WO2024017300A1

WO2024017300A1 - System for preparing mrna liposomes and use thereof

Info

Publication number: WO2024017300A1
Application number: PCT/CN2023/108173
Authority: WO
Inventors: 吴守亮; 毛叶飞; 徐丙良
Original assignee: 浙江健新原力制药有限公司
Priority date: 2022-07-19
Filing date: 2023-07-19
Publication date: 2024-01-25
Also published as: CN117597110A

Abstract

The present application relates to a system for preparing mRNA liposomes and use thereof. The system comprises a liquid preparation unit, an encapsulation unit, a liquid exchange unit, and a formulation unit according to the order in which they are connected. The system is a fully closed system. Using the system of the present application to produce mRNA liposomes can effectively avoid contamination and cross-contamination, reduce the frequency of sterilizing filtration, reduce the influence of the environment, equipment, personnel and other external factors on cell products, increase the yield, and improve the stability of products.

Description

A system for preparing mRNA liposomes and its application

Technical field

This application belongs to the field of liposome technology, and specifically relates to a system for preparing mRNA liposomes and its application.

Background technique

mRNA technology products are based on the "central dogma" of mRNA guiding protein synthesis. They are designed and synthesized in vitro containing mRNA sequences encoding specific antigens. After sequence optimization, chemical modification and purification, they are delivered to human cells in different ways and are translated by the body's cells. Produce proteins, induce immune responses, supplement body proteins, regulate immunity, etc., thereby preventing or treating diseases. It has a wide range of applications, including the preparation of infectious disease vaccines, tumor immunotherapy, monoclonal antibody drug substitution, and other protein drug substitution. At present, the fastest-growing and most widely used vaccines are preventive vaccines for infectious diseases. The mRNA vaccine represented by BNT162b2 produced by Pfizer has shined in the application of COVID-19.

There are many areas for improvement in the current production of mRNA drugs. For example: 1) Since the mRNA drug will be infused back into the patient, it is an injection grade. According to Pharmacopoeia IV, the level of sterility control is extremely high, so its preparation process requires very high sterility control of the environment, personnel, and equipment; 2 ) When the conventional production process produces different samples at the same time, it is easy to cause cross-contamination through equipment, environment, and personnel, which makes it impossible to achieve multi-variety collinear production of products. The production of gene therapy products itself has small scale, high quality requirements, and demand. Features such as multi-product collinear production; 3) The production process of mRNA liposomes mainly reduces the microbial load by adding sterilization and filtration processes, but sterilization and filtration will seriously affect the recovery rate of the product; 4) The production stage of mRNA liposomes requires The use of high-concentration ethanol solutions may cause safety hazards during the production process. Therefore, there is an urgent need in the market for a fully enclosed production process to ensure the quality and effectiveness of the product and promote the development of the entire gene therapy industry.

Contents of the invention

The purpose of this application is to provide a system for preparing mRNA liposomes and its application. When using the system of this application to prepare mRNA liposomes, the pipelines through which the raw materials, intermediate products and final products flow are fully closed, which can effectively avoid contamination and cross-contamination, reduce the number of sterilization filtrations, and reduce the environmental impact. , equipment, personnel and other external factors on cell products, and improve the quality and stability of the products.

In one aspect, the present application provides a system for preparing mRNA liposomes, which system follows the connection sequence. It includes liquid preparation unit, encapsulation unit, liquid replacement unit and preparation unit;

The system is a fully closed system.

The "unit" in this application may be an instrument or equipment, or a set of instruments or equipment capable of completing a certain work. These different units can form a system with specific functions.

As known in the art, "mRNA liposomes" are also known as "mRNA-liposome complexes", "mRNA-lipid nanocomplexes", "mRNA-liposome nanocomplexes", "lipid nanocomplexes" "Plastid-mRNA complex", etc., is a lipid nanoparticle (Lipid nanoparticle, LNP) prepared by methods including but not limited to film hydration method, extrusion method, homogenization method, microfluidics, etc. Regarding the types of lipids involved, the surface of LNP is mainly neutral lipids and PEGylated lipids as well as partially ionizable cationic lipids and cholesterol; inside the core, ionizable cationic lipids and cholesterol are present.

