Methods for producing and purifying viral vectors

Abstract

The present invention relates to a method for producing and purifying a viral vector (V), wherein the viral vector (V) is produced by cells in a fluid (F), and the fluid (F) containing the viral vector (V) is guided through a tangential flow filtration device (2) for purification, thereby purifying the viral vector (V) in the fluid (F). It is proposed that cells and cellular debris contained in the fluid (F) containing the viral vector (V) after producing the viral vector (V) are at least partially separated from the viral vector (V) by a fluidized bed centrifuge (5) before purification by the tangential flow filtration device (2).

Description

[Technical Field]

[0001] The present invention relates to a method for producing and purifying viral vectors according to the preamble of claim 1 and to a system for producing and purifying viral vectors according to the preamble of claim 9.

[0002] The method in question can be applied to different types of bioprocesses. For example, the proposed method can be used in the field of cell and gene therapy or for vaccine development. In general, the proposed method is not limited to a specific field, but rather can be applied to various fields of biotechnology.

[0003] The production of viral vectors by cells is well known in the art.Depending on the nature of the cells to be applied, different kinds of viral vectors can be produced, such as lentivirus vectors and gamma retrovirus vectors, retrovirus vectors including adenovirus vectors or adeno-associated virus vectors.Preferably, mammalian cell lines suitable for growth in suspension, such as HEK293 suspension cells, are used to produce viral vectors.For this purpose, cells are maintained in a fluid such as culture medium under certain conditions that allow viral vectors to be produced.

[0004] Thereafter, for example, when a certain number of viral vectors are present, the fluid containing the viral vectors and cells, as well as cellular debris from the cells, must be purified to obtain a purified viral vector, which can then be further used. For purification, particularly to separate cells and cellular debris from the viral vector, the fluid containing the cells, cellular debris, and viral vectors can be directed through a filter device for clarification, where the fluid passes through a filtration element such as a membrane. Here, larger impurities of a certain size, such as cells and cellular debris, are at least partially retained, while the fluid containing the viral vectors and smaller impurities pass through the filtration element. After filtering the fluid through such a "pass-through" filter device, a tangential flow filtration device is used to separate the remaining impurities from the viral vectors in the fluid and / or concentrate the viral vectors in the fluid. For this purpose, the fluid containing the viral vector is directed through the tangential flow filtration device for purification, and the remaining impurities are at least partially separated from the viral vector accordingly. As a result, the viral vector can be purified from larger and smaller impurities.

[0005] A known method for producing and purifying viral vectors in a fluid, which forms the basis of the present invention, is disclosed in International Publication No. 2021 / 252782. Here, a viral vector, such as a lentiviral vector, is produced by cells in a fluid, and a composition corresponding to the fluid containing the viral vector and cells and cell debris is first fed through a filtration device, in this case an alternating tangential flow filtration device, and the fluid containing the viral vector passes through the filtration element of the filtration device. The filtered fluid containing the viral vector is then fed through a single-pass tangential flow filtration system (SPTFF system). As a result, larger impurities, such as cells and cell debris, are separated from the fluid in the filtration device, and then smaller impurities are separated from the fluid. As a result, the viral vector is purified in the fluid, thus representing a purified viral vector composition.

[0006] Although known methods allow for successful purification of already produced viral vectors, the grade of purification is strongly influenced by, for example, the conditions of the SPTFF system membrane, operating parameters, membrane blocking and fouling, etc. Particularly over time, the separation performance of the SPTFF system can be reduced due to fouling of the SPTFF system as cells and cell debris block the membrane.

[0007] In addition to the resulting reduced separation performance of the SPTFF system, another problem with known methods is that the use of a pass-through filter can rapidly reduce the activity of purified viral vectors in the fluid, as some of the viral vectors may be destroyed as they pass through the filter. Furthermore, viral vectors may be trapped by the membrane and / or adsorbed to the membrane instead of passing through. Therefore, the resulting purified viral vector composition may contain only a small amount of active viral vector.

[0008] Therefore, there remains a need for further improvements of known methods.

[0009] The present invention is based on the problem of improving known methods so that the concentration and / or activity of purified viral vectors in a fluid can be increased.

[0010] The above problem is solved by the features of the characterizing part of claim 1.

[0011] A key realization of the present invention is that at least a portion of the cells and cell debris present in the fluid after viral vector production are separated from the viral vector by a fluidized bed centrifuge prior to purification by a tangential flow filtration device. Therefore, the fluidized bed centrifuge is positioned prior to the tangential flow filtration device. Separation by a fluidized bed centrifuge can serve as a kind of pretreatment step prior to purification by a tangential flow filtration device, i.e., separation and / or concentration. Separation by a fluidized bed centrifuge can be adjusted over a wide range as needed, particularly in terms of rotation speed, flow rate, centrifugation duration, etc., making it highly efficient and enabling very gentle separation. Therefore, in contrast to other centrifugation methods, separation by a fluidized bed centrifuge, i.e., centrifugation, results in a higher activity of the purified viral vector in the fluid. Furthermore, because cells and cell debris are gently separated from the fluid containing the viral vector by the fluidized bed centrifuge, further filtration by a pass-through filter can be prevented, resulting in a higher activity of the viral vector obtained after the overall purification process. Advantageously, due to the gentle separation by the fluidized bed centrifuge, the cells remain intact, allowing for the reuse of at least a portion of the cells. Overall, the proposed method increases the concentration and activity of viral vectors.

[0012] In particular, it is proposed that cells and cell debris contained in the fluid containing the viral vector after producing the viral vector are at least partially separated from the viral vector by a fluidized bed centrifuge prior to purification by a tangential flow filtration device, thereby increasing the concentration and activity of the purified viral vector in the fluid.

[0013] According to claim 2, enzymes and, additionally or alternatively, charged beads may be added to the fluid before the fluidized bed centrifuge. The enzymes can degrade cells, particularly cell membranes, cell debris, and / or DNA impurities, for example, to reduce the viscosity of the fluid and / or the molecular weight of proteins. Furthermore, if the viral vector is not released into the fluid by the cells, degrading the cells, particularly the cell membrane or cell wall, can release the viral vector into the fluid. Charged beads can also be used to improve the following separation process using a fluidized bed centrifuge, since at least some of the cells, cell debris, and / or DNA can be bound to them. The cells, cell debris, and / or DNA impurities are bound to the charged beads, and by separating the charged beads in the subsequent separation process, the bound cells, cell debris, and / or DNA are separated along with them.

