EP3565882A1

EP3565882A1 - Method and device assembly for providing a large amount of cell culture medium using disposable components

Info

Publication number: EP3565882A1
Application number: EP17817692.1A
Authority: EP
Inventors: Christian Grimm; Mario Becker; Christian MANZKE
Original assignee: Sartorius Stedim Biotech GmbH
Current assignee: Sartorius Stedim Biotech GmbH
Priority date: 2017-01-05
Filing date: 2017-12-04
Publication date: 2019-11-13
Also published as: CN110139924A; DE102017100150B3; WO2018127334A1; US20190352595A1

Abstract

The invention relates to a method for providing a large amount of cell culture medium (10), in particular for cultivating animal cells, for a large reactor system (12) using disposable components, having the following sequence of steps: a) producing a concentrated media stock solution (32), b) filling the concentrated media stock solution (32) into disposable containers (40), c) transporting the disposable containers (40) filled with media stock solution (32) to the large reactor system (12), d) aseptically connecting the disposable containers (40) to the large reactor system (12) by means of a disposable hose system (26), e) providing the media stock solution (32) by supplying high-purity water (24) and/or additives (44), and f) supplying the provided media stock solution (32) to the large reactor system (12).

Description

A method and apparatus arrangement for providing a large amount of cell culture medium using disposable components

The invention relates to a method for providing a large amount of cell culture medium, in particular for the cultivation of animal cells, for a large reactor system using disposable components. The invention further relates to a device arrangement which is set up for carrying out such a method.

The trends of recent years in the biopharmaceutical industry are increasingly in the direction of using single-use components. These are now not only used in the field of product and process development, but also in the field of clinical trial manufacturing (English: Clinical Trial Manufacturing, abbr .: CTM) for the approval process and even in the commercial "good manufacturing practice" (engl .: Good Manufacturing Practice, abbr .: GMP) in the production of pharmaceuticals.

In commercial production, such as in the cultivation of animal cells in a bioreactor, essentially two different approaches to the use of disposable components are introduced. On the one hand, operators are focusing on a complete one-way approach for new factories, locations or companies, especially in Asia, where the entire production chain is almost exclusively covered with disposable components. If, on the other hand, classic stainless steel appliances already exist, they should generally continue to be used due to the high purchase price. In this case, so-called hybrid systems are used consisting of existing stainless steel components and newly added disposable components. An example of this is the cultivation of animal cells in a bioreactor. Here, for example, the bioreactor is an existing stainless steel boiler with a volume of 2,000 to 50,000 liters, but the media and buffer production takes place in disposable mixing and storage containers. For both complete disposable and hybrid systems, a key challenge is to provide the appropriate amount of cell culture medium in a reactor with a working volume of several thousand liters. Since in disposable systems no rigid piping or the like is or is provided with a large media preparation tank, the medium must be used in manageable containers, stored and transported to the reactor. These containers normally have a capacity of only 50 to 1, 000 liters. For larger production reactors, therefore, a plurality of such containers must be brought to a reactor, aseptically connected and fed sterile filtered the reaction vessel. This is usually done sequentially and requires a lot of manual effort in properly connecting and disconnecting the container. In addition, each compound presents a potential risk of leakage or contamination of the reactor. The object of the invention is to provide a method which makes the process of providing a large amount of cell culture medium for a large reactor system using disposable components more efficient and less risky.

To achieve the object, a method for providing a large amount of cell culture medium, in particular for cultivating animal cells, for a large reactor system using disposable components, is provided with the following sequence of steps: a) producing a concentrated media stock solution, b) filling the concentrated D) aseptic connection of the disposable containers to the large reactor system by means of a disposable hose system, e) preparation of the media stock solution by feeding highly pure water and / or additives, and f) feeding the processed media strain into the large reactor system.

In this way, the media strain solution is provided in concentrated form and diluted on site in the reactor system to the required mixing ratio. Thus, the volume and the number of disposable containers required for transport are reduced, each of which must be connected to the reactor system. Thanks to the invention, even large quantities of cell culture medium using disposable components can be efficiently provided to a reactor system and the risks such as contamination of the reactor can be reduced.

