An apparatus for preparing plasmids

Abstract

The utility model relates to a kind of device for preparing plasmid, including purification module and ultrafiltration module, purification module includes first feeding unit, second feeding unit, chromatography unit, waste liquid container and collector, it is configured to at least have following three kinds of state: in first state, first feeding unit, chromatography unit, waste liquid container are sequentially communicated;In second state, the liquid inlet side of chromatography unit, liquid outlet side respectively with second feeding unit are communicated, to form circulation flow path and carry out multiple chromatography;In third state, second feeding unit, chromatography unit, collector are sequentially communicated.The device integrates the unit such as traditional dispersion independent purification, ultrafiltration in a unified device, layout is compact, and pollution risk in traditional open operating environment is eliminated;And, the circulation flow path configuration under the second state of purification module, so that feed liquid can circulate through chromatography column multiple times, realizes efficient, flexible multiple chromatography purification.

Description

Technical Field

[0001] This utility model relates to the field of plasmid preparation technology, specifically to an apparatus for preparing plasmids. Background Technology

[0002] Plasmids, as circular double-stranded DNA molecules, are fundamental carriers and key materials in modern biomedicine and industrial biotechnology. They play an irreplaceable role in gene therapy, cell therapy, viral vector production, mRNA vaccines, and recombinant protein drugs. Especially against the backdrop of explosive growth in the global mRNA technology platform and gene therapy industry, the market demand for high-quality, therapeutic-grade plasmids, as starting materials or key excipients, has reached an unprecedented scale.

[0003] However, large-scale plasmid production, especially scaling up from laboratory scale to commercial production compliant with Good Manufacturing Practices (GMP), faces significant challenges. These challenges primarily manifest in: yield, process stability, the complexity of downstream purification, and stringent quality control requirements. Currently, typical laboratory and small-scale plasmid preparation processes include: preparation and filtration of various solutions, adjustment of sample pH and conductivity, sample collection, multi-step chromatographic purification (such as affinity chromatography and ion exchange chromatography), ultrafiltration concentration and liquid exchange of intermediate products, and final pre-filtration and sterile filtration. This series of processes is not only cumbersome but also highly dependent on manual operation.

[0004] A more prominent problem is that existing technologies typically rely on laying out multiple independent functional devices (such as chromatography systems, ultrafiltration devices, and filter equipment) on a flat surface and connecting them with temporary tubing on a biosafety cabinet or workbench. This approach has two inherent drawbacks: First, it occupies a large amount of desktop space, has low system integration, and is difficult to standardize and scale up; second, and most importantly, the repeated manual connection, pipetting, and manipulation in open or semi-open spaces greatly increases the risk of product contamination by microorganisms, endotoxins, and other pollutants, seriously threatening the safety and quality uniformity of the final product and failing to meet the requirements of GMP closed production.

[0005] Therefore, there is an urgent need in this field for an integrated, closed plasmid preparation device to solve the problems of cumbersome operation, large space occupation, and high pollution risk in the existing technology, so as to meet the industry's urgent need for large-scale, efficient, and stable production of high-quality plasmids. Utility Model Content

[0006] The purpose of this invention is to provide an integrated, closed device for preparing plasmids, in order to solve the problems of cumbersome operation, large space occupation, and high risk of contamination in the prior art.

[0007] To achieve the above objectives, the technical solution adopted by this utility model is as follows: This invention provides an apparatus for preparing plasmids, comprising: A purification module includes a first feed unit, a second feed unit, a chromatography unit, a waste container, and a collector. The first feed unit has multiple first inlets connected to the inlet side of the chromatography unit via first inlet pipes. The second feed unit has multiple second inlets connected to the inlet side of the chromatography unit via second inlet pipes and connected to the outlet side of the chromatography unit via at least one return pipe. The chromatography unit has multiple chromatography columns arranged in parallel. The waste container and collector are connected to the outlet side of the chromatography unit via first outlet pipes and second outlet pipes, respectively. An ultrafiltration module includes an ultrafiltration unit connected to the collector, a sterilization filtration unit connected to the ultrafiltration unit, and a product container connected to the sterilization filtration unit. The purification module is configured to have at least the following three states: In the first state, the first feeding unit, the chromatography unit, and the waste liquid container are connected in sequence. In the second state, the inlet and outlet sides of the chromatography unit are connected to the second feed unit to form a circulation path for multiple chromatography steps. In the third state, the second feeding unit, the chromatography unit, and the collector are connected in sequence.

[0008] In some embodiments, the chromatography unit further includes a column position valve having multiple channels that are connected one-to-one with the chromatography column. The column position valve is configured to selectively open any one of its multiple channels, and when one channel is opened, the other channels are closed.

