High throughput LNP preparation systems and methods of use thereof

Abstract

The invention provides a high-flux LNP preparation system. The high-flux LNP preparation system comprises a water-phase flow path, an organic-phase flow path, and a mixed chip group, a dilution flow path and a collection flow path at the junction, each of the water phase flow path and the organic phase flow path comprises a sample injection valve, a sample injector, a continuous injection pump set and an output switching valve which are communicated in sequence; the input end of the sample injection valve is connected with a sample solution pipeline and a cleaning liquid gas pipeline; the output switching valve is used for guiding the fluid output by the continuous injection pump set into the mixing chip set; the hybrid chip group comprises a plurality of microfluidic hybrid chips which are connected in parallel and a hybrid chip selection valve group; the output end of the mixing chip set is connected with the dilution flow path integrating a dilution mixer and a dilution pump. The collection flow path is connected to the tail end of the dilution flow path and comprises a collection valve and a collector. The device is matched with a control system, serialized automatic operation for preparing the LNP can be achieved, automatic cleaning of the system is included, manual intervention is greatly reduced, the pollution risk is reduced, and high-efficiency and high-throughput microflow control preparation of the LNP can be achieved.

Description

Technical Field

[0001] This invention relates to the field of high-throughput LNP preparation, specifically to a high-throughput LNP preparation system and its usage method. Background Technology

[0002] Lipid nanoparticles (LNPs) are key delivery carriers for nucleic acid drugs (such as mRNA vaccines and siRNA drugs). With the expansion of mRNA-LNP applications—for example, different indications and routes of administration require sequence optimization and modification of mRNA, screening and establishing ionizable lipid libraries, screening the lipid composition and ratio of LNPs, and optimizing and screening LNP preparation processes. All of these are essential factors determining speed and success. However, these essential factors involve a significant amount of time-consuming and labor-intensive LNP preparation work, which is prone to errors. Therefore, there is an urgent need for automated high-throughput mRNA-LNP preparation systems.

[0003] Currently, R&D and process development still rely on manual methods such as changing syringes, replacing solutions, and cleaning chips, resulting in low efficiency in LNP preparation or screening. Some companies use automated pipetting workstations for high-throughput preparation of mRNA-LNPs, which can greatly improve the efficiency of mRNA-LNP preparation. However, automated pipetting workstations use impingement to promote the mixing of two solutions rather than a microfluidic mode. LNPs prepared using different principles will have differences in size, morphology, and internal structure, thus affecting their efficacy and toxicity. This makes it impossible to bridge to industrial production, rendering the high-throughput effort useless. There are also high-throughput mRNA-LNP systems based on microfluidics, such as Sunscreen, but the fluid dynamics of this system come from the agitation of immiscible liquids, which poses risks. High-throughput screening is also costly and causes environmental pollution. Furthermore, current pilot-scale tests use low flow rates and smaller channels (laminar flow or chaotic advection at low Reynolds numbers), while scale-up or production often uses T-mixers or cross-flow mixers (turbulent flow).

[0004] Therefore, there is an urgent need for a high-throughput automated LNP preparation system (with a wider range of Reynolds coefficients that can be screened) capable of automatically switching flow rates, offering a wide range of selectable flow rates, automatically switching between various mixer types, and covering a broader range of preparation volumes. This system should be able to meet the needs of multivariate and combinational high-throughput screening in small-scale trials, while also accommodating pilot-scale amplification, and can be used for bridging studies between small-scale and pilot-scale trials. Summary of the Invention

[0005] The purpose of this invention is to provide a high-throughput LNP preparation system that achieves wide-range, high-throughput automated preparation through the combination of parallel multi-range pump groups and microfluidic hybrid chip selection valve groups.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A high-throughput LNP preparation system is characterized by comprising an aqueous phase flow path, an organic phase flow path and a mixing chip assembly at the junction, a dilution flow path and a collection flow path; Both the aqueous phase flow path and the organic phase flow path include an injection valve, an injector, a flow direction switching valve, and a continuous injection pump assembly. The flow direction switching valve connects the continuous injection pump assembly to the injector or injection valve. The input end of the injection valve is connected to a sample solution pipeline and a cleaning fluid gas pipeline. The injector includes an injection needle cleaning station; The fluid output from the continuous injection pump assembly is introduced into the hybrid chipset; The hybrid chipset includes multiple microfluidic hybrid chips connected in parallel and a hybrid chip selection valve group; the output end of the hybrid chipset is connected to the dilution flow path that integrates a dilution mixer and a dilution pump. The collection flow path is connected to the end of the dilution flow path and includes a collection valve and a collector.

