WO2011144561A1

WO2011144561A1 - Methods for welding ethylene vinyl acetate (eva) tubing, tubing obtained thereby and use of such tubing for sterile transfer of content into a bioreactor

Info

Publication number: WO2011144561A1
Application number: PCT/EP2011/057862
Authority: WO
Inventors: Alfred Luitjens; Herman Van Herk; Agnieszka ÖNDER
Original assignee: Crucell Holland B.V.
Priority date: 2010-05-18
Filing date: 2011-05-16
Publication date: 2011-11-24

Abstract

The present invention relates to the field of cell culturing, in particular it relates to methods for welding tubing for instance of a cell freezing bag to a bioreactor and transferring the content of said bag into the bioreactor.

Description

Methods for welding ethylene vinyl acetate (EVA) tubing, tubing obtained thereby and use of such tubing for sterile transfer of content into a bioreactor

Background of the invention

Processes for culturing and infecting cells at large scale have been developed in order to satisfy the increased demand of cell based pharmaceutical products, e.g. vaccines. Process optimization is used to decrease process times and therewith the time to market and costs of said products.

In order to accelerate processes for culturing cells in large scale bioreactors, one strategy is to start the culture with a large inoculum. This can be achieved with the use of flexible storage container containing large volumes of cells. The use of said flexible storage containers in standard batch processes allow for substantial production time reduction. On one hand the flexible storage container allow for large volumes of cells to be inoculated at once, therewith reducing the preculture phase. On the other hand the use of flexible storage containers reduces the number of open handlings that are needed during the preculture phase, therewith reducing the contamination risks during the production process. Reported process failures due to contaminations are normally in range of 7%, and costs of these failures may exceed $1-2 billion (Eric S. Langer GEN, Vol.28, no 14).

Flexible storage containers, also named cell freezing bags are described in more detail in e.g. WO2006/069389. Said bags generally comprise at least a compartment for the cells and two tubes attached to it, one is the filling line which is used to fill the bag before storage, the other tube is the inoculation line, which is used to transfer the content of the bag into a bioreactor.

For optimal use in cell culture processes, it should be possible to store bags at typical cell suspension storage temperatures and weld the bags directly onto a bioreactor subsequently after thawing. Therefore said bags and in particular the attached tubing must be resistant to very low temperatures e.g. -70°C or -135°C, in order to allow for the cells they contain to be stored in respectively a standard cell freezer or the vapor phase of liquid Nitrogen. In addition, it should be possible, subsequently after thawing, to weld the attached tubing, in particular the inoculation line, to a similar tubing on a bioreactor.

A limitation of many commercially available cell freezing bags is the extensive use of polyvinyl chloride (PVC) tubing for filling and draining the bags. PVC tubing is brittle at typical cell suspension storage temperatures, and the plasticizer used in making PVC materials is known to leach out of the plastic and into the surrounding media. Both of these limitations make PVC an undesirable choice for these cell freezing bags. Another limitation of these cell freezing bags is that they are emptied via a Luer-Lock or membrane-covered port involving an open handling which is a potential source of contamination. A further limitation of commercially available cell freezing bags is that they often contain tubing that is not appropriate for sterile welding.

Ethylene vinyl acetate (EVA) is a suitable material for cell freezing bags commercially sold for this purpose. Moreover, methods for producing bags, wherein films of EVA are sealed to eachother have been described e.g. in Canadian patent 1138817. However, it could not have been derived from said patent that tubes (as opposed to films) made of EVA could be welded to eachother.

A widely used procedure in the art is to open thawed cell freezing bags in a laf cabinet by e.g. cutting the inoculation line (which cannot be welded) with a sterile blade and transfer the content of the bag in a sterile container which contains a tubing that can be welded sterilely to a bioreactor. This requires a trained person to perform an open handling which brings a considerable risk of contamination.

There is a need in the art for cell freezing bag tubing that consist of materials which are appropriate for storage at typical cell suspension storage temperatures and which can be welded sterilely.

