Multiport devices to connect the loop to one port of a bioreactor and in perfusion or concentrated fed-batch configurations for performing upstream steps in cell culture

Abstract

A multiport device (42) for connecting a loop (36), preferably a tangential flow filtration loop or a sensor loop, to a port (34) of a bioreactor (32), preferably a port of a single-use bioreactor. The multiport device is configured to be fixed to a port of the bioreactor. The multiport device comprises a first flow path (44) configured to withdraw fluid from the bioreactor and a second flow path (52) configured to supply fluid to the bioreactor. The first flow path has a first end (48) suitable for fluid connection with the bioreactor and a second end (50) suitable for connection with an inlet of the loop. The second flow path has an outer end (64) suitable for connection with an outlet of the loop and a mouth (62) suitable for fluid connection with the bioreactor. The mouth of the second flow path is at least 5 mm, preferably 10 mm, away from the first end of the first flow path.

Description

[Technical field]

[0001] The present invention relates to a multiport device for connecting a loop, preferably a tangential flow filtration loop, to one port of a bioreactor, preferably a single-use bioreactor.Furthermore, the present invention relates to a perfusion or concentrate fed-batch arrangement for carrying out upstream steps of cell culture. [Background technology]

[0002] In today's cell culture industry, high yields are required in the manufacturing process of biopharmaceuticals in order to reduce the cost of production and ultimately the cost of therapy. For several years, it has been particularly desirable to achieve high yields (high cell densities) in the upstream process of mammalian cell culture. Several developments have made it possible to apply hollow fiber or flat filtration devices for cell and / or product retention in order to obtain high cell densities and high product titers. The applied techniques are described as perfusion and concentrated fed-batch. Perfusion is well established in the market and has been used in the past, especially for sensitive products.

[0003] For the application of hollow fiber devices in tangential flow filtration (TFF) mode for perfusion / concentration fed-batch processes, it is important that at least two ports are required for connection to the bioreactor. This is especially important for single-use bioreactors, where the number of ports is usually limited. In single-use bioreactors, the ports are usually integrated by welding. However, the welds (seams) are associated with the risk of leakage. Therefore, it is desirable to have as few ports as possible. This is especially true for single-use bioreactors made of plastic.

[0004] To overcome the problem of the number of ports required, a technology called alternating tangential flow (ATF) has been developed. The basic concept of ATF is described, for example, in U.S. Patent No. 6,544,424. However, ATF has a general problem in that it cannot pump all of the contents of the hollow fiber filter unit into the bioreactor.

[0005] Another consideration is that ATF technology requires fairly complex and very expensive controls, and if ATF could be replaced by TFF, a simpler and less expensive control could be used.

[0006] In addition, the use of TFF method has the advantage of less stress on mammalian cells and less variable process conditions compared to ATF. Most importantly, TFF provides a more constant flow across the membrane surface, which reduces or delays fouling in the membrane and provides a more constant capacity throughout the perfusion. Fouling also leads to a reduced yield of the desired product in the permeate. Therefore, TFF is preferred over ATF. The challenge of providing the large number of ports required for the process remains.

[0007] From EP 3 460 036 A1 a system for transferring chemical, pharmaceutical and / or biological material to or from a container is known. The system comprises a disposable container. The disposable container has at least one port for accessing the interior of the container. Furthermore, the system comprises a transfer interface connectable to the at least one port. The transfer interface comprises a plurality of extendable transfer members for collecting samples from the disposable container in sterile conditions. Each transfer member has a corresponding biasing member for retracting the transfer member.

[0008] US Patent No. 7,875,448 shows a disposable bioreactor. The bioreactor comprises a vessel for holding a liquid culture, a diffuser disposed within the vessel, a discharge tube for removing the culture from the bottom of the vessel, and an inlet tube for returning at least a portion of the culture from the discharge tube through the diffuser to the vessel. The diffuser is disposed above the bottom of the vessel and is located completely within the liquid culture when the culture is held in the vessel. The diffuser disperses the culture returned to the vessel into a dispersed, wide stream. The diffuser mixes gas from a gas source outside the vessel with the returned culture before dispersing it into the vessel.

