Peptide Continuous Precipitation System

Abstract

This invention provides a continuous peptide precipitation system. The system includes: a main heat exchange module with an input and an output; a precipitation module comprising at least two mixing-precipitation maturation units connected in series, each unit including a cyclone mixer and a precipitation maturation unit; the inlet of the precipitation maturation unit being connected to the first outlet pipe of the cyclone mixer; the output of the main heat exchange module being connected to the first inlet pipe of the cyclone mixer in the first mixing-precipitation maturation unit; the first inlet pipes of the other cyclone mixers being connected to the outlet of the upstream precipitation maturation unit; and a second inlet pipe for inputting precipitation solvent into the cyclone mixer; and a filtration module including a vacuum filter and a waste bottle, optionally connected to the outlet of the precipitation maturation unit. The continuous peptide precipitation system according to this invention enables fully continuous operation, achieving continuous peptide precipitation with higher efficiency and simpler, more convenient operation.

Description

Technical Field

[0001] This invention relates to the field of bioseparation and purification technology, and more specifically, to a continuous polypeptide precipitation system. Background Technology

[0002] In recent years, peptides have emerged as a promising new type of drug. Unlike decades ago when naturally extracted, cell-derived peptides (such as thymosin and glutathione) were commonly used, peptide drugs have increasingly shifted towards using non-natural peptides with artificially modified or altered fragments (such as smegglutinin) to achieve better efficacy, reduce immune responses, and prolong drug circulation time compared to natural peptides. Therefore, peptide synthesis and post-processing technologies are gradually replacing traditional fermentation techniques, becoming increasingly important and indispensable in the research and development and production of novel peptide drugs. Among these post-processing steps, peptide precipitation, filtration, and washing play a crucial role in the research and production of peptide drugs, thus necessitating an efficient and user-friendly peptide precipitation technology and corresponding equipment.

[0003] Currently, peptide precipitation still employs conventional batch precipitation techniques. This involves adding a pre-prepared peptide lysis buffer into a precipitation tank, followed by the continuous dropwise addition of a precipitation solvent. During this process, the solution is continuously stirred, and heat generated during precipitation is removed via a heat exchanger to maintain a low temperature, thereby reducing peptide solubility and preventing heat damage. After the addition is complete, stirring continues for a period until complete precipitation. The solution containing the precipitate is then transferred to a vacuum filtration tank, where the solvent is removed by filtration. The precipitate is then washed several times with the solvent, dried, and recovered. This process involves multiple material transfers and operations, placing higher demands on equipment and manpower, making the peptide post-processing more complex, and to some extent limiting the progress of peptide drug research and production.

[0004] In the existing technology, the pyrolysis solution is pre-injected into the precipitation (crystallization) vessel, and then the precipitation solvent is slowly added dropwise to the precipitation (crystallization) vessel. The dropwise rate is mainly controlled by the temperature of the system in the vessel. After all the precipitation solvent has been added, the vessel is stirred for a long time before the precipitation (crystallization) operation is completed, which results in low operating efficiency. Summary of the Invention

[0005] The main objective of this invention is to provide a continuous peptide precipitation system that enables fully continuous operation of the system, achieves continuous peptide precipitation, is more efficient, and is simpler and more convenient to operate.

[0006] To achieve the above objectives, according to one aspect of the present invention, a continuous peptide precipitation system is provided, comprising: a main heat exchange module having an input end and an output end, the input end being used to input peptide lysis buffer and precipitation solvent into the main heat exchange module, and the output end being used to output a mixed solution that has undergone preliminary mixing in the main heat exchange module; a precipitation module including at least two mixing-precipitation maturation units arranged in series, each mixing-precipitation maturation unit including a cyclone mixer and a precipitation maturation unit, the cyclone mixer including a first inlet pipe, a second inlet pipe and a first outlet pipe, the inlet of the precipitation maturation unit being connected to the first outlet pipe of the cyclone mixer, the output end of the main heat exchange module being connected to the first inlet pipe of the cyclone mixer of the first mixing-precipitation maturation unit, the first inlet pipes of the other cyclone mixers being connected to the outlet of the upstream precipitation maturation unit, and the second inlet pipe of each cyclone mixer being used to input precipitation solvent into the cyclone mixer; and a filtration module including a suction filter and a waste bottle arranged in parallel, the suction filter and the waste bottle being selectively connected to the outlet of the precipitation maturation unit of the last mixing-precipitation maturation unit via a control valve.

[0007] Furthermore, the swirl mixer is a microfluidic mixer.

[0008] Furthermore, the microfluidic mixer includes a mixing chamber inlet channel and an outlet channel. The mixing chamber includes a cylindrical section. There are at least two inlet channels, which are connected to the mixing chamber along the tangential direction of the cylindrical section. The swirling direction of the solution entering the cylindrical section through each inlet channel is the same. The first inlet pipe and the second inlet pipe are both connected to the corresponding inlet channels.

[0009] Furthermore, the inlet channel has a rectangular cross-section, and its outer surface is tangent to the outer circumferential surface of the cylindrical section. Multiple inlet channels are evenly distributed around the cylindrical section.

[0010] Furthermore, the height of the inlet channel along the axial direction of the cylindrical section is the same as the axial height of the cylindrical section.

[0011] Furthermore, there are four import channels.

[0012] Furthermore, the outlet channel is located at one axial end of the mixing chamber and extends axially along the cylindrical section.

[0013] Furthermore, the mixing chamber also includes a conical section connected to the axial end of the cylindrical section and gradually narrowing away from the cylindrical section, with the outlet channel connected to the narrowing end of the conical section.

[0014] Furthermore, a protruding post is provided in the area between the inner surface of the inlet channel near the central axis of the cylindrical section and the outer peripheral surface of the outlet channel, and the axial height of the protruding post is less than the axial height of the cylindrical section.

