Protein purification equipment and production line

Abstract

The invention discloses protein purification equipment and a production line. The equipment comprises a power assembly, a chromatographic column, an input module, a first valve group, a monitoring module and a control module, the input module comprises a sample loading unit for inputting a sample stock solution and a buffer solution input unit for inputting a buffer solution; the power assembly is used for providing power; the chromatographic column is used as a functional reaction zone; the first valve group is respectively connected with the power assembly, the input module and the chromatographic column through a plurality of liquid paths and is used for controlling the on-off and the flow direction of each liquid path; the monitoring module monitors a liquid path and obtains liquid information; the control module receives liquid information of the monitoring module and controls the first valve group. Through the cooperative arrangement of the single power assembly and the first valve group, the structure utilization efficiency of the equipment is improved, redundant arrangement is reduced, the cost is reduced, the arrangement of the feeding module, the chromatographic column, the detection module and the control module is combined, an efficient and accurate purification result is ensured, and the problems of complex structure, difficult maintenance and the like are solved.

Description

Technical Field

[0001] This application relates to the field of life science technology, and in particular to a protein purification device and production line. Background Technology

[0002] Currently, there are many methods for purifying recombinant proteins produced by genetic engineering technology. The principle behind these purification methods utilizes differences in their physical and chemical properties. Physical properties include molecular size, shape, and solubility, while chemical properties include isoelectric point, hydrophobicity, and affinity for other molecules. Protein purification is broadly divided into two stages: coarse separation and fine purification. Coarse separation primarily separates the target protein from other cellular components such as RNA and DNA, using methods like ammonium sulfate precipitation. Fine purification distinguishes the target protein from other proteins of similar size and physicochemical properties.

[0003] Purification methods such as affinity chromatography use biomolecules as ligands and rely on the interaction between proteins and their ligands for separation. For example, antigens and antibodies, enzymes and enzyme inhibitors (or ligands), enzyme proteins and coenzymes, hormones and receptors, etc., have a specific affinity. Under certain conditions, they can bind tightly into complexes. When a protein mixture passes through an affinity chromatography column, only the target protein can specifically bind to the ligand, while other unbound proteins are eluted first. The target protein can be eluted by solvents containing a high concentration of free ligands, and this method is often used in metal chelate affinity chromatography and immunoaffinity chromatography.

[0004] Furthermore, the entire process of affinity chromatography involves a series of steps, thus placing extremely high demands on the relevant equipment, resulting in a highly complex and bulky overall structure. Existing chromatographic purification equipment on the market improves efficiency and reduces sample confusion or cross-contamination by incorporating driving components such as constant flow pumps and multiple parallel chromatographic pathways, or by using multi-channel valves to switch the liquid path upstream and downstream of the chromatographic column.

[0005] However, parallel chromatography pathways typically share only a few structures, such as the collection structure, resulting in low structural utilization efficiency and high costs. Multi-channel valve-type chromatography purification equipment employs two main methods: one uses rotary valves to switch multiple pathways upstream of the chromatography column, but these valve cores are complex, difficult to maintain, and the rotational switching method still carries the risk of material and pipeline contamination; the other uses different solenoid valves to switch multiple pathways upstream and downstream of the chromatography column, which is relatively complex to operate, install, and maintain. Furthermore, the difficulty in controlling different valves makes it challenging to establish a chromatography circulation loop. Summary of the Invention

[0006] This application proposes a protein purification device to effectively solve the technical problems in related technologies, such as low utilization efficiency and high cost of parallel chromatography pathway chromatography purification devices, complex valve core structure of multi-channel valve body type chromatography purification devices, difficult maintenance and risk of contamination, or difficulty in setting up chromatography circulation loops due to complex installation and maintenance.

[0007] This application also proposes a production line including the aforementioned protein purification equipment.

[0008] The first aspect of this application provides a protein purification device, including: a power component, a chromatography column, an input module, a first valve group, a monitoring module, and a control module;

[0009] The input module includes a sample loading unit and a buffer input unit. The sample loading unit is used to input the original sample solution, and the buffer input unit is used to input the buffer solution.

[0010] The power assembly is used to provide power for the flow of the liquid;

[0011] The chromatography column is used as a functional reaction zone for protein purification;

[0012] The first valve group is connected to the power component, the input module and the chromatography column through multiple liquid passages respectively. The first valve group is used to control the opening and closing and the flow direction of each liquid passage.

[0013] The monitoring module is used to monitor the liquid path and obtain liquid information;

[0014] The control module is used to receive liquid information from the monitoring module and issue control commands. The first valve group responds to the control commands so that the chromatography column can perform preset process steps of affinity chromatography respectively.

[0015] Furthermore, the protein purification device also includes a concentration detection module, which is connected to the chromatography column and used to measure the protein concentration of the effluent liquid. The control module is used to receive the measurement information from the concentration detection module and issue closed-loop control commands.

[0016] Furthermore, the concentration detection module includes an LED assembly, a first lens, a cuvette, a filter, a second lens, and a receiver assembly arranged in sequence. The cuvette is used to hold the measurement sample, the LED assembly is used to emit ultraviolet light of a preset wavelength, the receiver assembly is used to receive the ultraviolet light, and the control module calculates the protein concentration of the measurement sample based on the absorbance of the measurement sample at the preset wavelength.

[0017] Furthermore, the protein purification device also includes a waste liquid module and a collection module, and the control module controls the connection between the concentration detection module and the waste liquid module or the collection module based on the measurement information of the concentration detection module.

[0018] Furthermore, the waste liquid module includes a waste liquid tank, a pump body, and a waste liquid trough. The waste liquid trough and the waste liquid tank are respectively connected to the concentration detection module. The waste liquid trough is used to collect waste liquid with substandard concentration. The waste liquid tank is used to collect liquid. The waste liquid trough is connected to the waste liquid trough. The pump body is used to transport the liquid in the waste liquid trough to the waste liquid trough.

