WO2023025192A1 - 一种全封闭和全自动化的分装细胞的装置和方法 - Google Patents

一种全封闭和全自动化的分装细胞的装置和方法 Download PDF

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WO2023025192A1
WO2023025192A1 PCT/CN2022/114503 CN2022114503W WO2023025192A1 WO 2023025192 A1 WO2023025192 A1 WO 2023025192A1 CN 2022114503 W CN2022114503 W CN 2022114503W WO 2023025192 A1 WO2023025192 A1 WO 2023025192A1
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tube
cell
liquid
extraction
rehydration
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PCT/CN2022/114503
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English (en)
French (fr)
Inventor
王飞
谢莉
张丽
任加强
赵荻骏
张露亿
周依雯
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西比曼生物科技集团公司
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Priority to EP22860538.2A priority Critical patent/EP4393467A1/en
Publication of WO2023025192A1 publication Critical patent/WO2023025192A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J1/00—Containers specially adapted for medical or pharmaceutical purposes
    • A61J1/14—Details; Accessories therefor
    • A61J1/20—Arrangements for transferring or mixing fluids, e.g. from vial to syringe
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B3/00—Packaging plastic material, semiliquids, liquids or mixed solids and liquids, in individual containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, or jars
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B3/00—Packaging plastic material, semiliquids, liquids or mixed solids and liquids, in individual containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, or jars
    • B65B3/04—Methods of, or means for, filling the material into the containers or receptacles
    • B65B3/10—Methods of, or means for, filling the material into the containers or receptacles by application of pressure to material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B3/00—Packaging plastic material, semiliquids, liquids or mixed solids and liquids, in individual containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, or jars
    • B65B3/26—Methods or devices for controlling the quantity of the material fed or filled
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B63/00—Auxiliary devices, not otherwise provided for, for operating on articles or materials to be packaged
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B65/00—Details peculiar to packaging machines and not otherwise provided for; Arrangements of such details

Definitions

  • the invention relates to the field of biotechnology, in particular to a fully enclosed and fully automated cell subpackaging device and method.
  • Cellular immunotherapy is a kind of cell therapy that collects the patient's own immune cells, undergoes genetic modification and in vitro expansion and culture, and then enhances the targeting, lethality and persistence of immune cells.
  • tumor immunotherapy has performed very well clinically, bringing hope to the clinical cure of tumors.
  • the prepared immune cells need to be washed, concentrated, and distributed into multiple cryopreservation bags. Any intermediate link such as the process flow, equipment, facilities, and reagent selection of the cells will affect the quality of the cells, and then affect the clinical effect. . Therefore, a fully enclosed and fully automated immune cell packaging process can ensure product safety and batch-to-batch stability, reduce the impact of personnel and the environment, and improve the quality of immune cell therapy products.
  • Traditional cell subpackaging can only be carried out under artificial and non-closed conditions, which increases the risk of infection from contact with the external environment and greatly affects the safety of the product.
  • the purpose of the present invention is to provide a fully enclosed and automatic method for subpackaging immune cells, which can quickly obtain high-quality immune cells for clinical use.
  • a fully enclosed and fully automated device for distributing cells includes a sampling module, a liquid replenishment module and a liquid distribution module;
  • the sample injection module includes a cell sample container (such as a cell sample bag);
  • the fluid replacement module includes multiple fluid replacement devices, and the fluid replacement device includes multiple fluid replacement containers (such as fluid replacement bags (8-1, 8-2));
  • the liquid distribution module includes a liquid distributor, and the liquid distributor is provided with a sample inlet tube, a liquid outlet tube, an extraction tube, a waste liquid tube and a liquid replacement tube, and the sample inlet tube, the liquid outlet tube, the extraction tube
  • the pipe, the waste liquid pipe and the liquid replenishment pipe are connected, and valves are provided on the sampling pipe, liquid outlet pipe, extraction pipe, waste liquid pipe and liquid replenishment pipe;
  • the sample inlet tube is connected to the cell sample container (such as a cell sample bag), the liquid outlet pipe is connected to the cell freezing container (such as a cell freezing bag (7), and the extraction tube is connected to the extraction device (1) connected, the waste liquid pipe is connected with the waste liquid container (such as waste liquid bag (6)), and the described liquid replenishment pipe is connected with a plurality of liquid replenishment containers (such as liquid replenishment bag) successively through a plurality of branch pipes, and each A valve is arranged on the branch pipe.
  • the cell sample container such as a cell sample bag
  • the liquid outlet pipe is connected to the cell freezing container (such as a cell freezing bag (7)
  • the extraction tube is connected to the extraction device (1) connected
  • the waste liquid pipe is connected with the waste liquid container (such as waste liquid bag (6))
  • the described liquid replenishment pipe is connected with a plurality of liquid replenishment containers (such as liquid replenishment bag) successively through a plurality of branch pipes, and each A valve is arranged on the branch pipe.
  • the number of the rehydration container (eg, rehydration bag) is 1, 2, 3, 4, 5, 6, 7 or 8.
  • rehydration containers such as rehydration bags
  • two different rehydration containers such as rehydration bags
  • rehydration component 1 and rehydration component 2 respectively.
