One-way bioreactor sampling connecting device and application thereof

Abstract

The invention provides a one-way bioreactor sampling connecting device and application thereof, and relates to the technical field of bioreactor culture, the one-way bioreactor sampling connecting device comprises a sealing cover, a sampling pipe penetrating through the sealing cover, a sampling pipeline communicated with the sealing cover, and a pressure adjusting pipeline; a sample in the bioreactor is guided to a sample collecting device through a sampling pipeline, the sampling pipeline can enable liquid in the bioreactor to flow into the sample collecting device through a peristaltic pump, the sampling pipeline is closed, and the liquid cannot flow back into the bioreactor; the pressure adjusting pipeline can be used for carrying out pressure maintaining air tightness detection on the sampling bottle, and also can be used for sampling through the liquid outlet end of the sampling pipe after compressed air is introduced for pressurization to enable the pressure in the bottle to be positive pressure. The technical problems that in the prior art, repeated negative pressure pumping sampling is needed, flow cutoff is likely to happen, bubbles are generated, the biochemical characteristics of samples are affected, backflow is likely to be caused, and operation is complex and pollution is caused are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of bioreactor culture, and in particular to a unidirectional bioreactor sampling connection device and its application. Background Art

[0002] Bioreactor culture is the mainstream method for the research and development and production of biopharmaceuticals. During the culture process, it is necessary to frequently sample and detect parameters such as cells, metabolism, biochemistry, and expression. The current sampling methods include disposable multi-connected sampling bags, sampling bottles, and needleless samplers with check valves and Luer connectors. However, all of them have problems. Sampling bags are costly and cumbersome to operate; simple sampling bottles are cumbersome to operate, and repeatedly drawing negative pressure during sampling has a great impact on the biochemical properties of the sample (such as pH, pCO2, PO2); the existing sampling bottles for 50L reactors have a low safety factor, require repeated vacuum pumping, the liquid is prone to backflow, the operation is cumbersome, and it is easy to cause contamination; the operation of needleless samplers has high requirements for personnel operation, and if not operated carefully, the sample is prone to backflow, with extremely high risks, and it is very easy to cause bacterial contamination during sampling, resulting in a great waste of time and money costs. Sampling with a check valve and Luer connector easily causes the liquid in the pipeline to break and bubbles to generate, the sampling volume is too small, and there is a disadvantage of low cell density counting.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a unidirectional bioreactor sampling connection device to solve the technical problems in the prior art that it is necessary to repeatedly draw negative pressure for sampling, which is prone to flow interruption and bubble generation, affects the biochemical properties of the sample, and is prone to backflow, resulting in cumbersome operation and contamination.

[0005] Another purpose of the present invention is to provide the application of the above unidirectional bioreactor sampling connection device in bioreactor sampling or the preparation of a bioreactor sampling device.

[0006] A third purpose of the present invention is to provide a unidirectional bioreactor sampling device.

[0007] A fourth purpose of the present invention is to provide a sampling method using the above unidirectional bioreactor sampling device.

[0008] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted:

[0009] In the first aspect, the present invention provides a unidirectional bioreactor sampling connection device, which includes a sealing cover, a sampling tube penetrating the sealing cover, a sampling pipeline communicated with the sealing cover, and a pressure regulating pipeline;

[0010] The sealing cap is used to connect with the sample collection device;

[0011] The sampling tube is used to collect samples in the sample collection device;

[0012] The sampling pipeline is used to divert the sample in the bioreactor to the sample collection device;

[0013] The pressure regulating pipeline is used to regulate the pressure in the sample collection device.

[0014] Furthermore, clip pieces are provided on the sampling tube, the sampling pipeline and the pressure regulating pipeline.

[0015] Furthermore, a check valve is provided at the outlet of the sampling tube.

[0016] Furthermore, the vertical distance between the liquid inlet of the sampling tube and the sealing cap is greater than the vertical distance between the sample liquid level and the sealing cap.

[0017] Furthermore, a pressure regulator is provided at the free end of the pressure regulating pipeline.

[0018] In a second aspect, the present invention provides an application of the above-mentioned one-way bioreactor sampling connection device in bioreactor sampling or in preparing a bioreactor sampling device.