In step a), the concentrated media master solution can be matched to the volumes of subsequent mixing systems and / or the reactor volume. In this way, the preparation of the media strain solution simplifies, since, for example, existing mixing systems can be used without additional modification. Furthermore, tuning to the reactor volume allows the intended number of disposable containers to be completely emptied into the reactor with the concentrated media stock solution, thereby minimizing the remainder of media stock solution that is recovered as waste. Preferably, in step a), the concentrated media strain solution has a concentration of 5: 1 or higher. The higher the media master solution is concentrated, the lower the volume of media media solution required to prepare a particular solution in the reactor. Having a smaller volume also reduces the number of disposable containers needed to transport the media stem solution. This leads to a cost reduction, since less disposable containers are consumed and the transport is cheaper.

In a preferred embodiment, in step b) the concentrated media strain solution is filled into transportable disposable containers each having a volume of 1 liter to 500 liters, preferably 5 liters to 200 liters. Disposable containers of this size have the advantage that they can be handled by simple means, for example with roller and pallet trucks. Furthermore, with a fixed concentration of the media strain solution several Disposable containers, in particular of different sizes, are combined appropriately to provide exactly the required amount of media stock solution for the reactor system.

In a further preferred embodiment, the following steps take place between step a) and step b): g) filling the concentrated media strain solution into disposable containers, and h) preparing the media stem solution by supplying highly pure water and / or additives.

As a result, the produced concentrated media strain solution is stored at the manufacturer in disposable containers and adapted at a later date to the individual requirements of a reactor system. This has the advantage that a concentrated media master lot can be produced in large quantities and later used as a basis for different media master solutions with different requirements. In this case, in particular, additives of the media strain solution can also be supplied which have only a limited shelf life or service life. Furthermore, the use of disposable containers offers advantages through simplified quality assurance, in particular hygiene.

In step e) of the process according to the invention, unstable chemical components can be supplied to the media strain solution. By mixing these additives with a short shelf life just before feeding the media stem solution to the reactor system, the concentration of these time-critical additives in the reactor can be adjusted simply and effectively.

The disposable tube system may include a static mixer that dilutes the media strain solution, preferably with high purity water, particularly at a 1: 1 concentration. By means of the static mixer, the media strain solution can be easily diluted to an appropriate concentration (in particular, in-line dilution methods are possible). According to an advantageous embodiment, the media stock solution is sterile-filtered prior to step f) of the process according to the invention. This reduces the risk of contamination of the reactor system. The sterile filtration takes place in particular directly before step f) in order to also catch germs which have been introduced in preceding steps. Alternatively, the sterile filtration can be done earlier, for example, when introduced into the disposable hose system.

According to a further advantageous embodiment, sensors are arranged in the disposable tube system which are used to control at least one of steps e) and f) of the method according to the invention by means of an automation unit. The automated regulation of these steps ensures a particularly high quality of the prepared media stock solution.

The object of the invention is also achieved by a device arrangement for providing a large amount of cell culture medium, which is set up for carrying out the method according to the invention. With regard to the advantages of the device arrangement according to the invention, reference is made to the corresponding statements on the method according to the invention. In a preferred embodiment, the device arrangement according to the invention comprises an electronic monitoring and control device, in particular a SCADA system, which is set up to adjust the concentration of the concentrated media stock solution in step a). The automatic setting eliminates the need for manual intervention.

Particularly advantageous is the use of sensors for determining the pH and / or the conductivity of the concentrated media stock solution. The monitoring and control device may then regulate the adjustment of the concentration of the concentrated media stock solution based on the data of the sensors. Such automated control, taking into account the parameter values provided by the sensors, continuously provides accurate, reproducible results without incurring on-going Operation settings or readjustments must be made by a user.

In the reactor installation, an automation unit is preferably provided for the automated regulation of the preparation of the media stock solution and / or the supply of the prepared media stock solution into the large reactor system. Via the automation unit additives can be added automatically and with high accuracy to the diluted media stock solution.

According to an advantageous aspect of the invention, aseptic connections and a static mixer which dilutes the media stock solution, preferably with highly pure water, in particular to a 1: 1 concentration, can be used to realize a disposable hose system for the reactor system, in particular by a sensor for determining the flow rate can be supplemented. Under aseptic connections are to be understood here hose connections or connections of disposable components, which by means of aseptic connectors or z. B. can be realized by welding TPE tubing or similar measures.

Preferably, the disposable containers with gamma radiation can be sterilized, whereby they can be sterilized with little effort before filling and disposed of properly after emptying.

