BE1022269B1 - System, apparatus and method for the production of cells and / or cellular products - Google Patents

Abstract

The present invention provides a system and method for the production of cells and / or cell products. The system comprises at least one cell culture unit comprising at least one bioreactor for cell culture, at least one technical control unit for controlling cell growth parameters, said technical control unit is at least fluid-connected to the cell culture unit and at least one air treatment unit for treating the ambient air, said air treatment unit is fluidly connected to the cell culture unit. The system is characterized in that the system is self-contained and the total volume of the bioreactor is at most 1000 L.

Description

System, apparatus and method for the production of cells and / or cellular products

Technical Field The invention relates to methods and systems for the production of cells and / or cell products, such as viruses, proteins or peptides.

Background

With the increased use of cells and cell products, such as viruses, proteins and peptides, in clinical diagnosis and therapy, the need has arisen for more efficient, rapid, sterile production and purification processes.

Conventional approaches and tools for making cells or cell-based products typically involve many manual manipulations that are subject to variations even if conducted by skilled technicians. Small amounts of products secreted by cells are produced in different ways. T-flanges, roller bottles, agitated bottles or cell bags are manual processes using incubators or hot chambers to provide environments for cell growth and production. These processes are labor intensive, error prone and difficult to produce on a large scale.

The production of cells and / or products secreted by cells can be carried out using conventional stirred tank bioreactors or "special" bioreactors (fibers, microfibers, hollow fiber, ceramic composite material, fluidized bed, fixed bed, etc.). ). Currently available systems are versatile in nature and require considerable time for trained operators to install, load, empty, inoculate, operate, collect and unload.

Prior art techniques and systems utilize a large scale plant in which cells are cultured in batch bioreactors of, for example, 10,000 liters (L). Such large-scale systems are not suitable for placement in a normal laboratory.

In addition, prior art large scale systems require specific handling and specific installation such as specific lines for the supply of gas and / or medium to the bioreactor. Indeed, after a period of culture, the cells and / or the cell products of the batch are collected within about 8 hours. As a result, the 10,000 L suspension is clarified, the medium is exchanged (the cell culture medium is replaced by a buffer medium) by diafiltration, and the compounds are separated or purified by chromatography. An additional filtration step may follow.

A disadvantage of the prior art includes the use of a large filter, a large volume of buffer medium, and a considerable volume of purified water. These volumes represent a considerable cost in terms of the production of purified water and water storage. Another major drawback is the loss of yield in the clarification step which is an essential step of these large-scale systems to obtain a diafiltration which is efficient enough to exchange the cell culture medium within the limit of 8 hours. .

Another disadvantage of the printhead systems is the large investments required in terms of the necessary facilities and space, but also in terms of the material needed to produce the desired cells and / or cell products. In addition, the necessary energy input weighs heavily on the required budget. The huge investments required restrict development in the field, not only in the US and Europe, but also in developing countries.

The object of the current invention is to provide methods and systems for producing cells and / or cell products that overcome at least some of the aforementioned disadvantages and disadvantages. An object of the invention is to provide automated and integrated methods and systems for the production of cells and / or cellular products. Another object of the invention is to provide autonomous systems and small-scale installation for the production of cells and / or cellular products. Summary of the invention

In a first aspect, the present invention provides a system for producing cells and / or cell products. The system comprises at least one cell culture unit comprising at least one bioreactor for culturing cells, at least one technical control unit for controlling cell growth parameters, said technical control unit is at least fluidly connected to the cell. cell culture unit, and at least one air treatment unit for treating the ambient air, said air treatment unit is fluidly connected to the cell culture unit. The system is autonomous and the total volume of the bioreactor is at most 1000 L.

In a preferred embodiment, the system optionally includes at least one downstream unit which is fluidly connected to the cell culture unit, said downstream unit comprises a pluggable means selected from the group consisting of at least one means of filtration, at least one collection means, at least one dialysis means, at least one biomolecule purification means and at least one protein concentration unit or any combination thereof.

In a second aspect, the present invention provides an integrated automated method for the production of cells and / or cell products. The method comprises the steps of culturing cells in at least one bioreactor that is fluidly connected to a culture medium reservoir, said bioreactor being contained in a cell culture unit; providing a mixture of at least two gases to the bioreactor; and providing sterile ambient air to the cell culture unit; in which the total volume of the bioreactor is not more than 1000 L.

The system and / or method of the present invention does not require the use of a considerable number of large instruments such as large bioreactors. This is due to the small-scale installation of the system and the use of small bioreactor. Another advantage is the autonomy of the system. The latter should only be connected to an external reservoir of cell culture medium. No other specific tubing and fittings are required as gas lines. This simplifies the installation of the system which can therefore be installed and used in a normal laboratory in which only a connection for the system is required. Installation costs are also significantly reduced.

In addition, the present method and system are free from manual manipulation, thereby greatly reducing the risk of contamination. Moreover, the autonomous system of the invention makes it possible to carry out the complete process under high biosecurity circumstances thanks to the air treatment unit.

The systems and methods of the invention also permit rapid production of cells and / or cell products using significantly smaller equipment compared to prior art systems and methods. Another advantage is to allow high-throughput production of cells and / or cell products as compared to prior art methods and systems thereby reducing the costs of the final product. The present invention provides cheaper integrated and fully automated systems that cost at least 5 to 6 times less than conventional large scale systems. This ultimately results in a lower cost of investment and production, which is a considerable advantage.

Description of figures

Figure 1 shows an embodiment of the system of the invention; FIG. 2 represents an embodiment of the system of the invention in which the cell culture unit is connected to a downstream unit comprising a filtration means and a purification means.

Detailed description of the invention

The present invention provides methods, apparatus and systems for the production of cells and / or cell products or biomolecules such as viruses, proteins or peptides. The invention aspires specifically to provide a small scale system that can be implemented in a laboratory. The system and method have an optimal performance in terms of material input and product output. The current invention aspires to provide a fully integrated and automated methodology and system for the production of cells and / or biomolecules. By "proteins or peptides" and "cells and / or biomolecules", we refer to antibodies as well as antigens.

Unless otherwise stated, all terms used in the disclosure of the invention, including technical and scientific terms, have the meaning as generally understood by the ordinary skilled artisan to whom this invention belongs. With additional guidance, term definitions are included to better appreciate the teaching of the present invention.

As used in this document, the following terms have the following meanings: "One," "an," and "the" as used herein refer to both singular and plural referents unless the context say something else clearly. For example, "a compartment" refers to one or more compartments. "Approximate" as used herein with reference to a measurable value such as a parameter, a quantity, a time duration, and so on, means to encompass variations of +/- 20% or less, preferably +/- - 10% or less, more preferably +/- 5% or less, even more preferably +/- 1% or less, and still more preferably +/- 0.1% or less of and from the value specified, provided that such variations are appropriate to perform in the disclosed invention. However, it will be understood that the value to which the "about" modifier refers is also specifically disclosed. "Understand", "understand", and "understand" and "consist of" as used in this document are synonymous with "include", "include", "include" or "contain", "contain", "contains" and are inclusive or open terms that specify the presence of, for example, a component and do not exclude or exclude the presence of components, features, elements, members, additional steps, not mentioned, known in the art or revealed within it.

