A reusable filtration unit for a bioreactor and a method for providing reusable filtration of a culture of a bioreactor

Abstract

The present invention relates to a reusable filtration unit for a bioreactor. The invention furthermore relates to a method for providing reusable filtration of a culture of a bioreactor.

Description

[0001] Title of Invention

[0002] A reusable filtration unit for a bioreactor and a method for providing reusable filtration of a culture of a bioreactor.

[0003] Technical Field

[0004] The present invention relates to a reusable filtration unit for a bioreactor. The invention furthermore relates to a method for providing reusable filtration of a culture of a bioreactor.

[0005] Background

[0006] In continuous fermentation or cell culture processes, longer periods of high productivity are achieved by continuously removing unwanted constituents of the culture and adding new substrate and / or nutrients to the culture. The culture contains cell debris and / or metabolic products, which, if not removed, will impede the productivity due to thermodynamically unfavorable reactions, product pathways controlled by feedback inhibition, or product toxicity to the microorganism or cell culture. The metabolic products impeding productivity can be both the main product of the fermentation or cell culture process or metabolic biproducts.

[0007] A common way to remove unwanted constituents of a culture is by filtration, where cells are retained in the retentate, while substrate and other constituents of the culture are filtered off. Typically, a membrane is used for the filtration, where the membrane retains cells based on size exclusion. Examples of popular membranes for this purpose are polymeric membranes (e.g. PVDF) in spiral wound or hollow fiber membranes. Systems, which replace unwanted constituents of the culture with nutrients or substrate are sometimes called perfusion systems or perfusion membranes.

[0008] The membrane, along with the rest of the bioreactor system, is sterilized before the fermentation or cell culture process is initiated to reduce the risk of microbiological contamination. Commonly, a heated fluid, such as steam or heated water, is used as a cost-effective and efficient means to sterilize the membrane.

[0009] However, heating polymeric membranes to above a critical temperature, such as above 90°C, and repeated heating-cooling cycles results in membrane cracks and / or chemical deterioration of the membrane. Consequently, membranes often require replacement, adding additional material cost to the fermentation or cell culture process and causing production downtime.

[0010] To avoid excessive membrane repair or replacement, membranes can instead be treated at lower temperatures, such as at 80°C. An example of a system in which the maximum cleaning temperature is 80°C can be seen in CN207254117U. However, lower temperature treatment comes with an increased risk of microbiological contamination. In general, it is more desirable to sterilize the membrane at higher temperatures, such as temperatures in the range of 121-140°C, so that the culture stays highly productive for longer. Alternatively, single use membranes can be used, but their use adds additional cost and downtime.

[0011] In high-scale productions, single use membranes are especially impractical, as the downtime and costs related to membrane replacement rapidly scale with the size of the production. Filtration in continuous high-scale fermentation or cell cultures also lead to other challenges such as oxygen deprivation of the culture when passing through the filtration part of the system. In smaller scale productions, oxygen deprivation is a non-issue, since the smaller bioreactor system dimensions mean that oxygen provided to the culture in the bioreactor reach and oxygenate culture present in the filtration unit of the system. However, high-scale productions require larger amounts of culture to be filtrated, leading to larger and longer lines from the bioreactor to the filtration unit. This in combination with the rapid consumption of oxygen (e.g., typically within a few seconds) ultimately results in low levels or absence of oxygen in the filtration unit, increasing the production of undesirable metabolites.

[0012] Hence, there remains a need for a filtration method in continuous fermentation or cell culture process, which is suitable for high-scale production.

[0013] Summary of the invention

[0014] An object of the present invention is to address the above-mentioned drawbacks, and in particular to provide a reusable filtration unit for a bioreactor, by which longer periods of higher productivity can be achieved. In a first aspect, this and further objects are achieved by a reusable filtration unit for a bioreactor, which reusable filtration unit comprises a looped line, the looped line comprising a culture inlet valve, a culture outlet valve, a sterilization fluid inlet valve, a first cross-flow filter configured for filtering a culture in the looped line, the first cross-flow filter having a retentate side and a permeate side, wherein the reten- tate side faces the interior of the looped line and a heater configured for heating a sterilization fluid present in the looped line from an operation temperature to a sterilization temperature.