Summary of the invention

We have developed a method for welding two pieces of EVA tubing together. It was not known hitherto that EVA tubing could possibly be welded. Indeed, hitherto, the transfer of the content of stored EVA cell freezing bags into a bioreactor, required the procedure with open handlings described in the background section above. The instant invention enables the sterile transfer of content from the EVA cell freezing bag with EVA tubing without open handlings, therewith reducing the possibility for

contamination to virtually zero. The present invention relates to a method for preparing a piece of ethylene vinyl acetate (EVA) tubing comprising the step of welding a first piece of EVA tubing to a second piece of EVA tubing transversely of the axis of each EVA tubing at a temperature ranging between 96°C and 106°C. The method further comprises the steps of a) flattening a section of each piece of tubing to urge inside walls of each piece of tubing into contact, b) urging a hot cutting means, which has a temperature ranging between 96°C and 106°C, through the flattened section of each piece of tubing thereby temporarily sealing together the inside walls of each piece of tubing and providing molten tube ends, c) aligning the pieces of tubing to be connected with each other, d) joining the molten ends of said pieces of tubing together to form a joint between said pieces of tubing, and e) cooling the joint and then applying stress to the joint to open the weld in the tubing, thereby providing communication between the joined pieces of EVA tubing.

In a preferred embodiment one of said pieces of EVA tubing is connected to a cell freezing bag. In another preferred embodiment one of said pieces of EVA tubing is connected to a bioreactor.

Another aspect of the invention relates to a method for direct and sterile transfer of a substance into a bioreactor comprising the steps of: a) providing a cell freezing bag containing said substance and comprising a first piece of EVA tubing, b) providing a bioreactor comprising a second piece of EVA tubing, c) welding the first and second piece of EVA tubing sterilely together according to a method of any one of the preceding claims, and d) transferring the content of the cell freezing bag into said bioreactor.

In a preferred embodiment of the present invention said substance is a cell suspension. In another preferred embodiment said substance comprises virus.

Another aspect of the present invention relates to a piece of EVA tubing comprising a weld that was obtained by using a method as described hereabove. Another part of the invention relates to a combination of a cell freezing bag and a bioreactor characterized in that they are sterilely connected by a piece of EVA tubing comprising a weld that was obtained by using a method as described hereabove.

Brief description of the figures

FIG. 1. Standard virus production process compared with new process using weldable cell freezing bags with EVA tubing. FIG. 2. Cell growth in a 10L bioreactor after inoculation from a cell freezing bag. The cells were stored in a freezer, as a large volume high density (LVHD) working cell bank (WCB), in a cell freezing bag comprising an EVA tubing wich was directly and sterilely welded to an EVA tubing on said bioreactor.

Description of illustrative embodiments

"Bioreactor", as used herein, refers to a vessel containing a controlled environment for growing cells. Bioreactors are also referred to as fermentors. The "Wave" reactor is an example of a bioreactor; a bioreactor may comprise rigid materials (e.g. certain piped items) and/or a flexible container.

Ethylene vinyl acetate (EVA), is a flexible thermoplastic material. EVA is used as a thermoplastic material for making cell freezing bags, in particular for making the tubing attached to said bags, that is used for filling and draining the bags. EVA tubing is relatively cheap as compared to other thermoplastic material and is resistant to typical cell suspension storage temperatures (e.g. -70°C or -135°C).

"C-flex® tubing", as used herein refers to a type of thermoplastic elastomer that consists primarily of styrene ethylene-butylenestyrene block copolymer. C-flex® tubing is commonly used in the field of cell culturing since it can easily be welded with e.g. a SCD® welder. C-flex® is welded at a temperature around 200°C. C-flex® has two important disadvantages. It is expensive and it is not resistant to storage at typical cell suspension storage temperatures.

"Cell freezing bag", "flexible storage container", "storage bag" and "bag" are used interchangeably herein and refer to a flexible container, typically an ethylene vinyl acetate (EVA) fabric bag, which is usually used for storage of a cell suspension, which is typically a mammalian cell population. Said bags generally comprise at least a compartment for the cell suspension and two pieces of tubing attached to it, which are also made principally from EVA. One is the filing line which is used to fill the bags before storage, the other tube is the inoculation line, which is used to transfer the content of the bags into e.g. a bioreactor in a direct and sterile way. Instead of or in addition to cells, the bags may contain other contents that may be sterilely added to a bioreactor, e.g. viruses, culture medium components or additives, etc.

