Integrated urokinase automatic extraction system and extraction process thereof

Abstract

The application discloses an integrated urokinase full-automatic extraction system and an extraction process thereof, and belongs to the technical field of biological pharmacy. The application aims to solve the technical problems of large activity loss and poor batch consistency caused by discrete unit operation equipment and manual transfer in the existing urokinase extraction process. The application comprises: a closed fluid passage, which sequentially cascades a coarse filtration module, an online pH adjusting module, a tangential flow ultrafiltration module, a fluidized bed adsorption module and a gradient elution module along the material flow direction; and a central control unit, which is respectively connected with the control of each module and used for coordinating the sequential operation of each module according to preset process parameters. Through the deep integration of the closed fluid passage and the central control unit, the application eliminates the manual transfer link between traditional discrete unit operations, avoids the material exposure and the biological exposure risk of the operator, improves the batch process consistency, and significantly improves the activity retention rate of urokinase and the raw material utilization rate.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and specifically relates to an integrated fully automated urokinase extraction system and its extraction process. Background Technology

[0002] Urokinase is a proteolytic enzyme extracted from human urine and is widely used clinically for thrombolytic therapy in thrombotic diseases such as acute myocardial infarction and pulmonary embolism. Currently, industrial-scale urokinase extraction processes typically involve multiple unit operations, including coarse filtration, pH adjustment to precipitate impurities, ultrafiltration concentration, ion exchange or affinity chromatography purification, and gradient elution collection.

[0003] In existing industrial-scale urokinase extraction technologies, the various unit operations are discretely combined, with coarse filter tanks, pH adjustment reactors, ultrafiltration devices, and chromatography columns operating independently. Materials rely on manual transfer or open pipeline transport. This exposes the active urokinase protein to the external environment for extended periods, making it susceptible to microbial contamination and oxidative denaturation, and exposing operators to bio-exposure risks. Key process nodes such as pH adjustment endpoints, ultrafiltration termination timing, and elution peak cutting rely on operator experience, resulting in significant batch-to-batch variations. The relative standard deviation of urokinase activity content between batches typically exceeds 15%, failing to meet the batch-to-batch consistency requirements of the Chinese Pharmacopoeia for biological products. Manual transfer, equipment cleaning, and rebalancing between units consume substantial time; the cumulative exposure time of urokinase at room temperature can reach 4 to 8 hours, with activity retention generally below 60%, leading to low raw material utilization. Furthermore, while individual devices may be equipped with local programmable logic controllers (PLCs), the lack of a unified central control unit for closed-loop coordination across processes prevents the achievement of fully automated "one-click" operation from coarse filtration to gradient elution.

[0004] A search revealed that existing technologies mainly focus on improvements to single devices: CN202021981587.6 discloses a urine collection device for kidney disease patients, involving coarse urine filtration, but does not involve the integration of subsequent purification modules; CN202322989291.9 discloses a tangential flow ultrafiltration system, which optimizes the structure of the tangential flow ultrafiltration membrane package, but does not form a closed loop with upstream coarse filtration and downstream chromatography; the ÄKTA series chromatography system has automated control functions, and its original design was for a single chromatography process, lacking cascade interfaces for online pH adjustment and tangential flow ultrafiltration.

[0005] Therefore, there is a lack in the field of integrated systems that sequentially cascade coarse filtration, online pH adjustment, tangential flow ultrafiltration, fluidized bed adsorption, and gradient elution through a closed fluid passage and are uniformly coordinated by a central control unit. Summary of the Invention

[0006] The purpose of this invention is to provide an integrated, fully automated urokinase extraction system and its extraction process, which solves the technical problems of discrete unit operation equipment, reliance on manual transport leading to significant activity loss, and poor batch-to-batch consistency in existing urokinase extraction processes.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An integrated fully automated urokinase extraction system includes: A closed fluid passage is used to cascade the coarse filtration module, online pH adjustment module, tangential flow ultrafiltration module, fluidized bed adsorption module and gradient elution module sequentially along the material flow direction. The central control unit is connected to the coarse filtration module, the online pH adjustment module, the tangential flow ultrafiltration module, the fluidized bed adsorption module, and the gradient elution module respectively, in order to coordinate the operation of each module according to the preset process parameters. The central control unit is configured to perform the following steps: Step S1: Pass the raw material liquid into the coarse filtration module for preliminary filtration and impurity removal; Step S2: Pass the coarsely filtered liquid into the online pH adjustment module, and the central control unit automatically adjusts the pH of the liquid to the preset range according to the online pH monitoring value; Step S3: Pass the pH-adjusted feed solution into the tangential flow ultrafiltration module for concentration and buffer replacement; Step S4: Pass the ultrafiltration solution into the fluidized bed adsorption module so that the target protein is captured by the adsorption medium; Step S5: Control the gradient elution module to perform gradient elution of the adsorbed target protein and collect the target component.

[0008] Furthermore, the coarse filtration module includes a bag filter and a security filter arranged in series. The bag filter has a filtration accuracy of 50~100μm, and the security filter has a filtration accuracy of 10~20μm.

