Apparatus and method for operating a static mixing device - Patent Application 20070122997

Abstract

An apparatus for mixing two liquids is provided. The apparatus includes a static mixer and a first supply module (11) and a second supply module (12) for supplying the two liquids to the mixer. The supply modules include pressurized chambers (14a, 14b) for accommodating flexible containers holding the liquids to be mixed. When the chambers containing the flexible containers are pressurized, the liquids are forced into the static mixer. The pressurization is achieved by pressurized gas stored in pressure reservoir chambers (13a, 13b) connectable to the chambers holding the flexible containers. A related method of mixing two liquids is also provided.

Description

[Background technology]

[0001] Process automation and upscaling and downscaling of mixing processes are tasks that are routinely addressed in various fields of biotechnology and medicine.

[0002] Systems for the production of mixed fluids or products based on the combination of two or more components require a source of liquid raw materials, supply lines, a chamber where the mixing or reaction takes place, and an outlet for collecting the product. The chamber often represents the heart of such a system and can be bulky, for example, like a vessel, or miniaturized, as in microfluidic approaches. However, the system setup, conditions, and raw material quality also play a crucial role in these processes.

[0003] An example of a discontinuous mixing system is the device for mixing, storing, and homogenizing liquids, as disclosed in U.S. Patent No. 7,784,997. It comprises a rigid container fitted with a non-invasive pump. The container encloses a disposable bag with an orifice on the bottom used as a liquid outlet and additional orifices at the top for adding various liquids to produce the mixture. One of the top orifices is used to return the liquid (with the aid of a pump) to the inside of the bag, allowing for closed-loop circulation. This system is intended for single-use, avoiding the cleaning and sterilization steps required when using a rigid tank for mixing. It can handle bags with volumes ranging from 25 to 3,000 L, but does not appear to be suitable for producing smaller volumes, such as microliter or milliliter volumes.

[0004] EP 1146959 discloses an apparatus for the continuous production of encapsulated therapeutic compounds, showing an example of a precisely controlled metering system. The apparatus comprises a lipid phase storage means, an aqueous phase storage means, and a pressurized transfer means for transferring the phases to a mixing device, preferably a static mixer. The apparatus of EP 1146959 further comprises a premixing system. The phases are transferred from these means to the premixing chamber and the mixing chamber with the aid of a metering pump driven by a motor. Because this system is continuous, it can produce larger quantities of the desired product.

[0005] Despite these and other solutions to mixing systems and methods, there remains an incomplete need for methods and devices for producing small quantities of product that are fast, reliable, and can be performed under standardized conditions. This is especially true for applications in the field of personalized medicine.

[0006] It is therefore an object of the present invention to provide an apparatus and method for the production of mixed fluids or products based on the combination of two or more components that is flexible in terms of adapting to different types of products and that produces the desired product in a robust, rapid and reliable manner. These also aim to provide a high throughput approach. Further objects of the present invention will become apparent based on the following description, examples and claims of the invention. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 7,784,997 [Patent Document 2] European Patent No. 1146959 Summary of the Invention

[0008] In a first aspect of the present invention, there is provided an apparatus for operating a static mixer for mixing a first liquid and a second liquid, the static mixer comprising a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from the mixing of the first liquid and the second liquid, the apparatus further comprising: (a) a first supply module for supplying the first liquid to the first inlet, (b) a second supply module for supplying the second liquid to the second inlet, and (c) a pressure supply module disposed upstream of the first and second supply modules. Each of the first and second supply modules independently includes: (i) a pressure reservoir chamber for holding pressurized gas, the pressure reservoir chamber having an inlet and / or an outlet for the pressurized gas; (ii) a pressurizable substrate chamber for holding a flexible container, the flexible container having an interior space for holding a first liquid or a second liquid, respectively; and (iii) a connector for providing fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the fluid communication being via a connector for connecting the pressure reservoir chamber to the pressurizable substrate chamber. (iv) a means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the means having an open state and a closed state, the connector and the means being configured to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the pressurizable substrate chamber upon changing the state of the means from the closed state to the open state; and (v) a pressure sensor for measuring the pressure of the pressurized gas in the pressure reservoir chamber or in the supply module downstream of the pressure reservoir chamber.The pressure supply module comprises: (i) a gas inlet reversibly connectable to a pressurized gas source; (ii) a first gas outlet for supplying pressurized gas to the pressure reservoir chamber of the first supply module; (iii) a second gas outlet for supplying pressurized gas to the pressure reservoir chamber of the second supply module; (iv) a flow path for the pressurized gas to flow from the gas inlet to the first and / or second gas outlet, the flow path including a flow path divider; (v) at least one pressure amplifier disposed in the flow path; (vi) a first pressure control circuit adapted to control the pressure of the pressurized gas delivered to the pressure reservoir chamber of the first supply module; and (vii) a second pressure control circuit adapted to control the pressure of the pressurized gas delivered to the pressure reservoir chamber of the second supply module. Further features of the device are provided below in the detailed description of the invention.

[0009] According to another aspect of the present invention, there is provided an apparatus for mixing a first liquid and a second liquid, the apparatus comprising: a static mixer; and first and second supply modules adapted to supply the first and second liquids to the static mixer, each of the supply modules independently comprising: a substrate chamber for holding a flexible container having an interior space for holding the first or second liquid, respectively; a conduit for providing fluid communication between the interior space of the flexible container and a first inlet or a second inlet of the static mixer, respectively; a pressure reservoir chamber for holding pressurized gas, the pressure reservoir chamber having an inlet and an outlet for the pressurized gas; a pressure amplifier disposed upstream of the pressure reservoir chamber, the pressure amplifier having an inlet reversibly connectable to a pressurized gas source and an outlet for the pressurized gas fluidly connected to the inlet of the pressure reservoir chamber; and a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber. The connector comprises a means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber, the means having an open state and a closed state, the fluid communication allowing a flow of pressurized gas from the pressure reservoir chamber to the substrate chamber, for example, to apply pressure to an exterior surface of the flexible container and force the first or second liquid from the container into a conduit, and the connector and the means are further configured to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the substrate chamber upon changing the state of the means from the closed state to the open state.

[0010] In a further aspect, the present invention provides a method of mixing a first liquid and a second liquid. The method includes the steps of: (aa) providing a first flexible container holding a first liquid, the first flexible container being contained within a first pressurizable substrate chamber; (bb) providing a second flexible container holding a second liquid, the second flexible container being contained within a second pressurizable substrate chamber; (cc) providing a static mixer having a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from mixing the first and second liquids; (dd) independently pressurizing the first and second pressurizable substrate chambers with pressurized gas that applies pressure to outer surfaces of the first and second flexible containers to force the first and second liquids into the static mixer and mix the first and second liquids; and (ee) collecting the third liquid. Further features of the method are provided below in the detailed description of the invention.

[0011] In yet another aspect, the present invention provides a method of mixing a first liquid and a second liquid based on the use of an apparatus. In particular, the method includes the steps of: (aa) providing a first flexible container for holding a first liquid, the first flexible container being contained within a first pressurizable substrate chamber; (bb) providing a second flexible container for holding a second liquid, the second flexible container being contained within a second pressurizable substrate chamber; (cc) providing a static mixer having a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from mixing of the first liquid and the second liquid; (dd) independently pressurizing the first and second pressurizable substrate chambers with pressurized gas that applies pressure to outer surfaces of the first and second flexible containers to force the first and second liquids into the static mixer and mix the first and second liquids; and (ee) collecting the third liquid. Further features and embodiments of the method are disclosed below in the detailed description of the invention. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a flow chart showing the pneumatic circuit and components of one embodiment of the device (1). [Figure 2] 1 is a flow chart showing the pneumatic circuit and components of another embodiment of the device (1). [Figure 3] FIG. 1 is a front view of the device (1) in the direction of operation. [Figure 4] FIG. 4 is a perspective view of the device (1) shown in FIG. [Figure 5] FIG. 5 is a perspective view of the device (1) shown in FIG. 4 with the inserts (58a, 58b, 59a, 59b) installed. [Figure 6] FIG. 1 is a front view of an example of a frame (80) configured for use with apparatus (1), the frame comprising a static mixer assembled with other components provided for mixing a first liquid and a second liquid. [Figure 7]FIG. 1 is a perspective view of a typical static mixing device (70). DETAILED DESCRIPTION OF THE INVENTION