[0019] And / or, the collection module includes a collection rack, collection tubes, a driving component, and a cantilever. Multiple collection tubes are respectively disposed on the collection rack and are used to collect proteins with preset gradient requirements. The driving component is used to drive the cantilever to move so that each collection tube can collect the protein output by the concentration detection module.

[0020] And / or, the buffer input unit includes multiple buffer interfaces and a second valve group, the second valve group being used to control the on / off status of each of the buffer interfaces and the on / off status of the waste liquid module and the concentration detection module.

[0021] Furthermore, the liquid outflow end of the chromatography column is connected to the interface of the concentration detection module from bottom to top via a pipe.

[0022] Furthermore, the monitoring module includes a pressure sensor, which is used to detect the pressure information of the liquid path between the power assembly and the chromatography column, and the control module is used to receive the pressure information detected by the pressure sensor.

[0023] Furthermore, the sample loading unit includes sample loading tubes, sample loading channels, and sample loading components. The sample loading tubes are arranged in groups, with at least two groups. Multiple sample loading channels are arranged in parallel. One end of each sample loading channel is connected to the first valve group, and the other end of each sample loading channel is connected to each sample loading component. The sample loading component is used to load the protein stock solution from at least one group of sample loading tubes. The control module is used to control the sample loading component and the first valve group to achieve cyclic sample loading of each group of sample loading tubes.

[0024] Furthermore, the monitoring module includes a bubble sensor, which is used to detect the drainage information of each of the sample loading tubes. The control module controls the sample loading component and the first valve group according to the drainage information detected by the bubble sensor.

[0025] As can be seen from the above technical solutions, the embodiments of this application have at least the following beneficial effects: by cooperating with a single power component and the first valve group, the structural utilization efficiency of the equipment is improved, and the cost is reduced by reducing redundant settings. On this basis, by combining the settings of the feeding module, chromatography column, detection module and control module, efficient and accurate purification results are ensured while enabling multiple affinity chromatography process steps. It also solves as much as possible the technical problems in related technologies such as complex structure, difficult maintenance and risk of contamination, or difficulty in setting up chromatography circulation loops due to complex installation and maintenance.

[0026] A second aspect of this application provides a production line, including a protein purification apparatus as described in the first aspect of this application.

[0027] It is easy to understand that the production line in the second aspect embodiment of this application has the same technical effects as the protein purification equipment in the first aspect embodiment, and therefore will not be described again.

[0028] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of a structure provided in one embodiment of this application;

[0031] Figure 2 An internal schematic diagram provided for one embodiment of this application;

[0032] Figure 3 This is a schematic diagram of a concentration detection module provided in one embodiment of this application.

[0033] Figure label:

[0034] 100. Power components;

[0035] 200. Chromatography column;

[0036] 300. Input module; 310. Sample loading unit; 320. Buffer input unit; 321. Buffer interface; 322. Second valve assembly;

[0037] 400. First valve group;

[0038] 510. Pressure sensor; 520. Bubble sensor;

[0039] 600. Control module;

[0040] 700. Concentration detection module; 710. LED assembly; 720. First lens; 730. Cuvette; 740. Filter; 750. Second lens; 760. Receiver assembly;

[0041] 800. Waste liquid module; 810. Waste liquid tank; 820. Pump body;

[0042] 900. Collection module; 910. Collection rack; 920. Collection tube; 930. Drive assembly; 940. Cantilever. Detailed Implementation

[0043] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0044] See Figures 1 to 3 As shown, an embodiment of the first aspect of this application discloses a protein purification device, including a power component 100, a chromatography column 200, an input module 300, a first valve group 400, a monitoring module, and a control module 600;

[0045] The input module 300 includes a sample loading unit 310 and a buffer input unit 320. The sample loading unit 310 is used to input the original sample solution, and the buffer input unit 320 is used to input the buffer solution. The power assembly 100 is used to provide power for the flow of liquids. The chromatography column 200 is used as the functional reaction zone for protein purification. The first valve group 400 is connected to the power assembly 100, the input module 300, and the chromatography column 200 through multiple liquid paths. The first valve group 400 is used to control the opening and closing and flow direction of each liquid path. The monitoring module is used to monitor the liquid paths and obtain liquid information. The control module 600 is used to receive the liquid information from the monitoring module and issue control commands. The first valve group 400 responds to the control commands so that the chromatography column 200 can perform the preset process steps of affinity chromatography.

[0046] In the embodiments of this application, the structural utilization efficiency of the equipment is improved by cooperating with a single power component 100 and a first valve group 400, thereby reducing redundant settings and lowering costs. Based on this, the combination of a feeding module, a chromatography column 200, a detection module, and a control module 600 ensures efficient and accurate purification results while enabling multiple affinity chromatography process steps. It also solves as much as possible the technical problems in related technologies, such as complex structures, difficult maintenance, and risks of contamination, or difficulties in setting up chromatography circulation loops due to complex installation and maintenance.

[0047] Understandably, the single power unit 100, in conjunction with the first valve group 400, replaces the redundant design of multiple power units, reducing the number of parts and the complexity of the liquid path, lowering equipment costs and maintenance difficulty, and solving problems such as complex structure and difficult maintenance in related technologies. By switching the liquid path through the first valve group 400, different processes such as sample loading, buffer washing, and elution can be flexibly combined to adapt to various affinity chromatography requirements, reducing the need for additional equipment modifications and improving application scenario coverage. The monitoring module provides real-time feedback on liquid path information, and the control module 600 controls the first valve group 400 and the power unit 100 to ensure stable parameters such as sample loading volume, buffer ratio, and flow rate, achieving efficient and accurate protein purification. Furthermore, the centralized liquid control of the first valve group 400 reduces liquid path interfaces and exposed points, lowering the risk of contamination. At the same time, the simplified liquid path design facilitates the construction of chromatography loops, solving the problem of complex installation and maintenance in related technologies that makes it difficult to set up loops.