  • the rehydration component 1 includes compound electrolyte injection and human serum albumin aqueous solution
  • the rehydration component 2 includes CS10 frozen storage solution.
  • the cell sample container (such as a cell sample bag) contains cell samples.
  • the compound electrolyte injection includes 3-7 parts by weight of sodium chloride, 3-7 parts by weight of sodium gluconate, 2-6 parts by weight of sodium acetate, 0.1-0.8 parts by weight of potassium chloride, 0.1-0.6 parts by weight of magnesium chloride and 950-1050 parts by weight of water.
  • the concentration of human serum albumin in the aqueous solution of human serum albumin is 15-25% (v/v), preferably 18-22% (v/v).
  • the volume ratio of the compound electrolyte injection to the human serum albumin aqueous solution is 80-120:1, preferably 90-110:1, more preferably 95-105:1.
  • the CS10 cryopreservation solution includes DMSO aqueous solution.
  • the CS10 cryopreservation solution includes 8-12% (v/v) DMSO aqueous solution.
  • the pumping device is capable of sucking in liquid and pushing out liquid.
  • a method for preparing a cell suspension using the device as described in the first aspect of the present invention comprising the steps of:
  • the cell sample container (such as a cell sample bag) contains a cell sample
  • two different rehydration containers (such as a rehydration bag) respectively contain a rehydration component 1 and a rehydration component 2, wherein,
  • the rehydration component 1 includes compound electrolyte injection and human serum albumin aqueous solution, and the rehydration component 2 includes CS10 frozen storage solution;
  • the cell sample in the cell sample container (such as a cell sample bag) enters the extraction device through the injection tube and the extraction tube, and then the cell sample is discharged through the extraction tube and the extraction device.
  • the waste liquid tube enters into the waste liquid container (such as waste liquid bag), so as to perform pipeline rinsing on the sampling tube, extraction tube and waste liquid tube;
  • the cell sample in the cell sample container (such as a cell sample bag) enters the extraction device through the injection tube and the extraction tube, and then the cell sample is discharged through the extraction tube and The liquid outlet pipe enters into the cell freezing container (such as a cell freezing bag);
  • the rehydration component 1 in the rehydration container (such as a rehydration bag) enters the withdrawal device through the rehydration tube and the extraction tube, and then the rehydration component 1 passes through the extraction tube and the extraction tube through the discharge of the extraction device.
  • the liquid outlet pipe enters into the cell freezing container (such as a cell freezing bag);
  • the rehydration component 2 in the rehydration container (such as a rehydration bag) enters the withdrawal device through the rehydration tube and the extraction tube, and then through the discharge of the extraction device, the rehydration component 2 passes through the extraction tube and the liquid outlet tube into the cell freezing container (such as a cell freezing bag);
  • step (4) the rehydration component 1 and the rehydration component 2 enter into the cell freezing container (such as a cell freezing bag) sequentially, and the volume ratio of the rehydration component 1 and the rehydration component 2 is 1:1.
  • the cell freezing container such as a cell freezing bag
  • the method further includes step (5): counting the cells in the cell freezing container (such as a cell freezing bag) described in step (4), if the cell density is relatively large, follow the above steps ( Step 4) for rehydration, if the cell density is small, follow the steps of the above step (2) to replenish the cell sample, so as to obtain the cell suspension with the required cell density.
  • the cell freezing container such as a cell freezing bag
  • the method further includes the subpackaging step (6), which includes: connecting the cell freezing container (such as a cell freezing bag) containing the cell suspension to the sampling tube, The waste liquid pipe is connected with the sub-packaging bag;
  • the cell suspension in the cell freezing container (such as a cell freezing bag) enters the extraction device through the injection tube and the extraction tube, and then the cell suspension passes through the extraction tube and the waste through the discharge of the extraction device.
  • the liquid pipe enters into the subpackaging bag, and multiple subpackaging of the cell suspension is realized by changing the subpackaging bag.
  • the subpackaging step (6) is after the step (5).
  • the compound electrolyte injection includes 3-7 parts by weight of sodium chloride, 3-7 parts by weight of sodium gluconate, 2-6 parts by weight of sodium acetate, 0.1-0.8 parts by weight of potassium chloride, 0.1-0.6 parts by weight of magnesium chloride and 950-1050 parts by weight of water.
  • Fig. 1 is a structural schematic diagram of an embodiment of a fully enclosed and fully automated device for subpackaging cells, wherein 8-1 and 8-2 are liquid replacement bags, 5 is a sampling tube, 1 is a extraction device, 6 is a waste liquid bag and 7 is a cell freezing container (such as a cell freezing bag).
  • 8-1 and 8-2 are liquid replacement bags
  • 5 is a sampling tube
  • 1 is a extraction device
  • 6 is a waste liquid bag
  • 7 is a cell freezing container (such as a cell freezing bag).
  • the inventor accidentally developed a fully enclosed and fully automated device and method for subpackaging cells for the first time.
  • the method can perform cell subpackaging under closed conditions and reduce contact with the external environment.
  • the risk of infection, the fully enclosed and fully automated device and method for subpackaging cells described in the present invention can ensure the survival rate of subpackaged cells, and the precision of the subpackaged cells is high, thereby improving the quality of the subpackaged cells.