[0015] Furthermore, the conical segment is provided with a concave segment that curves downward toward the cylindrical segment. The cross-sectional area of ​​the concave segment decreases along the contraction direction of the conical segment. There are at least two concave segments, which are evenly arranged along the circumference of the conical segment.

[0016] Furthermore, the bottom surface of the concave section is flush with the top surface of the cylindrical section.

[0017] Furthermore, the sedimentation and maturation device is a plug flow maturation device.

[0018] Furthermore, the push-flow curing device is a spiral coil or a serpentine tube.

[0019] Furthermore, the continuous peptide precipitation system also includes a control module, which includes a controller and a data acquisition device. The data acquisition device is used to collect parameter information of the main heat exchange module, precipitation module, and filtration module. The controller controls the main heat exchange module, precipitation module, and filtration module based on the parameter information collected by the data acquisition device.

[0020] Applying the technical solution of this invention, the continuous peptide precipitation system includes a main heat exchange module. This module receives peptide lysis buffer and precipitation solvent at its input end, and after initial mixing and temperature control within the module, outputs a uniform premixed solution at its output end, providing stable initial conditions for subsequent continuous precipitation. The precipitation module consists of at least two series-connected mixing-precipitation maturation units. Each unit includes a cyclone mixer and a precipitation maturation unit. The cyclone mixer receives the mixed solution flowing from upstream through a first inlet pipe, and simultaneously introduces additional precipitation solvent in stages through a second inlet pipe. Efficient and instantaneous local mixing is achieved under the action of cyclone flow. The mixed solution enters the precipitation maturation unit through a first outlet pipe, completing stepwise precipitation and crystal nucleus maturation within a controllable residence time. Multiple mixing-precipitation maturation units are connected sequentially, with the outlet of the upstream precipitation maturation unit connected to the first inlet pipe of the downstream cyclone mixer. The interconnected inlet and outlet pipes form a continuous flow sedimentation path, enabling segmented and controllable sedimentation from initial nucleation to final stable particle size. This effectively avoids the problems of uneven mixing and wide particle size distribution caused by local oversaturation in traditional batch operations. The filtration module selectively guides the sediment slurry flowing out of the end sedimentation maturation unit outlet to a vacuum filter for solid-liquid separation or discharges it into a waste bottle through a control valve, achieving flexible switching of the production process. Through premixing in the main heat exchange module, segmented solvent injection and turbulent enhanced mixing in a multi-stage cyclone mixer, and steady-state maturation in a step-by-step sedimentation maturation unit, the system achieves continuous, homogenized, and precisely controlled particle size throughout the entire peptide precipitation process. This results in higher efficiency and simpler, more convenient operation, effectively solving the technical problems of batch operation, low mixing efficiency, and uncontrollable sedimentation particle size in existing technologies, and significantly improving the uniformity and process stability of peptide precipitation products. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 A schematic diagram of the structural principle of a continuous peptide precipitation system according to an embodiment of the present invention is shown;

[0023] Figure 2 A perspective view of a microfluidic mixer for a continuous peptide precipitation system according to an embodiment of the present invention is shown.

[0024] Figure 3 The morphology of the precipitate under different experimental conditions is shown in the embodiments of the present invention.

[0025] The above figures include the following reference numerals:

[0026] 1. Main heat exchange module; 2. Sedimentation module; 3. Filtration module; 4. Cyclone mixer; 5. Sedimentation maturation device; 6. First inlet pipe; 7. Second inlet pipe; 8. First outlet pipe; 9. Vacuum filter; 10. Waste liquid bottle; 11. Control valve; 12. Mixing chamber; 13. Inlet channel; 14. Outlet channel; 15. Cylindrical section; 16. Conical section; 17. Protruding column; 18. Concave section; 19. Controller; 20. Flow meter; 21. Pressure gauge; 22. Heat exchanger; 23. Microfluidic mixing heat exchange device; 24. Pump. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0028] See also Figures 1 to 2As shown, according to an embodiment of the present invention, a continuous peptide precipitation system includes: a main heat exchange module 1, which has an input end and an output end. The input end is used to input peptide lysis buffer and precipitation solvent into the main heat exchange module 1, and the output end is used to output a mixed solution that has undergone preliminary mixing in the main heat exchange module 1; a precipitation module 2, including at least two mixing-precipitation maturation units arranged in series. The mixing-precipitation maturation unit includes a cyclone mixer 4 and a precipitation maturation unit 5. The cyclone mixer 4 includes a first inlet pipe 6, a second inlet pipe 7, and a first outlet pipe 8. The precipitation maturation unit 5... The inlet is connected to the first outlet pipe 8 of the cyclone mixer 4, the output end of the main heat exchange module 1 is connected to the first inlet pipe 6 of the cyclone mixer 4 of the first mixing-precipitation maturation unit, the first inlet pipe 6 of the other cyclone mixers 4 is connected to the outlet of the upstream precipitation maturation unit 5, and the second inlet pipe 7 of each cyclone mixer 4 is used to input precipitation solvent into the cyclone mixer 4; the filtration module 3 includes a suction filter 9 and a waste liquid bottle 10 arranged in parallel, and the suction filter 9 and the waste liquid bottle 10 can be selectively connected to the outlet of the precipitation maturation unit 5 of the end mixing-precipitation maturation unit through the control valve 11.