Mentioning numeric ranges by a value box includes all numbers and fractions within that range, as well as the framing values mentioned. The term "% by weight" (percent by weight), herein and throughout the specification unless otherwise stated, refers to the relative weight of the respective component based on the total weight of the formulation.

The system and method of the invention will now be described with reference to the accompanying figures.

In a first aspect, the present invention provides a system for producing cells and / or cell products, comprising: - at least one cell culture unit 1 comprising at least one bioreactor 2 for culturing cells. Said bioreactor can be fixedly attached to the system, or removably attached to the system, at least one technical control unit 3 for controlling cell growth parameters, said technical control unit is at least fluidically connected to the cell culture unit 1, and - at least one air treatment unit 8 for the treatment of the ambient air, said air treatment unit is connected to fluid to the cell culture unit 1 (figure 1). The cell culture unit 1 and the technical control unit 3 have at least one common wall 21. Also, the cell culture unit 1 and the air treatment unit 8 have at least one common wall (20 in Figure 1).

The system is characterized in that it is autonomous and the total volume of the bioreactor is at most 1000 L. By total volume of the bioreactor is referred to the total volume of liquid that can be introduced into the bioreactor, thereby filling totally the bioreactor. By standalone refers to a system that is free of external connections for gas supply and / or external connections for sterilization of the system and / or external connections to take samples from the culture medium included in the bioreactor.

In a preferred embodiment, the total volume of the bioreactor is at most 980 L, at most 960 L, at most 940 L, at most 920 L, at most 900 L, at most 880 L, at most 860 L, at plus 840 L, not more than 820 L, not more than 800 L, not more than 780 L, not more than 760 L, not more than 740 L, not more than 720 L, not more than 700 L, not more than 680 L, not more than 660 L, plus 640 L, not more than 620 L, not more than 600 L, not more than 580 L, not more than 560 L, not more than 540 L, not more than 520 L, not more than 500 L, not more than 490 L, not more than 480 L, at plus 450 L, not more than 420 L, not more than 400 L, not more than 380 L, not more than 350 L, not more than 340 L, not more than 330 L, not more than 320 L, not more than 310 L, not more than 300 L, at plus 290 L, not more than 280 L, not more than 270 L, at most 260 L, at most 250 L, at most 240 L, at most 230 L, at most 220 L, at most 210 L, at most 200 L, at most 190 L, at most 180 L, at most 170 L, not more than 160 L, not more than 150 L, not more than 140 L, not more than 130 L, not more than 120 L or not more than 100 L, or any value between the previously mentioned values.

In a preferred embodiment, the total volume of the bioreactor is at least 0.5 L, preferably at least 1.5 L, preferably at least 3 L, more preferably at least 5 L, more preferably at least minus 10 L, most preferably at least 20 L, even more preferably at least 30 L. Preferably, the total volume of the bioreactor is at least 40 L, at least 50 L, at least 60 L, at least 70 L, at least 80 L, at least 90 L or any value between the previously mentioned values. The total volume of the bioreactor and the bioreactor itself are small compared to conventional bioreactors used for cell culture. This is advantageous in terms of the space required for the system and for ease of use. The cell culture unit enables the production of cell-derived cells and products in a closed, self-sustaining environment. The unit may comprise at least one bioreactor for cells and / or their product expansion with minimal need for interaction by a technician. The bioreactor may be attached to the system in a fixed manner, or may be removably attached to the system.

In a preferred embodiment, the technical control unit 3 comprises at least one movement means 5 for the movement of the bioreactor 2, said movement means 5 is mechanically and / or magnetically connected to the bioreactor 2 and provides a movement selected to from right to left, from top to bottom, rotating along a horizontal axis, rotating along a vertical axis, rocking motion along a tilted or inclined horizontal axis of the bioreactor or any combination of these. Said movement or displacement can be carried out in a continuous or discontinuous mode. Preferably, for rotational movement, the bioreactor is mounted to the wall 21 which is common to the cell culture unit 1 and the technical control unit 3 (Fig. 1).

Cells require oxygen during their growth phase in order to have optimal growth. The bioreactor can be moved, thereby increasing the oxygen transfer by a factor of at least 10 compared to conventional systems and processes and providing a gas balance in said bioreactor. The implementation of the bioreactor at the equilibrium gas is therefore performed. This in turn increases cell growth, which has a positive impact on the production of biomolecules. This makes it possible to direct cultures into a sensor-free bioreactor thereby providing a simple and less complicated bioreactor facility as well as a direct and simple methodology as compared to prior art bioreactors and methodologies. In addition, the use of a bioreactor free of sensors allows a considerable reduction in the risk of contamination. In addition, a sensor failure is no longer a problem, and repairs that were necessary in prior art systems due to said sensor failure are no longer required, leading to a sharp reduction in personnel and operating costs. .

Moving the bioreactor further improves cell harvesting. Indeed, collecting cells from a bioreactor containing supports such as fiber or microfiber bioreactors has been difficult to accomplish. Typically, the cells are tacky and attach to carriers or other cells and form aggregates. Movement of the bioreactor forces free cells thereby providing increased cell harvesting efficiency at high cell viabilities without the use of chemical or enzymatic release additives. The bioreactor may have a rigid or non-rigid outer body. The rigid outer body allows the bioreactor casing to be flexed causing a movement of microfibers. This movement enhances the release of cells that have attached to the interior of the bioreactor matrix.

In a preferred embodiment, the technical control unit 3 comprises at least one supply means 7, 9 connected to the bioreactor 2 and to a reservoir of culture medium 16 for feeding the bioreactor 2 in a culture medium (FIG. ). Said culture medium reservoir 16 is positioned outside the system and may be provided with wheels 17 for easy transportation of said reservoir. The culture medium reservoir 16 may be connected to a sterile filter 18 for discharge. Said culture medium tank may be provided with at least one heating and / or cooling means in order to supply the culture medium to bioreactor 2 at the desired temperature.

Preferably, the technical control unit 3 comprises more than one feed means for supplying the bioreactor 2 with any other element required for cell growth. Said elements are contained in at least one external reservoir and selected from lists comprising additives, cells, pH adjustment solutions or any combination thereof. The technical control unit may comprise 2, 3, 4, 5 or more feed means.

The feed means 7, 9 may be a peristaltic pump so that both the motor and the pump itself - also referred to as the pump head in this document - are positioned in the technical control unit 3. Both the motor and the the pump head can also be positioned in the cell culture unit 1. Preferably, the pump motor 7 is positioned in the technical control unit 3 while the pump head 9 is positioned in the control unit 3. cell culture 1 (Figure 1). This saves space in the cell culture unit and avoids having the moving part of the pump in said unit thereby minimizing the presence of particles.

If the pump is positioned in the cell culture unit 1, then a pipe or tube 13 is provided between said pump and the bioreactor 2 thereby connecting them to one another. It will be understood that the supply means 7, 9 is fluidly connected to the culture medium reservoir by at least one tube 22 or any equivalent means known to one skilled in the art. This makes it possible to feed the bioreactor 2 in culture medium (FIG. 1). In this case, the pipe or the tube 13 provided between said pump and the bioreactor 2 is the same as the tube 22 fluidly connecting the feed means 7, 9 to the culture medium reservoir (FIG. 1). The feed means 7, 9 is also fluidly connected to the bioreactor 2 by at least one inlet pipe 15 or any equivalent means known to those skilled in the art.