[0014] Advantageously, claim 3 further defines a separation using a fluidized bed centrifuge, the separation comprising a filling phase, a washing phase, and / or a discharge phase. During the filling phase, a fluid is filled into the chambers of the fluidized bed centrifuge, in particular into several chambers. Preferably, the filling is carried out while the centrifugation is already taking place. In this way, separation of the viral vector from at least a portion of the cells, cell debris, and / or DNA impurities contained in the fluid is advantageously already carried out. During the washing phase, a washing fluid is passed through the chambers, advantageously washing away the cells accumulated in the chambers. This allows the remaining fluid to be flushed out of the chambers by the washing fluid. Furthermore, it is possible to flush out the viral vector remaining in the chambers during the filling phase with the fluid and / or the washing fluid during the washing phase. During the discharge phase, the cells, cell debris, and / or DNA impurities that have accumulated in particular in one direction in the chambers of the fluidized bed centrifuge are discharged by the transport fluid, thereby flushing them out of the chambers, in particular as waste or for reuse.

[0015] After separation, the fluid containing the viral vectors can be guided from the fluidized bed centrifuge to a holding vessel as described in claim 4. In particular, if separation by fluidized bed centrifugation is applied periodically, the holding vessel provides an intermediate reservoir for the fluid. Furthermore, for example, a constant outflow of fluid after the holding vessel can be provided, or more separated fluid can be stored.

[0016] According to claim 5, an enzyme can be added to the fluid. This can at least partially degrade cells, particularly cell membranes and / or cell walls, cell debris, and / or DNA impurities that may or may not have been previously separated by the fluidized-bed centrifuge during separation. Furthermore, because the cells, cell debris, and / or DNA have already been separated or partially separated from the fluid, the enzyme can degrade them more efficiently, adsorbing only to the cells, cell debris, and / or DNA that have been separated or not and are still contained in the fluid. Furthermore, decomposition can reduce the viscosity of the fluid, thereby simplifying subsequent process steps. Generally, adding enzymes before and, additionally or alternatively, after the fluidized-bed centrifuge can degrade at least a portion of the cells, cell debris, and / or DNA impurities, which can generally improve the purification process. Enzymatic decomposition can prevent blocking of the separation filter elements of a tangential flow filtration device, particularly a single-pass tangential flow filter and / or a tangential flow filtration device.

[0017] Claim 6 advantageously further provides that the viral vector is concentrated in the retentate of the tangential flow filtration. Thus, impurities are separated and retained in the fluid, which flows out as permeate. This allows for a gentler purification method, as the viral vector does not pass through a separating filter element, such as a membrane, in the tangential flow filtration device.

[0018] According to one embodiment of claim 7, an ion exchange device is used for further purification of the viral vectors, which binds to the viral vectors and thus allows them to be obtained from the fluid, while further impurities in the fluid do not bind and are separated from the viral vectors.

[0019] According to claim 8, the fluids can be cooled and / or heated in different processing steps or rather components of the system. By controlling the temperature, the production and / or purification, in particular the separation, of the viral vectors can be optimized, in particular with regard to the activity of the viral vectors.

[0020] The equally important second teaching, as set forth in claim 9, relates to a system for producing and purifying viral vectors, particularly for carrying out the proposed method, in particular for carrying out the proposed method, wherein the viral vectors are produced by cells in a fluid, the system comprising a tangential flow filtration device for purifying the viral vectors produced by the cells in the fluid, and the system comprising a fluidized-bed centrifuge for separating at least a portion of cells and / or cell debris from the cells contained in the fluid after producing the viral vector and prior to purification by the tangential flow filtration device. The proposed system can be used to carry out the proposed method. Therefore, all descriptions given with respect to the proposed method are fully applicable. Furthermore, all descriptions below with respect to the proposed system are fully applicable to the proposed method.

[0021] The system according to claim 10 may include an initial holding container. The initial holding container allows for receiving and, in particular, temporarily holding fluids. It is intended that viral vectors also be produced in the initial holding container by cells in the fluid, particularly when the initial holding container is placed before a fluidized bed centrifuge. If the initial holding container is advantageously designed as a disposable initial holding container, contamination of different fluids with viral vectors can be avoided. Furthermore, the sterility of the cell cultures used to produce the viral vectors can be maintained. The initial holding container may include a mixing means for mixing and thus homogenizing the fluids received by the initial holding container.

[0022] Claims 11 and 12 further define a fluidized bed centrifuge, in particular a chamber for a fluidized bed centrifuge. In such a chamber, centrifugation can be performed simultaneously on a fluid flowing through the chamber. The preferred embodiment of claim 12 allows for a higher inflow velocity at the first flow opening at the tip, where the centrifugal force acting on the fluid is greater, and a lower outflow velocity at the second flow opening at the base, where the centrifugal force acting on the fluid is smaller, particularly during the filling and / or washing phases, resulting in improved separation performance.

[0023] The tangential flow filtration device may comprise at least one single-pass tangential flow filter for filtering the fluid (claim 13). By passing the fluid through the filter device in a single pass, no recirculation of the fluid is required.

[0024] Furthermore, the system may comprise an ion exchange device for purifying viral vectors according to claim 14. As already explained in the context of the method, the ion exchange device binds to the viral vectors and thus allows them to be obtained from the fluid, while further impurities in the fluid do not bind and are separated from the viral vectors.

[0025] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Brief explanation of the drawings]

[0026] [Figure 1] Schematic of the proposed integrated system for purifying viral vectors produced by cells in fluid. [Figure 2] Schematic of a further proposed integrated system in which the system includes a holding vessel after the fluidized bed centrifuge. [Figure 3] Schematic diagram of the different stages of separation by a fluidized bed centrifuge, a) shows the loading stage, b) shows the washing stage, and c) shows the draining stage. [Figure 4] 1 is a schematic diagram of a further proposed integrated system, where the system includes an advantageous embodiment of a tangential flow filtration device. [Figure 5] 1 is a schematic diagram of a further proposed integrated system, the system including an advantageous embodiment of a tangential flow filtration device, the system including a holding vessel; [Figure 6] Schematic of a further proposed integrated system in which diafiltration buffer is added to the fluid during the process. [Figure 7] Schematic diagram of a further proposed integrated system, in which the system comprises a further filter device.