As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide encoding at least one polypeptide. As used herein, mRNA includes modified and unmodified RNA. An mRNA may contain one or more coding and non-coding regions. The present invention can be used to encapsulate any mRNA.

The liquid preparation unit in this application can be a conventional liquid preparation tank in this field, optionally, it is equipped with a conventional stirring device or a magnetic stirring device. The functions of the liquid preparation unit in this application include but are not limited to dissolving an appropriate concentration of mRNA buffer in a slightly acidic solution to form an aqueous phase, and dissolving lipids in ethanol to form an organic phase. The liquid preparation unit can prepare the aqueous phase and the organic phase according to conventional preparation methods in the art, so that they have sufficient composition characteristics to form mRNA liposomes.

The preparation of the mRNA stock solution may be routine in the art, and may include, for example, a series of steps such as in vitro transcription, post-transcriptional modification, enzyme treatment, chromatography purification, concentration and medium replacement, and stock solution preparation. The instruments/equipment used to perform this series of steps can all be conventional in the art, and they constitute the unit for preparing the mRNA stock solution.

Optionally, the liquid preparation unit also has the function of remelting the original mRNA solution. Instruments/equipment used for refusion may be routine in the art. Various methods can be used to prepare mRNA solutions suitable for use in the present invention. In some embodiments, the mRNA can be dissolved directly in the buffer solutions described herein. In some embodiments, the mRNA solution can be produced by mixing the mRNA stock solution with a buffer solution before mixing with the organic phase for encapsulation.

In some embodiments, the liquid preparation unit can also mix the mRNA stock solution and the buffer solution.

The organic phase contains a mixture of lipids suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, a suitable organic phase is ethanol-based. For example, a suitable organic phase may contain a mixture of desired lipids dissolved in pure ethanol (ie, 100% ethanol). In another embodiment, a suitable organic phase is isopropyl alcohol based. in another In one embodiment, a suitable organic phase is based on dimethyl sulfoxide. In another embodiment, a suitable organic phase is a mixture of suitable solvents including, but not limited to, ethanol, isopropanol, and dimethyl sulfoxide.

In some embodiments, the organic phase includes one or more cationic lipids, one or more helper lipids, and one or more PEG-modified lipids. In some embodiments, the organic phase further includes one or more cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are cholesterol and/or PEGylated cholesterol. In some embodiments, the organic phase includes preformed lipid nanoparticles. In some embodiments, the organic phase is a suspension of preformed lipid nanoparticles.

A suitable organic phase may contain a mixture of various concentrations of the desired lipids. For example, a suitable organic phase may contain a mixture of the desired lipids at a total concentration equal to or greater than about 0.1 mg/ml, 1.0 mg/ml, 10 mg/ml, or 100 mg/ml.

Optionally, the liquid preparation unit also has the function of controlling the ratio of the aqueous phase and the organic phase. Any desired lipids can be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, a suitable organic phase contains a mixture of desired lipids including cationic lipids, accessory lipids (e.g., non-cationic lipids and/or cholesterol lipids), and/or PEG oxidized lipids. In some embodiments, a suitable organic phase contains a mixture of desired lipids including one or more cationic lipids, one or more accessory lipids (e.g., non-cationic lipids and /or cholesterol lipids) and one or more PEGylated lipids.

