CN117597110A

CN117597110A - System for preparing mRNA liposome and application thereof

Info

Publication number: CN117597110A
Application number: CN202380012307.0A
Authority: CN
Inventors: 吴守亮; 毛叶飞; 徐丙良
Original assignee: Zhejiang Jianxin Yuanli Pharmaceutical Co ltd
Current assignee: Zhejiang Jianxin Yuanli Pharmaceutical Co ltd
Priority date: 2022-07-19
Filing date: 2023-07-19
Publication date: 2024-02-23
Also published as: WO2024017300A1

Abstract

The present application relates to a system for preparing mRNA liposomes and uses thereof. The system sequentially comprises a liquid preparation unit, an encapsulation unit, a liquid exchange unit and a preparation unit according to the connection sequence; the system is a fully closed system. The system of the application is used for producing mRNA liposome, so that pollution and cross pollution can be effectively avoided, the times of sterilization and filtration are reduced, the influence of environment, equipment, personnel and other external factors on cell products is reduced, the yield is increased, and the stability of the products is improved.

Description

System for preparing mRNA liposome and application thereof

Technical Field

The application belongs to the field of liposome technology, and in particular relates to a system for preparing mRNA liposome and application thereof

Background

The mRNA technical product is based on the central rule of mRNA to guide protein synthesis, and mRNA sequences containing coded specific antigens are designed and synthesized in vitro, and are delivered to human cells in different modes through sequence optimization, chemical modification, purification and other processing, and the effects of generating protein, inducing immune response, supplementing organism protein, regulating immunity and the like are utilized by the translation of the organism cells, so that diseases are prevented or treated. The application field is wide, and the preparation method is mainly used for preparing infectious disease vaccines, tumor immunotherapy, monoclonal antibody drug substitution, other protein drug substitution and the like. The most rapidly advancing and most commonly used prophylactic vaccines against infectious diseases today, mRNA vaccines represented by BNT162b2 produced by pyroxene, are highly heterogeneous in the use of COVID-19.

At present, the production of mRNA drugs is still to be improved. For example, 1) since mRNA medicine is returned to the patient, the requirement on the sterility control level is very high according to the four parts of pharmacopoeia as the injection level is high, so the sterility control requirement on environment, personnel and equipment is very high in the preparation process; 2) The conventional production process is easy to produce cross contamination through equipment, environment and personnel when different samples are produced simultaneously, so that the multi-variety collinear production of the products cannot be realized, and the gene therapy products have the characteristics of small scale, high quality requirement, high-demand and multi-product collinear production and the like; 3) The mRNA liposome production process mainly reduces the microbial load by adding a sterilization filtering process, but the sterilization filtering can seriously influence the recovery rate of the product; 4) The production stage of mRNA liposome needs to use high-concentration ethanol solution, and potential safety hazards exist in the production process. Therefore, a set of totally-enclosed production process is needed in the market to ensure the quality and effect of the product and promote the development of the whole gene therapy industry.

Disclosure of Invention

The present application aims to provide a system for preparing mRNA liposome and application thereof. When the system of the application is used for preparing mRNA liposome, the pipelines through which raw materials, intermediate products and final products flow are in a fully-closed state, so that pollution and cross pollution can be effectively avoided, the times of sterilization and filtration are reduced, the influence of environment, equipment, personnel and other external factors on cell products is reduced, and the quality and stability of the products are improved.

In one aspect, the present application provides a system for preparing mRNA liposomes, comprising, in order of connection, a fluid dispensing unit, an encapsulation unit, a fluid exchange unit, and a formulation unit;

the system is a fully closed system.

The "unit" in this application may be one instrument or device or a collection of instruments or devices capable of performing a certain task. These different units may constitute a system with specific functions.