[0013] As described in the summary, a first aspect of the present invention relates to an apparatus for operating a static mixer for mixing a first liquid and a second liquid, the static mixer comprising a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from the mixing of the first liquid and the second liquid. The apparatus further comprises: (a) a first supply module for supplying the first liquid to the first inlet, (b) a second supply module for supplying the second liquid to the second inlet, and (c) a pressure supply module disposed upstream of the first and second supply modules. Each of the first and second supply modules independently includes: (i) a pressure reservoir chamber for holding pressurized gas, the pressure reservoir chamber having an inlet and / or an outlet for the pressurized gas; (ii) a pressurizable substrate chamber for holding a flexible container, the flexible container having an interior space for holding a first liquid or a second liquid, respectively; and (iii) a connector for providing fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the fluid communication being via a connector for connecting the pressure reservoir chamber to the pressurizable substrate chamber. (iv) a means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the means having an open state and a closed state, the connector and the means being configured to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the pressurizable substrate chamber upon changing the state of the means from the closed state to the open state; and (v) a pressure sensor for measuring the pressure of the pressurized gas in the pressure reservoir chamber or in the supply module downstream of the pressure reservoir chamber.The pressure supply module comprises (i) a gas inlet reversibly connectable to a pressurized gas source, (ii) a first gas outlet for supplying pressurized gas to the pressure reservoir chamber of the first supply module, (iii) a second gas outlet for supplying pressurized gas to the pressure reservoir chamber of the second supply module, (iv) a flow path for the pressurized gas to flow from the gas inlet to the first and / or second gas outlet, the flow path including a flow path divider, (v) at least one pressure amplifier disposed in the flow path, (vi) a first pressure control circuit adapted to control the pressure of the pressurized gas delivered to the pressure reservoir chamber of the first supply module, and (vii) a second pressure control circuit adapted to control the pressure of the pressurized gas delivered to the pressure reservoir chamber of the second supply module.

[0014] The inventors have discovered that the apparatus defined herein is surprisingly accurate in controlling the flow of first and second liquids into a static mixer at predetermined flow rates, enabling highly reproducible processing of two liquid substrates into a product, i.e., a third liquid composition. Furthermore, it enables aseptic processing of very small batches, avoiding the dead volume and startup losses of larger-scale apparatus for generating liquid flows, particularly those that use pumps and other flow or pressure control systems. The use of gas pressure applied to the exterior of a flexible container as the driving force for achieving controlled flow of liquid from such a flexible container into the static mixer also avoids the initial fluid pressure oscillations associated with the use of pumps. This advantage makes the claimed apparatus and process particularly useful for preparing small batches and short processing times. This is particularly true when gas pressure is provided suddenly by a pre-pressurized pressure reservoir chamber, rather than by a continuous flow of pressurized gas. It has been found that rapidly equalizing the pressure applied to the exterior surface of the flexible vessel minimizes start-up time to reach the required processing flow rate, improves efficiency with respect to substrate loss, and minimizes the amount of liquid product stream that forms under non-optimal flow mixing during start-up. Furthermore, because the pressurized gas does not contact the liquid, but rather the exterior surface of the flexible vessel, it does not potentially contaminate the liquid.

[0015] Given the absence of pumps and other peripheral hardware, the device can be designed to operate with disposable static mixers and fluid conduits, thus avoiding lengthy cleaning and sterilization cycles.

[0016] As used herein, an apparatus for operating a static mixer should be understood as an apparatus that is technically suitable and actually adapted or configured to operate a static mixer. Similarly, a static mixer for mixing a first liquid and a second liquid means a static mixer that is suitable and adapted to mix the first liquid and the second liquid.

[0017] In the context of this invention, a static mixer is any mixer or mixing device that does not rely on moving parts to perform the mixing process. An example of a very simple static mixer is a T-joint. Because static mixers are used to mix two liquids to produce a liquid mixture, i.e., a third liquid, they typically have at least two substrate inlets and a product outlet. In the context of this invention, these substrate inlets are referred to as a first inlet for receiving the first liquid and a second inlet for receiving the second liquid.

[0018] As used herein, a "feed module" refers to a group of device components designed, adapted, and / or configured to feed a substrate to a static mixer. According to the present invention, the device comprises at least two feed modules: a first feed module for feeding a first liquid to a first inlet of the static mixer, and a second feed module for feeding a second liquid to a second inlet. The design and configuration of these feed modules are key features that characterize the present invention. More specifically, the feed modules are adapted to accommodate a flexible container in which the liquid substrate is initially provided, from which it is fed to the mixing device. As used herein, a "flexible container" refers to any container capable of holding a liquid material having at least one flexible wall. In particular, the container exhibits a type of flexibility that allows for a significant reduction in the internal volume or space of the container, as in the case of a collapsible container, whose collapsibility is due to the flexibility of the container wall, similar to an infusion bag.

[0019] Each of the two supply modules independently includes a pressure reservoir chamber. Such a pressure reservoir chamber is designed and adapted to hold pressurized gas, such as pressurized air. Therefore, it typically includes a solid, pressure-resistant wall enclosing an interior space for holding the pressurized gas. The degree of pressure resistance of the wall should be selected taking into account the intended operating pressure. For example, the pressure reservoir chamber may be designed to operate at a pressure of up to about 50 bar, or up to about 25 bar. To fill the pressure reservoir chamber with pressurized gas and deliver the pressurized gas to the pressurizable substrate chamber, the pressure reservoir chamber exhibits at least one opening representing an inlet and an outlet. Optionally, the pressurized gas inlet and outlet may be independent of or different from each other.

[0020] Additionally, each of the two supply modules independently includes a pressurizable substrate chamber for holding a flexible container holding a first or second liquid. To operate the device and mix the two liquids, the substrate chamber is pressurized to expel, e.g., squeeze, the liquid from the flexible container and supply the liquid to the mixing device. Pressurization is achieved by pressurized gas initially contained in a pressure reservoir chamber of each supply module, which can flow into the substrate chamber to apply pressure to the exterior of the flexible container to initiate the mixing process.

[0021] As described above, the pressurizable substrate chamber is adapted to hold a flexible container containing a first or second liquid. To facilitate insertion and / or removal of such flexible containers, the substrate chamber of the first or second supply module can be provided with a means for opening and closing the substrate chamber. Preferably, both substrate chambers exhibit this feature. For example, the pressurizable substrate chamber can comprise a two-piece wall or housing, e.g., a main portion and a lid-like portion, and a means for hermetically securing the lid to the main portion.

[0022] Independently, as used in this context, means that each supply module component is present in each of the first and second supply modules, and the components of the first supply module may differ from each of the components of the second supply module with respect to optional features or dimensions of the components of the first supply module.

[0023] As described above, each supply module includes a connector for providing fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber. The fluid communication is such that pressurized gas can flow from the pressure reservoir chamber to the pressurizable substrate chamber. Preferably, the dimensions (e.g., inner diameter) of the connector are such that pressure equilibration between the pressure reservoir chamber and the pressurizable substrate chamber can occur very quickly. In other words, the connector's flow resistance should be low. For example, the inner diameter of the connector should be at least about 1 mm.

[0024] The means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, having an open state and a closed state, is configured to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the pressurizable substrate chamber upon changing the state of the means from the closed state to the open state, where the pressure equilibrium includes an abrupt increase in pressure in each of the first and second pressurizable substrate chambers from an initial pressure to a maximum process pressure, also referred to as a target process pressure.

[0025] In this context, the terms "instant" and "rapid" refer to a very rapid pressure change, with respect to the absolute duration required to reach the target process pressure. Typically, the duration is less than 10 seconds, in most cases substantially less than 10 seconds, or less than about 9, 8, 7, 6, 5, 3, or 2 seconds, or even less than about 1 second, respectively. In some preferred embodiments, the device is configured to achieve pressure equilibrium within about 1, 2, or 3 seconds, or within about 0.5, 1, 1.5, 2, 2.5, or 3 seconds. The duration of the pressure equilibrium phase may also depend on the dimensions of the device, including dead space in the fluid paths for the first and second fluids upstream of the static mixing device, or the duration of the mixing process. For example, for small batch sizes, e.g., less than 500 mL (i.e., of the third fluid), or mixing times, e.g., less than 1 minute, immediate pressure equilibration may mean a duration of, e.g., about 1 or 2 seconds or less, while for larger batch sizes or mixing times, e.g., 5-10 minutes, a duration of more than 2 seconds, such as 2-10 seconds, still represents immediate pressure equilibration.

[0026] Thus, the equilibration time is very short compared to the duration of the flow of each liquid from the flexible container to the static mixer, which is caused by such rapid pressure equilibration between the pressure reservoir chamber and the pressurizable substrate chamber, and is also very short compared to the total mixing time, i.e., the time required to produce a desired or predetermined amount of a third liquid by mixing the first and second liquids. In some embodiments, the pressure equilibration time is about 5% or less of the total mixing time, particularly about 3% or less of the total mixing time. In further embodiments, the pressure equilibration time is about 2% or less, about 1% or less, or even about 0.5% or less of the total mixing time.

