Multifunctional online sampler for microorganism and cell culture

Abstract

The utility model relates to a multifunctional on-line sampler system suitable for a microorganism and cell culture process. The multifunctional on-line sampler system comprises a sampling probe rod, an automatic sampler and an on-line analyzer, through precise switching of a multi-channel pipeline and a pinch valve, in cooperation with a peristaltic pump, an air filter, a cleaning liquid detector and a sample detector, sterile and quantitative online sampling of fermentation liquid or culture liquid is achieved, and sampling, emptying, backflow and cleaning can be automatically completed according to preset parameters. The system can be combined with a biochemical analyzer for use, meets the requirements of high-fermentation-frequency microbial fermentation and low-frequency animal cell culture sampling, effectively reduces manual intervention and pollution risks, improves the sampling precision and efficiency, and provides powerful support for real-time monitoring and optimization of a biological technological process.

Description

TECHNICAL FIELD

[0001] The utility model relates to the field of bioengineering technology especially relates to a multifunctional on -line sampler for microorganism and cell culture. BACKGROUND

[0002] In the process of microbial fermentation and animal cell culture, the culture solution needs to be sampled and analyzed to obtain key data such as glucose concentration, glutamine concentration, etc. to guide the culture process. The traditional sampling method has many problems, such as inaccurate sampling, easy to block, cannot meet the sampling needs of different scales and different frequencies, and has deficiencies in aseptic, sample backflow, etc. Therefore, an efficient, accurate and adaptive online sampler for various culture conditions is needed. SUMMARY

[0003] The utility model discloses a multifunctional on -line sampler for microorganism and cell culture to solve the shortcomings in prior art.

[0004] In order to achieve the above-mentioned purpose, the utility model adopts the following technical scheme:

[0005] A multifunctional on -line sampler for microorganism and cell culture, comprising an automatic sampler and an online analyzer, characterized in that:

[0006] The automatic sampler is connected with a sampling probe rod that can be inserted into the reactor through a first hose, a second hose and a third hose, a ball valve switch is arranged on the sampling probe rod, and the sampling channel of the fermentation broth or the culture solution is opened and closed through the ball valve switch;

[0007] The automatic sampler has a multi-channel pipeline and a four-way joint inside, and a pinch valve V1, a pinch valve V2, a pinch valve V3 and a pinch valve V4 are arranged on the corresponding channels of the four-way joint respectively;

[0008] One end of the automatic sampler is connected with the sampling probe rod through a sample input end hose, and the other end is connected to a peristaltic pump matched with the online analyzer through an output end hose;

[0009] The automatic sampler is also connected with a cleaning liquid hose and a cleaning liquid detector, and the cleaning liquid enters the pipeline through the pinch valve V4 to clean the sampling pipeline;

[0010] The automatic sampler is connected with an air hose and a one-way valve, and a first air filter and a second air filter are arranged on the air hose side to realize the aseptic guarantee and gas conduction of the air channel in the pipeline, which is used for sample backflow, emptying and drying pipeline;

[0011] A sample detector is arranged inside the automatic sampler to detect the sample liquid flow condition;

[0012] The shell of the automatic sampler is a plastic shell, provided with an indicator lamp and a cable joint, and can communicate with an online analyzer through a cable and a communication interface board to realize automatic control of the sampling process.

[0013] The ball valve switch output end is provided with a first hose joint.

[0014] The online analyzer is internally provided with a reaction pool and a sampling needle module, and the sampling needle module is used for receiving the sample delivered by the peristaltic pump and guiding the sample into the reaction pool for analysis.

[0015] Preferably, the sampling probe is connected to the reactor through a PG. threaded joint, can be inserted or removed in situ during fermentation and cell culture, and can adapt to in-situ sterilization operation.

[0016] Preferably, the first hose joint is detachably designed, which is used to quickly separate the first hose, the second hose and the third hose from the automatic sampler during off-site sterilization and cleaning.

[0017] Further, the cleaning liquid is stored in a sealed cleaning liquid bottle with a first air filter, and the cleaning liquid detector is used to detect the cleaning liquid passing condition to prevent insufficient cleaning liquid and abnormal pipeline blockage.