[0009] In some embodiments, the number of return lines is one less than the number of chromatography columns, and different return lines are connected to the same or different second inlets of the second feed unit.

[0010] In some specific embodiments, the number of chromatography columns is three.

[0011] In some embodiments, the first feeding unit includes a first inlet valve connected to the inlet side of the first liquid inlet pipeline and a first liquid inlet pump connected to the first liquid inlet pipeline, the first inlet valve having the plurality of first liquid inlets.

[0012] In some embodiments, the second feeding unit includes a second inlet valve connected to the inlet side of the second liquid inlet pipeline and a second liquid inlet pump connected to the second liquid inlet pipeline, the second inlet valve having the plurality of second liquid inlets.

[0013] In some implementations, the number of the first inlet and the number of the second inlet may be the same or different.

[0014] In some implementations, the number of the first liquid inlet and the number of the second liquid inlet are each independently 6 to 8, such as 6, 7 or 8.

[0015] In some implementations, when there are multiple return lines, the multiple return lines are connected to different second inlets.

[0016] In some embodiments, the apparatus further includes a detector located on the effluent side of the chromatography unit and in front of the waste container and the collector.

[0017] In some embodiments, the ultrafiltration unit includes a storage bottle, an ultrafilter, and a washing bottle; the inlet of the storage bottle is connected to the collector, and the outlet is connected to the inlet of the ultrafilter; the washing bottle is connected to the storage bottle; the ultrafilter has a first outlet and a second outlet, the first outlet being connected to a waste liquid collection container, and the second outlet being connected to the sterilization filtration unit.

[0018] In some specific embodiments, the ultrafilter is a tangential flow ultrafilter, and its filter membrane pack is a detachable structure.

[0019] In some embodiments, the device further includes a pressure monitoring module, which includes pressure sensors respectively disposed on the inlet and outlet sides of the ultrafiltration unit for monitoring the pressure at the corresponding locations.

[0020] Due to the application of the above technical solution, this utility model has the following advantages compared with the prior art: This invention, through the unique design of the purification module, especially the circulation flow path configuration in the second state, enables the feed solution to circulate through the chromatography column multiple times, achieving efficient and flexible multiple chromatography purification, improving the purification efficiency and recovery rate of the target plasmid, thereby effectively removing impurities and ensuring the quality of the final product.

[0021] This invention modularly integrates traditionally separate purification, ultrafiltration, and sterilization filtration units into a unified device. Through optimized piping design, it replaces numerous scattered pieces of equipment and temporary connections, greatly simplifying the overall structure, creating a compact layout, and significantly saving space. Furthermore, from the moment the sample enters the purification module until the final product is stored in the product container, the entire preparation process can be completed within sealed pipelines and units, completely eliminating the risk of microbial and endotoxin contamination common in traditional open operating environments. This provides reliable quality and safety assurance for the production of therapeutic plasmids. Attached Figure Description

[0022] Figure 1 This is a simplified structural diagram of the apparatus used for plasmid preparation in Example 1; Wherein: 1. First feeding unit; 11. First inlet valve; 111. First liquid inlet; 12. First liquid inlet pump; 13. First liquid inlet pipeline; 2. Second feeding unit; 21. Second inlet valve; 211. Second liquid inlet; 22. Second liquid inlet pump; 23. Second liquid inlet pipeline; 24. Return liquid pipeline; 25. Three-way valve; 3. Chromatography unit; 31. First chromatography column; 32. Second chromatography column; 33. Third chromatography column; 34. Column position valve; 35. Sample loading valve; 36. Sample dispensing valve; 37. Detector; 4. Waste liquid container; 5. Collector; 6. Ultrafiltration module; 61. Ultrafiltration unit; 611. Ultrafilter; 612. Storage bottle; 613. Washing solution bottle; 614. Waste liquid collection container; 62. Sterilization filtration unit; 63. Product container; 64. Pressure sensor; 65. Flow meter. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments shown in the accompanying drawings.

[0024] The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications. Implementation conditions not specified are standard conditions in this industry. The technical features involved in the various embodiments of this invention can be combined with each other as long as they do not conflict with each other.

[0025] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0026] In the description of the embodiments of this utility model, it should be understood that the terms "front" and "rear", etc., indicate the orientation or positional relationship based on the flow direction of the liquid, and are only for the convenience of describing the embodiments of this utility model and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the embodiments of this utility model.

[0027] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of embodiments of this utility model, "a plurality of" means two or more, unless otherwise explicitly specified.