[0009] Furthermore, the online pH adjustment module includes an online pH monitoring probe, an automatic acid-base feeding device, and a static mixer; the central control unit controls the automatic acid-base feeding device according to the feedback signal from the online pH monitoring probe to control the pH adjustment accuracy of the feed solution within ±0.1.

[0010] Furthermore, the tangential flow ultrafiltration module includes a circulation tank, a tangential flow membrane pack, a circulation pump, and a pressure / flow monitoring unit. The molecular weight cutoff of the tangential flow membrane pack is 10~30kDa. The central control unit automatically adjusts the rotation speed of the circulation pump according to the transmembrane pressure and the permeate flow rate.

[0011] Furthermore, the fluidized bed adsorption module includes a fluidized bed column, an adsorption medium, a flow rate controller, and an online ultraviolet (UV) monitor; the central control unit controls the process switching based on the penetration curve signal from the UV monitor.

[0012] Furthermore, the gradient elution module includes a gradient mixer, an elution pump, and a component collector; the central control unit automatically switches the component collector to the target component collection port based on the elution peak signal from the ultraviolet online monitoring instrument.

[0013] Furthermore, the sealed fluid passage uses sanitary 316L stainless steel pipes or medical-grade silicone tubing, and the connection and disconnection between each module are controlled by a pneumatic diaphragm valve, which is uniformly controlled by the central control unit.

[0014] Furthermore, the central control unit has an embedded process control algorithm, which includes sequential control, parameter closed-loop control, fault diagnosis and fail-safe mechanism, and data recording function.

[0015] Furthermore, the present invention also includes a raw material inlet, a target component collection port, and a waste liquid discharge port; the raw material inlet is connected to the inlet of the coarse filtration module, the target component collection port is connected to the target component outlet of the gradient elution module, and the waste liquid discharge port is connected to the waste liquid outlet of the fluidized bed adsorption module and the gradient elution module.

[0016] Furthermore, in step S2, the preset range is pH 8.5~9.5.

[0017] Furthermore, in step S3, the concentration factor is 510 times, and the transmembrane pressure is maintained at 0.15-0.25 MPa.

[0018] Furthermore, in step S4, the adsorption medium is DEAE-Sepharose or a specific affinity medium, and the loading flow rate is 150~250 cm / h.

[0019] Furthermore, in step S5, the gradient elution uses a linear gradient of 0~1M NaCl, and the elution volume is 15~25 column volumes.

[0020] Furthermore, the central control unit monitors the process parameters of each module in real time during operation. When any parameter exceeds a preset safety threshold, it automatically suspends operation and enters a safe state.

[0021] Furthermore, the closed fluid passage is equipped with an online flow matching and adjustment unit between the tangential flow ultrafiltration module and the fluidized bed adsorption module. This online flow matching and adjustment unit includes a buffer volume control tank, a replenishment pump, and an electromagnetic flow rate sensor. The central control unit is configured with a feedforward-feedback composite control algorithm, and the central control unit adjusts the flow rate based on the real-time permeate flow rate change rate of the tangential flow ultrafiltration module. and the liquid level scalar of the buffer volume-regulating tank. Calculate the set feed flow rate of the fluidized bed adsorption module. The calculation formula is: In the formula, This indicates the set feed flow rate of the fluidized bed adsorption module, in cm / h. The reference critical flow rate for maintaining the fluidization state of the adsorption medium is expressed in cm / h. The level feedback gain coefficient is dimensionless. The liquid level change rate of the buffer volume-regulating tank is expressed in mm / s; This is the flow feedforward gain coefficient, in units of ; This represents the real-time flow rate change rate of the permeate in the tangential flow ultrafiltration module, expressed in L / s. The real-time flow rate of the permeate is obtained by a flow meter installed on the permeate pipeline. The central control unit collects the instantaneous flow rate of the permeate at a sampling period of 50ms. And calculated using first-order backward difference In the formula ms, and These represent the nth and (n-1)th sampling times, respectively; the rate of change of the permeate flow rate. The flow rate of the concentrate entering the buffer volume-reducing tank The mapping function between them satisfies the conditions determined by calibration experiments. , The data is stored in the form of a lookup table built into the central control unit; the central control unit adjusts the speed of the replenishment pump via frequency conversion to ensure that the actual flow rate entering the fluidized bed adsorption module matches the set feed flow rate. The deviation is within ±5%.

[0022] Furthermore, the ultraviolet online monitoring instrument is a dual-wavelength, dual-channel ultraviolet detector that supports simultaneous acquisition of dual wavelengths, and the central control unit is embedded with a dynamic derivative peak cutting algorithm module; the dynamic derivative peak cutting algorithm module obtains the absorbance difference of the eluent at the first wavelength of 280 nm and the second wavelength of 214 nm. And calculate the absorbance difference. Second derivative with time The relation satisfies: The central control unit acquires data at a frequency of 10Hz. Discrete sequence, second derivative Calculated using the three-point central difference scheme: ,in s; where, This represents the real-time absorbance at a wavelength of 280 nm, expressed in AU. Indicates wavelength Real-time absorbance at nm, in AU; The background scattering correction coefficient is dimensionless. The second derivative of the absorbance difference, in units of 1000 ppm. ; Indicates time, in seconds; , , These represent the nth, (n+1)th, and (n-1)th sampling times, respectively; the central control unit detects the second derivative... Crossing zero and the absorbance difference When the concentration exceeds a preset threshold, a switching execution signal is sent to the gradient elution module, triggering the component collector to switch to the target component collection port.

