MXPA00006339A - Preparation of cells for production of biologicals - Google Patents

Abstract

The present invention relates to a method for the preparation of cells for use in the production of biologicals, by culturing cells up till a desired cell volume of a preproduction batch, where after in a repeated discontinous process:a) part of the cells of the preproduction batch is used for the preparation of at least one production batch, and b) the remaining part of the cells of the preproduction batch is used as a seed for the preparation of at least one subsequent preproduction batch.

Description

PREPARATION OF CELLS FOR THE PREPARATION OF BIOLOGICAL PRODUCTS DESCRIPTIVE MEMORY The present invention relates to a method for the preparation of cells for use in the preparation of biological products. For the production of biological products on, for example, cell lines, it will be necessary to prepare large quantities of cells using an extrapolation procedure in biological reactors. U.S. Patent No. 5,017,490 describes said extrapolation process which provides in particular the advantage of a low risk of transfer contamination. This method, however, is not suitable for binding dependent cells (therefore, not for cells that only grow if they are fixed to a substrate) or cells embedded in a substrate (eg, in porous vehicles). U.S. Patent No. 4,644,912 discloses a method for the preparation of fixation-dependent cells for the production of biological products (ie, viruses) starting with a cell sowing material and with subsequent transfers attempting to increase the consecutive volumes of reactors biological of 1 liter, 5 liters, 25 liters, 150 liters, and finally in a biological reactor of 1000 liters or in a multiplicity of biological reactors of 150 liters. In the middle of these transfer steps the cells were released from their vehicles with a diluted protease solution. In the final transfer, the virus was inoculated. Taking average cell cycle time of around 20-24 hours the transfer intervals will be approximately every 3-5 days. Therefore, in order to be able to expand the cells to sufficiently large cultures from a MWCS (manufacturer's bank of cells) the total extrapolation process will take several weeks, depending on the final volume of the biological reactor. In the above methods for cell preparation, each of the production batches should be prepared from MWCS. For the production of vast quantities of biological products it will be necessary to use several cultivation lines parallel to the larger container volumes. Said preparation process, therefore, takes a long time and requires the operation of a considerable number of biological reactors for the preparation of the cells as well as for the production of biological products. It is an object of the present invention to provide a faster performance in the preparation of cells for the production of biological products. Likewise, the present invention relates to a method for the preparation of cells to be used in the elaboration of biological products, cultivating cells until achieving a desired volume of cells of a preproduction lot, where after in a discontinuous repetition procedure: a) part of the cells of the preproduction lot is used for the preparation of at least one production lot, and b) the remaining part of cells of the preproduction lot is used as a seed for the preparation of at least one batch of Subsequent preproduction. In particular, the present invention relates to a method for the preparation of cells for use in the manufacture of biological products, by culturing cells to reach a desired volume of cells of a preproduction lot, where after in a discontinuous process of repetition: a) part of the cells of the preproduction lot is transferred to be used for the preparation of at least one production lot, and b) the remaining part of preproduction lot cells is transferred to be used as the seed for the preparation of at least one batch of subsequent preproduction. In a preferred embodiment of the present invention the first preproduction batch is prepared from a cell sowing material by at least one transfer. In another preferred embodiment of the present invention the cells that are prepared are binding dependent. In the latter case it will generally be necessary for the cells to be cultured on a substrate. Therefore it would be advisable that during the repetition procedure when a part of the lot is used for the preparation of a new batch, add an additional amount of substrate. In a preferred embodiment, each time before the addition of substrate at least part of the cells is released first from its original substrate. As used herein, the term "production lot" means a cell culture that is used for the production of biological products. As used herein, the term "preproduction lot" means a cell culture that is used in the process according to the present invention for the preparation of at least one production lot (as defined above) and a batch of subsequent preproduction. As used herein, the term, "biological product" means any substance or organism that can be produced from a cell culture. Examples of "biological products" are viruses and proteins such as enzymes. As used herein, the term "seed material" means an amount of certain type of defined species cells stored to be used as a seed from which all cultures of the same cell type are derived. As used herein, the term "attachment-dependent cells" means cells that by their own growth and / or propagation need to be fixed to a substrate as defined herein. As used herein, the term "substrate" means any particulate material useful for the attachment of cells. As used herein, the term "transfer step" means a sequence of activities in the propagation and production of cells that comprises at least the transfer of an appropriate amount of cells and an appropriate amount of culture medium into a container. of production, incubation of the container at conditions suitable for the growth and propagation of the cells for a sufficient time for effective growth and propagation of the cells. Optionally a transfer