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 from cells in a fluid (F) and wherein the fluid (F) comprising the viral vector (V) is conducted through a tangential flow filtration device (2) for purification, whereby the viral vector (V) is purified in the fluid (F). It is proposed that the cells and cell debris from the cells 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), which cells and cell debris are contained in a fluid (F) containing the viral vector (V) after production of the viral vector (V).

Description

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

[0002] The method can be applied to different types of biological processes. 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 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 properties of the cells used, different types of viral vectors can be produced, such as retroviral vectors, including lentiviral vectors and gamma retroviral vectors, adenoviral vectors or adeno-associated viral vectors. Preferably, a mammalian cell line (e.g., HEK293 suspension cells) suitable for growth in suspension is used to produce the viral vector. For this reason, under certain conditions of producing the viral vector, the cells are maintained in a fluid (e.g., culture medium).

[0004] In some embodiments, the viral vector of the present invention is purified by a filtration device. For example, a fluid containing viral vector and cells and cell fragments from cells must be purified to obtain purified viral vectors, which can then be used further. In order to purify, particularly in order to separate cells and cell fragments from viral vectors, the fluid containing cells, cell fragments and viral vectors can be guided through a filter device to clarify, wherein the fluid passes through a filter element, such as a membrane, etc. Here, at least partially retains larger impurities of a certain size, such as cells and cell fragments, wherein the fluid containing viral vectors and smaller impurities passes through a filter element. After filtering the fluid by such a "passing through" filter device, a tangential flow filtration device is used to separate the remaining impurities from the viral vector in the fluid and / or to concentrate the viral vector in the fluid. For this reason, the fluid containing viral vectors is guided through a tangential flow filtration device for purification, and therefore remaining impurities are at least partially separated from viral vectors. As a result, the viral vector can be purified from larger and smaller impurities.

[0005] A kind of known method for producing and purifying viral vector in fluid is disclosed in WO 2021 / 252782 A1, which constitutes the basis of the present invention.Here, viral vector (such as slow viral vector) is produced by the cell in fluid, and the composition corresponding to the fluid comprising viral vector and cell and cell debris is first fed through a filter, in this case, an alternating tangential flow filter, wherein the fluid comprising viral vector passes through the filter element of the filter.Then, the filtered fluid feed comprising viral vector is passed through a single-pass tangential flow filtration system (SPTFF system).As a result, larger impurities (such as cells and cell debris) are separated from fluid in a filter, and thereafter smaller impurities are separated from fluid.As a result, viral vector is purified in fluid, and therefore represents the viral vector composition of purification.

[0006] Although known methods already allow for a good way to purify produced viral vectors, the degree of purification is strongly influenced by the conditions of the SPTFF system, e.g. with regard to the membrane, operating parameters, clogging and fouling of the membrane, etc. In particular, over time, the separation performance of the SPTFF system may decrease due to fouling of the SPTFF system, as cells and cell debris clog the membrane.

[0007] In addition to the resulting reduced separation performance of the SPTFF system, another problem with the known methods is that the activity of the purified viral vector in the fluid is rapidly reduced by using a filter because a portion of the viral vector may be destroyed when passing through the filter. In addition, the viral vector may be trapped by the membrane and / or may be adsorbed onto the membrane rather than passing through it. Therefore, the purified viral vector composition obtained may contain only a small amount of active viral vector.

[0008] Therefore, there is still a need to further improve the known methods.

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

[0010] This problem is solved by the characterizing features of claim 1 .

[0011] The main realization of the present invention is that before purifying by tangential flow filtration device, at least part of the cells in the fluid after producing viral vector are separated from the cell debris from the cell by fluidized bed centrifuge, particularly separated from the viral vector. Therefore, the fluidized bed centrifuge is arranged before the tangential flow filtration device. The separation by the fluidized bed centrifuge can be used as a kind of pre-treatment step before purifying (i.e. separating and / or concentrating) by the tangential flow filtration device. Due to the separation by the fluidized bed centrifuge, it is possible to carry out a wide range of adjustments as needed, particularly with respect to the duration of rotation speed, flow velocity, centrifugation, etc., to carry out efficient and highly gentle separation. In contrast to other centrifugal methods, the separation by the fluidized bed centrifuge (i.e. centrifugation) therefore results in a higher activity of the viral vector purified in the fluid. In addition, because cells and cell debris are separated from the fluid comprising viral vector in a gentle manner by the fluidized bed centrifuge, it is possible to prevent the further filtration by using a filter, which results in a higher activity of the viral vector obtained after the whole purification process. In addition, due to the gentle separation by the fluidized bed centrifuge, cells are complete and it is advantageously possible to expect to reuse at least a portion of cells. In conclusion, the concentration as well as the activity of viral vectors were increased using the proposed method.

[0012] Specifically, it is proposed that cells and cell debris, which are present in the fluid containing the viral vector after viral vector production, be at least partially separated, in particular from the viral vector, by a fluidized bed centrifuge prior to purification by a tangential flow filtration device. This can thereby increase the concentration and activity of the purified viral vector in the fluid.

[0013] According to claim 2, enzymes and (in addition or as an alternative) charged beads can be added to the fluid before the fluidized bed centrifuge. Enzymes can degrade cells, particularly cell membranes, cell fragments and / or DNA impurities, such that, for example, the viscosity of the fluid is reduced and / or the molecular weight of the protein is reduced. In addition, if the cells do not release the viral vector into the fluid, degraded cells (particularly cell membranes or cell walls) can cause the viral vector to be released into the fluid. Charged beads can also be used to improve the subsequent separation process by the fluidized bed centrifuge, because at least a portion of cells, cell fragments and / or DNA can be combined on these. Because cells, cell fragments and / or DNA impurities are combined to the charged beads, and by separating the charged beads in the subsequent separation process, the combined cells, cell fragments and / or DNA are separated therefrom.