[0019] In a third aspect, the present invention provides a one-way bioreactor sampling device, including a sample collection device and the above-mentioned one-way bioreactor sampling connection device connected to the sample collection device.

[0020] In a fourth aspect, the present invention provides a sampling method using the above-mentioned one-way bioreactor sampling device, including the following steps:

[0021] A. Divert the sample in the bioreactor to the sample collection device through the sampling pipeline;

[0022] B. Increase the pressure in the sample collection device through the pressure regulating pipeline, so that the sample flows out along the sampling tube to complete the sampling.

[0023] Furthermore, after step A, it also includes closing the clip piece provided on the sampling pipeline.

[0024] Furthermore, before step B, it also includes performing an airtightness test through the pressure regulating pipeline.

[0025] A unidirectional bioreactor sampling connection device provided by the present invention diverts the sample in the bioreactor to a sample collection device through a sampling pipeline. The sampling pipeline can flow the liquid in the bioreactor into the sample collection device through a peristaltic pump. When the sampling pipeline is closed, the liquid cannot flow back into the bioreactor; the pressure regulating pipeline can perform a pressure-holding airtightness test on the sampling bottle, or compressed air can be introduced for pressurization so that the pressure in the bottle becomes positive pressure, and then sampling can be carried out through the liquid outlet end of the sampling tube. This solves the technical problems in the prior art that repeated negative pressure sampling is required, which is prone to flow interruption and bubble generation, affecting the biochemical characteristics of the sample, and is also prone to reverse flow, resulting in complicated operation and contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 It is a schematic structural diagram of a unidirectional bioreactor sampling connection device provided in Embodiment 1 of the present invention;

[0028] Figure 2 It is a schematic structural diagram of a unidirectional bioreactor sampling device provided in Embodiment 2 of the present invention;

[0029] Figure 3 It is a schematic side view structural diagram of a unidirectional bioreactor sampling device provided in Embodiment 2 of the present invention;

[0030] Figure 4 For Figure 3 It is a cross-sectional structural diagram in the A-A direction in

[0031] Figure 5 It is a schematic structural diagram of a unidirectional bioreactor sampling device provided in Comparative Example 3 of the present invention;

[0032] Figure 6 It is a schematic structural diagram of a unidirectional bioreactor sampling device provided in Comparative Example 4 of the present invention;

[0033] Figure 7 It is a schematic structural diagram of a unidirectional bioreactor sampling device provided in Comparative Example 5 of the present invention;

[0034] Figure 8 It is a schematic diagram of the working connection state of a unidirectional bioreactor sampling device provided in Embodiment 3 of the present invention.

[0035] Icon: 10 - Bioreactor;

[0036] 11 - Sealing cap;

[0037] 21 - Sampling tube; 22 - Check valve;

[0038] 31 - Sampling pipeline;

[0039] 41 - Pressure regulating pipeline; 42 - Pressure regulator;

[0040] 51 - Sample collection device;

[0041] 61 - Clip;

[0042] 71 - Luer connector. Detailed implementation manner

[0043] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non - restrictive.

[0044] Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well - known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0045] On the one hand, the present invention provides a one - way bioreactor sampling connection device, including a sealing cap 11, a sampling tube 21 passing through the sealing cap 11, a sampling pipeline 31 communicating with the sealing cap 11, and a pressure regulating pipeline 41;

[0046] The sealing cap 11 is used to connect with the sample collection device 51;

[0047] The sampling tube 21 is used to collect samples in the sample collection device 51;

[0048] The sampling pipeline 31 is used to divert samples in the bioreactor 10 to the sample collection device 51;

[0049] The pressure regulating pipeline 41 is used to regulate the pressure in the sample collection device 51.