[0015] The configuration of the reusable filtration unit thus allows for a culture from a bioreactor to be cross-flow filtered, and the configuration of the culture inlet valve, the culture outlet valve, and the sterilization fluid inlet valve enables that the looped line and the first cross-flow filter may be sealed off from the bioreactor where a sterilization fluid is provided via the sterilization inlet valve and heated from the operation temperature to the sterilization temperature. The ability to take the reusable filtration unit offline from the bioreactor so that the filtration unit can be sterilized whilst the bioreactor continues to operate is particularly advantageous. Further, heating the sterilization fluid from the operation temperature to the sterilization temperature may sterilize the looped line and the membrane of the first cross-flow filter. Importantly, the looped line may facilitate circulation of the sterilization fluid while it is heated, which has the effect that the temperature changes in the membrane of the first cross-flow filter may be finely controlled. The invented design shows that by employing a looped line to control the membrane temperature, such that the membrane is gradually heated, cracks and / or chemical deterioration of the membrane can be reduced or prevented.

[0016] In the context of the present disclosure a fermentation or cell culture process is a process where cells, microorganisms or a tissue, is cultivated for the production of a metabolic product, such as a small molecule or a macromolecule, or for the growth of the cells, microorganisms or a tissue itself.

[0017] In the context of the present disclosure, a bioreactor is a container, in which a fermentation or cell culture process takes place. In the context of the present disclosure, a bioreactor system is a system comprising a bioreactor.

[0018] In the context of the present disclosure, a culture comprises cells, such as mammalian cells, or biological organisms, such as microorganism, and a substrate for the biological organism.

[0019] In the context of the present disclosure, any mentioning of a sealing off may be a sterile sealing off.

[0020] In the context of the present disclosure, any mentioning of fluidly disconnecting and / or fluidly connecting may be a fluid connection or fluid disconnection made under sterile conditions, such that the bioreactor and / or the reusable filtration unit are not contaminated (i.e., kept sterile) where a fluid disconnection or fluid connection is made between the bioreactor and the reusable filtration.

[0021] The reusable filtration unit may be configured to service one or several bioreactors, or several filtration units may be configured to service one bioreactor.

[0022] In an embodiment, the reusable filtration unit furthermore comprises a pump for pumping culture into the looped line and / or for controlling the flow velocity where a fluid, which is circulated in the looped line.

[0023] In an embodiment, the reusable filtration unit furthermore comprises a drain, which drain is fluidly connected to the permeate side of the membrane. The drain may be used to collect or discard the permeate, depending on whether or not the permeate contains valuable metabolic products or valuable substrate.

[0024] In another embodiment, the membrane of the first cross-flow filter is a ceramic membrane or a steel membrane. In a specific version of this embodiment, the steel or ceramic membrane has an average pore size in the range of 0.1 micron to 5 microns. By using ceramic and steel membranes in combination with a looped line to control the membrane temperature, sterilization at temperatures as high as 180°C can be carried out without significantly impacting the service life of the first cross-flow filter. The higher temperatures advantageously lengthens the time that a continuous fermentation or cell culture process can run and / or decreases the frequency of membrane sterilization required for running the continuous fermentation or cell culture process.

[0025] The ceramic membrane or steel membrane may have an average pore size in the range of 700 dalton to 5 microns, such as 0.1 micron to 5 microns, preferably 0.1 microns to 1 micron, such that cells, and in some cases macromolecules, are retained by the membrane.

[0026] In an embodiment, the membrane is a nano filtration membranes, such as an AIO2 membrane, a TiO2 membrane, a zirconium oxide membrane, or a silicon dioxide membrane.

[0027] In an embodiment, the looped line furthermore comprises a circulation valve, which circulation valve is arranged downstream of the culture outlet valve and upstream of the culture inlet valve. By this configuration, flow of a retentate through the looped line may be blocked at the point of the looped line at which the circulation valve is arranged, i.e., the circulation valve may be used for blocking the retentate from circulating in the looped line. Importantly, where the circulation valve blocks the culture from circulating in the looped line, a culture drawn into the looped line only passes through the looped line a single time before reentering a bioreactor. Conversely, allowing the culture to recirculate during filtration would mean that at least a portion of the recirculated culture would stay in the looped line for at least another pass around the looped line, extending the time that the culture was not oxygenated. Hence, blocking circulation during filtration limits the oxygen deprivation that the culture experiences. Thus, during an operation mode involving filtration of culture, the culture can be treated in a single pass i.e. after filtration it will return to the bioreactor, rather than passing through the filter again. However, during a sterilization mode in which culture is not being filtered, but the filter is being sanitized, the circulation valve can be switched allowing sterilization fluid to recirculate and pass through the filter as many times as needed.

[0028] In an embodiment, the reusable filtration unit furthermore comprises a purging fluid unit, which comprises a purging fluid valve, the purging fluid valve being connected to the looped line. The purging fluid unit may be employed for supplying a purging fluid, such as sterile air, sterile water, or an inert gas such as nitrogen, to the looped line via the purging fluid valve. Where the purging fluid is supplied to the looped line, the purging fluid may push back a portion of culture present in the looped line to the bioreactor.