[0014] Advantageously, claim 3 further details the separation by a fluidized bed centrifuge, wherein the separation comprises a loading phase, a washing phase and / or a discharge phase. During the loading phase, the chambers (particularly several chambers) of the fluidized bed centrifuge are loaded with fluid. Preferably, loading is performed while centrifugation has already been performed. In this way, advantageously, separation of the viral vector from at least some of the cells, cell fragments and / or DNA impurities contained in the fluid has occurred. In the washing phase, a washing fluid passes through the chambers so that the cells accumulated in the chambers can be advantageously washed. Therefore, the remaining fluid may be washed out of the chambers by the washing fluid. In addition, the viral vectors retained in the chambers during the loading phase may be washed out with fluid and / or washing fluid during the washing phase. During the discharge phase, in the chambers of the fluidized bed centrifuge, cells, cell fragments and / or DNA impurities accumulated on one side, in particular, are discharged by the transport fluid so that these are washed out of the chambers, in particular as waste or for reuse.

[0015] According to claim 4, after separation, the fluid containing the viral vectors can be directed from the fluidized bed centrifuge into a holding container. In particular, if separation by the fluidized bed centrifuge is used cyclically, the holding container provides intermediate storage for the fluid. Furthermore, for example, a constant outflow of the fluid after the holding container can be provided, or larger quantities of separated fluid can be stored.

[0016] According to claim 5, enzyme can be added in the fluid.Therefore, can be at least partially degraded by the cell (particularly cell membrane and / or cell wall, cell debris and / or DNA impurity) that is separated in advance or not separated by fluid-bed centrifuge during separation.In addition, enzyme can be degraded in a more effective manner, because cell, cell debris and / or DNA are partly separated with fluid, so that enzyme will only be adsorbed on cell, cell debris and / or DNA, it is not separated and still is included in the fluid.In addition, by degrading, the viscosity of fluid can be reduced, and this can simplify subsequent method step. Usually can be before fluid-bed centrifuge and in addition or alternatively after fluid-bed centrifuge by adding enzyme to degrade at least part of cell, cell debris and / or DNA impurity, this can improve purification process usually.Can prevent tangential flow filtration device (particularly separation filter element of single-pass tangential flow filter and / or tangential flow filtration device) from blocking by the degraded of enzyme.

[0017] Claim 6 further advantageously specifies that the viral vectors are concentrated in the retentate of the tangential flow filtration. Impurities are thus separated and retained in the fluid that flows out as permeate. Purification can thus be performed in an even gentler manner, since the viral vectors do not pass through the separation filter element, such as a membrane, of the tangential flow filtration device.

[0018] According to the embodiment of claim 7, an ion exchange device is used to further purify the viral vectors. The ion exchange device is capable of binding the viral vectors and thus obtaining these viral vectors from the fluid, wherein other impurities in the fluid are not bound and are separated from the viral vectors.

[0019] According to claim 8, the fluid can be cooled and / or heated in different process steps or even components of the system. While regulating the temperature, the production and / or purification, in particular the separation, of the viral vector can be optimized, in particular with regard to the activity of the viral vector.

[0020] According to the second teaching of claim 9, which is equally important, a system for producing and purifying viral vectors, in particular a system for carrying out the proposed method, wherein in particular 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, wherein the system comprises a fluidized bed centrifuge for separating at least some of the cells and / or cell fragments from the cells contained in the fluid after the viral vectors are produced and before purification by the tangential flow filtration device. The proposed system can be used to carry out the proposed method. Therefore, all explanations given with respect to the proposed method are fully applicable. In addition, all following explanations with respect to the proposed system are fully applicable to the proposed method.

[0021] According to claim 10, the system can include an initial holding container. The initial holding container is capable of receiving and, in particular, temporarily holding a fluid. It is anticipated that viral vectors will also be produced from cells in the fluid within the initial holding container, in particular if the initial holding container is arranged upstream of the fluidized bed centrifuge. If the initial holding container is advantageously designed as a disposable initial holding container, contamination between different fluids and viral vectors can be avoided. In addition, the sterility of the cell culture used for producing the viral vector can be maintained. The initial holding container can include a mixing device for mixing and thus homogenizing the fluid received by the initial holding container.

[0022] Claims 11 and 12 further specify a fluidized bed centrifuge, in particular a chamber for a fluidized bed centrifuge. With such a chamber, centrifugation can occur simultaneously with the fluid flowing through the chamber. According to the preferred embodiment of claim 12, in particular during the loading and / or washing phases, a higher inflow velocity at the first flow opening at the tip, where the centrifugal forces acting on the fluid are greater, can be achieved, and a lower outflow velocity at the second flow opening at the base surface, where the centrifugal forces acting on the fluid are lower, can be achieved, 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, recirculation of the fluid is not required.

[0024] Furthermore, according to claim 14, the system can comprise an ion exchange device for purifying viral vectors. As already described in the context of the method, the ion exchange device is able to bind viral vectors and thus obtain these viral vectors from the fluid, wherein other impurities in the fluid are not bound and are separated from the viral vectors.

[0025] The following describes an embodiment of the present invention with reference to the accompanying drawings.

[0026] Figure 1 is a schematic diagram of the proposed integrated system for purifying viral vectors produced by cells in fluid,

[0027] Figure 2 is a schematic diagram of a further proposed integrated system, wherein the system comprises a holding vessel after the fluidized bed centrifuge,

[0028] Figure 3 is a schematic diagram of the different stages of separation by a fluidized bed centrifuge, wherein a) shows the loading stage, b) shows the washing stage and c) shows the discharge stage,

[0029] Figure 4 is a schematic diagram of a further proposed integrated system, wherein the system comprises advantageous embodiments of a tangential flow filtration device,

[0030] Figure 5 is a schematic diagram of a further proposed integrated system, wherein the system comprises an advantageous embodiment of a tangential flow filtration device, wherein the system comprises a holding container,

[0031] Figure 6 is a schematic diagram of a further proposed integrated system, wherein during the process, a diafiltration buffer is added to the fluid, and

[0032] Figure 7 is a schematic diagram of a further proposed integrated system, wherein the system comprises a further filter device.