[0018] Further, the online analyzer is connected to the automatic sampler through an RS-serial port, and the peristaltic pump is controlled by the command issued by the online analyzer to realize automatic operation of sampling from the reactor, cleaning liquid extraction, gas emptying and sample backflow.

[0019] Preferably, the pinch valve V1, the pinch valve V2, the pinch valve V3, the pinch valve V4 and the related hoses are made of high-temperature-resistant and high-pressure-sterilizable materials, which meet the sterile requirements in fermentation and cell culture and can maintain sealing and structural stability during sterilization.

[0020] Preferably, the sample detector is connected with a liquid detector and cooperates with the cleaning liquid detector to determine whether there is liquid passing in the sampling pipeline and to confirm the cleaning completion degree, so as to control the sampling frequency, sample volume and cleaning volume under the control of the online analyzer, and the second hose joint is arranged between the peristaltic pump and the liquid detector to quickly detach the pipeline during off-site sterilization and cleaning.

[0021] Preferably, displacement-resistant pipe clamps are arranged between the first hose, the second hose and the third hose to keep the position of the hoses stable during pipeline installation and operation, prevent the hoses from slipping and twisting to cause sampling errors.

[0022] The indicator light is preferably used for displaying the working state of the automatic sampler, including power-on state, sampling in progress, cleaning in progress and various state indications of fault alarm.

[0023] The utility model discloses the beneficial effect that:

[0024] 1. Improve sampling accuracy and stability: accurate sampling can be realized, the dead volume is small, consistent and accurate sampling, flushing time is reduced, the reliability of sampling data is ensured, and the key concentration data of perfusion culture of mammalian cells can be obtained, and the perfusion rate and timing, such as the best timing of induction and harvesting, can be adjusted.

[0025] 2. Adapt to various culture conditions: it can be matched with reactors from small laboratory scale to large production scale, and is suitable for sampling requirements of different frequencies such as microbial fermentation and animal cell culture. Whether it is microbial fermentation of multiple sampling in 1 hour or animal cell culture of sampling every day, it can show excellent performance.

[0026] 3. Ensure aseptic operation and reduce the risk of blockage: the sampling probe, the connecting hose, the air filter and the cleaning liquid bottle can be disassembled for high-pressure sterilization or in-situ sterilization, the risk of sampling blockage is reduced, the design of the 0.22um filter membrane is cancelled, the culture solution in the online sampling process can flow back to the reactor (no tank pressure), and the sampling for online analysis can be used in high-density cell or high-density microbial culture process.

[0027] 4. Simple operation and maintenance: simple operation and maintenance, convenient replacement, automatic sampling connecting hose can be cleaned and sterilized, pump pipe is replaced regularly, and online sampling pipeline design and manufacturing are durable, can resist temperature, pressure, viscosity and chemical composition under typical fermentation environmental conditions.

[0028] The above description is only a summary of the technical scheme of the utility model, in order to more clearly understand the technical means of the utility model, which can be implemented according to the content of the specification, and in order to make the above and other purposes, characteristics and advantages of the utility model more obvious and easy to understand, the following preferred embodiments are described in detail, and the accompanying drawings are described as follows. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The utility model proposes a kind of equipment overall structure and each module connection schematic view of multifunctional online sampler for microorganism and cell culture;

[0030] Figure 2 The utility model proposes a kind of automatic sampler internal detailed structure schematic view of multifunctional online sampler for microorganism and cell culture.

[0031] In the diagram: 1. First hose connector; 2. Ball valve switch; 3. Sampling probe; 4. Reactor; 5. First hose; 6. First air filter; 7. Cleaning fluid; 8. Cable; 9. Communication interface board; 10. Online analyzer; 11. Peristaltic pump; 12. Second hose connector; 13. Liquid detector; 14. Reaction tank; 15. Sampling needle module; 16. Second hose; 17. Automatic sampler; 18. Third hose; 1701. Pinch valve V1; 1702. Sample input. 1703. End hose; 1704. Plastic housing; 1705. Indicator light; 1706. Cable connector; 1707. Cleaning fluid hose; 1708. Cleaning fluid detector; 1709. Pinch valve V4; 1710. Four-way connector; 1711. Pinch valve V3; 1712. Air hose; 1713. Check valve; 1714. Second air filter; 1715. Pinch valve V2; 1716. Sample detector; 1717. Anti-displacement clamp; 1718. Output hose. Detailed Implementation

[0032] The technical solutions of the present utility model will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present utility model. Obviously, the described embodiments are only some embodiments of the present utility model, and not all embodiments.