"Welder" or "sterile weld device" are used interchangeably herein and refer to a device for welding (or joining) two thermoplastic tubes together. Welders used in the present invention are e.g. described in US4610670 included herein by reference. The welder comprises a cutting means, means adapted to heat said cutting means, a pair of mounting blocks adapted to receive, hold and flatten the tubes to be joined, means to provide movement between said blocks and said cutting means to a position such that the cutting means is between said blocks and traversing where the blocks are adapted to hold tubes, means for realigning the blocks to a position where two different tube ends are aligned with and facing each other, means to separate said blocks and said cutting means, and means for urging the mounting blocks together (see e.g. figures 1-3 of US4610670).

In certain embodiments of the present invention the tubes to be joined are flattened in an appropriate section so that the inside walls meet. Then the tubes are sequentially or simultaneously melted through by a hot cutting means with molten polymer temporarily sealing the resulting molten tube ends. Since the tubes are temporarily sealed viable airborne or surface bacteria are unable to find their way inside either of the tubes. The tubes are moved into alignment before or after the heated cutting means is slid away and then the molten tube ends are pushed together to form a joint. The joint is briefly cooled and then subjected to slight stress to open the temporary seal in each tube, hereby obtaining a weld and providing

communication between the joined pieces of tubing. The weld is sound and strong and a number of additional welds can be made in subsequent sterile connections with the same tube. The pieces of tubing that are welded together can either be filled with air or with a liquid. In both cases, the pieces of tubing are welded together in a sterile way, providing a fluid or air-to-air communication between the tubes.

"Weld" or "joint" are used interchangeably herein. A "joint" generally refers to a connection between two molten thermoplastic tubes. After said "joint" is cooled down and subjected to slight stress, a "weld" is obtained, which provides a communication between the tubes.

Sterile welding of tubing is common practice in biopharmaceutical production processes. Several sterile weld devices are commercially available, like e.g. the SCD IIB™ from Terumo biotech or the Bio Welder™ from Sartorius. These systems are designed to weld thermoplastic tubings like C-flex®, Sanipure®, Pharmed®, and Bioprene.

We have surprisingly found herein that EVA tubing can also be directly used for welding, leading to sterile and strong welds. This could be achieved using a known weld device, e.g. the Bio Welder® from Sartorius, wherein the hot cutting means has a temperature ranging between 96°C and 106°C. This temperature is in a completely different range than normally used for other types of tubing (e.g. C-flex® welds are made at about 200°C).

In certain embodiments the temperature of the hot cutting means ranges from about 96°C to about 105°C, 104°C, 103°C, 102°C, lOFC or 100°C.

In certain embodiments the temperature of the hot cutting means ranges from about 97°C, 98°C, 99°C, 100°C, 101°C or 102°C to about 106°C.

In certain embodiments, the hot cutting means has a temperature of 96°C, 97°C, 98°C, 99°C 100°C, 101°C, 102°C, 103°C, 104°C, 105°C or 106°C.

Flexible storage containers and use thereof

In certain embodiments, the present invention uses cell freezing bags or flexible storage containers, which are generally used for storing cell populations. The present invention can be used to generate master cell banks (MCB), manufacturer working cell banks (MWCB) and/or research cell banks (RCB's). The bag is typically constructed principally of ethylene vinyl acetate and it is designed to hold a sufficient volume of cell suspension to ensure that a bioreactor can be inoculated directly from its content. The bag is designed to be filled to a fraction of its maximum capacity so that when placed on its side, the cell suspension has a very thin cross-section of not more than about 10 mm. When cell freezing bags of the present invention are used, they will be placed on their side in a metal cassette (also referred to as a box or canister) in a substantially level orientation. If the cell suspension cross-section in the bag is substantially more than 10 millimeters, the cells adjacent to the bag surface may experience different freezing and thawing conditions than cells at the interior of the suspension, and may react differently over the course of freezing and thawing and when subsequently used in a bioreactor. The bag design includes a set of tubes that can be sterilely welded to the source or destination of the cells. These tubes allow the bags to be filled or emptied quickly with minimal risk of contamination.