Preferably, the culture medium is preheated to a temperature between 25 ° C and 37 ° C and / or mixed before transfer to the bioreactor. This ensures that the cells will not experience heat shock by coming into contact with the new medium - which would negatively affect their growth - as well as ensuring that all the nutrients in the medium are mixed and present in the necessary amounts.

Said preheating and / or mixing are performed in the culture medium reservoir 16 using at least one heating and / or cooling means and at least one mixing means (not shown). The mixing means may be housed inside or outside the tank 16. The heating and / or cooling means may be positioned between the tank 16 and the bioreactor 2. The culture medium may be a liquid comprising a well-defined mixture of salts, amino acids, vitamins and one or more protein growth factors. The culture medium is used to deliver nutrients to the cell and conversely, to remove or prevent toxic accumulation of metabolic waste.

In a preferred embodiment, the technical control unit 3 comprises at least one measuring means 6 for measuring a plurality of cell culture parameters. Said parameters are selected from, but not limited to, oxygen level, pH, temperature and cell biomass. Preferably, the measuring means comprises at least one transmitter and / or at least one sensor or any other means known to those skilled in the art. Said sensors may be optical sensors, light-based sensors, WiFi-based radio frequency sensors, or any other sensor known to those skilled in the art. Preferably, sensors that do not require a physical connection to the bioreactor or any other element of the system are used.

Preferably, the bioreactor itself is free of sensors. Said sensors may be arranged outside the bioreactor. This provides a simple and less complicated bioreactor facility as well as a straightforward and simple methodology in comparison to prior art bioreactors and methodologies. In addition, the use of a bioreactor free of sensors allows a considerable reduction in the risk of contamination. In another preferred embodiment, the bioreactor may be provided with at least one sensor for measuring at least one of the previously mentioned parameters.

In a preferred embodiment, the technical control unit 3 comprises at least one gas production means 4 which is fluidly connected to the bioreactor 2, said gas production means 4 comprises at least one gas mixing device ( not shown) to mix at least two different gases (Figure 1). The gas producing means 4 further comprises at least one means for producing oxygen (O2) (not shown) for the production of C> 2. Said O2 is preferably produced by enrichment from air using an oxygen concentrator for example. Said production of Ü2 can be carried out using zeolites included in the gas generating means 4. The gas generating means 4 further comprises at least one means for producing CO2 (not shown) for the production of CO2. Said CO2 production means may consist of single-use aluminum cans or any other means known to those skilled in the art. Preferably, said production of CO2 is carried out at a low pressure. Preferably, the gas generating means 4 is provided with an air pumping means for pumping air directly from the outside environment of the system. Said air pumping means may comprise at least one pipe having an end connected to the external environment or to the atmosphere as a laboratory atmosphere. For example, if the system is placed in a laboratory, then the gas producing means 4 is capable of pumping air from said laboratory. The cell culture unit 1 is also equipped with means for pumping air directly from the cell culture unit itself.

In a preferred embodiment, the gas mixing device mixes the gases (at least O2 and CO2) produced by the gas producing means 4 thereby obtaining a premixed gas. The premixed gas obtained is supplied to the bioreactor 2 using a single gas supply line 12. This further simplifies the installation of the system.

Gas such as pure oxygen or a gaseous mixture comprising oxygen is also provided through the inlet of the bioreactor. Oxygen is an essential requirement for the normal growth of mammalian cells. Preferably, said gas or gas mixture is supplied under pressure. In one embodiment, the cells will be exposed to dissolved oxygen concentrations of 300 μΜ or less (partial pressure 160 mmHg), preferably less than 200 μΜ, most preferably 20 to 150 μM.

In a preferred embodiment, gas or a gas mixture and culture medium will be intermingled before being supplied to the bioreactor. Then, the mixture of gas or gas mixture and the culture medium are supplied via a supply line (15 in FIG. 1). This provides an advantage that a cell medium with optimum oxygen concentration is delivered directly to the cells. In a further preferred embodiment, said gas or gas mixture is selected from air or oxygen. Preferably, air is used. Air should be viewed as a gaseous mixture comprising approximately 78% nitrogen, 21% oxygen and argon and carbon dioxide. The supply of air instead of pure oxygen or oxygen enriched atmospheres has the advantage that the system using the process may be free of highly concentrated oxygen supply units, which may otherwise involve a hazard. explosion or fire. These risks are also minimized and even non-existent using the system of the invention as said system is provided with its own means of producing C> 2.

The low solubility of oxygen in an aqueous medium (such as a cell culture medium) relative to its rate of consumption causes its feed rate to be a limiting factor for cell growth. In general, the oxygen transfer rate (OTR) in a fermentation device or a bioreactor is described by: OTR = K1_3 (Cgaz-Cliq), where OTR = oxygen transfer rate in pmol O2 l ^ h '1;

Ki_a = is the coefficient of oxygen transfer in h'1;

Cgaz = concentration O2 (equilibrium) in gaseous phase in μΜ;

Ciiq = concentration O2 in liquid phase in μΜ

Preferably, the oxygen transfer coefficient (Ki.a) in the current process is at least 20 h -1, preferably at least 30 h -1, more preferably at least 35 h -1. Said oxygen transfer coefficient is at most 100 h -1, preferably at most 50 h -1, more preferably at most 40 h -1.

A high oxygen transfer coefficient and therefore also a high OTR will have a positive influence on cell growth / health and thus on the desired end product yield. The inventors have found that an oxygen transfer coefficient as previously defined is particularly advantageous in terms of product yield, even using a rather small amount of starting cell culture.

In a preferred embodiment, the gas supply line differs from the medium supply line. The gas supply line may also be the same as the supply line in the medium as shown in FIG. 1. In said figure, the medium supply line 13 and the gas supply line 12 provide medium and gas at the same inlet pipe 15 in which the medium and the gas will be mixed together. This minimizes the risk of contamination and provides an easy system for connecting and disconnecting the bioreactor with the cell culture unit, thereby simplifying its separation from it if the bioreactor needs to be replaced for example.

In a preferred embodiment, the technical control unit 3 and / or the cell culture unit 1 comprises at least one sterile filter 23. Said filter is connected to fluid with the gas production means 4 and with the bioreactor, preferably, to the gas supply line 12 as shown in FIG. 1. As a result, the gaseous mixture flowing through the gas supply line 12 is passed through said sterile filter.

In a preferred embodiment, the air treatment unit 8 comprises at least one sterilization means for supplying sterile air to the cell culture unit. Said sterile air is preferably supplied to the cell culture unit 1 in a laminar flow (arrows a in FIG. 1). Said sterilization means may be a heating, ventilation and air conditioning system (HVAC system). The sterilization system provides sterile connections for a plurality of operations that are usually performed in a laminar flow hood. Said operations include, but are not limited to, cell seeding, virus infection, culture media and / or additive feed and culture medium sampling. Said culture medium may be supplemented with cultured cells and / or biomolecules from the bioreactor.

The completed culture medium, also referred to as supplemented medium in this document, refers to the supernatant of the bioreactor which may comprise a culture medium and / or cultured cells and / or their products. The supernatant of the bioreactor may be free of cells and / or their products. The cell product refers to biomolecules such as proteins, peptides, produced by cells and / or any other cellular biomolecules derived from lysis of cells such as cell membranes.