[0023] In addition, the present invention also discloses an integrated fully automated urokinase extraction process, including the use of an integrated fully automated urokinase extraction system as described above.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention reconstructs the urokinase extraction process by cascading a coarse filtration module, an online pH adjustment module, a tangential flow ultrafiltration module, a fluidized bed adsorption module, and a gradient elution module along a closed fluid pathway, and by setting up a central control unit to coordinate the operation of each module. This eliminates the manual transfer between traditional discrete unit operations, allowing materials to flow continuously in a completely closed environment, directly reducing the risk of microbial contamination and oxidative denaturation caused by exposure to active proteins, while also avoiding biological exposure for operators. The central control unit implements closed-loop scheduling of process parameters for each module, replacing critical operations such as pH adjustment endpoints, ultrafiltration termination timing, and elution peak cutting that rely on manual experience, significantly improving batch-to-batch process consistency. Zero-wait connection between modules shortens the total residence time of urokinase at room temperature, significantly improving activity retention and raw material utilization.

[0025] In a further improvement, this invention introduces an online flow matching and adjustment unit between the tangential flow ultrafiltration module and the fluidized bed adsorption module in a closed fluid passage, and runs a feedforward-feedback composite control algorithm. This technique utilizes a composite calculation of the permeate flow rate change rate and the scalar level of the buffer constant-volume tank to dynamically adjust the feed rate of the fluidized bed adsorption module. This effectively reconciles the mismatch between the nonlinear flow rate decay at the ultrafiltration product end and the rigid flow rate requirement at the fluidized bed feed end, thereby significantly narrowing the fluctuation range of the adsorption medium bed expansion height, significantly reducing the medium loss rate, and improving the dynamic binding capacity.

[0026] This invention upgrades the online UV monitor to a dual-wavelength, dual-channel UV detector and deploys a dynamic derivative peak-cutting algorithm. By subtracting baseline drift through the difference in absorbance between the two wavelengths and utilizing the zero-crossing feature of the second derivative, the inflection point of the elution peak is accurately identified. The cut-off collection is unaffected by batch-to-batch absolute concentration fluctuations, effectively removing shoulder peaks of high-molecular-weight impurity proteins. The final product purity and specific activity are significantly improved, and the relative standard deviation of batch-to-batch activity recovery is further reduced. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a diagram of the overall system architecture of the present invention.

[0029] Figure 2 This is a cascade connection diagram of the closed fluid passage of the present invention.

[0030] Figure 3 This is a control logic block diagram of the central control unit of the present invention.

[0031] Figure 4 This is a process curve from an embodiment of the present invention.

[0032] Figure 5 This is the elution chromatogram of Example 1 of the present invention.

[0033] Figure 6 This is the main process flow diagram of the present invention.

[0034] Figure 7 This is a flowchart of the feedforward-feedback composite control flow matching and adjustment sub-process of the present invention.

[0035] Figure 8 This is a flowchart of the dynamic derivative peak cutting, elution, and collection subprocess of the present invention. Detailed Implementation

[0036] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0037] The following disclosure provides many different implementations or examples for carrying out different structures of the embodiments of the present invention. To simplify the disclosure of the embodiments of the present invention, specific examples of components and arrangements are described below. Of course, these are merely examples and are not intended to limit the embodiments of the present invention. Furthermore, reference numerals and / or reference letters may be repeated in different examples of the embodiments of the present invention; such repetition is for simplification and clarity and does not in itself indicate a relationship between the various implementations and / or arrangements discussed.

[0038] The following is in conjunction with the appendix Figures 1-8 The embodiments of the present invention will be described in detail below.

[0039] Example 1: This example discloses an integrated fully automated urokinase extraction system, which includes a raw material inlet, a coarse filtration module, an online pH adjustment module, a tangential flow ultrafiltration module, a fluidized bed adsorption module, a gradient elution module, a target component collection port, a waste liquid discharge port, and a central control unit.

[0040] Each module is connected to a closed fluid passage via sanitary 316L stainless steel pipes or medical-grade silicone tubing. The pipes are equipped with pneumatic diaphragm valves, flow meters, pressure sensors, and temperature sensors, and the signals from each sensor are connected to the central control unit. The raw material inlet is connected to the inlet of the coarse filtration module, the target component collection port is connected to the target component outlet of the gradient elution module, and the waste liquid discharge port is connected to the waste liquid outlets of the fluidized bed adsorption module and the gradient elution module.