  • the inventors have completed the present invention.
  • the terms “comprising”, “comprising” and “containing” are used interchangeably to include not only open definitions, but also semi-closed, and closed definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • the fully enclosed and fully automated cell subpackaging device of the present invention includes a sampling module, a liquid replenishment module and a liquid distribution module;
  • the sample injection module includes a cell sample container (such as a cell sample bag);
  • the fluid replacement module includes multiple fluid replacement devices, and the fluid replacement device includes multiple fluid replacement containers (such as fluid replacement bags (8-1, 8-2));
  • the liquid distribution module includes a liquid distributor, and the liquid distributor is provided with a sample inlet tube, a liquid outlet tube, an extraction tube, a waste liquid tube and a liquid replacement tube, and the sample inlet tube, the liquid outlet tube, the extraction tube
  • the pipe, the waste liquid pipe and the liquid replenishment pipe are connected, and valves are provided on the sampling pipe, liquid outlet pipe, extraction pipe, waste liquid pipe and liquid replenishment pipe;
  • the sample inlet tube is connected to the cell sample container (such as a cell sample bag), the liquid outlet pipe is connected to the cell freezing container (such as a cell freezing bag (7), and the extraction tube is connected to the extraction device (1) connected, the waste liquid pipe is connected with the waste liquid container (such as waste liquid bag (6)), and the described liquid replenishment pipe is connected with a plurality of liquid replenishment containers (such as liquid replenishment bag) successively through a plurality of branch pipes, and each A valve is arranged on the branch pipe.
  • the cell sample container such as a cell sample bag
  • the liquid outlet pipe is connected to the cell freezing container (such as a cell freezing bag (7)
  • the extraction tube is connected to the extraction device (1) connected
  • the waste liquid pipe is connected with the waste liquid container (such as waste liquid bag (6))
  • the described liquid replenishment pipe is connected with a plurality of liquid replenishment containers (such as liquid replenishment bag) successively through a plurality of branch pipes, and each A valve is arranged on the branch pipe.
  • the number of the rehydration container (eg, rehydration bag) is 1, 2, 3, 4, 5, 6, 7 or 8.
  • the number of the rehydration container (such as a rehydration bag) is two, and two different rehydration containers (such as a rehydration bag) contain the rehydration component 1 and the rehydration component 2 respectively.
  • the rehydration component 1 includes compound electrolyte injection and human serum albumin aqueous solution
  • the rehydration component 2 includes CS10 frozen storage solution.
  • the compound electrolyte injection includes 3-7 parts by weight of sodium chloride, 3-7 parts by weight of sodium gluconate, 2-6 parts by weight of sodium acetate, 0.1-0.8 parts by weight of potassium chloride, 0.1-0.6 parts by weight Parts of magnesium chloride and 950-1050 parts by weight of water.
  • the concentration of human serum albumin in the aqueous solution of human serum albumin is 15-25% (v/v), preferably 18-22% (v/v).
  • the volume ratio of the compound electrolyte injection to the human serum albumin aqueous solution is 80-120:1, preferably 90-110:1, more preferably 95-105:1.
  • the CS10 cryopreservation solution includes DMSO aqueous solution.
  • the CS10 cryopreservation solution includes 8-12% (v/v) DMSO aqueous solution.
  • the cell sample container (such as a cell sample bag) contains cell samples.
  • the present invention also provides a method for subpackaging cells with a fully enclosed and fully automated cell subpackaging device as described in the present invention, said method comprising the steps of:
  • the cell sample container (such as a cell sample bag) contains a cell sample
  • two different rehydration containers (such as a rehydration bag) respectively contain a rehydration component 1 and a rehydration component 2, wherein,
  • the rehydration component 1 includes compound electrolyte injection and human serum albumin aqueous solution, and the rehydration component 2 includes CS10 frozen storage solution;
  • the cell sample in the cell sample container (such as a cell sample bag) enters the extraction device through the injection tube and the extraction tube, and then the cell sample is discharged through the extraction tube and the extraction device.
  • the waste liquid tube enters into the waste liquid container (such as waste liquid bag), so as to perform pipeline rinsing on the sampling tube, extraction tube and waste liquid tube;
  • the cell sample in the cell sample container (such as a cell sample bag) enters the extraction device through the injection tube and the extraction tube, and then the cell sample is discharged through the extraction tube and The liquid outlet pipe enters into the cell freezing container (such as a cell freezing bag);
  • the rehydration component 1 in the rehydration container (such as a rehydration bag) enters the withdrawal device through the rehydration tube and the extraction tube, and then the rehydration component 1 passes through the extraction tube and the extraction tube through the discharge of the extraction device.
  • the liquid outlet pipe enters into the cell freezing container (such as a cell freezing bag);
  • the rehydration component 2 in the rehydration container (such as a rehydration bag) enters the withdrawal device through the rehydration tube and the extraction tube, and then through the discharge of the extraction device, the rehydration component 2 passes through the extraction tube and the liquid outlet tube into the cell freezing container (such as a cell freezing bag);
  • step (4) the rehydration component 1 and the rehydration component 2 enter into the cell freezing container (such as a cell freezing bag) sequentially, and the volume ratio of the rehydration component 1 and the rehydration component 2 is 1:1.