In a preferred embodiment, the system further comprises at least one programmable control unit that is electromechanically connected to the system for controlling and / or controlling its operation. The programmable control unit is provided with an algorithm that sends instructions to the different units of the system thus ensuring its operation for the production of cells and / or the cell. The operator initiates the culture process through a user interface such as a touch screen interface. Said interface may be provided on the system itself or at a distance from said system.

In a preferred embodiment, a predetermined temperature is kept constant within the cell culture unit 1. Said predetermined temperature is about 37 ° C. Preferably, a predetermined pressure is also kept constant within the cell culture unit. The cell culture unit 1 acts similarly to a laminar flow hood thereby allowing air sampling for particle counting and bioburden evaluation. The cell culture unit is capable of extracting air from the external environment of the system as shown by the arrow b in FIG. 1. Said constant pressure allows the manipulation of microorganisms at Biosafety Level 2 as viruses.

The bioreactor used in the process and / or system of the invention may be any type of bioreactor. The bioreactor used preferably provides a culture area of at least 0.5 square meter m 2 per liter of bioreactor. The said bioreactor is preferably an infusion bioreactor. The bioreactor comprises at least one cell trapping system or supports selected from the list comprising fibers, microfibers, hollow microfibers, a hollow filter, a tangential flow filter, a decanter, microcarriers, microcarriers containing stirred containers or any combination thereof. The supports provide an excellent substrate for the cells to grow on.

Preferably, the bioreactor is provided with at least one admission for introducing gas and / or culture medium and at least one outlet for collecting the culture product and / or the medium contained in the bioreactor. At least one inlet manifold is provided for fluidically connecting the bioreactor via its inlet to a culture medium reservoir and / or a gaseous source. At least one outlet pipe is provided to fluidly connect the bioreactor, via its outlet, to a downstream unit and / or any other device.

Preferably, the supports present in the bioreactor provide a cell growth area of at least 10 square meters (m2), preferably at least 1000, more preferably at least 1200 square meters, more preferably at least 1500 square meters. m2, most preferably at least 1,800 m2. More preferably, the supports present in the bioreactor provide a cell growth area of at least 3 m 2 per L of bioreactor, preferably at least 4 m 2, more preferably at least 5 m 2, even more preferably at least 6 m 2. m2, most preferably at least 7 m2. The carriers may also provide a cell growth area of at least 8 m 2 per L of bioreactor, preferably at least 9 m 2, more preferably at least 10 m 2, even more preferably at least 11 m 2, most preferably at least 12 m2 per L of bioreactor or any value between the previously mentioned values. The cell growth surface of the bioreactor is also referred to herein as the bioreactor expression volume.

The carriers provide a cell growth area of at most 3,000 m2, preferably at most 2,800 m2, more preferably at most 2,500 m2, even more preferably at most 2,200 m2, most preferably at most 2,000 m2. Preferably, the supports present in the bioreactor provide a cell growth surface of at most 30 m 2 per l of bioreactor, preferably at most 26 m 2, more preferably at most 24 m 2, even more preferably at most 20 m 2, most preferably at most 19 m2. The supports may also provide a cell growth surface of at most 18 m 2 per L of bioreactor, preferably at most 17 m 2, more preferably at most 16 m 2, even more preferably at most 15 m 2, most preferably at plus 14 m2 per L of bioreactor or any value between the previously mentioned values.

The combination of carriers and movement of the bioreactor significantly increases the oxygen transfer coefficient in the bioreactor. Movement of the bioreactor, which is at least partially filled with culture medium, causes a portion of the supports to move from a liquid phase, in which they are in contact with the culture medium, to a gaseous phase, wherein they are not in contact with said medium. This oxygen transfer rate has increased at least 10-fold as compared to prior art bioreactors.

In a preferred embodiment, the bioreactor allows growth of cells with a density of 50,000 to 350,000 cells / cm 2 of support, preferably 100,000 to 250,000 cells / cm 2 of support, more preferably 150,000 to 200,000 cells / cm2 of support depending on the type of cell.

In a preferred embodiment, the bioreactor used in the method and / or the system of the invention is a small bioreactor. Said bioreactor may be a circular bioreactor having a diameter of at least 10 cm, preferably at least 20 cm, more preferably at least 40 cm and at most 50 cm, preferably at most 60 cm, more preferably at least plus 70 cm. Said bioreactor may also be a rectangular or square bioreactor having a height of at least 10 cm, preferably at least 20 cm, more preferably at least 40 cm, even more preferably at least 50 cm, most preferably at least 60 cm and at most 110 cm, preferably at most 100 cm, more preferably at most 80 cm, most preferably at most 70 cm. The width of said rectangular or square bioreactor is at least 40 cm, preferably at least 50 cm, more preferably at least 60 cm and at most 100 cm, preferably at most 90 cm, more preferably at most 80 cm, most preferably at most 70 cm.

In a preferred embodiment, the system is implemented in a single transportable chamber, suitable for a clean transportable room such as a laboratory. Preferably, the system is implemented in a small-scale cabinet that can be a transportable room or a clean transportable room. Preferably, the dimensions of the small-scale cabinet are 0.8 □ 1.6 □ 1.8 m3. The system, according to any embodiment of the invention, enables the production of cells and products derived from cells in a closed, self-sufficient environment. The operation of said system requires a minimal need for a technician interaction. The integration of components, functions and operations greatly reduces the labor and cost required to produce cells and / or a product derived from cells. The integrated system reduces the preparation and loading time and reduces the number of operator-induced errors that can cause a failure.

In a preferred embodiment, the system is provided with at least one extraction line 11 for extracting culture medium from the bioreactor. Said culture medium may be supplemented with cultured cells and / or cell products. The extraction line includes that sampling a sampling collector 10 to collect culture medium samples at any time during cell growth. Said samples are further analyzed thereby monitoring the evolution of cell growth.

The bioreactor of the system may be fluidly connected to at least one downstream unit which comprises different components or means suitable for the subsequent processing of supernatant, cultured cells and / or cell products. In a preferred embodiment, the downstream unit comprises a plug-in means selected from the group comprising at least one filtration means, at least one collection means, at least one dialysis means, at least one biomolecule purification means. and at least one protein concentration unit or any combination thereof.

In a preferred embodiment, the downstream unit comprises at least one collection means which is provided with at least one inlet and at least one outlet. Said means of the downstream unit may be connected to the cell culture unit of the system. The collection means comprises at least one tubing for directing the collected supernatant to another component of the downstream unit. The collection means further comprises at least one pump for extracting the supernatant from the bioreactor.

In a preferred embodiment, the downstream unit comprises at least one filtration means which is provided with at least one inlet and at least one outlet. Said means may be fluid-connected to the bioreactor or fluid-connected to the collection means of the downstream unit. Preferably, the filtering means comprises a filter which will selectively retain molecules based on their Dalton mass, for example. The filtration means may include viral hollow filters that can be used to filter and remove virus particles from the supernatant. In this case, viral filtration works on the principle of size exclusion. When a protein solution with possible viral contamination is introduced into these hollow filters, the smaller proteins enter the filter wall and make their way to the outside of the filter while the larger virus particles are retained.