[0029] In an embodiment, the sterilization fluid is water or hydrogen peroxide.

[0030] In an embodiment, the reusable filtration unit furthermore comprises a sterilization unit, which sterilization unit comprises a tank fluidly connected to the sterilization fluid inlet valve. In a particular version of this embodiment, the sterilization unit furthermore comprises a tank circulation line comprising a sterilization fluid circulation valve, the tank circulation line fluidly connecting the permeate side of the membrane to the tank. By this configuration, a cleaning solution may be circulated through the looped line, the first cross-flow filter, and the tank circulation line. In a particular version of this embodiment, the reusable filtration unit furthermore comprises a drain, which drain is fluidly connected to the permeate side of the membrane, and the sterilization fluid circulation valve may have an open position which blocks flow through the drain and allows flow through the tank circulation line, and a closed position which blocks flow through the tank circulation line and allows flow through the drain.

[0031] In an embodiment, the looped line furthermore comprises a flow regulation valve, which is arranged downstream of the first cross-flow filter and upstream of the culture outlet valve. By this design, flow over the membrane of the first cross-flow filter may be regulated, e.g., restricting the flow in the looped line via the flow regulation valve may increase the flow over the membrane and vice versa.

[0032] In an embodiment, the looped line furthermore comprises a flow transmitter arranged downstream of the pump and upstream of the first cross-flow filter, the flow transmitter being configured for transmitting a measured flow velocity to the pump, and wherein the pump is furthermore configured for adjusting its pumping speed based on the measured flow velocity. In a preferred embodiment, the pump is configured to compare the measured flow velocity with a reference flow velocity, and increase or decrease the pumping speed so as to approximate the reference flow velocity. Preferably, the reference flow velocity is a flow velocity at which a portion of culture travels through the looped line in less than 2 seconds, such that in less than 1.5, 1, or 0.5 seconds. Such low residence times can be especially beneficial when the system is used with cells that are particularly sensitive to oxygen deprivation, such as for example mammalian cells.

[0033] In an embodiment, the looped line length is in the range of 3-12 m.

[0034] In another embodiment, the reference flow velocity is in the range of 3 to 6 m / sec.

[0035] The cross-flow filter may have any suitable surface area. In an embodiment, the cross-flow filter has a surface area in the range of 10 cm to 50 m2.

[0036] In an embodiment, the looped line further comprises a cooler.

[0037] In some embodiments, the heater is configured for heating a sterilization fluid present in the looped line from an operation temperature, preferably the operation temperature is in the range of 15°C to 95°C, to a sterilization temperature which is in the range of 100°C to 180°C, preferably the sterilization temperature is in the range of l21°C to 180°C.

[0038] In some embodiments, the permeate side is connected to a drain, optionally via a permeate line. Thus, during operation mode the retentate can be returned to the bioreactor and the permeate is removed for disposal or otherwise further treatment. Thus, the permeate side can be considered to face a drain. Likewise, the reference to the retentate side facing the interior of the looped line means that retained matter i.e. retentate is able to travel through the looped line. In other words, there is a fluid connection between the retentate side and the looped line.

[0039] According to a second aspect of the present disclosure, a method for providing reusable filtration of a culture of a bioreactor is provided, the method comprising the steps of: providing a reusable filtration unit, the reusable filtration unit comprising a looped line, the looped line comprising a first cross-flow filter with a membrane having a retentate side and a permeate side, wherein the retentate side faces the interior of the looped line fluidly connecting the looped line to a bioreactor drawing a portion of culture of the bioreactor into the looped line and crossflow filtering the portion of culture by means of the first cross-flow filter thereby producing a retentate on the retentate side of the membrane and a permeate on the permeate side of the membrane moving the retentate via the looped line to the bioreactor fluidly disconnecting the looped line from the bioreactor providing a sterilization fluid to the looped line while circulating the sterilization fluid in the looped line, heating the sterilization fluid from an operation temperature to a sterilization temperature whereby the retentate side of the membrane is sterilized.

[0040] The filtration unit may be a filtration unit according to the first aspect of the invention.

[0041] The second aspect achieves at least the same objects and has at least the same advantages as the first aspect.

[0042] In an embodiment of the method of the present disclosure, the steps of: fluidly connecting the looped line to a bioreactor drawing a portion of culture of the bioreactor into the looped line and crossflow filtering the portion of culture by means of the first cross-flow filter thereby producing a retentate on the retentate side of the membrane and a permeate on the permeate side of the membrane moving the retentate via the looped line to the bioreactor fluidly disconnecting the looped line from the bioreactor providing a sterilization fluid to the looped line while circulating the sterilization fluid in the looped line, heating the sterilization fluid from an operation temperature to a sterilization temperature whereby the retentate side of the membrane is sterilized are performed while a fermentation or cell culture process is carried out by means of the bioreactor.