step can comprise separation of the cells from the culture medium and / or from the substrate after a sufficient time for effective growth and propagation of the cells. It will be apparent to those skilled in the art that the method according to the present invention differs essentially from methods known in the art, in that cells are produced in a continuous process rather than a batch process such as the present one. According to the patent applications EP0417531 and WO89 / 08701 the continuous culture systems can be used for the production of viruses as well. First, the cells are cultured in a first biological reactor, and after a certain cell density has been reached, the cells are fed continuously from said first biological reactor to a second biological reactor. In this second biological reactor, the viruses grow on the cells and subsequently these viruses are continuously removed from the second biological reactor. The basic method of work according to the present invention is to use a biological biological reactor from which the biological reactors (s) are fed with cells. When the cells are fixative-dependent, after each transfer step, the cells preferably need to be detached from their substrate. For this purpose, a trypsinization procedure has been developed in large biological reactors. The production cells are defined to a number of specific transfers and characterized for the so-called ECB (extended cell bank). The described method allows the production of high performance because the route of extrapolation of WCS to the production cells can be shortened and a smaller quantity of biological reactors is needed because the parallel production lines are no longer needed. Various embodiments of the present invention are illustrated in Figure 1. In a preferred embodiment the cells are expanded from one ampoule of a MWCS to the level of the first preproduction batch through one or more transfer steps. The size of the biological reactor used for said preproduction batch can vary in work volume from several liters to several hundred liters. After, a part, for example 10-20% of the expanded cells (for example, X transfer) are used to repopulate a biological reactor for the production of a subsequent preproduction batch (being the transfer number X + 1), while the group of cells is transferred (transfer X or X + 1) to a larger biological reactor in order to start production directly or to first populate it and subsequently start production. In classical serial production lines the cell duplication number derived from MWCS at the time of cultivation is known within certain limits. A maximum allowable generation number is established in the production system at the beginning. In the method according to the present invention the maximum number of cell transfers can be defined by ECB. The production transfer number (the number of cell transfers used before the production of the biological material), therefore, is irrelevant within the limits established by ECB. As a consequence, said maximum number of transfers must be obeyed in accordance with the regulatory restrictions. As a result, the particular batch of biological productions is the final product of a direct extrapolation route. In order to verify if the specifications of the cells in the ECB stage in the production are similar to MCB (master cell bank) it is necessary to perform specific validation for this purpose with respect to the characteristics of growth, freedom, foreign and endogenous agents in the different stages, cariological analysis of so-enzyme, and so on. Once said ECB is fully characterized it is possible to allow the production of products with cells in any number of transfers between DCM and ECB, since it can be assumed that the cells have not changed in their specifications. As a result, tests on MWCS can be limited to sterility tests. This is a particular advantage of the method according to the present invention. With the maximum transfer number determined it is possible to use cells at any stage. In order to minimize the time needed to expand the cells from MWCS to the biological production reactor, it would be an advantage to allow the massive start of cells. This can be done, for example, in one of the following ways: • The cells can be set to a specific transfer number for longer intervals at room temperature (17-32 ° C) and can be revitalized to measure expansion growth by increasing the temperature and changing the culture medium, or • Cells can freeze (Temp <-80 ° C) in bulk and then thawed before transferring them to a biological reactor of predetermined volume, thus significantly reducing the need for extrapolation route. The method according to the present invention can be carried out with cultures of animal cells and in particular with fixation-dependent cells. Suitable types of cells are, for example, hamster cells (CHO, BHK-1), monkey cells (Vero), bovine cells (MDBK), canine cells (MDCK), human cells (CaCo, A431) or cells of bird (CEF). A single unit of a plurality of units can be used as the biological reactor according to the present invention for example, stirred fermenters, fixed bed fermenters, fluidized bed fermenters, air extraction fermenters, or a hollow fiber reactor. The cells of the above times can and should even be cultured when they are fixed to a solid support, such as micro-vehicles or macro-vehicles in suspension, for example, in a fixed bed, a fluidized bed or in suspension, or in hollow fibers. The cells can also be embedded in a vehicle (eg, porous vehicle). In the course of the method according to the present invention, particularly when a solid support is used, the cells should be released from said solid support. This can be done by any method useful for detaching cells from a solid support. Advantageously, use can be made of a proteolytic enzyme solution. Optionally, this enzymatic release step can be preceded by one or more preconditioning steps, for example by treatment with PBS and / or EDTA, in order to increase the proteolytic efficiency, and / or to reduce the amount of proteolytic enzyme required.