The lipids in this application can be conventional in the art, including but not limited to cationic lipids (such as ALC-0315, CAS: 2036272-55-4), cholesterol, auxiliary lipids (such as DOPE), and polyethylene glycol. Alcoholized phospholipids (such as PEG2000-DMG), etc. Among them: the functions of the cationic lipid include, but are not limited to, binding to negatively charged mRNA and efficiently entrapping mRNA drugs. The functions of cholesterol include, but are not limited to, stabilizing the structure of the mRNA liposomes and improving the stability of the mRNA liposomes by regulating the fluidity of the membrane. The functions of the auxiliary lipid include, but are not limited to, stabilizing mRNA liposomes and improving the efficiency of delivering mRNA. The functions of the PEGylated phospholipids include, but are not limited to, improving the stability of mRNA liposomes, reducing their binding to plasma proteins, and prolonging their circulation time in the body.

According to various embodiments, the selection of cationic lipids, helper lipids, cholesterol-based lipids, and/or PEG-modified lipids comprising lipid nanoparticles, and the relative molar ratios of these lipids to each other are based on the selection Characteristics of the lipids, the nature of the intended target cells, and the characteristics of the mRNA to be delivered. Other considerations include, for example, the saturation of the alkyl chain and the size, charge, pH, pKa, fusogenicity, and toxicity of the selected lipid. Therefore, the molar ratio can be adjusted accordingly.

In some embodiments, the liquid preparation unit in this application is an explosion-proof liquid preparation unit. The function of the explosion-proof liquid dispensing unit This can include, but is not limited to, effectively avoiding safety hazards caused by using high-concentration ethanol solutions during the preparation of the organic phase. The explosion-proof liquid dispensing unit may be conventional in the field, such as a stainless steel explosion-proof liquid dispensing tank.

In this application, the functions of the encapsulation unit include but are not limited to controlling the injection temperature, pressure, flow rate and ratio of the two-phase solution, etc., and use the film hydration method, extrusion method, homogenization method, ultrasonic wave, shearing or Microfluidic mixing and other LNP preparation methods are used to prepare mRNA liposomes.

In certain embodiments, the encapsulation unit in this application is a microfluidic encapsulation unit, which prepares mRNA liposomes through a microfluidic mixing method. An example of the operation of the microfluidic encapsulation unit is that the organic phase and the aqueous phase are mixed by jet collision. At the same time, the ethanol in the organic phase is diluted, the pH of the solution changes, and the liposomes separate out to form lipid nanoparticles and interact with the mRNA. Formation of encapsulated complexes.

The structure of the microfluidic encapsulation unit can be conventional in the art, for example, it includes a high-pressure pump and a cavity. The high-pressure pump is used to form two jets of the aqueous phase and the organic phase, and the process is carried out in the cavity. Hedge. In certain embodiments, the flow rate of the liquid buffer during the preparation of mRNA liposomes through the microfluidic encapsulation unit is controllable. In certain embodiments, the diameter of at least one of the reservoirs, conduits, and/or connectors in the microfluidic chip can be controlled to achieve desired flow rates and resulting mixing properties. The larger the diameter of the conduit, the greater the flow of liquid through the conduit. In certain embodiments, flow limiting settings are employed in the microfluidic chip to achieve the desired flow rate and resulting mixing properties.

In certain embodiments, the process of preparing mRNA liposomes by the microfluidic encapsulation unit described in this application is sterile. In certain embodiments, the microfluidic encapsulation units described herein are not in a sterile environment. In certain embodiments, the microfluidic encapsulation unit allows reproducible encapsulation of mRNA in lipids. In certain embodiments, the microfluidic encapsulation unit allows reproducible production of lipid nanoparticles (LNPs).

The manufacturers of the microfluidic encapsulation unit can be selected from Genizer, Precision NanoSystems (PNI), PreciGenome, Dolomite Microfluidics, Myanna (Shanghai) Instrument Technology Co., Ltd., and Suzhou Aitson Pharmaceutical Equipment Co., Ltd., etc.

In the product obtained by the encapsulation unit, in addition to the mRNA liposome, there are also impurities such as unused mRNA or its fragments, unused lipids, ethanol, and possible microorganisms. The above-mentioned impurities are removed using the liquid exchange unit in this application to obtain purified mRNA liposomes, and preparations can be made according to the function or formula requirements of the mRNA liposomes, for example, adding ingredients to facilitate long-term stable storage of the product and Excipient ingredients (such as sucrose) that exert the medicinal effect.