[0029] Applying the technical solution of this embodiment, the continuous peptide precipitation system includes a main heat exchange module 1. This module receives peptide lysis buffer and precipitation solvent through its input end, and after completing preliminary mixing and temperature control internally, outputs a uniform premixed solution from its output end, providing stable initial conditions for subsequent continuous precipitation. The precipitation module 2 consists of at least two series-connected mixing-precipitation maturation units. Each unit includes a cyclone mixer 4 and a precipitation maturation unit 5. The cyclone mixer 4 receives the mixed solution flowing from upstream through a first inlet pipe 6, and simultaneously introduces additional precipitation solvent in stages through a second inlet pipe 7. Under the action of cyclone, efficient and instantaneous local mixing is achieved. The mixed solution enters the precipitation maturation unit 5 through a first outlet pipe 8, and completes stepwise precipitation and crystal nucleus maturation within a controllable residence time. Multiple mixing-precipitation maturation units are connected in sequence, with the outlet of the upstream precipitation maturation unit 5 connected to the first outlet pipe 8 of the downstream cyclone mixer 4. The inlet pipe 6 is connected to form a continuous flow sedimentation path, enabling the sedimentation process to be segmented and controllable from initial nucleation to final stable particle size. This effectively avoids the problems of uneven mixing and local oversaturation leading to wide particle size distribution in traditional batch operations. The filtration module 3 selectively guides the sediment slurry flowing out of the outlet of the end sedimentation ripening device 5 to the suction filter 9 for solid-liquid separation or discharges it into the waste liquid bottle 10 through the control valve 11, realizing flexible switching of the production process. Through the premixing of the main heat exchange module 1, the segmented solvent injection and turbulent enhanced mixing of the multi-stage cyclone mixer 4, and the steady-state ripening of the step-by-step sedimentation ripening device, the system realizes the continuity, homogenization and precise particle size control of the entire peptide precipitation process. It is more efficient and easier to operate, effectively solving the technical problems of batch operation, low mixing efficiency and uncontrollable precipitation particle size in the existing technology, and significantly improving the uniformity and process stability of peptide precipitation products.

[0030] In one embodiment, the control valve 11 is a three-way valve, and the filter 9 and the waste liquid bottle 10 are connected to the outlet of the sedimentation module 2 through the three-way valve.

[0031] In one embodiment, the swirl mixer 4 is a microfluidic mixer.

[0032] A microfluidic mixer is a microfluidic element that achieves molecular-level homogeneous mixing of lysis buffer and precipitation solvent within a flow channel at the micrometer to millimeter scale without external stirring, through a multi-inlet tangential flow and swirling cavity structure. The microfluidic mixer has no dead zones and no backmixing during the mixing process, and the mixing uniformity is quantified by the particle size distribution index. It can also simultaneously initiate the uniform nucleation of peptides, providing a stable and uniform initial supersaturated environment for subsequent controllable precipitation.

[0033] In this embodiment, the cyclone mixer 4 adopts a microfluidic mixer structure, which enables high-speed swirling disturbance of the peptide lysis solution and precipitation solvent within a microscale channel. The fluid completes molecular-level mixing in a very short time, significantly shortening the mixing time and eliminating local concentration gradients. This ensures that the precipitation nucleation process occurs synchronously in a uniform supersaturated environment, effectively suppressing large particle aggregation and heterogeneous nucleation phenomena. As a result, the particle size distribution of the obtained peptide precipitate is significantly narrowed and controllable. This microfluidic structure, combined with multiple mixing-precipitation maturation units arranged in series, realizes multi-stage continuous and precise mixing and maturation, avoiding precipitation quality fluctuations caused by uneven stirring in traditional batch mixing, and improving the stability and repeatability of the overall precipitation process.

[0034] The purpose of a microfluidic mixer is to rapidly mix a solution containing peptides with a poor solvent, reduce the solubility of the peptides in the whole system, and allow the peptides to precipitate from the solution.

[0035] See also Figure 2 As shown, in one embodiment, the microfluidic mixer includes a mixing chamber 12, an inlet channel 13, and an outlet channel 14. The mixing chamber 12 includes a cylindrical section 15. The number of inlet channels 13 is at least two. At least two inlet channels 13 are connected to the mixing chamber 12 along the tangential direction of the cylindrical section 15. The swirling direction of the solution entering the cylindrical section 15 through each inlet channel 13 is the same. The first inlet pipe 6 and the second inlet pipe 7 are both connected to the corresponding inlet channel 13. Figure 2 The diagram shown depicts the internal flow channels and chambers of a microfluidic mixer, omitting the external shell structure. The external shell structure can be selected as needed; only the internal flow channels and chambers require specific structural features. Figure 2 That's all that's required.

[0036] In this embodiment, the mixing chamber 12 is provided with a cylindrical section 15, and at least two inlet channels 13 are connected to the mixing chamber 12 along the tangential direction of the cylindrical section 15. This allows the peptide lysis solution from the first inlet pipe 6 and the precipitation solvent from the second inlet pipe 7 to be injected into the mixing chamber 12 at high speed in a tangential manner through the corresponding inlet channels 13, forming a swirling flow field rotating in the same direction. This swirling flow generates strong turbulence and shearing within the cylindrical section 15, achieving instantaneous homogeneous mixing of the two fluids on a millisecond timescale. This effectively eliminates the problems of local concentration gradients and uneven distribution of precipitant, thereby promoting the synchronous nucleation of peptide molecules in a uniform supersaturated environment. This provides a stable input fluid with narrow particle size distribution and high crystal nucleus density for the precipitation ripening device, significantly improving the uniformity and controllability of the precipitation product. The outlet channel 14 guides the swirling mixture smoothly into the precipitation ripening device along the axial direction, avoiding backflow and disturbance, and ensuring the continuity and stability of the precipitation process.

[0037] In one embodiment, the inlet channel 13 has a rectangular cross-section, the outer surface of the inlet channel 13 is tangent to the outer peripheral surface of the cylindrical segment 15, and multiple inlet channels 13 are evenly arranged around the cylindrical segment 15.

[0038] In this embodiment, the cross-section of the inlet channel 13 is designed to be rectangular, and its outer surface is tangent to the outer peripheral surface of the cylindrical segment 15. Multiple inlet channels 13 are evenly distributed along the circumference of the cylindrical segment 15, so that when the peptide lysis solution and the precipitation solvent enter the mixing chamber, they can form a uniform swirling field with stable and symmetrical momentum input in the tangential direction. The rectangular cross-section structure restricts the lateral diffusion of the fluid flow, and the tangent layout of the outer peripheral surface eliminates the disturbance edge when the fluid enters. The uniform circumferential arrangement ensures that the vortex intensity generated by each fluid in the cylindrical segment 15 is consistent and the phase is coordinated, thereby significantly improving the stability and repeatability of the mixing process, avoiding local oversaturation differences caused by uneven fluid distribution, and finally achieving narrowing of the particle size distribution and high consistency of the morphology of the peptide precipitation particles.