[0009] WO 2020 / 176990 relates to a tangential flow filtration system comprising a tangential flow filtration module and a reservoir. The reservoir has an inflow composition, such as a viscous fluid, solution, gel, paste, cream, or suspension. The system further comprises a tangential flow filtration inflow supply pipe configured to deliver the inflow composition from multiple depths in the reservoir to the tangential flow filtration module. The tangential flow filtration inflow supply pipe has multiple inlet ports arranged at different heights in the reservoir. The retentate return pipe is configured to return the retentate of the TFF from the tangential flow filtration module to the reservoir. Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of the present invention to enable a perfusion or concentrate fed-batch setup where a loop, in particular a TFF loop or a sensor loop, can be connected to a single port of a bioreactor. [Means for solving the problem]

[0011] According to one aspect of the invention, a multiport device for connecting a loop, preferably a tangential flow filtration loop or a sensor loop, to a port of a bioreactor, preferably a port of a single-use bioreactor, is configured to be fixed to the port of the bioreactor. The multiport device comprises a first flow path configured to withdraw a fluid from the bioreactor and a second flow path configured to supply a fluid to the bioreactor. The first flow path has a first end suitable for fluid connection with the bioreactor and a second end suitable for connection with an inlet of the loop. The second flow path has an outer end suitable for connection with an outlet of the loop and a mouth suitable for fluid connection with the bioreactor. The mouth of the second flow path is at least 5 mm, preferably 10 mm, away from the first end of the first flow path. Of course, the mouth of the second flow path is more reliably not directly interfering with any structural member in the bioreactor.

[0012] The main advantage of the present invention is that a bioreactor with only one port to which a cell retention device can be connected can be used to perform a perfusion or concentrate-fed-batch TFF process using a hollow fiber filtration unit, which would normally require two or more different ports to perform a perfusion or concentrate-fed-batch TFF process.

[0013] The mouth of the second flow path is at least 5 mm, preferably 10 mm, away from the first end of the first flow path. This ensures that the medium immediately returned from the loop to the bioreactor is not immediately sucked out again without a chance to mix properly with the remaining medium in the bioreactor. This is particularly important in the TFF system, where medium is simultaneously fed to and sucked out of the bioreactor, whereas in the ATF system medium is only fed and sucked out at one time. Furthermore, effective mixing of the returned medium in the bioreactor is important to save time and protect the cells. In general, it should be considered that the cells should not spend more time in the loop and should not pass through the pump more frequently than necessary.

[0014] In the context of the present invention, the term "flow path" should be understood to include only one fluid line or multiple fluid lines, meaning that the first and / or second flow paths may have more than one fluid line for the transfer of fluids from or to the bioreactor.

[0015] The multiport device of the present invention is preferably a one-piece device, possibly formed of the same material (i.e., not an interlocking assembly of parts), so that the multiport device can be used as a simple adapter between one available port of the bioreactor and the loop.

[0016] It has been found that a multi-port device design in which the distance between the first end of the first flow path and the mouth of the second flow path is at least three times the diameter of the port opening provides the desired effect of thorough mixing of the returned medium.

[0017] The second flow path may include at least one curved, bent or angled portion in order to position the mouth at a desired location in the bioreactor. However, the first flow path may also include at least one curved, bent or angled portion. In general, the curved or bent portion should be as smooth as possible to protect the cells in the flow path. This keeps the shear stress as low as possible. The angle of such a portion may usually be between 1 degree and 90 degrees, but is preferably between 2 degrees and 45 degrees, and particularly preferably between 5 degrees and 35 degrees.

[0018] According to a preferred design of the multiport device, the first flow path at least partially surrounds the second flow path, in which case part of the second flow path may be formed by a tube arranged inside a larger tube, which tube forms part of the first flow path.

[0019] According to another preferred design, the first flow passage is arranged next to the second flow passage, for example the first and second flow passages can be formed in different tubes, the walls of the tubes may be in contact with each other, especially in the region where the flow passages extend through the port.

[0020] The second flow path may include at least a flexible hose having a free end that forms a mouth of the second flow path. An advantage of a flexible hose in a bioreactor is its flexibility. Compared to a rigid tube, the risk of damage to the walls of the flexible bioreactor and components within the bioreactor during storage and transportation of the bioreactor in a non-deployed state can be significantly reduced.

[0021] The invention also provides a perfusion or concentration fed-batch arrangement for carrying out upstream steps of cell culture, comprising at least a bioreactor with ports, a loop, preferably a TFF loop or a sensor loop, a pump, a functional device, preferably a TFF unit such as a hollow fiber filtration unit or a sensor, and a multiport device as described above, which connects the inlet and the outlet of the loop to the bioreactor through the same port, with the advantages as described above.