As known in the art, "mRNA liposome", also referred to as "mRNA-liposome complex", "mRNA-lipid nanocomposite", "mRNA-liposome nanocomposite", "liposome-mRNA complex", etc., is a lipid nanoparticle (Lipid nanoparticle, LNP) prepared by means including, but not limited to, thin film hydration, extrusion, homogenization, microfluidics, etc. With respect to the type of lipids involved, the surface of LNP is primarily neutral lipids and pegylated lipids, and partially ionizable cationic lipids and cholesterol; inside the core, there are ionizable cationic lipids, cholesterol.

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

The dosing unit in the present application may be a dosing tank as is conventional in the art, optionally with conventional stirring means or magnetic stirring means. The functions of the partitioning unit herein include, but are not limited to, dissolving an appropriate concentration of mRNA buffer in a meta-acidic solution to form an aqueous phase, and dissolving lipids in ethanol to form an organic phase. The partitioning unit can be prepared by methods conventional in the art to provide the aqueous phase and the organic phase with sufficient compositional characteristics to form mRNA liposomes.

The preparation of the mRNA stock solution may be conventional in the art and may comprise, for example, a series of steps such as in vitro transcription, post-transcriptional modification, enzymatic treatment, chromatographic purification, concentrated stock solution exchange, stock solution preparation, etc. The instrumentation/equipment used to carry out the series of steps can be conventional in the art and constitute the unit for preparing the mRNA stock solution.

Optionally, the liquid preparation unit further has a function of re-melting the mRNA stock solution. The apparatus/device for the fusing may be conventional in the art. Various methods can be used to prepare mRNA solutions suitable for use in the present invention. In some embodiments, mRNA can be directly dissolved in a buffer solution as described herein. In some embodiments, the mRNA solution may be produced by mixing the mRNA stock solution with a buffer solution prior to mixing with the organic phase for encapsulation.

In some embodiments, the dosing unit may also mix the mRNA stock solution with a buffer solution.

The organic phase contains a lipid mixture suitable for forming lipid nanoparticles for encapsulating mRNA. In some embodiments, suitable organic phases are ethanol-based. For example, a suitable organic phase may contain a mixture of the desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable organic phase is isopropanol-based. In another embodiment, a suitable organic phase is dimethyl sulfoxide based. 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 comprises one or more cationic lipids, one or more helper lipids, and one or more PEG-modified lipids. In some embodiments, the organic phase further comprises 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 comprises preformed lipid nanoparticles. In some embodiments, the organic phase is a suspension of preformed lipid nanoparticles.

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

Optionally, the liquid dispensing unit further has a function of controlling the ratio of the aqueous phase and the organic phase. Any desired lipids may be mixed in any ratio suitable for encapsulating mRNA. In some embodiments, suitable organic phases contain a mixture of desired lipids including cationic lipids, helper lipids (e.g., non-cationic lipids and/or cholesterol lipids), and/or pegylated lipids. In some embodiments, a suitable organic phase contains a mixture of desired lipids including one or more cationic lipids, one or more helper lipids (e.g., non-cationic lipids and/or cholesterol lipids), and one or more pegylated lipids.

The lipids in this application may be conventional in the art, including but not limited to cationic lipids (e.g., ALC-0315, CAS: 2036272-55-4), cholesterol, helper lipids (e.g., DOPE), and pegylated phospholipids (e.g., PEG 2000-DMG), and the like. Wherein: the cationic lipids act to efficiently entrap mRNA drugs including, but not limited to, binding to negatively charged mRNA. The cholesterol effects include, but are not limited to, stabilization of mRNA liposome structure by regulating membrane fluidity, and improvement of mRNA liposome stability. The role of the helper lipid includes, but is not limited to, stabilizing mRNA liposomes, increasing the efficiency of mRNA delivery. The polyethylene glycol phospholipid has the effects of improving the stability of mRNA liposome, reducing the combination of mRNA liposome and plasma protein and prolonging the circulation time of mRNA liposome in vivo.