[0039] In this embodiment, the cross-section of the inlet channel 13 is designed as a rectangle, mainly considering the feasibility of the processing technology, processing cost and processing efficiency. Since the inlet channel in this embodiment is a microchannel structure with a channel size of micrometer to millimeter, the size is relatively small. If it is processed into a round hole, both the processing complexity and processing cost will increase exponentially. Therefore, considering the cost, processing feasibility and processing efficiency, a rectangular inlet channel 13 is selected.

[0040] Of course, if the technology and processing costs meet the requirements, a circular inlet channel or an inlet channel of other shapes can also be selected.

[0041] In one embodiment, there is one first inlet pipe 6 and at least one second inlet pipe 7, the sum of which is the same as the number of inlet channels 13. Both the first inlet pipe 6 and the second inlet pipe 7 have circular cross-sections, and the structure at the inlet position of the inlet channel is circular to facilitate docking with the first inlet pipe 6 and the second inlet pipe 7. In this embodiment... Figure 2 To illustrate the internal flow channel structure of the microfluidic mixer, the structure at the connection point is not shown. Therefore, a connection port is provided at the inlet end of the inlet channel 13. The connection port is connected to the inlet channel 13 and has a structure that facilitates connection with the inlet pipeline, such as a threaded structure. The cross-sectional shape of the connection port can also be circular or other shapes that are easy to adapt to the inlet pipeline.

[0042] In one embodiment, the height of the inlet channel 13 along the axial direction of the cylindrical segment 15 is the same as the axial height of the cylindrical segment 15.

[0043] In this embodiment, the height of the inlet channel 13 along the axial direction of the cylindrical section 15 is the same as the axial height of the cylindrical section 15. This ensures that the solution is uniformly injected throughout the entire axial range when it enters the mixing chamber 12 through the inlet channel 13. Combined with the fact that the inlet channel 13 is connected to the mixing chamber 12 along the tangential direction of the cylindrical section 15 and that multiple inlet channels 13 are uniformly arranged circumferentially on the cylindrical section 15, this ensures that the solution entering the mixing chamber 12 participates in tangential swirling motion throughout the entire axial direction, forming a complete and continuous three-dimensional swirling field. This avoids the axial velocity gradient and mixing unevenness caused by insufficient height of the inlet channel 13, significantly improving the mixing uniformity and efficiency of the peptide lysis solution and precipitation solvent in the microfluidic mixer. This provides a stable and controllable mixing basis for the formation of peptide precipitation products with narrow particle size distribution and high uniformity in the subsequent precipitation ripening unit 5.

[0044] In one embodiment, the height of the inlet channel 13 along the axial direction of the cylindrical section 15 may also be lower than the axial height of the cylindrical section 15.

[0045] In one embodiment, the number of inlet channels 13 is four.

[0046] In this embodiment, there are four inlet channels 13, and the four inlet channels 13 are connected to the mixing chamber along the tangential direction of the cylindrical section. This allows the peptide lysis solution and the precipitation solvent to be injected into the mixing chamber at high speed through each inlet channel 13 in the same swirling direction, forming four co-rotating fluid vortices. This significantly enhances the turbulence intensity of the fluid in the radial and tangential directions, breaking the common problems of flow field asymmetry and shear unevenness in traditional two-channel or single-channel swirling mixing. As a result, rapid and uniform mixing of precipitated components can be achieved at the microscale, with good controllability. This effectively promotes the synchronous nucleation of precipitated crystals in the initial stage, reduces the grain size distribution range, and improves the consistency and controllability of the precipitated products.

[0047] In one embodiment, the number of inlet channels 13 can be 2, 3 or 5, or other numbers can be selected as needed.

[0048] In practical applications, inlet channels 13 can be activated simultaneously, allowing different inlet channels 13 to be used concurrently to increase solvent delivery. Multiple inlet channels 13 can also be used to simultaneously introduce more different types of solvents. Inlet channels 13 can also be selected as needed; inactive inlet channels 13 can be blocked and opened when necessary.

[0049] In one embodiment, the outlet channel 14 is disposed at one axial end of the mixing chamber 12 and extends axially along the cylindrical segment 15.

[0050] In this embodiment, the outlet channel 14 is located at one axial end of the mixing chamber 12 and extends axially along the cylindrical section 15. This allows the solution introduced tangentially along the cylindrical section 15 via the inlet channel 13 to form a stable rotating flow field within the mixing chamber 12, and then smoothly exit along the axial direction. This avoids eddies or backflow caused by abrupt changes in the flow direction at the outlet, significantly reducing fluid resistance and improving the axial consistency of the flow field. This ensures that the solution entering the precipitation and ripening device 5 maintains good plug flow characteristics, enhances the uniformity and controllability of the precipitation process, and effectively improves the technical effect of narrow and adjustable particle size distribution of peptide precipitation.

[0051] In one embodiment, the mixing chamber 12 further includes a tapered section 16 connected to the axial end of the cylindrical section 15 and gradually tapering away from the cylindrical section 15, with an outlet channel 14 connected to the tapered end of the tapered section 16.

[0052] In this embodiment, a conical section 16 is added to the mixing chamber 12. The conical section 16 is connected to the axial end of the cylindrical section 15 and gradually narrows away from the cylindrical section 15. The outlet channel 14 is directly connected to the narrowing end of the conical section 16. This allows the swirling solution formed by the tangential liquid inlet in the cylindrical section 15 to converge smoothly along the gradually narrowing channel of the conical section 16 when it flows out. This effectively avoids the fluid backflow and violent turbulence disturbance caused by the sudden change in cross-section of the traditional straight outlet. As a result, the solution entering the sedimentation and ripening device 5 is always in a stable plug flow state, ensuring that the sediment particles are subjected to a uniform flow field during the ripening process, suppressing local aggregation, and significantly improving the consistency and controllability of the particle size distribution of the sediment products.