[0022] It should be noted that the pump used in such an arrangement is capable of providing a nearly constant flow across the surface of the membrane, in contrast to the ATF approach where the concept of flow reversal causes the flow to slow to a complete stop and then reverse direction.

[0023] In an advantageous embodiment of the arrangement according to the invention, the port is located in a first wall of the bioreactor and the mouth is located near a second, opposite wall. The first end preferably has a different distance to the first wall compared to the mouth. Since mixing means are usually provided in the bioreactor, even small distances will have a flow effect if the distance from the first wall to the first end and the distance from the first wall to the mouth are not the same.

[0024] If the composition of the medium contained in the bioreactor changes with height, it may be advantageous to withdraw medium from one level of the medium and return it at a different level. This can be achieved by design and positioning of a multiport device in which the first end of the first flow path and the mouth of the second flow path are located at different heights within the bioreactor.

[0025] In a perfusion or concentrated fed-batch configuration, the bioreactor will usually have a mixing means such as a stirrer or impeller, in which case the mouth is preferably located very close to the mixing means, at least closer than the peripheral wall of the bioreactor.

[0026] If a mixing means is provided, the mixing means induces a flow in the bioreactor to achieve more reliable mixing. The mouth is preferably oriented such that the medium fed from the loop through the second flow path to the bioreactor flows through the mouth in a direction that essentially corresponds to the direction of flow in the bioreactor. However, in some cases it may be more useful if the mouth is oriented such that the medium from the loop is fed in a direction essentially opposite to the direction of flow.

[0027] According to advanced aspects of the invention, the multiport device can perform further additional functions. In particular, the multiport device can be used to deliberately influence the flow behavior of the medium in the bioreactor in a desired manner. This can be achieved by the design of baffles or flow guides surrounding the secondary flow path. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows a diagram of a perfusion setup using TFF as a method for retaining cells. [Diagram 2] FIG. 2 shows a perfusion setup using ATF as a method for retaining cells. [Diagram 3]FIG. 3 shows a simplified TFF perfusion setup with a multiport device according to the present invention. [Figure 4] FIG. 4 is a diagram showing a first embodiment of a multiport device according to the present invention. [Diagram 5] FIG. 5 is a schematic cross-sectional view of a second embodiment of a multiport device. [Figure 6] FIG. 6 is a schematic cross-sectional view showing a first modification of the third embodiment of the multiport device. [Figure 7] FIG. 7 is a schematic cross-sectional view showing a second modification of the third embodiment of the multiport device. [Figure 8a] 8a to 8c are cross-sectional views taken along line VIII-VIII in FIG. 7, showing other possible embodiments. [Figure 8b] 8a to 8c are cross-sectional views taken along line VIII-VIII in FIG. 7, showing other possible embodiments. [Figure 8c] 8a to 8c are cross-sectional views taken along line VIII-VIII in FIG. 7, showing other possible embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] A basic perfusion setup for upstream cell culture is shown in Figure 1. The setup includes a bioreactor 10 and a TFF loop 12. The bioreactor 10 has a means for mixing the cell culture fluid contained within the bioreactor 10. The TFF loop 12 includes a pump 14 for circulating the fluid from the bioreactor 10 through a hollow fiber filtration unit 16 for retaining the cells.

[0030] Cell culture medium is withdrawn from the bioreactor 10 through a first port 18 formed in the wall of the bioreactor 10 and returned to the bioreactor 10 through a second port 20 formed at another location in the wall of the bioreactor. The first port 18 can be considered as the outlet port of the bioreactor 10 or the inlet port of the TFF loop 12, while the second port 20 can be considered as the inlet port of the bioreactor 10 or the outlet port of the TFF loop 12. (For simplicity, the filtrate / permeate outlet of the TFF filter is not shown in any of the figures.)

[0031] 2 shows an alternative perfusion arrangement comprising a bioreactor 22 and an ATF module 24. The ATF module 24 includes a hollow fiber filter 26 and a diaphragm pump 28. The diaphragm pump 28 is used to draw cell culture medium from the bioreactor 22 through the hollow fiber filter 26 and back to the bioreactor 22 via the same route. This means that only one port 30 is needed to alternately drain the medium from the bioreactor 22 and return the filtered medium to the bioreactor 22.