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

In certain embodiments, the dosing unit in the present application is an explosion-proof dosing unit. The explosion-proof liquid preparation unit has the functions of effectively avoiding potential safety hazards caused by using high-concentration ethanol solution in the preparation process of an organic phase, but is not limited to the explosion-proof liquid preparation unit. The explosion-proof liquid preparation unit can be conventional in the art, such as a stainless steel explosion-proof liquid preparation 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, and preparing mRNA liposomes by LNP preparation methods such as thin film hydration, extrusion, homogenization, ultrasound, shearing, or microfluidic mixing.

In certain embodiments, the encapsulation unit in the present application is a microfluidic encapsulation unit that prepares mRNA liposomes by a microfluidic mixing method. An example of the operation of the microfluidic encapsulation unit is that the organic phase is mixed with the aqueous phase by jet collision hedging, while the ethanol in the organic phase is diluted, the pH of the solution is changed, and the liposomes precipitate out to form lipid nanoparticles and form an encapsulated complex with mRNA.

The structure of the microfluidic encapsulation unit may be conventional in the art, for example comprising a high pressure pump for forming the aqueous and organic phases into two jets and a cavity in which the counter-flushing is performed. In certain embodiments, the flow rate of the liquid hedging during the preparation of mRNA liposomes by the microfluidic encapsulation unit is controllable. In certain embodiments, the diameter of at least one of the reservoirs, conduits, and/or junctions in the microfluidic chip may be controlled to achieve a desired flow rate and resulting mixing properties. The larger the diameter of the conduit, the greater the flow of liquid through the conduit. In certain embodiments, a flow-restricting arrangement is employed in the microfluidic chip to achieve the desired flow rate and resulting mixing properties.

In certain embodiments, the microfluidic encapsulation unit described herein is sterile in the process of preparing mRNA liposomes. In certain embodiments, the microfluidic encapsulation units described herein are not in a sterile environment. In certain embodiments, the microfluidic encapsulation unit allows for reproducible encapsulation of mRNA in lipids. In certain embodiments, the microfluidic encapsulation unit allows for reproducible production of Lipid Nanoparticles (LNPs).

The manufacturer of the microfluidic encapsulation unit may be selected from Genizer, precision NanoSystems (PNI), prescgenome, dolomite Microfluidics, michaux (Shanghai) instruments and technologies, inc. Of pharmaceutical equipment, su zhou Ai Tesen, etc.

In addition to mRNA liposomes, impurities such as unused mRNA or fragments thereof, unused lipids, ethanol, and possibly microorganisms are present in the product obtained by the encapsulation unit. The impurities are removed by using the liquid exchange unit in the application to obtain purified mRNA liposome, and preparation liquid can be prepared according to the function or formula requirement of the mRNA liposome, for example, auxiliary ingredients (such as sucrose) which are beneficial to long-term stable storage and drug effect exertion of the product are added.

In certain embodiments, the liquid exchange unit in the present application is a tangential flow filtration liquid exchange unit. Tangential flow filtration (Tangential Flow Filtration, TFF), also known as cross-flow filtration (Cross Flow Filtration), refers to a form of filtration in which the direction of liquid flow is perpendicular to the direction of filtration, and by pressure driven membrane separation is performed according to molecular size. The pore size of the membranes in the tangential flow filtration unit described herein can be dependent on the particle size of the mRNA liposomes produced.

In TFF, concentration and liquid exchange are accomplished by tangential flow principles.

The main advantage of tangential flow filtration is that instead, impermeable retentate or "cake" that can collect within the filter and clog the filter during conventional "dead-end" filtration is 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 important in a typical TFF process: transmembrane pressure differential, feed rate, and permeate flow rate. The transmembrane pressure differential is the force that pushes the fluid through the filter with the permeable molecules. The feed rate (also referred to as cross-flow rate) is the rate at which the solution flows through the feed channel and through the filter. The feed rate determines the force to clear molecules that might otherwise plug 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 may increase the pressure across the filter, resulting in an increase in filtration rate, while also potentially increasing the risk of filter plugging or fouling. The principles, theory and apparatus for TFF are described in Michaels et al, "Tangential Flow Filtration" in Separations Technology, pharmaceutical and Biote chnology Applications (W.P. Olson, interferm Press, inc., buffalo Grove, 1995). See also U.S. patent nos. 5,256,294 and 5,490,937 for a description of high performance tangential flow filtration (HP-TFF).