[0007] Furthermore, the injector includes a multi-axis moving module, a liquid aspiration needle mounted on the moving module, and a multi-specification sample bottle rack, well plate, and injection needle cleaning station set within the moving stroke of the liquid aspiration needle.

[0008] Furthermore, the continuous injection pump group in both the aqueous phase flow path and the organic phase flow path includes at least a first-range pump, a second-range pump, and a third-range pump arranged in parallel; the maximum single-stroke volume of the first-range pump, the second-range pump, and the third-range pump increases sequentially. The flow direction switching valve includes three switching states; In the first state, the flow direction switching valve is configured to connect any range pump in the continuous injection pump group to the injection valve.

[0009] In the second state, a communication path is established between the injection valve and the injector; In the third state, the flow direction switching valve is configured to connect any range pump in the continuous injection pump assembly to the injector.

[0010] Furthermore, an output switching valve is provided between the continuous injection pump assembly and the hybrid chipset; The output switching valve enables one of the first range pump, the second range pump, or the third range pump to establish a communication path with the hybrid chipset.

[0011] Furthermore, the hybrid chip selection valve assembly includes an inflow selection valve assembly disposed at the inflow end of the hybrid chip assembly and an outflow selection valve disposed at the outflow end of the hybrid chip assembly. The inflow selection valve assembly includes a first inflow selection valve and a second inflow selection valve respectively corresponding to the aqueous phase flow path and the organic phase flow path; the input end of the first inflow selection valve is connected to the output switching valve of the aqueous phase flow path, and the input end of the second inflow selection valve is connected to the output switching valve of the organic phase flow path; the output ends of the first inflow selection valve and the second inflow selection valve are respectively connected to the corresponding inlets of the plurality of microfluidic mixing chips. The input end of the outflow selection valve is connected to the outlet of each microfluidic mixing chip, and the output end of the outflow selection valve can be selectively connected to the dilution flow path or to the collector.

[0012] Furthermore, the dilution mixer in the dilution flow path has a microchannel structure; Furthermore, the injector includes sample containers with various different capacities; The collector includes collection containers with various different capacity sizes.

[0013] It also includes instructions for using the high-throughput LNP preparation system, including the following steps: Preparation steps: S1 path configuration: Based on the target preparation volume, the control system locks the corresponding range of the continuous injection pump group in the aqueous phase flow path and the organic phase flow path, and at the same time conducts the specified microfluidic mixing chip through the mixing chip selection valve group; S2 Liquid Aspiration Preparation: Control the flow direction switching valve to establish a flow path between the continuous injection pump group and the injector or injection valve; drive the injector to move to the designated sample position or switch the injection valve to the corresponding valve port, and the continuous injection pump group runs in reverse to aspirate the designated sample, wherein the aspirated volume is controlled to be greater than the preset preparation volume; subsequently, control the continuous injection pump group to run in the forward direction to expel excess sample and any air bubbles that may be present at the front end of the tubing; S3 Mixing Preparation: The continuous injection pump group of the aqueous phase flow path and the organic phase flow path advances synchronously in the forward direction according to a preset flow rate ratio, driving the two-phase fluids into the mixing chip group to mix and form LNP; S4 Online Dilution: Start the dilution pump to deliver buffer solution to the dilution mixer to dilute the LNP flowing through the dilution path in real time; S5 Collection: The waste liquid is first discharged through the collection flow path, and then the LNP is collected to the designated location through the collection valve or collector; Cleaning steps: C1 Discharge of waste liquid: Drives the continuous injection pump assembly to discharge residual liquid in the pump chamber, mixer assembly, dilution flow path and collection flow path; C2 Aspiration of cleaning medium: Control the flow direction switching valve to establish a connection between the continuous injection pump group and the injection valve; Control the injection valve to connect to the cleaning liquid gas pipeline, and the continuous injection pump group to run in reverse to draw the cleaning liquid gas into the pump chamber; C3 Flushing Injector: Controls the flow direction switching valve to establish a connection between the continuous injection pump assembly and the injector; the continuous injection pump assembly operates in the forward direction, pumping the cleaning fluid gas in the pump chamber into the injector and discharging it to the waste liquid area; C4 Rinsing the LNP Preparation System Flow Path: Referring to step C2, control the flow direction switching valve to establish a connection flow path between the continuous injection pump group and the injection valve to draw in cleaning liquid gas; after liquid aspiration, control the flow path connection relationship to establish a connection flow path between the continuous injection pump group and the mixing chip group; referring to the flow path control logic of preparation steps S3 to S5, drive the continuous injection pump group to run in the forward direction, so that the cleaning liquid gas flows sequentially through the mixing chip group, the dilution flow path and the collection flow path to perform online rinsing of the above pipelines; at the same time, control the collection valve or collector at the end of the collection flow path to switch to the waste discharge position to discharge the cleaning waste liquid.