EXAMPLE 1 Cell detachment and separation of the vehicles before transfer to the next biological reactor MDCK4 cell line binding dependent cells were cultured at 37 ° C on Cytodex-3 micro-vehicles (Pharmacia, Uppsala, Sweden) (5 g vehicles / l) in a stirred 4 liter biological reactor ("biological biological reactor"). "). The culture medium was EpiSerf (Life Technologies, Paisly, Scotland). The growth continued until a maximum of 5 x 106 cells / ml of culture was obtained. The cells were detached from the vehicles by trypsinization in a trypsin-EDTA solution (Life Technologies, Paisly, Scotland). After establishing the vehicles, 80% of the detached cells were transferred to another 3 biological reactors of similar size. The last "production" biological reactors have vehicles (cellular substrate) added by the front. The cells were allowed to repopulate the vehicles and were subsequently used for production in these biological production reactors. The rest of the cells in the "mother biological reactor" were allowed to repopulate the rest of the Cytodex-3 vehicles and were cultured at the desired cell density.

EXAMPLE 2 Cell detachment without separation of vehicles before transfer to the next biological reactor The cell culture was performed as described in Example 1, however after the trypsinization, 80% of the detached cells including the vehicles were transferred to the 3 biological production reactors. Additionally, suitable vehicles were added to all biological reactors.

EXAMPLE 3 Cell detachment without separation of vehicles after transfer to the next biological reactor The cell culture was performed as described in Example 1, however, 80% of cells still adhered were transferred to a biological reactor of similar size which was then used directly for production generation. The remaining cells on micro-vehicles in the mother fermenter were detached by trypsinization, after which new vehicles were added and the cells were allowed to repopulate the substrates.

EXAMPLE 4 Starting from frozen massive cells In this experiment part of the culture was used to recharge the mother fermenter and some daughter fermenters by lot and part of the culture was used to freeze cells in mass. Frozen bulk cells (14.4 X 108 cell total) were inoculated into a 3 liter mother fermenter starter culture containing 5 grams of Cytodex per liter and EpiSerf medium, and after which they were incubated at 37 ° C. The residual cryoconservatives were removed by a medium change on day 1. On day 2, trypsinization was performed, 50% of the cells were frozen en masse and the rest of the cells were inoculated into micro-vehicles in a subsequent fermentor. From table 1 it can be deduced that the cells continued to grow at a normal speed between day 2 and 3. On day 4 the content of the mother fermenter was detached from trypsin and recharged by batch in new micro-vehicles (10 g / l) in two other fermenters next to the mother fermentor. On day 5, the plating efficiency was approximately 85%.