The reactor system may comprise a disposable bioreactor made of a suitable plastic or a stainless steel reactor. That is, the method is also suitable for hybrid reactor systems with existing stainless steel equipment.

Further advantages and features will become apparent from the following description taken in conjunction with the accompanying drawings. The single FIGURE shows a schematic representation of the devices used and the sequence of the method according to the invention. In the figure, the device arrangement according to the invention and the method according to the invention, by means of which a large amount of Cell culture medium 10 is provided in the form of a stock solution in a large reactor system 12, shown schematically.

The large reactor system 12 comprises a reactor 14 and is part of a reactor unit 16, which further comprises an automation unit 18, a sterile filtration unit 20 and a WFI (Water For Injection) unit 22, which provides high purity water 24. The reactor system 12, the automation unit 18 and the WFI system 22 are connected to each other via a disposable hose system 26, which has a static mixer 28 and a plurality of sensors 30. The sensors 30 are provided in particular for determining the flow rates, the pH values and the conductivity. Also, the pressure determination is of interest, especially with regard to a safety shutdown. Furthermore, sensors 30 may be provided for optical spectroscopic identification and quantitative analysis methods. The reactor 14 is a stainless steel reactor with a working volume of 10,000

Liters, which, in contrast to the one-way components used in the process, will continue to be used for subsequent productions. The reactor system 16 is therefore a hybrid system.

In a first step of the method according to the invention, a concentrated media stock solution 32 is produced outside the reactor system 16, for example at a media manufacturer. The concentrated media stock solution 32 is recovered from powdered and / or liquid components 34 which are provided in tubular bags 36.

The concentrated media stock solution 32 can be produced in a disposable mixing system (not shown).

The concentration of the concentrated media stock solution 32 is adjusted via a SCADA (Supervisory Control and Data Acquisition) system 38 and controlled by means of sensor data of the pH value and the conductivity. The concentrated media stock solution 32 is produced with a particularly high concentration of 10: 1, ie each volume unit of the media stock solution 32 contains only very little water. In a next step, the concentrated media stock solution 32 is filled into sterile disposable containers 40. The disposable containers 40 each hold a volume of about 200 liters and can be transported by a person with a trolley 42. Furthermore, the disposable containers 40 are sterilizable with gamma radiation, whereby they can be sterilized in a simple manner before filling.

Thereafter, the disposable containers 40 filled with concentrated media stock solution 32 are intermediately stored before being transported to the reactor system 12. In an optional step, the concentrated media stock solution 32, in particular shortly before being transported to the reactor system 12, is further diluted and / or further additives are added to the concentrated media stock solution 32 in order to meet the individual requirements of the reactor system 12 or of the user of the media stock solution 32 , The additives may in particular have components with a limited shelf life or service life, which is why they are not suitable for intermediate storage and therefore are only supplied shortly before transport.

Alternatively, further additives of the concentrated media stock solution 32 can also be mixed directly during production, in particular if the properties of the concentrated media stock solution 32 do not change adversely until they are fed into the reactor system 12.

After this preparation of the concentrated media stock solution 32, it is again filled into corresponding, sterilized disposable container 40. The concentration of the concentrated media stock solution 32 is used in the

Production in the first step or in the optional step in the preparation of the media stock solution 32 matched to the volume of the reactor 14 and the automation unit 18 so that the required amount of cell culture medium 10 can be provided by the concentrated media stock solution 32 without modification of the reactor system 16.

After filling the prepared or prepared concentrated media stock solution 32 and after the optional intermediate storage, the transported to the media system 32 filled disposable container 40 to the reactor system 12.

In a subsequent step, the disposable containers 40 are aseptically connected to the reactor system 12 by means of the disposable tube system 26. The media master solution 32 is processed in a next step.

Here, the medium strain solution 32 is diluted with high purity water 24 from the connected WFI plant 22 by means of the static mixer 28 to a 1: 1 concentration. In addition, the thinned media tree solution 32 is metered with additives 44 via the automation unit 18. These additives 44 contain, in particular, unstable chemical components which, due to their short lifetime, are added to the media stem solution 32 only at this point in time in order to be able to ensure a certain concentration of these additives 44 in the reactor system 12.

Of course, less critical additives 44 can be supplied in this step, the media strain solution 32 or additives 44, which are available only to the user of Medienstammlosung 32, for example, due to licenses or safety regulations.