[0033] Please also refer to Figure 1 and Figure 2 , Figure 1 This diagram illustrates the overall connection of the online sampling system of this invention. Figure 2 A schematic diagram of the internal structure of the automatic sampler 17 is shown.

[0034] In this invention, the multifunctional online sampler mainly includes an automatic sampler 17 and an online analyzer 10 that works with it. It can be applied to bioprocesses such as microbial fermentation and animal cell culture to achieve online quantitative sampling and analysis of samples.

[0035] Structure and connection relationships:

[0036] like Figure 1 As shown, the automatic sampler 17 is connected to the sampling probe 3, which can be inserted into the reactor 4, via a first hose 5, a second hose 16, and a third hose 18. The sampling probe 3 is equipped with a ball valve switch 2 to control the opening and closing of the sampling channel for the fermentation broth or culture medium. The sampling probe 3 is installed at the side or top opening of the reactor 4 via a PG13.5 threaded connector, allowing for in-situ insertion or removal during fermentation or cell culture, while simultaneously meeting in-situ sterilization requirements.

[0037] A first hose joint 1 is provided at the output end of the ball valve switch 2, which is detachable for easy separation of the first hose 5, the second hose 16 and the third hose 18 from the automatic sampler 17 during sterilization and cleaning maintenance.

[0038] The online analyzer 10 is connected with the automatic sampler 17 through a cable 8 and a communication interface board 9 in RS-485 serial communication connection, and the analyzer 10 sends a command to control the opening and closing of the valves in the peristaltic pump 11 and the automatic sampler 17 according to a preset program, so as to realize online sampling, cleaning and maintenance.

[0039] The internal structure and function of the automatic sampler:

[0040] As shown in Figure 2 The automatic sampler 17 has multiple channels inside, which are collected through a four-way joint 1709, and pinch valves V11701, V21714, V31710 and V41708 are respectively arranged on the channels, so as to realize flexible regulation and control of the sample liquid, cleaning liquid and air channel through precise switching of these valves. The shell of the automatic sampler 17 is made of plastic shell 1703, which is light and easy to clean. The shell is provided with an indicator lamp 1704 for displaying the working state (power on, sampling, cleaning, fault alarm), and a cable joint 1705 for connecting the cable 8.

[0041] One end of the automatic sampler 17 is connected with the sampling probe 3 through a sample input hose 1702 to extract the sample from the reactor 4, and the other end is connected to the peristaltic pump 11 through an output hose 1717. The other side of the peristaltic pump 11 is connected to the third hose 18, and finally connected to the reaction cell 14 and the sampling needle module 15 of the online analyzer 10, so as to realize biochemical analysis of the sample entering the analyzer.

[0042] The automatic sampler 17 is also connected with a cleaning liquid hose 1706 and a cleaning liquid detector 1707, and the cleaning liquid 7 is provided by a sealed cleaning liquid bottle with a first air filter 6. When cleaning the pipeline is needed, the cleaning liquid can be pumped into the pipeline through the opening of the pinch valve V41708 to realize efficient online cleaning. The cleaning liquid detector 1707 is used to monitor whether the cleaning liquid passes normally to ensure that the cleaning process is stable and reliable.

[0043] The automatic sampler 17 is also provided with an air hose 1711 and a one-way valve 1712, and a second air filter 1713 is arranged at the side of the air hose 1711. These air passage elements are used to empty the sample sampling pipeline by drawing air after sample sampling, or to backflow the residual sample under the condition of a pressureless reactor. In addition, the pipeline can also be dried after cleaning by the introduction of air. During off-site or in-situ sterilization, the entire pipeline can be independently subjected to high-pressure sterilization by quickly separating the pipeline at the second hose joint 12 and adding a manual pipe clamp.