[0050] The sample in the bioreactor 10 is diverted to the sample collection device 51 through the sampling pipeline 31. The sampling pipeline 31 can allow the liquid in the bioreactor 10 to flow into the sample collection device 51 through a peristaltic pump. When the sampling pipeline 31 is closed, the liquid cannot flow back into the bioreactor 10; the pressure regulating pipeline 41 can perform a pressure-holding airtightness test on the sampling bottle, or compressed air can be introduced for pressurization so that the pressure in the bottle becomes positive pressure and then sampling is performed through the liquid outlet end of the sampling tube 21. This solves the technical problems in the prior art that repeated negative pressure sampling is required, which is prone to flow interruption and generation of bubbles, thus affecting the biochemical characteristics of the sample, and is also prone to backflow, resulting in complicated operation and contamination.

[0051] In some specific embodiments, in order to facilitate the control of the opening or closing of the sampling tube 21, the sampling pipeline 31, and the pressure regulating pipeline 41, clip pieces 61 are provided on the sampling tube 21, the sampling pipeline 31, and the pressure regulating pipeline 41.

[0052] In some specific embodiments, a check valve 22 is provided at the outlet of the sampling tube 21.

[0053] After the fluid in the sampling tube 21 flows to the sampling end, the check valve 22 realizes fluid check.

[0054] To ensure normal sampling. In some specific embodiments, the vertical distance between the liquid inlet of the sampling tube 21 and the sealing cover 11 is greater than the vertical distance between the sample liquid level and the sealing cover.

[0055] In some specific embodiments, a pressure regulator 42 is provided at the free end of the pressure regulating pipeline 41.

[0056] The pressure inside the sample collection device 51 communicated with the pressure regulating pipeline 41 is controlled by the pressure regulator 42 to keep it within a certain range. Compressed air can be introduced to increase the pressure, so that the liquid flows out smoothly along the sampling tube 21 and the liquid does not flow back. At the same time, the pressure regulator 42 can also prevent external pollutants from entering, thus avoiding contamination. When airtightness detection is required, it can also be used for airtightness detection.

[0057] Specifically, the pressure regulator 42 can be a breather.

[0058] Among them, the size of the one-way bioreactor sampling connection device can be set according to the size and model of the specific sample collection device 51 and the bioreactor used.

[0059] According to another aspect of the present invention, there is also provided the application of the above-mentioned one-way bioreactor sampling connection device in bioreactor sampling or in the preparation of a bioreactor sampling device.

[0060] Among them, the bioreactor includes various types such as stirred type, jet type or rocking type, etc.

[0061] According to another aspect of the present invention, there is also provided a unidirectional bioreactor sampling device, including a sample collection device 51 and the above-mentioned unidirectional bioreactor sampling connection device connected to the sample collection device 51.

[0062] According to another aspect of the present invention, there is also provided a sampling method using the above-mentioned unidirectional bioreactor sampling device, including the following steps:

[0063] A. Guide the sample in the bioreactor to the sample collection device through the sampling pipeline 31;

[0064] B. Increase the pressure in the sample collection device 51 through the pressure regulation pipeline 41, so that the sample flows out along the sampling tube 21 to complete the sampling.

[0065] During use: Use a peristaltic pump to fill the sampling pipeline 31 with liquid and flow it into the sample collection device 51, close the sampling pipeline 31 to prevent liquid backflow, introduce compressed air through the pressure regulation pipeline 41 to make the sample collection device 51 under positive pressure, and flow to the sampling end through the sampling tube 21 to complete the sampling. The sampling end can be connected to a 15 mL centrifuge tube to form a component, which not only makes the structure more compact and is convenient for assembly, but also can be connected to a section of silica gel tube for more flexible liquid outlet.

[0066] It should be noted that there is a sequence for the clips 61 in the pressure regulation pipeline 41 and the sampling tube 21 during use. When the pressure is sufficient, first close the clip 61 on the pressure regulation pipeline 41 to prevent the liquid in the sampling bottle from being blown back into the bioreactor. As the clip 61 is closed, after the sample is guided to the sample collection device 51 in step A, quickly close the clip on the sampling pipeline 31, then open the clip on the pressure regulation pipeline 41, increase the pressure in the sample collection device 51 through the pressure regulation pipeline 41 after increasing the pressure, so that the sample flows out along the sampling tube 21. After the liquid flows out, immediately close the clip 61 on the sampling tube 21, and then close the clip 61 on the pressure regulation pipeline 41 to complete the sampling.