[0027] Specifically, a method for producing and purifying a viral vector V from and / or in a fluid F is proposed. This method can be performed using a system 1, as exemplarily shown in FIG. 1 . The viral vector V is produced by cells in the fluid F. For example, the viral vector V, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a gammaretroviral vector, can be produced by cells, such as HEK-293 cells, in a fluid F, such as a cell culture medium. Preferably, the cells grow in suspension in a serum-free medium and release the produced viral vector into the medium. As exemplarily shown in FIG. 1 , the viral vector V can be produced in a holding vessel 12 of the system 1. The holding vessel 12 can be designed as a holding tank. Alternatively, the viral vector V can be pre-produced in the fluid F, for example, in a batch process, and then introduced into the system 1.

[0028] The term "viral vector" refers to any type of virus. For example, viral vector V can be a virus-based tool used to transfer and integrate a transgene of interest into target cells. Viral vectors can be produced by cells. Preferably, mammalian cells are used as the cells for producing viral vectors. It is even more preferable that the cells are adapted to grow in suspension. In a particularly preferred embodiment, suspension-adapted HEK293 cells are used to produce viral vectors. For viral vector production, cells are preferably maintained in a fluid F, such as a culture medium, and in an engineered culture environment. The term "culture medium" refers to a specially designed growth medium that provides the nutrients required by each cell to produce the viral vector. The term "culture environment" refers to the complex influence of all physical, chemical, and / or biological parameters, such as temperature, pH, and nutrient concentration, that act on one or more cells and determine their physiology and survival. The composition of the fluid F can change over time due to cell growth and metabolism. Therefore, the term "fluid" should be understood broadly and refers to any type of fluid, particularly liquids such as suspensions and dispersions. The composition of Fluid F may change during the process, for example, due to cell growth and metabolism and / or ongoing separation and / or purification. Fluid F may contain cells, at least some of the components of the culture medium (e.g., micronutrients, salts, etc.), as well as components produced by the cells, before entering the fluidized bed centrifuge 5. The components produced by the cells may include viral vectors, but may also include cellular metabolic products such as lactate, cell debris and / or DNA.

[0029] "DNA" has to be understood broadly in the context of this application. DNA includes in particular chromatin and / or plasmid DNA from transfection and / or released nucleic acid.

[0030] "Cellular debris" should be understood broadly in the context of the present application. Cellular debris includes, in particular, cells without an intact outer cell membrane or cell wall, especially dead cells, cell fragments such as parts of the cell membrane or cell wall, and / or cellular proteins, especially cellular proteins released from cells when the cell membrane or cell wall is not intact.

[0031] The terms "purifying" and "purification" in the context of the present application should be understood broadly. During or after purification, impurities may be separated from fluid F, and / or products and / or outputs in fluid F may be concentrated, and / or outputs may be separated from fluid F. The output may be a viral vector. The impurities may be cells and / or cell debris.

[0032] In the proposed method, a fluid F containing a viral vector V for purification is guided through a tangential flow filtration device 2, whereby the viral vector V is purified in the fluid F. Since the viral vector V is preferably the product to be obtained, the tangential flow filtration device 2 allows for gentle purification of the viral vector V. By using the tangential flow filtration device 2, impurities in the fluid F, such as cells and / or cell debris, can be filtered out. As exemplarily shown in FIG. 1 , the purified viral vector V in the fluid F can exit the tangential flow filtration device 2 as a retentate R, and thus the viral vector V is partially, particularly mostly, suppressed by the tangential flow filtration device 2 and does not flow through any separation filter element 4 or the like of the tangential flow filtration device 2. However, alternatively, the fluid F containing the viral vector V can exit the tangential flow filtration device 2 as a permeate P, and thus the viral vector V can partially, particularly mostly, flow through the separation filter element 4 or the like of the tangential flow filtration device 2. In the context of tangential flow filtration device 2, purification may include separating impurities from viral vector V and / or concentrating viral vector V in fluid F. By using tangential flow filtration device 2, purification may result in viral vector V of higher purity and / or higher concentration.

[0033] The term "impurity" refers to any component present in the fluid that is not the product. Thus, components can be cells, cell debris, DNA, media components, etc.

[0034] In the proposed method, it is essential that, after producing the viral vector V, cells and / or cell debris contained in the fluid F containing the viral vector V are at least partially separated from the viral vector V by a fluidized-bed centrifuge 5, particularly before purification by the tangential flow filtration device 2. During the production of the viral vector V, cells partially die, for example, due to enzymes added to the fluid F or naturally. When dead, the cells may contain intracellular components, particularly proteins and / or DNA, in the fluid. The cell debris includes, among other things, dead cells. Separation by the fluidized-bed centrifuge 5 allows a significant portion of impurities in the fluid F, such as cells and cell debris, to be separated prior to purification by the tangential flow filtration device 2, thus improving overall purification.

[0035] In a preferred embodiment of the method, as shown in FIG. 1, fluid F is supplied to a disposable initial holding container 12 to produce viral vector V. Fluid F may be a culture medium. Preferably, cells are added to fluid F, preferably in the initial holding container 12 or beforehand, so that viral vector V is produced by the cells, particularly in the initial holding container 12. For homogenization reasons, as shown in FIG. 1, fluid F in the initial holding container 12 is preferably at least temporarily mixed, for example, by a mixing means 14 of the initial holding container 12. The mixing means 14 may be a mixer, such as a dynamic mixer. Further preferably, the initial holding container 12 is equipped with temperature means for manipulating the temperature and / or aeration means for aerating fluid F in the initial holding container 12.

[0036] Preferably, an enzyme is added to the fluid F prior to the fluidized-bed centrifuge, preferably in the initial holding vessel 12, to degrade cells and / or cell debris and / or DNA impurities and / or reduce their molecular size. Enzymatic degradation of cells, cell debris, and / or DNA is common in the art, for example, to reduce the viscosity of a fluid and / or reduce the molecular weight of proteins. Enzymes such as nucleases, such as benzonase, can be used to degrade DNA. Furthermore, the viral vector V is preferably released from the cells into the fluid F, especially if it has not yet been transported by the cells into the fluid F. Because the viral vector V is preferably a product obtained by the overall purification process, the viral vector V must be released into the fluid F before the cells and cell debris are separated by the fluidized-bed centrifuge 5. Alternatively or additionally, charged beads CB are added to the fluid F so that cells, particularly cell membranes or cell walls, and / or cell debris and / or DNA impurities bind to the charged beads CB. The charged beads CB can improve the separation performance of the fluidized bed centrifuge 5, since the charged beads CB with bound, i.e. adsorbed, cells and / or cell debris and / or DNA can be more easily separated by the following separation, i.e. by the following centrifugation.