[0014] Through the above structure, the present invention has the following beneficial effects: This invention enables automated and continuous operation of LNP preparation, dilution, collection, system cleaning, and preparation parameters, all without human intervention. It allows for high-throughput screening of LNP formulations (including lipid structure and composition screening) and preparation processes. Combined with AI and / or ML, it can significantly improve the screening or evolution speed of lipid libraries, RNA sequences or chemical modifications, formulation compositions, and LNP preparation and dilution processes.

[0015] To address the varying flow rates and production demands in actual production processes, this invention utilizes a continuous injection pump assembly in conjunction with a hybrid chip selection valve assembly and multiple microfluidic hybrid chips connected in parallel to construct a high-throughput preparation platform with a wide range and multiple modes.

[0016] The continuous injection pump assembly covers a wide flow rate range from pilot-scale to pilot-scale, thus achieving coverage of fluid states with different Reynolds numbers. The assembly can switch between single-injection and continuous-injection modes, and the multi-volume injection pumps can meet the needs of high-throughput LNP screening preparation across a wide volume range. Through a mixing chip selection valve assembly, the system can automatically switch between different mixers. This design enables the system to meet both high-throughput multivariate screening and simulate the mixing conditions of industrial production, effectively solving the problem of disconnect between R&D and production and reducing the risk of process scale-up.

[0017] Meanwhile, this invention provides precise direct positive pressure drive through a continuous injection pump assembly, ensuring controlled physical mixing of the two-phase fluids within the microfluidic mixing chip. Its nucleation mechanism is completely consistent with that of industrial production equipment, guaranteeing the comparability of key quality attributes of LNPs such as particle size, PDI, and encapsulation efficiency. Compared to batch blowing or indirect top-feeding, this invention ensures controllable, scalable, and bridging LNP quality and process while achieving high-throughput LNP production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a piping diagram according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the injector structure in one embodiment of the present invention; Figure 3 This is a schematic diagram of the flow path during preparation step S1 in the preparation process. Figure 4 This is a schematic diagram of the flow path during preparation step S2 in the preparation process; Figure 5 This is a schematic diagram of the flow path during preparation step S3 in the preparation process. Figure 6 This is a schematic diagram of the flow path during cleaning step C2 in the cleaning process. Figure 7 This is a schematic diagram of the flow path during cleaning step C3 in the cleaning process. Figure 8 This is a three-dimensional structural diagram of an embodiment of the present invention.

[0020] Explanation of reference numerals in the attached figures: 1. Aqueous phase flow path; 2. Organic phase flow path; 3. Injector; 31. Multi-axis moving module; 32. Aspiration needle; 33. Sample bottle rack; 34. Injector cleaning station; 4. Injection valve; 41. Flow direction switching valve; 5. Continuous injection pump assembly; 51. First range pump; 52. Second range pump; 53. Third range pump; 6. Output switching valve; 7. Mixing chip assembly; 71. Microfluidic mixing chip; 72. Mixing chip selection valve assembly; 721. First inflow selection valve; 722. Second inflow selection valve; 723. Outflow selection valve; 81. Dilution flow path; 82. Dilution mixer; 83. Dilution pump; 84. Mechanical check valve; 9. Collection flow path; 91. Collection valve; 92. Collector. Detailed Implementation

[0021] The accompanying drawings are for illustrative purposes only and should not be construed as limiting the scope of this patent. To better illustrate this embodiment, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual dimensions of the product.