[0043] In an embodiment of the method of the present disclosure, the steps of drawing a portion of culture of the bioreactor into the looped line and cross- flow filtering the portion of culture by means of the first cross-flow filter thereby producing a retentate on the retentate side of the membrane and a permeate on the permeate side of the membrane moving the retentate via the looped line to the bioreactor are completed in less than 2 seconds.

[0044] Oxygen is provided to the culture when it is present the bioreactor. However, additional oxygen is not provided as the cells travel through the looped line, which can cause cell stress, as the oxygen present in the culture is usually consumed within seconds. Thus, to minimize stress caused by lack of oxygen, the portion of culture may be pumped from the culture inlet valve to the culture outlet valve in less than 2 seconds, such as less than 1.5, 1 or 0.5 seconds. Such low residence times can be especially beneficial when the system is used with cells that are particularly sensitive to oxygen deprivation, such as for example mammalian cells.

[0045] In an embodiment of the method of the present disclosure, the method further comprises the step of: priorto the step of providing a sterilization fluid to the filtration unit, providing a cleaning solution to the looped line.

[0046] As such, a more effective sterilization can be achieved, as build-up of cells or cell debris is removed from the membrane and / or the looped line. For an even more effective cleaning and sterilization, a purging step can be carried out prior to the cleaning and sterilization, where a purging fluid, such as sterile air, is supplied to the looped line to move the majority of the portion of culture back to the bioreactor. By this method, more valuable culture, e.g. cells and substrate is saved, while the cleaning solution need not be used to remove nearly as much cells or cell debris.

[0047] In a preferred embodiment of the method of the present disclosure, the method furthermore comprises the step of after the step of providing a cleaning solution to the looped line, circulating the cleaning solution in the looped line while heating the cleaning solution.

[0048] In a specific version of this embodiment, the cleaning solution is heated to a temperature is in the range of 45°C to 80°C, such as between 60-80°C. Heating and circulating the cleaning solution provides a more effective cleaning, such that cell material build-up is removed from the membrane and / or the looped line prior to sterilization.

[0049] The choice of cleaning solution depends on production type and fouling, and suitable cleaning steps and cleaning solution are well known in the art.

[0050] In an embodiment of the method according to the present disclosure, the operation temperature is in the range of 15°C to 95°C and the sterilization temperature is in the range of 100°C to 180°C, preferably in the range of 121°C to 180°C.

[0051] In an embodiment of the method according to the present disclosure, the step of heating the sterilization fluid comprises heating the sterilization fluid from the operation temperature to the sterilization temperature at a rate of less than 20°C per minute, preferably at a rate of less than 10°C per minute.

[0052] In an embodiment of the method of the present disclosure, the method furthermore comprises the step of:

[0053] - after the step of heating the sterilization fluid, holding the sterilization fluid at the sterilization temperature for a period of time. In a specific version of this embodiment, the period of time is less than 30 min, preferably less than 20 min, such as in the range of 20 to 15 min, even more preferably 15 min. In a preferred version of this embodiment, the fluid is held at sterilization temperature while the sterilization fluid is circulated in the looped line.

[0054] In an embodiment of the method according to the present disclosure, the method further comprises the step of: after the step of heating the sterilization fluid, cooling the sterilization fluid from the sterilization temperature to the operation temperature.

[0055] The sterilization fluid is preferably cooled at a rate of less than 20°C per minute, preferably at a rate of less than 10°C per minute.

[0056] Slower rates of heating and cooling is particularly advantageous where the method also employs a ceramic membrane or a steel membrane, as this method results in membranes with a very long services life. The ceramic membrane or steel membrane may have an average pore size in the range of 700 dalton to 5 microns, such as 0.1 micron to 5 microns, preferably 0.1 microns to 1 micron, such that cells, and in some cases macromolecules, are retained on the membrane.

[0057] In an embodiment of the method of the present disclosure, the method further comprises the step of: after the step of cooling the sterilization fluid from the sterilization temperature to the operation temperature, fluidly connecting the looped line to the bioreactor.

[0058] After fluidly reconnecting the looped line, the filtration unit may again be used for providing filtration of the culture of the bioreactor. Essentially, the overall process of filtrating a culture of the bioreactor with the reusable filtration unit, fluidly disconnecting the reusable filtration unit from the bioreactor, sterilizing the membrane, and reconnecting the looped line to the bioreactor, may be iterated for a number of times, such as 3 to 100 times, so as to prolong the time that a fermentation or cell culture can be maintained. Importantly, as the fluid connection and / or disconnection may be made under sterile conditions, sterility may be maintained throughout the process iterations.

