Novel lentiviral vector downstream purification pipeline system

Abstract

The utility model discloses a novel lentiviral vector downstream purification pipeline system. The novel lentiviral vector downstream purification pipeline system comprises a first peristaltic pump, a sterilization filter, a second peristaltic pump, a hollow fiber column, a first container, a second container, a third container, a first air filter, a second air filter, a third air filter and a pipeline clamp. According to the pipeline system provided by the utility model, the concentration step of the lentiviral vector can be carried out in a closed sterile environment, and sterilization and filtration are carried out when the concentration of the viral vector is relatively low, so that the recovery rate of the viral vector is improved.

Description

Technical Field

[0001] The utility model relates to a purification pipeline system, in particular to a novel downstream purification pipeline system for lentiviral vectors. Background Art

[0002] Gene therapy refers to introducing exogenous normal genes into target cells to correct or compensate for diseases caused by defective and abnormal genes, enabling long-term expression and tissue-specific expression of therapeutic proteins without drug intervention, radiotherapy, or surgical treatment. And this emerging treatment method can target various diseases such as monogenic genetic diseases, cardiovascular diseases, immunodeficiency diseases, and cancers. Gene therapy includes in vivo gene therapy and ex vivo gene therapy (such as CAR-T).

[0003] The global commercialization process of cell gene therapy (CGT) drugs has been accelerating continuously, and there are nearly 2,000 in-progress CGT clinical trial projects. Driven by technology, capital, and policies, the CAR-T cell therapy market has witnessed rapid growth, and the Chinese market size is expected to increase to 28.9 billion in 2030.

[0004] The core of gene therapy (including CAR-T therapy) is the vector. Lentivirus vector (LV) is a very effective tool with many unique advantages in gene transfection and is also widely used in gene therapy. Therefore, preparing stable and high-purity lentiviral vectors is a key step in gene therapy. The production of viral vectors is one of the most critical sub-sectors in the cell and gene therapy contract research and manufacturing organization (CDMO) market. According to a report in The New York Times, the production of viral vectors accounts for more than one-third of the R & D investment in CGT.

[0005] Due to the relatively large size of lentiviral vectors (80 - 120 nm), highly concentrated lentiviral vectors are prone to aggregation, resulting in their inability to pass through a sterilizing filter (0.2 μm). Therefore, sterilizing filtration cannot be used for filtration and sterilization, causing difficulties in achieving aseptic operation in the downstream purification and concentration steps of lentiviral vectors, resulting in a low recovery rate of lentiviral vectors and restricting the recovery rate of lentiviral vectors. Optimizing the purification process and increasing the recovery rate of viral vectors can significantly reduce the R & D cost. Summary of the Utility Model

[0006] In order to overcome the difficulties caused by the aggregation of lentiviral vectors, which makes it impossible to implement filtration sterilization and causes difficulties in the downstream purification process of lentiviral vectors, the utility model provides a novel downstream purification pipeline system for lentiviral vectors, which can carry out the concentration step of lentiviral vectors in a closed and aseptic environment, perform sterilizing filtration when the concentration of viral vectors is relatively low, and increase the recovery rate of viral vectors.

[0007] To achieve the above object, the technical solution adopted by the utility model is:

[0008] A novel downstream purification pipeline system for lentiviral vectors, comprising a first peristaltic pump, a sterilizing filter, a second peristaltic pump, a hollow fiber column, a first container, a second container, a third container, a first air filter, a second air filter, a third air filter and a pipeline clamp;

[0009] One end of the pipeline on the first peristaltic pump is used for sample injection, and the other end is connected to the sample injection end of the sterilizing filter. The sample outlet end of the sterilizing filter is connected to the first end of the pipeline on the second peristaltic pump, and the second end of the pipeline on the second peristaltic pump is connected to the inlet end of the hollow fiber column;

[0010] The first container and the second container are connected to the pipeline between the sterilizing filter and the second peristaltic pump through a branch pipeline;

[0011] Through pipelines, the first air filter, the first container and the second container are all connected to the reflux end of the hollow fiber column, the third container is connected to the permeate end of the hollow fiber column, and the second air filter is connected to the container mouth of the first container;

[0012] A sampling pipeline is further provided at the container mouth of the first container, and a third air filter is provided on the sampling pipeline;

[0013] The peristaltic direction of the second peristaltic pump can be switched by clockwise rotation and counterclockwise rotation, that is, it can make the substances in the pipeline flow from the first end to the second end, and can also make the substances in the pipeline flow from the second end to the first end;

[0014] The pipeline clamp is used to block the pipeline flow path.