[0041] The coarse filtration module consists of bag filters and security filters connected in series. The bag filters have a filtration accuracy of 50μm to 100μm, and the security filters have a filtration accuracy of 10μm to 20μm. The feed liquid enters the coarse filtration module through the feed inlet to remove large particulate suspended solids, flocculent precipitates, and other impurities. An online turbidity monitor is installed at the outlet of the coarse filtration module; when the outlet turbidity exceeds 5 NTU, the central control unit issues an alarm and automatically switches the filter bag.

[0042] The online pH adjustment module includes an online pH monitoring probe, an automatic acid / alkali feeding device, and a static mixer. The coarsely filtered liquid flows into the online pH adjustment module. The online pH monitoring probe detects the pH value of the liquid in real time. The central control unit controls a peristaltic pump to automatically add acid or alkali solution according to a preset pH target value. After uniform mixing by the static mixer, the solution is output, with pH adjustment accuracy controlled within ±0.1.

[0043] The tangential flow ultrafiltration module includes a circulation tank, a tangential flow membrane pack, a circulation pump, and a pressure / flow monitoring unit. The tangential flow membrane pack has a molecular weight cutoff of 10kDa to 30kDa, effectively retaining urokinase with a molecular weight cutoff of approximately 54kDa. The pH-adjusted feed solution enters the circulation tank and, driven by the circulation pump, flows tangentially through the membrane pack, completing concentration, removal of small molecule impurities, and buffer replacement. The central control unit adjusts the circulation pump speed in real time based on the circulation tank level, transmembrane pressure, and permeate flow rate. When the concentration factor reaches a preset value, the pump automatically stops and proceeds to the next process.

[0044] The fluidized bed adsorption module includes a fluidized bed column, adsorption medium, flow rate controller, and UV online monitoring instrument. The concentrated feed solution enters from the bottom of the fluidized bed column, and the adsorption medium is kept in a fluidized state at an appropriate flow rate. Urokinase is captured by the adsorption medium, while impurities and waste liquid are discharged through the waste liquid outlet. The UV online monitoring instrument continuously monitors the absorbance of the outlet fluid at 280 nm; the central control unit automatically switches to washing buffer for column washing when the breakthrough curve reaches a preset threshold.

[0045] The gradient elution module includes a gradient mixer, an elution pump, and a component collector. After washing, the central control unit controls the gradient mixer to perform elution according to a preset program, while an online UV monitor tracks the elution peaks in real time. When the target peak appears, the central control unit automatically switches the collection valve to the target component collection port, while non-target components are discharged through the waste liquid discharge port.

[0046] The central control unit consists of a process control server, a human-machine interface, and distributed input / output modules. It embeds process control algorithms, makes real-time decisions based on online monitoring data from each module, and issues instructions to each actuator, realizing sequential control, parameter closed-loop control, fault diagnosis and fail-safe mechanisms, and data logging functions. Sequential control ensures that steps S1 to S5 switch automatically in a fixed order; parameter closed-loop control implements PID closed-loop adjustment of key process variables in each module; the fault diagnosis and fail-safe mechanism automatically pauses the system and enters a safe state when any module malfunctions, while triggering an alarm; the data logging function automatically saves process parameter curves for each batch, meeting the data integrity requirements of Good Manufacturing Practices (GMP).

[0047] To facilitate understanding by those skilled in the art, Table 1 lists the key hardware components, process parameters, and monitoring logic of each module in the integrated urokinase fully automated extraction system.

[0048] Table 1. System component hardware composition, parameter thresholds and control logic correspondence table;

[0049] The closed fluid pathway uses sanitary 316L stainless steel pipes or medical-grade platinum-cured silicone tubing, connected by Tri-Clamp quick-connect clamps or aseptic welding. The overall airtightness of the system meets the sanitary standards for biopharmaceutical equipment. The pneumatic diaphragm valves between modules are centrally controlled to ensure unidirectional flow of materials within the preset pathways and prevent cross-contamination.

[0050] In practice, 1000 liters of human urine sample is taken and passed through a coarse filtration module, sequentially through a 50μm bag filter and a 20μm security filter. The turbidity of the filtered solution is 3 NTU. The solution then enters the online pH adjustment module. Based on the feedback signal from the online pH monitoring probe, the central control unit drives a peristaltic pump to add acid or alkali solution dropwise. After thorough mixing by a static mixer, the pH of the solution is adjusted to 9.0 ± 0.1. The solution adjusted to the target pH then enters the tangential flow ultrafiltration module, where it is concentrated using a membrane with a molecular weight cutoff of 30 kDa. The transmembrane pressure is maintained between 0.15 MPa and 0.25 MPa, and the solution is concentrated to 100 liters. The concentrate is then transferred to the fluidized bed adsorption module, using DEAE-Sepharose FastFlow adsorption medium as the loading medium at a flow rate of 200 cm / h. After adsorption, the sample was washed with 5 column volumes of equilibration buffer, followed by elution with a linear gradient of 0 mol / L to 0.5 mol / L sodium chloride, for a total elution volume of 20 column volumes. The absorbance at 280 nm was monitored in real-time using an online UV monitor. The central control unit automatically switched the component collection valve to the target component collection port based on the elution peak signal to collect the target component.