[0048] In some embodiments, a single power unit 100 provides uniform fluid flow power. The first valve group 400 connects the power unit 100, the sample loading unit 310, the buffer input unit 320, and the chromatography column 200 through multiple fluid paths, realizing the control of fluid path opening and / or flow direction. The control module 600 receives fluid path information such as fluid flow status and concentration from the monitoring module, and sets control commands automatically or manually, which are then sent to the first valve group 400 to switch different fluid path combinations, enabling the chromatography column 200 to execute preset process steps. Furthermore, the chromatography column 200 is the functional reaction zone in protein affinity chromatography, capable of realizing various processes of protein affinity chromatography, including: equilibration, sample loading, washing, elution, and regeneration.

[0049] The following will combine Figures 1 to 3 The protein purification equipment disclosed in the embodiments of this application will be explained and described in detail.

[0050] Understandably, in order to make the switching timing of each preset process step of the chromatography column 200 more accurate and to provide judgment for the entire protein purification affinity chromatography system process, in some embodiments of this application, the protein purification device further includes a concentration detection module 700. The concentration detection module 700 is connected to the chromatography column 200 and is used to measure the protein concentration of the effluent liquid. The control module 600 is used to receive the measurement information from the concentration detection module 700 and issue closed-loop control commands.

[0051] Understandably, the concentration detection module 700 is connected to the chromatography column 200, collects protein concentration data of the effluent liquid and feeds it back to the control module 600. The control module 600 combines the liquid path information and concentration data from the monitoring module to form a closed-loop control logic, and sends precise commands to the first valve group 400 and the power component 100. By dynamically adjusting at least one of the liquid path on / off, flow direction, flow rate or buffer ratio, the chromatography column 200 is always adapted to changes in protein concentration in each process step such as sample loading, equilibration, and elution, thus achieving intelligent collaboration.

[0052] In some embodiments, real-time monitoring of protein concentration enables the control module 600 to dynamically adjust key parameters such as elution flow rate and buffer ratio, avoiding incomplete purification or loss of target protein due to concentration fluctuations. When the concentration detection module 700 detects that the target protein concentration has reached different preset thresholds, such as when the target protein concentration meets the standard or elution is complete, the control module 600 automatically instructs the first valve group 400 to switch process steps, improving resource utilization. Furthermore, based on the concentration distribution characteristics of different proteins, concentration feedback can dynamically adjust the process rhythm to adapt to the chromatography requirements of different concentration samples and different affinity strengths, thereby flexibly adapting to various purification application scenarios.

[0053] Exemplary, in some embodiments, reference is made to Figure 3The concentration detection module 700 includes an LED assembly 710, a first lens 720, a cuvette 730, a filter 740, a second lens 750, and a receiver assembly 760 arranged in sequence. The cuvette 730 is used to hold the measurement sample, the LED assembly 710 is used to emit ultraviolet light of a preset wavelength, and the receiver assembly 760 is used to receive ultraviolet light. The control module 600 calculates the protein concentration of the measurement sample based on the absorbance of the measurement sample at the preset wavelength. Understandably, the first lens 720 is used for collimation of ultraviolet light, and the second lens 750 is used for focusing ultraviolet light. An optical detection architecture is adopted, consisting of LED emission, lens collimation, cuvette 730 light transmission, filter purification, lens focusing, and receiver acquisition. The LED component 710 emits ultraviolet light of a preset wavelength (e.g., 280nm) characteristic of protein absorption. This light is collimated into parallel light by the first lens 720, penetrates the cuvette 730 containing the sample, and is filtered by the filter 740 to remove stray light, allowing only the target wavelength ultraviolet light to pass through. After being focused by the second lens 750, it is captured by the receiver component 760 and converted into an electrical signal. The control module 600 calculates the sample absorbance based on the signal, derives the protein concentration, and combines this with the liquid path information from the monitoring module to form a closed-loop command. This dynamically controls the liquid path switching of the first valve group 400 and the flow rate adjustment of the power component 100, adapting the purification process of the chromatography column 200 to changes in protein concentration, achieving a balance between detection accuracy and a simplified overall equipment design.

[0054] In some embodiments, to achieve better light transmission, the first lens 720 and the second lens 750 are semi-circular lenses.

[0055] It should be noted that, in order to avoid contamination of the target gradient protein and the waste liquid generated during the protein purification process by the waste liquid of the intermediate gradient during the collection of waste liquid of different gradient protein purification, in some embodiments of this application, the protein purification device further includes a waste liquid module 800 and a collection module 900. The control module 600 controls the concentration detection module 700 to connect to the waste liquid module 800 or the collection module 900 according to the measurement information of the concentration detection module 700.

[0056] Understandably, the concentration detection module 700 acquires protein concentration data and feeds it back to the control module 600. The control module 600 presets a concentration threshold, such as the minimum concentration of the target protein, and issues a diversion command after comparing it with the measured data. The liquid path is switched through the first valve group 400. If the concentration output by the concentration detection module 700 does not meet the standard, the waste liquid containing miscellaneous proteins is connected to the waste liquid module 800. If the concentration meets the standard, the liquid containing the target protein solution is connected to the collection module 900. This prevents the target protein from being lost with the waste liquid and also prevents the collection liquid from being contaminated by miscellaneous proteins. This solves the problems of easy errors and difficulty in ensuring purity in manual diversion and improves the quality of purified products.

[0057] In some embodiments, the control module 600 can adjust the threshold according to the concentration characteristics of different proteins to adapt to various affinity chromatography processes. Furthermore, it can recycle unqualified solutions through valve groups to achieve the effect of compatibility between the diversion logic and the chromatography circulation loop, thereby improving the versatility of the equipment.