[0033] A method for producing and purifying a viral vector V, in particular a method for producing and purifying a viral vector V from and / or in a fluid F, is proposed. Figure 1The 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, a gamma-retroviral vector, etc.) can be produced by cells (such as HEK-293 cells) in the fluid F (such as a cell culture medium). Preferably, the cells are grown in suspension in a serum-free culture medium and the produced viral vector is released into the culture medium. Figure 1 As shown in the example, the viral vector V can be produced in the holding container 12 of the system 1. The holding container 12 can be designed as a holding tank. Alternatively, the viral vector V can be produced in the fluid F in advance, for example in a batch process, and can be introduced into the system 1 thereafter.

[0034] The term "viral vector" refers to all kinds of viruses. For example, viral vector V can be a tool based on viruses for transferring and integrating transgenes of interest into target cells. Viral vectors can be produced by cells. Preferably, mammalian cells are used as cells for producing viral vectors. Further preferably, cells are suitable for suspension growth. In a particularly preferred embodiment, HEK293 cells adapted to suspension are used to produce viral vectors. For viral vector production, cells are preferably maintained in a fluid F (e.g., culture medium) and a controlled culture environment. The term "culture medium" refers to a specially designed growth medium that supplies the nutrients required for each cell to produce viral vectors. The term "culture environment" currently refers to the complex influence of the entirety of all physical, chemical and / or biological parameters (e.g., temperature, pH, and nutrient concentration), which act on one or more cells and determine their physiology and survival. The composition of fluid F can change over time through the growth and metabolism of cells. Therefore, the term "fluid" should be broadly understood and refers to all kinds of fluids, particularly liquids, such as suspensions, dispersions, etc. The composition of fluid F may change during the method, for example, due to the growth and metabolism of cells and / or ongoing separation and / or purification. Fluid F may contain cells, at least some components of the culture medium (e.g., micronutrients, salts, etc.), and components produced by the cells before entering the fluidized bed centrifuge 5. Components produced by the cells may include viral vectors, but may also include cellular metabolites such as lactate, cell debris, and / or DNA.

[0035] In the context of this application, "DNA" must be understood in a broad sense. DNA also comprises, in particular, chromatin and / or plasmid DNA from transfection and / or free nucleic acids.

[0036] In the context of the present application, "cell debris" must be understood in a broad sense. Cell debris particularly comprises cells without an intact outer cell membrane or cell wall, in particular dead cells, cell fragments, such as parts of the cell membrane or cell wall and / or cell proteins, in particular cell proteins released from cells when the cell membrane or cell wall is not intact.

[0037] In the context of this application, the terms "purifying" and "purification" must be understood in a broad sense. During or during purification, impurities may be separated from the fluid F and / or the product, and / or the product may be concentrated in the fluid F and / or the product may be separated from the fluid F. The product may be a viral vector. Impurities may be cells and / or cell debris.

[0038] By using the proposed method, a fluid F containing a 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. Since the viral vector V is preferably the product to be obtained, the tangential flow filtration device 2 can gently purify the viral vector V. By using the tangential flow filtration device 2, impurities (such as cells and / or cell debris) in the fluid F can be filtered out. Figure 1 As exemplarily shown in FIG, the purified viral vector V in the fluid F can flow out of the tangential flow filtration device 2 as a retentate R, and thus the viral vector V is partially (particularly mostly) retained by the tangential flow filtration device 2 and does not flow through any separation filter element 4, etc., of the tangential flow filtration device 2. However, alternatively, the fluid F containing the viral vector V can also flow out of 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, etc., of the tangential flow filtration device 2. In the context of the tangential flow filtration device 2, purification can include separating impurities from the viral vector V and / or concentrating the viral vector V in the fluid F. By using the tangential flow filtration device 2, purification can therefore result in a higher purity and / or a higher concentration of the viral vector V.

[0039] The term "impurity" refers to any component present in a fluid that is not a product. Thus, a component may be cells, cell debris, DNA, culture medium components and / or the like.

[0040] For the proposed method, it is essential that after the viral vector V is produced, the cells and / or cell fragments contained in the fluid F containing the viral vector V are at least partially separated, in particular separated from the viral vector V, by a fluidized bed centrifuge 5 and then purified by a tangential flow filtration device 2. While the viral vector V is being produced, the cells partially die, for example due to enzymes added to the fluid F, or die naturally. When dying, the cells may allow intracellular components (in particular proteins and / or DNA) to enter the fluid. The cell fragments particularly contain dead cells. Due to the separation by the fluidized bed centrifuge 5, most of the impurities (such as cells and cell fragments) in the fluid F can be separated before being purified by the tangential flow filtration device 2, and thus the overall purification can be improved.

[0041] In a preferred embodiment of the method, and as Figure 1 As shown, in order to produce the viral vector V, a fluid F is fed into an initial holding container 12, which is particularly disposable. The fluid F may be a culture medium. Preferably, cells are added to the fluid F in the initial holding container 12 or in advance, so that the viral vector V is produced by the cells, particularly in the initial holding container 12. For homogenization reasons, and as Figure 1 As shown, the fluid F in the initial holding container 12 is preferably at least temporarily mixed, for example, by a mixing device 14 of the initial holding container 12. The mixing device 14 can be a mixer, such as a dynamic mixer, etc. Further preferably, the initial holding container 12 includes a temperature device for controlling the temperature and / or an aeration device for aerating the fluid F in the initial holding container 12.