[0059] In an embodiment of the method according to the present disclosure, the looped line further comprises a second cross-flow filter, the second cross-flow filter having a retentate side and a permeate side, wherein the retentate side faces the interior of the looped line, and wherein the first cross-flow filter and the second crossflow filter are arranged in parallel or in series on the looped line.

[0060] Other reusable filtration units with even more cross-flow filters are also possible, such as three or more cross-flow filters in parallel or in series. In general, filtration units with cross-flow filters arranged in parallel allow for more gentle handling of cells, as such systems have a smaller ratio of flow over the membrane to filtration surface area. On the other hand, filtration units with cross-flow filters arranged in series generally provide higher throughout, with higher filtration capacity. Filtration units with cross-flow filters arranged in series also offer a simpler design as compared to the parallel configuration. The parallel configuration requires splitting the looped line into two or more conduits for the parallel flow, whereas cross-flow filters arranged in series are all connected to the same conduit (the looped line).

[0061] Presently preferred embodiments and further advantages will be apparent from the subsequent detailed description and drawings. A person skilled in the art will appreciate that any one of the above aspects of the disclosure and embodiments thereof may be combined with any one or more aspects of the disclosure and embodiments. For example, the methods of the second aspect may be performed on apparatus according to the first aspect.

[0062] Brief Description of Drawings

[0063] In the following description embodiments of the invention will be described with reference to the drawings, in which:

[0064] FIG. 1 is a schematic of a bioreactor system which employs a reusable filtration unit according to an embodiment of the present invention.

[0065] FIG. 2 is a schematic of a bioreactor system which employs a reusable filtration unit according to another embodiment of the present invention, wherein the reusable filtration unit includes two cross-flow filters arranged in series.

[0066] FIG. 3 is a schematic of a bioreactor system which employs a reusable filtration unit according to another embodiment of the present invention, wherein the reusable filtration unit includes two cross-flow filters arranged in parallel.

[0067] Detailed description

[0068] The present invention will now be described in more detail hereinafter with reference to the accompanying drawings, in which embodiments of the invention is shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness.

[0069] Referring first to FIG. 1 of the drawings, which shows a bioreactor system 100 which employs a reusable filtration unit 200 according to an embodiment. The reusable filtration unit 200 comprises a looped line 201, the looped line 201 comprising a culture inlet valve 211, a culture outlet valve 212, a circulation valve 213, a sterilization fluid inlet valve 311, a first cross-flow filter 221 configured for filtering a culture (not shown) in the looped line 201, the first cross-flow filter 221 having a membrane (not shown) with a retentate side 221a and a permeate side 221b, wherein the retentate side 221a faces the interior of the looped line 201 and the permeate side is fluidly connected to a permeate line 202, the permeate line 202 comprising a drain 203, a heater 232 configured for heating a sterilization fluid (not shown) present in the looped line 201 from an operation temperature to a sterilization temperature, a cooler 231 for cooling the a sterilization fluid (not shown) present in the looped line 201 from a sterilization temperature to an operation temperature, and a looped line pump 241 configured for pumping a fluid in the looped line. The reusable filtration unit 200 is furthermore fluidly connected to a bioreactor 500, which bioreactor 500 comprises a bioreactor tank 551 with a bioreactor tank inlet 551a and a bioreactor tank outlet 551b, allowing a culture (not shown) present in the bioreactor tank 551 to flow through the bioreactor tank outlet 551a to the bioreactor line 501, and back to the bioreactor tank 551 through the bioreactor inlet 551b.

[0070] Where the filtration unit 100 is employed to filter a portion of a culture present in the bioreactor (operational mode), the looped line pump 241 draws a portion of a culture present in the bioreactor line 501 into the looped line 201 through the culture inlet valve 211. The portion of culture then flows through the looped line 201 and is cross-flow filtered by the first cross-flow filter 221 producing a retentate (not shown) in the looped line 221 and a permeate (not shown) at the permeate side 221b of the membrane. The permeate flows through the a permeate line 202 and out of the drain 203. The retentate flows via the looped line 201 and out of the culture outlet valve 212 to enter the bioreactor line 501. In operational mode, the circulation valve 213 is in a closed position, blocking the flow of retentate through the looped line 213 at the point of the looped line 201 where the circulation valve 213 is arranged, i.e., the circulation valve blocks the retentate from circulating in the looped line 201.