[0037] In this application, the terms "before" or "after" each component of system 1, such as fluidized bed centrifuge 5, refer to the flow of fluid F and therefore the order in which fluid F flows through each component of system 1.

[0038] Additionally, the fluid F containing cells and / or cell debris can be treated mechanically and / or via temperature change to lyse the cells, where they may release intracellular viral vectors V and / or DNA into the fluid F.

[0039] Alternatively or additionally, a cell disrupting agent is preferably added to fluid F, particularly in initial holding vessel 12 and / or intermediate holding vessel 13, where cells can be lysed such that particularly viral vector V and / or DNA and / or other intracellular components, such as intracellular proteins and metabolites, are released into fluid F. The cell disrupting agent can be a detergent such as sodium dodecyl sulfate, Triton X100, Tween, and / or an enzyme such as lysozyme.

[0040] In a further preferred embodiment, the separation of, in particular, cells and / or cell debris and / or DNA impurities and / or cell debris from the viral vector V by the fluidized bed centrifuge 5 comprises a loading stage LP, in which a fluid F containing the viral vector V, cells and / or cell debris passes through a chamber 6 of the fluidized bed centrifuge 5 during centrifugation, and at least a portion of the cells and / or cell debris accumulates unilaterally in the chamber 6 due to the acting centrifugal force, in particular due to the balance between the fluid flow force and the centrifugal force. During the loading stage LP, the cells and / or cell debris can be at least partially separated from the fluid F containing the viral vector V.

[0041] Furthermore, by using the fluidized bed centrifuge 5, less shear stress is exerted on the cells, resulting in fewer cells losing their cellular integrity, thus preventing the release of cellular debris and / or DNA and / or other intracellular components into the fluid F.

[0042] As exemplarily shown in FIGS. 1 and 3, the fluidized-bed centrifuge 5 comprises a chamber 6. A fluid F is guided from a holding vessel 12 into the chamber 6 and can pass through the chamber 6 during a filling stage LP (see FIG. 3a). The flow of the fluid F results in a fluid force acting on, in particular, the viral vectors V and / or cells and / or cellular debris, which are at least partially transported or transported together with the fluid F. At the same time, the chamber 6 rotates so that a centrifugal force acts on the fluid F, in particular the viral vectors V and / or cells and / or cellular debris. As shown schematically, the chamber 6 can rotate around a substantially vertical axis. However, it is also entirely possible for the chamber 6 to rotate around a substantially horizontal axis. Since the acting fluid force and the acting centrifugal force act in opposite directions, due to the acting centrifugal force, at least a portion of the cells and / or cellular debris accumulate unilaterally in the chamber 6 because the fluid force acting on them is lower than the centrifugal force acting on them, respectively. Because the fluid force acting on the viral vector V is greater than the centrifugal force acting on the viral vector V, the viral vector V passes through chamber 6 and is separated from at least a portion of the cells and / or cellular debris remaining in chamber 6. The use of charged beads CB, which are also separated by the fluidized bed centrifuge, can facilitate this separation. The flow rate of fluid F and / or the rotation speed of fluidized bed centrifuge 5 may be adjusted. The flow rate and / or rotation speed of fluid F should preferably be selected so that the cells and / or cellular debris remain at least partially, preferably mostly, in chamber 6 during centrifugation, especially during the loading and washing stages.

[0043] Preferably, separation by the fluidized-bed centrifuge 5 includes a washing step WP, in which a washing fluid WF passes through chamber 6 during centrifugation to wash the cells accumulated in chamber 6. The washing step WP optimizes separation by effectively removing any remaining fluid F containing the viral vector V, as well as any remaining non-cellular impurities, from chamber 6. Additionally, any viral vector V that may remain in chamber 6 may be separated from at least a portion of the cells and / or cell debris by the flow of washing fluid WG through chamber 6, and the washing fluid WF containing the viral vector V may flow out of chamber 6. The viral vector V may be prevented from passing through the chamber 6 during the loading step LP, for example, by accumulated cells and / or cell debris, and may be washed out of chamber 6 by the washing fluid WF during the washing step WP. The washing fluid WF may be a fluid F containing the viral vector V or a fluid F free of the viral vector V. Following the fluidized-bed centrifuge 5, the washing fluid WF may be mixed with the fluid F that passed through chamber 6 during the loading step, or may be further purified separately. The washing stage WP is exemplarily shown in FIG. 3b).

[0044] Alternatively or additionally, separation using the fluidized-bed centrifuge 5 includes a discharge phase DP in which a transport fluid TF is introduced into the chamber 6, and cells and / or cell debris accumulated unilaterally in the chamber 6 flow out of the chamber together with the transport fluid TF. During the discharge phase DP, the cells and / or cell debris can be transported from the chamber 6 by the transport fluid TF so that the chamber 6 is emptied. Because separation using the fluidized-bed centrifuge 5 is relatively gentle on cells, the cells can be advantageously reused for further production of viral vectors V. Due to the gentle separation process using the fluidized-bed centrifuge 5, the cells can remain viable. Preferably, during the discharge phase DP, the transport fluid TF is introduced into the chamber 6 such that its flow direction is opposite to the flow direction of the fluid F during the loading phase LP and / or the washing fluid WF during the washing phase WP, as exemplarily shown in FIG. 3c). More preferably, during the discharge phase DP, the chamber 6 rotates so that the centrifugal force acting thereon supports the discharge of the cells and / or cell debris, as exemplarily shown in FIG. 3c).

[0045] In a further preferred embodiment of the method, the fluid F containing the viral vector V is guided from the fluidized bed centrifuge 5 to an intermediate holding vessel 13, as exemplarily shown in FIG. 2 . The intermediate holding vessel 13 allows for temporary storage of the fluid F containing the viral vector V that has already passed through the fluidized bed centrifuge 5. The intermediate holding vessel 13 may be designed as a holding tank. Preferably, the fluid F flows discontinuously into the intermediate holding vessel 13 and flows continuously out of the intermediate holding vessel 13. Since the separation by the fluidized bed centrifuge 5 can be discontinuous, the intermediate holding vessel 13 preferably compensates for the fluctuating fluid flow from the fluidized bed centrifuge 5, so that the discontinuous outflow fluid F from the fluidized bed centrifuge 5 is temporarily stored, preferably allowing a continuous flow of the fluid F from the intermediate holding vessel 13. Thus, preferably, the fluid F containing the viral vector V flows discontinuously into the intermediate holding vessel 13 and flows continuously out of the intermediate holding vessel 13. Preferably, the intermediate holding vessel 13 is arranged after the fluidized bed centrifuge 5 and before the tangential flow filtration device 2. An enzyme may be added to the fluid F in the intermediate holding container 13, for example, as shown in FIG.