[0051] In this embodiment, the fully automated operation took 1.8 hours, the urokinase activity retention rate was 87.3%, the specific activity reached 65,000 IU / mg, and the inter-batch relative standard deviation of three consecutive batches was 4.2%.

[0052] Example 2: This example verifies the system's adaptability to high-viscosity raw materials. High-viscosity raw materials, having undergone low-temperature precipitation treatment at 2-8℃, with a dynamic viscosity greater than 50 cP, were used. The central control unit automatically added an equal volume of Tris-HCl equilibrium buffer (pH 9.0) to pre-dilute the raw material solution based on the online viscometer reading. After pre-dilution, the dynamic viscosity of the solution decreased to approximately 25 cP. The diluted solution was then fed into the coarse filtration module. Subsequent online pH adjustment, tangential flow ultrafiltration, fluidized bed adsorption, and gradient elution steps were identical to those in Example 1. During operation, neither the coarse filtration module nor the tangential flow ultrafiltration module experienced clogging. The total system operating time was 2.2 hours, and the urokinase activity retention rate was 85.1%, indicating that the system has good adaptability to high-viscosity raw materials.

[0053] Example 3: This example tests the Fail-Safe fault response mechanism. During the operation of Example 1, an abnormal increase in transmembrane pressure in the tangential flow ultrafiltration module was artificially simulated. When the transmembrane pressure sensor reading reached 0.36 MPa, exceeding the safety threshold of 0.35 MPa, the central control unit automatically closed the feed pneumatic diaphragm valve of the tangential flow ultrafiltration module within 3 seconds, stopped the circulation pump, and simultaneously switched the fluidized bed adsorption module to low-speed circulation of the equilibrium buffer, so that the adsorption medium and the captured target protein were in a standby equilibrium state, and a red alarm popped up on the human-machine interface. After the fault was cleared, the operator clicked the continue button, and the central control unit automatically retrieved the real-time process parameters stored before the shutdown, including the transmembrane pressure setpoint, circulation pump speed, valve opening status, and buffer circulation flow rate of the fluidized bed adsorption module, resuming operation from the breakpoint in step S3 without having to repeat the feed pretreatment and adsorption medium equilibration operation. Activity testing of the target components collected before and after the fault showed no significant difference, confirming that the Fail-Safe mechanism effectively protected the intermediate product.

[0054] Example 4: Based on the hardware architecture of Example 1, this example addresses the fluid dynamics mismatch problem in the cascade process of the tangential flow ultrafiltration module and the fluidized bed adsorption module by introducing an online flow matching and adjustment unit and running a feedforward-feedback composite control algorithm for process optimization.

[0055] The membrane flux of a tangential flow ultrafiltration module decreases non-linearly with increasing concentration factor and the thickness of the concentration polarization layer on the membrane surface. During the increase in concentration factor from 1 to 10, the typical flux decrease can reach 40% to 60% of the initial flux. Fluidized bed adsorption modules require the feed flow rate to be strictly maintained within the critical fluidization range corresponding to the selected DEAE-Sepharose FastFlow adsorption medium, i.e., 170 to 250 cm / h, to maintain a stable expansion rate of the adsorption medium. Flow rates below 170 cm / h lead to fluidized bed collapse and reduced mass transfer efficiency; flow rates above 250 cm / h cause the adsorption medium to be lost with the waste liquid. Conventional constant-rate pumping methods cannot simultaneously accommodate the flow rate decrease at the ultrafiltration product end and the rigid flow rate requirements at the fluidized bed feed end.

[0056] An online flow matching and regulating unit is connected between the concentrate outlet of the tangential flow ultrafiltration module and the bottom inlet of the fluidized bed adsorption module in a closed fluid passage. The online flow matching and regulating unit includes a 50L 316L stainless steel buffer tank. A radar level gauge is installed on the side wall of the buffer tank, and a variable frequency cam rotor replenishment pump is connected to the bottom outlet. An electromagnetic flow rate sensor is installed at the end of the pipeline. A bypass of the replenishment pump is connected to a balance buffer storage tank, controlled by a proportional-integral electric valve, for replenishing the system with the base liquid.

[0057] The central control unit acquires the instantaneous flow rate reading from the flow meter on the permeate line of the tangential flow ultrafiltration module at a sampling period of 50ms. The rate of change of permeate flow rate was calculated using first-order backward difference. The calculation formula is: In the formula ms, and These represent the nth and (n-1)th sampling times, respectively. The rate of change of the permeate flow rate... The flow rate of the concentrate entering the buffer volume-determining tank The mapping function between them satisfies the conditions determined by calibration experiments. Calibration was performed on the selected 30kDa membrane pack model and urine matrix conditions, using the mapping function. Stored in the form of a lookup table built into the central control unit. The radar level gauge synchronously provides feedback on the scalar level in the buffer volume control tank. The feedforward-feedback composite control algorithm built into the central control unit is based on the input variables. , and Calculate the set feed flow rate of the fluidized bed adsorption module.