In a preferred embodiment, the downstream unit comprises at least one purification means which is provided with at least one inlet and at least one outlet. Said means may be fluidly connected to the bioreactor or connected to fluid by the collection means or by means of filtration of the downstream unit. Preferably, the purification means comprises at least one selection device. Said selection device may be a chromatography column such as affinity chromatography, ion exchange chromatography (for example anion or cation), hydrophobic interaction chromatography, exclusion-diffusion chromatography (SEC), immunoaffinity chromatography. which is a column filled with an affinity resin, such as anti-IgM resin, Protein A, Protein G, or an anti-IgG resin. The anion exchange exploits charge differences between the different products contained in the collected supernatant. The neutrally charged product passes onto the anion exchange chromatography column cartridge without being retained, while charged impurities are retained. The size of the column may vary based on the type of purified protein and / or the volume of the solution from which said protein is to be purified. The downstream unit can be customized according to the needs of the user and can be powered by a combination of any of the aforementioned means. Thus, the user is provided with multiple finished product possibilities, cells, filtered cells, filtered cell products, purified cellular products or biomolecules. The user can choose and connect the different downstream compartments according to the desired end product.

In a preferred embodiment, a waste collection container is provided in which metabolic waste is removed from the bioreactor. The container is connected to the bioreactor and can be positioned within the culture unit and / or within the system control unit. Said container may also be disposed outside the system while being connected to the bioreactor. In this case, those skilled in the art are aware of the connections required to ensure the removal of waste.

In a second aspect, the present invention provides an integrated automated method for producing cells and / or cell products comprising the steps of: - culturing cells in at least one bioreactor that is fluidly connected to a culture medium reservoir said bioreactor being contained in a cell culture unit; supplying the bioreactor with a mixture of at least two gases; and - supplying sterile ambient air into the cell culture unit; in which the total volume of the bioreactor is not more than 1000 L.

In a preferred embodiment, the total volume of the bioreactor is at most 980 L, at most 960 L, at most 940 L, at most 920 L, at most 900 L, at most 880 L, at most 860 L, at plus 840 L, not more than 820 L, not more than 800 L, not more than 780 L, not more than 760 L, not more than 740 L, not more than 720 L, not more than 700 L, not more than 680 L, not more than 660 L, plus 640 L, not more than 620 L, not more than 600 L, not more than 580 L, not more than 560 L, not more than 540 L, not more than 520 L, not more than 500 L, not more than 490 L, not more than 480 L, at plus 450 L, not more than 420 L, not more than 400 L, not more than 380 L, not more than 350 L, not more than 340 L, not more than 330 L, not more than 320 L, not more than 310 L, not more than 300 L, at plus 290 L, not more than 280 L, not more than 270 L, not more than 260 L, not more than 250 L, not more than 240 L, not more than 230 L, not more than 220 L, not more than 210 L, not more than 200 L, at plus 190 L, not more than 180 L, not more than 170 L, not more than 160 L, not more than 150 L, not more than 140 L, not more than 130 L, not more than 120 L or not more than 100 L, or any value between the previously mentioned values es.

In a preferred embodiment, the total volume of the bioreactor is at least 0.5 L, preferably at least 1.5 L, preferably at least 3 L, more preferably at least 5 L, even more preferably at least 10 L, most preferably at least 20 L, even more preferably at least 30 L. Preferably, the total volume of the bioreactor is at least 40 L, at least 50 L, at least 60 L, at least 70 L, at least 80 L, at least 90 L or any value between the previously mentioned values. The total volume of the bioreactor and the bioreactor itself are small compared to conventional bioreactors used for cell culture. This is advantageous in terms of the space required for the system and for ease of use.

In a preferred embodiment, the method of the invention is suitable for being performed by a system as previously described and in any embodiment of the present invention. Preferably, the volume of culture medium provided to the bioreactor for culturing cells is sufficient to fill at least about half of the bioreactor expression volume. By volume of bioreactor expression refers to the volume of bioreactor used for the expression of available surface. For example, if the total volume of the bioreactor is 300 L and its expression volume is 10 L, then about 5 L of culture medium is supplied to the bioreactor while the remaining volume of about 295 L circulates between the bioreactor and the reservoir of culture medium.

In a preferred embodiment, the bioreactor is moved or moved during cell culture. Said movement or movement is selected from right to left, up and down, rotating along a horizontal axis, rotating along a vertical axis, rocking along a tilted horizontal axis or inclined bioreactor or any combination thereof. Said movement or displacement can be carried out in a continuous or discontinuous mode.

Preferably, the bioreactor in which cells are cultured is provided with supports selected from the list comprising fibers, microfibers, hollow microfibers, a hollow filter, a tangential flow filter, a decanter, microcarriers, containers agitated containing microcarriers or any combination thereof. The supports provide an excellent substrate for the cells to grow on. The displacement or the movement of the bioreactor makes it possible to obtain free cells of the support - before collecting the cells from the bioreactor for example.

In a preferred embodiment, a predetermined temperature and / or a predetermined pressure are held constant in the cell culture unit. Said predetermined pressure is about -2 to -5 mbar, preferably -3 to -4 mbar. Preferably, the predetermined temperature of the cell culture unit is between 20 ° C and 40 ° C, more preferably between 25 ° C and 37 ° C. The operating temperature of the downstream unit may be between 0 ° C and 25 ° C, more preferably between 1 ° C and 20 ° C, even more preferably between 2 ° C and 10 ° C, most preferably about 4 ° C. The temperature of the two units is maintained by cooling and / or heating units and the maintenance of the temperature can be checked by sensors.

Preferably, the method of the current invention further comprises the steps of culturing cells at a density of at least 50 million cells per ml and fluidly connecting the cell culture unit and / or the bioreactor to a downstream unit. Preferably, at least one sensor is provided for measuring the cell density inside the bioreactor. Preferably, the bioreactor allows growth of cells at a high density. Said density is at least 80 million cells / ml, more preferably at least 100 million cells / ml, most preferably at least 200 million cells / ml. Said density can reach 600, 500, 400 or 300 million cells / ml.

In a preferred embodiment of the current invention, the completed medium of the bioreactor is transferred from the bioreactor to or collected in the downstream unit. Said bioreactor and the downstream unit are fluidly connected to each other. A pump may be provided to transfer the supernatant to the downstream unit. The downstream unit may comprise a filtering means and / or a collection means and / or a dialysis means and / or a means for purifying biomolecules such as the purification of peptides or proteins. In its simplest form, said downstream unit comprises only a means for collecting the desired finished product, without any prior filtration / purification / dialysis step. The components of the downstream unit are easily connected to or disconnected from said unit and can therefore be easily replaced, cleaned or sterilized. The downstream unit may be customized according to the needs and wishes of the users and may be provided with a combination of any of the previously mentioned units. The user is thus provided with multiple finished product possibilities, cells, filtered cells, filtered cell products, purified cell products or biomolecules. The user can choose and connect the different compartments downstream according to the desired end product.