[0046] It should be noted that in the context of the present application, the term "second" in combination with each component of system 1 does not necessarily have to be understood as meaning that the embodiment, in particular system 1, comprises at least two of the respective components. Furthermore, the term "second" is chosen for distinction. Thus, system 1 may comprise a second component but not necessarily a respective first component.

[0047] According to a further preferred embodiment, enzymes are preferably added to fluid F after the fluidized-bed centrifuge 5 and / or before the tangential flow filtration device 2, resulting in the degradation of cells and / or cell debris and / or DNA impurities, in particular, thereby releasing viral vectors and / or reducing their molecular size. As already mentioned above, the degradation of cells, cell debris, in particular cellular proteins, and / or DNA impurities with enzymes is common in the art. Adding enzymes to fluid F after the fluidized-bed centrifuge 5 and / or before the tangential flow filtration device 2 can result in higher degradation performance, since the cells and / or cell debris are already at least partially separated from fluid F. As a result, less enzyme may be required to degrade the remaining cell debris and / or DNA impurities. Preferably, enzymes are added to the initial holding vessel 12 and / or the intermediate holding vessel 13.

[0048] Preferably, the viral vector V is concentrated in the fluid F exiting the tangential flow filtration device 2 as a concentrate R, and impurities are present in the fluid exiting the tangential flow filtration device 2 as a permeate P. Preferably, the viral vector V is at least partially retained by the tangential flow filtration device 2, particularly the single-pass tangential flow filter 3 and / or the separation filter element 4. As a result, the viral vector V, which is preferably the product of the entire purification process, does not pass through the separation filter element 4, e.g., a membrane, and thus the activity of the viral vector V exiting the tangential flow filtration device 2 can be maintained. The viral vector V is purified because the impurities are separated from the viral vector V, i.e., filtered, and the concentration of the viral vector V in the fluid F exiting the tangential flow filtration device 2 increases. As can be seen from FIG. 1 , for example, a concentrate R stream and a permeate P stream exit the tangential flow filtration device 2. The permeate P may contain at least a portion of impurities, particularly cells and / or cell debris and / or DNA impurities. The concentrate R may contain viral vectors V so that the concentrate R can be further purified after the tangential flow filtration device 2.

[0049] For further purification, a preferred embodiment of the proposed method includes guiding the fluid F containing the viral vector V through an ion exchanger 15, preferably after the tangential flow filtration device 2, to purify the viral vector V. The ion exchanger 15 preferably includes an ion exchange material 16, preferably an anion exchange material, more preferably a weak anion exchange material. Alternatively, the ion exchange material 16 may be a weakly hydrophobic material. It is further preferred that a monolith with a defined channel size distribution is used. In a particularly preferred embodiment, the channel size is in the 6 μm range. This allows larger particles, such as remaining cellular debris, to pass through the ion exchanger 15 instead of blocking the channels. In an exemplary embodiment, CIMmultus® OH, CIMmultus® SO3, or CIMmultus® QA can be used. For example, as shown in FIG. 1, the ion exchanger 15 may be positioned after the tangential flow filtration device 2. Here, the fluid F containing the viral vector V is guided from the tangential flow filtration device 2 to the ion exchanger 15. As fluid F flows through ion exchange device 15, viral vectors V are bound to ion exchange material 16. Impurities still present in fluid F may have a different charge relative to viral vectors V, so that the impurities at least partially do not bind to ion exchange material 16 and are therefore separated from viral vectors V. The impurities exit ion exchange device 15 along with fluid F flowing through ion exchange device 15.

[0050] In particular, the viral vector V, which is the product of the overall process, can be obtained as an eluate, preferably by using a suitable elution reagent, and more preferably, a further fluid F, particularly free of impurities and having an adjusted pH and / or conductivity, is guided through an ion exchanger 15, where the viral vector V is released from the ion exchange material 16. On the other hand, in the embodiment of FIG. 1, if the viral vector V is initially bound and the impurities are not bound to the ion exchange material 16, as shown in FIG. 1, the impurities may be bound and the viral vector V may not be bound to the ion exchange material 16. This can be achieved by adjusting the pH of the fluid F before entering the ion exchanger 15, particularly by a pH shift, and / or by selecting a different ion exchange material 16. In this case, the viral vector V flows through the ion exchanger 15, and the impurities can be eluted from the ion exchanger 15.

[0051] The pH and / or conductivity of fluid F can be adjusted before ion exchange device 15. The adjustment can control when viral vectors V bind to ion exchange material 16 or when impurities bind to ion exchange material 16. The pH is preferably adjusted so that viral vectors V first bind to ion exchange material 16 and are then obtained in fluid F as an eluate. Preferably, the pH and / or conductivity adjustment can be performed by tangential flow filtration device 2, in particular single-pass tangential flow filter 3, so that the pH and / or conductivity of fluid F flowing through tangential flow filtration device 2 is adjusted.

[0052] Furthermore, the fluid F containing the viral vector V is preferably guided through a further filtration device 19 after the tangential flow filtration device 2 and / or before the ion exchange device 15, and the viral vector V is purified by the further filtration device 19. The further filtration device 19 can further improve the overall purification process. Certain impurities that may not be removed by the tangential flow filtration device 2 and / or the fluidized bed centrifuge 5 can be removed by the further filtration device 19. Preferably, the fluid F containing the viral vector V is depth filtered when guided through the further filtration device 19. The further filtration device 19 is exemplarily shown in FIG. 7.

[0053] Preferably, the method includes at least partially cooling and / or heating Fluid F in the initial holding vessel 12 and / or in the fluidized bed centrifuge 5 and / or in the intermediate holding vessel 13 and / or in the tangential flow filtration device 2 and / or in the ion exchange device 15 and / or in the fluid line for guiding Fluid F. Viral vectors V are often temperature-sensitive. Therefore, at least partially controlling the temperature of Fluid F by heating or cooling within the process can result in higher activity of the purified viral vector V and therefore higher performance of the process. Preferably, the temperature of Fluid F is set to 4°C to 37°C, more preferably the temperature of Fluid F is set to 4°C.