[0027] In other words, the initial pressure equilibration occurs rapidly, and in one embodiment, the pressure equilibration requires only a fraction of a second (e.g., less than 1 second, e.g., 0.5 seconds or less) and is already complete or nearly complete by the time the respective first or second liquid first reaches the static mixer.

[0028] According to another preferred embodiment, the time required for pressure equilibration is such that no more than about 5% of the batch or desired volume of the third liquid is produced in the static mixer before the maximum process pressure is reached. In even more preferred embodiments, no more than about 3%, 2%, or 1% of the batch or desired volume of the third liquid is produced in the static mixer before the maximum process pressure is reached.

[0029] Instantaneous pressure equilibration between the pressure reservoir chamber and the pressurizable substrate chamber involves a rapid and significant pressure increase in the substrate chamber, as the pressurized gas suddenly exerts pressure on the exterior surface of the flexible container, and a corresponding rapid pressure decrease in the pressure reservoir chamber. As will be appreciated by those skilled in the art, the pressure equilibration achieved by instantaneous pressure equilibration is dynamic in that the pressures in the pressure reservoir chamber and the substrate chamber, which are essentially the same after equilibration, may change slightly over time. For example, as the respective first or second liquids flow out of the flexible container, the pressure may decrease slightly, thus reducing the overall volume of the flexible container and increasing the gas space in the substrate chamber. Other factors, such as slight temperature changes, may cause the equilibrium pressure to decrease or increase slightly.

[0030] In some embodiments, the total decrease in equilibrium pressure caused by the flow of liquid from the flexible container into the static mixer is about 10% or less of the initial equilibrium pressure. In this way, the pressure drop during the mixing process may not substantially affect the mixing process itself. As will be appreciated by those skilled in the art, the total equilibrium pressure drop during the mixing process can be minimized, for example, by selecting a pressure reservoir chamber with a large internal volume relative to the volume of liquid being pushed from the flexible container into the static mixer. For example, the internal volume of the pressure reservoir chamber may be at least about 10 times the volume of liquid originally held by the flexible container, or the internal volume of the pressure reservoir chamber may be at least about 15 times, or even about 20 times or more, the volume of liquid originally held by the flexible container.

[0031] In another embodiment, the drop in equilibrium pressure caused by the flow of liquid from the flexible container into the static mixer is no more than 5%, 4%, 3%, 2%, or 1% of the initial equilibrium pressure. In preferred embodiments, the total equilibrium pressure drop during the mixing process is minimized, or in some embodiments, even negligible.

[0032] As described above, each supply module further comprises a pressure sensor for measuring the pressure of the pressurized gas in the respective supply module at or downstream of the pressure reservoir chamber. In some embodiments, the pressure sensor is configured to measure the gas pressure directly in the respective pressure reservoir chamber. Alternatively, the pressure sensor may be located, for example, in the connector, preferably upstream of the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber.

[0033] As used herein, a pressure supply module is understood to refer to a group of device components designed and adapted to supply pressurized gas to a supply module, particularly to the pressure reservoir chamber of the supply module. As described above, the pressure supply module includes a gas inlet, first and second gas outlets, and a flow path for the pressurized gas to flow from the gas inlet to the first and / or second gas outlets. The gas inlet, reversibly connectable to a pressurized gas source, represents the upstream end of the flow path, while the first and second gas outlets, configured to supply pressurized gas to the pressure reservoir chambers of the first and second supply modules, respectively, represent the downstream end of the flow path. The gas inlet can represent any type of inlet or connector, such as a tube connector, connectable to a pressurized gas source. The gas outlet can be embodied, for example, by a tube end that can be connected to or form the inlet of the pressure reservoir chamber. In some embodiments, the gas inlet of the pressure supply module is connected to a pressurized gas source. In some related embodiments, the gas inlet of the pressure supply module is connected to a pressurized gas source that supplies gas at a pressure of about 6-10 bar.

[0034] The flow path divider is configured to divide the flow path downstream of the flow path divider into two subpaths, one terminating in a first gas outlet and the other terminating in a second gas outlet. Such a flow path divider may be, for example, a simple T-joint or Y-joint.

[0035] As described above, at least one pressure amplifier is disposed in the flow path. For example, a single pressure amplifier may be disposed upstream of the flow path divider to increase the gas pressure delivered to both the first and second gas outlets. Alternatively or additionally, two pressure amplifiers may be disposed in the flow path downstream of the flow path divider, one of which is fluidly connected to the first gas outlet and the other of which is fluidly connected to the second gas outlet. In this regard, the term "fluidly connected" refers to a combination of two pressure amplifiers.

[0036] A pressure amplifier, sometimes called a pneumatic amplifier or pressure booster, is a device that receives an input gas (e.g., air) at a specific pressure and delivers an output gas with a pressure higher than the input gas pressure. Generally, a pressure amplifier may be understood as a pump. A pressure amplifier may be powered by externally supplied (e.g., electrical) energy. Alternatively, an amplifier may be a purely mechanical device powered by a portion of the input compressed gas supply, thereby circulating and pumping the remainder of the supply to a higher output pressure. Pressure may be generated, for example, by using an area-differential piston assembly, based on the principle that low-pressure gas applied to a large area produces high-pressure gas over a corresponding smaller area.

[0037] In some embodiments, the pressure amplifier used in the apparatus is a mechanical device adapted to amplify gas pressure so that the output pressure is twice the input pressure. For example, the pressure amplifier can generate gas having a pressure of up to about 20 bar from a supply gas having a pressure of up to about 10 bar. In some further embodiments, the pressure amplifier is adapted to increase the pressure of pressurized gas received from a pressurized gas source to which the pressure amplifier is connectable or is connected by at least about 50%, where the percentage is the pressure of the pressurized gas received from the pressurized gas source.

[0038] The use of a pressure amplifier is particularly advantageous in that it allows the apparatus to operate at pressures of about 16-20 bar, which are useful or required in some mixing processes, while still using conventional pressurized air sources that typically provide pressures of about 8-10 bar or less.

[0039] As described above, the pressure supply module includes at least two pressure control circuits, one of which is adapted to control the pressure of the pressurized gas delivered by the pressure supply module to the pressure reservoir chamber of the first supply module, and another of which is adapted to control the pressure of the pressurized gas delivered by the pressure supply module to the pressure reservoir chamber of the second supply module.

[0040] In some preferred embodiments, the pressure control circuits used in the device are electrical or electronic control circuits, i.e., they include a controller that receives an electrical signal as an input and provides control via an electrical output signal. Typically, an electronic microcontroller is used for this purpose. For the avoidance of doubt, a single microcontroller may be used to simultaneously control both the first and second pressure control circuits.

[0041] In some embodiments, each of the first and second pressure control circuits of the pressure supply module comprises a valve and / or an electric pressure regulator disposed between the flow path divider and the respective first or second gas outlet. In some further embodiments, each pressure control circuit includes both a valve and an electric pressure regulator. For example, the valve may be a non-regulating valve and may be disposed downstream of the electric pressure regulator but upstream of the respective gas outlet.

[0042] The electric pressure regulator, which may also be referred to as an electronic pressure regulator or simply a pressure regulator or electronic regulator (e.g., FIGS. 1 and 2), is preferably controlled by a microcontroller as described above. The electric pressure regulator provides independently selectable predetermined output pressures for each gas outlet. In some embodiments, both the valve and the electric pressure regulator are configured to be operated by the microcontroller.

[0043] The microcontroller may be configured to receive a signal from a pressure sensor arranged to measure the gas pressure in each of the first or second supply modules, in other words, the microcontroller controls the pressure regulator depending on the actual pressure in each supply module, in particular the pressure in the pressure reservoir chamber to which the supply module is connected, i.e. depending on the signal received by the pressure sensor of the supply module.

[0044] In some further embodiments, the pressure supply module further comprises a pressure reservoir chamber disposed in the flow path for holding pressurized gas. According to some further preferred embodiments, this additional pressure reservoir chamber may be disposed in the flow path upstream of the flow path divider. The inventors have found that such a pressure reservoir chamber disposed in an upstream portion of the pressure supply module can substantially dampen pressure fluctuations and allow for very precise control of the gas pressure within the pressure supply module. In such a configuration, for example, pressure control within ±0.01 bar of a preset pressure of 2 bar has been achieved.