[0053] In one embodiment, a protruding post 17 is provided in the area between the inner surface of the inlet channel 13 near the central axis of the cylindrical section 15 and the outer peripheral surface of the outlet channel 14, and the axial height of the protruding post 17 is less than the axial height of the cylindrical section 15.

[0054] In this embodiment, by setting a protruding post 17 in the mixing chamber 12, the backflow path formed by the high-speed swirling discharge of the solution at the axial end of the mixing chamber 12 can be effectively blocked. This effectively breaks the boundary layer of the fluid inside the swirling mixer, reduces the risk of fluid and sediment accumulation caused by low boundary flow velocity, and avoids the retention or resuspension of precipitated seed crystals or microparticles near the outlet channel 14. This structure, together with the stable swirling field formed by the multiple tangentially entering solutions in the cylindrical section 15, forces the fluid to be discharged uniformly and directionally along the axial direction, significantly suppressing the generation of local low flow velocity areas and eddies. This maintains the stability of the crystal nucleus growth environment during the precipitation process, ensures that the particle size distribution of the precipitated product is narrow and uniform, and improves the controllability and repeatability of the precipitation process.

[0055] In one embodiment, the tapered segment 16 is provided with a concave segment 18 that is recessed towards the cylindrical segment 15. The cross-sectional area of ​​the concave segment 18 decreases along the contraction direction of the tapered segment 16. There are at least two concave segments 18, and the at least two concave segments 18 are evenly arranged along the circumference of the tapered segment 16.

[0056] In this embodiment, by providing a concave section 18 that curves downward toward the cylindrical section 15 on the conical section 16, the concave section 18 can form a compression protrusion on the conical section 16 that bulges toward the central axis of the cylindrical section 15. After the swirling solution enters the conical section 16 from the cylindrical section 15, its rotational momentum will be gradually guided and compressed along the circumference by the concave section 18, effectively breaking the boundary layer of the fluid inside the swirling mixer, reducing the risk of fluid and sediment accumulation caused by low boundary flow velocity, and enabling the mixed solution to form a stable axial convergence flow state in the conical section 16, ensuring that the solution flows uniformly and smoothly toward the outlet channel 14, thereby significantly improving the mixing efficiency and the consistency of sediment distribution, creating favorable conditions for the homogeneous growth of sediment particles in the subsequent sedimentation maturation tank 5, and realizing continuous and highly stable control of the sedimentation process.

[0057] In one embodiment, the bottom surface of the concave segment 18 is flush with the top surface of the cylindrical segment 15.

[0058] In this embodiment, the conical segment 16 is provided with a concave segment 18 that curves downward toward the cylindrical segment 15. The bottom surface of the concave segment 18 is flush with the top surface of the cylindrical segment 15. This allows the precipitating solvent flowing out of the inlet channel 13 to be guided by the concave curved surface structure of the concave segment 18 before it has fully diffused after high-speed tangential flow into the mixing chamber. It is then directionally converged and evenly distributed in the inner cavity of the cylindrical segment, effectively suppressing local disturbances and uneven circumferential distribution of the fluid at the outlet of the inlet channel. This significantly improves the mixing uniformity of the precipitating solvent and the peptide lysis solution in the initial stage of the mixing chamber, thereby providing a stable and controllable nucleation environment for the subsequent precipitation maturation process, and ultimately achieving narrowing and improved uniformity of the precipitate particle size distribution.

[0059] In one embodiment, the bottom surface of the concave section 18 is disposed above the outlet of at least a portion of the inlet channel 13. The concave section 18 being disposed above the outlet of at least a portion of the inlet channel 13 means that when the number of concave sections 18 is less than the number of inlet channels 13, the concave sections 18 cooperate with a portion of the inlet channels 13, while no concave sections 18 are disposed above the other inlet channels 13.

[0060] In some embodiments, a concave section 18 may be provided above each inlet channel 13.

[0061] In this embodiment, the mixing chamber 12 is composed of a cylindrical section 15 and a conical section 16. A protruding post 17 is provided in the cylindrical section 15, and a concave section 18 is provided in the conical section 16. When the solution enters the mixing chamber 12, it first enters the cylindrical section 15. Since the solution enters the cylindrical section 15 tangentially, a swirling flow can be formed. Since the protruding post 17 is located between the inner surface of the inlet channel 13 and the outer peripheral surface of the outlet channel 14, and the bottom surface of the concave section 18 is located above the outlet channel 14, when the solution flows in the cylindrical section 15, it is constrained by both the protruding post 17 and the concave section 18. By setting the protruding post 17 and the concave section 18, the boundary layer of the fluid inside the swirling mixer is effectively broken, reducing the risk of fluid and sediment accumulation caused by low boundary flow velocity.

[0062] The main structure of the precipitation ripening device 5 is a flow channel structure with a certain volume, which allows the solution to be held within it for a period of time. During this time, the solution can flow within the flow channel structure. The main function of the precipitation ripening device 5 is to allow the peptide precipitate to gradually grow and homogenize in size during operation, ultimately completing the precipitation of the peptide. In addition, the precipitation module 2 is designed to use several mixing-ripening units connected in series to complete the stepwise precipitation function. Taking the simplest operating condition as an example, there are two mixing-ripening units, forming a two-stage ripening structure. The volume of precipitation solvent added to each stage is the same. In the first stage unit, the peptide solution flowing out of the main heat exchange module mixes with half the volume of precipitation solvent, and a smaller precipitate is formed. This precipitate is ripened and homogenized and then flows into the second stage unit, where it mixes with the other half of the precipitation solvent. After mixing, the precipitate further precipitates, and new precipitates grow around the first stage precipitation generation unit to obtain larger precipitates. After precipitation is completed in the second stage precipitation unit, the liquid flows into the filtration unit for filtration and recovery. By adjusting the number of mixing-precipitation maturation units and the ratio of precipitation solvent between units, this equipment can effectively regulate parameters such as the size and morphology of the precipitate. Furthermore, each mixing-precipitation maturation unit employs independent pool-type cooling or jacketed cooling for temperature control, or all mixing-precipitation maturation units share a single pool-type cooling or jacketed cooling system for temperature control, ensuring the entire precipitation process is conducted at the designed temperature.