[0058] In some embodiments, for example, the waste liquid module 800 includes a waste liquid tank, a pump body 820, and a waste liquid reservoir 810. The waste liquid reservoir 810 and the waste liquid tank are respectively connected to the concentration detection module 700. The waste liquid tank 810 is used to collect waste liquid with substandard concentration, and the waste liquid reservoir is used to collect liquid. The waste liquid tank 810 is connected to the waste liquid reservoir, and the pump body 820 is used to transport the liquid in the waste liquid reservoir to the waste liquid reservoir. It is understood that the waste liquid tank 810 is used to avoid collecting waste liquid from intermediate gradients during protein purification at different gradients, which could lead to contamination of the target gradient protein. The waste liquid reservoir is used to collect waste liquid generated during protein purification.

[0059] It should be noted that after the liquid is determined to be unqualified, the waste liquid flows into the waste liquid tank 810 connected to the concentration detection module 700. Part of it can be recycled, and the remaining part is independently transported by the pump body 820 to stably transport the waste protein waste liquid temporarily stored in the waste liquid tank 810 to the waste liquid pool for centralized storage and subsequent treatment, thereby improving the continuity of the purification process and the convenience of operation.

[0060] In some embodiments, the pump body 820 is a diaphragm pump, which is more effective at treating waste liquid.

[0061] In some embodiments, for example, the collection module 900 includes a collection rack 910, collection tubes 920, a drive component 930, and a cantilever 940. Multiple collection tubes 920 are respectively mounted on the collection rack 910 and used to collect proteins with preset gradient requirements. The drive component 930 drives the cantilever 940 to move, so that each collection tube 920 can collect the protein output by the concentration detection module 700. It is understood that the multiple collection tubes 920 correspond to collecting different concentration gradients and can collect target proteins of different purities or concentration ranges. The drive component 930 drives the cantilever 940 to switch movements, so that proteins of preset concentrations can reach the appropriate collection tube 920 after being output by the concentration detection module 700, further reducing the risk of sample contamination and cross-contamination. The multiple collection tubes 920 are integrated into the collection rack 910, and the cantilever 940 and drive component 930 have a simple structure. Maintenance only requires replacing the collection tubes 920 or calibrating the drive accuracy, reducing maintenance costs.

[0062] In some embodiments, multiple collection tubes 920 are arrayed on the collection rack 910. Each collection tube 920 corresponds to a different protein concentration gradient requirement. The control module 600 receives real-time data from the concentration detection module 700, identifies the gradient level of the current outflowing liquid, and sends a command to the drive component 930 to drive the cantilever 940 to move, so that the collection tube 920 corresponding to the gradient is connected to the output end of the concentration detection module 700.

[0063] For example, in some embodiments, the buffer input unit 320 includes multiple buffer interfaces 321 and a second valve group 322. The second valve group 322 is used to control the on / off state of each buffer interface 321 and the on / off state of the waste liquid module 800 and the concentration detection module 700. Specifically, the second valve group 322 is used to control the on / off state of each buffer interface 321 and the on / off state of the waste liquid pool and the concentration detection module 700.

[0064] Understandably, the buffer input unit 320 adapts to different types of buffers through multiple buffer interfaces 321. The second valve group 322 is instructed by the control module 600 to switch the connection between each buffer interface 321 and the main liquid path. At the same time, it integrates the control of the connection between the waste liquid tank and the concentration detection module 700. That is, the second valve group 322 simultaneously controls the connection between the buffer interface 321 and the connection between the waste liquid tank and the concentration detection module 700. This avoids cross-contamination of different buffers or waste liquid backflow contaminating the main liquid path, while reducing the number of parts and the complexity of the liquid path, further improving the structural utilization efficiency and reducing costs.

[0065] In some embodiments, the second valve group 322 can work with the control module 600 and the first valve group 400 to control the flow path of the buffer solution and / or waste liquid. This simplifies the construction and control of the chromatography loop while ensuring timely discharge of waste liquid, thus solving the problem of difficulty in setting up the loop in related technologies.

[0066] For example, the liquid outlet of the chromatography column 200 is connected to the interface of the concentration detection module 700 from bottom to top via a pipe. This effectively avoids the formation of a cavity or gas when the liquid flows through the concentration detection module 700. By connecting the lower interface of the concentration detection module 700 from bottom to top, the liquid can flow through the concentration detection module 700 in a full tube, ensuring that the data measured by the concentration detection module 700 is continuous and accurate. This avoids the formation of air bubbles in the liquid tube, which could lead to misjudgment that the protein washing or elution process has been completed, resulting in waste of raw materials.

[0067] In some embodiments, the monitoring module includes a pressure sensor 510, which detects the pressure information of the liquid path between the power assembly 100 and the chromatography column 200. The control module 600 receives the pressure information detected by the pressure sensor 510. It is understood that the pressure sensor 510 can monitor the main liquid path pressure in real time. When a blockage occurs in the pipeline or the first valve group 400 becomes stuck, causing a sudden pressure increase, the control module 600 can quickly instruct the power assembly 100 to stop or the valve group to switch, reducing the risk of failure.

[0068] In some embodiments, one end of the pressure sensor 510 is connected to a liquid pipe exiting from the power assembly 100, and the other end is connected to a liquid pipe exiting from the chromatography column 200. By setting a certain pressure value as the basis for clogging, when the chromatography column 200 becomes clogged and the liquid pressure is too high, the pressure sensor 510 triggers an alarm, and the equipment stops operating to prevent liquid from continuing to be drawn into the chromatography tube, causing liquid to overflow from the chromatography tube and contaminate the equipment and the surrounding environment, thus maintaining stable equipment operation.