In certain embodiments, the full closure described herein may be achieved by a disposable closure process. For example, the different units are connected/disconnected using sterile connectors/sterile blockers. Wherein, according to the different units connected, the aseptic connector can be an aseptic connector or an aseptic pipe connecting machine; depending on the connection unit disconnected, the blocker may be a sterile disconnect or a sterile tube sealer.

Another advantage of the present invention is that the area of the sterile field, particularly the area of the core sterile production area, is reasonably designed, and the sterility assurance cost is reduced. In certain embodiments, the formulation unit is in the class C region. In certain embodiments, the liquid change unit and the formulation unit are in the class C zone. In certain embodiments, the liquid change unit and the formulation unit are in the class C zone.

In certain embodiments, the system is an explosion-proof fluid dispensing unit, a sterile connector/sterile disconnect, a microfluidic encapsulation unit, a sterile tube takeover machine/sterile tube sealer, a TFF fluid change unit, a sterile tube takeover machine/sterile tube sealer, and a liposome formulation unit in that order of connection. The order of connection of the units described in this application is preferably consistent with the steps in a mass production.

In certain embodiments, the connection between the dosing unit and the encapsulation unit is achieved by a sterile connector.

In certain embodiments, the disconnection between the dosing unit and the encapsulation unit is achieved by a sterile disconnect.

In certain embodiments, the connection between the fluid mixing and packaging unit and the liquid change unit is achieved by a sterile nipple machine.

In certain embodiments, the disconnection between the fluid mixing and packaging unit and the change unit is achieved by a sterile tube sealer.

In certain embodiments, the connection between the liquid change unit and the preparation unit is achieved by a sterile adapter machine.

In certain embodiments, the disconnection between the change unit and the preparation unit is achieved by a sterile tube sealer.

The use of the linker and blocker described herein enables the efficient attachment of different units, enabling the preparation of mRNA liposomes to be the first in the art to achieve an integrated production process for mRNA liposomes, overcoming the drawbacks of lack of integration and attachment between devices for different suppliers' quality checks.

The aseptic connector/aseptic disconnect as described herein allows for quick and easy completion of aseptic connection and disconnection, even in non-sterile environments. In certain embodiments, the sterile connector/sterile disconnect may filter the solution as a sterile filter. In certain embodiments, the system does not include additional sterile filters after the aseptic connector/aseptic disconnect.

In certain embodiments, a filter is provided after the anti-burst dosing unit as a sterile filter. In certain embodiments, the explosion-proof dosing unit is followed by a 0.22 μm filter cartridge to filter out residual microorganisms from the solution to a sterility level of sal=10e-6. In certain embodiments, in the system, the number of other sterilizing filters connected after the anti-burst dispensing unit is less than 5. In certain embodiments, in the system, the number of other sterilizing filters connected after the anti-burst dispensing unit is less than 3. In certain embodiments, in the system, the number of other sterilizing filters connected after the anti-burst dispensing unit is less than 1.

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

Optionally, the system in the present application further comprises a unit for preparing plasmid DNA stock solution and a unit for preparing mRNA stock solution upstream of the dosing unit. The connection/disconnection between the units for preparing the stock solution and the liquid preparation unit is realized through a sterile connector/sterile disconnect.

The plasmid DNA stock solution can be prepared as is conventional in the art, and can include, for example, fermentation culture, harvest clarification, purification by purification, linearization, filtration and packaging. The equipment used to carry out the series of steps may be conventional in the art, e.g., bacteria culture fermenters, CO ₂ Incubator, centrifuge, and apparatus for tangential flow filtration or depth filtration, etc., which constitute the apparatus for producing plasmid DAnd a unit of NA stock solution.

Optionally, the system in the present application further comprises a unit for preparing a cell therapy related product using the prepared mRNA liposome downstream of the preparation unit.