[0063] In other embodiments, the number of mixing-aging units may be three or more, and the flow rate ratio of the precipitating solvent in each stage may be adjusted as needed.

[0064] In one embodiment, the sedimentation and maturation device 5 is a plug flow maturation device.

[0065] In this embodiment, the precipitation ripening device 5 adopts a plug flow structure. Its inlet is directly connected to the first outlet pipe 8 of the cyclone mixer 4, and its outlet is connected to the control valve 11 of the filter module 3. This allows the polypeptide precipitate particles, which have been initially mixed and precipitated by the cyclone mixer 4, to move stably in a single direction during the ripening stage, without axial backmixing or flow velocity disturbance. This significantly suppresses collision aggregation and uneven growth between particles, achieving precise control of the precipitate particle size during the ripening process. This plug flow design, together with multiple mixing-precipitation ripening units arranged in series, ensures that each stage of precipitate is ripened in a uniform, continuous, and non-backflowing flow field. This avoids the problems of wide particle size distribution and large size dispersion caused by uneven fluid mixing in traditional intermittent or fully mixed flow ripening devices. As a result, the final precipitate product has a narrower particle size distribution and better uniformity, providing a reliable physical property basis for the stable operation and high-yield separation of the subsequent filter module 3.

[0066] In one embodiment, the push-flow curing device is a spiral coil or a serpentine tube.

[0067] In this embodiment, when the plug flow ripening device adopts a spiral coil or serpentine tube structure, it can effectively ensure that the solution moves forward stably in an approximately piston flow manner during the ripening process, significantly suppressing fluid backmixing and turbulent disturbances. This allows the precipitated peptide particles to grow uniformly under constant flow rate gradient and thermodynamic conditions, thereby further narrowing the particle size distribution range of the precipitate. The aspect ratio and bending radius of the spiral coil or serpentine tube can be precisely designed according to process requirements, extending the ripening path and enhancing heat exchange uniformity. This avoids inconsistent nucleation caused by local overcooling or overheating. At the same time, its compact structure facilitates integration into a series-connected mixing-precipitation ripening unit. It works in synergy with the step-by-step feeding mode of the cyclone mixer 4 to achieve continuous and controllable processes from mixing, nucleation to ripening, ultimately improving the consistency and yield of the precipitated products.

[0068] In one embodiment, the sedimentation and maturation device 5 can also be a fully mixed flow reactor or the like.

[0069] In one embodiment, the continuous peptide precipitation system further includes a control module, which includes a controller 19 and a data acquisition device. The data acquisition device is used to acquire parameter information of the main heat exchange module 1, the precipitation module 2 and the filtration module 3. The controller 19 controls the main heat exchange module 1, the precipitation module 2 and the filtration module 3 according to the parameter information acquired by the data acquisition device.

[0070] In this embodiment, the control module includes a controller 19 and a data acquisition device. The data acquisition device monitors in real time the temperature and flow rate of the main heat exchange module 1, the mixing intensity and residence time of each cyclone mixer in the precipitation module 2, and the filtration pressure and waste liquid flow rate of the filtration module 3. These parameters are then fed back to the controller 19. The controller 19 dynamically adjusts the heat exchange rate of the main heat exchange module 1, the precipitation solvent input flow rate and pressure of each cyclone mixer, and the opening and closing sequence of the control valves between the filtration module 3 and the waste liquid bottle according to the preset process window. This achieves closed-loop automatic control of the entire peptide precipitation process, ensuring that the precipitation solvent and peptide lysis solution maintain optimal mixing uniformity and nucleation rate in each mixing-precipitation maturation unit. This effectively reduces the particle size distribution range of the precipitation, improves product consistency, and avoids batch fluctuations or over-precipitation caused by human intervention delays. This ensures that the system can stably, efficiently, and repeatedly complete peptide precipitation and separation operations under continuous operation.

[0071] In one embodiment, the data acquisition device includes a thermometer 20, a pressure gauge 21, a turbidity meter, a pH meter, a particle size analyzer, etc.

[0072] In one embodiment, the main heat exchange module 1 further includes a microfluidic mixing heat exchange device 23, which is used to mix the peptide lysis solution and the precipitation solvent, and release the heat generated during the mixing process of the peptide lysis solution and the precipitation solvent.

[0073] The microfluidic mixing heat exchanger 23 is the core component of the main heat exchange module 1, used to handle the strong exothermic reaction during the initial mixing of the lysis solution and the precipitation solvent in the precipitation process. This microfluidic mixing heat exchanger 23 has a larger specific surface area and uses materials resistant to strong acid corrosion and with higher thermal conductivity (such as SiC), enabling it to achieve superior heat exchange efficiency. Within the main heat exchange module 1, two pumps 24 respectively introduce the peptide lysis solution and the precipitation solvent (such as MTBE) into the microfluidic heat exchanger, where initial mixing and heat release are completed, and the solvent flows out from the rear end into the precipitation module 2. By rationally controlling the volume of the precipitation solvent introduced in this stage, precipitation can be prevented from occurring in this stage while maximizing heat release.

[0074] In one embodiment, the structure of the microfluidic mixing heat exchanger 23 is similar to that of the swirl mixer 4, and will not be described in detail here.

[0075] In one embodiment, the microfluidic mixing heat exchanger 23 may also employ other existing mixed-flow heat dissipation structures to achieve initial mixing and heat dissipation of the pyrolysis liquid and the precipitating solvent.

