Double pump harvesting method and apparatus

Abstract

The present invention relates to the technical field of bioengineering, and in particular to a double-pump harvesting method and apparatus. The double-pump harvesting method includes concentration and formulation. The concentration step involves using two pumps, a first pump connected at one end to an initial sample container and at the other end to a centrifuge container, and a second pump connected at one end to the centrifuge container and at the other end to an intermediate product container and a waste liquid container, and a continuous-flow concentration step in which the flow rate of the second pump is increased while the difference in flow rate between the second pump and the first pump during concentration is set as a flow rate threshold. The present invention achieves a cyclic continuous-flow concentration process using a specific double-pump process, and in combination with subsequent cyclic formulation, achieves the function of formulation in multiple formulation bags without manual operation, resulting in a cell formulation method that improves formulation efficiency and accuracy, and is of great significance in the field of cellular pharmaceutical manufacturing.

Description

[Technical Field]

[0001] <Cross reference> This application claims priority to a Chinese patent application entitled "Double Pump Harvesting Method and Apparatus" filed on December 28, 2022, with application number 202211701908.6, the entire contents of which are incorporated herein by reference in their entirety. The present invention relates to the technical field of bioengineering, and in particular to a double pump harvesting method and apparatus. [Background technology]

[0002] With the continuous advancement of medical technology, the field of modern medicine is gradually shifting from molecular therapy to cell therapy, which refers to the transplantation or importation of normal or bioengineered human autologous or allogeneic cells into the patient's body, so that the newly imported cells can replace the original damaged cells or exert stronger immune killing function, thereby restoring the function of diseased cells or enhancing the ability of immune cells to fight specific diseases. Summary of the Invention [Problem to be solved by the invention]

[0003] Currently, the process of producing cell drugs includes cell culture and concentration and washing. When the cells after concentration and washing are used for cryopreservation, retransportation, testing, etc., they need to be formulated and dispensed. When the number and volume of dispensing bags are relatively large, the manual calculations and operations are tedious and complicated, resulting in low efficiency and relatively high costs. [Means for solving the problem]

[0004] The present invention provides a double pump harvesting method and apparatus to solve the problems existing in the prior art.

[0005] In a first aspect, the present invention provides a double pump harvesting method comprising a concentrate and a formulation, The concentration is A step of concentrating using two pumps, wherein one end of a first pump is connected to an initial sample container and the other end is connected to a centrifuge container, and one end of a second pump is connected to the centrifuge container and the other ends are connected to an intermediate product container and the waste liquid container; and performing continuous flow concentration by increasing the flow rate of the second pump while setting the difference in flow rate between the second pump and the first pump during concentration as a flow rate threshold.

[0006] Preferably, the flow rate threshold is 100 to 200 ml / min.

[0007] Furthermore, the flow rate of the first pump is 100 to 200 ml / min, and / or the flow rate of the second pump is 200 to 300 ml / min.

[0008] Furthermore, the first pump transfers the initial sample from the initial sample container into the centrifuge container, followed by centrifugation, and the second pump transfers the supernatant from the centrifuge container to the waste container, thereby forming the circulating continuous flow concentration until the volume of the cell solution in the centrifuge container falls below a concentration threshold, preferably 70 to 150 mL.

[0009] Furthermore, in the process of the cyclic continuous flow concentration, the concentration volume is preset, and the number of circulations is determined according to the concentration volume. For example, if the preset concentration volume is 1000 mL, the number of circulations = initial sample volume / 1000 mL, and is rounded down.

[0010] Furthermore, the concentration may be determining a concentration method according to an initial sample volume; using cyclic continuous flow concentration if the initial sample volume is greater than a predetermined multiple of the volume of the initial sample container, and otherwise using standard continuous flow concentration; Further includes:

[0011] Preferably, the preset multiple is 12 times.

[0012] Furthermore, during the concentration process, the initial sample in the initial sample container is moved to a centrifuge container, and an air bubble sensor is activated when the amount of the initial sample in the initial sample container is reduced to a preset percentage.

[0013] Preferably, the preset percentage is 30%.