  • the cell freezing container such as a cell freezing bag
  • the compound electrolyte injection includes 3-7 parts by weight of sodium chloride, 3-7 parts by weight of sodium gluconate, 2-6 parts by weight of sodium acetate, 0.1-0.8 parts by weight of sodium chloride Potassium, 0.1-0.6 parts by weight of magnesium chloride and 950-1050 parts by weight of water.
  • the method further includes step (5): counting the cells in the cell freezing container (such as a cell freezing bag) described in step (4), if the cell density is relatively large, according to the above Replenish the liquid in the step (4). If the cell density is low, supplement the cell sample according to the above step (2), so as to obtain the cell suspension with the required cell density.
  • the cell freezing container such as a cell freezing bag
  • the method also includes a packing step (6), and the step includes: carrying out a cell freezing container (such as a cell freezing bag) containing the cell suspension with a sampling tube Connect, and the waste liquid pipe is connected with the sub-packaging bag;
  • a cell freezing container such as a cell freezing bag
  • the cell suspension in the cell freezing container (such as a cell freezing bag) enters the extraction device through the injection tube and the extraction tube, and then the cell suspension passes through the extraction tube and the waste through the discharge of the extraction device.
  • the liquid pipe enters into the subpackaging bag, and multiple subpackaging of the cell suspension is realized by changing the subpackaging bag.
  • the subpackaging step (6) is after the step (5).
  • the fully enclosed and fully automated cell subpackaging device and method of the present invention can perform cell subpackaging under closed conditions, reducing the risk of infection from contact with the external environment.
  • the device and method for packaging cells can ensure the survival rate of the subpackaged cells, and the precision of the subpackaged cells is high, thereby improving the quality of the subpackaged cells.
  • the cell preparation method of the present invention can quickly prepare immune cells, reduces the cost of the enterprise, improves production capacity, and is suitable for industrial production. Meanwhile, the immune cells prepared by the cell preparation method of the present invention have high quality and can ensure clinical efficacy.
  • the amount of positive T cells in the final product bag is 150 ⁇ 10 6 cells, and the frozen storage volume is 40ml; the amount of positive T cells in the cell bag for detection is 37.5 ⁇ 10 6 cells, and the frozen storage volume is 10ml;
  • the positive rate was 20.71%; the theoretical cryopreservation density was 19.918 ⁇ 10 6 cells/ml.
  • Component 1 Add the HSA (human serum albumin) aqueous solution with a concentration of 20% (v/v) to the compound electrolyte injection (every 1000ml of water contains 5.26g of sodium chloride, 5.02g of sodium gluconate, and 5.02g of sodium acetate) 3.68g, potassium chloride 0.37g, magnesium chloride 0.30g), and then filled into the component 1 bag; wherein the volume ratio of 20% (v/v) HSA aqueous solution and compound electrolyte injection is 1:100.