[0067] In some specific embodiments, after step A, it further includes closing the clip 61 provided on the sampling pipeline 31.

[0068] In some specific embodiments, before step B, it further includes performing an airtightness inspection through the pressure regulation pipeline 41.

[0069] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0070] Embodiment 1

[0071] Combined with Figure 1 For illustration, a unidirectional bioreactor sampling connection device includes a sealing cover 11, a sampling tube 21 passing through the sealing cover 11, a sampling pipeline 31 communicating with the sealing cover 11, and a pressure regulating pipeline 41;

[0072] The sealing cover 11 is used to connect with a sample collection device 51; the sampling tube 21 is used to collect samples in the sample collection device 51; the sampling pipeline 31 is used to divert the samples in the bioreactor 10 to the sample collection device 51; the pressure regulating pipeline 41 is used to adjust the pressure in the sample collection device 51.

[0073] Clamping pieces 61 are provided on all of the sampling tube 21, the sampling pipeline 31, and the pressure regulating pipeline 41. A check valve 22 is provided at the outlet of the sampling tube 21. The vertical distance between the liquid inlet of the sampling tube 21 and the sealing cover 11 is greater than the vertical distance between the sample liquid level and the sealing cover.

[0074] A pressure regulator 42 is provided at the free end of the pressure regulating pipeline 41. In this embodiment, the pressure regulator 42 is a respirator.

[0075] Comparative Example 1

[0076] Different from Embodiment 1, a check valve 22 is not provided on the sampling tube 21.

[0077] Comparative Example 2

[0078] Different from Embodiment 1, the pressure regulating pipeline 41 is not provided. A Luer connector 71 is connected to the liquid outlet end of the check valve 22, and sampling is performed with a needleless sampler.

[0079] Embodiment 2

[0080] Combined with Figures 2 to 4 For illustration, a unidirectional bioreactor sampling device includes a sample collection device 51 and the unidirectional bioreactor sampling connection device provided in Embodiment 1 connected to the sample collection device 51.

[0081] Comparative Examples 3-4

[0082] Combined with Figure 5 And Figure 6For illustration, different from Example 2, the unidirectional bioreactor sampling connection device provided in Comparative Example 1 and Comparative Example 2 was respectively used to replace the unidirectional bioreactor sampling connection device provided in Example 1.

[0083] Comparative Example 5

[0084] Combined with Figure 7 For illustration, the unidirectional bioreactor sampling device includes a sampling tube 21, a check valve 22 provided at the free end of the sampling tube, a Luer connector 71 connected to the liquid outlet of the check valve, and sampling is performed using a needleless sampler.

[0085] Example 3

[0086] Combined with Figure 8 For illustration, the sampling method of the unidirectional bioreactor sampling device provided in Example 2 includes the following steps:

[0087] 1. Under sufficient pressure, first close the clip 61 on the pressure regulating pipeline 41 to prevent the liquid in the sampling bottle from being blown back into the bioreactor. As this clip 61 is closed, use a peristaltic pump to fill the sampling pipeline 31 with liquid and flow it into the sample collection device 51, and quickly close the clip 61 provided on the sampling pipeline 31 to prevent liquid backflow;

[0088] 2. Open the clip 61 of the pressure regulating pipeline 41 to perform an airtightness inspection through the pressure regulating pipeline 41.

[0089] 3. After increasing the pressure, open the clip 61 on the sampling tube 21, and introduce compressed air through the pressure regulating pipeline 41 to make the sample collection device 51 under positive pressure. The sample is taken through the sampling tube 21 flowing through the check valve to the sampling end. Immediately close the clip 61 on the sampling tube 21, and then close the clip 61 on the pressure regulating pipeline 41.

[0090] Comparative Example 6

[0091] Different from Example 2, the unidirectional bioreactor sampling device provided in Comparative Example 3 was used.

[0092] Comparative Example 7

[0093] Using the unidirectional bioreactor sampling device provided in Comparative Example 4, the sampling method includes the following steps:

[0094] 1. Use a peristaltic pump to fill the sampling pipeline 31 with liquid and flow it into the sample collection device 51, and close the clip 61 provided on the sampling pipeline 31 to prevent liquid backflow;

[0095] 2. Open the clip 61 on the sampling pipeline 31 to keep the sample collection device 51 in communication with the outside world. Connect a needleless sampler to the Luer connector and draw a sample through the sampling tube 21.