In a preferred embodiment, the downstream unit receives supplemented medium or medium supplemented with biomolecules from said continuous mode bioreactor. Preferably, the downstream unit receives at most 1000 ml / min of medium supplemented with biomolecules from said continuous mode bioreactor. Preferably, the transfer of the supplemented medium is initiated when a predetermined cell density is reached within the bioreactor. Said predetermined cell density is at least 30 million / ml, preferably 40 million / ml, more preferably 50 million / ml, most preferably 60 million / ml. In a preferred embodiment, parallel to the transfer of the supplemented medium from the bioreactor to the downstream unit, culture medium is added from the internal reservoir of culture medium to said bioreactor so as to maintain the initial volume of the culture medium. in the bioreactor. For example, if at the beginning of the process the bioreactor contained 80 L of culture medium, once the transfer of completed medium from the bioreactor to the downstream unit is initiated, new culture medium is added to the bioreactor in a sufficient volume of to maintain a volume of 80 L in said bioreactor. If the transfer of medium supplemented from the bioreactor to the downstream unit is carried out in continuous mode, the addition of new culture medium from the internal reservoir of culture medium in the bioreactor will also be carried out in continuous mode. The method and system of the present invention thus allows the treatment of the completed culture medium in the downstream unit parallel to the growth of the cells in the bioreactor. This provides several advantages over processes in which cells are cultured in large bioreactors containing large volumes of cell culture followed by stopping said cell culture after a certain period of time or reaching a certain concentration and then starting the downstream processes of the large volume of cell culture. Among the advantages one can mention a considerable increase of yield and thus a considerable reduction of cost.

In a preferred embodiment, the medium supplemented with biomolecules received by the downstream unit undergoes at least one process selected from the group consisting of filtration, collection, dialysis, biomolecule purification and protein concentration or any combination of them.

The supplemented medium is preferably collected continuously at a predetermined small volume flow rate. Said volume flow rate is at least 100 ml / min, preferably at least 150 ml / min, more preferably at least 200 ml / min, most preferably at least 250 ml / min. Said volume ratio is at most 1000 ml / min, preferably at most 800 ml / min, more preferably at most 600 ml / min, most preferably at most 400 ml / min. The supernatant collection can also be carried out in a discontinuous manner. The collected supernatant can then be subjected to a subsequent treatment selected from simple harvesting, filtering, purification of molecules, storage or any combination thereof. The treatment of small volumes of supplemented medium greatly reduces the yield loss and improves the quality and the efficiency of treatment, for example a better filtration and / or purification quality. In addition, no increase in the scale of operations performed in the downstream unit is necessary thus avoiding wasting time and money to proportionally increase said operations. The continuous collection mode can be initiated by the operator based on the product concentration. The collection continues until a preprogrammed time interval has elapsed or until the operator manually completes the harvest using a user interface provided in the system of the invention.

In a preferred embodiment, unruptured cultured cells are mass-collected from the bioreactor into a bag provided in the downstream unit. The cells may be hybridoma cells, transfected or transformed cells, or stably transfected cultured cells. In order to obtain the cells without their substrate (the fibers), the bioreactor may be subjected to discontinuous or continuous agitation prior to collection. Said agitation ranges from 10 to 150 Hz at an amplitude of 1 to 5 mm, preferably from 20 to 100 Hz at an amplitude of 1 to 5 mm. In the case where the bioreactor is provided with supports, the agitation will separate the cells from said supports and bring them into the supernatant. The collection of the supernatant is performed using a collection means comprising at least one pump. The bag and / or downstream unit may be designed to maintain the collected supernatant at the same temperature as the temperature of the culture medium or at a different temperature. The collected cells can be maintained in the bag of the downstream unit at a temperature of about 4 ° C. Mass-cultured cultured cells may be filtered using filtering means from the downstream unit before directing said cells into the bag.

In a preferred embodiment, the cultured cells are infected and then ruptured / lysed at a location designed for this purpose in the downstream unit. The supernatant comprising the cell debris and the desired products is then collected using the collection means from the bioreactor. Collection rates are as previously mentioned. The supernatant can be collected and stored for later use in a bag provided in the downstream unit as previously mentioned. The collected supernatant may be subjected to filtration using filtration means prior to storage in a bag of the downstream unit. Alternatively, the recovered supernatant may be filtered and / or subjected to a purification step to separate a specific molecule, such as an antibody, from said supernatant.

Purification can be performed using a purification means of the downstream unit. Said means may be an automated means for obtaining a purified biological product such as proteins, purified antibodies, from the supernatant. In a preferred embodiment, the purification means comprises at least one or any combination of the following: a selection device such as a purification chromatography column (affinity purification, ion exchange, etc. .), a sequence of purification columns or membrane absorbers, at least one liquid reservoir, a device for flowing the liquid from the reservoirs and in the selection device, a device for diverting the effluent from the selection device. The purification means is capable of being installed in the small-scale system of the invention of cabinet size via a simple or "snap-in" or "quick-load" movement and includes mechanical and mechanical interfaces. to communicate with the other components of the system of the invention. It will be appreciated that the buffers and solutions required to carry out the purification process or step may be provided in at least one bag. Said bag can be positioned inside or outside the downstream unit and is naturally provided with the necessary connections to ensure its connection to the purification unit.

Fig. 2 shows an embodiment of the system which is designed for collecting, filtering and purifying at least one cellular product, such as a protein or a peptide. The cell culture unit 1 is fluidly connected to the downstream unit 30 through the outlet tubing 35. The culture unit 1 is as previously described. The outlet tubing directs the supernatant to a filtering means 37. A pump, or collection means, may be provided to collect the supernatant from the bioreactor. The pump can be programmed to begin supernatant collection from a predetermined time period from the start of the culture. The pump may be programmed to collect a predetermined volume of supernatant in an automated continuous mode. The collected filtered supernatant is then directed to a purification means 32 of the downstream unit 30 via at least one tubule 31. The purified cellular product obtained can be stored in a reservoir connected to the purification means or directed via at least one tubular 39, to another downstream component for later applications or to simply be collected by the user.

In a preferred embodiment, the purification means, for example, an affinity column, and / or the filtering means are connected to multiple liquid reservoirs. The reservoirs each contain liquid, such as a wash buffer, an elution buffer, or a neutralization solution, for delivery by means of purification and / or filtration means. The purifying means further comprises a pre-purified or pre-sterilized device for pouring liquid from the reservoirs into the chromatography column for example. For example, pre-sterilized valves and tubing that connect the reservoirs to the column can be used.

Those skilled in the art know the purification using a chromatography column and can be carried out using the appropriate buffers to elute the desired biomolecules. Upon elution of the desired biomolecule, the eluted purified protein may be automatically deposited in a pre-sterilized, disposable collection vessel provided in the downstream unit and removed from the purification means. Alternatively, the eluted purified protein may undergo subsequent automated processing. A purified protein, for example, an antibody, is substantially free of host cell contaminants such as host cell proteins, nucleic acids, and endotoxins.

In a preferred embodiment, the eluted protein is transferred to different solutions. The transfer occurs automatically using a pre-sterilized diafiltration module. Diafiltration is the fractionation process that cleans smaller molecules through a membrane and retains the molecules of interest in the retentate. Diafiltration can be used to remove salts or exchange buffers. In discontinuous diafiltration, the solution is concentrated, and the lost volume is replaced by new buffer. Concentrating a sample at half its volume and adding new buffer four times can remove more than 96% of the salt. In a continuous diafiltration, the sample volume is maintained by the influx of new buffer while the salt and the old buffer are removed. At least 99% of the salt can be removed by adding up to seven volumes of new buffer during continuous diafiltration. Specifically, the diafiltration module is used to further purify the protein (e.g. antibody) and uses the tangential flow filtration principle whereby molecules of greater than 50,000 Daltons (e.g., antibodies, such as IgG and IgM) can not pass through the membrane, but small molecules, such as buffers, can pass through. As a result, the diafiltration module can be used to exchange one buffer with another and is a more effective substitute for dialysis. Diafiltration can be used to neutralize the pH and as a concentration step (to concentrate the cell product).