  • HSA human serum albumin
  • Component 2 Fill the CS10 cryopreservation solution into the component 2 bag, wherein the CS10 cryopreservation solution is DMSO aqueous solution, and the volume fraction of DMSO is 10%.
  • Kit CT-49.1 a fully enclosed and fully automated device for distributing cells
  • open Kit CT-49.1 a fully enclosed and fully automated device for distributing cells
  • close the clips use a sterile pipette to connect the harvested cell sample bags to position 5, and the 250ml cell cryopreservation bags to At position 7, component 1 bag is at position 8-1, component 2 bag is at position 8-2, and waste liquid bag is at position 6.
  • Figure 1 The schematic diagram of the pipeline position of Kit CT-49.1 is shown in Figure 1;
  • i. Cell counting After concentrating the cell suspension, connect a 5ml syringe to a 250ml cryopreservation bag containing the cell suspension, shake the cryopreservation bag well, and extract about 0.2ml of the cell suspension for cell counting;
  • the volume of component 1 to be added the volume of the cell suspension in the cryopreservation bag + the volume of component 1 to be added;
  • Pipeline rinsing input component 1 rinsing volume 10ml, open the pipe clamp, and start the program;
  • Pipeline rinsing input component 2 rinsing volume 10ml, open the pipe clamp, and start the program;
  • Rinse the pipeline input the rinse volume of the final product as 10ml, open the pipeline clamp, and start the program;
  • ii. Cell bag dispensing for detection Before dispensing, first calculate the dispensing volume according to the amount of cells filled in the cell bag for testing and the freezing storage density, then connect the 50ml cell freezing storage bag to position 6, and input the calculated dispensing volume , click " ⁇ " to open the pipeline clamp and start the program. Repeat this step twice to obtain three bags of cell bags for testing;
  • Dispensing of the final product Before dispensing, first calculate the dispensing volume according to the amount of cells filled in the final product and the frozen storage density of the final product, then connect the 250ml cell freezing bag to position 6, input the calculated dispensing volume, and click " ⁇ ", open the pipe clamp and start the program. Get a bag of final product;

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Abstract

一种全封闭和全自动化的分装细胞的装置和方法。该装置包括进样模块、补液模块和液体分配模块。该装置能够在封闭的条件下进行细胞分装，降低与外界环境接触感染的风险，且保证分装细胞的存活率，分装得到的细胞精密度高，从而提高分装的细胞质量。

Description

一种全封闭和全自动化的分装细胞的装置和方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种全封闭和全自动化的分装细胞的装置和方法。
背景技术
细胞免疫治疗是一种采集患者自身免疫细胞，经过基因修饰和体外扩增培养，进而增强免疫细胞靶向性、杀伤力以及持久性的细胞疗法。在近年来的肿瘤免疫治疗临床上有着很好的表现，给临床治愈肿瘤带来了希望。然而制备得到的免疫细胞需要洗涤、浓缩并分装到多个冻存袋中，分装细胞的工艺流程、设备设施和试剂选择等任何一个中间环节都会影响分装细胞的质量，进而影响临床效果。因此，一个全封闭和全自动化的分装免疫细胞工艺可以保证产品的安全性和批次间的稳定性，减少人员和环境带来的影响，提高免疫细胞治疗产品的质量。传统的细胞分装只能在人工且非封闭的条件下进行，增加了与外界环境接触感染的风险，极大影响了产品的安全性。