[0045] In some embodiments, the flow path in the pressure supply module upstream of the flow path divider includes both a pressure amplifier and a downstream pressure reservoir chamber (see, e.g., FIG. 1 ). Optionally, the pressure supply module may include an additional valve disposed in the flow path between the pressure reservoir chamber and the flow path divider. This valve may also be configured to be operated by a microcontroller. Again, for the avoidance of doubt, the microcontroller controlling the pressure of the pressurized gas delivered to the first gas outlet may be the same microcontroller controlling the pressure of the second gas outlet. This valve, like other valves used in the device, may be a non-regulating valve, i.e., it may only completely (but not partially) block fluid communication between upstream and downstream components relative to the valve position. However, this valve may also be selected as a three-way valve that allows venting of a pneumatic element (e.g., a pressure reservoir chamber disposed upstream of the valve).

[0046] In some embodiments, the pressure supply module includes two pressure amplifiers arranged downstream of the flow path divider, with a first pressure amplifier fluidly connected to the first gas outlet and a second pressure amplifier fluidly connected to the second gas outlet (see, for example, FIG. 2). In this case, it may not be necessary to provide a pressure amplifier upstream of the flow path divider. In a related embodiment, an electric pressure regulator is arranged between each of the two pressure amplifiers and the flow path divider. Furthermore, additional valves may be arranged upstream and / or downstream of each of the two pressure amplifiers. Again, such additional valves are preferably non-adjustable but designed as three-way valves to allow venting of adjacent pneumatic elements.

[0047] In some further embodiments, the pressure supply module may further include a flow path branch arranged to bypass at least one pressure amplifier and a check valve arranged at the flow path branch. Such an arrangement is beneficial for shortening the time required for the pressure reservoir chambers of the first and second supply modules to reach the desired gas pressure. The amplifier can be bypassed initially, i.e., while the gas pressure in the supply module is still lower than the pressure provided by the pressurized gas source, thereby avoiding the inherent flow reduction of the supply module. Only after the pressure in the pressure reservoir chamber reaches the pressure provided by the pressurized gas source is the pressure amplifier used to further increase the pressure until the desired pressure in the pressure reservoir chamber is reached.

[0048] In some preferred embodiments, the static mixer includes or represents a T-mixer, a Y-mixer, a vortex mixer, a baffled static mixer, a microfluidic mixing device, a multi-inlet vortex mixer (MIVM), or a jet impingement reactor. As used herein, a T-mixer and a Y-mixer are mixing devices that include a T-joint or a Y-joint, respectively, and function to mix two liquids together at such a T-joint or Y-joint. A static vortex mixer is a precisely designed device for continuous mixing of liquids, typically based on a baffle-like structure shaped to generate vortices, which are regions in a liquid mixture where the flow rotates around an axis parallel to the overall flow direction. Therefore, such vortex mixers can also be understood as a special type of baffled static mixer. A microfluidic mixing device, as used in the context of the present invention, is any static mixing device whose fluid conduits typically have diameters of 2 or 3 mm or less, often less than 1 mm, and can be designed to provide a relatively large interface (compared to the diameter of the fluid conduits) between two liquid substrates, for example, by splitting the fluid stream into multiple microfluidic streams before the substrates are brought into contact with each other. A multi-inlet vortex mixer (MIVM) is a special type of static vortex mixer with three or more fluid inlets.

[0049] As described above, the first and / or second supply modules of the device independently include a pressure sensing means or pressure sensor that may be disposed with or housed within the pressure reservoir chamber. Alternatively, the pressure sensing means may be disposed outside the pressure reservoir chamber but in fluid communication with the pressure reservoir chamber, for example, in a fluid path downstream of the pressure reservoir chamber. Optionally, the pressure sensing means may also be part of or connected to a pressure amplifier, provided that the pressure sensing means is arranged to sense pressure at its outlet in fluid communication with the pressure reservoir chamber. Preferably, each of the first and second supply modules independently includes a pressure sensing means. Optionally, a supply module may include two or more pressure sensing means.

[0050] In some embodiments, the pressure sensing means comprises or is connected to a transducer that generates an electrical signal in response to the pressure it senses, thereby enabling a control loop to be used to actuate and operate a valve, an electrical pressure regulator, or a pressure amplifier in response to the signal received from the respective pressure sensing means.

[0051] Thus, in some embodiments, the apparatus includes a controller configured to control the gas pressure supplied to the first and second supply modules in response to signals received from the respective pressure sensing means. A separate controller, which may be a computer, need not be provided for each supply module. In other words, a single controller may be used to receive signals from each pressure sensing device and to actuate or operate each electrical pressure regulator or valve in response to those signals.

[0052] Such a feedback or control loop can be used to maintain the pressure during the mixing process within narrow boundaries, such as within ±5% of the target process pressure reached at initial pressure equilibrium, i.e., while the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber is in an open state. In this regard, the criterion for whether a percentage is at the target process pressure.

[0053] In some embodiments, the pressure supply module is adapted to maintain a selected pressure in the pressure reservoir chamber within a range of approximately 2-20 bar when the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, e.g., by a pressure control circuit, is in an open state. The selected pressure may differ between the two supply modules, i.e., the pressure is independently selected for each pressure reservoir chamber. For example, for the first supply module, the apparatus may be operable to maintain the operating pressure in the pressure reservoir chamber at a first selected pressure within a range of approximately 2-5 bar, and for the second supply module, the apparatus may be operable to maintain the operating pressure in the pressure reservoir chamber at a second selected pressure within a range of approximately 6-16 bar. Preferably, each operating pressure is maintained within, e.g., approximately ±5% (or less, e.g., ±3%) of the respective selected pressure.

[0054] The inventors have found that apparatus according to these preferred embodiments not only allows for very rapid initiation of a stable mixing process, which is particularly important for very small batches, but also allows for a very high degree of control over product quality by minimizing fluctuations in operating pressure due to parameters such as dead volume or slight changes in temperature.

[0055] The equilibrium and time to equilibrium described herein can be achieved by changing the state of the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber from its closed state to its open state. As understood in this context, a closed state refers to a fully closed state, such as a fully closed valve state that substantially prevents fluid flow, and an open state refers to a fully open state that does not substantially restrict fluid flow. In a preferred embodiment, the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber has only these two states, i.e., a fully closed state and a fully open state. In other words, in this embodiment, even if there is an intermediate state, such as a pressure adjustment function, such an intermediate state is not used in carrying out the processes described herein. Therefore, in a preferred embodiment, neither the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber nor the connector generally includes a pressure adjustment member.

[0056] Optionally, the open state is such that the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber are at least about 1 mm apart in the open state. 2 , or at least about 2 mm 2 , 3mm 2 , 4mm 2 , and even 5mm 2 The pressure vessel may also be characterized by having a fluid path for pressurized gas having a relatively large cross-sectional area, such as a pressure vessel.

[0057] The means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber may be integrated into the connector, or the connector may be located as part of the outlet of the pressure reservoir chamber or the inlet of the substrate chamber, which provides fluid communication between the pressure reservoir chamber and the substrate chamber.

[0058] In one preferred embodiment, the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber is the only means for controlling the flow of pressurized gas between the pressure reservoir chamber and each pressurizable substrate chamber.

[0059] Additionally, in some preferred embodiments, the device includes first and second opposing components, at least one of which includes a first cavity and a second cavity, and the opposing components are configured to be secured to one another such that one of the pressurizable substrate chambers is formed to include the first cavity and the other of the pressurizable substrate chambers is formed to include the second cavity.

[0060] In this context, a counter part is to be understood as a solid structural component of the device having the dimensions and physical properties necessary to provide a portion of a respective pressurizable substrate chamber.

[0061] In an alternative embodiment, first and second opposing parts are provided which, when secured together, form one of the pressurizable substrate chambers, and third and fourth opposing parts are provided which, when secured together, form the other of the pressurizable substrate chambers, again requiring that at least one of the first and second opposing parts and at least one of the third and fourth opposing parts comprise a cavity which, when the respective opposing parts are secured together, forms a chamber which can function as a pressurizable substrate chamber.

[0062] In some embodiments, the first and second opposing components together form both pressurizable substrate chambers, i.e., the pressurizable substrate chamber of the first supply module and the pressurizable substrate chamber of the second supply module. Cavities, which may also be understood as recesses, in the first and / or second opposing components provide the space necessary to accommodate the flexible containers containing the first and second liquids. These cavities may be located only on the first opposing component, only on the second opposing component, or on both the first and second opposing components. In the latter case, the pressurizable substrate chamber is formed from a cavity or recess in the first opposing component that merges with a cavity or recess in the second opposing component when the opposing components are secured together.