In some embodiments, the liquid exchange unit in this application is a tangential flow filtration liquid exchange unit. Tangential Flow Filtration (TFF), also known as Cross Flow Filtration, refers to the flow of liquid The filtration form is perpendicular to the filtration direction. It is driven by pressure and performs membrane separation based on molecular size. The cutoff pore size of the membrane in the tangential flow filtration unit described in this application can be determined based on the particle size of the prepared mRNA liposomes.

In TFF, concentration and liquid replacement are accomplished through the tangential flow principle.

The main advantage of tangential flow filtration is that the non-permeable retentate or "filter cake" that can collect within the filter and clog it during traditional "dead-end" filtration is instead transported along the surface of the filter. This advantage makes tangential flow filtration particularly suitable for large-scale purification of mRNA liposomes.

At least three process variables are important in a typical TFF process: transmembrane pressure differential, feed rate, and permeate flow rate. The transmembrane pressure differential is the force that pushes fluid through the filter, carrying permeable molecules with it. Feed rate (also called cross-flow rate) is the rate at which solution flows through the feed channel and through the filter. The feed rate determines the force that removes molecules that might otherwise clog or foul the filter and thereby limit the filtrate flow rate. The permeate flow rate is the rate at which permeate is removed from the system. For a constant feed rate, increasing the permeate flow rate increases the pressure across the filter, resulting in an increase in filtration rate, and may also increase the risk of filter clogging or fouling. The principles, theory, and apparatus for TFF are described in Michaels et al., "Tangential Flow Filtration" in Separations Technology, Pharmaceutical and Biotechnology Applications (W.P. Olson, ed., Interpharm Press, Inc., Buffalo Grove, 1995). See also the description of high performance tangential flow filtration (HP-TFF) in US Pat. Nos. 5,256,294 and 5,490,937.

In some embodiments, the total sealing described in this application can be achieved through a one-time sealing process. For example, use sterile connectors/sterile interrupters to connect/disconnect different units. Wherein, depending on the connected unit, the aseptic connector can be a sterile connector or a sterile takeover machine; depending on the disconnected connection unit, the blocker can be a sterile disconnector or a sterile disconnector. Bacteria sealing tube machine.

Another advantage of the present invention is that the area of the sterile area, especially the core sterile production area, is reasonably designed to reduce the cost of sterility assurance. In certain embodiments, the formulation unit is in the Class C zone. In some embodiments, the fluid exchange unit and the preparation unit are in the Class C zone. In some embodiments, the fluid exchange unit and the preparation unit are in the Class C zone.

In some embodiments, the system, in order of connection, is an explosion-proof liquid preparation unit, a sterile connector/sterile disconnector, a microfluidic encapsulation unit, a sterile take-over machine/a sterile tube sealing machine, TFF liquid changing unit, sterile tube taking machine/sterile sealing machine and liposome preparation unit. The connection sequence of each unit described in this application is preferably consistent with the steps in large-scale production.

In some embodiments, the connection between the liquid preparation unit and the encapsulation unit is achieved through a sterile connector.

In some embodiments, the disconnection between the dispensing unit and the encapsulating unit is achieved by a sterile disconnect.

In some embodiments, the connection between the fluid mixing and encapsulation unit and the liquid exchange unit is achieved through a sterile pipe machine.

In some embodiments, the disconnection between the fluid mixing and encapsulation unit and the fluid exchange unit is accomplished by a sterile sealing machine.

In some embodiments, the connection between the liquid changing unit and the preparation unit is realized through a sterile pipe machine.

In some embodiments, the disconnection between the liquid exchange unit and the preparation unit is achieved by a sterile sealing machine.