[0096] Comparative Example 8

[0097] Using the unidirectional bioreactor sampling device provided in Comparative Example 5, the sampling method includes connecting a needleless sampler to a Luer connector and extracting a sample through a sampling tube 21.

[0098] Application Example

[0099] The model of the unidirectional bioreactor used in this application example is the JYSS-CUR5-50 L jet reactor, and the sample is ST-BA cells. Samples in the unidirectional bioreactor were sampled according to the methods of Example 3 and Comparative Examples 6-8 respectively, 10 times each, and the number of occurrences of flow interruption, bubbles and backflow was recorded. The specific results are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] Among them, less than 10 bubbles are considered as a small amount of bubbles, and more than 10 bubbles are considered as a relatively large amount of bubbles.

[0104] As can be seen from Table 1, there was no flow interruption or backflow in Example 3, and only a small amount of bubbles occurred; in Comparative Example 6, due to the lack of a check valve 22, flow interruption and backflow occurred, and the number of flow interruptions was the largest. The flow interruption and backflow led to the generation of bubbles; in Comparative Example 7, due to passive sampling, when the needleless sampler completed suction in sequence, the liquid reflux led to flow interruption and backflow, generating a large amount of bubbles. In Comparative Example 8, sampling was directly taken from the bioreactor 10. During the process, due to the insufficient suction force of the needleless sampler and the inability to continuously complete a sufficient amount of sampling, flow interruption and a relatively large number of bubbles occurred. It can be seen from this that Example 3 has well solved the problems of flow interruption, bubbles and backflow that occur in sampling in the prior art.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A unidirectional bioreactor sampling connection device, characterized in that It includes a sealing cover (11), a sampling tube (21) passing through the sealing cover (11), a sampling pipeline (31) communicating with the sealing cover (11), and a pressure regulating pipeline (41); The sealing cover (11) is used for connecting with a sample collection device (51); The sampling tube (21) is used for collecting samples in the sample collection device (51); The sampling pipeline (31) is used for diverting samples in the bioreactor (10) to the sample collection device (51); The pressure regulating pipeline (41) is used for regulating the pressure in the sample collection device (51).

2. The unidirectional bioreactor sampling connection device according to claim 1, characterized in that, Clip pieces (61) are provided on the sampling tube (21), the sampling pipeline (31), and the pressure regulating pipeline (41).

3. The unidirectional bioreactor sampling connection device according to claim 2, characterized in that, A check valve (22) is provided at the outlet of the sampling tube (21).

4. The unidirectional bioreactor sampling connection device according to claim 3, characterized in that, The vertical distance between the liquid inlet of the sampling tube (21) and the sealing cover (11) is greater than the vertical distance between the sample liquid level and the sealing cover.

5. The one-way bioreactor sampling connection device according to claim 4, characterized in that, A pressure regulator (42) is provided at the free end of the pressure regulating pipeline (41).

6. Application of the one-way bioreactor sampling connection device according to any one of claims 1 to 5 in bioreactor sampling or in preparing a bioreactor sampling device.

7. A unidirectional bioreactor sampling device, characterized in that, It includes a sample collection device (51) and the one-way bioreactor sampling connection device according to any one of claims 1 to 5 connected to the sample collection device (51).

8. A sampling method using the sampling device of the unidirectional bioreactor according to claim 7, characterized in that, It includes the following steps: A. Divert the samples in the bioreactor to the sample collection device through the sampling pipeline (31); B. Increase the pressure in the sample collection device (51) through the pressure regulating pipeline (41) to make the samples flow out along the sampling tube (21) to complete the sampling.

9. The sampling method according to claim 8, characterized in that, After step A, it further includes closing the clip piece (61) provided on the sampling pipeline (31).

10. The sampling method according to claim 8, wherein Before step B, it further includes performing an airtightness test through the pressure regulating pipeline (41).

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