[0058] The mathematical model of the feedforward-feedback composite control algorithm is as follows: ; In the formula, This indicates the set feed flow rate of the fluidized bed adsorption module, in cm / h. The reference critical flow rate for maintaining the fluidization state of the adsorption medium is expressed in cm / h. The level feedback gain coefficient is dimensionless. The liquid level change rate of the buffer constant volume tank is expressed in mm / s; the flow feedforward gain coefficient is expressed in units of... ; This represents the real-time flow rate change rate of the permeate in the tangential flow ultrafiltration module, expressed in L / s.

[0059] In practice, The flow rate was set at 180 cm / h, a value determined based on the critical fluidization parameters of the selected DEAE-Sepharose FastFlow adsorption medium in a urine matrix. Level feedback gain coefficient. Set to 2.5, flow feedforward gain coefficient The value is set to 0.8. During the initial operation of the tangential flow ultrafiltration module, the membrane flux is at its maximum, and a large amount of concentrate enters the buffer volumetric tank, causing a rapid rise in the liquid level scalar. This is related to the feedback term in the algorithm. When the value is positive and dominant, the central control unit instructs the replenishment pump to increase its speed, thereby increasing the feed rate to the fluidized bed adsorption module. In the later stages of operation, membrane flux decay leads to a change in the permeate flow rate. A negative step occurs, feedforward term Early intervention in the calculation, adjusting the liquid level scalar downwards before a substantial drop occurs. When the available concentrate supply to the system is insufficient to maintain At this time, the central control unit opens the bypass proportional-integral electric valve and precisely pumps in the equilibrium buffer to replenish the volume according to the flow rate difference, so as to maintain the fluid dynamic balance of the fluidized bed.

[0060] A continuous operation experiment was conducted using 1500 liters of human urine as raw material. The control group had its feedforward-feedback composite control algorithm disabled and used a constant-level PID single-loop control for the replenishment pump, with the same hardware configuration as the experimental group. The experimental group had its feedforward-feedback composite control algorithm enabled. An ultrasonic interface analyzer placed on the outer wall of the fluidized bed column was used to record the bed expansion height of the adsorption medium in real time, and the number of resin particles in the effluent was detected by post-column sampling to assess the medium loss rate. After elution, the total active units of the target component were measured, and the dynamic binding capacity was calculated.

[0061] Table 2 Comparison of Flow Matching Regulation and Control Effects;

[0062] Note: The fluctuation range of bed expansion height is expressed as the relative deviation based on the set expansion height; the media loss rate is the percentage of the mass of resin lost in the post-column effluent to the total mass of the initial packed media; the dynamic binding capacity is calculated based on the 10% breakthrough point.

[0063] The feedforward-feedback composite control algorithm compressed the fluctuation range of bed expansion height to within -2.1% to +2.1%. Under stable fluid shear force, the adsorption medium formed a uniformly distributed pore network. The medium loss rate decreased to near the equipment detection limit. The stabilization of the hydrodynamic state eliminated the channeling effect within the bed, increasing the dynamic binding capacity of urokinase from 14.3 mg / mL to 19.8 mg / mL. The total system time was reduced from 2.8 h to 2.5 h, avoiding the dead zone time caused by waiting for liquid level accumulation at the end of the membrane flux decline.

[0064] Example 5: Based on the hardware architecture of Example 1, this example upgrades the UV online monitoring instrument to a dual-wavelength dual-channel UV detector to address the peak cutting accuracy issue of the gradient elution module, and runs a dynamic derivative peak cutting algorithm for comparative testing.

[0065] The UV online monitoring instrument in Example 1 is a standard single-wavelength detector that relies on an absolute absorbance threshold to trigger component collection. In this example, the UV online monitoring instrument is replaced with a dual-wavelength, dual-channel UV detector that supports simultaneous dual-wavelength acquisition. A dynamic derivative peak cutting algorithm module is deployed in the central control unit. The remaining hardware configuration is identical to that of Example 1, including the cascaded fluid pathways of the coarse filtration module, online pH adjustment module, tangential flow ultrafiltration module, fluidized bed adsorption module, and gradient elution module. In Example 5, no online flow matching adjustment unit is provided between the tangential flow ultrafiltration module and the fluidized bed adsorption module; this part follows the direct cascade method of Example 1.

[0066] Urokinase contains isoenzyme structures with varying activities and high-molecular-weight polymers. During linear gradient elution with 0 to 1 M NaCl, the NaCl concentration continuously increases from 0 mol / L to 1 mol / L, causing the buffer medium's refractive index to monotonically increase from approximately 1.3330 to approximately 1.3410. This results in a significant baseline drift during conventional 280 nm single-wavelength UV monitoring, with drift amplitudes reaching 15 to 25 mAU. The elution behavior of high-molecular-weight contaminating proteins highly overlaps with that of urokinase, forming indistinguishable shoulders at the elution peak. Traditional cleavage methods relying on absolute absorbance thresholds are highly susceptible to contaminating the target component with these leading contaminating proteins, or to prematurely closing the collection valve due to baseline drift, leading to the loss of the effective component.

