process as claimed in claim 2, according to which the biological agent is an infectious agent and the treatment of the cell population in step c) is performed by infecting the cell population with said infectious agent in an infection medium.

4. The process as claimed in claim 3, in which the infectious agent is the rabies virus and the infection medium is a viral infection medium free of any product of animal origin.

5. The process as claimed in one of claims 1 to 4, according to which the number of cell passages which are performed in the same culture vessel is 2, 3 or 4.

6. The process as claimed in one of claims 1 to 5, according to which the concentration of microcarriers in the culture medium during the first cell passage is < 1 g/1, and preferably < 0.5 g/1.

7. The process as claimed in one of claims 1 to 6, according to which the enzymatic treatment employs a solution containing a proteolytic enzyme.

8. The process as claimed in one of claims 1 to 7, according to which each subsequent cell passage is carried out while increasing the volume of the culture medium.

9. The process as claimed in one of claims 1 to 8, according to which the first cell passage is performed in a culture medium volume which is between 1/5 and half the working volume of the culture vessel.

10. The process as claimed in one of claims 1 to 9, according to which the culture medium is free of serum of animal origin.

11. The process as claimed in one of claims 1 to 10, according to which the culture medium is free of any product of animal origin.

12. The process as claimed in one of claims 1 to 11, according to which the protein concentration in the culture medium is <15 mg/1.

13. The process as claimed in one of claims 1 to 12, according to which the culture medium also contains a cell protection agent.

14. The process as claimed in claim 13, according to which the cell protection agent is a polyvinylpyrrolidone or a poloxamer.

15. The process as claimed in one of claims 1 to 14, according to which the culture vessel is a bioreactor which has a working volume of between 3 and 3000 liters, preferably of between 20 and 1000 liters, and particularly preferably between 20 and 500 liters.

16. The process as claimed in claim 15, according to which the culture vessel is a single-use bioreactor.

17. The process as claimed in one of claims 1 to 16, according to which the adherent cells are Vero cells.

18. The process as claimed in one of claims 1 and 5 to 17, according to which the cell population which is harvested in step c) contains at least 60 times the amount of cells that were initially introduced in step a) of the process.

19. A process for producing adherent cells, according to which: a. a stock of adherent cells is thawed, then

b. the thawed adherent cells are subjected to the process as claimed in one of claims 1 and 5 to 18.

20. A process for producing adherent cells, according to which, after having produced adherent cells in a first culture vessel as claimed in one of claims 1 and 5 to 19:

a. the cell population harvested after having subjected said cell population to an enzymatic treatment in order to detach the cells from the microcarriers is transferred into a second culture vessel which has a larger working volume and which contains a culture medium containing a larger amount of micropores than the amount of micropores that was present during the final cell passage performed in the first culture vessel, and

b. steps b) and c) of the process as claimed in one of claims 1 and 5 to 19 are
implemented in this second vessel.

21. The process as claimed in claim 20, according to which steps a) and b) of claim 20 are repeated again in a third culture vessel which has a larger working volume than the working volume of the second culture vessel.

22. The use of the cells which have been produced according to the process of one of claims 1 and 5 to 21, for the production of biological agents.

23. A process for producing adherent cells in a culture vessel which contains microcarriers in a culture medixrai, according to which the amount of cells produced is increased by a factor > 60 by carrying out successive cell passages in a single culture vessel.