[0014] Furthermore, the formulation comprises: adding a first predetermined resuspension volume of the resuspension to the centrifuge vessel to resuspend the cells, and then determining the formulation process based on a preset total formulation volume; employing a single-dose formulation when the preset total formulation volume is 250 mL or less; employing a circulating formulation when the preset total formulation volume exceeds 250 mL; Includes.

[0015] Furthermore, the circulating formulation comprises: adding a first predetermined amount of preparation to an intermediate container, then transferring the resuspended cells in the centrifuge container to the intermediate container, washing the centrifuge container, transferring the washing liquid after washing to the intermediate container, and adding a second predetermined amount of preparation to the intermediate container so that the total volume of the cell solution in the intermediate container becomes the sum of the preset total formulation volume and compensation volume; dispensing the cell solution in the intermediate container into formulations according to a preset number of formulation dispenses and a preset dispensing volume; Includes.

[0016] In a second aspect, the present invention provides a double pump harvesting device comprising a first pump and a second pump. the first pump has one end connected to an initial sample container and the other end connected to a centrifuge container, the second pump has one end connected to the centrifuge container and the other ends connected to an intermediate product container and a waste liquid container; The flow rate of the second pump is increased while the difference in flow rate between the second pump and the first pump during concentration is set as a flow rate threshold, and circulatory continuous flow concentration is performed.

[0017] In a third aspect, the present invention provides a cell preparation system comprising the double pump harvesting device.

[0018] The present invention has the following beneficial effects: The present invention uses a special double-pump process to achieve continuous cyclic concentration at a higher flow rate in the subsequent concentration and formulation process. Combined with the subsequent cyclic formulation process, it significantly improves formulation efficiency and accuracy, allowing up to nine products to be formulated per cycle, exceeding existing formulation efficiencies. The double-pump harvesting provided by the present invention significantly improves the manufacturing efficiency of cell medicines, which is of great significance. [Brief explanation of the drawings]

[0019] In order to more clearly describe the technical solutions of the present invention or the prior art, the following briefly introduces drawings necessary for describing the embodiments or the prior art. The drawings in the following description are some embodiments of the present invention, and it is obvious to those skilled in the art that other drawings can be obtained based on these drawings without any creative efforts. [Figure 1] 1 is a schematic diagram of a double pump harvesting device according to an embodiment of the present invention. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0020] In order to clarify the objectives, technical aspects and advantages of the present invention, the technical aspects of the present invention will be described clearly and completely below with reference to the drawings in the present invention, but it is clear that the described embodiments are only a part of the embodiments of the present invention, and are not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative labor fall within the protection scope of the present invention.

[0021] The present invention provides a double pump harvesting process including concentration and formulation, The concentration is a step of concentrating using two pumps, wherein one end of the first pump is connected to an initial sample container and the other end is connected to a centrifuge container, and one end of the second pump is connected to the centrifuge container and the other ends are connected to the intermediate product container and the waste liquid container; and performing circulatory continuous flow concentration by increasing the flow rate of the second pump while setting the difference in flow rate between the second pump and the first pump during concentration as a flow rate threshold.

[0022] Taking the apparatus shown in Figure 1 as an example, the process is as follows: Step 1: Consumable self-test, including consumable input, consumable seal, discharged waste detection, self-test criteria, bubble / pressure sensor performance, continuous flow detection.

[0023] Step 2: Parameter setting involves inputting the initial sample volume (0 to 99,999 mL), determining the concentration method for the input initial sample volume, and determining the concentration conditions according to the capacity of the centrifuge container 3. Specifically, if the input initial sample volume is 12 times or more the capacity of the centrifuge container 3, circulating continuous flow concentration is used; otherwise, standard continuous flow concentration is used; for example, if the capacity of the centrifuge container 3 is 1 L, circulating continuous flow concentration is used if the initial sample volume is 12 L or more. As the centrifuge container 3, multiple types of devices capable of centrifuging, such as a centrifuge cup, can be selected.

[0024] When performing continuous flow concentration, the initial sample is injected from the initial sample container 4 into the centrifuge container 3 via the first pump 1, and during this process, the remaining volume of the initial sample in the initial sample container 4 is monitored. When the remaining volume falls below a volume threshold, an air bubble sensor 12 is activated, which can be used to monitor whether the initial sample has been completely output from the initial sample container 4.