[0044] The automatic sampler 17 is internally provided with a sample detector 1715 and a liquid detector 13, which work in cooperation with the cleaning liquid detector 1707 to determine whether liquid passes through the sampling pipeline, whether the sample has reached the analyzer, and whether the cleaning process is completed. The feedback signals of these detectors can be processed by the online analyzer 10 to automatically adjust the operation of the peristaltic pump 11 and the state of each pinch valve, so as to realize closed-loop control and automatic operation. The hose joint is also provided with a displacement prevention pipe clamp 1716 to keep the hose position stable and avoid deviation of the flow path or leakage of liquid caused by displacement of the hose during operation or sterilization.

[0045] Operation process:

[0046] Sampling process:

[0047] First, the set parameters (sampling frequency, sampling volume, cleaning volume, flow path length) of fermentation or cell culture are pre-programmed in the online analyzer 10.

[0048] When the set sampling period is reached, the analyzer 10 sends a command to the automatic sampler 17 through RS-485 to control the opening and closing of the pinch valve V11701, the pinch valve V31710, etc. and the rotation of the peristaltic pump 11, so as to draw the sample from the reactor 4. The sample passes through the sampling probe 3, the ball valve switch 2, the first hose 5, the second hose 16, the third hose 18, and the internal passage of the automatic sampler 17, and finally enters the sampling needle module 15 of the online analyzer 10 and is introduced into the reaction cell 14 for analysis. The sample detector 1715 and the liquid detector 13 monitor the passing of the sample in real time.

[0049] Cleaning and emptying:

[0050] After sampling is completed, the pinch valve V41708 can be opened according to the set program to introduce the cleaning liquid 7 into the pipeline through the peristaltic pump 11 to clean the entire sampling flow path. The cleaning liquid detector 1707 is used to ensure that the cleaning liquid passes through smoothly. After cleaning, the corresponding valves can be opened to introduce air into the pipeline to blow out the residual cleaning liquid, so as to ensure the sterile and dry state of the pipeline.

[0051] Backflow and emptying:

[0052] For the case of no internal pressure in the reactor, the corresponding air passage valve can be opened after sampling is completed to allow the sample to flow back into the reactor 4, thereby reducing sample waste. If the reactor has pressure, the relevant valve can be closed to maintain the passage seal, and the sample does not flow back.

[0053] Off-site and in-situ sterilization operation:

[0054] When sterilization is required, the pipeline can be quickly disassembled at the first hose joint 1 and the second hose joint 12, and a manual pipe clamp is added for sealing. The entire pipeline, together with the sampling probe 3, the cleaning liquid bottle, and the peristaltic pump pipeline, is sterilized off-site, or the sampling probe 3 is kept in place and the reactor 4 is sterilized separately.

[0055] All valves, hoses, and related components are made of high-temperature-resistant, high-pressure steam sterilizable materials, which can maintain sealing and structural stability during sterilization.

[0056] Example 1:

[0057] Microbial fermentation sampling example

[0058] In the application of monitoring and controlling the microbial fermentation process, the multifunctional online sampler of the present application can be used in cooperation with the AP-100 online biochemical analyzer. The specific operation steps are as follows:

[0059] Pre-set parameters: According to the production process and experimental requirements, set the sampling interval, single sampling volume, cleaning liquid volume, sampling pipeline length, and liquid detection method, etc. in the operation interface of the AP-100 online biochemical analyzer. If high-frequency sampling is required (for example, multiple times per hour), the sampling period can be shortened accordingly, and the pipeline cleaning amount and the peristaltic pump speed can be adjusted appropriately.

[0060] Sampling probe installation and sterilization: The sampling probe is inserted into the side port or top port of the microbial fermentation reactor through the PG13.5 threaded joint, and is connected to the automatic sampler of the present application. Before the fermentation is started, the reactor interior and the related pipeline are sterilized in-situ or off-site to ensure the initial sterile state of the system.

[0061] Online automatic sampling: During the fermentation operation, when the sampling time set in the analyzer is reached, the analyzer issues an instruction to the automatic sampler through the RS-485 interface. The pinch valve and the peristaltic pump in the automatic sampler act according to the pre-set program in sequence:

[0062] First, the residual liquid or air in the pipeline is emptied by opening the corresponding pinch valve and operating the peristaltic pump.