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

When the system is adopted to prepare mRNA liposome, the following operations can be sequentially carried out: explosion-proof butt joint, fluid butt joint, sample sampling, buffer solution preservation and transfer and sample solution preservation and transfer.

Unless otherwise indicated, the manipulation of each unit in this application may be well-established in the art, e.g., two-phase formulation using a partitioning unit, encapsulation of mRNA using an encapsulation unit.

The system is a totally-enclosed system, a disposable sealing process can be adopted in the use process, pollution and cross pollution are effectively avoided, the times of sterilization and filtration are reduced, the influence of environment, equipment, personnel and other external factors on cell products is reduced, the yield is increased, and the stability of the products is improved. Compared with the prior art, the system reasonably divides each unit into an explosion-proof area and a C-level area. In certain embodiments, the explosion-proof liquid dispensing unit and the encapsulation unit comprise an explosion-proof zone, and the liquid exchange unit and the formulation unit comprise a class C zone. The system can provide an explosion-proof production environment when the explosion-proof liquid preparation unit is contained, and is effectively explosion-proof. The purification, preparation and packaging processes are performed in a clean zone of class C or higher, so that the purification, preparation and packaging processes are free from microbial contamination. Sterile connection/disconnection is adopted between the explosion-proof area and the C-level area, repeated sterilization treatment can be carried out for many times without equipment of the C-level area in the whole process flow, and the production process is greatly simplified.

In certain embodiments, sterilization of the equipment in the class C zone is not required after the explosion proof docking, and/or fluid docking step. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 10. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 5. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 4. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 3. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 2. In certain embodiments, the number of sterilization treatments to the equipment in the class C zone during each batch process is less than 1.

In certain embodiments, the systems as described herein may be used in a continuous production process. In certain embodiments, the frequency of sterilization treatment of the equipment in the class C zone is less than 10 times per day in a continuous production process. In certain embodiments, the sterilization process for the equipment in the class C zone is performed less than 5 times per day in a continuous production process. In certain embodiments, the sterilization process for the equipment in the class C zone is performed less than 4 times per day in a continuous production process. In certain embodiments, the sterilization process for the equipment in the class C zone is performed less than 3 times per day in a continuous production process. In certain embodiments, the sterilization process for the equipment in the class C zone is performed less than 2 times per day in a continuous production process. In certain embodiments, the sterilization process for the equipment in the class C zone is performed less than 1 time per day in a continuous production process.

The system for preparing mRNA liposome provides a new idea for the collaborative development of mRNA liposome integrated equipment.

The foregoing is a summary of the application and there may be cases where details are simplified, summarized and omitted, so those skilled in the art will recognize that this section is merely illustrative and is not intended to limit the scope of the application in any way. This summary is not 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-mentioned and other features of the present application will be more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. It is appreciated that these drawings depict only several embodiments of the present application and are therefore not to be considered limiting of its scope. The contents of the present application will be more specifically and more specifically described with reference to the accompanying drawings.

Drawings

FIG. 1 shows a system for preparing mRNA liposomes in example 1 of the present application.

Fig. 2 shows a liquid storage bag for transferring liquid to be treated from a liquid anti-explosion dosing unit to an encapsulation unit.

Fig. 3 shows a TFF liquid change unit.

Fig. 4 shows a sample reservoir for transferring a sample.

Fig. 5 shows a formulation reservoir for transfer and filling of the formulation.

Detailed Description

The illustrative embodiments described in the detailed description, drawings, and claims are not meant 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 the present application. It will be readily understood that the aspects of the present application, as generally described herein, and illustrated in the figures, could be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated as part of this application.

Examples

In order that the present application may be more fully understood, the following examples are presented. 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: construction of a System for preparing mRNA liposomes

The system mainly comprises an explosion-proof liquid preparation unit (1), a microfluidic encapsulation unit (2), a TFF liquid exchange unit (3) and a preparation unit (4). Wherein the explosion-proof liquid preparation unit and the microfluidic encapsulation unit form an explosion-proof area, and the TFF liquid exchange unit and the preparation unit form a C-level area (see figure 1).

