Recombinant cell construction and function verification system

Abstract

The invention provides a recombinant cell construction and function verification system, which belongs to the technical field of biomedical engineering, and mainly comprises a reactor array, a material supplementing module, a sampling module, a detection module and a control unit, according to the system, by establishing an automatic feedback mechanism of online sampling, real-time detection and closed-loop feeding, the post-translational modification state of a recombinant cell can be obtained, accurate dynamic regulation and control of key quality attributes of target protein are achieved, and the culture efficiency and the sampling amount of a sampling module are remarkably improved through a reactor array, so that the quality of the target protein is improved. The yield and the quality stability of the complex recombinant protein are ensured.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a system for constructing and validating recombinant cells. Background Technology

[0002] With the development of biotechnology, recombinant protein drugs are playing an increasingly important role in the treatment of diabetes, obesity, and neurodegenerative diseases. Recombinant protein technology is used to create therapeutic fusion proteins that express the properties of natural peptides. The process involves cell engineering steps including transcription, translation, and post-translational modifications, and its technology directly affects the physicochemical properties, biological activity, and yield of the drug. For example, the GLP-1 fusion protein, which currently exhibits specific post-translational modifications, has a hydroxylation modification at lysine 34 (K34), which significantly improves protein yield and biological activity.

[0003] In existing recombinant cell construction and screening processes, cell screening and process validation typically rely on a pattern of culture, endpoint sampling, offline purification, and detection. This pattern suffers from feedback lag and cannot provide real-time information on the cells' ability to hydroxylate proteins during culture. Consequently, the optimization of process parameters relies heavily on trial and error, making it difficult to capture the optimal cellular physiological window.

[0004] Therefore, there is an urgent need for a system that can monitor key quality attributes online in order to enable the rapid construction and functional verification of high-quality recombinant cells. Summary of the Invention

[0005] This invention provides a recombinant cell construction and functional verification system to address the shortcomings of existing technologies in real-time monitoring of key quality attributes, and to achieve precise dynamic regulation of key quality attributes of target proteins.

[0006] This invention provides a system for constructing and validating recombinant cells, comprising:

[0007] The reactor array includes multiple independent cell culture chambers, each equipped with an independent temperature control unit, stirring and oscillation device, and fluid interface;

[0008] The feeding module is connected to the cell culture chamber via a microfluidic pipeline. The feeding module includes at least a first pump group for delivering basal culture medium and a second pump group for delivering modification and regulatory factors.

[0009] A sampling module, located downstream of the reactor array, is used to periodically extract trace amounts of culture medium from each culture chamber and perform cell retention and aseptic filtration.

[0010] The detection module, which is in fluid communication with the sampling module, includes a sensor unit whose surface is modified with ligands that specifically recognize specific post-translational modification domains of the target protein;

[0011] The control unit is electrically connected to both the detection module and the feeding module, and is used to receive the detection data from the detection module and adjust the feeding rate of the second pump group based on the detection data.

[0012] According to the present invention, a recombinant cell construction and functional verification system is provided, wherein the sensor unit includes a first channel and a second channel, the first channel being modified with a capture molecule targeting the Fc domain of the target fusion protein for detecting the protein expression level; the second channel being modified with a specific antibody targeting the hydroxylated lysine epitope for detecting the modification integrity or biological activity of the protein.

[0013] According to the recombinant cell construction and functional verification system provided by the present invention, the second pump group includes multiple independent micro-injection pumps, which are respectively connected to storage tanks containing ferrous ion solution, α-ketoglutarate solution and ascorbic acid solution.

[0014] According to the recombinant cell construction and functional verification system provided by the present invention, the sampling module further includes a microfilter and a cell reflux pipeline. The molecular weight cutoff of the microfilter allows recombinant proteins to pass through but retains cells. One end of the cell reflux pipeline is connected to the microfilter, and the other end of the cell reflux pipeline is connected to the cell culture chamber. The cell reflux pipeline is used to return the retained cells to the cell culture chamber to maintain the cell density in the reactor array.

[0015] According to the recombinant cell construction and functional verification system provided by the present invention, the control unit is further configured to determine the ratio of the binding response value of the target protein and the ligand detected by the detection module, and obtain the real-time hydroxylation rate; the control unit is further configured to adjust the working mode of the second pump group according to the real-time hydroxylation rate to adjust the delivery parameters of each micro-injection pump.