[0063] In some embodiments, the first opposing component is substantially stationary, and the second opposing component is movable so that the corresponding opposing components can be secured to one another and the cavity forms a pressurizable substrate chamber. In some embodiments, the opposing components are rotatable and hingedly connectable or connected to the first opposing component. The hinge connection may be located on any side of the first and / or second opposing components.

[0064] In some preferred embodiments, the first and second opposing parts have a vertical operating orientation and the hinge connection is located on the underside or end of the second opposing part, while in alternative embodiments, the hinge connection is located on one of the vertical sides or on the upper side of the second opposing part.

[0065] Advantageously, at least one circumferential gasket may be provided between the first and second opposing parts, allowing each pressurizable substrate chamber to be separately sealed. In other words, each pressurizable substrate chamber formed when the first and second opposing parts are secured to one another is individually sealed by at least one circumferential gasket. Such a gasket may be provided, for example, on either of the opposing parts. Sealing of the two chambers may be achieved by a single gasket, provided that the shape allows each chamber to be separately sealed. Alternatively, a separate circumferential gasket may be provided to seal each pressurizable substrate chamber. Optionally, at least one circumferential gasket is provided on each of the first and second opposing parts. In any case, because the operating pressures in the substrate chambers are often different from each other, each pressurizable substrate chamber must be separately sealed, even if only by a single gasket.

[0066] In some preferred embodiments, at least two circumferential gaskets are provided between the first and second opposing parts to separately seal each of the pressurizable substrate chambers, and frames for holding the first liquid and the second liquid flexible containers are sealed between the at least two circumferential gaskets. Thus, the device is adapted to hold such frames used to hold the flexible containers, and the frames are "sandwiched" and sealed between the first and second opposing parts. One side of the frame is sealed to the first opposing part by at least one circumferential gasket, and the other side of the frame is sealed to the second opposing part by at least another circumferential gasket, each gasket shaped to separately seal the pressurizable substrate chambers.

[0067] In some further embodiments, the pressurizable substrate chamber and the pressure reservoir chamber of each of the first and second supply modules are fluidly connected through openings in the first or second opposing parts, respectively, which openings are gas outlets of the respective pressure supply modules. Each opening is arranged and located in an area of ​​the opposing parts that becomes part of one of the pressurizable substrate chambers when the two opposing parts are secured together. In some preferred embodiments, these openings for supplying pressurized gas to the pressurizable substrate chambers are housed in the substantially stationary first opposing part.

[0068] A particular advantage of the present invention is that the pressure in the pressure reservoir chamber of each supply module and the volume ratio between the pressure reservoir chamber and the pressurizable substrate chamber can be preselected to achieve a desired pressure in the pressurizable substrate chamber and a desired flow rate at which a given liquid is forced out of a given flexible container and fed to the mixing device. As a result, the use of pumps or process controls that rely on flow meters can be avoided. As is commonly known to those skilled in the art, the internal pressures of two chambers fluidly connected to each other (such as the pressure reservoir chamber and the substrate chamber when the valves on the connectors between them are opened) will be identical, and the resulting pressure in the chamber can be accurately calculated and predicted based on the initial pressure in the chamber and the internal volume of the chamber before establishing the fluid connection.

[0069] The actual flow rate of liquid ejected from the flexible container contained within the substrate chamber will, of course, depend on other parameters, such as the viscosity of the liquid and the flow resistance of the fluid path downstream of the flexible container. However, for a given liquid and a given flow path, the pressure required to achieve a specific flow rate can be easily determined experimentally. Once known, the feed module can be easily configured to achieve such target pressure, i.e., by calculating and selecting a specific chamber volume and starting pressure of the pressure reservoir chamber. If the batch size changes and the volume of the flexible container and pressurizable substrate chamber changes, the feed module can be easily reconfigured to achieve the same target pressure and flow rate, for example, by adapting the starting pressure or the internal volume of the pressure reservoir chamber. This versatility is another advantage of the present invention.

[0070] As mentioned above, one preferred static mixer for which the apparatus is adapted to operate is a jet impingement reactor. The function of a jet impingement reactor involves injecting two fluid streams, e.g., a first stream of a first liquid to be mixed and a second stream of a second liquid, into a reactor cavity through a nozzle so that the streams collide in a turbulent mixing zone. Preferably, the first and second streams are injected from diametrically opposed positions in the reactor, preferably so that the streams collide substantially head-on, i.e., at an angle of substantially about 180°. Examples of jet impingement reactors include confined impinging jet (CIJ) reactors and microjet reactors (MJR).

[0071] In one preferred embodiment, the static mixer is a jet impingement reactor having a mixing chamber defined by the inner surface of a mixing chamber wall, the mixing chamber having a substantially spheroidal overall shape, and the mixing chamber preferably comprises: a first fluid inlet and a second fluid inlet, the first fluid inlet and the second fluid inlet being arranged at opposite positions on a first central axis of the reaction mixing chamber, e.g., facing each other, and each of the first fluid inlet and the second fluid inlet comprising a nozzle; a fluid outlet disposed at a third location, the third location being on a second central axis of the chamber, the second central axis being perpendicular to the first central axis; and Equipped with The distance between the nozzle of the first fluid inlet and the nozzle of the second fluid inlet is equal to or less than the diameter of the mixing chamber along the first central axis. Furthermore, such a jet impingement reactor can have further features as described in International Patent Application Publication No. WO 2023 / 025736, which is incorporated herein by reference.

[0072] When using an apparatus for operating a static mixer, it is usually necessary to provide conduits to provide fluid communication between the interior space of the flexible container and each inlet of the static mixer. Specifically, the conduit of the first supply module provides a fluid connection between the interior space of the flexible container holding a first liquid and the first inlet, and the conduit of the second supply module provides a fluid connection between the interior space of the flexible container holding a second liquid and the second inlet of the static mixer. The conduits can include or represent tubes that can be flexible, such as tubes made of an elastomeric (polymeric) material, or rigid, such as metal tubes.

[0073] In one embodiment, the conduits associated with the first and / or second supply modules include means for, preferably reversibly, blocking fluid communication between the interior space of the flexible container and the respective inlets of the static mixer. Optionally, the means is a valve. If the conduits are flexible tubing, such as plastic tubing, the fluid communication may alternatively be first blocked by a tubing clamp, in which case the respective fluid connection is created by opening or removing the clamp, thereby allowing fluid to flow from the flexible container to the static mixer when the substrate chamber is pressurized.

[0074] In one embodiment, there is no means for interrupting the fluid connection disposed in the conduit associated with the first and / or second supply modules. The conduit may, for example, include a connection achieved by engagement of a connecting piece initially introduced as part of the outlet of the respective flexible container with a complementary connecting piece in a downstream portion of the conduit leading to the respective inlet of the static mixing device.

[0075] To initiate pressurization of the substrate chamber, the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber is opened or removed, the connector is opened or released, and the fluid connection is created. As used herein, a connector is any part, conduit, pipe, or tube that allows pressurized gas to flow from the pressure reservoir chamber to or between the pressurizable substrate chamber. The means may be a clamp or a valve if the connector material is flexible. In a preferred embodiment, the means is a valve.

[0076] The pressure reservoir chamber may be designed to be relatively large to achieve sufficient pressure within the pressurizable substrate chamber without pressurizing the pressure reservoir chamber too high, for example, by opening a valve on the connector, before fluidly connecting the two chambers. For example, the pressure reservoir chamber may have a larger volume than the substrate chamber with which it is fluidly connected. In this context, volume should be understood as the internal volume of the respective chamber. Optionally, the volume of the pressure reservoir chamber may be twice the volume of the respective substrate chamber, or even larger. In some embodiments, the ratio of the volume of the pressure reservoir chamber to the volume of the pressurizable substrate chamber is at least about 2, or at least about 3, or at least about 4, particularly at least about 5, and optionally at least about 10. In other preferred embodiments, this ratio is in the range of about 2 to about 10. In further embodiments, this ratio is in the range of about 10 to about 100, e.g., about 20 to about 50. For example, the pressure reservoir chamber may have an internal volume of about 2 to about 10 liters, or about 5 to about 30 liters, and the corresponding substrate chamber may have an internal volume of about 0.3 to about 3 liters, or about 0.3 to about 1 liter. In further embodiments, the pressure reservoir chamber has a substantially cylindrical or cylindrical overall shape.