[0069] In some embodiments of this application, the sample loading unit 310 includes sample loading tubes, sample loading channels, and sample loading components. The sample loading tubes are arranged in groups of at least two, and multiple sample loading channels are arranged in parallel. One end of each sample loading channel is connected to a first valve group 400, and the other end of each sample loading channel is connected to a sample loading component. The sample loading component is used to load the protein stock solution from at least one group of sample loading tubes. The control module 600 is used to control the sample loading component and the first valve group 400 to achieve cyclic sample loading of each group of sample loading tubes. It can be understood that at least two groups of sample loading tubes are loaded alternately to achieve continuous sample supply. Combined with the diversion design of the parallel loading channels, this can meet the purification needs of large batches of protein stock solution. The control module 600 can adjust the sample loading component and the first valve group 400 to achieve reflux and reloading of unqualified samples, simplifying the construction of the chromatography loop. Simultaneously, multiple groups of sample loading tubes and parallel sample loading channels can adapt to the batch loading of different types of protein samples.

[0070] In some embodiments, there are two sets of sample loading tubes, each set having multiple tubes of the same or different specifications, and eight sample loading channels connected in parallel. Only one power unit 100 is needed to provide power, improving the efficiency of purifying similar samples by 8 times, and each sample loading channel supports sample processing of 100ml-2L.

[0071] In some embodiments, the monitoring module includes a bubble sensor 520, which is used to detect the liquid drainage information of each sample loading tube. The control module 600 controls the sample loading component and the first valve group 400 according to the liquid drainage information detected by the bubble sensor 520. It is understood that the bubble sensor 520 is used to detect the liquid drainage of each sample loading tube. When the liquid in one of the sample loading tubes is emptied, and only gas passes through, the bubble sensor 520 will send a signal to the system, facilitating further processing.

[0072] In other embodiments, the bubble sensor 520 is divided into a buffer solution bubble sensor 520 and a stock solution bubble sensor 520. The stock solution bubble sensor 520 is used to detect the drainage information of each sample tube and achieve the above-mentioned effect. The buffer solution bubble sensor 520 is used to detect the drainage information of the buffer solution interface 321 and achieve a similar effect as described above. Further details are not provided here.

[0073] In some embodiments, the sample loading tubes are divided into two rows, A tubes and B tubes, with more than two in number, preferably eight in each row. The groove on the bubble sensor 520 in the A tube row is used to fix the liquid tube connected to the A tube and detect the liquid discharge of the A tube. The bubble sensor 520 in the B tube row detects the liquid discharge of the B tube.

[0074] In some embodiments, the power unit 100 is an injection pump that simultaneously provides power for the liquid flow of each parallel feeding channel.

[0075] In one specific embodiment, the buffer interface 321 is connected to the buffer solution through a tubing, and the sample loading unit 310 includes a sample loading machine and A and B groups of sample loading tubes set in the sample loading rack. After the chromatography column 200 is loaded with chromatography solution, the preparation work is completed.

[0076] During equilibration: the second valve group 322, the first valve group 400, and the power unit 100 operate simultaneously, drawing buffer solution from one of the buffer tanks at the buffer interface 321 and introducing it into the chromatography column 200 for column equilibration. As the buffer solution flows, it passes through the second valve group 322, the buffer bubble sensor 520, the first valve group 400, the power unit 100, the pressure sensor 510, the chromatography column 200, and the concentration detection module 700. Waste solution is discharged to the corresponding waste solution tank through the waste outlet via the first valve group 400 and the second valve group 322.

[0077] During automated sample loading: First, the original solution is drawn from either sample loading tube A or sample loading tube B. As the solution flows, it passes through the original solution bubble sensor 520, the first valve group 400, the power assembly 100, the pressure sensor 510, and the chromatography column 200, causing the target protein to be adsorbed into the chromatography tube. Non-target protein waste liquid is discharged through the concentration detection module 700 and the first valve group 400. Furthermore, since some of the target protein drawn into the chromatography column 200 may not be absorbed and may flow away, to avoid waste, the unabsorbed target protein and other impurities are drawn together and stored in a backup sample loading tube (either sample loading tube B or sample loading tube A). After washing and elution, the automated sample loading process is repeated from the liquid in sample loading tubes B or A. Through multiple cycles of sample loading, sample utilization is maximized.

[0078] During the cleaning process: When the concentration detection module 700 reads a stable peak value from the liquid automatically loaded in the previous step, the corresponding buffer solution is drawn from one of the interfaces of the buffer interface 321 to wash out the non-target protein impurities contained in the chromatography column 200. The buffer solution enters from the buffer interface 321 and flows all the way to the chromatography column 200 to wash out the non-target proteins and discharges to the waste liquid outlet for collection in the waste liquid pool. When the buffer solution flows, it passes through the second valve group 322, the buffer bubble sensor 520, the first valve group 400, the power component 100, the pressure sensor 510, the chromatography column 200, and the concentration detection module 700. The waste liquid is discharged into the corresponding waste liquid pool through the first valve group 400 and the second valve group 322.

[0079] During elution: When the concentration reading from the concentration detection module 700 reaches a stable minimum value, the corresponding buffer solution is drawn from one of the interfaces of the buffer interface 321 to elute the target protein contained in the chromatography column 200 and collect it onto the collection module 900. The buffer solution enters from the buffer interface 321, flows through the chromatography column 200 to elute the target protein, and is discharged onto the collection rack 910. The liquid then passes through the second valve group 322, the buffer bubble sensor 520, the first valve group 400, the power unit 100, the pressure sensor 510, the chromatography column 200, and the concentration detection module 700. The first valve group 400 collects the target protein onto the collection tube 920 of the collection rack 910. During collection, if different gradient proteins are required, the intermediate gradient non-target proteins need to be discharged into the waste liquid tank 810. Another pump 820, preferably a diaphragm pump, is used as the power source to draw the protein from the waste liquid tank 810 to the waste liquid outlet and discharge it into the corresponding waste liquid pool.