[0016] According to the recombinant cell construction and functional verification system provided by the present invention, when the real-time hydroxylation rate is lower than a first preset threshold, the control unit is used to control the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate to maintain the reduction environment, and after a preset time interval, control the micro-injection pumps corresponding to the ferrous ion solution and the α-ketoglutaric acid solution to simultaneously increase the delivery rate of the ferrous ion solution and the α-ketoglutaric acid solution according to the stoichiometric ratio.

[0017] According to the recombinant cell construction and functional verification system provided by the present invention, when the real-time hydroxylation rate is greater than or equal to a second preset threshold, the control unit is used to control each micro-injection pump in the second pump group to reduce the delivery rate to avoid cytotoxicity; the second preset threshold is greater than the first preset threshold.

[0018] According to the recombinant cell construction and functional verification system provided by the present invention, when the detection module detects that the oxidation level of the target protein is higher than a third preset threshold, the control unit is used to control the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate of the ascorbic acid solution, and / or the control unit is used to control the temperature control unit to reduce the temperature of the cell culture chamber.

[0019] According to the recombinant cell construction and functional verification system provided by the present invention, the control unit is further configured to control the temperature control unit to reduce the temperature of the cell culture chamber when the cell density information fed back by the detection module meets the preset conditions, so as to reduce the temperature of the cell culture chamber from a first temperature to a second temperature at a preset rate, and maintain it at the second temperature until the end of the culture.

[0020] According to the recombinant cell construction and functional verification system provided by the present invention, the fluid interface is further provided with a laminar flow mixing zone, which is used to premix the modification regulatory factors before they enter the cell culture chamber.

[0021] The recombinant cell construction and functional verification system provided by this invention establishes an automated feedback mechanism for online sampling, real-time detection, and closed-loop feeding, thereby obtaining the post-translational modification status of recombinant cells and achieving precise dynamic control of key quality attributes of target proteins. While significantly improving culture efficiency through reactor arrays and sampling volume through sampling modules, it ensures the yield and quality stability of complex recombinant proteins. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in this invention 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the recombinant cell construction and functional verification system provided by the present invention;

[0024] Figure 2 This is a schematic diagram of the reactor array provided by the present invention;

[0025] Figure 3 This is a schematic diagram of the feeding module provided by the present invention;

[0026] Figure 4 This is a schematic diagram of temperature changes at different reaction stages in the reactor array provided by the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] It should be noted that the terms "first," "second," etc., used in this specification are only used to distinguish similar objects and should not be construed as a specific order or sequence. Furthermore, the terms "comprising" and "having," and any variations thereof, are used to indicate a non-exclusive inclusion relationship.

[0029] The following is combined with Figures 1-4 The present invention describes a recombinant cell construction and functional verification system.

[0030] It should be noted that the recombinant cell construction and functional verification system provided in this embodiment of the invention is an integrated platform combining hardware and software, used to culture and screen host cells transfected with the target gene in the laboratory or pilot scale, and to verify their ability to express the target protein, especially proteins with specific post-translational modifications, under specific process conditions.

[0031] This embodiment provides a system for constructing and validating recombinant cells, such as... Figure 1 As shown, the system mainly includes a reactor array, a feeding module, a sampling module, a detection module, and a control unit.

[0032] The reactor array is the core site for cell growth. In some embodiments, the array comprises multiple independent cell culture chambers. For example, a microbioreactor with a working volume of 15 mL to 50 mL.

[0033] like Figure 2 As shown, each cell culture chamber is equipped with an independent temperature control unit, a stirring and oscillation device, and a fluid interface. The temperature control unit can be an independent Peltier element or an independent water jacket wrapped around the outside of each chamber, enabling precise temperature control from 20℃ to 40℃ with an accuracy of ±0.1℃, and supporting rapid temperature change operations.

[0034] The stirring and oscillation device can be a suspended magnetic stirring paddle or an eccentric track oscillation mechanism equipped in each chamber, and the stirring rate can be independently adjusted by the control unit.