[0028] The following disclosure provides many different implementations or examples for different structures of the embodiments of the present invention. To simplify the disclosure of the embodiments of the present invention, specific examples of components and arrangements are described below. Of course, these are merely examples and are not intended to limit the embodiments of the present invention. Furthermore, reference numerals and / or reference letters may be repeated in different examples of the embodiments of the present invention; such repetition is for simplification and clarity and does not in itself indicate a relationship between the various implementations and / or arrangements discussed.

[0029] Example 1: This utility model provides an apparatus for preparing plasmids, such as... Figure 1 As shown, it includes a purification module and an ultrafiltration module 6. The purification module includes a first feed unit 1, a second feed unit 2, a chromatography unit 3, a waste container 4, and a collector 5. The first feed unit 1 has multiple first inlets 111, which are connected to the inlet side of the chromatography unit 3 through a first inlet pipe 13; the second feed unit 2 has multiple second inlets 211, which are connected to the inlet side of the chromatography unit 3 through a second inlet pipe 23, and connected to the outlet side of the chromatography unit 3 through at least one return pipe 24; the chromatography unit 3 has multiple chromatography columns arranged in parallel; the waste container 4 and the collector 5 are connected to the outlet side of the chromatography unit 3 through a first outlet pipe and a second outlet pipe, respectively. The ultrafiltration module 6 includes an ultrafiltration unit 61 connected to the collector 5, a sterilization filtration unit 62 connected to the ultrafiltration unit 61, and a product container 63 connected to the sterilization filtration unit 62. The purification module is configured to have at least the following three states: in the first state, the first feed unit 1, the chromatography unit 3, and the waste liquid container 4 are connected in sequence; in the second state, the inlet side and outlet side of the chromatography unit 3 are connected to the second feed unit 2 respectively to form a circulation path for multiple chromatographys; in the third state, the second feed unit 2, the chromatography unit 3, and the collector 5 are connected in sequence.

[0030] In this embodiment, the chromatography unit 3 preferably has three parallel chromatography columns, namely a first chromatography column 31, a second chromatography column 32, and a third chromatography column 33. The three chromatography columns may be of the same or different models, and the specific selection can be made according to the actual production scale and process requirements. The number of return lines 24 is one less than the number of chromatography columns, that is, there are two return lines 24. Each return line 24 has a valve that can control the opening and closing of the line, and the valve is preferably a three-way valve 25. As a further preferred embodiment, the two return lines 24 are connected to different second inlets 211. The chromatography unit 3 also includes a column position valve 34, which has multiple channels, and the multiple channels are connected to the chromatography columns one by one. The column position valve 34 is configured to selectively open any one of its multiple channels. When one channel is opened, the other channels are closed. Its specific structure can be referred to the prior art.

[0031] During operation, the sample enters the second feed unit 2 through the second inlet 211 and then flows into the first chromatography column 31 for primary chromatography. The outflowing liquid returns to the second feed unit 2 via a return line 24 and is then pumped into the second chromatography column 32 for secondary chromatography. This process is repeated once, with the liquid returning again via another return line 24 and entering the third chromatography column 33 to complete the third chromatography. After chromatography, the liquid flows into the collector 5. In other embodiments, the number of chromatography columns and return lines 24 can be reduced or increased to perform fewer or more chromatography steps.

[0032] The main advantages of the above design are: 1) Achieving efficient multi-step purification: Through the cascaded path of "chromatography-reflux-re-chromatography," a single sample can automatically and continuously undergo three independent chromatographic purification processes within a closed system. This greatly enhances the removal capacity of impurities such as host RNA, proteins, and endotoxins, significantly improving the purity and quality of the final plasmid product and effectively meeting the stringent standards for therapeutic-grade plasmids. 2) Optimized structure and reduced costs: The "N chromatography columns with N-1 return lines" scheme ensures multi-step purification while minimizing the complexity of the piping system. This not only makes the device structure more compact and reduces internal dead volume but also lowers hardware manufacturing costs and the complexity of system cleaning and validation. 3) Enhanced automation and reliability: The "selective conduction" function of the column position valve ensures precise switching of the fluid path. The entire process requires no manual intervention, simplifying operation and completely avoiding the contamination risks associated with open operation, ensuring stability and reproducibility between production batches.

[0033] In the aforementioned tandem chromatography process, a column cleaning and equilibration step is also included. Specifically, the equilibration solution and / or eluent can enter the first feed unit 1 through the first inlet 111, and then flow through the target chromatography column to be put into use, performing thorough cleaning and condition equilibration. The waste liquid generated during this process is collected in the waste liquid container 4. In this way, the consistency of chromatography column performance is ensured, and the target plasmid has a stable and high binding efficiency on the chromatography column, while timely cleaning minimizes cross-contamination between different purification cycles. At the same time, the entire production process is kept completely closed from beginning to end, completely eliminating the risk of contamination introduced by manual handling between multiple chromatography steps.