The use of linkers and blockers described in this application can effectively connect different units, enabling the preparation of mRNA liposomes to realize an integrated production process of mRNA liposomes for the first time in this field, overcoming the quality inspection of different suppliers. The device lacks integration and connection defects between devices.

Sterile connectors/sterile disconnectors as described in this article allow for quick and easy sterile connection and disconnection, even in non-sterile environments. In certain embodiments, the sterile connector/disconnector can act as a sterile filter to filter the solution. In certain embodiments, the system does not include additional sterile filters after the sterile connector/sterile disconnector.

In some embodiments, a filter is provided as a sterilizing filter after the explosion-proof liquid preparation unit. In some embodiments, a 0.22 μm filter element is installed after the explosion-proof liquid preparation unit to filter and remove residual microorganisms in the solution, and the sterility level is SAL=10E-6. In some embodiments, in the system, the number of other sterilizing filters connected after the explosion-proof liquid preparation unit is less than 5. In some embodiments, in the system, the number of other sterilizing filters connected after the explosion-proof liquid preparation unit is less than 3. In some embodiments, in the system, the number of other sterilizing filters connected after the explosion-proof liquid preparation unit is less than 1.

In certain embodiments, the number of sterile filters in the system is less than 5. In certain embodiments, the number of sterile filters in the system is less than 4. In certain embodiments, the number of sterile filters in the system is less than 3. In certain embodiments, the number of sterile filters in the system is less than 2. In certain embodiments, the number of sterile filters in the system is less than 1.

Optionally, the system in this application further includes a unit for preparing plasmid DNA stock solution and a unit for preparing mRNA stock solution upstream of the liquid preparation unit. The connection/disconnection between such units for preparing raw solutions and between them and the liquid dispensing unit is achieved through sterile connectors/sterile disconnectors.

The preparation of the plasmid DNA stock solution may be routine in the art, and may include steps such as fermentation culture, harvesting and clarification, refining and purification, linearization, and filtration and aliquots. The instruments/equipment used to perform this series of steps can all be conventional in the art, for example Bacterial culture fermentation tanks, CO ₂ incubators, centrifuges, and equipment for tangential flow filtration or depth filtration, etc., constitute the unit for preparing plasmid DNA stock solution.

Optionally, the system in the present application further includes a unit downstream of the preparation unit for preparing cell therapy-related products using the prepared mRNA liposomes.

In another aspect, the present application provides a method for preparing mRNA liposomes, which includes preparing using a system as described in the first aspect.

When using the system to prepare mRNA liposomes, the following operations can be performed in sequence: explosion-proof docking, fluid docking, sample sampling, buffer storage and transfer, and sample solution storage and transfer.

Unless otherwise specified, the operation of each unit in this application can be a mature process in the field, such as using a liquid preparation unit to prepare two phases and using an encapsulation unit to encapsulate mRNA.

The system described in this application is a fully closed system. A one-time closed process can be used during use to effectively avoid contamination and cross-contamination, reduce the number of sterilization filtrations, and reduce the impact of the environment, equipment, personnel, and other external factors on cell products. impact, increase production and improve product stability. Compared with the existing technology, the system described in this application reasonably divides each unit into an explosion-proof area and a Class C area. In some embodiments, the explosion-proof liquid preparation unit and the encapsulation unit constitute an explosion-proof area, and the liquid replacement unit and the preparation unit constitute a Class C area. When the system of this application contains an explosion-proof liquid dispensing unit, it can provide an explosion-proof production environment and effectively prevent explosions. The purification, preparation, and packaging processes are carried out in clean areas of Class C or higher, so that there is no microbial contamination during the purification, preparation, and packaging processes. Aseptic connection/disconnection is adopted between the explosion-proof area and the C-level area. The equipment in the C-level area does not need to be sterilized multiple times during the entire process, which greatly simplifies the production process.