[0035] Each chamber has a sterile fluid interface located at its top; different fluid interfaces can be connected to feed lines, sampling lines, and reflux lines. The feed module can be connected to the cell culture chamber via microfluidic tubing.

[0036] like Figure 3 As shown, the feeding module includes at least a first pump assembly for delivering the basal culture medium and a second pump assembly for delivering the modification and regulation factors. For example, the feeding module is connected to the chamber via microfluidic tubing such as polytetrafluoroethylene or medical-grade silicone tubing.

[0037] The first pump set can be a high-precision peristaltic pump used to deliver basic culture media, such as those containing glucose and amino acids.

[0038] The second pump assembly may include multiple independent micro-injection pumps. In some embodiments, it may include at least three independent micro-injection pumps, each connected to one of three reservoirs: reservoir A, reservoir B, and reservoir C.

[0039] Storage tank A can contain ferrous ion solution, such as FeSO4; storage tank B can contain α-ketoglutaric acid solution; and storage tank C can contain ascorbic acid solution.

[0040] For the complex enzymatic reaction of lysine hydroxylation, the second pump unit in the feeding module of this system is a precision multi-component delivery system.

[0041] like Figure 3 As shown, the second pump group can include three independently controlled micro-injection pumps, each connected to a dedicated reservoir. Ferrous ions provide an essential cofactor for the active site of lysyl hydroxylase. α-Ketoglutarate, as a co-substrate in the hydroxylation reaction, directly participates in the reaction cycle. Ascorbic acid, as a reducing agent, maintains the ferric ions in the active site in a divalent reduced state, preventing oxidative inactivation. In this embodiment, the independent tubing design allows the control unit to perform individual, decoupled flow control of these three key components according to the real-time metabolic needs of the cells.

[0042] In some embodiments, the fluid interface is further provided with a laminar flow mixing zone for premixing the modification regulatory factors before they enter the cell culture chamber.

[0043] Before the aforementioned modification and regulation factors enter the culture chamber, a laminar mixing zone is integrated at the fluid interface, and the interior can be designed with herringbone or serpentine flow channels.

[0044] Because ferrous ions and low-pH ascorbic acid, if directly added to the culture medium, can create a localized high-concentration zone at the droplet point, leading to instantaneous cell death or non-specific oxidation upon contact, the laminar flow mixing zone allows these trace factors to be premixed and diluted with a small amount of basal medium delivered by the first pump group before entering the main reaction system. This design ensures that the modification and regulatory factors enter the cellular environment in a uniform and gentle manner, significantly reducing cytotoxicity.

[0045] Understandably, after the system in this embodiment of the invention is started, the reactor array begins to operate. Each independent cell culture chamber simulates a miniature bioreactor environment for culturing engineered cells expressing target recombinant proteins, such as the GLP-1 fusion protein. Independent temperature control units and stirring / oscillating devices provide suitable growth temperature and dissolved oxygen mass transfer environment for the cells. At this time, the first pump group in the feeding module can continuously or intermittently deliver basal culture medium into the chamber according to a preset basic process curve to maintain the basal metabolism and proliferation requirements of the cells.

[0046] The sampling module can be located downstream of the reactor array to periodically extract trace amounts of culture medium from each culture chamber and perform cell retention and sterile filtration.

[0047] Understandably, in order to achieve continuous monitoring without consuming too many cells, the sampling module can have a cell reinfusion function.

[0048] In some embodiments, the sampling module further includes a microfilter and a cell reflux line. The microfilter has a molecular weight cutoff that allows recombinant proteins to pass through but retains cells. One end of the cell reflux line is connected to the microfilter, and the other end is connected to the cell culture chamber. The cell reflux line is used to return the retained cells to the cell culture chamber to maintain the cell density within the reactor array.

[0049] The microfilter can be a hollow fiber membrane module (HFF) with a pore size of 0.2 μm or a molecular weight cutoff of 500 kDa. A pump, such as a peristaltic pump, can be configured to extract the culture medium, which flows through the microfilter. In this case, the liquid permeating through the microfilter is a sterile, clear liquid containing secreted recombinant proteins and is sent to the detection module. The liquid retained by the microfilter contains a high-density cell concentrate. A cell reflux tubing can return the retained liquid to the culture chamber in real time.