[0063] Then, the sampling valve is opened, and the sample is drawn from the fermentation broth according to the set volume and sent to the reaction cell of the analyzer.

[0064] The whole sampling process does not need manual intervention, can be carried out in a timed, quantitative and fast manner.

[0065] Emptying and cleaning: after sampling is completed, the automatic sampler opens the relevant pinch valve according to the preset program, introduces sterile air and cleaning liquid into the pipeline, discharges the residual sample and flushes and dries the pipeline. The sample detector and the cleaning liquid detector monitor the liquid flow in the pipeline in real time, ensuring thorough cleaning.

[0066] Continuous circulation: the above-mentioned sampling and cleaning steps can be repeated periodically during the fermentation process, so that the analyzer can obtain the biochemical parameters of the fermentation broth in real time, and assist the operator or the control system to optimize and control the process.

[0067] Through the embodiment, the secondary pollution risk and labor intensity caused by manual sampling can be greatly reduced, and online rapid feedback and precise regulation under high-density fermentation conditions can be realized.

[0068] Example two:

[0069] Animal cell culture sampling embodiment

[0070] In biopharmaceutical, vaccine production or cell biology research, the key parameters of the cell culture process (such as metabolic products, nutrient consumption rate, cell density, etc.) need to be monitored by regular online sampling. The utility model is also suitable for such scenes.

[0071] Parameter setting: on the AP-100 online biochemical analyzer, set the sampling frequency (such as daily sampling), single sampling volume, cleaning volume, pipeline length and liquid detection mode according to the requirements of cell culture. Since the cell culture process usually does not need high-frequency continuous sampling, the sampling interval can be appropriately extended and the cleaning frequency can be reduced.

[0072] Sampling probe rod installation and sterility assurance: insert the sampling probe rod into the side port or top port of the cell culture reactor, and set a ball valve switch on the probe rod. Use in-situ sterilization or off-site sterilization to ensure that the sampling probe rod, pipeline, cleaning liquid bottle and automatic sampler maintain a sterile state throughout the process.

[0073] Online sampling operation: when the set daily sampling time point is reached, the analyzer sends a sampling instruction to the automatic sampler. The automatic sampler starts the peristaltic pump, opens the corresponding pinch valve, and draws out the cell culture liquid according to the predetermined volume, and transports it to the online analyzer for detection.

[0074] Sample backflow and emptying: if the cell culture reactor has no internal pressure, the gas channel can be opened according to the process requirements to make a small amount of culture liquid remaining in the sample input end backflow to the reactor, thereby minimizing the waste of culture liquid. If there is pressure inside the reactor, the backflow step is skipped to avoid affecting the internal environment of the reactor.

[0075] Pipeline cleaning: after sampling is completed, the automatic sampler introduces an appropriate amount of cleaning liquid into the pipeline by opening the pinch valve V4, flushes and empties the sampling passage, and restores the sterile and clean state. The sample detector and the cleaning liquid detector monitor the entire process to ensure that the cleaning is thorough and in place, providing a sterile guarantee for the next sampling.

[0076] The embodiment realizes the simplification of operation steps, the reduction of manual intervention and the risk of pollution, the improvement of sampling accuracy and data reliability in the animal cell culture process. By integrating the online detection result with the culture control system, the cell culture conditions can be adjusted and optimized in time, so that the cell growth is stabilized, the culture yield is improved and the product quality is improved.