[0025] Additionally, the remaining volume of the initial sample in the initial sample container 4 can be monitored in various ways, for example using a weight sensor 13 to activate an air bubble sensor 12 when the weight drops below a volume threshold of the initial weight.

[0026] Furthermore, the volume threshold is a preset threshold, and is preferably 20 to 40%, and more preferably 30%.

[0027] Step 3: Concentration; Based on the determination of the concentration method in the previous step, standard continuous flow concentration or circulating continuous flow concentration is performed, and the steps of standard continuous flow concentration are as follows: Standard continuous flow concentration is performed with standard continuous flow concentration parameters of centrifuge vessel 3 centrifuge rotation speed 2200 rpm, first pump 1 flow rate 220 mL / min, and second pump 2 flow rate 400 mL / min. After a preset volume of liquid is poured into the centrifuge container 3 and centrifuged, the first pump 1 pumps the liquid in at a flow rate of 220 mL / min, and the second pump 2 pumps the liquid out at a flow rate of 400 mL / min. The flow rate of the first pump 1 is lower than that of the second pump 2, and the flow rate difference between the two pumps is within a threshold flow rate, achieving continuous flow concentration. Both pumps operate continuously, and the flow rate difference provides the concentration effect. The flow rate threshold is preferably 100-200 mL / min, and can be selected depending on the initial sample volume in actual applications.

[0028] Additionally, the degree of liquid injection is monitored by the air bubble sensor 12 until the liquid injection is terminated and standard continuous flow concentration is completed.

[0029] Circulating continuous flow concentration parameters: 1. Determining the number of cycles for concentration: The number of circulations is determined according to the concentration volume of each cycle. For example, if the concentration volume of each cycle is 5 L, the number of circulations = initial sample volume / 5 L. For example, if the concentration volume of each cycle is 4 L, the number of circulations = initial sample volume / 4 L.

[0030] Each concentration is performed using a continuous flow method with a constant flow rate difference, similar to the standard continuous flow concentration described above. The first pump 1 aspirates the initial sample from the initial sample container 4 into the centrifuge container 3. Next, after centrifugation, the second pump 2 aspirates the supernatant into the waste container 8. When the remaining volume of the cell solution in the centrifuge container 3 reaches 140 to 145 mL (this volume can be preset according to the volume of the centrifuge container 3; for example, the remaining volume can be controlled to 140 to 145 mL when a large centrifuge container is used, and 70 to 75 mL when a small centrifuge container is used), it is aspirated into the intermediate container 7. The intermediate container 7 used in this embodiment of the present invention is preferably an intermediate product bag.

[0031] 2. During the final circulation concentration, the air bubble sensor 12 is enabled to monitor the progress.

[0032] 3. Finishing: The cell fluid in the intermediate vessel 7 may be further concentrated by continuing to repeat the continuous flow concentration process described above.

[0033] Step 4: Washing; After concentration is complete, the cell fluid remaining in the centrifuge container 3 is washed. This washing involves using the first pump 1 to suck the washing fluid from the washing fluid container 5 and fusing it with other components in the cell fluid, followed by continuing centrifugation and using the second pump 2 to suck the supernatant into the waste fluid container 8. The degree of washing can be determined according to the volume of the centrifuge container 3; for example, when a large centrifuge container is used, the residual volume can be controlled to 140 to 145 mL, and when a small centrifuge container is used, the residual volume can be controlled to 70 to 75 mL.

[0034] Furthermore, the number of washes and the volume per wash can be preset, for example, 0 to 10 washes and 0 to 175 mL per wash.

[0035] Step 5: Cryopreservation solution washing; After the previous washing step, you can choose to wash using a cryopreservation solution. The procedure is the same as the previous washing step, but the number of washes can be set to 0 to 5, and the volume per wash can be set to 0 to 400 mL (for large centrifuge containers) or 0 to 175 mL (for small centrifuge containers).

[0036] Similarly, the degree of cleanliness can be determined according to the volume of the centrifuge container 3. For example, when a large centrifuge container is used, the residual volume can be controlled to 90 to 100 mL, and when a small centrifuge container is used, the residual volume can be controlled to 40 to 45 mL.