The explosion-proof liquid distribution unit (1) and the microfluidic encapsulation unit (2) are connected/disconnected by adopting a sterile connector/sterile disconnect.

Fig. 2 provides a reservoir bag for transferring liquid to be encapsulated from a liquid anti-burst dispensing unit to an 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 liquid inlet conduit (52) into the liquid storage bag (51). The filter (57) can filter the liquid flowing in the liquid dispensing unit. The reservoir (51) is equipped with a sampling head (55). The sampling head can conveniently extract the solution in the liquid storage bag (51). Wherein the liquid inlet conduit (52) and the liquid outlet conduit (54) of the liquid storage bag can be thermoplastic pipes, including but not limited toSilicone tube,Andetc. Thermoplastic tubing can be used for aseptic welding for liquid transfer if desired. The port of the liquid outlet conduit (54) of the liquid storage bag is provided with an air filter head (56) for preventing pollutants, particularly bacteria, in the air from entering. The liquid storage bag liquid inlet conduit (52) and the liquid storage bag liquid outlet conduit (54) are provided with pipe clamps (53) for controlling the opening and closing of the pipelines.

The liquid storage bag (51) can be connected with the microfluidic encapsulation unit (2) through a liquid outlet pipe (54) of the liquid storage bag.

And the microfluidic encapsulation unit and the TFF liquid exchange unit are connected/disconnected by adopting a sterile pipe connecting machine/sterile pipe sealing machine. Fig. 3 shows a TFF liquid change unit. Wherein the liquid inlet conduit (32) can be connected with/disconnected from the microfluidic encapsulation unit (2) through a sterile pipe connecting machine/a sterile pipe sealing machine. Wherein the reflux liquid conduit (33) can be connected/disconnected with the preparation unit (3) through a sterile pipe connecting machine/a sterile pipe sealing machine. mRNA liposomes prepared by the microfluidic encapsulation unit flow into a tangential flow filter (31) through a feed conduit (32). Permeate flows into and out of the tangential flow filter (31) through permeate conduit (34) to filter impurities in the formulation.

The liquid can be transferred between the microfluidic encapsulation unit (2) and the TFF liquid exchange unit (3) through a sample liquid storage bag (61) as shown in fig. 4. The sample reservoir inlet conduit (62) and the sample reservoir outlet conduit (64) may be thermoplastic tubes that may be used for aseptic welding to transfer fluids. The liquid outlet of the sample liquid storage bag liquid inlet conduit (62) and the liquid outlet of the microfluidic encapsulation unit (2) can be connected/disconnected through a sterile pipe connecting machine/a sterile pipe sealing machine, and the sample liquid storage bag liquid outlet conduit (64) and the liquid inlet conduit (32) can be connected/disconnected through the sterile pipe connecting machine/the sterile pipe sealing machine.

The TFF liquid exchange unit (3) and the preparation unit (4) are connected/disconnected by adopting a sterile pipe connecting machine/a sterile pipe sealing machine. Liquid can also be transferred between the TFF liquid exchange unit (3) and the preparation unit (4) through a preparation liquid storage bag (71) as shown in figure 5. Wherein, the preparation liquid storage bag liquid inlet conduit (72) and the TFF liquid exchange unit (3) can be connected/disconnected through a sterile pipe connecting machine/a sterile pipe sealing machine. The preparation in the preparation reservoir (71) flows to the filling needle (75) via a filling pump tube (74), wherein the filling pump tube (74) can be equipped with a peristaltic pump for pumping the liquid. The beta bag (76) is sleeved outside the filling pump pipe (74) and the filling needle (75), and the bag body is connected with the part where the filling pump pipe (74) is contacted in a welding mode so as to ensure that the beta bag is in a fully-closed state; a beta valve (77) of a rapid transfer interface (RTP) is attached to the beta bag to be connected aseptically to the formulation unit (4).

Example 2: methods of using systems for preparing mRNA liposomes

1. Explosion-proof butt joint

And (5) butting all parts of the explosion-proof area through an explosion-proof joint.