[0015] In some embodiments, it further includes a first pressure gauge; the first pressure gauge is arranged on the pipeline between the second peristaltic pump and the hollow fiber column.

[0016] In some embodiments, it further includes a second pressure gauge; the second pressure gauge is arranged on the pipeline connected to the reflux end of the hollow fiber column.

[0017] In some embodiments, it further includes a third pressure gauge; the third pressure gauge is arranged on the pipeline between the first peristaltic pump and the sterilizing filter.

[0018] In some embodiments, the first air filter is connected to the first container, the second container and the hollow fiber column through a first four-way pipe.

[0019] In some embodiments, the branch pipeline is a three-way pipe; the three-way pipe includes a first three-way pipe and a second three-way pipe; the first container is connected to the pipeline between the sterilizing filter and the second peristaltic pump through the first three-way pipe; the second container is connected to the pipeline between the sterilizing filter and the second peristaltic pump through the second three-way pipe.

[0020] In some embodiments, the branch pipe is a second four-way pipe; the first container and the second container are connected to the pipe between the sterilizing filter and the second peristaltic pump through the second four-way pipe.

[0021] In some embodiments, some or all of the pipes for connection are silicone tubes.

[0022] In some embodiments, the length of the pipe between adjacent components is 1 - 100 cm, or 2 - 50 cm, or 3 - 30 cm, or 5 - 25 cm. Components that can be referred to as such in this text are the first peristaltic pump, the sterilizing filter, the second peristaltic pump, the hollow fiber column, the first container, the second container, the third container, the first air filter, the second air filter, the third air filter, the first pressure gauge, the second pressure gauge, the third pressure gauge, the first four-way pipe, the first three-way pipe, and the second three-way pipe; excluding pipe clamps.

[0023] In some embodiments, the novel downstream purification pipeline system of the lentiviral vector is set to be in a sterile state before use, and the sterile state is achieved through gamma-ray irradiation in combination with the sterilizing filter, the first air filter, the second air filter, and the third air filter.

[0024] In some embodiments, the first container is a liquid storage bottle; the second container is a liquid storage bag; the third container is a waste liquid bag.

[0025] In some embodiments, the number of pipe clamps is ≥5 or ≥6 or ≥7.

[0026] Compared with the prior art, the beneficial effects of the present utility model are as follows: Through the novel downstream purification pipeline system of the lentiviral vector disclosed in the present utility model, the concentration step of the lentiviral vector can be carried out in a closed and sterile environment, and sterilizing filtration is carried out when the concentration of the viral vector is relatively low, improving the recovery rate of the viral vector. Through the synergistic effect of the pipe clamps, the first peristaltic pump, and the second peristaltic pump, the present utility model can realize the flow of fluid in different pipelines, and realize operations such as exhaust, rinsing, top washing, ultrafiltration of the second container, transfer to the first container, ultrafiltration of the first container, circulation of the first container, sampling, and recovery of the final product. The pressure of the corresponding parts of the pipeline can be monitored through the pressure gauges (including the first pressure gauge, the second pressure gauge, and the third pressure gauge). The pipeline of the novel downstream purification pipeline system of the lentiviral vector disclosed in the utility model is simple, clear, not complex, the usage method is simple and easy to operate, easy to control, and the concentration effect of the lentiviral vector is good.

[0027] The following will further illustrate the concept, specific structure, and technical effects of the present utility model in conjunction with the drawings to fully understand the purpose, features, and effects of the present utility model. Description of the Drawings

[0028] Figure 1It is a flowchart of the use of a novel downstream purification pipeline system for lentiviral vectors according to an embodiment of the present utility model.

[0029] Figure 2 It is a schematic structural diagram of a novel downstream purification pipeline system for lentiviral vectors according to Embodiment 1 of the present utility model.

[0030] Figure 3 It is an exhaust flow path diagram of Embodiment 1. Figure 3-11 The arrows in it indicate the liquid flow direction, and the red color indicates the connected flow path.

[0031] Figure 4 It is a rinsing flow path diagram of Embodiment 1.

[0032] Figure 5 It is a sterilization top-washing filtration flow path diagram of Embodiment 1.

[0033] Figure 6 It is a second container ultrafiltration flow path diagram of Embodiment 1.

[0034] Figure 7 It is a flow path diagram of transferring from the second container to the first container in Embodiment 1.

[0035] Figure 8 It is a first container ultrafiltration flow path diagram of Embodiment 1.

[0036] Figure 9 It is a first container circulation flow path diagram of Embodiment 1.

[0037] Figure 10 It is a first container sampling flow path diagram of Embodiment 1.