[0050] Understandably, the sampling module allows the system to perform high-frequency sampling analysis without reducing cell density, which is beneficial for maintaining a high cell density.

[0051] As the culture process progresses, the sampling module can begin its work. It can extract minute amounts of culture medium from each culture chamber at set time intervals, such as once per hour, using precise fluid control. To avoid affecting cell density within the reactor and subsequent culture, a microfilter blocks cells, and a cell return pipeline returns cells to the reactor, allowing only the clear supernatant containing secreted target proteins to flow downstream. This ensures the purity of the sample and prevents cell debris from clogging subsequent sensors.

[0052] The detection module is in fluid communication with the sampling module. The detection module includes a sensor unit whose surface is modified with ligands that specifically recognize specific post-translational modified domains of the target protein.

[0053] Understandably, filtered samples can enter the detection module. The sensor unit inside the detection module can be based on SPR (Surface Plasmon Resonance) technology or BLI (Bio-Layer Interferometry) technology, with specific ligands pre-modified on the surface of the sensor unit.

[0054] Ligands can precisely capture target proteins in samples that have specific post-translational modification domains, such as specific hydroxylated lysine sites. Sensor units can convert the binding interactions between biomolecules into optical or electrical signals, thereby quantitatively or qualitatively reflecting the key quality attributes of the product at the current time point, such as the modification ratio or the content of specific active substances.

[0055] The control unit is electrically connected to both the detection module and the feeding module, and is used to receive the detection data from the detection module and adjust the feeding rate of the second pump group based on the detection data.

[0056] The control unit receives real-time data from the detection module and compares the detected modification level with a preset target value. If a specific modification of the target protein, such as hydroxylation, is insufficient, the control unit calculates the deviation and sends a command to the feeding module to precisely adjust the feeding rate of the second pump unit. Based on this, the second pump unit can change the amount of modification regulator added. Dynamic adjustment can alter the intracellular enzymatic reaction microenvironment, prompting the cell to synthesize a more highly modified target protein in the next stage of metabolism.

[0057] The recombinant cell construction and functional verification system provided in this embodiment of the invention establishes an automated feedback mechanism for online sampling, real-time detection, and closed-loop feeding, thereby obtaining the post-translational modification status of recombinant cells and achieving precise dynamic control of key quality attributes of target proteins. While significantly improving culture efficiency through reactor arrays and sampling volume through sampling modules, it ensures the yield and quality stability of complex recombinant proteins.

[0058] In some embodiments, the sensor unit includes a first channel and a second channel, wherein the first channel is modified with a capture molecule targeting the Fc domain of the target fusion protein for detecting protein expression levels; and the second channel is modified with a specific antibody targeting a hydroxylated lysine epitope for detecting the integrity of protein modification or biological activity.

[0059] In this embodiment, the sensor unit is designed with a dual-channel parallel detection mechanism, which can simultaneously acquire data on both the quantity and quality of the target protein.

[0060] The first channel can serve as a reference benchmark. Its surface can be modified with broad-spectrum capture molecules targeting the Fc domain of the target fusion protein, such as Protein A, Protein G, or anti-human IgG Fc nanobodies. Since the GLP-1 fusion protein contains the Fc fragment, regardless of its post-translational modification state, as long as the protein is successfully expressed and secreted by the cells, it will be captured by this channel and generate a binding response signal, which can directly reflect the total protein expression level in the current culture system.

[0061] The second channel serves as a specific screening window, its surface modified with a specific antibody targeting the hydroxylated lysine epitope. This antibody can only recognize and bind to protein molecules that have undergone specific hydroxylation modifications, such as K34 hydroxylation. For unmodified or incompletely modified proteins, it produces little or no signal. The signal intensity of the second channel represents the concentration of the effectively modified protein.

[0062] By comparing the signal differences between the two channels, such as calculating the ratio, the quality attributes of the product can be obtained in real time. The dual-channel design enables simultaneous decoupled monitoring of protein expression levels and modification integrity, avoiding the misjudgment of modification levels due to fluctuations in total protein content in traditional single-channel detection. This provides an accurate data foundation for subsequent calculation of real-time hydroxylation rate, ensuring that the screened cell lines not only have high yields but also correct active conformations.