[0071] The membrane of the first cross-flow filter 221 is a ceramic membrane with an average pore size of 0.2 micron, such that cells of the portion of culture are retained on the retentate side 221a of the membrane when the culture is filtered by the first cross-flow filter 221. The resulting permeate is thus substantially cell free and contains, beside a fraction of substrate of the portion culture, metabolic products of the cells. The retentate, in turn, contains substantially all cell of the portion of culture and the remaining fraction of the substrate containing also metabolic products of the cells.

[0072] In one embodiment the cell culture may comprise a microorganism such as fungus, yeast or bacteria.

[0073] The fungal cells may be one of the genera Aspergillus and Trichoderma, in particular fungal cells of one of the Species Aspergillus niger var. niger, Aspergillus niger var. awamori, Aspergillus nidulans, Aspergillus Oryzae Tri choderma reisei and Trichoderma viride.

[0074] The yeast cells may be one of the genera Saccharomyces, Kluyveromyces, Hansenula and Pichia, in particular yeast cells of one of the Species Saccharomyces cerevisiae, Saccharomyces carlbergensis, Kluyveromyces lactis, Kluyveromyces marx- ianus, Hansenula polymorpha and Pichia I storis.

[0075] The bacteria may be one of the bacterial genera Bacillus, Lactobacillus and Streptococcus, such as bacteria of the species Bacillus Subtilis.

[0076] Oxygen is provided to the culture in the bioreactor 500, while no oxygen is provided to the culture when it is present in the reusable filtration unit 200. To minimize stress caused by lack of oxygen, the looped line pump 241 is configured for pumping the portion of culture from the culture inlet valve 211 to the culture outlet valve 212 in 0.5 to 2 seconds.

[0077] When the filtration unit 100 is to be sterilized (sterilization mode), the culture inlet valve 211 and the culture outlet valve 212 are moved from open positions to closed positions, such that a fluid present in the bioreactor line 501 cannot flow into or out of the looped line 201, while fluid present in the looped line 201 is still allowed to pass through the looped line 201. Furthermore, the circulation valve 213 is moved from the closed position to an open position, allowing flow of a fluid through the looped line 213 at the point of the looped line 201 where the circulation valve 213 is arranged. As such, during sterilization mode, a fluid is allowed to circulate in the looped line 201. Next, a sterilization fluid (not shown), such as water or hydrogen peroxide, is introduced to the looped line 201 via the sterilization fluid inlet valve 311, which sterilization fluid is circulated in the looped line 201 by means of the looped line pump 241. The heater 232 heats the sterilization fluid from an operation temperature which preferably is a temperature in the range of 15°C to 95°C to a sterilization temperature, which is preferably in the range of 100°C to 180°C. Preferably, the sterilization fluid is heated from the operation temperature to the sterilization temperature at a rate of less than 10°C per minute. While the sterilization fluid is heated, the sterilization fluid is circulated in the looped line 201. A portion of the heated sterilization fluid penetrates the ceramic membrane, such that the ceramic membrane is heated to the sterilization temperature. Then, the portion of sterilization fluid, which penetrated the ceramic membrane of the first cross-flow filter 221, flows via the permeate line 202 to the drain 203.

[0078] The looped line 201 and the membrane of the first cross-flow filter 221 are subjected to the sterilization fluid at the sterilization temperature for up to 30 min, thereby sterilizing the looped line 201 and the membrane of the first cross-flow filter 221. The sterilization fluid is then cooled by means of the cooler 231 from the sterilization temperature to the operation temperature, whereby the membrane of the first cross-flow filter 221 is also cooled from the sterilization temperature to the cooling temperature. Preferably, the sterilization fluid is cooled from the sterilization to the operation temperature at a rate of less than 10°C per minute. After the sterilization fluid has cooled to the operation temperature, circulation valve 213 is moved from the open position to the closed position, and the culture inlet valve 211 and the culture outlet valve 212 are moved from closed positions to open positions, such that culture present in the bioreactor line 501 is again allowed to flow into the looped line 201 via the culture inlet valve 211 and out of the looped line 201 and back to the bioreactor line 501 via the culture outlet valve 212. The operational model is then resumed, and the filtration unit 100 is again employed to filter a portion of a culture present in the bioreactor.

[0079] The reusable filtration unit 200 furthermore comprises a purging fluid unit 400, which purging fluid unit 400 comprises a purging fluid valve 411. Preferably, the purging fluid unit 400 supplies a purging fluid (not shown), such as sterile air, to the looped line 201 via the purging fluid valve 411 to push back to the bioreactor line 501 substantially all culture present in the looped line 201 before sterilization fluid is supplied to the looped line 201. When the purging fluid is supplied via the purging fluid valve 411, the culture inlet valve 211 and the culture outlet valve 212 are both moved to their open positions, such that culture present in the looped line 201 may be pushed back to the bioreactor line 501.