When a bioreactor is to be inoculated, a cell freezing bag according to the invention can be thawed and welded sterilely to said bioreactor. It is the merit of the present invention to provide for a flexible storage container that, subsequently after thawing, can be directly and sterilely welded to a bioreactor. This was achieved with the use of EVA tubing which can be stored at temperatures resistant to typical cell suspension storage temperatures (e.g. -70°C or -135°C) and which can be welded sterilely when being heated at a temperature ranging from 96°C to 106°C.

According to the present invention, the inoculation of the bioreactor is achieved by sterilely welding the inoculation line of the cell freezing bag, which consists essentially of EVA, with an EVA tubing connected to the bioreactor.

In one embodiment of the present invention, said EVA tubing on the bioreactor side is directly connected to the bioreactor e.g. when said bioreactor is a wave bag and said EVA tubing is mounted on the wave bag before sterilization of the wave bag and all its tubing connections.

In another embodiment of the present invention, said EVA tubing is not directly connected to the bioreactor e.g. when said bioreactor is a glass bioreactor. Instead it can be connected to another piece of tubing e.g. a C-flex® tubing, that had previously been connected to the bioreactor before sterilization (at e.g. 121°C). In order to do so in a sterile manner, a C-flex® - EVA tube set, that is a piece of C- flex® tubing connected to a piece of EVA tubing, can be assembled and sterilized first e.g. by Gamma irradiation. Subsequently the C-flex® - EVA tube set can be welded sterilely to the C-flex® tubing on the bioreactor leaving the EVA part of the tube set unconnected. After thawing a cell freezing bag according to the invention, the EVA tubing of the bag can be directly and sterilely welded to the remaining free EVA tubing on the bioreactor, using the methods of the invention.

In both embodiments an open handling is circumvented. Hitherto, the content of a cell freezing bag that was stored in a freezer could not be welded directly and sterilely to a bioreactor. Instead, its content had to be transferred in another container that comprised a tubing that could be welded directly to a bioreactor e.g. a C-flex® tubing. Said transfer had to take place in a laf hood requiring an open handling and therewith increasing the risk of contamination.

The introduction of an "EVA-EVA weld" into a cell culture or virus production process according to the invention allows the complete production process to become fully closed, and thereby substantially reducing the risk of contamination. Furthermore, the first steps of the preculture are deleted from the process, which results in shorter production times.

The cell freezing bags are typically stored in freezers at typical cell suspension storage temperatures (e.g. -70°C). The bags can also be stored in the gas phase of a freezer containing liquid Nitrogen. The temperature in said gas phase is around -135°C. Storage in the vapor phase prevents transfer of contaminants that can occur during storage in the liquid Nitrogen. Because of its flexibility at low temperature, and in turn the reduced possibility of low temperature fracture, the EVA fabric provides additional protection to the contents of the cell freezing bag during freezing, long-term storage, and thawing.

The cell freezing bag may be designed to hold approximately between 10 mL and 1 L, more particularly 10 mL and 300 mL, more particularly between 20 mL and 200 mL, more particularly between 50 mL and 150 mL, more particularly approximately 100 mL of cell suspension. The cell densities contained in the cell freezing bags are generally similar to the cell densities in vials that are currently used. The cell freezing bag can thus contain an increased volume of cell suspension as compared to vials that are commonly used as a cell bank. Because of this volume difference, the bags can hold, in certain embodiments, approximately 100 times more cells than a vial. In addition, the cells may be stored in the cell freezing bags at very high cell densities, allowing even more cells to be stored in the same volume.

Processes for culturing cells at high cell densities are disclosed in e.g. Yallop et al. or WO 2005/095578. Said reference also discloses bioreactors that contain high cell density cultures. The content of such bioreactors can be drained in several bags in order to obtain a high cell density cell bank which is contained into different cell freezing bags.

In a certain embodiment, the frozen cell suspension has a cell density of 30xl0⁶ cells/mL; thus, in this embodiment, 30xl0⁹ cells total may be stored in a single bag. A single cell freezing bag contains enough cells to allow for direct inoculation of a large bioreactor. Indeed, bioreactors with working volumes up to 50L can be inoculated with a single bag according to the invention. Herewith, a lengthy preculture process as was commonly used hitherto is circumvented. Fig. 1 shows the comparison of a standard preculture process which contains a multitude of steps, with a single preculture step allowed by the flexible storage containers.