[0042] Preferably, before fluid bed centrifuge, preferably in initial holding container 12, enzyme is added in fluid F so that cell and / or cell debris and / or DNA impurity degradation and / or molecular size are reduced. It is common in the art by enzyme degradation of cells, cell debris and / or DNA, for example to reduce the viscosity of fluid and / or reduce the molecular weight of protein. In order to degrade DNA, enzyme such as nuclease (nuclease), for example nuclease (benzonase) can be used. In addition, if particularly viral vector V is not yet transported in fluid F by cell, viral vector V is preferably released into fluid F from cell. Because viral vector V is preferably the product that should be obtained by whole purification process, before separating cells and cell debris by fluid bed centrifuge 5, viral vector V must be released into fluid F. Alternatively or in addition, charged pearl CB is added in fluid F so that cell (particularly cell membrane or cell wall) and / or cell debris and / or DNA impurity are attached to charged pearl CB. The charged beads CB can improve the separation performance of the fluidized bed centrifuge 5 , since the charged beads CB with bound (ie adsorbed) cells and / or cell debris and / or DNA can be separated more easily by subsequent separation (ie by subsequent centrifugation).

[0043] In this application, the terms “before” or “after” the corresponding components of the system 1 (such as the fluidized bed centrifuge 5 , etc.) refer to the flow of the fluid F, and therefore refer to the order in which the fluid F flows through the corresponding components of the system 1 .

[0044] Furthermore, the fluid F containing cells and / or cell fragments can be treated mechanically and / or via temperature changes to cause cell lysis. Here, the cells can release intracellular viral vectors V and / or DNA into the fluid F.

[0045] Alternatively or in addition, a cell disrupting agent is preferably added to the fluid F, in particular in the initial holding container 12 and / or the intermediate holding container 13. Here, the cells can be lysed so that in particular the viral vector V and / or DNA and / or other intracellular components (such as intracellular proteins and metabolites) are released into the 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.

[0046] In another preferred embodiment, the separation of, in particular, cells and / or cell fragments and / or DNA impurities and / or cell fragments from the viral vectors V by means of the fluidized bed centrifuge 5 comprises a loading phase LP, in which, during centrifugation, the fluid F containing the viral vectors V, cells and / or cell fragments passes through the chamber 6 of the fluidized bed centrifuge 5, wherein at least a portion of the cells and / or cell fragments accumulates unilaterally in the chamber 6 due to the acting centrifugal forces (in particular due to the balance between the fluid flow forces and the centrifugal forces). During the loading phase LP, the cells and / or cell fragments can be at least partially separated from the fluid F containing the viral vectors V.

[0047] Additionally, by using a fluidized bed centrifuge 5 , less shear stress may be exerted on the cells, which results in fewer cells losing their integrity and thus prevents the release of cell debris and / or DNA and / or other intracellular components into the fluid F.

[0048] like Figure 1 and Figure 3 As shown schematically in FIG, the fluidized bed centrifuge 5 comprises a chamber 6. During the loading phase LP, a fluid F can be led from the holding container 12 to the chamber 6 and through the chamber 6, see Figure 3a). The flow of fluid F generates a flow force that acts in particular on the viral vectors V and / or cells and / or cell fragments, thereby at least partially transporting them along with the fluid F. Simultaneously, chamber 6 rotates, causing centrifugal force to act on fluid F, and in particular on the viral vectors V and / or cells and / or cell fragments. As schematically depicted, chamber 6 can rotate about a substantially vertical axis. However, it is also possible for chamber 6 to rotate about a substantially horizontal axis. Because the flow force and the centrifugal force act in opposite directions, at least a portion of the cells and / or cell fragments accumulate on one side of chamber 6 due to the centrifugal force, because the flow force acting on these cells and / or cell fragments is lower than the centrifugal force acting on these cells and / or cell fragments. Because the flow force acting on the viral vectors V is greater than the centrifugal force acting on the viral vectors V, the viral vectors V pass through chamber 6 and are separated from at least a portion of the cells and / or cell fragments retained in chamber 6. This separation can be enhanced by using charged beads CB that are also separated by a fluidized bed centrifuge. It is possible to adjust the flow rate of the fluid F and / or the rotation speed of the fluidized bed centrifuge 5. The flow rate of the fluid F and / or the rotation speed must preferably be selected such that the cells and / or cell debris remain at least partially (preferably mostly) in the chamber 6 during centrifugation (in particular during the loading and washing phases).

[0049] Preferably, the separation by the fluidized bed centrifuge 5 includes a washing phase WP, in which a washing fluid WF is passed through the chamber 6 during centrifugation, wherein the cells accumulated in the chamber 6 are washed. By means of the washing phase WP, the separation can be optimized to the effect that any remaining fluid F containing the viral vectors V and remaining other impurities in addition to the cells can be removed from the chamber 6. In addition, it is possible that, due to the flow force exerted by the washing fluid WG flowing through the chamber 6, the viral vectors V that may remain in the chamber 6 can be separated from at least a portion of the cells and / or cell fragments, and the washing fluid WF containing the viral vectors V can flow out of the chamber 6. For example, during the loading phase LP, the passage of the viral vectors V through the chamber may be hindered by accumulated cells and / or cell fragments, and during the washing phase WP, the viral vectors V are washed out of the chamber 6 by the washing fluid WF. The washing fluid WF can be the fluid F containing the viral vectors V or the fluid F not containing the viral vectors V. The washing fluid WF can be mixed with the fluid F after the fluidized bed centrifuge 5, which passes through the chamber 6 during the loading phase, or can be further purified separately. The washing phase WP is exemplarily shown in Figure 3 b).

[0050] Alternatively or in addition, the separation by the fluidized bed centrifuge 5 comprises a discharge phase DP, in which a transport fluid TF is introduced into the chamber 6, wherein cells and / or cell fragments accumulated on one side 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 fragments may be transported out of the chamber 6 together with the transport fluid TF, so that the chamber 6 is emptied. Since the separation by using the fluidized bed centrifuge 5 is relatively gentle on the cells, the cells may advantageously be reused for further production of viral vectors V. Due to the gentle separation process by the fluidized bed centrifuge 5, the cells may be viable. Preferably, during the discharge phase DP, the transport fluid TF is introduced into the chamber 6 so that the 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, e.g. Figure 3 c) is exemplarily shown. Further preferably, during the discharge phase DP, the chamber 6 is rotated so that due to the acting centrifugal force, the emptying of cells and / or cell debris is supported, as shown in FIG. Figure 3 c) is exemplarily shown.