[0038] Figure 11 It is a final product recovery flow path diagram of Embodiment 1.

[0039] Figure 12 It is a schematic structural diagram of a novel downstream purification pipeline system for lentiviral vectors according to Embodiment 2 of the present utility model.

[0040] Figure 13 It is an exhaust flow path diagram of Embodiment 2. Figure 13-21 The arrows in it indicate the flow direction, and the red color indicates the connected flow path.

[0041] Figure 14 It is a rinsing flow path diagram of Embodiment 2.

[0042] Figure 15 It is a sterilization top-washing filtration flow path diagram of Embodiment 2.

[0043] Figure 16 It is a second container ultrafiltration flow path diagram of Embodiment 2.

[0044] Figure 17It is the flowchart of the flow path from the second container to the first container in Example 2.

[0045] Figure 18 It is the ultrafiltration flow path diagram of the first container in Example 2.

[0046] Figure 19 It is the circulation flow path diagram of the first container in Example 2.

[0047] Figure 20 It is the sampling flow path diagram of the first container in Example 2.

[0048] Figure 21 It is the flowchart of the final product recovery in Example 2. Detailed implementation manners

[0049] In order to make the technical means, creative features, achieved purposes and functions realized by the utility model easy to understand, the present utility model will be further described below in conjunction with specific drawings. However, the present utility model is not limited to the following implemented cases.

[0050] It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in this technology to understand and read, and are not used to limit the limited conditions under which the present utility model can be implemented. Therefore, they do not have substantial technical significance. Any modification of the structure, change of the proportional relationship or adjustment of the size, without affecting the functions that the present utility model can produce and the purposes that can be achieved, should still fall within the scope that can be covered by the technical content disclosed by the present utility model.

[0051] Figure 1 It shows the usage flowchart of the novel downstream purification pipeline system of lentiviral vector disclosed by the present utility model. From Figure 1 It can be seen that when using the pipeline system involved in the present utility model for downstream purification of lentiviral vector, it is necessary to go through operations of exhaust 100, rinsing 101, sterilizing filtration 102, top washing 103, ultrafiltration of the second container 104, transfer of substances from the second container to the first container 105, ultrafiltration of the first container 106, circulation of the first container 107, sampling 108, and final product recovery 109.

[0052] Example 1

[0053] Figure 2 It shows the novel downstream purification pipeline system of the present embodiment, including a first peristaltic pump 1, a sterilizing filter 3, a second peristaltic pump 13, a hollow fiber column 15, a first container 8, a second container 10, a third container 17, a first air filter 12, a second air filter 4, a third air filter 7, a first pressure gauge 14, a second pressure gauge 16, a third pressure gauge 2 and a pipe clamp; the pipe clamp is used to block the pipeline flow path.

[0054] One end of the pipeline on the first peristaltic pump 1 is used for sample injection, and the other end is connected to the sample injection end 31 of the sterilizing filter 3. The sample outlet end 32 of the sterilizing filter 3 is connected to the first end of the pipeline on the second peristaltic pump 13, and the second end of the pipeline on the second peristaltic pump 13 is connected to the inlet end 151 of the hollow fiber column 15;

[0055] On the pipeline between the sterilizing filter 3 and the second peristaltic pump 13, a first container 8 and a second container 10 are connected through a branch pipeline;

[0056] Through pipelines, the first air filter 12, the first container 8 and the second container 10 are all connected to the return end 153 of the hollow fiber column 15, the third container 17 is connected to the permeate end 152 of the hollow fiber column 15, and the second air filter 4 is connected to the container opening of the first container 8;

[0057] A sampling pipeline 6 is also provided at the container opening of the first container 8, and a third air filter 7 is provided on the sampling pipeline 6;

[0058] The peristaltic direction of the second peristaltic pump 13 can be switched by clockwise rotation and counterclockwise rotation, that is, it can make the substances in the pipeline flow from the first end to the second end, and can also make the substances in the pipeline flow from the second end to the first end.

[0059] The first pressure gauge 14 is arranged on the pipeline between the second peristaltic pump 13 and the hollow fiber column 15. The second pressure gauge 16 is arranged on the pipeline connected to the return end 153 of the hollow fiber column 15. The third pressure gauge 2 is arranged on the pipeline between the first peristaltic pump 1 and the sterilizing filter 3.

[0060] The first air filter 12 is connected to the first container 8, the second container 10 and the hollow fiber column 15 through the first four-way pipe 11.