In a preferred embodiment, the collection means and / or the filtration means and / or the purification means include at least one control device for controlling the circulating medium: unfiltered collected supernatant, filtered supernatant, purified product and elected, etc. The control device may be a probe or a sensor for measuring conductivity and / or pH and / or absorbance at a particular wavelength of said circulating medium. One or more pressure sensors may be included to control the circulating medium pressure with respect to excessive pressures, or to control the pump speed, for example, to maintain the pump speed of the collection means for example at desired pressure.

In a preferred embodiment, the system is adapted to the desired product. This means that if cells are to be mass-supplied, the system will include a downstream unit in which at least one collection bag is provided. If the filtered cells are to be provided, the system will comprise the cell culture unit and the downstream unit in which a filtering means and at least one collection bag are provided. If a specific protein is to be provided, the system will include the cell culture unit and the downstream unit in which at least one filtering means and purification means are provided.

In a preferred embodiment, the method and system of the present invention are free of closed loops or recycling loops. This means that the completed culture medium is returned to the bioreactor at no stage of the process as after passing through the downstream unit. This is advantageous because it considerably reduces the risk of contamination. In addition, it simplifies the setup and installation of the system thereby reducing costs.

In a preferred embodiment, the method is entirely controlled by a programmable controller. This greatly limits human intervention thereby greatly reducing the risk of contamination and errors. The operator may initiate some actions of the cell culture unit and / or the downstream unit as the culture process and / or the collection process and / or the purification process by a user interface as an interface to touch screen on the transportable chamber and / or the cell culture unit.

In a preferred embodiment, cells (mammalian or insect cells) and a suitable culture medium are introduced into the bioreactor. A suitable culture medium refers to the composition of the medium that is required for cell growth. Said compositions are known to those skilled in the art and generally include salts, vitamins, amino acids, sugars, or any combination thereof. The culture medium is preferably supplied to the bioreactor from an external reservoir of culture medium, that is to say not contained in the system of the invention. Preferably, the culture medium is preheated before being supplied to the bioreactor. The preheating temperature of the culture medium is 20 to 40 ° C, preferably 25 to 38 ° C, more preferably 30 to 37 ° C. In a most preferred embodiment, said culture medium is preheated to about 37 ° C.

In a preferred embodiment, the cells are cultured in the bioreactor for a period of time which may vary from a few hours to several days depending on the cells cultured. The cultivation time period is at least 4 hours, at least 10 hours, at least 24 hours, at least 5 days, at least 7 days or any intermediate time. The cultivation period is not more than 70 days, not more than 60 days, not more than 50 days, not more than 40 days, not more than 25 days, not more than 20 days, not more than at most 10 days or any intermediate time.

Depending on the final product, viral transduction or the introduction of viral vectors may be used. Viral replication competent vectors or replicons have been used for a long period of time as an alternative expression system to increase the yields of therapeutic proteins in mammalian cells. The target gene (s) can be expressed under the transcriptional control of viral promoters so that mRNAs accumulate at extremely high levels in the cytoplasm after transfection and when replication, giving large amounts of target proteins. The viral infection can lead to a transduction process without lysis of the cultured cells or lysis of the cultured cells thereby providing the contents of the cells in the supernatant of the bioreactor. Alternatively, hybridoma cells or stably transfected cells may be cultured to produce the desired protein or peptide as an antibody or an antibody fragment.

The method and / or system of the present invention may be used for the cultivation of any cell line and / or for the production of any desired protein and peptide. Examples of preferred cells used in the current system include non-exhaustively Vero cells, CHO cells, Hek293T cells, COS cells, 293T cells, HeLa cells, Hep-2 cells, MCF-7 cells, U373 cells or any other cell line. Examples of viral replication systems include, but are not limited to, polyoma viruses, lentiviral systems, retroviral systems, adenoviral systems, adeno-associated viruses.

In a preferred embodiment of the current invention, the completed medium of the bioreactor is transferred from the bioreactor to or collected in a downstream unit. Said downstream unit is positioned outside the system of the invention and may have a wall in common with any unit of the system, preferably with the cell culture unit. It will be understood that the bioreactor and the downstream unit are fluidly connected to each other. A pump may be provided to transfer the completed medium to the downstream unit. The completed culture medium, also referred to as supplemented medium in this document, refers to the culture medium of the bioreactor which may comprise cultured cells and / or their products. The cell product refers to biomolecules such as proteins, peptides, produced by cells and / or any other cell biomolecules derived from cell membrane lysis.

In a preferred embodiment, at least one feed means 7, 9, connected to the bioreactor 2 and to a culture medium reservoir 16, ensures that the bioreactor 2 is provided with a culture medium (FIG. 1). The transfer of culture medium can be carried out continuously and / or at a constant rate and / or at variable rates. Said transfer of medium may also be carried out discontinuously and / or at a constant rate and / or at variable rates.

In a preferred embodiment, the method further comprises the step of measuring physical and / or chemical parameters of the cell culture and / or culture medium. Said parameters are selected from the group consisting of temperature, pH, salinity, acidity or any combination thereof. Measurements can be made on samples taken from the culture medium before being injected into the bioreactor and / or from the culture medium taken in the bioreactor during cell growth which could include cells and / or products. Such sampling could be done using the collectors of the system.

In a preferred embodiment, the method and / or system of the present invention is used for the production of viral vaccine. For this purpose adherent cells are preferably used and they are cultured in a bioreactor having any trapping system, preferably microfibres. The liner preferably provides for at least 0.5 m2 of growth area per L of bioreactor or any of the previously mentioned growth area values. The cells grow to at least 100,000 to 250,000 cells / cm 2 depending on the cell type. Preferred volumes of culture medium used are about 0.3 ml medium / cm 2 for cell growth and 0.3 ml medium / cm 2 for virus production. Examples of bioreactor volumes and areas are summarized in Table 1 below. It will be understood that any possible combination of values from Table 1 is also included in this application.

Table 1: Examples of Bioreactor Culture Medium Volumes and Growth Surfaces for Viral Vaccine Production

In a preferred embodiment, the method and / or system of the present invention is used for the production of antibodies. For this purpose, preferably suspended and / or adherent cells are used and cultured in a bioreactor filled with microfibers or comprising another trapping system. The liner preferably provides for at least 0.5 m2 of growth area per L of bioreactor or any of the previously mentioned growth area values. The cells grow to at least 100 μg / ml of bioreactor depending on the cell type. Preferably, the bioreactor is supplemented with a volume of culture medium each day. Said volume is preferably up to 1.5 times the volume of initial culture medium introduced into the bioreactor. The process can be done up to 30 days. Examples of bioreactor volumes and areas are summarized in Table 2 below. It will be understood that any possible combination of values in Table 2 is also included in this application.

Table 2: Examples of bioreactor culture medium volumes for antibody production

The method and / or system of the invention provide for the production of monoclonal antibodies, recombinant proteins or any other biological molecules secreted by cells. The production of viral vaccine and the production of antibodies using the method and / or system of the present invention, and in particular the use of the volumes of growth medium and growth surfaces mentioned above, allow (i) to perform the production process at small and intermediate scales and to implement clinical and / or commercial productions of vaccines, genetic vectors or oncolytic viruses. For example for antibodies, a small scale production of 10 to 50 L and an intermediate production of about 250 L can be achieved using the system and / or the method according to any embodiment of the invention.