[0022] It will be understood by those skilled in the art that certain well-known structures and their descriptions may be omitted in the accompanying drawings. The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] like Figure 1-8 The high-throughput LNP preparation system shown includes an aqueous phase flow path 1, an organic phase flow path 2, a hybrid chip assembly 7 at the junction of the two, a dilution flow path 81, and a collection flow path 9. Both the aqueous phase flow path 1 and the organic phase flow path 2 include an injection valve 4, an injector 3, a flow direction switching valve 41, and a continuous injection pump assembly 5; The flow switching valve 41 connects the continuous injection pump group 5 to the injector 3 or the injection valve 4. The input end of the injection valve 4 is connected to the sample solution pipeline and the cleaning fluid gas pipeline; The injector 3 includes an injection needle cleaning station 34; The fluid output from the continuous injection pump unit 5 is introduced into the mixing chip unit 7; The hybrid chipset 7 includes multiple microfluidic hybrid chips 71 connected in parallel and a hybrid chip selection valve group; the output of the hybrid chipset 7 is connected to a dilution flow path 81 that integrates a dilution mixer 82 and a dilution pump 83. The collection flow path 9 is connected to the end of the dilution flow path 81 and includes a collection valve 91 and a collector 92.

[0024] Collector 92 includes a waste discharge outlet.

[0025] Specifically, the injection valve 4 is a multi-position rotary valve. Its common end is connected to the inlet of the continuous injection pump group 5 through the flow switching valve 41; its selector ends are respectively connected to the cleaning fluid gas line and the sample solution line; the cleaning fluid gas line can be used to input substances such as water, alcohol, alkali, and compressed air.

[0026] Specifically, the dilution pump 83 is preferably a continuous flow plunger pump. The dilution pump 83 delivers the buffer solution to the dilution mixer 82 through pipelines to perform on-the-spot dilution of LNP to stabilize the particle size and adjust the pH of the external aqueous phase of LNP, etc. Different dilution solutions can be selected to perform online dilution of LNP, or the LNP can be set to pass through the dilution mixer 82 without online dilution according to the process settings.

[0027] The injector 3 includes a multi-axis moving module 31, a liquid extraction needle 32 mounted on the moving module, and a multi-size sample bottle rack 33, a well plate, and a needle cleaning station 34 set within the moving stroke of the liquid extraction needle 32.

[0028] Specifically, the sample bottle holder 33 and the well plate can accommodate sample solutions of various sizes, such as 2ml, 5ml, and 10ml.

[0029] The continuous injection pump group 5 in the aqueous phase flow path 1 and the organic phase flow path 2 each includes at least a first-range pump 51, a second-range pump 52 and a third-range pump 53 arranged in parallel; the maximum single stroke volume of the first-range pump 51, the second-range pump 52 and the third-range pump 53 increases sequentially. Specifically, the continuous injection pump group 5 is configured in parallel. The maximum single-stroke volume of each pump increases sequentially to cover different flow rates, precision, and pressure requirements. It includes a first-range pump 51 (e.g., a 1ml injection pump, used in single-injection mode for 1-4ml preparation), a second-range pump 52 (e.g., a 5ml injection pump, used in single-injection mode for 4-10ml preparation), and a third-range pump 53 (e.g., a 10ml or 25ml injection pump, used in single-injection mode for large-volume preparation). Any injection pump in continuous injection mode can achieve large (unlimited volume) volume preparation.

[0030] The flow direction switching valve 41 includes three switching states; The first state is used to establish a flow path between the first range pump 51, the second range pump or the third range pump 53 and the injection valve 4. In the second state, the injection valve 4 and the injector 3 establish a connection flow path; The third state is used to establish a connection path between the first range pump 51, the second range pump 52, or the third range pump 53 and the injector 3.

[0031] Specifically, the flow direction switching valve 41 is preferably a multi-position multi-way selector valve. The flow direction switching valve 41 is connected to the output end of the injection valve 4 and the injector 3 through a pipeline, serving as the main inlet for the fluid; the flow direction switching valve 41 is also connected to the inlet ends of the first range pump 51, the second range pump 52 and the third range pump 53 respectively.

[0032] An output switching valve 6 is provided between the continuous injection pump group 5 and the hybrid chip group 7; the output switching valve 6 enables one of the first range pump 51, the second range pump 52 or the third range pump 53 to establish a flow path with the hybrid chip group 7.

[0033] Understandably, in this embodiment, the flexible selection and continuous delivery of the range pump are achieved through the combined control of the state of the flow direction switching valve 41 and the output switching valve 6.