[0067] The gradient elution module's outlet piping is equipped with a dual-wavelength, dual-channel ultraviolet detector supporting simultaneous dual-wavelength acquisition. The dual-wavelength, dual-channel ultraviolet detector employs a holographic concave grating for beam splitting, with a flow cell optical path of 5 mm. The central control unit's data bus synchronously reads the digital signals from the dual-wavelength, dual-channel ultraviolet detector at wavelengths of 280 nm and 214 nm at a frequency of 10 Hz.

[0068] In the initial stage of signal processing, high-frequency noise caused by air bubbles in the pipeline is removed by moving average filtering. The filtering window width is 5 sampling points, corresponding to a 0.5-second time window.

[0069] Subsequently, the dynamic derivative peak cutting algorithm module obtains the absorbance difference, and the calculation model is as follows: In the formula, This represents the real-time absorbance at a wavelength of 280 nm, expressed in AU. This represents the real-time absorbance at a wavelength of 214 nm, expressed in AU. is the background scattering correction coefficient, which is dimensionless. The values ​​were determined based on blank NaCl gradient elution data: A linear gradient elution of 0 to 1 M NaCl was run without sample loading, and baseline signals for the 280 nm and 214 nm channels were recorded. The results were then fitted using the least squares method. make Minimizing the baseline drift variance in this embodiment The calibration value is 0.35. The difference between the two wavelengths physically subtracts the baseline drift caused by changes in refractive index. Protein peptide bonds exhibit strong absorption at 214 nm, and aromatic amino acid residues show characteristic absorption at 280 nm; the absorbance difference is... It can amplify the spectral response differences between the target protein and the polymer.

[0070] To accurately locate the cutting inflection point of the elution peak, the dynamic derivative peak cutting algorithm module... Real-time second derivative calculations are performed. The central control unit acquires data at a frequency of 10Hz. Discrete sequence, second derivative Calculated using the three-point central difference scheme: ;in s, in the formula The second derivative of the absorbance difference, in units of 1000 ppm. The zero-crossing point of the second derivative curve corresponds to the inflection point of the original elution peak curve, that is, the extreme position of the rate of change of the target protein concentration in the eluent.

[0071] After the elution program is started, the 0 to 1M NaCl linear gradient solution gradually desorbs the protein on the adsorption medium. The curve begins to rise when the elution volume reaches 12 column volumes, at which point the central control unit's monitoring logic determines... When the concentration exceeds the set threshold of 50 mAU, the peak initiation identification marker is triggered. As elution proceeds, a shoulder-like peak composed of high-molecular-weight impurities appears. Slower growth rate, first derivative From positive to negative, second derivative During the initial dip below zero, high-purity urokinase has not yet appeared in the eluent, and the central control unit maintains the pneumatic diaphragm valve in waste liquid discharge mode. Once the main peak begins elution... After a sharp rise, it crosses the peak, and the first derivative... From positive to negative, second derivative The peak reverses again and crosses zero. The dynamic derivative peak cutting algorithm module recognizes the zero-crossing feature and immediately sends a control pulse. The pneumatic diaphragm valve actuates, and the component collector instantly switches to the target component collection port to begin receiving the core active peak. After elution enters the tailing stage, The changes tend to be gradual and Once the temperature drops below 20mAU, the central control unit resets the valve, ending the collection process.

[0072] The dynamic derivative peak cutting algorithm module transforms threshold judgment into curvature feature recognition. The cutting action is based on the second derivative. Zero-crossing feature triggering, The physical meaning of zero point corresponds to the extreme position of the concentration change rate on the elution peak curve, which is unrelated to the overall shift in absolute concentration levels between batches. The cutting logic is controlled by the inherent mathematical characteristics of protein elution kinetics.

[0073] Three batches of concentrated human urine were compared under the same elution gradient. The conventional group was collected according to a fixed 280 nm UV threshold (activated above 100 mAU, deactivated below 50 mAU); the dynamic cutting group was controlled by a central control unit executing the second-derivative logic described above. After ultrafiltration and desalting, the purity and high molecular weight impurity content of the collected solution were determined by size exclusion high-performance liquid chromatography (HPLC), and specific activity was determined using a chromogenic substrate method.

[0074] Table 3. Comparison of the effects of different elution peak cutting methods;

[0075] Note: SEC-HPLC purity was calculated using the 280nm chromatographic peak area normalization method; high molecular weight impurity content was the percentage of the total peak area of ​​urokinase with retention times shorter than the main peak; specific activity was detected using the chromogenic substrate method of the Chinese Pharmacopoeia (2020 edition); RSD was calculated from the results of 3 independent batches.

[0076] In Example 5 above, the specific activity of urokinase was determined using the chromogenic substrate method according to the Chinese Pharmacopoeia (2020 edition). Purity was determined by SEC-HPLC using size exclusion high-performance liquid chromatography (column: TSKgel G3000SWXL, mobile phase: 0.1 mol / L phosphate buffer pH 6.8, flow rate: 0.5 mL / min, detection wavelength: 280 nm). The content of high molecular weight impurities was calculated using the peak area normalization method of the SEC-HPLC chromatogram. In Example 4, the dynamic binding capacity of urokinase was determined using frontier analysis, and the media loss rate was calculated as the ratio of the number of resin particles in the post-column effluent to the total amount of sample media.