[0061] The branch pipeline is a three-way pipe; the three-way pipe includes the first three-way pipe 5 and the second three-way pipe 9; the first container 8 is connected to the pipeline between the sterilizing filter 3 and the second peristaltic pump 13 through the first three-way pipe 5; the second container 10 is connected to the pipeline between the sterilizing filter 3 and the second peristaltic pump 13 through the second three-way pipe 9. On the pipeline where the flow path needs to be controlled, a silicone tube is used, and the silicone tube can be clamped by a pipeline clamp to control the size of the flow path and completely disconnect the flow path. Therefore, the pipeline used for connection can be set as a silicone tube at the place where the flow path needs to be disconnected, and it can be all silicone tubes or partially silicone tubes.

[0062] The novel downstream purification pipeline system of the lentiviral vector is set to be in a sterile state before use, and the sterile state is achieved through gamma-ray irradiation in combination with a sterilizing filter 3, a first air filter 12, a second air filter 4, and a third air filter 7. The first container 8 is a liquid storage bottle; the second container 10 is a liquid storage bag; the third container 17 is a waste liquid bag. The number of pipeline clamps is ≥6.

[0063] The pipeline length between adjacent components is 1 - 100 cm, or 2 - 50 cm, or 3 - 30 cm, or 5 - 25 cm. In this embodiment, it is preferably about 20 cm.

[0064] The usage method of the novel downstream purification pipeline system of the lentiviral vector includes the following steps:

[0065] S1. Exhaust: Block the pipeline connecting the first container 8 and the second container 10 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the second container 10; turn on the first peristaltic pump 1 to pump the preparation buffer solution into the novel downstream purification pipeline system of the lentiviral vector, discharge the air in the sterilizing filter 3 and the hollow fiber column 15 from the first air filter 12, and then block the liquid flow through the first air filter 12 with a pipeline clamp, and the waste liquid is recovered by the third container 17. The flow path diagram of this step is shown in Figure 3 。

[0066] S2. Rinsing: Block the pipeline connecting the first container 8 and the second container 10 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the second container 10; continue to pump the preparation buffer solution into the sterilizing filter 3 and the hollow fiber column 15 with the first peristaltic pump 1, and the rinsing volume is not less than 18 L / m 2 , detect the pH of the waste liquid in the third container 17, and if the difference from the pH of the preparation buffer solution is within 0.1, it is regarded as the end of the balance, and the rinsing is completed. The flow path diagram of this step is shown in Figure 4 。

[0067] S3. Sterilizing filtration and top washing: Block the pipeline connecting the first container 8 and the hollow fiber column 15 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the hollow fiber column 15; pump the solution containing the lentiviral vector through the first peristaltic pump 1, and after sterilizing filtration through the sterilizing filter 3, obtain the sterilized lentiviral vector solution and store it in the second container 10. The flow path diagram of this step is shown in Figure 5 。

[0068] S4. Ultrafiltration of the second container 10: Block the pipeline connecting the sterilizing filter 3 and the first container 8 with a pipeline clamp, so that the substances in the pipeline cannot enter the sterilizing filter 3, the first container 8, the second air filter 4 and the sampling pipeline 6; Turn off the first peristaltic pump 1, turn on the second peristaltic pump 13 and the peristaltic direction is such that the substances in the pipeline flow from the first end 131 to the second end 132, so as to pump the sterilized lentiviral vector solution in the second container 10 to the hollow fiber column 15 for concentration, obtaining the concentrated lentiviral vector solution, storing it in the second container 10, and discharging the filtrate to the third container 17; When it is concentrated to 1 / 5 to 1 / 10 of the original volume, the ultrafiltration of the second container 10 is completed. The flow path diagram of this step is shown in Figure 6 。

[0069] S5. Transfer of substances from the second container 10 to the first container 8: Block the pipeline connecting the sterilizing filter 3, the third container 17 and the first air filter 12 with a pipeline clamp, as well as the sampling pipeline 6, and the pipeline connecting the second container 10 through a branch pipeline (here it is the second three-way pipe 9), and the pipeline connecting the first four-way pipe 11 and the first container 8, so that the substances in the pipeline cannot enter the sterilizing filter 3, the third container 17, the first air filter 12, the sampling pipeline 6 and the second container 10; The first peristaltic pump 1 remains in the closed state, continue to turn on the second peristaltic pump 13 but the peristaltic direction is such that the substances in the pipeline flow from the second end 132 to the first end 131, so as to transfer the concentrated lentiviral vector solution in the second container 10 to the first container 8 through the hollow fiber column 15, and communicate with the atmosphere through the second air filter 4. The flow path diagram of this step is shown in Figure 7 。The pipeline connected to the second air filter 4 is in an open state, so that the pipeline is connected to the atmosphere, but no liquid will flow through.