[0080] During the automatic sample loading process, some target proteins may not be bound by the chromatography column 200, resulting in flow cytometry errors. The three steps of automatic sample loading, washing, and elution are repeated. The automatic sample loading, together with two sets of sample tubes, A and B, are used to achieve back-and-forth sample loading through the system.

[0081] During regeneration: After the target protein extraction is complete, the chromatography solvent can be drawn from the chromatography column 200 through the buffer interface 321 to achieve chromatography solvent regeneration and avoid waste. At this time, the second valve group 322, the first valve group 400, and the power unit 100 are opened simultaneously, drawing buffer from one of the buffer tanks of the buffer interface 321 into the chromatography column 200 to achieve chromatography solvent regeneration. During liquid flow, it passes through the second valve group 322, the buffer bubble sensor 520, the first valve group 400, the power unit 100, the pressure sensor 510, the chromatography column 200, and the concentration detection module 700, and finally discharges the waste liquid into the corresponding waste liquid pool through the first valve group 400 and the second valve group 322.

[0082] It is understood that the protein purification device provided in this application can realize multiple processes of protein affinity chromatography, including: equilibration, sample loading, washing, elution, and regeneration functions. Moreover, the device has a compact structure, making it easy to place in a biosafety cabinet or clean bench. Furthermore, plasmid extraction can be achieved with simple changes to the purification program without requiring any other structural modifications.

[0083] Furthermore, the protein purification equipment allows for the parallel use of multiple loading channels, requiring only one syringe pump for power. This significantly increases the efficiency of purifying similar samples. Without altering the structure, the loading volume can be changed simply by replacing the loading tubes, and each channel supports sample processing from 100ml to 2L. Moreover, each loading channel is equipped with two sample feeders, and loading tubes A and B can be cyclically loaded. This avoids the problem of incomplete target protein extraction during a single pass through the chromatography column (200°C). The lost protein liquid is transferred from loading tube A to B and then back to A repeatedly to ensure efficient column chromatography and prevent sample waste.

[0084] In some embodiments, this protein purification device features high compatibility, supporting 200 mL chromatography columns of 3 mL, 12 mL, 30 mL, 55 mL, and 100 mL. A preferred design uses a spring-clamping mechanism to clamp the chromatography tubes, accommodating various tube sizes for easy replacement. Furthermore, the device enables fully automated operation of the experimental process from sample loading to regeneration without manual intervention, ensuring efficient and accurate purification results and guaranteeing high consistency in protein purification results for each iteration while maintaining the same program.

[0085] Furthermore, a display screen, preferably a 10-inch touchscreen, can be provided for users to program and set different experimental steps and parameters according to their experimental needs. The collection mode supports manual collection as well as automatic collection based on volume, threshold, and slope. The screen will also display the current process step, completed steps, pending process steps, running time, and estimated remaining time.

[0086] This protein purification equipment includes a waste liquid tank 810 and a waste liquid reservoir. The waste liquid tank 810 is used to collect waste proteins from different target gradient protein intervals. Then, a pump 820, preferably a diaphragm pump, discharges non-target proteins into the waste liquid reservoir. This prevents non-target proteins from contaminating the target proteins in the collection tube 920, greatly improving the purity of the collected proteins. Furthermore, the protein purification equipment features real-time UV monitoring throughout the process, enabling automatic peak collection and supporting 6 external liquid elutions and 6-10 gradient elution collections. Finally, the outer shell of this protein purification equipment has a convenient disassembly design; by separating the shell at the corresponding locations, disassembly, maintenance, and inspection are easy, making operation simple.

[0087] The protein purification apparatus of this application is described in detail below with reference to a specific embodiment. It should be noted that the following embodiment is merely an exemplary description and should not be construed as limiting the embodiments of this application.

[0088] See Figures 1 to 3 As shown, the protein purification equipment in this embodiment, based on affinity chromatography as its core principle, integrates multi-module monitoring and precise control functions, including:

[0089] The sample loading assembly is used to fix the sample loading tubes. It consists of two rows of A and B tubes, with eight tubes in each row (more than two). All A tubes can be filled with the stock solution from which protein is to be extracted, while B tubes remain empty; or B tubes can be filled with the stock solution from which protein is to be extracted, while A tubes remain empty. When there are eight sample loading channels, tubes A and B of #1 are connected to the stock solution bubble sensor 520 via Luer connectors, and then connected to the two designated valve ports of the corresponding first valve group 400. The connection method for tubes A and B of #2-#8 is the same as that for the liquid path of #1.

[0090] The original solution bubble sensor 520 is used to detect the liquid discharge of sample tubes A and B. When the liquid in tube A or tube B is emptied and only gas passes through, the bubble sensor 520 will send a signal to the system to detect the liquid discharge during sample A loading.

[0091] The first valve group 400 is used to control the flow of liquid and can change the direction of liquid flow, for example, allowing it to flow to the waste liquid tank 810, waste liquid pool, sample loading tubes A and B, centrifuge tubes on the collection rack 910, etc. Specifically, the first valve group 400 contains a total of 8 solenoid valves, forming an 8-port solenoid valve array. When there are 8 sample loading channels, 8 8-port solenoid valve arrays are used, in which 7 valve ports are open and 3 valve ports are blocked and not working. In this configuration, valve port #1 of the eight-panel solenoid valves connects to the buffer solution exiting from valve group 322; valve port #2 connects to the drain connector of cantilever 940; valve port #3 connects to the inlet of power assembly 100; valve ports #4 of the eight eight-panel solenoid valves converge and connect to the outlet of power assembly 100; valve ports #5, #9, and #10 are blocked and do not work; valve port #6 connects to the liquid pipe exiting from tube A on the sample holder and passing through bubble sensor 520; valve port #7 connects to the liquid pipe exiting from tube B on the sample holder and passing through bubble sensor 520; and valve port #8 connects to concentration detection module 700. In some embodiments, the valve port interface order of the eight-panel solenoid valves is not unique and can be changed according to requirements.