According to the invention cell freezing bags usually contain cell densities between lOxlO⁶ and 150xl0⁶ cells/ml. In one embodiment said cell freezing bags contain cell densities between lOxlO⁶ and 80x10⁶ cells/ml, preferably between 20x10⁶ and 40xl0⁶ cells/ml and even more preferably between 25xl0⁶ and 35xl0⁶ cells/ml. In a preferred embodiment of the present invention, the storage bags are filled with high cell density culture and contain cell cultures with cell densities up to 30xl0⁶ cells/ml. Large volume high density (LVHD) WCB can thus be obtained and according to the invention such LVDH WCBs can be sterily transferred into a bioreactor.

In order to efficiently produce a cell bank, several bags can be assembled on a large filling line which allows for many bags to be filled at once. The filling line is usually made of C-fiex® and can be connected to a bioreactor in order to drain the content of the bioreactor in the cell freezing bags. The design of cell freezing bags typically is such that all tubing that can become brittle at low temperature (e.g. C- fiex®) is sealed off from the bag after filling. The only tubing that remains connected to the bag after filling is ethylene vinyl acetate (EVA) tubing, which can resist storage at ultra low temperature. Subsequently after storage in a freezer said bags can be used to inoculate a bioreactor. The present invention shows that cell freezing bags with EVA tubes, can be used for storing high cell density cultures and subsequently be used to inoculate bioreactors with large working volumes up to 50L.

In other embodiments, the flexible storage containers can be used to store virus seeds. Virus seeds contained in cell freezing bags can be obtained by draining a bioreactor that was infected by a virus and in which said virus has propagated. After thawing, the bags can be used to infect cell cultures in a bioreactor in a direct and sterile manner. Herewith the safety of the infection process is substantially increased. Also, flexible storage containers which contain up to 500ml of virus suspension allow for the infection of bioreactors with high working volumes up to 2000 L.

Hitherto the infection of a bioreactor required several open handlings, enhancing the risk of contaminations. The use of said cell freezing bags in standard infection processes allow for substantial production time reduction. The use of cell freezing bags reduces the number of open handlings that are needed during the preparation of a virus solution for infection, therewith reducing the contamination risks during the virus production process.

A part from the propagation of viruses, the cells in bioreactors can be used to produce biological material, e.g. recombinant proteins and antibodies. Examples

Example 1: EVA - EVA tube welding

EVA tubing was hitherto considered unsuitable for direct welding. We herein surprisingly show that EVA tubing can be welded, and provide the conditions.

Welding temperatures for EVA tubing were tested in the range of 86°C to 110°C. For this experiment the Biowelder™ from Sartorius was used. Standard set points of the Bio Welder™ were used in this experiment. The dimensions of the EVA tubing used in the experiment were: ¼" x 5/16". The weld was examined on:

• Alignment of the welded tubes

· Strength of the connection

• Leakage

The best welds between two pieces of EVA tubing were obtained at a blade temperature ranging between 96°C and 106°C.

Example 2: Production of a working cell bank from a 10L bioreactor Vials containing PER.C6® cells were thawed and propagated in serum free culture medium in a humidified incubator at 37°C and 10% C02. Subculture was performed every 3 to 4 days in T-flasks and roller bottles until a sufficient cell density was reached to inoculate a 10L bioreactor at a cell density of 0.58xl0⁶ viable cells/ml. Cells in the 10L bioreactor were propagated in serum free medium at 37°C, a DO of 40% and a pH of 7.3. When a cell density of approximately 2.15xl0⁶ viable cells was reached (~60 hours post inoculation) an ATF system (Furey et al.) was started. 212 hours post inoculation of the 10L bioreactor a cell density was reached of 30xl0⁶ viable cells/mL. At this moment the cell suspension was concentrated with a factor of 2 to a 5L volume. This concentration step was performed with the ATF system in approximately 1 hour. Subsequently, 5L freezing medium was added to the bioreactor. This freezing medium consists of 15% DMSO, 5% Sucrose and 80% serum free medium. During addition of the freezing medium the ATF system was stopped and the bioreactor was cooled with cooling blocks. After addition of freezing medium the cell freezing bags were connected to the bioreactor and filled with lOOmL high cell density suspension. In total 90 bags were filled. The bags were frozen in a blast freezer until they reached a temperature of -40°C. At this point the bag were taken out of the blast freezer and transported on dry ice to the liquid nitrogen storage containers. The bags were stored in the vapor phase of liquid nitrogen until further use.