[0037] Step 6: Resuspension; 1. Obtain the volume of the concentrated cell solution. At this time, the concentrated and washed cell solution is placed in the centrifuge container 3, and the resuspension is aspirated from the resuspension container 6 into the centrifuge container 3. The volume of the resuspension is set to a preset volume, for example, 0 to 250 mL. Then, mixing is performed. The mixing time can also be set in advance, for example, 300 s.

[0038] 2. After mixing is complete, the next step of sampling and counting can be carried out to calculate the cell density.

[0039] Step 7: Dispensing the formulation; The dispensing process is determined by the total formulation volume, a formulation threshold is set, and if the total formulation volume exceeds the threshold, a circulating formulation is executed, and if the total formulation volume does not exceed the threshold, a single formulation is executed. For example, if the formulation threshold is set to 250 mL, a single formulation is executed if the total formulation volume is 250 mL or less. If the total formulation volume is greater than 250 mL, a circulating formulation is executed.

[0040] Single dose: The volume of the second resuspension is preset, and the resuspension is aspirated from the resuspension container 6 into the centrifuge container 3, and may be, for example, 0 to 250 mL. Similarly, the mixing time is preset, and after mixing, a sampling count is performed to calculate the cell density, and the cell liquid can then be aspirated directly into the product container (9, 10, or 11). The number and volume of dispensing bags can be preset; for example, 1 to 9 bags are dispensed, with the volume of each bag being 1 to 600 mL. Circulating drugs: The total volume of the preparation to be injected into the intermediate container 7 is set in advance, and the volume to be injected into the intermediate container 7 the first time is set. The preparation is then aspirated into the centrifuge container 3 via the resuspension container 6, and after the cells are resuspended, the preparation is injected directly into the intermediate container 7. Cup washing is then performed, with the number of washes set to 0 to 5 and the volume per wash set to 0 to 200 mL, and the washing solution is injected directly into the intermediate container 7. After washing, a sampling count is performed again to determine the cell density, and the volume of the preparation to be injected into the intermediate container 7 the second time is set, this time with a compensation volume added. The compensation volume is calculated by compensating 1 mL for each additional dispensing bag.

[0041] Furthermore, after the above steps are completed, the formulation is dispensed from the intermediate container 7 into product containers (9, 10, or 11), the number and volume of which are preset, for example, the number of dispensing bags is 1 to 9 bags, and the volume of each bag is 10 to 5000 mL.

[0042] Furthermore, each product container corresponds to one valve, for example, product container 9 corresponds to valve 14, product container 10 corresponds to valve 15, and product container 11 corresponds to valve 16. In actual application, after a product container is filled, the valve can be closed, the product container can be replaced, and then the valve can be opened to achieve continuous dispensing.

[0043] While conventional processes can generally only process small batches of cells, the double-pump process provided by the present invention can process large amounts of cells by flexibly using two different concentration methods. Its high flow rate allows for higher cell concentration efficiency, reduces cell damage caused by long-term centrifugation, and improves cell yield and viability. The following are the cell yield and viability results for cells processed using the double-pump harvesting method provided in an embodiment of the present invention. In actual application, the preferred parameters are used. The results are shown in the following table:

[0044] JPEG2025536095000002.jpg62170

[0045] In an embodiment of the present invention, ten formulations were carried out using a double pump harvesting process, with a formulation volume of 1500 mL, and the input, display, and error monitoring results for valves 14, 15, and 16 were as follows:

[0046] JPEG2025536095000003.jpg63170

[0047] JPEG2025536095000004.jpg55170

[0048] JPEG2025536095000005.jpg58170

[0049] JPEG2025536095000006.jpg55170

[0050] The examples of the present invention are further compared with formulations that do not use the conventional double-pump harvesting process, and the effectiveness results of the formulations using the conventional single-pump harvesting process are shown in the following table, which shows that the error of the single-pump process formulation is significantly higher than that of the double-pump harvesting process provided by the examples of the present invention.