[0032] In Figure 3, a perfusion arrangement similar to that shown in Figure 1 is shown. However, the bioreactor 32 in Figure 3, like the arrangement shown in Figure 2, only has one port 34 for providing the essential fluid connection with a TFF loop 36. The TFF loop 36 has a pump 38 and a hollow fiber filtration unit 40. In practice, both the outlet of the bioreactor (or the inlet of the TFF loop) and the inlet of the bioreactor (or the outlet of the TFF loop) are provided by a multi-port device 42 attached to the port 34.

[0033] The flow rate of the pump 38 has to be selected accordingly to ensure sufficient tangential flow on the membrane of the filtration unit 40. An increased overflow may be required during the process, so the flow rate has to be adjusted accordingly. The optimum flow rate depends on the specifications of the membrane used. Typical values ​​are up to 15000 L / h, depending on the size of the TFF device.

[0034] Another method for selecting an appropriate flow rate for pump 38 is to achieve a certain bioreactor / vessel volume exchange per day (vvd). Typical values ​​are 0.5-10 vvd, with a preferred range of 0.5-3 vvd.

[0035] 4 illustrates a first exemplary embodiment of a multi-port device 42. The multi-port device 42 includes a first flow path 44 surrounded by a pipe 46. A first end 48 of the first flow path 44 is adapted to connect to a port 34 of a bioreactor 32. An opposite second end 50 of the first flow path 44 is adapted to connect to an inlet of a TFF loop 36. In particular, the second end 50 can be configured as, for example, a hose barb or a tri-clamp connection.

[0036] The multi-port device 42 also includes a second flow path 52 formed by a first tube 54 and a second tube 56. An outer end 64 of the second flow path 52 is suitable for connection to an outlet of the TFF loop 36 ("outside" meaning outside the bioreactor 32). Similar to the second end 50 of the first flow path 44, the outer end 64 of the second flow path 52 may be configured as, for example, a barb fitting or a tri-clamp connection.

[0037] The first tube 54 extends through the wall of the pipe 46 into the interior of the pipe 46 and is connected to a second tube 56 inside the pipe 46. A first portion 58 of the second tube 56 extends within the pipe 46 towards the first end 48 of the pipe 46. A second portion 60 of the second tube 56 protrudes from the pipe 46. A mouth 62 is the terminus of the second flow path 52.

[0038] The multi-port device 42 is a one-piece device, i.e., the multi-port device 42 has no parts that can come apart or come loose. The multi-port device 42 can be a reusable part made, for example, from stainless steel, but can also be a single-use part made from a suitable plastic material.

[0039] When the multiport device 42 is secured to the bioreactor 32, a first end 48 of a first flow path 44, which may have a flange, is fluid-tightly connected to a port 34 of the bioreactor 32. The first flow path 44 of the multiport device 42 is used to withdraw cell culture medium from the bioreactor 32 and deliver it to the TFF loop 36, while the second flow path 52 is used to return the filtered cell culture medium from the TFF loop 36 back to the bioreactor 32.

[0040] With the multiport device 42 in a fixed position, the mouth 62 of the second flow path 52 is secured to a defined position in the bioreactor 32 by the elongated second portion 60 of the second tube 56. The defined position has a defined minimum distance of at least 5 mm, preferably at least 10 mm, from the first end 48 of the first flow path 44. The cell culture medium is withdrawn from the bioreactor 32 through the first flow path 44 and sent to the TFF loop 36. The filtered cell culture medium is returned from the TFF loop 36 to the bioreactor 32 through the second flow path 52.

[0041] In this way, it is possible to prevent the filtered culture fluid (retentate) immediately after being returned to the bioreactor 32 from being immediately sent back to the TFF loop 36. Both the mouth 62 of the second flow path 52 and the first end 48 of the first flow path 44 are located in positions that do not contact or directly interfere with any structural components within the bioreactor 32.

[0042] The distance between the mouth 62 of the second flow passage 52 and the first end 48 of the first flow passage 44, as well as the location and orientation of the mouth 62, can be selected as desired depending on at least one of the following factors: the distance to the second wall 66 opposite the first wall 68 in which the port 34 of the bioreactor 32 is formed; - the level of the fluid in the bioreactor 32 Height from the bottom of the bioreactor 32 - Position relative to the mixing means (agitator, impeller) provided in the bioreactor 32 -Direction of flow caused by the mixing means

[0043] The second flow passage 52 may be shaped as appropriate to meet desired requirements. In particular, the second portion 60 of the second tube 56 may include curved portions, bent portions, angled portions, etc. Additionally, the second portion 60 of the second tube 56 may be designed and positioned to function as a baffle (flow breaker) or flow guide itself.