因此，本领域需要开发一种全封闭自动化分装免疫细胞的方法，能够快速获得高质量的临床使用免疫细胞。
发明内容
本发明的目的在于提供一种全封闭自动化分装免疫细胞的方法，能够快速获得高质量的临床使用免疫细胞。
本发明第一方面，提供一种全封闭和全自动化的分装细胞的装置，所述的装置包括进样模块、补液模块和液体分配模块；
所述的进样模块包括细胞样品容器(如细胞样品袋)；
所述的补液模块包括多个补液器，所述的补液器包括多个补液容器(如补液袋(8-1、8-2))；
所述的液体分配模块包括液体分配器，所述的液体分配器设有进样管、出液管、抽出管、废液管和补液管相连，所述的进样管、出液管、抽出管、废液管和补液管是相通的，且所述的进样管、出液管、抽出管、废液管和补液管上均设有阀门；
所述的进样管与所述的细胞样品容器(如细胞样品袋)相连，所述的出液管与细胞冷冻容器(如细胞冷冻袋(7))相连，所述的抽出管与抽出装置(1)相连，所述废液管与废液容器(如废液袋(6))相连，所述的补液管通过多个分支管依次与多个补液容器(如补液袋)连接，且各个分支管上设有阀门。
在另一优选例中，所述补液容器(如补液袋)的个数为1、2、3、4、5、6、7或8个。
在另一优选例中，所述补液容器(如补液袋)的个数为2个，2个不同的补液容器(如补液袋)分别含有补液组分1和补液组分2。优选地，所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液。
在另一优选例中，所述的细胞样品容器(如细胞样品袋)含有细胞样品。
在另一优选例中，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
在另一优选例中，所述的人血清白蛋白水溶液中的人血清白蛋白的浓度为15-25％(v/v)，较佳地18-22％(v/v)。
在另一优选例中，所述的复方电解质注射液与所述人血清白蛋白水溶液的体积比为80-120:1，较佳地90-110:1，更佳地95-105:1。
在另一优选例中，所述的CS10冻存液包括DMSO水溶液。
在另一优选例中，所述的CS10冻存液包括8-12％(v/v)DMSO水溶液。
在另一优选例中，所述的抽出装置能够吸入液体和推出液体。
本发明第二方面，提供一种用如本发明第一方面所述的装置制备细胞悬液的方法，所述的方法包括步骤：
(1)所述的细胞样品容器(如细胞样品袋)含有细胞样品，2个不同的补液容器(如补液袋)分别含有补液组分1和补液组分2，其中，
所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液；
(2)通过抽出装置的抽吸使细胞样品容器(如细胞样品袋)中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和废液管进入到废液容器(如废液袋)中，从而对进样管、抽出管和废液管进行管路润洗；
(3)通过抽出装置的抽吸使细胞样品容器(如细胞样品袋)中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
(4)通过抽出装置的抽吸使得补液容器(如补液袋)中补液组分1经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分1经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
通过抽出装置的抽吸使得补液容器(如补液袋)中补液组分2经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分2经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
在步骤(4)中，补液组分1和补液组分2依次进入到细胞冷冻容器(如细胞冷冻袋)中，且补液组分1和补液组分2的体积比为1：1。
在另一优选例中，所述的方法还包括步骤(5)：对步骤(4)所述的细胞冷冻容器(如细胞冷冻袋)的细胞进行计数，如果细胞密度较大，按照上述步骤(4)的步骤进行补液，如果细胞密度较小，按照上述步骤(2)的步骤进行补充细胞样品，从而得到所需细胞密度的细胞悬液。
在另一优选例中，所述的方法还包括分装步骤(6)，所述的步骤包括：将含有所述细胞悬液的细胞冷冻容器(如细胞冷冻袋)与进样管进行连接，所述废液管与分装袋进行连接；
通过抽出装置的抽吸使细胞冷冻容器(如细胞冷冻袋)中的细胞悬液经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞悬液经抽出管和废液管进入到分装袋中，通过更换分装袋实现细胞悬液的多次分装。
在另一优选例中，所述的分装步骤(6)在步骤(5)后。
在另一优选例中，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
附图仅用于示例性说明，不能理解为对本实用新型的限制；为了更好说明本实 施例，附图某些部件会有省略、放大或缩小，并不代表实际产品的尺寸；对于本领域技术人员来说，附图中某些公知结构及其说明可能省略是可以理解的；相同或相似的标号对应相同或相似的部件；附图中描述位置关系的用语仅用于示例性说明，不能理解为对本专利的限制。
图1为全封闭和全自动化的分装细胞的装置实施例的结构示意图，其中，8-1、8-2为补液袋、5为进样管、1为抽出装置、6为废液袋和7为细胞冷冻容器(如细胞冷冻袋)。
具体实施方式
本发明人通过广泛而又深入的研究，首次意外开发一种全封闭和全自动化的分装细胞的装置和方法，所述的方法能够在封闭的条件下进行细胞分装，降低与外界环境接触感染的风险，本发明所述的全封闭和全自动化的分装细胞的装置和方法能够保证分装细胞的存活率，分装得到的细胞精密度高，从而提高分装的细胞质量。在此基础上，发明人完成了本发明。
术语
除非另有定义，否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包括”、“包含”与“含有”可互换使用，不仅包括开放式定义，还包括半封闭式、和封闭式定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
装置
为了便于说明，以下参考附图1进一步描述本发明所述的全封闭和全自动化的分装细胞的装置，在发明中应当理解的是，附图并不限定本发明的范围。
代表性地，本发明所述的全封闭和全自动化的分装细胞的装置包括进样模块、补液模块和液体分配模块；
所述的进样模块包括细胞样品容器(如细胞样品袋)；
所述的补液模块包括多个补液器，所述的补液器包括多个补液容器(如补液袋(8-1、8-2))；
所述的液体分配模块包括液体分配器，所述的液体分配器设有进样管、出 液管、抽出管、废液管和补液管相连，所述的进样管、出液管、抽出管、废液管和补液管是相通的，且所述的进样管、出液管、抽出管、废液管和补液管上均设有阀门；
所述的进样管与所述的细胞样品容器(如细胞样品袋)相连，所述的出液管与细胞冷冻容器(如细胞冷冻袋(7))相连，所述的抽出管与抽出装置(1)相连，所述废液管与废液容器(如废液袋(6))相连，所述的补液管通过多个分支管依次与多个补液容器(如补液袋)连接，且各个分支管上设有阀门。