2. Fluid docking;

the liquid preparation unit, the encapsulation unit, the liquid exchange unit and the preparation unit are in butt joint through fluid pipelines.

3. Sampling of samples

Sampling heads (55) and (65) can sample and monitor samples.

4. Buffer preservation and transfer

After pumping the liquid into the reservoir (51), the reservoir clamp (53) may be closed to store the buffer. For transferring the solution, the reservoir tube clamp (53) may be opened and the buffer transferred by sterile docking through the reservoir tube (54).

5. Sample fluid preservation and transfer

After the sample is pumped into the sample reservoir (61), the sample reservoir clamp (63) may be closed to store the sample fluid. When transferring the solution, the sample fluid bag clamp (63) can be opened and the sample fluid can be transferred by sterile docking through the sample fluid bag outlet conduit (64).

Example 3: procedure for preparing mRNA liposomes

And 1, re-melting the stock mRNA in an anti-explosion liquid preparation unit (1) to prepare an aqueous phase containing the mRNA solution and an organic phase for encapsulating mRNA.

And 2, filtering the solution prepared by the explosion-proof liquid preparation unit by a filter (57) and flowing the filtered solution into a liquid storage bag (51).

Step 2.1. Optionally, the sampling head (55) is opened to sample and detect the solution in the reservoir bag (51).

Step 3. The solution in the reservoir bag (51) is pumped into an encapsulation unit (2), such as a microfluidic encapsulation unit. The encapsulation unit encapsulates the mRNA to form a liposome.

And 4, introducing the encapsulated liposome solution into a liquid exchange unit (3). The liquid inlet conduit (32) may be directly connected to the microfluidic encapsulation unit, alternatively liquid may be transferred through the sample reservoir (61). The tangential flow filter (31) performs a fluid change operation on the liposomes.

Step 4.1. Optionally, the sampling head (65) is opened to sample and detect the solution in the sample reservoir (61).

And 5, introducing the liposome solution after liquid exchange into a preparation liquid storage bag (71). The liposomes are filled into separate bottles via a filling pump tube (74) and a filling needle (75).

The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present application and are not meant to limit the scope of the invention, but to limit the scope of the invention.

Claims

A system for preparing mRNA liposome, which is characterized by comprising a liquid preparation unit, an encapsulation unit, a liquid exchange unit and a preparation unit in sequence according to a connection sequence; wherein the system is a fully closed system.
The system of claim 1, wherein the dosing unit is an explosion-proof dosing unit.
The system of claim 1 or 2, wherein the encapsulation unit encapsulates by film hydration, extrusion, homogenization, or microfluidic mixing.
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 group consisting of: genizer, precision NanoSystems (PNI), prescgenome, dolomite Microfluidics, michaux instrument technologies, inc.
The system of any of the preceding claims, wherein the liquid exchange unit is a tangential flow filtration liquid exchange unit.
A system according to any of the preceding claims, wherein the connection between the different units is achieved by means of sterile connectors and the disconnection is achieved with a sterile blocker.
The system of claim 8, wherein the sterile connector is a sterile connector or a sterile nipple machine; the blocker is a sterile breaker or a sterile tube sealer.
The system of claim 9, wherein the connection between the dosing unit and the fluid mixing and packaging unit is achieved by a sterile connector and the disconnection is achieved by a sterile disconnect.
The system according to claim 9 or 10, wherein the connection between the fluid mixing encapsulation unit and the change unit, and between the change unit and the preparation unit, is achieved by a sterile tube-taking machine, and the disconnection is achieved by a sterile tube-sealing machine.
The system of claim 1, wherein the dispensing unit is not in a sterile environment.
The system of claim 1, wherein the dosing unit is followed by a sterilizing filter.
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 comprise a sterilizing filter.
The system of claim 1, wherein the encapsulation unit is not followed by a sterilizing filter.
Use of the system according to any of the preceding claims for the preparation of mRNA liposomes.