[0070] S6. Ultrafiltration of the first container 8: Block the pipeline connecting the sterilizing filter 3, the second container 10 and the first air filter 12 with a pipeline clamp, as well as the sampling pipeline 6, so that the substances in the pipeline cannot enter the sterilizing filter 3, the second container 10, the first air filter 12 and the sampling pipeline 6; The first peristaltic pump 1 remains in the closed state, continue to turn on the second peristaltic pump 13 and reverse the peristaltic direction to be such that the substances in the pipeline flow from the first end 131 to the second end 132, so as to concentrate the concentrated lentiviral vector solution in the first container 8 to the required volume through the hollow fiber column 15, communicate with the atmosphere through the second air filter 4, and discharge the filtrate to the third container 17. The flow path diagram of this step is shown in Figure 8 。The pipeline connected to the second air filter 4 is in an open state, so that the pipeline is connected to the atmosphere, but no liquid will flow through.

[0071] S7. First container circulation: Block the pipelines connecting the sterilizing filter 3, the second container 10, the third container 17 and the first air filter 12, as well as the sampling pipeline 6, with pipe clamps, so that the substances in the pipelines cannot enter the sterilizing filter 3, the second container 10, the third container 17, the first air filter 12 and the sampling pipeline 6; The first peristaltic pump 1 remains closed, and the second peristaltic pump 13 continues to be opened with the peristaltic direction such that the substances in the pipeline can flow from the first end 131 to the second end 132, thereby circulating and rinsing the lentiviral vector solution in the hollow fiber column 15, and communicating with the atmosphere through the second air filter 4. The flow path diagram of this step is shown in Figure 9 . The pipeline connected to the second air filter 4 is in an open state, so that the pipeline is connected to the atmosphere, but no liquid will flow through.

[0072] S8. Sampling: Both the first peristaltic pump 1 and the second peristaltic pump 13 remain closed, open the sampling pipeline 6, and sample in a laminar flow hood, communicating with the atmosphere through the second air filter 4 and the third air filter 7. The flow path diagram of this step is shown in Figure 10 . The pipelines connected to the second air filter 4 and the third air filter 7 are both in an open state, so that the pipelines are connected to the atmosphere, but no liquid will flow through. Liquid will not flow out from the second air filter 4 and the third air filter 7. The third air filter 7 connects the sampling pipeline 6 to the atmosphere, facilitating sampling. In this step, the second air filter 4 mainly connects the first container 8 to the atmosphere.

[0073] S9. Final product recovery: Block the pipelines connecting the sterilizing filter 3, the second container 10 and the third container 17, as well as the sampling pipeline 6, and the pipeline connecting the first four-way pipe 11 to the first container 8 with pipe clamps, so that the substances in the pipelines cannot enter the sterilizing filter 3, the second container 10, the third container 17, the sampling pipeline 6 and enter the first container 8 through the pipeline connecting the first four-way pipe 11 to the first container 8; The first peristaltic pump 1 remains closed, open the second peristaltic pump 13 with the peristaltic direction such that the substances in the pipeline can flow from the second end 132 to the first end 131, thereby recovering the final product into the first container 8, and communicating with the atmosphere through the first air filter 12 and the second air filter 4. The flow path diagram of this step is shown in Figure 11 . The pipelines connected to the first air filter 12 and the second air filter 4 are both in an open state, so that the pipelines are connected to the atmosphere. Liquid will not flow out from the first air filter 12 and the second air filter 4. The residual liquid in the pipeline connected to the first air filter 12 is recovered into the first container 8.

[0074] Various flow paths of the new lentiviral vector downstream purification pipeline system are realized through various combinations of pipe clamps, the switch and peristaltic direction of the first peristaltic pump, the switch and peristaltic direction of the second peristaltic pump, the depth of the pipeline extending into the first container and the second container, and the switches at the inlet end, permeate end and reflux end of the hollow fiber column.

[0075] Example 2

[0076] Figure 12 The novel downstream purification pipeline system of the lentiviral vector in this example is shown, including a first peristaltic pump 1, a sterilizing filter 3, a second peristaltic pump 13, a hollow fiber column 15, a first container 8, a second container 10, a third container 17, a first air filter 12, a second air filter 4, a third air filter 7, a first pressure gauge 14, a second pressure gauge 16, a third pressure gauge 2 and a pipe clamp; the pipe clamp is used to block the pipeline flow path.