[0077] The above is only the preferred specific implementation of the present application, but the protection scope of the present application is not limited to this, any skilled person in the art can make equivalent replacement or change according to the technical scheme and the application concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. A multifunctional on-line sampler for microbial and cell culture, comprising an automatic sampler (17) and an on-line analyzer (10), characterized in that: the automatic sampler (17) is connected with a sampling probe (3) which can be inserted into the inside of a reactor (4) through a first hose (5), a second hose (16) and a third hose (18), and the sampling probe (3) is provided with a ball valve switch (2) for controlling the opening and closing of the sampling channel of the fermentation broth or culture broth; the automatic sampler (17) has a multi-channel pipeline and a four-way joint (1709) inside, and the four-way joint (1709) is respectively provided with pinch valves V1 (1701), V2 (1714), V3 (1710) and V4 (1708) on the corresponding channels; one end of the automatic sampler (17) is connected with the sampling probe (3) through a sample input end hose (1702), and the other end is connected to a peristaltic pump (11) matched with the on-line analyzer (10) through an output end hose (1717); the automatic sampler (17) is also connected with a cleaning liquid hose (1706) and a cleaning liquid detector (1707), and the cleaning liquid (7) enters the pipeline under the control of the pinch valve V4 (1708) to clean the sampling pipeline; the automatic sampler (17) is connected with an air hose (1711) and a one-way valve (1712), and the air hose (1711) is provided with a first air filter (6) and a second air filter (1713) to ensure the sterility of the air channel in the pipeline and the gas conduction, which is used for sample backflow, emptying and drying the pipeline; the automatic sampler (17) is provided with a sample detector (1715) inside for detecting the flow of the sample liquid; the shell of the automatic sampler (17) is a plastic shell (1703) provided with an indicator light (1704) and a cable connector (1705), which can communicate with the on-line analyzer (10) through a cable (8) and a communication interface board (9) to realize the automatic control of the sampling process; the output end of the ball valve switch (2) is provided with a first hose connector (1); the on-line analyzer (10) is provided with a reaction cell (14) and a sampling needle module (15) inside, and the sampling needle module (15) is used for receiving the sample delivered by the peristaltic pump (11) and introducing it into the reaction cell (14) for analysis. The sampling probe (3) is connected with the reactor (4) through a PG13.5 threaded connector, which can be inserted or removed in situ during fermentation and cell culture, and can adapt to in-situ sterilization operation. The first hose connector (1) is designed to be detachable, which is used to quickly separate the first hose (5), the second hose (16) and the third hose (18) from the automatic sampler (17) during off-site sterilization and cleaning. The cleaning liquid (7) is stored in a sealed cleaning liquid bottle with a first air filter (6), and the cleaning liquid detector (1707) is used to detect the passing of the cleaning liquid to prevent insufficient cleaning liquid and abnormal blockage of the pipeline. ​ ​ ​ ​ ​ ​ 2. The multi-functional on-line sampler of claim 1, wherein: ​ 3. The multi-functional on-line sampler according to claim 1 or 2, characterized in that: ​ 4. The multi-functional on-line sampler of claim 1, wherein: ​ 5. The multi-functional on-line sampler of claim 1, wherein: The online analyzer (10) is connected with the automatic sampler (17) through RS-485 serial port, and the peristaltic pump (11) is controlled by the instruction from the online analyzer (10), so that the automatic operation of sample extraction, cleaning liquid extraction, gas exhaust and sample backflow from the reactor (4) is realized.

6. The multi-functional on-line sampler of claim 1, wherein: The pinch valve V1 (1701), the pinch valve V2 (1714), the pinch valve V3 (1710), the pinch valve V4 (1708) and the related hoses are made of high-temperature-resistant and high-pressure-sterilizable materials, meet the sterile requirements in fermentation and cell culture, and can keep sealing and structural stability during sterilization.

7. The multi-functional on-line sampler of claim 1, wherein: The sample detector (1715) is connected with the liquid detector (13) and cooperates with the cleaning liquid detector (1707), is used for judging whether liquid passes in the sampling pipeline and confirming the cleaning degree, so that the parameters of sampling frequency, sample volume and cleaning volume are controlled under the control of the online analyzer (10), and the second hose joint (12) is arranged between the peristaltic pump (11) and the liquid detector (13) and is used for quickly disassembling the pipeline during off-site sterilization and cleaning.

8. The multi-functional on-line sampler of claim 1, wherein: The first hose (5), the second hose (16) and the third hose (18) are provided with the anti-displacement hose clamp (1716), so that the hose position is kept stable during pipeline installation and operation, and sampling error caused by hose slipping and twisting is prevented.

9. The multi-functional on-line sampler of claim 1, wherein: The indicator light (1704) is used for displaying the working state of the automatic sampler (17), including power-on state, sampling in progress, cleaning in progress and fault alarm.

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