[0051] In another 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 container 13, such as Figure 2 As shown exemplarily in . The intermediate holding container 13 is capable of temporarily storing the fluid F containing the viral vector V that has passed through the fluidized bed centrifuge 5. The intermediate holding container 13 can be designed as a holding tank. Preferably, the fluid F flows discontinuously into the intermediate holding container 13 and flows out of the intermediate holding container 13 continuously. Since the separation through the fluidized bed centrifuge 5 may be discontinuous, the intermediate holding container 13 preferably compensates for the changing fluid flow from the fluidized bed centrifuge 5 so that the discontinuous outflow fluid F flowing out of the fluidized bed centrifuge 5 is temporarily stored, and preferably enables the fluid F to flow out of the intermediate holding container 13 continuously. Therefore, preferably, the fluid F containing the viral vector V flows discontinuously into the intermediate holding container 13 and flows out of the intermediate holding container 13 continuously. Preferably, the intermediate holding container 13 is arranged after the fluidized bed centrifuge 5 and before the tangential flow filtration device 2. Components can be added to the fluid F in the intermediate holding container 13, for example, Figure 2 As shown, enzymes may be added.

[0052] It should be noted that, in the context of the present application, the term "second" in conjunction with a corresponding component of system 1 does not necessarily mean that the embodiment (in particular, system 1) actually includes at least two corresponding components. Moreover, the term "second" is chosen for reasons of distinction. For this reason, system 1 may include a second component, but not necessarily also the corresponding first component.

[0053] According to another preferred embodiment, preferably after fluidized bed centrifuge 5 and / or before tangential flow filtration device 2, enzyme is joined among the fluid F, cell and / or cell debris and / or DNA impurity degraded, especially, thereby discharge viral vector, and / or molecular size is reduced.As previously mentioned, with enzyme degradation cell, cell debris, particularly cellular protein and / or DNA impurity are common in this area.After fluidized bed centrifuge 5 and / or before tangential flow filtration device 2, enzyme is joined among the fluid F and may cause higher degradation performance, because cell and / or cell debris have at least partially separated with fluid F.The result is that the required enzyme of residual cell debris and / or DNA impurity in degraded may be less.Preferably, enzyme can be joined among the initial maintenance container 12 and / or the intermediate maintenance container 13.

[0054] Preferably, the viral vector V is concentrated in the fluid F, which flows out of the tangential flow filtration device 2 as a retentate R, while impurities are present in the fluid, which flows out of 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, in particular by the single-pass tangential flow filter 3 and / or the separation filter element 4, etc. As a result, the viral vector V (preferably the product of the entire purification process) does not pass through the separation filter element 4, such as a membrane, etc., and thus the activity of the viral vector V leaving the tangential flow filtration device 2 can be maintained. Due to the separation (i.e., filtration) of the impurities from the viral vector V, the viral vector V is purified, and the concentration of the viral vector V in the fluid F flowing out of the tangential flow filtration device 2 increases. For example, as Figure 1 As can be seen in FIG, a retentate R stream and a permeate P stream leave the tangential flow filtration device 2. The permeate P may contain at least some impurities, in particular cells and / or cell debris and / or DNA impurities. The retentate R may contain viral vectors V, so that the retentate R can be further purified after the tangential flow filtration device 2.

[0055] For further purification, a preferred embodiment of the proposed method comprises guiding the fluid F containing the viral vector V through an ion exchange device 15, preferably after the tangential flow filtration device 2, and purifying the viral vector V. The ion exchange device 15 preferably comprises an ion exchange material 16, preferably an anion exchange material, more preferably a weak anion exchange material. Alternatively, the ion exchange material 16 can be a weakly hydrophobic material. It is further preferred to use a monolith with a defined channel size distribution. In a particularly preferred embodiment, the channel size is in the range of 6 μm. This allows larger particles (e.g., remaining cell debris) to pass through the ion exchange device 15 instead of blocking the channels. In an exemplary embodiment, a monolith with a defined channel size distribution can be used. OH, SO3 or QA. For example, Figure 1As shown, the ion exchange device 15 can be arranged after the tangential flow filtration device 2. Here, the fluid F containing the viral vectors V is guided from the tangential flow filtration device 2 to the ion exchange device 15. When the fluid F flows through the ion exchange device 15, the viral vectors V bind to the ion exchange material 16. Since impurities still present in the fluid F may have a different charge than that of the viral vectors V, the impurities at least partially do not bind to the ion exchange material 16 and are thus separated from the viral vectors V. The impurities flow out of the ion exchange device 15 together with the fluid F that has flowed through the ion exchange device 15.

[0056] The viral vector V (in particular the product of the entire process) can preferably be obtained as an eluate, in particular by using a suitable elution reagent, wherein further preferably a further fluid F with an adjusted pH and / or conductivity (in particular a fluid F without any impurities) is passed through the ion exchange device 15 and the viral vector V is released from the ion exchange material 16. Figure 1 In the embodiment of the present invention, the viral vectors V first bind to the ion exchange material 16 while the impurities do not. Alternatively, the impurities may bind to the ion exchange material 16 while the viral vectors V may not. This can be achieved by adjusting the pH of the fluid F before entering the ion exchange device 15 (particularly changing the pH) and / or by selecting a different ion exchange material 16. In this case, the viral vectors V flow through the ion exchange device 15 while the impurities can be eluted from the ion exchange device 15.

[0057] The pH and / or conductivity of the fluid F can be adjusted before the ion exchange device 15. If the viral vectors V bind to the ion exchange material 16 or if impurities bind to the ion exchange material 16, the pH and / or conductivity can be adjusted by adjusting the pH. Preferably, the pH is adjusted so that the viral vectors V first bind to the ion exchange material 16 and are then obtained as eluate in the fluid F. Preferably, the pH and / or conductivity adjustment can be performed by the tangential flow filtration device 2, particularly in the single-pass tangential flow filter 3, so that the pH and / or conductivity of the fluid F flowing through the tangential flow filtration device 2 is adjusted.