[0077] As will be understood by those skilled in the art, preparing an apparatus for producing a batch of liquid mixture (i.e., a third liquid) by mixing two liquid substrates (i.e., a first liquid and a second liquid) involves filling the pressure reservoir chambers of the first and second supply modules with pressurized gas. The amount of pressurized gas used for this purpose, or the pressure the pressure reservoir chambers should have at the start of batch production, is selected with consideration of the volume of that chamber and the corresponding pressurizable substrate chamber, taking into account the viscosity of the respective liquids, the flow resistance of the flow paths, and the desired flow rate. Typical initial pressures in the pressure reservoir chambers may be, for example, in the range of about 2 bar to 20 bar. Other pressures may also be used depending on the selection of pressurized gas used.

[0078] Additionally, the pressure may be selected to force the first and second liquids from the containers into their respective conduits and into the static mixer at a flow rate ranging from about 10 to about 200 ml / min.

[0079] Generally, any technically suitable pressurized gas or gas mixture can be used. Examples of potentially useful gases include, but are not limited to, inert gases such as nitrogen, oxygen, air, helium, carbon dioxide, nitrous oxide, diethyl ether, n-butane, isobutane, heptafluoropropane, tetrafluoroethane, dichlorodifluoromethane, or propane. Preferred gases include nitrogen, air, and carbon dioxide. Pressurized air is particularly preferred.

[0080] According to further preferred embodiments, the dimensions of each pressurizable substrate chamber are selected so that its internal volume (or internal space) is slightly larger than the total or external volume of the flexible container to be held. For example, the flexible container, at its initial dimensions, i.e., when filled with liquid substrate and before fluid communication with the static mixer is established or when inserted into the pressurizable substrate chamber, can fill at least about 30%, or at least about 50%, of the total internal volume of each substrate chamber. As used herein, the total internal volume of the chamber, as used in these percentages, should be understood as the total internal volume of the substrate chamber when empty. In further preferred embodiments, the initial total volume of the flexible container is at least about 60%, or at least about 70%, or in the range of about 60% to about 95% of the total internal volume of the pressurizable substrate chamber.

[0081] The dimensions of the two flexible containers holding the first and second liquids, respectively, may also differ from each other. This arises from the somewhat typical situation where one of the two liquids must be dispensed at a greater volume or flow rate than the other to obtain the desired product. Therefore, in another preferred embodiment, the dimensions of the two substrate chambers holding the flexible containers are also different from each other. This applies in particular to the internal volumes of the chambers, which may differ from each other. In one embodiment, the difference between the internal volumes of the substrate chambers of the first and second supply modules is a factor ranging from about 1.5 to about 10, or from about 2 to about 5. In this context, internal volume refers to the total internal volume of the respective chambers, i.e., the total internal volume when empty.

[0082] As mentioned above, flexible containers are used to hold the liquid substrate, i.e., the first and second liquids that are mixed to form the third liquid using the device of the present invention. Preferably, the containers are flexible enough to be at least partially collapsible. In other words, their internal volume can change substantially depending on the shape of the container wall at a given moment.

[0083] In a further preferred embodiment, the flexible container is a flexible plastic bag, similar to an infusion bag. The bag is preferably disposable. Preferably, the bag contains the first or second liquid in sterile form. As noted above, one advantage of the present invention is that it enables easy, rapid, and versatile aseptic production of small batches, which is substantially facilitated by the use of sterile raw materials and disposable containers that do not need to be cleaned or sterilized between two batch production campaigns.

[0084] In a further preferred embodiment, the apparatus is configured and / or oriented so that the liquid processed in or obtained by use of the apparatus has an overall flow direction upward, i.e., against gravity. For example, conduits for providing fluid communication between the interior space of the flexible container and the first and second inlets of the static mixer may be oriented such that their downstream ends are higher than their upstream ends. For the avoidance of doubt, the upstream end of the conduit is the end connected to the flexible container, and the downstream end of the conduit is connected to the first or second inlet of the static mixer. In this context, a higher position means a position higher than a relatively low position relative to the horizontal axis of the apparatus in its normal operating orientation.

[0085] Similarly, the static mixing device or static mixer may be oriented so that its outlet is higher than its inlet. This orientation of the static mixer, combined with the aforementioned orientation of the conduit, is also advantageous. In this case, the overall liquid flow from the flexible container to the outlet of the static mixer is upward. This flow direction has been found to reduce or even avoid the entrapment of air bubbles in the liquid mixture representing the product, i.e., the third liquid, especially when the product contains a surface-active compound, as in the case of producing liposomes or lipid nanoparticles described below in the context of the method according to the present invention. Optionally, the inlet of the product container may also be positioned higher than the outlet of the static mixer.

[0086] According to a further preferred embodiment, the device is fully adapted for aseptic processing. This includes providing all substrate- or product-contacting surfaces in a sterile form. This further includes the use of a sterile container for receiving the product prepared using the device, i.e., the third liquid. Therefore, the outlet of the static mixer is preferably fluidly connectable or actually connected to a container for receiving the third liquid, which container is optionally a flexible container. This connection may include sterile tubing between the outlet of the static mixer and the container. This product container is also referred to as a first container for receiving the third liquid. To avoid any doubt, the term "first container" does not imply any processing sequence; for example, if the device is configured with a first and second container for receiving the third liquid, the process may be performed such that the second container receives a certain amount of the third liquid before the first container.

[0087] Thus, the product container or first container may be a sterile flexible bag aseptically connected to the outlet. In this context, aseptically connected means that the third liquid is connected tightly enough to prevent contamination with, for example, airborne contaminants, particularly microbiological contaminants. Those skilled in the art will appreciate that these preferences further facilitate aseptic small-batch production in that the product can be easily removed from the device, for example, by clamping the connecting tubing to seal the product container and then disconnecting the container upstream of the clamp.

[0088] Optionally, the connection between the mixing device outlet and the product container may include a filter, such as a sterile filter having a filter membrane with an effective pore size of 0.2 μm or 0.22 μm, capable of removing microbiological contaminants from the third liquid. Of course, such filters may also be used in other locations within the device, such as the inlet for pressurized gas or in a conduit providing fluid communication between the interior space of the flexible container holding the liquid substrate and the corresponding inlet of the static mixer. Such a filter may also be located at the inlet of the flexible container, allowing for aseptic filling of the substrate into the container. These filters may be removable under sterile conditions so that they can be removed when the container is placed in the substrate chamber.

[0089] In a further preferred embodiment, the device comprises means for reversibly interrupting the fluid connection between the outlet of the static mixer and the first container for receiving the third liquid. For example, a pinch valve is suitable as the means for interrupting the fluid connection. Both electrically and pneumatically operated pinch valves are suitable in the context of implementing the present invention.

[0090] In a further embodiment, the first container for receiving the third liquid comprises a first and a second fluid inlet, the first fluid inlet being connectable to the outlet of the static mixer, and the second fluid inlet being adapted to fill the first container with a quantity of liquid diluent. In this context, it is clear that "connectable" means fluidly connectable. This embodiment is advantageous when it is desired to modify the composition of the third liquid after discharge from the static mixer by adding one or more additional components. Typically, these one or more additional components are added in liquid form, i.e., in the form of a liquid diluent. The second fluid inlet allows for the introduction of such a liquid diluent either before or after the first container receives the third liquid.

[0091] It should be noted that the liquid diluent may have other functions, or may have an important function entirely different from diluting the third liquid. For example, the liquid diluent may serve to adjust the pH of the product to a specific value. For this purpose, it may contain a pH adjuster, such as an acid, base, buffer salt, or buffer system. Alternatively or additionally, the liquid diluent may contain a tonicity adjuster, such as sodium chloride, a sugar, a sugar alcohol, or any other osmotically active compound. The liquid diluent may also contain a lyophilization aid, such as a sugar or sugar alcohol. Examples of suitable lyophilization aids include, but are not limited to, trehalose, sucrose, glucose, mannitol, and sorbitol. Any combination of pH adjusters, tonicity adjusters, and / or lyophilization aids may also be used.

[0092] To facilitate the aseptic introduction of a liquid diluent into the container through one of the fluid inlets, an in-line sterile filtration means can be disposed upstream of each fluid inlet. Such a sterile filtration means typically comprises a filter housing and a filter, such as a filter membrane with an effective pore size of, for example, 0.2 or 0.22 μm. After aseptically introducing the liquid into the container, the sterile filtration means can be aseptically removed by melt-clamping or melt-sealing the inlet upstream of the filtration means while simultaneously removing the filtration means.

[0093] In addition to the product container or the first container, there may also be a third liquid, i.e., a second container for receiving a quantity of the third liquid. In other words, the outlet of the static mixer may be fluidly connectable to the second container for receiving the third liquid. Optionally, the second container is also a flexible container, such as a bag-like container. The outlet of the static mixer may be fluidly connected to both the first and second containers, for example, via a T-joint.