[0044] FIG. 5 shows a detail of a second embodiment of the multiport device 42. In this embodiment, the first flow channel 44 and the second flow channel 52 are arranged next to each other. Furthermore, both flow channels 44, 52 have angled sections on the inside and outside of the bioreactor 32. Generally, flat angles (less than 90 degrees) are preferred to protect the cells as much as possible from shear stress. Depending on the actual assembly, it may be advantageous to provide an angled section only on one of the two flow channels 44, 52 and / or only on one of the sections of the flow channels 44, 52 (either the inside or the outside section). Of course, any other shape providing a "soft" curved or bent section may be used.

[0045] Figures 6 and 7 show two variations of the third embodiment of the multiport device 42. In the variation shown in Figure 6, the flow paths 44, 52 are arranged next to each other, while in the variation shown in Figure 7, the second flow path 52 is at least partially surrounded by the first flow path 44, similar to the configuration of the first embodiment shown in Figure 4.

[0046] In both variations, the multiport device 42 includes a flexible hose 70 attached to the inner end 72 of the second tube 56 (with "inside" meaning inside the bioreactor 32). The flexible hose 70 thus forms part of the second flow path 52. The free end of the flexible hose 70 inside the bioreactor 32 is the mouth 62 of the second flow path 52 (not shown in Figures 6 and 7). The flexibility of the hose 70 significantly reduces the risk of damage to the flexible bioreactor walls and internal components of the bioreactor 32 during storage and transport.

[0047] 4, 6 and 7, the mouth 62 is inside the bioreactor 32 and the first end 48 is located directly on the first wall 68. While such an embodiment is preferred, the reverse is also possible.

[0048] Figures 8a, 8b and 8c are cross-sectional views along line VIII-VIII in Figure 7 showing other possible implementations and possible designs of the first flow channel 44 and the second flow channel 52. The possible designs are not limited to the embodiment shown in Figure 7 but can also be used in other embodiments or variants of the multiport device 42.

[0049] In Fig. 8a, the first flow path 44 and the second flow path 52 are formed as a single fluid line. In particular, the fluid line of the first flow path 44 is defined by a pipe 46. The pipe 46 surrounds a second tube 56 which forms the fluid line of the second flow path 52. Of course, the fluid lines of the first flow path 44 and the second flow path 52 could also be located next to each other, for example as shown in Figs. 5 and 6.

[0050] 8b shows an example where the first flow path 44 is formed by a plurality of individual fluid lines 74. The individual fluid lines 74 may be arranged within the pipe 46, around the second tube 56, or in any other suitable manner. The individual fluid lines 74 may recombine to form a single fluid line, for example after the branching of the second flow path in the variation shown in FIG.

[0051] Alternatively or additionally, the second flow path 52 may be split into multiple individual fluid lines 76, as shown, for example, in Figure 8c. Such a configuration may be used, for example, to supply liquid to the bioreactor 32 at different locations or elevations.

[0052] As discussed above, the division of the first flow path 44 and / or the second flow path 52 into individual fluid lines 74 and / or 76 may also be performed in other embodiments or variations of the multi-port device 42 .

[0053] Of course, it is also possible to combine certain features of the above-mentioned embodiments. Furthermore, the multiport device 42 can also include additional flow paths that allow access to the interior of the bioreactor 32. Such additional flow paths can be used to supply other fluids to the cell culture medium, to remove samples of the cell culture medium, to insert sensors or probes, etc.

[0054] The description of the preferred embodiment focuses on the TFF module. However, the skilled person will recognize that the concept of the present invention can also be used in connection with other loops in which the culture medium circulates, such as a loop containing a sensor (sensor loop). Thus, such an arrangement for carrying out the upstream steps of cell culture comprises a bioreactor 32 having a port 34, a loop 36, preferably a sensor loop, a pump 38, a functional device, preferably a sensor, and a multiport device 42. The multiport device 42 connects the inlet and the outlet of the loop 36 to the bioreactor 32 via the same port 34. [Explanation of symbols]