[0080] The reusable filtration unit 200 furthermore comprises a sterilization unit 300, which sterilization unit 400 comprises a tank 352 fluidly connected to the sterilization fluid inlet valve 311 via a tank outlet line 302. The tank 351 is furthermore fluidly connected a tank circulation line 302, which tank circulation line 302 in turn is connected to the permeate line 202 via a sterilization fluid circulation valve 312.

[0081] Preferably, the sterilization unit 400 supplies a cleaning solution (not shown), such as sodium hydroxide, to the looped line 201 via the sterilization fluid inlet valve 311 before sterilization fluid is supplied to the looped line 201. When the cleaning solution is supplied to the looped line 201 via the sterilization fluid inlet valve 311, the culture inlet valve 211 and the culture outlet valve 212 are both moved to their closed positions, such that the cleaning solution does not flow to the bioreactor line. Furthermore, the circulation valve 213 is moved from the closed position to an open position, allowing flow of the cleaning solution through the looped line 213 at the point of the looped line 201 where the circulation valve 213 is arranged. The supplied cleaning solution penetrates the membrane of the first cross-flow filter 221, and enters the permeate line 202.

[0082] Preferably, the sterilization unit 400 supplies the cleaning solution to the looped line 201 via the sterilization fluid inlet valve 311 after substantially all culture in the looped line 201 has been pushed back to the bioreactor line 501 by the purging fluid unit 400.

[0083] Preferably, where the sterilization unit 400 supplies the cleaning solution to the looped line 201, the sterilization fluid circulation valve 312 is moved from a closed position, which does not allow fluid to flow from the permeate line 202 to the tank circulation line 302, to an open position, which blocks fluid from entering the drain 203 and allow fluid to flow from the permeate line 202 to the tank circulation line 302. The sterilization line pump 341 and / or the looped line pump 241 then pumps the cleaning solution, such that the cleaning solution circulates around the looped line 201, then to the permeate line 202, the tank circulation line 302, the tank 351, the tank outlet line 301, and finally back to the looped line 202. While the cleaning solutions is circulated, the cleaning solution is heated by the heater 232 to a temperature in the range of 25°C to 95°C.

[0084] The looped line 201 furthermore comprises a flow regulation valve 214 for regulating a pressure over the membrane of the first cross-flow filter 221. Restricting the flow by means of the flow regulation valve 214 increases the flow of fluid through the membrane of the first cross-flow filter 221, and decreases the circulating (cross) flow in the looped line 201.

[0085] The looped line 201 furthermore comprises a flow transmitter 215 for measuring the flow velocity of a fluid in the looped line 201 and transmitting the velocities to the looped line pump 241. The pumping activity of the looped line pump 241 is regulated based on the velocities measurements received from the flow transmitter 215.

[0086] Referring now to FIG. 2 of the drawings, which shows a schematic of a bioreactor system 100 which employs a reusable filtration unit 200 according to another embodiment of the present invention, wherein the reusable filtration unit 200 includes a first cross-flow 221 and a second cross flow filter 222 arranged in series on the looped line 201.

[0087] Referring now to FIG. 3 of the drawings, which shows a schematic of a bioreactor system 100 which employs a reusable filtration unit 200 according to another embodiment of the present invention, wherein the reusable filtration unit 200 includes a first cross-flow 221 and a second cross flow filter 222 arranged in parallel. At the junction 261, the looped line 201 splits into a first conduit 201a and second conduit 201b, which are joined again at junction 262. A retentate side 221a of the membrane of the first cross filter 221 faces the interior of the first conduit 201a, and a retentate side 221b of the membrane of the second cross filter 222 faces the interior of the second conduit 201b.