[0058] Furthermore, it is preferred that the fluid F containing the viral vector V is passed through an additional filtering device 19, preferably after the tangential flow filtration device 2 and / or before the ion exchange device 15, wherein the viral vector V is purified by the additional filtering device 19. The additional filtering device 19 can further improve the overall purification process. Certain impurities that cannot be removed by the tangential flow filtration device 2 and / or by the fluidized bed centrifuge 5 can be removed by the additional filtering device 19. Preferably, when the fluid F containing the viral vector V is passed through the additional filtering device 19, it is depth-filtered. The additional filtering device 19 is at Figure 7 exemplarily shown in FIG.

[0059] Preferably, the method includes at least partially cooling and / or heating the 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 conducting the fluid F. Viral vectors V are generally temperature sensitive. Therefore, at least partially regulating the temperature of the fluid F by heating or cooling during the method can result in higher activity of the purified viral vector V and, therefore, higher performance of the method. Preferably, the temperature of the fluid F is set between 4°C and 37°C, more preferably, the temperature of the fluid F is set to 4°C.

[0060] In addition to the above method, as a second teaching, a system 1 is described with reference to the accompanying drawings. Since the above method can be performed using the proposed system 1, all explanations and features of the method are applicable to the system 1, and vice versa.

[0061] The system 1 for purifying viral vectors V produced by cells in a fluid F comprises a tangential flow filtration device 2 for purifying viral vectors V produced by cells in a fluid F. Figure 1 、 2 , 4, 5 and 6, and preferably, this is necessary, the system 1 includes a fluidized bed centrifuge 5 for separating cells and / or cell debris contained in the fluid F after the production of the viral vector V and before purification by the tangential flow filtration device 2. The fluidized bed centrifuge 5 is located before the tangential flow filtration device 2, so that the fluid F containing the viral vector V can first flow through the fluidized bed centrifuge 5 and then flow through the tangential flow filtration device 2. Since impurities (such as cells and cell debris) have been partially separated by the fluidized bed centrifuge 5, the overall performance of the purification process can be improved.

[0062] In general, the performance of the separation process through the fluidized bed centrifuge 5 can be improved by adjusting the flow rate through the fluidized bed centrifuge 5, wherein a lower flow rate can result in higher performance than when a higher flow rate is utilized, particularly when smaller particles are retained within the chamber 6. Furthermore, the performance of the purification process (i.e., separation process) through the tangential flow filtration device 2 can be improved by adjusting the flow rate through the tangential flow filtration device 2, wherein a lower flow rate can result in higher performance. Improved separation performance can lead to improved overall purification performance.

[0063] According to a preferred embodiment, system 1 includes an initial holding container 12. Initial holding container 12 can enable the production of viral vectors V in a fluid F. Fluid F can be a culture medium. Preferably, cells are added to fluid F within initial holding container 12 or before fluid F is introduced into initial holding container 12. Alternatively, fluid F already containing viral vectors V can be filled into the initial holding container and temporarily stored in initial holding container 12. Preferably, initial holding container 12 is designed as a disposable initial holding container 12. This reduces the risk of contamination because initial holding container 12 can be replaced within system 1 for each run of purified viral vector V. Further preferably, initial holding container 12 includes at least one mixing device 14 for mixing the fluid F received by initial holding container 12. Here, fluid F is homogenized, which can result in a higher yield of viral vector V and / or higher activity of the purified viral vector V and / or higher overall performance of the purification process. Enzymes and / or charged beads CB can be added to fluid F in initial holding container 12.

[0064] In another preferred embodiment, the system 1 comprises an intermediate holding vessel 13. The intermediate holding vessel 13 can buffer the discontinuous flow of fluid F from the fluidized bed centrifuge 5. In addition, enzymes and / or charged beads CB can be added to the fluid F in the intermediate holding vessel 13. The intermediate holding vessel 13 can comprise a mixing device 14 for homogenization. Figure 2 An embodiment of the system 1 comprising an intermediate holding container 13 is shown by way of example.

[0065] Advantageously, the fluidized bed centrifuge 5 comprises at least one chamber 6 through which the fluid F to be centrifuged can flow, e.g. Figure 3 As previously described in the context of the method, a fluid F containing viral vectors V and impurities flows through the chamber 6 of the fluidized bed centrifuge 5, wherein the fluid F is centrifuged simultaneously, in particular so that at least part of the impurities are separated from the fluid F.

[0066] Regarding the design of the chamber 6, it is preferred that the chamber 6 is conical, having a tip 7 and a base surface 8 facing the tip 7, as shown Figure 3a, 3b and 3c. Due to the changed cross section, the conical design of the chamber 6 makes it possible to simply adjust the flow of the fluid F through the chamber 6 during centrifugation, in particular to make the fluid flow rate near the tip 7 faster than the fluid flow rate near the base surface 8. Preferably, a first flow opening 9 for allowing the fluid F to flow into or out of the chamber 6 is arranged at the tip 7 of the chamber 6. In addition or as an alternative, a second flow opening 10 for allowing the fluid F to flow into or out of the chamber 6 is arranged at the base surface 8 of the chamber 6. The first flow opening 9 and / or the second flow opening 10 can have a dual function as an inflow and outflow opening. For example, the fluid F can enter the chamber 6 during the loading phase LP, as shown in FIG. Figure 3 As shown in Figure a. Here, fluid F containing viral vectors V flows into chamber 6 via first flow opening 9. In addition, fluid F flows out of chamber 6 via second flow opening 10. Thus, first flow opening 9 enables inflow into chamber 6, and second flow opening 10 enables outflow from chamber 6. During washing phase WP, washing fluid WF can flow into chamber 6 via first flow opening 9 and out of chamber 6 via second flow opening 10. During discharge phase DP, transport fluid TF can flow out of chamber 6 via first flow opening 9 and into chamber 6 via second flow opening 10, since the flow direction of transport fluid TF can be opposite to the flow direction of fluid F and / or washing fluid WF.