Example 3: recovery of a large volume high density WCB stored in a liquid nitrogen freezer

A 10L bioreactor was prepared according to standard procedures. One port was dedicated to be used as inoculation port for a bag containing a large volume high density WCB, which was prepared as disclosed in example 2 and which was stored in the vapor phase of Nitrogene at -135 °C. The inoculation port consisted of C-flex® tubing. After sterilization of the 10L bioreactor at 121°C in an autoclave, the dedicated inoculation port was connected with a SCD tube welder to a C-flex® - EVA tube set. Said C-flex® - EVA tube set was previously sterilized by gamma irradiation at Isotron (Ede, the Netherlands). 10L of serum free medium was transferred to the 10L bioreactor. After medium transfer, a cell freezing bag containing a large volume high density WCB was quickly thawed in a water bath of 37°C. After thawing the cell freezing bag was connected to the inoculation port of the 10L bioreactor. The weld between the EVA tube of the 10L inoculation port and the EVA tube of the cell freezing bag was performed with a Bio Welder™ from Sartorius. The blade temperature of the Bio Welder™ was 104°C. After the weld was made the cell suspension of the cell freezing bag was transferred to the 10L bioreactor. The cell suspension was propagated in serum free medium at 37°C, a DO of 40% and a pH of 7.3 for 7 days. Fig. 2 represents the growth curve of the cell suspension and shows that cells originating from a cell freezing bag that was stored in a freezer at -135 °C could successfully be transferred and cultured into a bioreactor.

References

Furey J. Scale-up of a cell culture perfusion process - A low-shear filtration system that inhibits filter-membrane fouling. Genetic Engineering News. Vol. 22, No. 7, April 2002.

Yallop C, Crowley J, Cote J, Hegmans-Brouwer K, Lagerwerf F, Gagne R, Martin

JC, Oosterhuis N, Opstelten DJ, Bout A. Per.C6 cells for the manufacture of biopharmaceutical proteins. Modern Biopharmaceuticals - Design, Development and Optimization. Vol. 3, 2005

Claims

A method for preparing a piece of ethylene vinyl acetate (EVA) tubing

comprising a weld from a first piece of EVA tubing and a separate second piece of EVA tubing, the method comprising the step of welding the first and second piece of EVA tubing together transversely of the axis of each EVA tubing at a temperature ranging between 96°C and 106°C.

2. A method according to claim 1 comprising the steps of:

a) flattening a section of the first and second piece of tubing to urge inside walls of the first and second piece of tubing into contact,

b) urging a hot cutting means, which has a temperature ranging between 96°C and 106°C, through the flattened section of each piece of tubing thereby temporarily sealing together the inside walls of each piece of tubing and providing molten tube ends,

c) aligning the pieces of tubing to be connected with each other, d) joining the molten ends of said pieces of tubing together to form a joint between said pieces of tubing, and

e) cooling the joint and then applying stress to the joint to open the weld in the tubing, thereby providing communication between the joined pieces of EVA tubing.

3. A method according to claim 1 or 2 characterized in that one of said pieces of EVA tubing is connected to a cell freezing bag.

4. A method according to any one of claims 1 or 3 characterized in that one of said pieces of EVA tubing is connected to a bioreactor.

5. A method for direct and sterile transfer of a substance into a bioreactor

comprising the steps of:

a) providing a cell freezing bag containing said substance and comprising a first piece of EVA tubing,

b) providing a bioreactor comprising a second piece of EVA tubing, c) welding the first and second piece of EVA tubing sterilely together according to a method of any one of the preceding claims, and d) transferring the content of the cell freezing bag into said bioreactor.

6. A method according to claim 5 wherein said substance is a cell suspension.

7. A method according to claim 6 wherein said substance comprises virus.

8. A piece of EVA tubing comprising a weld, obtainable by a method according to any one of claims 1 to 4.

9. A combination of a cell freezing bag and a bioreactor characterized in that they are sterilely connected by a piece of EVA tubing according to claim 8.