[0051] JPEG2025536095000007.jpg50170

[0052] Finally, the above embodiments are used only to explain the technical solutions of the present invention, and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may be modified or some technical features may be replaced with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. [Industrial Applicability]

[0053] The present invention provides a double-pump harvesting method and apparatus. The double-pump harvesting method includes concentration and formulation. The concentration step involves using two pumps, one end of the first pump connected to an initial sample container and the other end to a centrifuge container, and one end of the second pump connected to the centrifuge container and the other end to an intermediate product container and a waste liquid container, and a continuous-flow concentration step in which the flow rate of the second pump is increased while the difference in flow rate between the second pump and the first pump during concentration is set as a flow rate threshold. The present invention achieves a cyclic continuous-flow concentration process using a specific double-pump process, and in combination with subsequent cyclic formulation, achieves the function of formulation in multiple formulation bags without manual operation, resulting in a cell formulation method that improves formulation efficiency and accuracy. This is of great significance in the field of cellular pharmaceutical manufacturing and has great economic value and potential for future applications. [Explanation of symbols]

[0054] 1: First pump 2: Second pump 3: Centrifugal container 4: Initial sample container 5: Cleaning container 6:Resuspension container 7: Intermediate container 8: Waste liquid container 9:Product container 10:Product container 11:Product container 12: Air bubble sensor 13: Weight sensor 14: Valve 15: Valve 16: Valve.

Claims

1. 1. A double pump harvesting method comprising: Includes concentrates and formulations The concentration is a step of concentrating using two pumps, wherein one end of a first pump is connected to an initial sample container and the other end is connected to a centrifuge container, and one end of a second pump is connected to the centrifuge container and the other ends are connected to an intermediate product container and the waste liquid container; a step of performing continuous flow concentration by increasing the flow rate of the second pump while setting a flow rate difference between the second pump and the first pump during concentration as a flow rate threshold; A double pump harvesting method comprising:

2. The flow rate of the first pump is 100 to 200 ml / min, and / or the flow rate of the second pump is 200 to 300 ml / min. The double pump harvesting method of claim 1.

3. The first pump moves the initial sample from the initial sample container to the centrifuge container, and then performs centrifugation; the second pump moves the supernatant in the centrifuge container to the waste container; and further forms the continuous flow concentration until the volume of the cell solution in the centrifuge container falls below a concentration threshold.

3. The double pump harvesting method according to claim 1 or 2.

4. In the process of the circulating continuous flow concentration, a concentration volume is preset, and the number of circulations is determined according to the concentration volume.

4. The double pump harvesting method of claim 3.

5. The enrichment may further comprise: Determining a concentration method according to an initial sample volume; using cyclic continuous flow concentration if the initial sample volume is greater than a predetermined multiple of the volume of the initial sample container, and otherwise using standard continuous flow concentration; The double pump harvesting method of any one of claims 1 to 4, further comprising:

6. In the concentration process, the initial sample in the initial sample container is transferred to a centrifuge container, and when the amount of the initial sample in the initial sample container is reduced to a predetermined percentage, an air bubble sensor is activated. The double pump harvesting method of claim 1.

7. The formulation comprises: adding a first predetermined resuspension volume to the centrifuge vessel to resuspend the cells, and then determining the formulation process based on a preset total formulation volume; employing a single-dose formulation when the preset total formulation volume is 250 mL or less; employing a circulating formulation when the preset total formulation volume exceeds 250 mL; The double pump harvesting method of claim 1, further comprising:

8. The circulating formulation comprises: adding a first predetermined amount of preparation to an intermediate container, then transferring the resuspended cells in the centrifuge container to the intermediate container, washing the centrifuge container, transferring the washing liquid after washing to the intermediate container, and adding a second predetermined amount of preparation to the intermediate container so that the total volume of the cell solution in the intermediate container becomes the sum of the preset total formulation volume and compensation volume; Dispensing the cell solution in the intermediate container into formulations according to a preset number of formulation dispenses and a preset dispensing volume; The double pump harvesting method of claim 7, further comprising:

9. 1. A double pump harvesting device comprising a first pump and a second pump, the first pump has one end connected to an initial sample container and the other end connected to a centrifuge container, the second pump has one end connected to the centrifuge container and the other ends connected to the intermediate product container and the waste liquid container, The flow rate of the second pump is increased while the flow rate difference during concentration between the second pump and the first pump is set as a flow rate threshold, and circulating continuous flow concentration is performed. A double pump harvesting device characterized by:

10. 10. A double pump harvesting device according to claim 9, A cell preparation system characterized by:

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