[0054] In addition to the above method, as a second teaching, a system 1 is described in connection with the drawings. The above method can be implemented using the proposed system 1, so all descriptions and features of the method can be adapted to the system 1, and vice versa.

[0055] System 1 for purifying viral vector V produced by cells in fluid F includes a tangential flow filtration device 2 for purifying viral vector V produced by cells in fluid F. As can be seen from FIGS. 1 , 2 , 4 , 5 , and 6 , system 1 preferably includes, but is not limited to, a fluidized-bed centrifuge 5 for separating cells and / or cell debris contained in fluid F after producing viral vector V and before purification by tangential flow filtration device 2. Fluidized-bed centrifuge 5 is positioned before tangential flow filtration device 2 so that fluid F containing viral vector V can first flow through fluidized-bed centrifuge 5 and then flow through tangential flow filtration device 2. Impurities such as cells and cell debris have already been partially separated by fluidized-bed centrifuge 5, thereby improving the overall performance of the purification process.

[0056] In general, the performance of the separation process using the fluidized bed centrifuge 5 may be enhanced by adjusting the flow rate through the fluidized bed centrifuge 5, with lower flow rates resulting in higher performance, particularly since smaller particles are retained within the chamber 6 compared to when higher flow rates are utilized. Additionally, the performance of the purification process, i.e., the separation process, using the tangential flow filtration device 2 may be enhanced by adjusting the flow rate through the tangential flow filtration device 2, with lower flow rates resulting in higher performance. Improved separation performance may lead to improved overall purification performance.

[0057] According to a preferred embodiment, the system 1 includes an initial holding container 12. The initial holding container 12 can produce a viral vector V in a fluid F. The fluid F can be a culture medium. Preferably, cells are added to the fluid F in the initial holding container 12 or before introducing the fluid F into the initial holding container 12. Alternatively, a fluid F already containing the viral vector V can be loaded into the initial holding container 12 and temporarily stored therein. Preferably, the initial holding container 12 is designed as a disposable initial holding container 12. Thus, the initial holding container 12 can be changed within the system 1 for each run of purified viral vector V, thereby reducing the risk of contamination. More preferably, the initial holding container 12 includes at least one mixing means 14 for mixing the fluid F received by the initial holding container 12. Here, the fluid F is homogenized, which can result in a higher production rate of the viral vector V and / or a higher activity of the purified viral vector V and / or a higher overall performance of the purification process. Enzymes and / or charged beads CB can be added to the fluid F in the initial holding vessel 12 .

[0058] In a further preferred embodiment, the system 1 comprises an intermediate holding vessel 13. The intermediate holding vessel 13 can buffer the discontinuous flow of the fluid F from the fluidized bed centrifuge 5. Furthermore, it is possible to add enzymes and / or charged beads CB to the fluid F in the intermediate holding vessel 13. The intermediate holding vessel 13 may comprise a mixing means 14 for homogenization. Figure 2 exemplarily shows one embodiment of the system 1 comprising the intermediate holding vessel 13.

[0059] Advantageously, the fluidized bed centrifuge 5 comprises at least one chamber 6 through which the fluid F to be centrifuged can flow, as shown in particular in Figures 3a, 3b and 3c. As described above in connection with the method, the chamber 6 of the fluidized bed centrifuge 5 is flowed by the fluid F containing the viral vectors V and impurities, while the fluid F is centrifuged in such a way that at least a portion of the impurities is separated from the fluid F.

[0060] Regarding the design of chamber 6, it is preferable that chamber 6 be conical, having a tip 7 and a base surface 8 facing the tip 7, as shown in particular in Figures 3a, 3b, and 3c. The conical design of chamber 6 allows for simple control of the flow of fluid F through chamber 6 during centrifugation, particularly because the cross section changes such that the fluid flow rate is faster near the tip 7 compared to the base surface 8. Preferably, a first flow opening 9 for the inflow or outflow of fluid F into or out of chamber 6 is located at the tip 7 of chamber 6. Additionally or alternatively, a second flow opening 10 for the inflow or outflow of fluid F into or out of chamber 6 is located at the base surface 8 of chamber 6. The first flow opening 9 and / or the second flow opening 10 may have a dual function as an inflow and outflow opening. For example, fluid F may enter chamber 6 during the filling stage LP, as shown in Figure 3a. Here, fluid F containing viral vector V flows into chamber 6 via the first flow opening 9. Furthermore, fluid F is exiting chamber 6 via second flow opening 10. Thus, first flow opening 9 allows entry into chamber 6, and second flow opening 10 allows exit from chamber 6. During the cleaning phase WP, cleaning fluid WF may enter chamber 6 via first flow opening 9 and exit chamber 6 via second flow opening 10. During the draining phase DP, the flow direction of transport fluid TF may be opposite to the flow direction of fluid F and / or cleaning fluid WF, so that transport fluid TF may exit chamber 6 via first flow opening 9 and enter chamber 6 via second flow opening 10.

[0061] According to a further preferred embodiment, chamber 6 is disposed on a rotating means 11 that rotates during centrifugation. As a result, during centrifugation of fluids, particularly fluid F, washing fluid WF, and / or transport fluid TF, the centrifugal force exerted by fluid-bed centrifuge 5 on the fluid, particularly impurities, is greater at tip 7 of chamber 6 than at base 8. This can improve centrifugation performance, particularly due to the higher centrifugal force acting at tip 7, where the fluid flow may be higher, than at base 8, where the fluid flow may be lower. As a result of the fluid flow velocity gradient and centrifugal force gradient formed within chamber 6, particles with higher density and / or larger diameter may accumulate near tip 7, while particles with lower density and / or smaller diameter may accumulate toward base 8. Therefore, it is possible to manipulate the retention of particles with a particular size and / or density within chamber 6 by the rotation speed and / or flow rate of fluid F.