[0094] One particular advantage of such a configuration is that the second vessel can be used for product disposal, such as product obtained at the very beginning of the operation of the apparatus or at the end of a batch process. This can be particularly useful when there is a risk that the product will achieve its target specifications only after the early stages of the batch process or before the stage of the batch process.

[0095] In order to use both the first and second containers without interrupting the mixing process, means can be arranged for reversibly interrupting the fluid connection between the outlet of the static mixer and the second container for receiving the third liquid. In one preferred embodiment, the apparatus is configured to operate with the first and second containers for receiving the third liquid in fluid connection with the outlet of the static mixer, and the means for reversibly interrupting the fluid connection is arranged upstream of each of the two containers and downstream of a location where the fluid paths to the two containers branch, for example, downstream of a location where a T-shaped fitting is used.

[0096] Again, a pinch valve is suitable as a means for blocking the fluid connection between the static mixer outlet and the second container for receiving the product, which may represent a waste container. Both electrically and pneumatically actuated pinch valves are suitable in the context of practicing the present invention.

[0097] In one embodiment, the device is configured to operate the pinch valve in response to a parameter measured in the third liquid produced by the mixing process. For example, if the third liquid is a liposome dispersion or a dispersion containing lipid nanoparticles, the important product parameter can be represented by the particle size measured in the third liquid. Therefore, the device can be equipped with a means for in-line particle size measurement of the third liquid. In this context, in-line means that the measurement is performed in real time during the mixing process without interrupting the process or the flow of the liquid and without the need to withdraw a sample of the third liquid for measurement purposes.

[0098] In one embodiment, the in-line particle size measuring means is located in a downstream portion of the static mixer, at the outlet of the static mixer, or downstream of the outlet of the static mixer, in fluid connection with the outlet of the static mixer, which should be understood such that at least a sensor that senses a signal from which particle size is determined is located at one of the specified locations.

[0099] The in-line particle size measuring means may include a transparent sensing or measuring window to allow transmission of an optical signal from the third liquid to a sensor that is not in fluid communication with the third liquid. For example, the transparent measuring window may be provided in the form of a glass or plastic capillary tube.

[0100] These features allow for in-line particle size measurement of the third liquid by optical methods such as light scattering or laser diffraction. Sensing a signal emitted or returned by the third liquid while it is flowing through the device requires the presence of a transparent portion or window in one of the walls of the structure through which the third liquid flows. Such a transparent portion or window may be located downstream of the static mixer, at the static mixer outlet, or downstream of the static mixer outlet, such as near the static mixer outlet, in fluid communication with the static mixer outlet. If two containers for receiving the third liquid are used, it may preferably be located upstream of the location where the fluid paths to these two containers separate. For example, a capillary tube made of glass or transparent plastic can form part of the structure that directs the third liquid from the static mixer to the product container. Such a capillary tube may be part of the mixer itself. Indeed, according to one embodiment of the present invention, the static mixing device is made of glass or plastic. Transparent glass or plastic static mixing devices are also preferred. In this context, transparent means sufficiently transparent to allow transmission of an optical signal for particle size measurement.

[0101] Those skilled in the art will understand that the guidance provided herein referring to the location of the in-line particle size measurement means primarily refers to the location where the measurement means, e.g., a sensor, interacts with the third fluid. Other components of the measurement means, such as a laser beam generator, may be located at specific distances relative to the designated location.

[0102] In one embodiment, the transparent region, e.g., a glass or plastic capillary, has an inner surface coated with a layer of a material that has low protein or nucleic acid binding. Alternatively, the capillary may be made of a type or grade of glass or plastic that has low protein or nucleic acid binding.

[0103] Particularly preferred are static mixers made of plastic. In this context, the term "plastic" should be understood to mean that the mixer is made predominantly or primarily of polymeric material, but does not exclude the presence of certain amounts of non-polymeric materials. Furthermore, the primary material from which the static mixer is made can represent a (e.g., thermoplastic) polymeric material that further contains one or more additives, such as glass fibers, ceramic fillers, plasticizers, antioxidants, colorants, antibacterial agents, antistatic agents, UV stabilizers, flame retardants, etc.

[0104] As mentioned above, it may be desirable to position and orient the apparatus components so that the primary overall direction of liquid flow during the mixing process is upward, i.e., from lower to higher. Thus, in one embodiment, the first and / or second containers for receiving the third liquid may also be positioned higher than the outlet of the static mixer to achieve an upward liquid flow downstream of the static mixer. More precisely, the inlets of the first and / or second containers may be positioned higher than the outlet of the static mixer to achieve an upward liquid flow direction from the static mixer to the product and / or waste containers.

[0105] Because the third liquid, or the mixture obtained by combining the third liquid with the liquid diluent, may require further processing, it may be advantageous to use the first and / or second containers that further comprise an outlet for withdrawing the liquid, this being particularly true for the first container.

[0106] In a further aspect, the present invention provides an apparatus for mixing a first liquid and a second liquid, the apparatus comprising: (a) a static mixer; (b) a first supply module for supplying the first liquid; and (c) a second supply module for supplying the second liquid to the static mixer. The static mixer itself is characterized by having a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from the mixing of the first liquid and the second liquid. Further, each of the first and second supply modules independently comprises: (i) a substrate chamber for holding a flexible container having an interior space for holding a first liquid or a second liquid, respectively; (ii) a conduit for providing fluid communication between the interior space of the flexible container and the first or second inlet of the static mixer, respectively; (iii) a pressure reservoir chamber for holding pressurized gas, having an inlet and an outlet for pressurized gas; (iv) a pressure amplifier disposed upstream of the pressure reservoir chamber, the pressure amplifier comprising an inlet reversibly connectable to a pressurized gas source and an outlet for pressurized gas fluidly connected to the inlet of the pressure reservoir chamber; and (v) a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber, the connector comprising means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber, the fluid communication being for enabling flow of pressurized gas from the pressure reservoir chamber to the substrate chamber so as to apply pressure to an outer surface of the flexible container and force the first or second liquid from the container into the conduit. The means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber has an open state and a closed state, and the connector and the means are further configured to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the substrate chamber upon changing the state of the means from the closed state to the open state.

[0107] In some embodiments, the apparatus disclosed above is adapted to carry out the methods described below.

[0108] In a further aspect, the present invention provides a method for mixing a first liquid and a second liquid, such as to obtain a third liquid, the method being essentially characterized in that an apparatus as described above is used for mixing the liquids.

[0109] In a related embodiment, a method of mixing a first liquid and a second liquid includes the steps of: (aa) providing a first flexible container holding a first liquid, the first flexible container being contained within a first pressurizable substrate chamber; (bb) providing a second flexible container holding a second liquid, the second flexible container being contained within a second pressurizable substrate chamber; and (cc) providing a second flexible container having: (i) a first inlet for receiving the first liquid; (ii) providing a static mixer having a second inlet for receiving a second liquid, and (iii) an outlet for discharging a third liquid resulting from the mixing of the first and second liquids, (dd) independently pressurizing the first and second pressurizable substrate chambers with pressurized gas that applies pressure to the outer surfaces of the first and second flexible containers, respectively, to force the first and second liquids into the static mixer to mix the first and second liquids, and (ee) collecting the third liquid. In this context, independently means that each pressurizable substrate chamber is independently pressurized with the pressurized gas, such that the gas pressure may differ between the two pressurizable substrate chambers.

[0110] As will be appreciated by those skilled in the art, steps (aa), (bb) and (cc) may be performed in any order, and optionally simultaneously. Steps (dd) and (ee), which follow steps (aa), (bb) and (cc), may be performed essentially simultaneously or substantially overlapping in time, in that step (ee) may begin and end slightly after step (dd) begins and ends.

[0111] Step (ee) may advantageously comprise collecting at least a portion of the third liquid in a first container for receiving the third liquid, said first container being arranged in fluid connection with the outlet of the static mixer.

[0112] As described above in the context of the description of the apparatus features, step (dd) can include a first substep characterized by a sudden or rapid increase in the pressure in each of the first and second pressurizable substrate chambers. In preferred embodiments, the pressure in the substrate chambers increases from the initial pressure to a target process pressure (also referred to as a maximum process pressure) within less than about 2 seconds and / or less than about 5% of the total time required to mix the first and second liquids. Again, the target process pressure is selected independently for each pressurizable substrate chamber.

[0113] In some preferred embodiments, the first substep includes changing the state of the means for reversibly blocking fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber from a closed state to an open state to create pressure equilibrium between each pressure reservoir chamber and the respective pressurizable substrate chamber within a period of about 2 seconds or less.