Subsequently, in a further step, the prepared media strain solution 32 is passed into the sterile filtration device 20 and sterilized by filtration.

In the last step, the sterile-filtered, processed media strain solution 32 is fed to the reactor system 12 and is now available, for example, for the cultivation of animal cells.

The automation unit 18 monitors all the above-mentioned steps that take place within the reactor system 16 and regulates the entire process by means of the sensor data which are transmitted to the automation unit 18 by the sensors 30. This ensures high process reliability.

In this way, the method according to the invention makes it possible to provide a large amount of stock solution in a large reactor system 12, which in particular is part of a hybrid system, and at the same time the transport effort and the risk of contamination of the reactor system 12 to reduce. In the embodiment described above, the method according to the invention provides 10,000 liters of cell culture medium by means of a few disposable containers with a capacity of only 200 liters each.

Claims

claims

1 . A method for providing a large amount of cell culture medium (10), in particular for cultivating animal cells, for a large reactor system (12) using disposable components, comprising the following sequence of steps: a) producing a concentrated media stem solution (32), b) filling c) transporting the disposable container (40) filled with media stem solution (32) to the large reactor system (12), d) aseptically connecting the disposable containers (40) to the large reactor system (12) a disposable tubing system (26), e) conditioning the medium stem solution (32) by supplying high purity water (24) and / or additives (44), and f) feeding the processed media stem solution (32) into the large one

Reactor system (12).

2. The method according to claim 1, characterized in that in step a) the concentrated media strain solution (32) on the volumes of subsequent mixing systems (18) and / or the reactor volume is tuned.

3. The method according to any one of the preceding claims, characterized in that in step a) the concentrated media strain solution (32) has a concentration of 5: 1 or higher.

4. The method according to any one of the preceding claims, characterized in that in step b) the concentrated Medienstammlosung (32) in transportable disposable containers (40) each having a volume of 1 L to 500 L, preferably 5 L to 200 L is filled.

5. The method according to any one of the preceding claims, characterized in that between step a) and step b) the following steps are carried out: g) filling the concentrated media stock solution (32) into disposable containers (40); and h) preparing the media stock solution (32) by supplying high purity water and / or additives.

6. The method according to any one of the preceding claims, characterized in that in step e) unstable chemical components of the media stock solution (32) are supplied.

7. The method according to any one of the preceding claims, characterized in that the disposable hose system (26) comprises a static mixer (28) which dilutes the media stock solution (32), preferably with high purity water (24), in particular to a 1: 1 concentration.

8. The method according to any one of the preceding claims, characterized in that the media strain solution (32) is sterile-filtered prior to step f).

9. The method according to any one of the preceding claims, characterized in that in the disposable hose system (26) sensors (30) are arranged, which are used to control at least one of steps e) and f) by means of an automation unit (18).

10. Device arrangement for providing a large amount of cell culture medium (10), characterized in that the device arrangement is arranged for carrying out the method according to one of the preceding claims.

1 1. Device arrangement according to Claim 10, characterized by an electronic monitoring and control device, in particular a SCADA system (38), which is set up to adjust the concentration of the concentrated media stock solution (32) in step a).

12. A device arrangement according to claim 1 1, characterized by sensors for determining the pH and / or the conductivity of the concentrated media stock solution, wherein the monitoring and control means is adapted to adjust the concentration of the concentrated media stock solution (32) based on data of To regulate sensors.

13. Device arrangement according to one of claims 10 to 12, characterized by an automation unit (18) for the automated control of the preparation of the Medienstammlosung (32) and / or the supply of the processed Medienstammlosung (32) in the large reactor system (12).

14. Device arrangement according to one of claims 10 to 13, characterized by a disposable tube system (26) with aseptic connections, a static mixer (28) which dilutes the media stem solution (32), preferably with ultrapure water (24), in particular to a 1: 1 concentration.

15. Device arrangement according to claim 14, characterized by a

Sensor (30) for determining the flow rate.

16. The method according to any one of claims 10 to 15, characterized in that the disposable containers (40) are sterilized with gamma radiation.

17. The method according to any one of claims 10 to 16, characterized in that the reactor system (12) comprises a disposable bioreactor (14) made of a suitable plastic.

18. The method according to any one of claims 10 to 16, characterized in that the reactor system (12) comprises a reactor (14) made of stainless steel.