[0088] List of reference numerals

[0089] 100 bioreactor system

[0090] 200 reusable filtration unit

[0091] 201 looped line

[0092] 202 permeate line

[0093] 203 drain

[0094] 211 culture inlet valve

[0095] 212 culture outlet valve

[0096] 213 circulation valve

[0097] 214 flow regulation valve

[0098] 215 flow transmitter

[0099] 221 first cross-flow filter

[0100] 221a retentate side of first cross-flow filter

[0101] 221b permeate side of first cross-flow filter

[0102] 222 second cross-flow filter

[0103] 222a retentate side of first cross-flow filter

[0104] 222b permeate side of first cross-flow filter

[0105] 231 cooler

[0106] 231a cooler inlet

[0107] 231b cooler outlet

[0108] 232 heater

[0109] 232a heater inlet

[0110] 232b heater outlet

[0111] 241 looped line pump

[0112] 300 sterilization fluid unit

[0113] 301 tank inlet line

[0114] 302 tank outlet line

[0115] 303 tank circulation line

[0116] 311 sterilization fluid inlet valve

[0117] 312 sterilization fluid circulation valve

[0118] 341 sterilization line pump

[0119] 352 tank

[0120] 400 purging fluid unit

[0121] 411 purging fluid valve

[0122] 500 bioreactor

[0123] 501 bioreactor line

[0124] 551 bioreactor tank

[0125] 551a bioreactor tank outlet

[0126] 551a bioreactor tank inlet

Claims

C L A I M S1. A reusable filtration unit for a bioreactor, wherein the reusable filtration unit comprises a looped line, the looped line comprising a culture inlet valve, a culture outlet valve, a sterilization fluid inlet valve, a first cross-flow filter configured for filtering a culture in the looped line, the first cross-flow filter having a membrane with a retentate side and a permeate side, wherein the retentate side faces the interior of the looped line, and a heater configured for heating a sterilization fluid present in the looped line from an operation temperature to a sterilization temperature.

2. The reusable filtration unit of claim 1, wherein the looped line furthermore comprises a pump.

3. The reusable filtration unit of any one of the claims 1 or 2, wherein the membrane of the first cross-flow filter comprises a ceramic membrane or a steel membrane.

4. The reusable filtration unit of claim 1 to 3, wherein the looped line furthermore comprises a circulation valve, which circulation valve is arranged downstream of the culture outlet valve and upstream of the culture inlet valve.

5. The reusable filtration unit according to any previous claim, wherein the heater is configured for heating a sterilization fluid present in the looped line from an operation temperature, preferably the operation temperature is in the range of 15°C to 95°C, to a sterilization temperature which is in the range of 100°C to 180°C, preferably the sterilization temperature is in the range of 121°C to 180°C.

6. The reusable filtration unit according to any previous claim, wherein the permeate side is connected to a drain, optionally via a permeate line.

7. A method for providing reusable filtration of a culture of a bioreactor, the method comprising the steps of: providing a reusable filtration unit such as the reusable filtration unit of any of claims 1 to 6, the reusable filtration unit comprising a looped line, the looped line comprising a first cross-flow filter with a membrane having a retentate side and a permeate side, wherein the retentate side faces the interior of the looped line;fluidly connecting the looped line to a bioreactor; drawing a portion of culture of the bioreactor into the looped line and crossflow filtering the portion of culture by means of the first cross-flow filter thereby producing a retentate on the retentate side of the membrane and a permeate on the permeate side of the membrane; moving the retentate via the looped line to the bioreactor; fluidly disconnecting the looped line from the bioreactor; providing a sterilization fluid to the looped line; and while circulating the sterilization fluid in the looped line, heating the sterilization fluid from an operation temperature to a sterilization temperature whereby the retentate side of the membrane is sterilized.

8. The method of claim 7, wherein the steps of drawing a portion of culture of the bioreactor into the looped line and crossflow filtering the portion of culture by means of the first cross-flow filter thereby producing a retentate on the retentate side of the membrane and a permeate on the permeate side of the membrane moving the retentate via the looped line to the bioreactor are completed in less than 2 seconds.

9. The method of claim 5 or 6 further comprising the step of: prior to the step of providing a sterilization fluid to the looped line, providing a purging fluid to the looped line.

10. The method according to claim 7, wherein in the step of providing a purging fluid to the looped line, culture and / or retentate present in the looped line is driven into the bioreactor by the purging fluid.

11. The method of any one of the claims 5 to 8 further comprising the step of: priorto the step of providing a sterilization fluid to the filtration unit, providing a cleaning solution to the looped line.

12. The method of claim 9 further comprising the step of: after the step of providing a cleaning solution to the looped line, circulatingthe cleaning solution in the looped line while heating the cleaning solution.

13. The method according to any one of the claims 5 to 10, wherein the operation temperature is in the range of 15°C to 95°C and the sterilization temperature is in the range of 100°C to 180°C, preferably in the range of 121°C to 180°C.

14. The method according to any one of the claims 5 to 11, wherein the membrane is a ceramic membrane or a steel membrane.

15. The method according to any one of the claims 5 to 12, wherein in the step of heating the sterilization fluid, the sterilization fluid is heated from the operation temperature to the sterilization temperature at a rate of less than 20°C per mi- nute, preferably at a rate of less than 10°C per minute.

16. The method according to any one of the claims 5 to 13 further comprising the step of: after the step of heating the sterilization fluid, cooling the sterilization fluid from the sterilization temperature to the operation temperature.

17. The method according to claim 14, wherein in the step of cooling the sterilization fluid, the sterilization fluid is cooled at a rate of less than 20°C per minute, preferably at a rate of less than 10°C per minute.

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