[0034] When a specific flow range is required (taking the first-range pump 51 as an example), the control system drives the output switching valve 6 to establish a unique flow path between the first-range pump 51 and the hybrid chipset 7, while simultaneously disconnecting or cutting off the output paths of the second-range pump 52 and the third-range pump 53. Simultaneously, the control flow direction switching valve 41 switches to the first state according to the sample injection requirements and connects specifically to the inlet of the first-range pump 51. Through this dual locking of the input and output ends, the system achieves exclusive use of the specific flow range pump and physically isolates other idle flow range pumps, effectively preventing pressure fluctuations or fluid contamination caused by the dead volume of idle pump chambers in the current main flow path.

[0035] Specifically, the first-range pump 51, the second-range pump 52, and the third-range pump 53 are all continuous injection pumps. Taking the first-range pump 51 as an example, when continuous injection is required, it includes a pair of pump units A and B. When pump unit A performs a reverse suction stroke, the flow direction switching valve 41 is switched to the first state (or the third state) to open the input flow path of pump unit A, while the output switching valve 6 is controlled to cut off the output flow path of pump unit A. Simultaneously, pump unit B, at the same range, performs a forward push stroke. At this time, the flow direction switching valve 41 is controlled to cut off the input flow path of pump unit B, and the output switching valve 6 is controlled to open the flow path between pump unit B and the mixing chip assembly 7. The two operate in an alternating complementary "suction and push" state, and a smooth switch is completed before the piston stroke ends, thereby eliminating the flow interruption phenomenon caused by the single-pump suction return stroke and ensuring that the two-phase fluid continuously enters the mixing chip assembly 7 at a constant flow rate ratio.

[0036] The hybrid chip selection valve assembly 72 includes an inflow selection valve assembly disposed at the inflow end of the hybrid chip assembly 7 and an outflow selection valve 723 disposed at the outflow end of the hybrid chip assembly 7. The inflow selection valve assembly includes a first inflow selection valve 721 and a second inflow selection valve 722 respectively corresponding to the aqueous phase flow path 1 and the organic phase flow path 2; the input end of the first inflow selection valve 721 is connected to the output switching valve 6 of the aqueous phase flow path 1, and the input end of the second inflow selection valve 722 is connected to the output switching valve 6 of the organic phase flow path 2; the output ends of the first inflow selection valve 721 and the second inflow selection valve 722 are respectively connected to the corresponding inlets of multiple microfluidic mixing chips 71. The input of the outflow selection valve 723 is connected to the outlet of each microfluidic mixing chip 71, and the output of the outflow selection valve 723 can be selectively connected to the dilution flow path 81 or to the collector 92.

[0037] Specifically, the hybrid chipset 7 is located at the intersection of the aqueous phase flow path 1 and the organic phase flow path 2, and the microfluidic hybrid chipset 71 has multiple chips with different flow channel structures or sizes connected in parallel.

[0038] The hybrid chip selection valve assembly 72 includes an inlet selection valve assembly at the front end and an outlet selection valve 723 at the rear end. The inlet selection valve assembly receives fluid from the output switching valve 6 and distributes it to the designated chip; the outlet selection valve 723 collects the chip products. The hybrid core can be automatically replaced by rotating the valve position via a program, without the need for manual disassembly and assembly.

[0039] The dilution mixer 82 in the dilution flow path 81 has a microchannel structure; A mechanical check valve 84 is provided between the dilution pump 83 and the dilution mixer 82; the outlet end of the dilution mixer 82 is connected to the collection valve 91 or the collector 92 for receiving and discharging the fluid after online dilution.

[0040] A mechanical check valve 84 is provided between the dilution pump 83 and the dilution mixer 82 to prevent the diluent from flowing freely due to potential energy or capillary action when the dilution flow path 81 is not in operation.

[0041] Specifically, the LNP fluid treated by the dilution mixer 82 flows into the collection path 9. The outlet of the dilution mixer 82 can be selectively connected to the collection valve 91 or the collector 92 via a flow path switching mechanism. The specific operating process includes the following two conditions: 1. Operating Condition 1: Connecting to Collection Valve 91 (Waste Discharge or Continuous Large-Volume Collection Mode); When the user sets the single large-batch preparation mode, the control system drives the flow path switching, so that the outlet of the dilution mixer 82 is connected to the collection valve 91, and directly and continuously introduces the liquid into the externally connected large-capacity container or collection bottle through the large-diameter pipeline.