In some embodiments, the equipment in the Class C area does not need to be sterilized after the explosion-proof docking and/or fluid docking steps. In some embodiments, the equipment in the Level C zone is sterilized less than 10 times during each intermittent production process. In some embodiments, the equipment in the Level C zone is sterilized less than 5 times during each intermittent production process. In some embodiments, the equipment in the Level C zone is sterilized less than 4 times during each intermittent production process. In some embodiments, the equipment in the Level C zone is sterilized less than three times during each intermittent production process. In some embodiments, the equipment in the Level C zone is sterilized less than twice during each intermittent production process. In some embodiments, the equipment in the Level C zone is sterilized less than once during each intermittent production process.

In certain embodiments, systems as described herein can be used in continuous production processes. In some embodiments, The frequency of sterilization treatment of equipment in the Class C area during continuous production is less than 10 times per day. In some embodiments, the equipment in the Level C zone is sterilized less than 5 times per day during the continuous production process. In some embodiments, the equipment in the Level C zone is sterilized less than 4 times per day during the continuous production process. In some embodiments, the equipment in the Level C zone is sterilized less than three times per day during the continuous production process. In some embodiments, the equipment in the Level C zone is sterilized less than 2 times per day during the continuous production process. In some embodiments, the equipment in the Level C zone is sterilized less than once per day during the continuous production process.

The system for preparing mRNA liposomes provided in this application provides new ideas for the cooperative development of integrated equipment for mRNA liposomes.

The above is an overview of the present application, and there may be situations where simplifications, generalizations, and details are omitted. Therefore, those skilled in the art should realize that this part is only illustrative and is not intended to limit the scope of the present application in any way. This Summary is neither intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

The above and other features of the present application will be more fully understood from the following description and appended claims, taken in conjunction with the accompanying drawings. It will be understood that these drawings only depict several embodiments of the present application and therefore should not be considered as limiting the scope of the present application. By referring to the accompanying drawings, the contents of this application will be explained more clearly and in detail.

Description of drawings

Figure 1 shows the system used to prepare mRNA liposomes in Example 1 of the present application.

Figure 2 shows a liquid storage bag that transfers liquid to be liquid from the liquid explosion-proof dispensing unit to the encapsulation unit.

Figure 3 shows the TFF fluid exchange unit.

Figure 4 shows a sample storage bag used to transfer samples.

Figure 5 shows a formulation storage bag for transferring and filling formulations.

Detailed ways

The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter of this application. It will be understood that various aspects of the subject matter generally described in this application and illustrated in the accompanying drawings may be A variety of configurations, substitutions, combinations, and designs can be made in various aspects, and all of these clearly form part of the content of this application.

Example

In order that the present application may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting in any way.

Example 1: Composition of a system for preparing mRNA liposomes

The system mainly consists of an explosion-proof liquid preparation unit (1), a microfluidic encapsulation unit (2), a TFF liquid replacement unit (3) and a preparation unit (4). Among them, the explosion-proof liquid preparation unit and microfluidic encapsulation unit constitute the explosion-proof area, and the TFF liquid replacement unit and preparation unit constitute the C-level area (see Figure 1).

The explosion-proof liquid preparation unit (1) and the microfluidic encapsulation unit (2) are connected/disconnected using a sterile connector/sterile disconnector.

Figure 2 provides a liquid storage bag that transfers the liquid to be encapsulated from the liquid explosion-proof dispensing unit to the encapsulation unit (2). Liquid (such as mRNA solution) flows from the explosion-proof liquid preparation unit (1) through the filter (57) and the liquid storage bag inlet conduit (52) into the liquid storage bag (51). The filter (57) can filter the liquid flowing into the dosing unit. The liquid storage bag (51) is equipped with a sampling head (55). The sampling head can conveniently extract the solution from the liquid storage bag (51). Among them, the liquid storage bag inlet conduit (52) and the liquid storage bag outlet conduit (54) can be thermoplastic tubes, including but not limited to silicone tube, and wait. If required, thermoplastic tubing can be used for aseptic welding for liquid transfer. The port of the liquid storage bag outlet conduit (54) is equipped with an air filter head (56) to prevent the entry of pollutants in the air, especially bacteria. Pipe clamps (53) are installed on the liquid storage bag liquid inlet conduit (52) and the liquid storage bag liquid outlet conduit (54) to control the opening and closing of the pipelines.