在本发明的一个优选例中，所述补液容器(如补液袋)的个数为1、2、3、4、5、6、7或8个。
在本发明的另一优选例中，所述补液容器(如补液袋)的个数为2个，2个不同的补液容器(如补液袋)分别含有补液组分1和补液组分2。优选地，所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液。
优选地，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
在另一优选例中，所述的人血清白蛋白水溶液中的人血清白蛋白的浓度为15-25％(v/v)，较佳地18-22％(v/v)。
在另一优选例中，所述的复方电解质注射液与所述人血清白蛋白水溶液的体积比为80-120:1，较佳地90-110:1，更佳地95-105:1。
在另一优选例中，所述的CS10冻存液包括DMSO水溶液。
在另一优选例中，所述的CS10冻存液包括8-12％(v/v)DMSO水溶液。
在本发明的另一优选例中，所述的细胞样品容器(如细胞样品袋)含有细胞样品。
方法
本发明还提供一种用如本发明所述的全封闭和全自动化的分装细胞的装置分装细胞的方法，所述的方法包括步骤：
(1)所述的细胞样品容器(如细胞样品袋)含有细胞样品，2个不同的补液容器(如补液袋)分别含有补液组分1和补液组分2，其中，
所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液；
(2)通过抽出装置的抽吸使细胞样品容器(如细胞样品袋)中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和废液管进入到废液容器(如废液袋)中，从而对进样管、抽出管和废液管进行管路润洗；
(3)通过抽出装置的抽吸使细胞样品容器(如细胞样品袋)中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
(4)通过抽出装置的抽吸使得补液容器(如补液袋)中补液组分1经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分1经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
通过抽出装置的抽吸使得补液容器(如补液袋)中补液组分2经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分2经抽出管和出液管进入到细胞冷冻容器(如细胞冷冻袋)中；
在步骤(4)中，补液组分1和补液组分2依次进入到细胞冷冻容器(如细胞冷冻袋)中，且补液组分1和补液组分2的体积比为1：1。
在本发明的一个优选例中，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
在本发明的一个优选例中，所述的方法还包括步骤(5)：对步骤(4)所述的细胞冷冻容器(如细胞冷冻袋)的细胞进行计数，如果细胞密度较大，按照上述步骤(4)的步骤进行补液，如果细胞密度较小，按照上述步骤(2)的步骤进行补充细胞样品，从而得到所需细胞密度的细胞悬液。
在本发明的一个优选例中，所述的方法还包括分装步骤(6)，所述的步骤包括：将含有所述细胞悬液的细胞冷冻容器(如细胞冷冻袋)与进样管进行连接，所述废液管与分装袋进行连接；
通过抽出装置的抽吸使细胞冷冻容器(如细胞冷冻袋)中的细胞悬液经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞悬液经抽出管和废液管进入到分装袋中，通过更换分装袋实现细胞悬液的多次分装。
在另一优选例中，所述的分装步骤(6)在步骤(5)后。
本发明的主要优点主要包括：
本发明所述的全封闭和全自动化的分装细胞的装置和方法能够在封闭的 条件下进行细胞分装，降低与外界环境接触感染的风险，本发明所述的全封闭和全自动化的分装细胞的装置和方法能够保证分装细胞的存活率，分装得到的细胞精密度高，从而提高分装的细胞质量。
本发明的细胞制备方法能够快速制备免疫细胞，降低了企业的成本，提高了生产产能，适合工业化生产，同时本发明的细胞制备方法制备的免疫细胞具有高质量，能够保证临床疗效。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
1.实验方案
a)冻存细胞量需求：终产品袋阳性T细胞量为150×10 6cells，冻存体积为40ml；检测用细胞袋阳性T细胞量为37.5×10 6cells，冻存体积为10ml；细胞阳性率为20.71％；理论冻存密度19.918×10 6cells/ml。
b)试剂配制：
i.组分1：将浓度为20％(v/v)的HSA(人血清白蛋白)水溶液加入到复方电解质注射液(每1000ml水含氯化钠5.26g、葡萄糖酸钠5.02g、醋酸钠3.68g、氯化钾0.37g、氯化镁0.30g)中，再灌装到组分1袋中；其中浓度为20％(v/v)的HSA水溶液与复方电解质注射液的体积比1：100。
ii.组分2：将CS10冻存液灌装到组分2袋中，其中CS10冻存液为DMSO水溶液，DMSO的体积分数为10％。
c)分装前管路准备
i.安全柜中打开Kit CT-49.1(全封闭和全自动化的分装细胞的装置)并关闭夹子，使用无菌接管机分别连接收获的细胞样品袋于5号位置，250ml细胞冻存袋于7号位置，组分1袋于8-1号位置，组分2袋于8-2号位置，废液袋于6号位置。Kit CT-49.1管路位置示意图如图1；
ii.按照图1安装以上管路于Sepax上；
d)Culture Wash程序浓缩细胞悬液
i.细胞计数：浓缩细胞悬液后，将5ml注射器连接到含有细胞悬液的250ml冻存袋上，摇匀此冻存袋后，抽取约0.2ml细胞悬液进行细胞计数；
e)Dilution(分装)程序配制终产品：最终冻存液添加配比为组分1:组分2＝50％:50％。
i.组分体积计算：根据上步细胞计数结果，细胞悬液体积，终产品灌装密度与终产品灌装细胞量，计算组分1与组分2各自应添加体积。其中，组分1应添加体积＝冻存袋内细胞悬液体积+还需添加的组分1体积；
ii.管路润洗：输入组分1润洗体积10ml，打开管路夹，启动程序；
iii.分装组分1：输入分装体积＝还需添加的组分1体积，点击“√”，打开管路夹，启动程序；
iv.管路润洗：输入组分2润洗体积10ml，打开管路夹，启动程序；
v.分装组分2：输入分装体积＝组分2应添加的总体积，点击“√”，打开管路夹，启动程序；
f)终产品分装
i.管路润洗：输入终产品润洗体积10ml，打开管路夹，启动程序；
ii.检测用细胞袋分装：分装前，先按检测用细胞袋灌装细胞量与冻存密度计算分装体积，再连接50ml细胞冻存袋于6号位置，输入计算的分装体积，点击“√”，打开管路夹，启动程序。重复此步骤两次，即得三袋检测用细胞袋；
iii.终产品分装：分装前，先按终产品灌装细胞量与终产品冻存密度计算分装体积，再连接250ml细胞冻存袋于6号位置，输入计算的分装体积，点击“√”，打开管路夹，启动程序。即得一袋终产品；
iv.排除上述三袋检测用细胞袋和一袋终产品袋内的空气，对袋子进行封合；