[0042] 2. Operating Condition 2: Connecting Collector 92 (High-throughput Collection Mode); When the user sets the mode to multi-formula screening or small-volume multi-part preparation, the control system drives the flow path switching to connect the outlet of dilution mixer 82 with collector 92.

[0043] At this point, the LNP finished fluid flowing out of the dilution mixer 82 is guided to the injection needle of the collector 92. Based on preset formula coordinate information, the collector 92 drives the injection needle to move in the X / Y / Z axis directions, precisely positioning it above the designated well of the target collection container (such as a 96-well deep-well plate, centrifuge tube rack, or vial array). Fluid is injected quantitatively into the corresponding collection well or vial according to a preset volume, and then automatically moves to the next position, thus achieving high-throughput, automated array-type collection.

[0044] The injector 3 includes sample containers with various different capacities.

[0045] Specifically, sample containers include SBS-compliant microplates (such as 96-well, 24-well, and 48-well plates). For scale-up validation of single formulations or small-batch production scenarios, sample containers also include large-capacity individual sample vials (such as 2ml, 5ml, 7ml, and 10ml).

[0046] Collector 92 also includes collection containers with a variety of different capacity sizes.

[0047] Preparation steps: The preparation process of this system is detailed below: I. Preparation steps (S1-S5) S1 path configuration: such as Figure 3 As shown, the user sets the formula in the software (e.g., 10 ml of aqueous phase). The control system automatically determines and locks the use of the corresponding continuous injection pump group 5 (e.g., second-range pump 52), and at the same time controls the operation of the mixing chip selection valve group 72 to conduct the microfluidic mixing chip 71 of the specified specification (e.g., medium throughput), and controls the flow path connection to point to the mixing chip group 7.

[0048] S2 Preparation for aspiration: as follows Figure 4 As shown, this step includes addressing the injector 3 and liquid aspiration.

[0049] Flow path establishment: Control flow direction switching valve 41 to switch to the third state to establish a direct flow path between continuous injection pump group 5 and injector 3 (liquid aspiration needle 32).

[0050] Addressing liquid aspiration: The multi-axis moving module 31 of the driving injector 3 drives the aspiration needle 32 to move horizontally and vertically downward to the bottom of the sample carrier.

[0051] Negative pressure aspiration: Drive the continuous injection pump group 5 to run in reverse, drawing the sample into the pump chamber through the aspiration needle 32. During this process, control the aspiration volume to be slightly larger than the set volume (excessive aspiration).

[0052] Exhaust and isolation: The continuous injection pump unit 5 is then driven to run in a micro-volume forward direction to expel air bubbles. After aspiration is completed, the flow direction switching valve 41 is switched to the second state (connected output / mixing flow path) or the intermediate cut-off position to physically disconnect the pump from the needle, prevent leakage, and prepare for subsequent injection.

[0053] S3 mixture preparation: such as Figure 5 As shown, the continuous injection pump group 5, which controls the aqueous and organic phases, propels them synchronously in the forward direction according to the flow rate ratio (FRR) and total flow rate (TFR) set in the formulation. The fluid is pressurized and pushed out, entering the microfluidic mixing chip 71 through the output flow path, where it is mixed in a controlled collision within the microchannel to form the initial LNP product.

[0054] S4 Online Dilution: Dilution pump 83 starts synchronously or with a delay in conjunction with the preparation pump assembly. The LNP mixture immediately enters the dilution path 81 after flowing out of the chip. The buffer solution delivered by dilution pump 83 merges with the LNP in the dilution mixer 82, performing immediate dilution and stabilization.

[0055] S5 Collection: The fluid flowing out of the outlet of the dilution mixer 82 is first discharged through the control of the collection flow path 9, and after the fluid flow rate and composition are stable, the valve is switched or the pipeline is moved to collect the finished product LNP into the designated container.

[0056] To prevent cross-contamination between batches, the system performs an automated cleaning procedure between preparation tasks or after the sequence is completed. This step includes the following steps: C1 Discharge of waste liquid: Drives the continuous injection pump group 5 to run in the forward direction, emptying any excess liquid from the previous batch that may remain in the pump chamber and flow path.

[0057] C2 suction cleaning medium: such as Figure 6 As shown, the system enters the cleaning fluid loading mode.