[0067] According to another preferred embodiment, chamber 6 is arranged on a rotating device 11 that rotates during centrifugation so that during centrifugation of the fluid (particularly fluid F, washing fluid WF, and / or transfer fluid TF) by fluidized bed centrifuge 5, the centrifugal force acting on the fluid (and particularly the impurities) is greater at tip 7 of chamber 6 than at base surface 8. This improves the performance of the centrifugation, particularly because the centrifugal force acting at tip 7, where the fluid flow rate is likely to be higher, is higher than at base surface 8, where the fluid flow rate is likely to be lower. Due to the fluid flow rate gradient and the centrifugal force gradient that develop within chamber 6, particles with a higher density and / or a larger diameter accumulate near tip 7, while particles with a lower density and / or a smaller diameter accumulate toward base surface 8. Consequently, the retention of particles of a certain size and / or density within chamber 6 can be controlled by the rotational speed and / or the flow rate of fluid F.

[0068] Advantageously, the tangential flow filtration device 2 comprises at least one single-pass tangential flow filter 3 for filtering the fluid F, such as Figure 1As can be seen by way of example in the figure. The single-pass tangential flow filter 3 can be used as a pretreatment for the fluid F before it enters the ion exchange device 15. The single-pass tangential flow filter 3 can include a separation filter element 4, such as a membrane, serving as a filter element for the single-pass tangential flow filter 3. The separation filter element 4 can 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 more preferably at least 300 kDa ("kDa" means kilodalton, 1 Da = 1 g / mol).

[0069] It is also possible and preferred that the tangential flow filtration device 2 comprises two single-pass tangential flow filters 3, for example Figure 4 As can be seen, here and 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, making it possible to adjust the filtration process. In addition, the use of more than one single-pass tangential flow filter 3 may lead to higher purification performance, such as higher separation performance and / or higher concentration of viral vectors V after the tangential flow filtration device 2.

[0070] like Figure 5 As shown in the embodiment of FIG, and preferably, after separation by the single-pass tangential flow filter 3, the pH and / or conductivity of the fluid F can be adjusted (in particular, changed) before separation by the subsequent single-pass tangential flow filter 3. Here and preferably, the pH and / or conductivity can be adjusted in an intermediate holding container 13, in particular by adding a buffer solution, which is arranged between the single-pass tangential flow filters 3. Adjusting the pH and / or conductivity can produce improved conditions in the subsequent ion exchange device 15.

[0071] According to another preferred embodiment, Figure 6 As exemplarily shown in FIG, the pH and / or conductivity of the fluid F can be adjusted (in particular, changed) during separation by the single-pass tangential flow filter 3. Here, a buffer solution is added directly to the fluid F while it flows through the single-pass tangential flow filter 3. The single-pass tangential flow filter 3 is preferably designed such that a buffer solution can be added to the fluid F during purification (in particular, separation) by the single-pass tangential flow filter 3.

[0072] In another advantageous embodiment, the system 1 (in particular the tangential flow filtration device 2) comprises a first prefilter, which is arranged before the single-pass tangential flow filter 3 and / or before the separation filter element 4. Due to the first prefilter, clogging of the single-pass tangential flow filter 3 and / or the separation filter element 4 can be avoided, since larger impurities can be filtered out by the first prefilter. The first prefilter can be designed as a safety prefilter.

[0073] Furthermore, the system 1 (in particular the ion exchange device 15) can include a second pre-filter, which is arranged upstream of the ion exchange material 16. Due to the second pre-filter, negative effects of the ion exchange material 16, such as clogging, can be avoided because larger impurities can be filtered out by the second pre-filter. The second pre-filter can be designed as a safety pre-filter.

[0074] Furthermore, it is preferred that the system 1 comprises an ion exchange device 15 for purifying the viral vector V. As described in the context of the method, the ion exchange device 15 is capable of further purifying the viral vector V, wherein the viral vector V can be obtained directly with the circulating fluid F or as an eluate. Preferably, the ion exchange device 15 comprises at least one ion exchange material 16. Here, anion exchange materials as well as cation exchange materials are generally possible. It is further preferred that the ion exchange material 16 is an ion exchange resin, in particular anion exchange resin or cation exchange resin, which is particularly arranged in a column or formed as a membrane. The ion exchange device 15 preferably comprises an ion exchange material 16, preferably an anion exchange material, more preferably a weak anion exchange material. Alternatively, the ion exchange material 16 can be a weakly hydrophobic material. It is further preferred to use a monolith with a defined channel size distribution. In a particularly preferred embodiment, the channel size is in the range of 6 μm. This allows larger particles (e.g. remaining cell debris) to pass through the ion exchange device 15 instead of blocking the channel. In an exemplary embodiment, it is possible to use OH, SO3 or QA.

[0075] like Figure 7 It can be seen that it is preferred that the system 1 includes an additional filtering device 19, which is preferably arranged after the tangential flow filtration device 2 and / or before the ion exchange device 15. The additional filtering device 19 may include at least one additional filter 20. The additional filter 20 may in particular be a second pre-filter. Preferably, the additional filter 20 (in particular the second pre-filter) is designed as a depth filter. Typically, the additional filter 20 may include an additional separation filter element 21. If the additional filter 20 is designed as a depth filter, the additional separation filter element 21 may include a depth filter material. Here, the depth filter material is preferably designed so that the average retention rate 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.

[0076] It is further preferred that the system 1 comprises at least one pumping device 17. The one or more pumping devices 17 can be designed as peristaltic pumps. Preferably, the system 1 comprises several pumping devices 17, which are respectively 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, such as Figure 1 As shown in the example.