v.将封合的袋子转移至程序降温仪内，通过程序降温仪进行冻存，冻存后转移至液氮冰箱储存。
2.实验结果
a)浓缩细胞悬液计数
使用细胞计数仪检测浓缩的细胞样品袋内细胞密度和活率，如表1。浓缩后的
细胞量为181.5×10 6cells/ml×(12.2ml-0.2ml)＝2178×10 6cells。
表1.浓缩细胞悬液计数
Figure PCTCN2022114503-appb-000001
Figure PCTCN2022114503-appb-000002
b)终产品配制
计算得到的组分体积如表2，总体积为54.7ml+54.7ml＝109.4ml。
表2.组分体积
组分名称 分装体积(ml)
组分1 54.7
组分2 54.7
c)终产品分装
使用细胞计数仪分别检测分装到三个检测用细胞袋和一袋终产品袋内细胞密度和活率，如表4、表5、表6和表7。检测用细胞袋1阳性细胞量为19.25×10 6cells/ml×10ml×20.71％＝39.87×10 6cells；检测用细胞袋2阳性细胞量为18.8×10 6cells/ml×10ml×20.71％＝38.93×10 6cells；检测用细胞袋3阳性细胞量为16.5×10 6cells/ml×10ml×20.71％＝34.17×10 6cells；终产品袋阳性细胞量为20.033×10 6cells/ml×40ml×20.71％＝165.95×10 6cells。细胞分装汇总结果如表8。与理论冻存密度相比，三袋检测用细胞和一袋终产品的细胞密度均相差不大，最大相差17.16％；与理论阳性细胞量相比，最大相差10.63％，均在可接受范围之内，且各袋细胞活率几乎没有差异。因此，本工艺使用Sepax配制的终产品体积和总细胞量准确，细胞几乎没有损失，表明使用Sepax对收获的细胞进行终产品的分装是可行的，实现了分装免疫细胞工艺流程的全封闭自动化。
表4.检测用细胞袋1细胞计数
Figure PCTCN2022114503-appb-000003
Figure PCTCN2022114503-appb-000004
表5.检测用细胞袋2细胞计数
Figure PCTCN2022114503-appb-000005
表6.检测用细胞袋3细胞计数
Figure PCTCN2022114503-appb-000006
表7.终产品细胞计数
Figure PCTCN2022114503-appb-000007
表8.细胞分装汇总结果
Figure PCTCN2022114503-appb-000008
Figure PCTCN2022114503-appb-000009
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种全封闭和全自动化的分装细胞的装置，其特征在于，所述的装置包括进样模块、补液模块和液体分配模块；
    所述的进样模块包括细胞样品容器；
    所述的补液模块包括多个补液器，所述的补液器包括多个补液容器；
    所述的液体分配模块包括液体分配器，所述的液体分配器设有进样管、出液管、抽出管、废液管和补液管相连，所述的进样管、出液管、抽出管、废液管和补液管是相通的，且所述的进样管、出液管、抽出管、废液管和补液管上均设有阀门；
    所述的进样管与所述的细胞样品容器相连，所述的出液管与细胞冷冻容器相连，所述的抽出管与抽出装置相连，所述废液管与废液容器相连，所述的补液管通过多个分支管依次与多个补液容器连接，且各个分支管上设有阀门。
  2. 如权利要求1所述的装置，其特征在于，所述补液容器的个数为2个，2个不同的补液容器分别含有补液组分1和补液组分2，其中，所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液。
  3. 如权利要求2所述的装置，其特征在于，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
  4. 如权利要求2所述的装置，其特征在于，所述的人血清白蛋白水溶液中的人血清白蛋白的浓度为15-25％(v/v)。
  5. 如权利要求1所述的装置，其特征在于，所述的抽出装置能够吸入液体和推出液体。
  6. 如权利要求2如权利要求1所述的装置，其特征在于，所述的复方电解质注射液与所述人血清白蛋白水溶液的体积比为80-120:1，较佳地90-110:1，更佳地95-105:1。
  7. 如权利要求2所述的装置，其特征在于，所述的CS10冻存液包括8-12％(v/v)DMSO水溶液。
  8. 一种用如权利要求1所述的装置制备细胞悬液的方法，其特征在于，所述的方法包括步骤：
    (1)所述的细胞样品容器含有细胞样品，2个不同的补液容器分别含有补 液组分1和补液组分2，其中，
    所述补液组分1包括复方电解质注射液和人血清白蛋白水溶液，所述补液组分2包括CS10冻存液；
    (2)通过抽出装置的抽吸使细胞样品容器中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和废液管进入到废液容器中，从而对进样管、抽出管和废液管进行管路润洗；
    (3)通过抽出装置的抽吸使细胞样品容器中的细胞样品经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞样品经抽出管和出液管进入到细胞冷冻容器中；
    (4)通过抽出装置的抽吸使得补液容器中补液组分1经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分1经抽出管和出液管进入到细胞冷冻容器中；
    通过抽出装置的抽吸使得补液容器中补液组分2经补液管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使补液组分2经抽出管和出液管进入到细胞冷冻容器中；
    在步骤(4)中，补液组分1和补液组分2依次进入到细胞冷冻容器中，且补液组分1和补液组分2的体积比为1：1。
  9. 如权利要求8所述的方法，其特征在于，所述的方法还包括步骤(5)：对步骤(4)所述的细胞冷冻容器的细胞进行计数，如果细胞密度较大，按照上述步骤(4)的步骤进行补液，如果细胞密度较小，按照上述步骤(2)的步骤进行补充细胞样品，从而得到所需细胞密度的细胞悬液。
  10. 如权利要求8所述的方法，其特征在于，所述的方法还包括分装步骤(6)，所述的步骤包括：将含有所述细胞悬液的细胞冷冻容器与进样管进行连接，所述废液管与分装袋进行连接；
    通过抽出装置的抽吸使细胞冷冻容器中的细胞悬液经进样管和抽出管进入到抽出装置中，然后再通过抽出装置的排出使细胞悬液经抽出管和废液管进入到分装袋中，通过更换分装袋实现细胞悬液的多次分装。
  11. 如权利要求8所述的方法，其特征在于，所述的复方电解质注射液包括3-7重量份氯化钠、3-7重量份葡萄糖酸钠、2-6重量份醋酸钠、0.1-0.8重量份氯化钾、0.1-0.6重量份氯化镁和950-1050重量份水。
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