[0092] The power unit 100 is one of the power sources providing power to the system. Exemplarily, it can be an 8-port syringe pump with eight glass tube injectors, acting as a transfer station reservoir. A single push rod causes all eight injectors to simultaneously operate, allowing liquid to enter and exit the system. All liquids first enter the device through this syringe pump. It has two rows of valve ports, eight in each row. One row connects to the liquid lines from the eight 8-port solenoid valves, and the other row connects to the liquid lines from the pressure sensor 510. When the syringe pump receives a working signal, it begins to push the glass tube injectors to absorb or expel liquid. Simultaneously, the eight-port solenoid valves open, allowing liquid to enter the syringe pump's glass tube injectors or emptying the liquid inside the glass tube injectors.

[0093] Pressure sensor 510 is the system's pressure detection system. By setting a certain pressure value as the basis for determining blockage, when the chromatography column 200 becomes blocked and the liquid pressure in the liquid path becomes too high, pressure sensor 510 triggers an alarm, and the equipment stops operating. This prevents liquid from continuing to be drawn into the chromatography tube, causing liquid to overflow and contaminate the equipment and the surrounding environment, thus maintaining stable equipment operation. One end of pressure sensor 510 is connected to the liquid line from the injection pump, and the other end is connected to the liquid line from the chromatography column 200.

[0094] Column 200 is the functional reaction zone in affinity chromatography for protein purification. Equilibration, loading, washing, elution, and regeneration are all completed within this column. The liquid in column 200 flows through a pipe to the lower interface of concentration detection module 700. Connecting to the lower interface of concentration detection module 700 prevents the formation of a cavity or air pocket as the liquid flows through it. This connection ensures the liquid flows fully through the concentration detection module 700, guaranteeing continuous and accurate data. It also prevents air bubbles from misinterpreting the process as completed protein washing or elution, thus avoiding waste of raw materials.

[0095] The concentration detection module 700 is used to determine the concentration of a protein by measuring its ultraviolet light absorption at a wavelength of 280 nm, primarily relying on the absorption characteristics of tyrosine, tryptophan, and phenylalanine in the protein. At 280 nm, the absorbance (A280) of a protein is directly proportional to its concentration. Therefore, by measuring the absorbance of the sample at this wavelength, the protein concentration can be calculated. The main components of this module include: an LED assembly 710, a semi-circular lens, a cuvette 730, a filter 740, and a PD receiver assembly 760. The lower interface of the concentration detection module 700 connects to the liquid outlet of the chromatography column 200, detecting the protein concentration in the liquid flowing out of the chromatography column 200. The upper interface connects to the #8 port of eight 8-cell solenoid valves. As the most important detection module in the entire system, it converts the light signal into an electrical signal to determine the protein concentration, providing a basis for the entire protein purification affinity chromatography process. In some embodiments, the concentration detection module 700 is a UV280 module.

[0096] The second valve group 322 is used to control the inflow and outflow of buffer solution and waste liquid. This second valve group 322 is an 8-port solenoid valve, with 6 valve ports connected to the buffer solution pipeline, 1 valve port connected to the waste liquid discharge pipeline, and 1 valve port connected to the pipeline of the bubble sensor 520. The liquid path is divided into two paths by a three-way valve. One path is then split to the corresponding valve ports of the four 8-port solenoid valves on the left, and the other path is then split to the corresponding valve ports of the four 8-port solenoid valves on the right. Alternatively, two valve ports can be connected to one bubble sensor 520 each, and then connected to the corresponding valve ports of the 8-port solenoid valves on the left and right sides through the liquid pipeline. The other valve ports are blocked with plugs and do not work. This allows the bubble sensor 520 to detect the liquid status of the buffer solution and to work in conjunction with the first valve group 400.

[0097] Furthermore, to prevent waste liquid from intermediate gradients during the purification of proteins at different gradients from contaminating the target gradient protein, a waste liquid tank 810, piping, and a diaphragm pump are also included. The waste liquid tank 810 is connected to the diaphragm pump via piping. When the diaphragm pump is working, it can pump away the waste liquid from the waste liquid tank 810 and discharge it into port #4 of the 8-panel solenoid valve.

[0098] The collection module mainly includes a centrifuge tube collection rack 910, liquid tubes, a cantilever 940, a linear slide rail, a synchronous belt, a stepper motor, a drag chain, and sensors. The collection rack 910 is used to collect proteins with different gradient requirements and can expand the collection quantity.

[0099] Understandably, the protein purification equipment of this application can realize multiple processes of protein affinity chromatography: equilibration, sample loading, washing, elution, and regeneration. The purification program can be modified to achieve plasmid extraction. The plasmid extraction principle is as follows: the negatively charged phosphate groups on the plasmid DNA bind to the cationic groups on the anion exchange packing material under low pH and low salt concentration conditions, adsorbing the DNA onto the packing material. After washing away impurities such as proteins, RNA, and endotoxins with a medium salt concentration solution, the plasmid DNA is finally eluted with a high pH and high salt concentration solution, and then precipitated with isopropanol to obtain high-purity plasmid DNA.

[0100] The device features eight parallel loading channels, with the potential for further channel expansion. It requires only one syringe pump for power, resulting in an eightfold increase in purification efficiency for similar samples. Each channel supports sample processing from 100ml to 2L. The loading rack is independently detachable and external to the device, and its structure can be replaced as needed. Each channel is equipped with two sample loaders, and A and B loading devices can be cyclically used to ensure efficient column pass-through and prevent sample waste.