[0077] One end of the pipeline on the first peristaltic pump 1 is used for sample injection, and the other end is connected to the sample injection end 31 of the sterilizing filter 3. The sample outlet end 32 of the sterilizing filter 3 is connected to the first end of the pipeline on the second peristaltic pump 13, and the second end of the pipeline on the second peristaltic pump 13 is connected to the inlet end 151 of the hollow fiber column 15;

[0078] The first container 8 and the second container 10 are connected to the pipeline between the sterilizing filter 3 and the second peristaltic pump 13 through a branch pipeline;

[0079] Through the pipeline, the first air filter 12, the first container 8 and the second container 10 are all connected to the reflux end 153 of the hollow fiber column 15. The third container 17 is connected to the permeate end 152 of the hollow fiber column 15, and the second air filter 4 is connected to the container mouth of the first container 8;

[0080] A sampling pipeline 6 is also provided at the container mouth of the first container 8, and a third air filter 7 is provided on the sampling pipeline 6;

[0081] The peristaltic direction of the second peristaltic pump 13 can be switched by rotating clockwise and counterclockwise, that is, it can make the substances in the pipeline flow from the first end to the second end, and can also make the substances in the pipeline flow from the second end to the first end.

[0082] The first pressure gauge 14 is arranged on the pipeline between the second peristaltic pump 13 and the hollow fiber column 15. The second pressure gauge 16 is arranged on the pipeline connected to the reflux end 153 of the hollow fiber column 15. The third pressure gauge 2 is arranged on the pipeline between the first peristaltic pump 1 and the sterilizing filter 3.

[0083] The first air filter 12 is connected to the first container 8, the second container 10 and the hollow fiber column 15 through a first four-way pipe 11.

[0084] In this example, all the pipelines used for connection are silicone tubes, which is convenient for the pipe clamp to control the flow path to be unblocked or disconnected.

[0085] The novel downstream purification pipeline system for lentiviral vectors is set to be in a sterile state before use, and the sterile state is achieved through gamma-ray irradiation in combination with a sterilizing filter 3, a first air filter 12, a second air filter 4, and a third air filter 7. The first container 8 is a liquid storage bottle; the second container 10 is a liquid storage bag; the third container 17 is a waste liquid bag. The number of pipeline clamps is ≥6.

[0086] The branch pipeline is a second four-way pipe; the first container and the second container are connected to the pipeline between the sterilizing filter and the second peristaltic pump through the second four-way pipe.

[0087] The pipeline length between adjacent components is 1-100 cm, or 2-50 cm, or 3-30 cm, or 5-25 cm. In this embodiment, it is preferably about 17 cm.

[0088] The usage method of the novel downstream purification pipeline system for lentiviral vectors includes the following steps:

[0089] S1. Exhaust: Block the pipeline connecting the first container 8 and the second container 10 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the second container 10; turn on the first peristaltic pump 1 to pump the preparation buffer solution into the novel downstream purification pipeline system for lentiviral vectors, discharge the air in the sterilizing filter 3 and the hollow fiber column 15 from the first air filter 12, and then block the liquid from flowing through the first air filter 12 with a pipeline clamp, and the waste liquid is recovered by the third container 17. The flow path diagram of this step is shown in Figure 13 .

[0090] S2. Rinsing: Block the pipeline connecting the first container 8 and the second container 10 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the second container 10; continue to pump the preparation buffer solution into the sterilizing filter 3 and the hollow fiber column 15 with the first peristaltic pump 1, and the rinsing volume is not less than 18 L / m 2 , detect the pH of the waste liquid in the third container 17, and consider the balance to be completed when the difference from the pH of the preparation buffer solution is within 0.1, and the rinsing is completed. The flow path diagram of this step is shown in Figure 14 .

[0091] S3. Sterilizing filtration and top washing: Block the pipeline connecting the first container 8 and the hollow fiber column 15 with a pipeline clamp, so that the substances in the pipeline cannot enter the first container 8, the second air filter 4, the sampling pipeline 6, and the hollow fiber column 15; pump the solution containing lentiviral vectors through the first peristaltic pump 1, and after sterilizing filtration through the sterilizing filter 3, obtain the sterilized lentiviral vector solution and store it in the second container 10. The flow path diagram of this step is shown in Figure 15 .