TABLE 1 EXAMPLE 5 Transfer of the mother fermenter to peguefia scale to the large scale production fermenter Cells were extrapolated on a large scale in 65-liter and 550-liter fermenters (50-liter and 250-liter work volume, respectively) using a micro-vehicle of 5 g density of Cytodex per liter. As can be seen in Table 2, 90% of the total cells are transferred to the large scale fermentor from a 50 liter fermentor culture with 800,000 cells / ml, of which 69% proved viable. The same was found in the 50 liter mother fermentor; about 69% of the repropagation cells showed to be viable. The procedure was the following: On day O, the vehicles were allowed to stand in the 50 liter culture, after which the supernatant was removed (culture medium) and replaced by PBS. The contents of the fermenter were stirred for 5-15 minutes. The supernatant was removed after re-establishing the vehicles. This step can be repeated if necessary. After this step was repeated with PBS / EDTA (0.4 grams EDTA / liter PBS). Again the culture was stirred for 5-15 minutes, the vehicles were allowed to stand, the supernatant was removed, and the passage of PBS / EDTA was repeated until the cells had turned round and were ready to be detached from trypsin. Then trypsin (0.025% final concentration) was added to the PBS / EDTA and incubated for 5-15 minutes. Then the supernatant containing cells (after establishing now "natural" vehicles) was transferred (as in example 9) or the mixture of cells plus vehicles was transferred (80% of the total mixture). After transferring the cells to the 550-liter fermentor, the rest of the cells (ie, 10% of the viable cells) were allowed to repopulate the vehicles still present in the fermenter after filling the 50-liter fermenter with medium. culture. About 70% of the cells proved to be viable.

TABLE 2 EXAMPLE 6 Analogous to Example 5, however, 80% of the culture of the cells bound to vehicle was transferred from the biological biological reactor to the biological production reactor. The production was started after the addition of virus. 20% of the remaining cells and vehicles in the mother biological reactor were trypsinized and detached and after the addition of the new substrate in the mother biological reactor it was allowed to repopulate the mother biological reactor while the production continues in the reactor biological production physically separated.

EXAMPLE 7 Large scale cultivation initiated from frozen massive cells Massive frozen cells were thawed and inoculated in a 10 liter fermenter (work volume) (vehicle density) Cytodex 5 g / l; EpiSerf culture medium) at an inoculation density of 1 x 106 cells / ml. After binding, the culture medium was replaced to remove residual cryoprotectants. After day 1 the amount of viable cells bound to the vehicles was 0.45x106 cells / ml from which growth started.

At a density of 2.8 × 10 6 cells / ml the cells were detached from their vehicles by trypsinization and 80% was transferred to a fermentor with a working volume of 50 liters (vehicles 5 g / l). As can be seen from table 3, on day 1 the number of viable cells after freezing the cells was approximately 45%. Of the total amount of cells transferred, the viability after trypsin release was 71.4%.

TABLE 3

Claims (6)

NOVELTY OF THE INVENTION CLAIMS

1. - Method for the preparation of cells for use in the elaboration of biological products, cultivating cells up to a desired cell volume of a preproduction lot, where after in a discontinuous repetition procedure: a) part of the cells of the preproduction lot is used for the preparation of at least one production batch, and b) the remaining part of cells of the preproduction batch was used as a seed for the preparation of at least one batch of subsequent preproduction.

2. Method according to claim 1, further characterized in that in the repetitive batch process; a) part of the cells of the preproduction lot are transferred to be used for the preparation of at least one production lot, and b) the remaining part of cells of the preproduction lot is transferred to be used as a seed for the preparation of minus one subsequent preproduction batch.

3. Method according to claim 1 or 2, further characterized in that a first batch of preproduction is prepared from a planting material of cells by at least one transfer step.

4. - Method according to claim 1-3, further characterized in that the cells are dependent on fixation.

5. Method according to claim 2, further characterized in that the cells are dependent on binding, the cells are cultured on a substrate, and before each transfer step the cells are released from that substrate.

6. Method of compliance with any of claims 1-5, further characterized in that the biological product of interest is a virus.