[0058] Flow path switching: Control the front-end injection valve 4 to switch to the cleaning solution port (such as pure water or ethanol), and at the same time control the flow direction switching valve 41 to switch to the first state (establish the connection between the continuous injection pump group 5 and the injection valve 4).

[0059] Liquid aspiration: Drive the continuous injection pump group 5 to run in reverse, directly drawing in sufficient cleaning liquid gas from the injection valve 4 into the pump chamber.

[0060] C3 flushing injector 3: as shown Figure 7 As shown (covering needle movement, needle insertion, and spraying actions).

[0061] Needle position movement: Drive the injector 3 to move the aspiration needle 32 and insert it into the injection needle cleaning station 34.

[0062] Backwashing: Control flow switching valve 41 switches to the third state (connecting the pump set and injector 3). Drive the continuous injection pump set 5 to run in the forward direction. The high-pressure cleaning fluid in the pump chamber flows in the reverse direction through the flow switching valve 41 and the aspiration needle 32, and is sprayed at high speed from the needle tip into the cleaning station to complete the cleaning of the inner wall of the needle.

[0063] C4 Rinsing the LNP Preparation System Flow Path: After repeating step C2 to draw in the cleaning solution, control the flow direction switching valve 41 to switch to the second state (connecting the output flow path). Drive the continuous injection pump group 5 to run in the forward direction, so that the cleaning solution flows sequentially through the microfluidic mixing chip 71, the diluent mixer 82, and the collection line 9 according to the preparation path S3-S5. At the same time, control the collection line 9 to switch to the waste discharge end to discharge the waste liquid. This step can be repeated to thoroughly remove lipid residues in the microfluidic mixing chip 71.

[0064] This process uses high-speed fluid to flush the inner walls of the tubing and the needle tip, completely removing any residual sample. The system can cycle through steps C2 to C4 (liquid aspiration-valve shut-off-flushing) as needed to achieve multiple cleaning cycles.

[0065] For cleaning the hybrid chip assembly 7 and other downstream flow paths after the output of the continuous injection pump assembly 5, the fluid drive and path control logic is consistent with the preparation process (S3 to S5). Specifically, the injection valve 4 and / or the dilution flow path 81 (input) are switched to the cleaning liquid source or the purge gas source, and then the continuous injection pump assembly 5 and the dilution pump 83 are driven to run in the forward direction. The cleaning medium or gas is used to replace the preparation raw materials, and the entire system flow path is flushed or purged online according to the preparation path.

Claims

1. A high-throughput LNP preparation system, characterized in that, This includes aqueous phase flow path, organic phase flow path, and the mixing chip assembly, dilution flow path, and collection flow path at the junction; Both the aqueous phase flow path and the organic phase flow path include an injection valve, an injector, a flow direction switching valve, and a continuous injection pump assembly. The flow direction switching valve connects the continuous injection pump assembly to the injector or injection valve. The input end of the injection valve is connected to a sample solution pipeline and a cleaning fluid gas pipeline. The injector includes an injection needle cleaning station; The fluid output from the continuous injection pump assembly is introduced into the hybrid chipset; The hybrid chipset includes multiple microfluidic hybrid chips connected in parallel and a hybrid chip selection valve group; the output end of the hybrid chipset is connected to the dilution flow path that integrates a dilution mixer and a dilution pump. The collection flow path is connected to the end of the dilution flow path and includes a collection valve and a collector.

2. The high-throughput LNP preparation system according to claim 1, characterized in that, The injector includes a multi-axis moving module, a liquid extraction needle mounted on the moving module, and a multi-size sample bottle rack, well plate, and injection needle cleaning station set within the moving stroke of the liquid extraction needle.

3. The high-throughput LNP preparation system according to claim 1, characterized in that, The continuous injection pump group in both the aqueous phase flow path and the organic phase flow path includes at least a first-range pump, a second-range pump, and a third-range pump arranged in parallel; the maximum single-stroke volume of the first-range pump, the second-range pump, and the third-range pump increases sequentially. The flow direction switching valve includes three switching states; In the first state, the flow direction switching valve is configured to connect any range pump in the continuous injection pump group to the injection valve.

4. Second state: Establish a flow path between the injection valve and the injector; In the third state, the flow direction switching valve is configured to connect any range pump in the continuous injection pump assembly to the injector.