[0034] The first feeding unit 1 includes a first inlet valve 11 connected to the inlet side of the first liquid inlet pipeline 13 and a first liquid inlet pump 12 connected to the first liquid inlet pipeline 13. The first inlet valve 11 has the aforementioned plurality of first liquid inlets 111. In this embodiment, the number of first liquid inlets 111 is seven, namely first liquid inlet a, first liquid inlet b, first liquid inlet c, first liquid inlet d, first liquid inlet e, first liquid inlet f, and first liquid inlet g. The second feeding unit 2 includes a second inlet valve 21 connected to the inlet side of the second liquid inlet pipeline 23 and a second liquid inlet pump 22 connected to the second liquid inlet pipeline 23. The second inlet valve 21 has the aforementioned plurality of second liquid inlets 211. The number of second liquid inlets 211 is also seven, namely second liquid inlet a, second liquid inlet b, second liquid inlet c, second liquid inlet d, second liquid inlet e, second liquid inlet f, and second liquid inlet g. Each first liquid inlet 111 and second liquid inlet 211 is connected to a feed pipe for connecting to the material source; and the feed pipe is equipped with a valve, preferably a solenoid valve, which can automatically control opening and closing.

[0035] The chromatography unit 3 also includes a sample loading valve 35 disposed on its inlet side, a sample outlet valve 36 disposed on its outlet side, and a detector 37 located between its outlet side and the waste container 4. The detector 37 includes, but is not limited to, one or more of UV detection, pH detection, and conductivity detection devices.

[0036] The ultrafiltration unit 61 includes a storage bottle 612, an ultrafilter 611, and a washing bottle 613. The inlet of the storage bottle 612 is connected to the collector 5 for receiving the chromatographically purified sample; its outlet is connected to the inlet of the ultrafilter 611. The washing bottle 613 is connected to the storage bottle 612 and is used to introduce washing solution or replacement buffer into the system during ultrafiltration. The ultrafilter 611 has a first outlet and a second outlet. The first outlet is connected to a waste collection container 614 for discharging the filtrate containing impurities; the second outlet is connected to a sterile filtration unit 62 for guiding the ultrafiltered product liquid into subsequent processes. The ultrafilter 611 is preferably a tangential flow ultrafilter 611, and its filter membrane pack is a detachable structure. The sterile filtration unit 62 can refer to existing technologies in the art. To further improve the accuracy of process control and process reproducibility, a pressure monitoring module is also added to the device. This module includes pressure sensors 64 respectively located on the inlet side, reflux side and outlet side of the ultrafiltration unit 61, for real-time monitoring of key process parameters such as transmembrane pressure.

[0037] Working principle: 1. Purification: 1.1 First step of chromatography: First chromatography column 31: Sepharose 6 Fast Flow, experimental flow rate: 90 cm / h; first inlet a: equilibration buffer 5CV; first inlet b: elution buffer for elution; collection parameters: 1000~100 mAU, collected into second inlet a.

[0038] 1.2 Second step chromatography: Second chromatography column 32: PlasmidSelect Xtra, experimental flow rate: 90 cm / h; equilibration buffer is introduced into the first inlet c for 5CV; elution buffer is introduced into the first inlet d for elution; collection parameters: 2000~100 mAU, collected into the second inlet b.

[0039] 1.3 Third step chromatography: Third chromatography column 33: Capto Q ImpRes, experimental flow rate: 90 cm / h; equilibration buffer is introduced into the first inlet e to equilibrate 5CV; elution buffer is introduced into the first inlet f for elution; collection parameters: 200~100 mAU, collected into collector 5.

[0040] 2. Ultrafiltration: The purified intermediate is pumped into ultrafiltration unit 61 for ultrafiltration concentration. Based on the central control data, it is concentrated to 2.0 mg / mL. The remaining solution volume is calculated based on the difference between the permeate volume measured by flow meter 65 and the total intermediate volume in storage bottle 612. The buffer solution in the eluent is pumped into ultrafiltration unit 61 to replace the original buffer solution with 8 times its original volume. After the buffer replacement is complete, ultrafiltration concentration continues to the desired volume. The pressure in the inlet, reflux, and permeate pipes of ultrafiltration unit 61 is monitored by pressure sensor 64, and the pressure must not exceed 10 Psi.