[0076] The process of the technical solution of this application is described below: The peptide lysis buffer and precipitation solvent are input through the input end of the main heat exchange module 1. After preliminary mixing and heat exchange in the main heat exchange module 1, the premixed solution is output from the output end of the main heat exchange module 1. The premixed solution flows into the first inlet pipe 6 of the cyclone mixer 4 of the mixing-precipitation maturation unit at the beginning of the precipitation module 2. At the same time, the precipitation solvent is input through the second inlet pipe 7 of the cyclone mixer 4. In the cyclone mixer 4, the peptide lysis buffer and precipitation solvent are respectively connected through the inlet channels tangentially along the cylindrical section 15. 13 is injected at high speed into the cylindrical section 15 of the mixing chamber 12 in the same swirling direction, forming a swirling field rotating in the same direction, achieving millisecond-level instantaneous homogeneous mixing. The mixed solution is smoothly discharged along the axial direction through the outlet channel 14. The outlet channel 14 extends along the axial direction of the cylindrical section 15 and is connected to the concave end of the conical section 16 of the mixing chamber 12. The conical section 16 is provided with at least two concave sections 18 evenly arranged circumferentially. The bottom surface of the concave section 18 is flush with the top surface of the cylindrical section 15 and is located above at least part of the outlet of the inlet channel 13, so that the solution flows circumferentially within the conical section 16. The solution is directed and evenly distributed to avoid backflow and disturbance. After entering the precipitation and ripening unit 5, the mixed solution is stably advanced in the plug flow structure to complete the first stage of precipitation and crystal nucleus ripening. The outlet of the precipitation and ripening unit 5 is connected to the first inlet pipe 6 of the cyclone mixer 4 of the next mixing-precipitation ripening unit. At the same time, another part of the precipitation solvent is input through the second inlet pipe 7 of the downstream cyclone mixer 4, repeating the above-mentioned tangential injection, cyclone mixing, axial discharge and plug flow ripening process. This process is carried out in series step by step until the final precipitation is completed in the end mixing-precipitation ripening unit. After the precipitated slurry flows out from the outlet of the precipitator 5 of the end mixing-precipitation maturation unit, it is selectively guided to the filter 9 or the waste bottle 10 by the control valve 11. At the initial stage of equipment startup, the control valve 11 guides the fluid to the waste bottle 10. When the precipitate is about to flow out, the control valve 11 switches to the filter 9 and starts the filtration, so that the peptide solid is retained above the filter 9 and the solution is discharged through the filter 9. After the precipitate has completely flowed out, it continues to be rinsed for 1-2 minutes. Then the control valve 11 switches back to the waste bottle 10 to complete the solid-liquid separation. Finally, the peptide precipitate is recovered from above the filter 9.

[0077] Example

[0078] This embodiment follows Figure 1 The schematic diagram shows the construction of a complete continuous peptide precipitation system. This system was used to process the lysis buffer (i.e., peptide solution, with trifluoroacetic acid as the main solvent) in peptide solid-phase synthesis and to recover intact peptide precipitates.

[0079] In the experiment, methyl tert-butyl ether (MTBE) was selected as the precipitant. MTBE was sequentially introduced into the main heat exchange module 1 and the precipitation module 2. Visual observation clearly showed that the liquid flowing out of the main heat exchange module 1 remained clear and transparent. In the precipitation module 2, the liquid flowing out from the first stage mixing-maturation unit was slightly turbid, and the liquid flowing out from the second stage mixing-maturation unit was completely turbid. This experimental phenomenon is consistent with the design expectation of the device: no precipitation occurs in the main heat exchange module 1, fine seed crystals precipitate in the first stage of the precipitation module 2, and a large amount of precipitate precipitates in the second stage. After treatment by the filtration module 3, the dried white precipitate was recovered, which met the expectations for peptide precipitation recovery and had low solvent residue (when the solvent residue is high, the precipitate appears yellow). After testing, the purity and recovery rate of the recovered peptide were comparable to those of traditional peptide precipitation schemes, while the precipitation recovery time was significantly reduced (to less than 1 / 10), showing a significant advantage over traditional schemes.

[0080] Furthermore, this embodiment experimentally demonstrates the device's ability to control precipitate size. Here, a preferred device structure is one in which the precipitation module 2 contains two mixing-aging units. By adjusting the ratio of the precipitation solvent input to the two units, this embodiment successfully regulates the final precipitate size.

[0081] In the experiment, three experimental conditions were selected: 1. The flow rate of the first-stage mixing-maturation unit was higher than that of the second-stage mixing-maturation unit; 2. The flow rate of the first-stage mixing-maturation unit was equal to that of the second-stage mixing-maturation unit; 3. The flow rate of the first-stage mixing-maturation unit was less than that of the second-stage mixing-maturation unit. The size of the precipitate was characterized by microscopic observation. Figure 3 It is evident that the largest precipitate size was obtained under experimental condition 1, followed by experimental condition 2, and the smallest precipitate size was obtained under experimental condition 3, demonstrating the device's ability to control the precipitate size.

[0082] The technical solution of this application embodiment has the following effects:

[0083] 1. The equipment in this embodiment is a fully continuous operating system, rather than a batch operating system. It has higher efficiency, smaller size and footprint, lower operating and maintenance costs, and can quickly meet the demand for increased production.

[0084] 2. The fully continuous operation equipment in this embodiment is easier to operate, easier to achieve in a closed environment, and more suitable for the environmental requirements of the oligonucleotide field.

[0085] 3. By using microfluidic components to enhance heat exchange and mixing efficiency, higher quality sediment can be obtained.

[0086] 4. The peptide precipitation efficiency is higher, the time is shorter, and the operation is simpler. The contact time between peptides and TFA is shortened from hours to minutes, which greatly reduces the risk of peptide deterioration.

[0087] 5. Through rapid mixing with a microfluidic mixer and subsequent plug flow precipitator, the precipitation process can be controlled more precisely, resulting in precipitated products with more uniform peptide size distribution and larger particle size.