The method and / or system according to the invention are particularly useful for the production of biologically similar antibodies. The term "biologically similar" antibodies should be understood as "generic" versions of "native" antibodies that have the same amino acid sequence as these "originating" antibodies, but which are produced from different clones and / or different manufacturing processes.

In a preferred embodiment, the method and / or system of the present invention allows the production of vaccine quantities of about 940 850 cc roll-in bottles which are for the majority of vaccines a commercial scale production. In a preferred embodiment, the method and / or system of the present invention allows the production of antibody amounts up to 360 g, preferably up to 400 g which are also for a majority of antibiotics a production. on a commercial scale.

The method and / or system can be used for the production of: - anti-inflammatory biomolecules or any antibodies such as infliximab, adalimumab, basiliximab, daclizymab, omalizumab, palivizumab and abciximab; - anti-cancer biomolecules such as gemtuzumab, alemtuzumab, rituximab, transuzumab, nimotuzumab, cetuximab, bevacizumab; - human vaccines such as, but not limited to, polio vaccine (IPV), Rotavirus vaccine, influenza vaccine, Yellow Fever vaccine, Varicella vaccine, Measles, Mumps, Rubella vaccine , Hepatitis and Rabies; - Veterinary vaccines such as, but not limited to, the vaccine against Marek's disease and the vaccine against Newcastle disease. The methodology and system can also be used for the production of antibody-RSV-based vaccine; and formulations thereof.

Those skilled in the art will appreciate that the necessary tubing and / or pumps can be provided within the system to achieve the required connections between the different units and / or means of the system. In addition, the system may be provided with a plurality of switching valves used to route the fluids between said different compartments. In addition, software may be provided to direct the system and / or the method according to one embodiment of the invention.

Although the present invention has been described with reference to preferred embodiments thereof, many modifications and variations can be made by a person of ordinary skill without departing from the scope of this invention. is defined by the appended claims.

Claims (25)

Amended Claims - Amendments Accepted A system for the production of cells and / or cellular products, comprising: at least one cell culture unit comprising at least one bioreactor for culturing cells, the total volume of the bioreactor is at most 1000 L; at least one technical control unit for controlling cell growth parameters, said technical control unit is at least fluidly connected to the cell culture unit, and - at least one air treatment unit for treating the cell culture unit. ambient air, said air treatment unit is fluidly connected to the cell culture unit, characterized in that the air treatment unit comprises at least one sterilization means for supplying sterile air to the cell culture unit. The system of claim 1, wherein the total volume of the bioreactor is at most 900 L, preferably at most 800 L, more preferably at most 700 L, even more preferably at most 450 L, most preferably not more than 300 L, even more preferably not more than 250 L, even more preferably not more than 200 L 3. System according to any one of the preceding claims, wherein the total volume of the bioreactor is at least 1.5 L, preferably at least 3 L, more preferably at least 10 L, even more preferably at least 30 L. L, most preferably at least 50 L, even more preferably at least 60 L A system according to any one of the preceding claims, wherein the technical control unit comprises at least one moving means for moving the bioreactor, said moving means is mechanically and / or magnetically connected to the bioreactor and provides motion selected from right-to-left, up-down, rotational along a horizontal axis, rotating along a vertical axis, rocking motion along an inclined or tilted horizontal axis of the bioreactor or any combination of these. 5. System according to any one of the preceding claims, wherein the technical control unit comprises at least one supply means connected to the bioreactor and to a culture medium reservoir for supplying the bioreactor with the culture medium, said reservoir. of culture medium is positioned outside the system. The system of any preceding claim, wherein the technical control unit comprises at least one measuring means for measuring a plurality of cell culture parameters. 7. System according to any one of the preceding claims, wherein the technical control unit comprises at least one gas generating means which is fluidly connected to the bioreactor, said gas generating means comprises at least one mixing device. of gas for mixing at least two different gases. The system of any preceding claim, wherein a predetermined temperature is kept constant within the cell culture unit. The system of any preceding claim, wherein a predetermined pressure is maintained constant within the cell culture unit. The system according to any one of the preceding claims, wherein the bioreactor comprises at least one cell trapping system selected from the list comprising microfibers, a hollow filter, a tangential flow filter, a decanter or the like. any combination of these. The system according to any one of the preceding claims, wherein said cell trapping system provides a cell growth area of at least 1000 m2, preferably at least 100 m2, more preferably at least 10 m2 per square meter. L of bioreactor. A system according to any one of the preceding claims, optionally comprising at least one downstream unit which is fluidly connected to the cell culture unit, said downstream unit comprises a pluggable means selected from the group comprising at least one filtration means, at least one collection means, at least one dialysis means, at least one biomolecule purification means and at least one protein concentration unit or any combination thereof. 13. System according to any one of the preceding claims, further comprising at least one programmable control unit which is electromechanically connected to the system for controlling and / or controlling its operation. System according to any one of the preceding claims, wherein said system is implemented in a single transportable chamber, suitable for a transportable clean room. An integrated automated method for producing cells and / or cellular products comprising the steps of: a. culturing cells in at least one bioreactor that is fluidly connected to a culture medium reservoir, said bioreactor being contained in a cell culture unit; b. providing a mixture of at least two gases to the bioreactor; and c. supply of sterile ambient air to the cell culture unit; in which the total volume of the bioreactor is not more than 1000 L. The process according to claim 15, wherein the total volume of the bioreactor is at most 900 L, preferably at most 800 L, more preferably at most 700 L, even more preferably at most 450 L, most preferably not more than 300 L, even more preferably not more than 250 L, even more preferably not more than 200 L The method of any of claims 15 and 16, wherein the total volume of the bioreactor is at least 1.5 L, preferably at least 3 L, more preferably at least 10 L, even more preferably at least minus 30 L, most preferably at least 50 L, even more preferably at least 60 L. 18. A method according to any one of claims 15 to 17, wherein the bioreactor is moved during cell culture, said motion is selected from right to left, up and down, rotating along a horizontal axis, rotating along a vertical axis, rocking motion along an inclined or tilted horizontal axis of the bioreactor or any combination thereof. The method of any one of claims 15 to 18, wherein a predetermined temperature is kept constant in the cell culture unit. The method of any one of claims 15 to 19, wherein a predetermined pressure is maintained constant in the cell culture unit. The method of any one of claims 15 to 20, further comprising the steps of fluid binding said bioreactor with a downstream unit and / or growing cells at a density of at least 50 million cells per ml. The method of any one of claims 15 to 21, wherein said downstream unit receives supplemented culture medium from said bioreactor in a continuous mode, said supplemented culture medium comprises culture medium and / or cultured cells and or products of said cultured cells comprising proteins, peptides and / or any other cellular biomolecules derived from cell lysis such as cell membranes. The method of any one of claims 15 to 22, wherein said downstream unit receives not more than 1000 ml / min of supplemented culture medium from said bioreactor. 24. The method of any of claims 15 to 23. wherein the supplemented culture medium received by the downstream unit undergoes at least one process selected from the group consisting of filtration, collection, dialysis, biomolecule purification and protein concentration or any combination thereof. this. 25. The method of any one of claims 15 to 24, wherein said method is fully controlled by a programmable controller.