[0041] 3. Filtration: After ultrafiltration, the product is pumped into the sterilization filtration unit 62 for pre-filtration and sterilization filtration, and the pressure shall not exceed 1 bar.

[0042] The above embodiments are only for illustrating the technical concept and features of this utility model, and are intended to enable those skilled in the art to understand the content of this utility model and implement it accordingly. They should not be construed as limiting the scope of protection of this utility model. All equivalent changes or modifications made in accordance with the spirit and essence of this utility model should be included within the scope of protection of this utility model.

Claims

1. An apparatus for preparing plasmids, characterized in that, include: The purification module includes a first feed unit (1), a second feed unit (2), a chromatography unit (3), a waste container (4), and a collector (5). The first feed unit (1) has multiple first inlets (111), which are connected to the inlet side of the chromatography unit (3) via a first inlet pipe (13). The second feed unit (2) has multiple second inlets (211), which are connected to the inlet side of the chromatography unit (3) via a second inlet pipe (23) and to the outlet side of the chromatography unit (3) via at least one return pipe (24). The chromatography unit (3) has multiple chromatography columns arranged in parallel. The waste container (4) and the collector (5) are connected to the outlet side of the chromatography unit (3) via a first outlet pipe and a second outlet pipe, respectively. The ultrafiltration module (6) includes an ultrafiltration unit (61) connected to the collector (5), a sterilization filtration unit (62) connected to the ultrafiltration unit (61), and a product container (63) connected to the sterilization filtration unit (62). The purification module is configured to have at least the following three states: In the first state, the first feeding unit (1), the chromatography unit (3), and the waste liquid container (4) are connected in sequence; In the second state, the inlet side and outlet side of the chromatography unit (3) are connected to the second feed unit (2) to form a circulating flow path for multiple chromatography steps; In the third state, the second feeding unit (2), the chromatography unit (3), and the collector (5) are connected in sequence.

2. The apparatus for preparing plasmids according to claim 1, characterized in that, The chromatography unit (3) also includes a column position valve (34), which has multiple channels that are connected to the chromatography column one by one. The column position valve (34) is configured to selectively open any one of its multiple channels, and when one channel is opened, the other channels are closed.

3. The apparatus for preparing plasmids according to claim 1, characterized in that, The number of return lines (24) is one less than the number of chromatography columns, and different return lines (24) are connected to the same or different second inlets (211) of the second feed unit (2).

4. The apparatus for preparing plasmids according to claim 3, characterized in that, The number of chromatography columns is three.

5. The apparatus for preparing plasmids according to claim 1, characterized in that, The first feeding unit (1) includes a first inlet valve (11) connected to the inlet side of the first liquid inlet pipeline (13) and a first liquid inlet pump (12) connected to the first liquid inlet pipeline (13), wherein the first inlet valve (11) has the plurality of first liquid inlets (111); and / or, The second feeding unit (2) includes a second inlet valve (21) connected to the inlet side of the second liquid inlet pipeline (23) and a second liquid inlet pump (22) connected to the second liquid inlet pipeline (23). The second inlet valve (21) has the plurality of second liquid inlets (211).

6. The apparatus for preparing plasmids according to claim 5, characterized in that, The number of the first liquid inlet (111) and the second liquid inlet (211) may be the same or different; and / or, The number of the first liquid inlet (111) and the second liquid inlet (211) are each independently 6 to 8; and / or, When there are multiple return lines (24), the multiple return lines (24) are connected to different second inlets (211).

7. The apparatus for preparing plasmids according to claim 1, characterized in that, The device also includes a detector (37) located on the liquid outlet side of the chromatography unit (3) and in front of the waste container (4) and the collector (5).

8. The apparatus for preparing plasmids according to claim 1, characterized in that, The ultrafiltration unit (61) includes a storage bottle (612), an ultrafilter (611), and a washing bottle (613); the inlet of the storage bottle (612) is connected to the collector (5), and the outlet is connected to the inlet of the ultrafilter (611); the washing bottle (613) is connected to the storage bottle (612); the ultrafilter (611) has a first outlet and a second outlet, the first outlet is connected to the waste liquid collection container (614), and the second outlet is connected to the sterilization filtration unit (62).

9. The apparatus for preparing plasmids according to claim 8, characterized in that, The ultrafilter (611) is a tangential flow ultrafilter (611), and its filter membrane pack is a detachable structure.

10. The apparatus for preparing plasmids according to claim 1, characterized in that, The device also includes a pressure monitoring module, which includes pressure sensors (64) respectively disposed on the inlet side and outlet side of the ultrafiltration unit (61) for monitoring the pressure at the corresponding locations.

Images