[0062] Advantageously, the tangential flow filtration device 2 comprises at least one single-pass tangential flow filter 3 for the filtration of the fluid F, as can be seen exemplarily in FIG. 1 . The single-pass tangential flow filter 3 may act as a pretreatment for the fluid F before it enters the ion exchange device 15. The single-pass tangential flow filter 3 may comprise a separation filter element 4, such as a membrane, which acts as the filtering element of the single-pass tangential flow filter 3. The separation filter element 4 may be designed as a hollow fiber membrane or a flat membrane. Preferably, the separation filter element 4 is designed to have a molecular weight cut-off of at least 500 kDa, preferably at least 400 kDa, and even more preferably at least 300 kDa ("kDa", kilodalton, 1 Da = 1 g / mol).

[0063] The tangential flow filtration device 2 can also preferably include two single-pass tangential flow filters 3, as shown in FIG. 4 . Preferably, the single-pass tangential flow filters 3 are arranged in series. Separation, i.e., filtration, by each single-pass tangential flow filter 3 can be performed under different conditions so that the filtration process can be controlled. Furthermore, using two or more single-pass tangential flow filters 3 can result in higher purification performance, such as higher separation performance and / or higher concentration of the viral vector V after the tangential flow filtration device 2.

[0064] As shown and preferred in the embodiment of Figure 5, the pH and / or conductivity of fluid F may be adjusted, in particular shifted, before or after separation by the following single-pass tangential flow filter 3. Here, and preferably, the pH and / or conductivity may be adjusted, in particular by adding a buffer solution, to the interior of an intermediate holding vessel 13 arranged between the single-pass tangential flow filters 3. Adjustment of the pH and / or conductivity may result in improved conditions in the subsequent ion exchange unit 15.

[0065] According to another preferred embodiment, exemplarily shown in Figure 6, the pH and / or conductivity of fluid F may be adjusted, in particular shifted, during separation by single-pass tangential flow filter 3. Here, a buffer solution is added directly to fluid F as it flows through single-pass tangential flow filter 3. Single-pass tangential flow filter 3 is preferably designed such that a buffer solution can be added to fluid F during purification, in particular separation, by single-pass tangential flow filter 3.

[0066] In a further advantageous embodiment, the system 1, in particular the tangential flow filtration device 2, comprises a first pre-filter arranged before the single-pass tangential flow filter 3 and / or before the separation filter element 4. Due to the first pre-filter, larger impurities can be filtered out by the first pre-filter, so that blocking of the single-pass tangential flow filter 3 and / or the separation filter element 4 can be avoided. The first pre-filter may be designed as a safety pre-filter.

[0067] Furthermore, the system 1, and in particular the ion exchange device 15, may be equipped with a second pre-filter arranged before the ion exchange material 16. The second pre-filter allows larger impurities to be filtered out by the second pre-filter, thereby avoiding adverse effects such as blocking of the ion exchange material 16. The second pre-filter may be designed as a safety pre-filter.

[0068] Furthermore, the system 1 preferably includes an ion exchange device 15 for purifying the viral vector V. As described in connection with the method, the ion exchange device 15 allows for further purification of the viral vector V, which can be obtained directly in the flow-through fluid F or as an eluate. Preferably, the ion exchange device 15 includes at least one ion exchange material 16. Here, anion exchange materials as well as cation exchange materials are generally possible. The ion exchange material 16 is preferably an ion, particularly anion or cation exchange resin, arranged in a column or formed as a membrane. The ion exchange device 15 preferably includes an ion exchange material 16, preferably an anion exchange material, more preferably a weak anion exchange material. Alternatively, the ion exchange material 16 may be a weakly hydrophobic material. It is further preferred that a monolith with a defined channel size distribution is used. In a particularly preferred embodiment, the channel size is in the 6 μm range. This allows larger particles, such as residual cellular debris, to pass through the ion exchange device 15 instead of blocking the channels. In an exemplary embodiment, CIMmultus® OH, CIMmultus® SO3 or CIMmultus® QA may be used.

[0069] As shown in FIG. 7, the system 1 preferably includes an additional filtration device 19, preferably disposed after the tangential flow filtration device 2 and / or before the ion exchange device 15. The additional filtration device 19 may include at least one additional filter 20. It is particularly possible for the additional filter 20 to be a second pre-filter. The additional filter 20, particularly the second pre-filter, is preferably designed as a depth filter. Generally, the additional filter 20 can include an additional separation filter element 21. If the additional filter 20 is designed as a depth filter, the additional separation filter element 21 can include a depth filtration material. Here, the depth filter material is preferably designed so that the average retention of the additional separation filter element 21 is at least 0.05 μm and at most 15.0 μm, preferably at least 0.1 μm and at most 10.0 μm, more preferably at least 0.2 μm and at most 8.0 μm.

[0070] The system 1 further preferably comprises at least one pumping device 17. The pumping device 17 or pumping devices 17, respectively, may be designed as peristaltic pumps. Preferably, the system 1 comprises several pumping devices 17, which are arranged before the fluidized bed centrifuge 5 and / or after the fluidized bed centrifuge 5 and before the tangential flow filtration device 2 and / or after the tangential flow filtration device 2 and before the ion exchange device 15, respectively, as exemplarily shown in FIG.

[0071] Preferably, the system 1 comprises a fluid line 18 for conducting the fluid F within the system 1. Preferably, the fluid line 18 is arranged so that the fluid F can be conducted to and from components of the system 1, such as the fluidized bed centrifuge 5 and / or the tangential flow filtration device 2 and / or the ion exchange device 15 and / or the initial holding vessel 12 and / or the intermediate holding vessel 13. Further preferably, the fluid line 18 is designed as a tube and / or a pipe, respectively. Further preferably, the fluid line 18 may be designed as a tube and / or a pipe if the pump device 17 or pump devices 17 are designed as a peristaltic pump or peristaltic pumps.

[0072] In the drawings, the direction of flow of fluid F is generally from left to right. As can be seen exemplarily in Figure 1, within system 1, a fluidized bed centrifuge 5 is arranged before the tangential flow filtration device 2 with respect to the flow of fluid F. Thus, fluid F enters fluidized bed centrifuge 5 before tangential flow filtration device 2. In other words, separation by fluidized bed centrifuge 5 is performed before purification, i.e., separation and / or concentration such as filtration by tangential flow filtration device 2.