[0063] In some embodiments, the control unit is further configured to determine the ratio of the binding response values ​​of the target protein and the ligand detected by the detection module, and to obtain the real-time hydroxylation rate; the control unit is further configured to adjust the working mode of the second pump group according to the real-time hydroxylation rate to adjust the delivery parameters of each micro-injection pump.

[0064] The control unit can receive raw signals from the first and second channels of the sensor unit in real time and calculate the ratio of their binding response values ​​using an internal algorithm. This ratio can be converted into a real-time hydroxylation rate after calibration using a standard curve, thus providing a direct reflection of the current product's modification quality percentage.

[0065] In this situation, based on this real-time calculated percentage, the control unit can initiate a PID control or fuzzy control algorithm to dynamically adjust the operating mode of the second pump group. For example, if the hydroxylation rate decreases, the algorithm will automatically calculate the required cofactor compensation amount and convert it into pulse frequency or injection speed commands for the micro-injection pump to change the delivery parameters, thereby adjusting the feeding rate.

[0066] In some embodiments, when the real-time hydroxylation rate is lower than a first preset threshold, the control unit is used to control the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate to maintain the reduction environment, and after a preset time interval, control the micro-injection pumps corresponding to the ferrous ion solution and the α-ketoglutaric acid solution to simultaneously increase the delivery rate of the ferrous ion solution and the α-ketoglutaric acid solution according to the stoichiometric ratio.

[0067] For situations where the real-time hydroxylation rate is low, i.e., the modification is insufficient, the control unit can execute a phased activation and supplementation control strategy.

[0068] When the hydroxylation rate is detected to be below a first preset threshold, such as 15%, the system first determines that the enzyme's active site may be inactivated due to an excessively strong intracellular oxidative environment. Therefore, the control unit prioritizes instructing the micro-injection pump corresponding to ascorbic acid to increase the delivery rate. As a strong reducing agent, ascorbic acid can rapidly reduce oxidized ferric ions back to active ferrous ions, thereby activating the existing lysyl hydroxylase. After a preset time interval allows the reduction reaction to proceed sufficiently, if the modification rate still does not meet the target, the system determines that the absolute amount of the modification regulatory factor is insufficient. At this time, the control unit instructs the corresponding micro-injection pump to synchronously increase the delivery rate of the ferrous ion pump and the α-ketoglutarate pump at a specific stoichiometric ratio, such as 1:1, to provide sufficient substrate for the enzymatic reaction.

[0069] Understandably, this hierarchical feedback strategy aligns with the catalytic mechanism of enzymes and effectively avoids the cytotoxic risks that may arise from blindly adding metal ions. By prioritizing the restoration of the reducing environment before replenishing the substrate, the system can achieve the most efficient restoration of modification capacity with minimal material consumption.

[0070] It should be noted that excessive ferrous ions can generate harmful free radicals through the Fenton reaction, while excessive α-ketoglutarate may interfere with the tricarboxylic acid cycle metabolic flux in cells.

[0071] In some embodiments, when the real-time hydroxylation rate is greater than or equal to a second preset threshold, the control unit is used to control each micro-injection pump in the second pump group to reduce the delivery rate to avoid cytotoxicity; the second preset threshold is greater than the first preset threshold.

[0072] Understandably, the system can introduce a toxicity avoidance mechanism for situations where the real-time hydroxylation rate is too high or has already reached the target. When the real-time hydroxylation rate is greater than or equal to a second preset threshold, such as 90%, the modification is close to saturation, and the current concentration of the modification regulator is sufficient or even potentially excessive. At this point, the control unit will immediately issue an instruction to reduce the delivery rate of all micro-injection pumps in the second pump group, or even stop delivery altogether.

[0073] In this embodiment, cell poisoning and resource waste caused by excessive feeding can be effectively prevented, ensuring that while obtaining high-quality products, the long-term activity and growth homeostasis of host cells are maintained, thus extending the effective production cycle.

[0074] In some embodiments, when the detection module detects that the oxidation level of the target protein is higher than a third preset threshold, the control unit is used to control the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate of the ascorbic acid solution, and / or the control unit is used to control the temperature control unit to reduce the temperature of the cell culture chamber.