The liquid storage bag (51) can be connected to the microfluidic encapsulation unit (2) through the liquid storage bag outlet conduit (54).

The microfluidic encapsulation unit and the TFF liquid change unit, as well as the TFF liquid change unit and the preparation unit, are connected/disconnected using a sterile takeover machine/sterile tube sealing machine. Figure 3 shows the TFF fluid exchange unit. The liquid inlet conduit (32) can be connected/disconnected from the microfluidic encapsulation unit (2) through a sterile tube taking machine/sterile tube sealing machine. The reflux fluid conduit (33) can be connected/disconnected from the preparation unit (3) through a sterile tube taking machine/sterile tube sealing machine. The mRNA liposomes prepared by the microfluidic encapsulation unit flow into the tangential flow filter (31) through the liquid inlet conduit (32). The permeate flows into and out of the tangential flow filter (31) through the permeate conduit (34) to filter impurities in the preparation.

The sample storage liquid shown in Figure 4 can also be passed between the microfluidic encapsulation unit (2) and the TFF liquid replacement unit (3). The bag (61) transfers liquid. Both the sample liquid storage bag liquid inlet conduit (62) and the sample liquid storage bag liquid outlet conduit (64) can be thermoplastic tubes, which can be used for aseptic welding to transfer liquid. Among them, the liquid inlet conduit (62) of the sample liquid storage bag and the liquid outlet of the microfluidic encapsulation unit (2) can be connected/disconnected through a sterile take-over machine/sterile sealing machine, and the liquid outlet of the sample liquid storage bag The conduit (64) and the liquid inlet conduit (32) can be connected/disconnected through a sterile tube taking machine/sterile tube sealing machine.

The TFF liquid changing unit (3) and the preparation unit (4) are connected/disconnected using a sterile tube taking machine/sterile tube sealing machine. Liquid can also be transferred between the TFF liquid changing unit (3) and the preparation unit (4) through the preparation liquid storage bag (71) as shown in Figure 5. Among them, the liquid inlet conduit (72) of the preparation liquid storage bag can be connected/disconnected from the TFF liquid exchange unit (3) through a sterile tube taking machine/sterile tube sealing machine. The preparation in the preparation storage bag (71) flows to the filling needle (75) through the filling pump tube (74), where a peristaltic pump can be installed on the filling pump tube (74) to pump liquid. The β bag (76) is placed outside the filling pump tube (74) and the filling needle (75). The contact areas between the bag body and the filling pump tube (74) are connected by welding to ensure that the β bag is fully enclosed. ; The beta valve (77) of the rapid transfer interface (RTP) is attached to the beta bag so as to be aseptically connected to the preparation unit (4).

Example 2: Method of use of system for preparing mRNA liposomes

1. Explosion-proof docking

Connect all components in the explosion-proof area through explosion-proof joints.

2. Fluid docking;

Connect the components of the liquid preparation unit, encapsulation unit, liquid replacement unit and preparation unit through fluid pipelines.

3. Sample sampling

The sampling heads (55) and (65) can take samples for monitoring.

4. Buffer storage and transfer

After the liquid is pumped into the storage bag (51), the pipe clamp (53) of the storage bag can be closed to store the buffer solution. When transferring the solution, the pipe clamp (53) of the liquid storage bag can be opened and the buffer can be transferred through the sterile docking of the liquid storage bag outlet conduit (54).

5. Storage and transfer of sample solutions

After pumping the sample into the sample liquid storage bag (61), the sample liquid storage bag pipe clamp (63) can be closed to store the sample liquid. When transferring the solution, the pipe clamp (63) of the sample storage bag can be opened and connected aseptically through the outlet conduit (64) of the sample storage bag to transfer the sample liquid.