[0077] Preferably, system 1 includes a fluid line 18 for conducting fluid F within system 1. Preferably, fluid line 18 is arranged so that fluid F can be conducted to and from components of system 1, such as fluidized bed centrifuge 5 and / or tangential flow filtration device 2 and / or ion exchange device 15 and / or initial holding vessel 12 and / or intermediate holding vessel 13. Further preferably, fluid line 18 is designed as a tube and / or pipe, respectively. Further preferably, if one or more pumping devices 17 are designed as one or more peristaltic pumps, fluid line 18 can be designed as a tube and / or pipe.

[0078] In the accompanying drawings, the flow direction of the fluid F is generally from left to right. Figure 1 As can be seen by way of example in FIG, within the system 1, with respect to the flow of the fluid F, the fluidized bed centrifuge 5 is arranged before the tangential flow filtration device 2. Therefore, the fluid F enters the fluidized bed centrifuge 5 before the tangential flow filtration device 2. In other words, separation by the fluidized bed centrifuge 5 occurs before purification (i.e., separation, such as filtration) and / or concentration by the tangential flow filtration device 2.

[0079] In addition, if Figure 2 As can be seen in FIG, further components of the system 1 (such as an intermediate holding container 13) can be arranged between the fluidized bed centrifuge 5 and the tangential flow filtration device 2. Figure 1 As can be seen in the example, within the system 1, with respect to the flow of the fluid F, the tangential flow filtration device 2 is preferably arranged before the ion exchange device 15. Therefore, the fluid F enters the tangential flow filtration device 2 before the ion exchange device 15. Further components of the system 1 (such as further holding containers, etc.) can be arranged between the tangential flow filtration device 2 and the ion exchange device 15. An initial holding container 12, such as, for example, Figure 1 Fluid F may enter the system 1 in an 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 wherein the fluid (F) containing the viral vector (V) is directed through a tangential flow filtration device (2) for purification, whereby the viral vector (V) is purified in the fluid (F), It is characterized by: Prior to purification by the tangential flow filtration device (2), the cells and cell fragments therefrom are at least partially separated from the viral vector (V) by a fluidized bed centrifuge (5), and the cells and cell fragments are contained in the fluid (F) comprising the viral vector (V) after production of the viral vector (V).

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

3. The method according to claim 1 or 2, characterized in that The separation by the fluidized bed centrifuge (5) comprises a loading phase (LP) in which, during centrifugation, the fluid (F) comprising the viral vector (V), cells and / or cell fragments passes through a chamber (6) of the fluidized bed centrifuge (5), wherein at least a portion of the cells and / or cell fragments accumulates unilaterally in the chamber (6) due to the acting centrifugal force, and / or, The separation by means of the fluidized bed centrifuge (5) comprises a washing phase (WP) in which a washing fluid (WF) passes through the chamber (6) during centrifugation, wherein cells accumulated in the chamber (6) are washed, and / or, The separation by the fluidized bed centrifuge (5) comprises a discharge phase (DP) in which a transport fluid (TF) is introduced into the chamber (6), wherein the cells and / or the cell fragments accumulated on one side in the chamber (6) flow out of the chamber (6) together with the transport fluid (TF).

4. The method according to any one of the preceding claims, characterized in that The fluid (F) containing the viral vector (V) is directed from the fluidized bed centrifuge (5) into an intermediate holding container (13), preferably, the fluid (F) flows discontinuously into the intermediate holding container (13) and flows continuously out of the intermediate holding container (13).

5. The method according to any one of the preceding claims, characterized in that An enzyme is added to the fluid (F) so that the cells and / or the cell fragments and / or the DNA impurities are degraded, in particular, thereby releasing the viral vector (V), and / or reducing the molecular size. Preferably, the enzyme is added to the fluid after the fluidized bed centrifuge (5) and / or before the tangential flow filtration device (2).

6. The method according to any one of the preceding claims, characterized in that The viral vector (V) is concentrated in the fluid (F), which flows out of the tangential flow filtration device (2) as a retentate (R), and impurities are separated into the fluid, which flows out of the tangential flow filtration device (2) as a permeate (P).

7. The method according to any one of the preceding claims, characterized in that Preferably, after the tangential flow filtration device (2), the fluid (F) containing the viral vector (V) is led through an ion exchange device (15) and the viral vector (V) is purified.

8. The method according to any one of the preceding claims, characterized in that The fluid (F) in the initial holding container (12) and / or in the fluidized bed centrifuge (5) and / or in the intermediate holding container (13) and / or in the tangential flow filtration device (2) and / or in the ion exchange device (15) and / or in the fluid line (18) for conducting the fluid (F) is at least partially cooled and / or heated.

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

10. The system according to claim 9, characterized in that The system (1) comprises an initial holding container (12), which is preferably arranged before the fluidized bed centrifuge (5). Preferably, the initial holding container (12) is designed as a disposable initial holding container. Further preferably, the initial holding container (12) has at least one mixing device (14) for mixing the fluid received by the initial holding container (12), and / or, The system (1) comprises an intermediate holding container (13), which is preferably arranged after the fluidized bed centrifuge (5). Preferably, the intermediate holding container (13) is designed as a disposable intermediate holding container. Further preferably, the intermediate holding container (13) has at least one mixing device (14) for mixing the fluid received by the intermediate holding container (13).

11. The 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. The system according to claim 11, wherein: 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 allowing a fluid (F) to flow 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 allowing a fluid (F) to flow 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 device (11), which rotates during centrifugation so that during centrifugation of the fluid (F) by 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. 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 filtering the fluid (F).

14. The system according to any one of claims 9 to 13, characterized in that 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, wherein the ion exchange resin consists of a porous matrix in the form of spherical particles or polymer membranes, preferably, the ion exchange material (16) is formed as an anion exchange material, in particular, as an anion exchange resin or an anion exchange monolith, wherein the anion exchange resin consists of a porous matrix in the form of spherical particles or polymer membranes.

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