[0092] S4. Ultrafiltration of the second container 10: Block the pipeline connecting the sterilizing filter 3 and the first container 8 with a pipe clamp, so that the substances in the pipeline cannot enter the sterilizing filter 3, the first container 8, the second air filter 4 and the sampling pipeline 6; Turn off the first peristaltic pump 1, turn on the second peristaltic pump 13 and the peristaltic direction is such that the substances in the pipeline can flow from the first end 131 to the second end 132, so as to pump the sterilized lentiviral vector solution in the second container 10 to the hollow fiber column 15 for concentration, obtaining the concentrated lentiviral vector solution, storing it in the second container 10, and discharging the filtrate to the third container 17; When it is concentrated to 1 / 5 to 1 / 10 of the original volume, the ultrafiltration of the second container 10 is completed. The flow chart of this step is shown in Figure 16 。

[0093] S5. Transfer of substances from the second container 10 to the first container 8: Block the pipelines connecting the sterilizing filter 3, the third container 17 and the first air filter 12, and the sampling pipeline 6 with pipe clamps, as well as the pipeline connecting the second container 10 through a branch pipeline (here it is the second four-way pipe 18), and the pipeline connecting the first four-way pipe 11 and the first container 8, so that the substances in the pipeline cannot enter the sterilizing filter 3, the third container 17, the first air filter 12, the sampling pipeline 6 and the second container 10; The first peristaltic pump 1 remains closed, and the second peristaltic pump 13 continues to be turned on but the peristaltic direction is such that the substances in the pipeline can flow from the second end 132 to the first end 131, so as to transfer the concentrated lentiviral vector solution in the second container 10 to the first container 8 through the hollow fiber column 15, and communicate with the atmosphere through the second air filter 4. The flow chart of this step is shown in Figure 17 。The pipeline connected to the second air filter 4 is in an open state, so that the pipeline is connected to the atmosphere, but no liquid will flow through.

[0094] S6. Ultrafiltration of the first container 8: Block the pipelines connecting the sterilizing filter 3, the second container 10 and the first air filter 12, and the sampling pipeline 6 with pipe clamps, so that the substances in the pipeline cannot enter the sterilizing filter 3, the second container 10, the first air filter 12 and the sampling pipeline 6; The first peristaltic pump 1 remains closed, and the second peristaltic pump 13 continues to be turned on and the peristaltic direction is adjusted to be such that the substances in the pipeline can flow from the first end 131 to the second end 132, so as to concentrate the concentrated lentiviral vector solution in the first container 8 to the required volume through the hollow fiber column 15, communicate with the atmosphere through the second air filter 4, and discharge the filtrate to the third container 17. The flow chart of this step is shown in Figure 18 。The pipeline connected to the second air filter 4 is in an open state, so that the pipeline is connected to the atmosphere, but no liquid will flow through.

[0095] S7. First container circulation: Block the pipelines connecting the sterilizing filter 3, the second container 10, the third container 17, and the first air filter 12, as well as the sampling pipeline 6, with pipeline clamps, so that the substances in the pipelines cannot enter the sterilizing filter 3, the second container 10, the third container 17, the first air filter 12, and the sampling pipeline 6; The first peristaltic pump 1 remains closed, and the second peristaltic pump 13 is continuously opened with a peristaltic direction that enables the substances in the pipelines to flow from the first end 131 to the second end 132, thereby circulating and rinsing the lentiviral vector solution in the hollow fiber column 15, and communicating with the atmosphere through the second air filter 4. The flow path diagram of this step is shown in Figure 19 . The pipeline connected to the second air filter 4 is in an open state, enabling the pipeline to be connected to the atmosphere, but no liquid will flow through.

[0096] S8. Sampling: Both the first peristaltic pump 1 and the second peristaltic pump 13 remain closed, the sampling pipeline 6 is opened, and sampling is carried out in a laminar flow hood, communicating with the atmosphere through the second air filter 4 and the third air filter 7. The flow path diagram of this step is shown in Figure 20 . The pipelines connected to the second air filter 4 and the third air filter 7 are both in an open state, enabling the pipelines to be connected to the atmosphere, but no liquid will flow through. Liquid will not flow out from the second air filter 4 and the third air filter 7. The third air filter 7 enables the sampling pipeline 6 to be connected to the atmosphere, facilitating sampling. In this step, the second air filter 4 mainly enables the first container 8 to be connected to the atmosphere.

[0097] S9. Final product recovery: Block the pipelines connecting the sterilizing filter 3, the second container 10, and the third container 17, as well as the sampling pipeline 6, and the pipeline connecting the first four-way pipe 11 to the first container 8 with pipeline clamps, so that the substances in the pipelines cannot enter the sterilizing filter 3, the second container 10, the third container 17, the sampling pipeline 6, and enter the first container 8 through the pipeline connecting the first four-way pipe 11 to the first container 8; The first peristaltic pump 1 remains closed, and the second peristaltic pump 13 is opened with a peristaltic direction that enables the substances in the pipelines to flow from the second end 132 to the first end 131, thereby recovering the final product into the first container 8, and communicating with the atmosphere through the first air filter 12 and the second air filter 4. The flow path diagram of this step is shown in Figure 21 . The pipelines connected to the first air filter 12 and the second air filter 4 are both in an open state, enabling the pipelines to be connected to the atmosphere. Liquid will not flow out from the first air filter 12 and the second air filter 4. The liquid remaining in the pipeline connected to the first air filter 12 is recovered into the first container 8.