[0075] It should be noted that when the detection module detects a decrease in the binding force of oxidation-sensitive sites and finds that the oxidation level of the target protein exceeds a third preset threshold, such as 5%, the control unit can activate a dual antioxidant strategy.

[0076] In some examples, the control unit can instruct the second pump set to significantly increase the delivery of ascorbic acid solution, utilizing its antioxidant properties to directly remove free radicals in the culture system.

[0077] In other examples, the control unit can instruct the temperature control unit to rapidly reduce the temperature of the cell culture chamber, for example, from 37°C to 30°C-32°C. Cooling can significantly reduce the chemical reaction rate of non-enzymatic oxidation reactions.

[0078] In other examples, the control unit can instruct the second pump group to significantly increase the delivery of ascorbic acid solution, and the control unit can also instruct the temperature control unit to rapidly reduce the temperature of the cell culture chamber.

[0079] Understandably, the antioxidant control strategies described above can solve the problem of oxidative side effects caused by highly active iron ions. While ensuring the normal lysine hydroxylation process, they can protect key protein sites from oxidative damage to the greatest extent possible, thereby ensuring the safety and efficacy of the final drug molecule.

[0080] In some embodiments, the oxidation of W31 / M251 can also be addressed in the following ways. W31 / M251 are specific tryptophan / methionine sites on the target GLP-1 fusion protein.

[0081] Since lysyl hydroxylase is a dioxygenase, it requires oxygen to participate in the reaction, but oxygen is also a factor that leads to the oxidation of W31 cells. When an oxidation signal is detected, the system's gas regulation module can be used to reduce the partial pressure of oxygen in the incoming gas, thereby reducing the dissolved oxygen concentration. The gas regulation module can include a stirring and oscillating device or a gas delivery device such as a gas supply pipe connected to a gas source. To prevent cell asphyxiation due to low oxygen partial pressure, the control unit can synchronously increase the rotation speed of the stirring and oscillating device, thereby improving the mass transfer coefficient. This allows oxygen to be rapidly transferred from the gas phase to the cells even under low oxygen partial pressure, reducing the time oxygen remains dissolved in the liquid phase.

[0082] During the intense hydroxylation reaction that occurs while the second pump unit delivers α-ketoglutarate, the control unit can maintain the oxygen partial pressure within a specific window of 40%-60% saturation using the gas regulation module. This window provides sufficient oxygen for the hydroxylase while precisely controlling the oxygen partial pressure to prevent excessive oxidation of tryptophan, thus achieving a precise process balance at the biochemical reaction mechanism level.

[0083] The control unit is also used to control the temperature control unit to reduce the temperature of the cell culture chamber when the cell density information fed back by the detection module meets the preset conditions, so as to reduce the temperature of the cell culture chamber from the first temperature to the second temperature at a preset rate, and maintain it at the second temperature until the end of the culture.

[0084] refer to Figure 4 The diagram illustrates temperature changes at different reaction stages within the reactor array. In the early stages of culture, the control unit can maintain a primary temperature, such as 36.5℃-37℃, to promote rapid cell division and proliferation. Simultaneously, the control unit continuously monitors cell density data from the sampling module or online probe.

[0085] When the received cell density reaches preset conditions, such as entering the late logarithmic growth phase or reaching a specific VCD value, the control unit automatically triggers a cooling program. The temperature control unit can be controlled to smoothly reduce the temperature of the culture chamber to a second temperature, which can be 30℃-32℃, at a preset gentle rate, such as 0.5℃ / 30min, and maintain this low temperature for the remainder of the culture cycle.

[0086] In some embodiments, temperature control can be specifically performed in the following three stages.

[0087] Phase 1: After inoculation, the temperature control unit is set to the first temperature of 37℃. The sampling module provides feedback on cell density information.

[0088] Phase Two: When the cell density reaches a preset condition, such as 10 × 10⁻⁶ 6 When cells / mL or when entering the late logarithmic growth phase, the control unit automatically triggers a cooling program.

[0089] Phase 3: The temperature control unit smoothly reduces the temperature to a second temperature, such as 30℃, at a preset rate, such as 0.5℃ / 30min, and maintains it until the end.