[0088] 6. By adjusting the number of precipitation units and the feeding ratio of precipitation solvent, the particle size of peptide products can be controlled as needed, which is beneficial for subsequent operations such as filtration and washing.

[0089] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0090] It should be noted that the terms "first," "second," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in sequences other than those illustrated or described herein.

[0091] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide continuous precipitation system, characterized in that, include: The main heat exchange module (1) has an input end and an output end. The input end is used to input peptide lysis solution and precipitation solvent into the main heat exchange module (1), and the output end is used to output the mixed solution that has been initially mixed in the main heat exchange module (1). The precipitation module (2) includes at least two mixing-precipitation maturation units arranged in series. The mixing-precipitation maturation unit includes a cyclone mixer (4) and a precipitation maturation unit (5). The cyclone mixer (4) includes a first inlet pipe (6), a second inlet pipe (7), and a first outlet pipe (8). The inlet of the precipitation maturation unit (5) is connected to the first outlet pipe (8) of the cyclone mixer (4). The output end of the main heat exchange module (1) is connected to the first inlet pipe (6) of the cyclone mixer (4) of the first mixing-precipitation maturation unit. The first inlet pipe (6) of the other cyclone mixers (4) is connected to the outlet of the upstream precipitation maturation unit (5). The second inlet pipe (7) of each cyclone mixer (4) is used to input precipitation solvent into the cyclone mixer (4). The filtration module (3) includes a suction filter (9) and a waste bottle (10) arranged in parallel. The suction filter (9) and the waste bottle (10) are selectively connected to the outlet of the sedimentation maturer (5) of the end of the mixing-sedimentation maturer unit via a control valve (11). The main heat exchange module also includes a microfluidic mixing heat exchange device (23), which is used to mix the peptide lysis solution and the precipitation solvent, and release the heat generated during the mixing process of the peptide lysis solution and the precipitation solvent; The cyclone mixer (4) receives the mixed solution flowing from upstream through the first inlet pipe (6), and at the same time introduces additional precipitation solvent in stages through the second inlet pipe (7). Under the action of cyclone, efficient and instantaneous local mixing is achieved. The mixed solution enters the precipitation ripening device (5) through the first outlet pipe (8) and completes step-by-step precipitation and crystal nucleus ripening within a controllable residence time. Multiple mixing-sedimentation maturation units are connected in sequence. The outlet of the upstream sedimentation maturation unit (5) is connected to the first inlet pipe (6) of the downstream cyclone mixer (4) to form a continuous flow sedimentation path, so that the sedimentation process can be segmented and controllable from initial nucleation to final particle size stabilization.

2. The polypeptide continuous precipitation system of claim 1, wherein, The swirl mixer (4) is a microfluidic mixer.

3. The polypeptide continuous precipitation system of claim 2, wherein, The microfluidic mixer includes a mixing chamber (12), an inlet channel (13), and an outlet channel (14). The mixing chamber (12) includes a cylindrical section (15). There are at least two inlet channels (13). At least two inlet channels (13) are connected to the mixing chamber (12) along the tangential direction of the cylindrical section (15). The swirling direction of the solution entering the cylindrical section (15) through each inlet channel (13) is the same. The first inlet pipe (6) and the second inlet pipe (7) are both connected to the corresponding inlet channel (13).

4. The continuous polypeptide precipitation system according to claim 3, characterized in that, The inlet channel (13) has a rectangular cross-section. The outer surface of the inlet channel (13) is tangent to the outer circumferential surface of the cylindrical segment (15). Multiple inlet channels (13) are evenly arranged around the cylindrical segment (15).

5. The continuous polypeptide precipitation system according to claim 4, characterized in that, The height of the inlet channel (13) along the axial direction of the cylindrical segment (15) is the same as the axial height of the cylindrical segment (15).

6. The continuous polypeptide precipitation system according to claim 3, characterized in that, The number of import channels (13) is four.

7. The continuous polypeptide precipitation system according to claim 3, characterized in that, The outlet channel (14) is located at one axial end of the mixing chamber (12) and extends axially along the cylindrical section (15).

8. The continuous polypeptide precipitation system according to claim 7, characterized in that, The mixing chamber (12) further includes a conical section (16) which is connected to the axial end of the cylindrical section (15) and gradually narrows away from the cylindrical section (15). The outlet channel (14) is connected to the narrowed end of the conical section (16).

9. The continuous polypeptide precipitation system according to claim 7 or 8, characterized in that, A protruding post (17) is provided in the area between the inner surface of the inlet channel (13) near the central axis of the cylindrical section (15) and the outer peripheral surface of the outlet channel (14). The axial height of the protruding post (17) is less than the axial height of the cylindrical section (15).

10. The continuous polypeptide precipitation system according to claim 8, characterized in that, The conical segment (16) is provided with a concave segment (18) that is recessed into the cylindrical segment (15). The cross-sectional area of ​​the concave segment (18) decreases along the contraction direction of the conical segment (16). There are at least two concave segments (18), and at least two concave segments (18) are evenly arranged along the circumference of the conical segment (16).

11. The continuous polypeptide precipitation system according to claim 10, characterized in that, The bottom surface of the concave section (18) is flush with the top surface of the cylindrical section (15).

12. The continuous polypeptide precipitation system according to claim 1, characterized in that, The sedimentation and maturation device (5) is a push flow maturation device.

13. The continuous polypeptide precipitation system according to claim 12, characterized in that, The push-flow curing device is a spiral coil or a serpentine tube.

14. The continuous polypeptide precipitation system according to claim 1, characterized in that, The continuous polypeptide precipitation system further includes a control module, which includes a controller (19) and a data acquisition device. The data acquisition device is used to acquire parameter information of the main heat exchange module (1), the precipitation module (2) and the filtration module (3). The controller (19) controls the main heat exchange module (1), the precipitation module (2) and the filtration module (3) according to the parameter information acquired by the data acquisition device.

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