5. The high-throughput LNP preparation system according to claim 3, characterized in that, An output switching valve is provided between the continuous injection pump assembly and the hybrid chipset; The output switching valve enables one of the first range pump, the second range pump, or the third range pump to establish a communication path with the hybrid chipset.

6. The high-throughput LNP preparation system according to claim 4, characterized in that, The hybrid chip selection valve assembly includes an inflow selection valve assembly disposed at the inflow end of the hybrid chip assembly and an outflow selection valve disposed at the outflow end of the hybrid chip assembly. The inflow selection valve assembly includes a first inflow selection valve and a second inflow selection valve respectively corresponding to the aqueous phase flow path and the organic phase flow path; the input end of the first inflow selection valve is connected to the output switching valve of the aqueous phase flow path, and the input end of the second inflow selection valve is connected to the output switching valve of the organic phase flow path; the output ends of the first inflow selection valve and the second inflow selection valve are respectively connected to the corresponding inlets of the plurality of microfluidic mixing chips. The input end of the outflow selection valve is connected to the outlet of each microfluidic mixing chip, and the output end of the outflow selection valve can be selectively connected to the dilution flow path or to the collector.

7. The high-throughput LNP preparation system according to claim 1, characterized in that, The dilution mixer in the dilution flow path has a microchannel structure; A mechanical check valve is provided between the dilution pump and the dilution mixer; the outlet end of the dilution mixer is connected to the collection valve or the collector for receiving and discharging the fluid after online dilution.

8. The high-throughput LNP preparation system according to claim 1, characterized in that, The injector includes sample containers with various different capacities; The collector includes collection containers with various different capacity sizes.

9. A method of using a high-throughput LNP preparation system, applicable to the high-throughput LNP preparation system as described in any one of claims 1-7, characterized in that, Includes the following steps: Preparation steps: S1 path configuration: Based on the target preparation volume, the control system locks the corresponding range of the continuous injection pump group in the aqueous phase flow path and the organic phase flow path, and at the same time conducts the specified microfluidic mixing chip through the mixing chip selection valve group; S2 Liquid Aspiration Preparation: Control the flow direction switching valve to establish a flow path between the continuous injection pump group and the injector or injection valve; drive the injector to move to the designated sample position or switch the injection valve to the corresponding valve port, and the continuous injection pump group runs in reverse to aspirate the designated sample, wherein the aspirated volume is controlled to be greater than the preset preparation volume; subsequently, control the continuous injection pump group to run in the forward direction to expel excess sample and any air bubbles that may be present at the front end of the tubing; S3 Mixing Preparation: The continuous injection pump group of the aqueous phase flow path and the organic phase flow path advances synchronously in the forward direction according to a preset flow rate ratio, driving the two-phase fluids into the mixing chip group to mix and form LNP; S4 Online Dilution: Start the dilution pump to deliver buffer solution to the dilution mixer to dilute the LNP flowing through the dilution path in real time; S5 Collection: The waste liquid is first discharged through the collection flow path, and then the LNP is collected to the designated location through the collection valve or collector; Cleaning steps: C1 Discharge of waste liquid: Drives the continuous injection pump assembly to discharge residual liquid in the pump chamber, mixer assembly, dilution flow path and collection flow path; C2 Aspiration of cleaning medium: Control the flow direction switching valve to establish a connection between the continuous injection pump group and the injection valve; Control the injection valve to connect to the cleaning liquid gas pipeline, and the continuous injection pump group to run in reverse to draw the cleaning liquid gas into the pump chamber; C3 Flushing Injector: Controls the flow direction switching valve to establish a connection between the continuous injection pump assembly and the injector; the continuous injection pump assembly operates in the forward direction, pumping the cleaning fluid gas in the pump chamber into the injector and discharging it to the waste liquid area; C4 Rinsing the LNP Preparation System Flow Path: Referring to step C2, control the flow direction switching valve to establish a connection flow path between the continuous injection pump group and the injection valve to draw in cleaning liquid gas; after liquid aspiration, control the flow path connection relationship to establish a connection flow path between the continuous injection pump group and the mixing chip group; referring to the flow path control logic of preparation steps S3 to S5, drive the continuous injection pump group to run in the forward direction, so that the cleaning liquid gas flows sequentially through the mixing chip group, the dilution flow path and the collection flow path to perform online rinsing of the above pipelines; at the same time, control the collection valve or collector at the end of the collection flow path to switch to the waste discharge position to discharge the cleaning waste liquid.

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