[0101] Furthermore, the device of this application has high compatibility, supporting 200 mL of chromatography columns of 3 mL, 12 mL, 30 mL, 55 mL, and 100 mL. Preferably, the chromatography tubes are clamped by a spring-loaded clamping mechanism for easy replacement. It achieves fully automated operation of the experimental process from sample loading to regeneration without manual intervention, ensuring efficient and accurate purification results and guaranteeing high consistency of protein purification results for each iteration while maintaining the same program. It includes eight 8-unit solenoid valve groups, each controlling one channel independently. Even if one channel malfunctions, such as a problem with one bubble sensor 520, the other seven channels continue to operate normally. Through real-time UV monitoring throughout the process, it achieves automatic peak collection and supports six external liquid elutions and six to ten gradient elutions. In some embodiments, the device can be designed so that the entire top cover can be disassembled into multiple independent plates, preferably four. Liquid interface ports can be arranged on the front and rear top cover plates. When the top plate needs to be disassembled for maintenance or to check the operation of the syringe pump, the middle top cover plate can be directly removed without affecting the top liquid channels or requiring the removal of liquid tubing, making it simple and convenient. Furthermore, to facilitate observation of the infusion pump's operation from the front (e.g., checking the pump when air bubbles are generated in the endotracheal tube), the front housing is designed as a detachable front housing assembly plus a sheet metal outer shell on the left side. When checking the pump's operation, simply remove two screws to detach the detachable front housing assembly. Disassembly is simple and convenient.

[0102] The second aspect of this application discloses a production line, which can be a production line for the efficient purification of biological macromolecules such as recombinant proteins, antibodies, and enzymes in the fields of life sciences, molecular biology, or medicine. The production line includes: the protein purification equipment of the first aspect of this application.

[0103] It is easy to understand that the production line in the second aspect embodiment of this application has the same technical effects as the protein purification equipment in the first aspect embodiment, and therefore will not be described again.

[0104] In the description of this application, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0105] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "multiple" means two or more. It should be noted that the term "and / or" used herein is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. Similarly, at least one of A or B can also represent: A alone, A and B simultaneously, or B alone.

[0106] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0107] In the foregoing description of this specification, references to terms such as "one embodiment," "another embodiment," or "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Claims

1. A protein purification device, characterized in that, include: Power assembly, chromatography column, input module, first valve group, monitoring module, and control module; The input module includes a sample loading unit and a buffer input unit. The sample loading unit is used to input the original sample solution, and the buffer input unit is used to input the buffer solution. The power assembly is used to provide power for the flow of the liquid; The chromatography column is used as a functional reaction zone for protein purification; The first valve group is connected to the power component, the input module and the chromatography column through multiple liquid passages respectively. The first valve group is used to control the opening and closing and the flow direction of each liquid passage. The monitoring module is used to monitor the liquid path and obtain liquid information; The control module is used to receive liquid information from the monitoring module and issue control commands. The first valve group responds to the control commands so that the chromatography column can perform preset process steps of affinity chromatography respectively.

2. The protein purification apparatus according to claim 1, characterized in that: The protein purification equipment further includes a concentration detection module, which is connected to the chromatography column and used to measure the protein concentration of the effluent liquid. The control module is used to receive the measurement information from the concentration detection module and issue closed-loop control commands.

3. The protein purification apparatus according to claim 2, characterized in that: The concentration detection module includes an LED assembly, a first lens, a cuvette, a filter, a second lens, and a receiver assembly arranged in sequence. The cuvette is used to hold the measurement sample, the LED assembly is used to emit ultraviolet light of a preset wavelength, and the receiver assembly is used to receive the ultraviolet light. The control module calculates the protein concentration of the measurement sample based on the absorbance of the measurement sample at the preset wavelength.

4. The protein purification apparatus according to claim 2, characterized in that: The protein purification equipment further includes a waste liquid module and a collection module. The control module controls the connection between the concentration detection module and the waste liquid module or the collection module based on the measurement information from the concentration detection module.

5. The protein purification apparatus according to claim 4, characterized in that: The waste liquid module includes a waste liquid tank, a pump body, and a waste liquid reservoir. The waste liquid reservoir and the waste liquid tank are respectively connected to the concentration detection module. The waste liquid tank is used to collect waste liquid with a concentration that does not meet the standard. The waste liquid reservoir is used to collect liquid. The waste liquid tank is connected to the waste liquid reservoir. The pump body is used to transport the liquid in the waste liquid tank to the waste liquid reservoir. And / or, the collection module includes a collection rack, collection tubes, a driving component, and a cantilever. Multiple collection tubes are respectively disposed on the collection rack and are used to collect proteins with preset gradient requirements. The driving component is used to drive the cantilever to move so that each collection tube can collect the protein output by the concentration detection module. And / or, the buffer input unit includes multiple buffer interfaces and a second valve group, the second valve group being used to control the on / off status of each of the buffer interfaces and the on / off status of the waste liquid module and the concentration detection module.

6. The protein purification apparatus according to claim 2, characterized in that: The liquid outlet of the chromatography column is connected to the interface of the concentration detection module from bottom to top via a pipe.

7. The protein purification apparatus according to claim 1, characterized in that: The monitoring module includes a pressure sensor, which is used to detect the pressure information of the liquid path between the power component and the chromatography column, and the control module is used to receive the pressure information detected by the pressure sensor.

8. The protein purification apparatus according to claim 1, characterized in that: The sample loading unit includes sample loading tubes, sample loading channels, and sample loading components. The sample loading tubes are arranged in groups, with at least two groups. Multiple sample loading channels are arranged in parallel. One end of each sample loading channel is connected to the first valve group, and the other end of each sample loading channel is connected to each sample loading component. The sample loading component is used to load the protein stock solution from at least one group of sample loading tubes. The control module is used to control the sample loading component and the first valve group to achieve cyclic sample loading of each group of sample loading tubes.

9. The protein purification apparatus according to claim 8, characterized in that: The monitoring module includes a bubble sensor, which is used to detect the drainage information of each sample loading tube. The control module controls the sample loading component and the first valve group according to the drainage information detected by the bubble sensor.

10. A production line, characterized in that, include: The protein purification apparatus as described in any one of claims 1 to 9.

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