Example 3: Steps for preparing mRNA liposomes

Step 1. Remelt the original mRNA solution in the explosion-proof liquid preparation unit (1), and prepare the aqueous phase containing the mRNA solution and the organic phase used to encapsulate the mRNA.

Step 2. The solution prepared by the explosion-proof liquid preparation unit is filtered by the filter (57) and then flows into the liquid storage bag (51).

Step 2.1. Optionally, open the sampling head (55) to sample and detect the solution in the liquid storage bag (51).

Step 3. Pump the solution in the liquid storage bag (51) into the encapsulation unit (2), such as a microfluidic encapsulation unit. The encapsulation unit encapsulates the mRNA to form liposomes.

Step 4. Introduce the encapsulated liposome solution into the liquid replacement unit (3). The liquid inlet conduit (32) can be directly connected to the microfluidic encapsulation unit. Alternatively, the liquid can also be transferred through the sample storage bag (61). The tangential flow filter (31) performs a liquid exchange operation on the liposomes.

Step 4.1. Optionally, open the sampling head (65) to sample and detect the solution in the sample storage bag (61).

Step 5. Introduce the changed liposome solution into the preparation storage bag (71). The liposomes are filled into the dispensing bottle through the filling pump tube (74) and the filling needle (75).

The above-mentioned specific embodiments further describe the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above-mentioned are only specific embodiments of the present application and are not intended to limit the present application. Within the spirit and principles of this application, any modifications, equivalent replacements, improvements, etc. shall be included in the protection scope of this application.

Claims

A system for preparing mRNA liposomes, characterized in that it includes a liquid preparation unit, an encapsulation unit, a liquid exchange unit and a preparation unit in order of connection; wherein the system is a fully closed system.
The system of claim 1, wherein the liquid dispensing unit is an explosion-proof liquid dispensing unit.
The system according to claim 1 or 2, wherein the encapsulation unit is encapsulated by a film hydration method, an extrusion method, a homogenization method or a microfluidic mixing method.
The system of claim 3, wherein the encapsulation unit is a fluid mixing encapsulation unit.
The system of claim 4, wherein the fluid mixing encapsulation unit is a microfluidic encapsulation unit.
The system of claim 5, wherein the manufacturer of the microfluidic encapsulation unit is selected from the following group: Genizer, Precision NanoSystems (PNI), PreciGenome, Dolomite Microfluidics, Maianna (Shanghai) Instrument Technology Co., Ltd. and Suzhou Aitson Pharmaceutical Equipment Co., Ltd.
The system according to any one of the preceding claims, wherein the fluid exchange unit is a tangential flow filtration fluid exchange unit.
System according to any one of the preceding claims, characterized in that the connection between different units is achieved by sterile connectors and the disconnection is achieved by sterile interrupters.
The system according to claim 8, wherein the aseptic joint is a sterile connector or a sterile tube take-over machine; the blocker is a sterile disconnector or a sterile tube sealing machine.
The system according to claim 9, wherein the connection between the liquid preparation unit and the fluid mixing and encapsulation unit is realized through a sterile connector, and the disconnection is realized through a sterile disconnector.
The system according to claim 9 or 10, characterized in that the connections between the fluid mixing and encapsulation unit and the liquid exchange unit, and between the liquid exchange unit and the preparation unit are through sterile pipes. The disconnection is achieved by a sterile tube sealing machine.
The system of claim 1, wherein the liquid preparation unit is not in a sterile environment.
The system according to claim 1, wherein a sterilizing filter is provided behind the liquid preparation unit.
The system of claim 1, wherein the encapsulation unit is not in a sterile environment.
The system of claim 1, wherein the encapsulation unit does not contain a sterilizing filter.
The system of claim 1, wherein a sterilizing filter is not provided after the encapsulation unit.
Use of a system according to any one of the preceding claims for the preparation of mRNA liposomes.