[0055] 10 Bioreactor 12. TFF Loop 14 Pump 16 Hollow fiber filtration unit 18 First Port 20 2nd Port 22 Bioreactor 24 ATF Module 26 Hollow fiber filter 28 Diaphragm Pump 30 Ports 32 Bioreactor 34 Ports 36 TFF Loop 38 Pump 40 Hollow fiber filtration unit 42 Multiport Devices 44 First Stream 46 Pipe 48 First end 50 Second end 52 Second Flow Path 54 First Tube 56 Second Tube 58 Part 1 60 Part 2 62 Mouth 64 Outer end 66 2nd wall section 68 1st wall 70 Flexible Hose 72 Inner end 74 Individual fluid lines of the first flow path 76 Individual fluid lines of the second flow path

Claims

1. A multiport device (42) for connecting a loop (36), preferably a tangential flow filtration loop or a sensor loop, to one port (34) of a bioreactor (32), preferably one port (34) of a single-use bioreactor (32), The multi-port device (42) is configured to be fixed to the port (34) of the bioreactor (32), The multiport device (42) is A first channel (44) configured to extract fluid from the bioreactor (32), A second channel (52) configured to supply fluid to the bioreactor (32), Equipped with, The first flow path (44) has a first end (48) suitable for fluid connection with the bioreactor (32) and a second end (50) suitable for connection with the inlet of the loop (36), The second flow path (52) has an outer end portion (64) suitable for connection to the outlet portion of the loop (36) and an opening portion (62) suitable for fluid connection to the bioreactor (32), The opening (62) of the second flow path (52) is at least 5 mm, preferably 10 mm, away from the first end (48) of the first flow path (44). Multiport device (42).

2. The multi-port device (42) according to claim 1, which is an integral molding device.

3. The multiport device (42) according to claim 1 or 2, wherein the distance between the first end (48) of the first flow path (44) and the opening (62) of the second flow path (52) is at least three times the diameter of the opening of the port (34).

4. The multiport device (42) according to claim 1 or 2, wherein the first channel (44) and / or the second channel (52) includes at least one curved portion, bent portion, or angled portion.

5. The multiport device (42) according to claim 1 or 2, wherein the first channel (44) surrounds the second channel (52) at least in part.

6. The multiport device (42) according to claim 1 or 2, wherein the first channel (44) is located next to the second channel (52).

7. The second flow path (52) includes at least a flexible hose (70), The free end of the flexible hose (70) forms the opening (62) of the second flow path (52). The multiport device (42) according to claim 1 or 2.

8. A configuration for perfusion or concentrated feed-add method for performing upstream processes in cell culture, A bioreactor (32) having a port (34), A loop (36), preferably a TFF loop or a sensor loop, Pump (38) and A functional device, preferably a TFF unit such as a hollow fiber filtration unit (40), or a sensor, A multiport device (42) according to claim 1 or 2, Includes, The multi-port device (42) connects the inlet and outlet of the loop (36) to the bioreactor (32) via the same port (34). A configuration for perfusion or concentrated flow-addition.

9. The bioreactor (32) has a first wall portion (68) and a second wall portion (66) opposite to the first wall portion (68), The port (34) is located in the first wall portion (68), The opening (62) is located closer to the second wall (66) than to the first wall (68). The arrangement of the perfusion or concentrated feed addition method according to claim 8.

10. The perfusion or concentration flow addition arrangement according to claim 8, wherein the first end (48) of the first flow channel (44) and the opening (62) of the second flow channel (52) are located at different heights within the bioreactor (32).

11. The bioreactor (32) has a mixing means, The opening (62) is located closer to the mixing means than to the peripheral wall of the bioreactor (32). The arrangement of the perfusion or concentrated feed addition method according to claim 8.

12. The bioreactor (32) has a mixing means that causes a flow within the bioreactor (32), The opening (62) is oriented such that the culture medium supplied from the loop (36) to the bioreactor (32) via the second flow path (52) flows through the opening (62) in a direction that is essentially corresponding to the direction of the flow. The arrangement of the perfusion or concentrated feed addition method according to claim 8.

13. The bioreactor (32) has a mixing means that causes a flow within the bioreactor (32), The opening (62) is oriented such that the culture medium supplied from the loop (36) to the bioreactor (32) via the second flow path (52) flows through the opening (62) in a direction essentially opposite to the direction of the flow. The arrangement of the perfusion or concentrated feed addition method according to claim 8.

14. The perfusion or concentrated flow addition arrangement according to claim 8, wherein the second flow path (52) is surrounded by a baffle or flow guide.

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