[0077] The test data showed that the method in Example 1 introduced a large amount of elution shoulder material, with high molecular weight impurities accounting for 12.7% of the collected components, severely diluting the specific activity of the active ingredient. The dual-wavelength differential and second-derivative inflection point finding technology removed the leading and tailing segments containing impurities, resulting in a final product chromatographic purity of 96.1%, a reduction in high molecular weight impurity content to 1.8%, and an increase in specific activity from 58,000 IU / mg to 95,000 IU / mg. The system does not rely on manual interpretation of the chromatograms by operators; the dynamic derivative peak cutting algorithm module completes peak shape identification and physical action command issuance, reducing the relative standard deviation of inter-batch activity recovery to 3.2%, meeting the process validation standards for large-scale pharmaceutical manufacturing. The integrated system has a closed and timely data link, avoiding the time lag diffusion caused by manual handling of eluent in discrete systems.

[0078] Table 4 summarizes the operating conditions and results of Examples 1 to 3. Tables 4 and 5 provide comparative data for the newly added Example 4 and Example 5, respectively.

[0079] Table 4 Summary of Operating Conditions and Results of the Examples;

[0080] Table 5 provides a horizontal comparison of the key indicators of the present invention with existing discrete segmentation extraction processes.

[0081] Table 5 Comparison of key indicators between the present invention and existing processes;

[0082] The comparative data in Table 5 show that the present invention, by utilizing the deep integration of a closed fluid pathway and a central control unit, solves the technical problems of large activity loss and poor batch-to-batch consistency in urokinase extraction from the system architecture level, achieving unexpected technical results.

[0083] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An integrated fully automated urokinase extraction system, characterized in that, include: A closed fluid passage is used to cascade the coarse filtration module, online pH adjustment module, tangential flow ultrafiltration module, fluidized bed adsorption module and gradient elution module sequentially along the material flow direction. The central control unit is connected to the coarse filtration module, the online pH adjustment module, the tangential flow ultrafiltration module, the fluidized bed adsorption module, and the gradient elution module respectively, in order to coordinate the operation of each module according to the preset process parameters. The central control unit is configured to perform the following steps: Step S1: Pass the raw material liquid into the coarse filtration module for preliminary filtration and impurity removal; Step S2: Pass the coarsely filtered liquid into the online pH adjustment module, and the central control unit automatically adjusts the pH of the liquid to the preset range according to the online pH monitoring value; Step S3: Pass the pH-adjusted feed solution into the tangential flow ultrafiltration module for concentration and buffer replacement; Step S4: Pass the ultrafiltration solution into the fluidized bed adsorption module so that the target protein is captured by the adsorption medium; Step S5: Control the gradient elution module to perform gradient elution of the adsorbed target protein and collect the target component.

2. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The coarse filtration module includes a bag filter and a security filter arranged in series. The bag filter has a filtration accuracy of 50~100μm, and the security filter has a filtration accuracy of 10~20μm.

3. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The online pH adjustment module includes an online pH monitoring probe, an automatic acid-base feeding device, and a static mixer; the central control unit controls the automatic acid-base feeding device according to the feedback signal from the online pH monitoring probe to control the pH adjustment accuracy of the feed solution within ±0.

1.

4. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The tangential flow ultrafiltration module includes a circulation tank, a tangential flow membrane pack, a circulation pump, and a pressure / flow monitoring unit. The tangential flow membrane pack has a molecular weight cutoff of 10~30kDa. The central control unit automatically adjusts the rotation speed of the circulation pump according to the transmembrane pressure and the permeate flow rate.

5. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The fluidized bed adsorption module includes a fluidized bed column, an adsorption medium, a flow rate controller, and an online ultraviolet (UV) monitor; the central control unit controls the process switching based on the penetration curve signal from the UV monitor.

6. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The gradient elution module includes a gradient mixer, an elution pump, and a component collector; the central control unit automatically switches the component collector to the target component collection port based on the elution peak signal from the ultraviolet online monitoring instrument.

7. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The sealed fluid passage uses sanitary 316L stainless steel pipes or medical-grade silicone tubing. The connection and disconnection between each module are controlled by a pneumatic diaphragm valve, which is uniformly controlled by the central control unit.

8. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, The central control unit has an embedded process control algorithm, which includes sequential control, parameter closed-loop control, fault diagnosis and fail-safe mechanism, and data recording function.

9. The integrated urokinase fully automated extraction system according to claim 1, characterized in that, It also includes a raw material inlet, a target component collection port, and a waste liquid discharge port; the raw material inlet is connected to the inlet of the coarse filtration module, the target component collection port is connected to the target component outlet of the gradient elution module, and the waste liquid discharge port is connected to the waste liquid outlet of the fluidized bed adsorption module and the gradient elution module.

10. An integrated, fully automated urokinase extraction process, characterized in that, This includes the use of an integrated urokinase fully automated extraction system as described in any one of claims 1-9.

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