[0073] Furthermore, as can be seen in FIG. 2, further components of system 1, such as an intermediate holding vessel 13, may be disposed between fluidized bed centrifuge 5 and tangential flow filtration device 2. As can be further exemplarily seen in FIG. 1, within system 1, tangential flow filtration device 2 is preferably disposed before ion exchange device 15 with respect to the flow of fluid F. Thus, fluid F enters tangential flow filtration device 2 before ion exchange device 15. Further components of system 1, such as a further holding vessel, may be disposed between tangential flow filtration device 2 and ion exchange device 15. Prior to fluidized bed centrifuge 5, an initial holding vessel 12 may preferably be disposed, for example, as shown in FIG. 1. Fluid F may enter system 1 in initial holding vessel 12.

Claims

1. A method for producing and purifying a viral vector (V), wherein the viral vector (V) is produced by cells in a fluid (F), and the fluid (F) containing the viral vector (V) is guided through a tangential flow filtration device (2) for purification, whereby the viral vector (V) is purified in the fluid (F); the cells and cell debris contained in the fluid (F) containing the viral vector (V) after producing the viral vector (V) are at least partially separated from the viral vector (V) by a fluidized bed centrifuge (5) before purification by the tangential flow filtration device (2), method.

2. 2. The method according to claim 1, characterized in that, prior to the fluidized bed centrifuge (5), an enzyme is added to the fluid (F), so that the cells and / or the cell debris and / or DNA impurities are degraded, in particular the viral vectors (V) are released from the cells into the fluid (F) and / or their molecular size is reduced, and / or charged beads (CB) are added to the fluid (F), so that the cells and / or the cell debris and / or the DNA impurities bind to the charged beads (CB).

3. the separation by the fluidized bed centrifuge (5) comprises a loading stage (LP) in which the fluid (F) containing the viral vectors (V), cells and / or cell debris passes through a chamber (6) of the fluidized bed centrifuge (5) during centrifugation, and at least a portion of the cells and / or cell debris accumulates unilaterally in the chamber (6) due to the centrifugal force acting thereon; and / or the separation by the fluidized bed centrifuge (5) comprises a washing step (WP) in which a washing fluid (WF) is passed through the chamber (6) during centrifugation, so that the cells accumulated in the chamber (6) are washed; and / or 3. The method according to claim 1 or 2, characterized in that the separation by the fluidized bed centrifuge (6) comprises a discharge stage (DP) in which a transport fluid (TF) is introduced into the chamber (6), and the cells and / or cell debris accumulated unilaterally in the chamber (6) flow out of the chamber (6) together with the transport fluid (TF).

4. 4. The method according to claim 1, wherein the fluid (F) containing the viral vector (V) is guided from the fluidized bed centrifuge (5) to an intermediate holding vessel (13), preferably with a discontinuous flow into the intermediate holding vessel (13) and a continuous flow out of the intermediate holding vessel (13).

5. 5. The method according to any one of claims 1 to 4, characterized in that an enzyme is added to the fluid (F), preferably after the fluidized bed centrifuge (5) and / or before the tangential flow filtration device (2), so that the cells and / or the cell debris and / or DNA impurities are degraded, in particular so that the viral vectors (V) are released and / or the molecular size is reduced.

6. 6. The method according to claim 1, wherein the viral vector (V) is concentrated in the fluid (F) leaving the tangential flow filtration device (2) as a concentrate (R) and impurities are separated in the fluid leaving the tangential flow filtration device (2) as a permeate (P).

7. 7. The method according to any one of claims 1 to 6, characterized in that the fluid (F) containing the viral vector (V) is guided through an ion exchange device (15), preferably after the tangential flow filtration device (2), to purify the viral vector (V).

8. 8. The method according to claim 1, wherein the fluid (F) in the initial holding vessel (12) and / or the fluidized bed centrifuge (5) and / or the intermediate holding vessel (13) and / or the tangential flow filtration device (2) and / or the ion exchange device (15) and / or the fluid line (18) for guiding the fluid (F) is at least partially cooled and / or heated.

9. A system for producing and purifying viral vectors (V), in particular for carrying out the method according to any one of claims 1 to 8, comprising a tangential flow filtration device (2) for purifying viral vectors (V) produced by cells in a fluid (F), the system (1) comprising a fluidized bed centrifuge (2) for separating cells and / or cell debris from the cells contained in the fluid (F) after producing the viral vector (V) and before purification by the tangential flow filtration device (2); A system characterized by:

10. the system (1) comprises an initial holding vessel (12), preferably arranged before the fluidized bed centrifuge (5), preferably the initial holding vessel (12) is designed as a disposable initial holding vessel, further preferably the initial holding vessel (12) has at least one mixing means (14) for mixing the fluids received by the initial holding vessel (12), and / or The system (1) comprises an intermediate holding vessel (13), preferably arranged after the fluidized bed centrifuge (5), preferably the intermediate holding vessel (13) is designed as a disposable intermediate holding vessel, more preferably the intermediate holding vessel (13) has at least one mixing means (14) for mixing the fluids received by the intermediate holding vessel (13). The system of claim 9 .

11. 11. A system according to claim 9 or 10, characterized in that the fluidized bed centrifuge (5) has at least one chamber (6), preferably four chambers (6), through which the fluid (F) to be centrifuged can flow.

12. 12. The system according to claim 11, characterized in that the chamber (6) is conical with a tip (7) and a base surface (8) facing the tip (7), preferably a first flow opening (9) for the inflow or outflow of a fluid (F) into or out of the chamber (6) is arranged at the tip (7) of the chamber (6) and / or a second flow opening (10) for the inflow or outflow of a fluid (F) into or out of the chamber (6) is arranged at the base surface (8) of the chamber (6), preferably the chamber (6) is arranged on a rotating means (11) that rotates during centrifugation, so that during centrifugation of a fluid (F) in the fluidized bed centrifuge (5), the centrifugal force acting on the fluid is greater at the tip (7) of the chamber (8) than at the base surface (8).

13. 13. The system according to any one of claims 9 to 12, characterized in that the tangential flow filtration device (2) comprises at least one single-pass tangential flow filter (3) for the filtration of the fluid (F).

14. 14. The system according to claim 9, wherein the system (1) comprises an ion exchange device (15) for purifying the viral vector (V), preferably the ion exchange device (15) comprises at least one ion exchange material (16), in particular an ion exchange resin or an ion exchange monolith, the ion exchange resin being composed of a porous matrix in the form of spherical particles or a polymer membrane, preferably the ion exchange material (16) is formed as an anion exchange material, in particular an anion exchange resin or an anion exchange monolith, the anion exchange resin being composed of a porous matrix in the form of spherical particles or a polymer membrane.

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