[0090] Under these conditions, high temperature promotes rapid cell division, while low temperature inhibits cell division, promotes the synthesis of metabolic flow-directing proteins, and lysyl hydroxylase exhibits better folding and stability and fewer oxidative side reactions at low temperatures.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A system for constructing and validating the function of recombinant cells, characterized in that, include: The reactor array includes multiple independent cell culture chambers, each equipped with an independent temperature control unit, stirring and oscillation device, and fluid interface; The feeding module is connected to the cell culture chamber via a microfluidic pipeline. The feeding module includes at least a first pump group for delivering basal culture medium and a second pump group for delivering modification and regulatory factors. A sampling module, located downstream of the reactor array, is used to periodically extract trace amounts of culture medium from each culture chamber and perform cell retention and aseptic filtration. The detection module, which is in fluid communication with the sampling module, includes a sensor unit whose surface is modified with ligands that specifically recognize specific post-translational modification domains of the target protein; The control unit is electrically connected to both the detection module and the feeding module, and is used to receive the detection data from the detection module and adjust the feeding rate of the second pump group based on the detection data.

2. The recombinant cell construction and functional verification system according to claim 1, characterized in that, The sensor unit includes a first channel and a second channel. The first channel is modified with a capture molecule targeting the Fc domain of the target fusion protein for detecting the protein expression level. The second channel is modified with a specific antibody targeting the hydroxylated lysine epitope for detecting the integrity of the protein modification or its biological activity.

3. The recombinant cell construction and functional verification system according to claim 1, characterized in that, The second pump set includes multiple independent micro-injection pumps, which are respectively connected to reservoirs containing ferrous ion solution, α-ketoglutaric acid solution and ascorbic acid solution.

4. The recombinant cell construction and functional verification system according to claim 1, characterized in that, The sampling module also includes a microfilter and a cell reflux line. The microfilter has a molecular weight cutoff that allows recombinant proteins to pass through but retains cells. One end of the cell reflux line is connected to the microfilter, and the other end is connected to the cell culture chamber. The cell reflux line is used to return the retained cells to the cell culture chamber to maintain the cell density within the reactor array.

5. The recombinant cell construction and functional verification system according to claim 3, characterized in that, The control unit is also used to determine the ratio of the binding response value between the target protein and the ligand detected by the detection module, and to obtain the real-time hydroxylation rate; the control unit is also used to adjust the working mode of the second pump group according to the real-time hydroxylation rate to adjust the delivery parameters of each micro-injection pump.

6. The recombinant cell construction and functional verification system according to claim 5, characterized in that, When the real-time hydroxylation rate is lower than a first preset threshold, the control unit controls the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate to maintain the reduction environment, and after a preset time interval, controls the micro-injection pumps corresponding to the ferrous ion solution and the α-ketoglutaric acid solution to simultaneously increase the delivery rate of the ferrous ion solution and the α-ketoglutaric acid solution according to the stoichiometric ratio.

7. The recombinant cell construction and functional verification system according to claim 6, characterized in that, When the real-time hydroxylation rate is greater than or equal to a second preset threshold, the control unit is used to control each micro-injection pump in the second pump group to reduce the delivery rate to avoid cytotoxicity; The second preset threshold is greater than the first preset threshold.

8. The recombinant cell construction and functional verification system according to claim 3, characterized in that, When the detection module detects that the oxidation level of the target protein is higher than a third preset threshold, the control unit is used to control the micro-injection pump corresponding to the ascorbic acid solution in the second pump group to increase the delivery rate of the ascorbic acid solution, and / or the control unit is used to control the temperature control unit to reduce the temperature of the cell culture chamber.

9. The recombinant cell construction and functional verification system according to any one of claims 1-8, characterized in that, The control unit is also used to control the temperature control unit to reduce the temperature of the cell culture chamber when the cell density information fed back by the detection module meets the preset conditions, so as to reduce the temperature of the cell culture chamber from the first temperature to the second temperature at a preset rate, and maintain it at the second temperature until the end of the culture.

10. The recombinant cell construction and functional verification system according to claim 1, characterized in that, The fluid interface is also provided with a laminar flow mixing zone, which is used to premix the modification regulatory factors before they enter the cell culture chamber.

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