[0098] Various flow paths of the new downstream purification pipeline system for lentiviral vectors are realized through various combinations of pipeline clamps, the switch and peristaltic direction of the first peristaltic pump, the switch and peristaltic direction of the second peristaltic pump, the depth of the pipeline extending into the first container and the second container, and the switches at the inlet end, permeate end, and reflux end of the hollow fiber column.

[0099] The preferred specific embodiments of the present utility model have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present utility model without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in this technical field based on the concept of the present utility model through logical analysis, reasoning, or limited experiments on the basis of the prior art shall fall within the protection scope determined by the claims.

Claims

1. A novel downstream purification pipeline system for lentiviral vectors, characterized in that, The device comprises a first peristaltic pump, a sterilizing filter, a second peristaltic pump, a hollow fiber column, a first container, a second container, a third container, a first air filter, a second air filter, a third air filter and a pipe clamp; One end of the pipe on the first peristaltic pump is used for sample injection, and the other end is connected to the sample injection end of the sterilizing filter. The sample outlet end of the sterilizing filter is connected to the first end of the pipe on the second peristaltic pump, and the second end of the pipe on the second peristaltic pump is connected to the inlet end of the hollow fiber column. The first container and the second container are connected to the pipeline between the sterilizing filter and the second peristaltic pump through a branch pipeline; The first air filter, the first container, and the second container are all connected to the reflux end of the hollow fiber column through a pipeline, the third container is connected to the permeate end of the hollow fiber column, and the second air filter is connected to the container port of the first container; A sampling pipe is further provided at the container opening of the first container, and the third air filter is provided on the sampling pipe; The peristaltic direction of the second peristaltic pump can be switched by rotating clockwise or counterclockwise, that is, the material in the pipeline can flow from the first end to the second end, and can also flow from the second end to the first end; The pipeline clamp is used to block the pipeline flow path.

2. The novel downstream purification pipeline system for lentiviral vectors according to claim 1, characterized in that, It also includes a first pressure gauge, a second pressure gauge and a third pressure gauge; the first pressure gauge is arranged on the pipe between the second peristaltic pump and the hollow fiber column; the second pressure gauge is arranged on the pipe connected to the reflux end of the hollow fiber column; the third pressure gauge is arranged on the pipe between the first peristaltic pump and the sterilizing filter.

3. The novel downstream purification pipeline system of the lentiviral vector according to claim 1, wherein, The first air filter is connected to the first container, the second container and the hollow fiber column through a first four-way pipe.

4. The novel downstream purification pipeline system for lentiviral vectors according to claim 1, characterized in that, The branch pipe is a tee; the tee includes a first tee and a second tee; the first container is connected to the pipe between the sterilizing filter and the second peristaltic pump through the first tee; the second container is connected to the pipe between the sterilizing filter and the second peristaltic pump through the second tee.

5. The novel downstream purification pipeline system for lentiviral vectors according to claim 1, characterized in that, The branch pipeline is a second four-way pipe; the first container and the second container are connected to the pipeline between the sterilizing filter and the second peristaltic pump through the second four-way pipe.

6. The novel downstream purification pipeline system of lentiviral vector according to claim 1, wherein, Part or all of the pipes used for connection are silicone pipes.

7. The novel lentiviral vector downstream purification pipeline system according to claim 1, characterized in that: The length of the pipe between adjacent components is 1-100 cm, or 2-50 cm, or 3-30 cm, or 5-25 cm.

8. The novel lentiviral vector downstream purification pipeline system according to claim 1, characterized in that: The novel lentiviral vector downstream purification pipeline system is configured to be in a sterile state before use, and the sterile state is achieved by gamma-ray irradiation in combination with the sterilizing filter, the first air filter, the second air filter and the third air filter.

9. The novel downstream purification pipeline system of the lentiviral vector according to claim 1, wherein The first container is a liquid storage bottle; the second container is a liquid storage bag; and the third container is a waste liquid bag.

10. The novel downstream purification pipeline system for lentiviral vectors according to any one of claims 1-9, characterized in that, The number of the pipe clamps is ≥5.

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