[0114] As noted above, the abrupt nature of this pressure increase can also be explained in relation to the duration of the overlapping step (ee) of collecting the third liquid. In preferred embodiments, the duration of the abrupt pressure increase in each of the first and second pressurizable substrate chambers to the initial equilibrium pressure is less than about 10% of the duration of step (ee), or less than about 5%, less than about 2%, or even less than about 1% of the duration of step (ee). As will be appreciated, the duration of step (ee) substantially reflects the time it takes for the third liquid to actually be produced within the static mixing device from the mixing of the first and second liquids.

[0115] In further related embodiments, the initial pressure is about ambient pressure and the target process pressure is in the range of greater than 1 bar to 16 bar. Also preferred are target process pressures in the range of about 2 to 12 bar, such as about 2 to 5 bar, or 4 to 8 bar, or 3 to 10 bar, respectively.

[0116] As mentioned above, pressurization of the substrate chambers is achieved by pressurized gas exerting pressure on the exterior surface of each pressurizable substrate chamber. In a preferred embodiment, this pressurized gas is provided by a pressure reservoir chamber, as already explained. In this case, the target process pressure in the substrate chamber also corresponds to the initial equilibrium pressure reached upon pressure equilibrium between the pressure reservoir chamber and the respective substrate chamber.

[0117] The pressurizing step (dd) can further include a second sub-step, characterized in that a target process pressure is maintained in each of the first and second pressurizable substrate chambers for a selected period of time. In some related embodiments, the target process pressure is maintained in each of the first and second pressurizable substrate chambers for at least 50% of the total time required to mix the first and second liquids. In even more preferred embodiments, the target process pressure is maintained for at least 80% or at least 90% of the total time required to mix the first and second liquids.

[0118] In this context, reaching a target process pressure (as in the first substep of step (dd)) or maintaining a target process pressure (as in the second substep of step (dd)) should be understood to allow for technically acceptable tolerances. For example, the target process pressure can be considered to be reached or maintained if the actual pressure during the method is within about ±10%, particularly about ±5%, of the preselected target process pressure. For example, if the preselected target process pressure of a pressurizable substrate chamber is 6.0 bar, the pressure will be maintained if the actual pressure remains within the range of 5.7 to 6.3 bar. It should be noted that the inventors have found that much more accurate pressure control is possible when using the apparatus described herein above to perform the method.

[0119] As described above, according to some preferred embodiments, after the maximum or target process pressure is reached in each of the first and second pressurizable substrate chambers, pressures that do not deviate by more than 10% or 5% from the respective maximum process pressures are maintained in the first and second pressurizable substrate chambers until the desired volume of the third liquid is discharged from the static mixer. Also preferred are embodiments in which the pressure in each pressurizable substrate chamber is maintained at or below about 3% of the maximum process pressure (or target process pressure, or initial equilibrium pressure). The end of the mixing process is achieved when the desired amount of the third liquid is produced. Typically, this coincides with the point at which all or a majority of the first and / or second liquids, such as at least about 95% of the first and / or second fluids initially provided in the first and / or second flexible containers, have been expelled into the static mixer.

[0120] In other words, the maximum process pressure or initial equilibrium pressure is also the target pressure maintained after equilibrium throughout the mixing process and preparation of a desired volume or batch of the third liquid. To terminate the mixing process, the means for reversibly blocking fluid communication between each pressure reservoir chamber and each pressurizable substrate chamber may simply be changed from its open state back to its closed state.

[0121] Thus, in a related embodiment, each of the first and second pressurizable substrate chambers is connected to a first and second pressure reservoir chamber for holding pressurized gas by a connector for providing fluid communication between the pressure reservoir chamber and the respective pressurizable substrate chamber, and means having an open state and a closed state are arranged for reversibly blocking fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber, and prior to performing the pressurization step (dd), each pressure reservoir chamber is in a pressurized state such that its pressure is about 2 to 20 times higher than the pressure of the respective pressurizable substrate chamber, and the means for reversibly blocking fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber is in a closed state.

[0122] For the avoidance of doubt, the first pressurizable substrate chamber is connected to the first pressure reservoir chamber via a first connector, and the second pressurizable substrate chamber is connected to the second pressure reservoir chamber via a second connector. A first means is configured to reversibly block fluid communication between the first pressure reservoir chamber and the first pressurizable substrate chamber, and this means may be associated with the first connector. A second means is configured to reversibly block fluid communication between the second pressure reservoir chamber and the second pressurizable substrate chamber, and this means may be associated with the second connector. There is no fluid communication between the first pressurizable substrate chamber or the first pressure reservoir chamber and the second pressurizable substrate chamber or the second pressure reservoir chamber. Furthermore, there is no fluid contact between the pressurized gas and the first or second liquid. When used in this context, the term "respectively" means "corresponding," "connected," or "associated."

[0123] In a further related embodiment, the pressurizing step (dd) comprises flowing essentially all of the first and second liquids from their respective flexible containers through a static mixer to produce a third liquid, e.g., over a period of at least about 5 seconds, preferably at least 10 seconds, e.g., from about 10 seconds to about 15 minutes. Those skilled in the art will appreciate that the duration of liquid flow will vary depending not only on flow rate but also on batch size.

[0124] The function of the pressure supply module, as described above, is important for maintaining the gas pressure in the pressure reservoir chamber and the pressurizable substrate chamber after the initial equilibration in the first substep of the pressurization step (dd). During operation, the gas inlet of the pressure supply module preferably remains connected to a pressurized gas source. Through the action of at least one pressure amplifier disposed in the flow path of the pressure supply module and through control by the pressure control circuit, additional pressurized gas is delivered to the pressure reservoir chamber, such as to accurately maintain the target process pressure. As described above, the accuracy of pressure control can be further improved by using an additional pressure reservoir disposed in the flow path of the pressure supply module.

[0125] As described above, in some preferred embodiments, the device includes a conduit for providing fluid communication between the interior space of the flexible container and the first or second inlet of the static mixer, the conduit having an upstream end connected to the flexible container and a downstream end connected to the first or second inlet of the static mixer, the downstream end being oriented higher than the upstream end. In other words, at least the flexible container and the static mixer are positioned so that liquid flow occurs in an upward direction.

[0126] Regarding optional or preferred features of the static mixer, chamber, and other apparatus features, reference is made to the disclosures above. In other words, the embodiments and preferences provided for the apparatus provided in accordance with the present invention are also applicable to the present method, and as a result, preferred embodiments of the present method are characterized by the use of preferred apparatus features to perform mixing of two fluids. Furthermore, optional and preferred process features are specifically described in the context of the apparatus, as necessary to explain their function.

[0127] For example, in some embodiments, the method can further include filling the first container with a liquid diluent before or after the third liquid is received in the first container. To fill the container with the liquid diluent, the container can at least initially include an additional inlet and a means for in-line sterile filtration associated with the additional inlet. Thus, the method of the present invention can preferably include filling the first container with the liquid diluent through the additional inlet of the container and the associated sterile filter. The liquid diluent is preferably an aqueous liquid composition. It may further include, for example, a pH adjuster, a tonicity adjuster, a lyophilization aid, or any combination thereof.

[0128] Whether or not the third liquid collected in the first (or product) vessel downstream of the static mixer is combined with a liquid diluent, further processing steps may follow, for example, to further purify or characterize the product, or to make the product more stable for easier handling, storage, and shipping. Preferably, such further processing is also carried out under sterile conditions.

[0129] For example, further processing can include tangential flow filtration, chromatography, freezing, or lyophilization of the third liquid or the mixture of the third liquid and the liquid diluent collected according to step (ee). Tangential flow filtration or chromatography can be useful for product concentration purposes, in the sense that the concentration of any particles generated by the mixing of the first liquid with the second liquid, such as liposomes or lipid nanoparticles, is increased, or for removing or reducing the concentration of certain solutes, such as free molecules of biologically active components, i.e., molecules not incorporated into liposomes or lipid nanoparticles. Freezing or lyophilization can be useful for converting the third liquid or the liquid mixture of the third liquid and the liquid diluent into a solid composition that can typically be stored for long periods of time. These processes or process steps themselves and their implementation are generally known to those skilled in the art.

[0130] As mentioned above, the apparatus can include a means for in-line particle size measurement of the third liquid. Therefore, a preferred embodiment of the method of the present invention utilizes this feature of the apparatus to perform an in-line particle size measurement on the third liquid before it is received by the first container. In this way, it can be ensured that the important product parameter, i.e., the target particle size, is actually achieved by the product in the form of the third liquid when it is collected in the product container. This embodiment is particularly relevant for the production of small batches of liquid products containing liposomes or lipid nanoparticles.

[0131] The in-line particle size measurement step may also be advantageous when working with a further preferred embodiment of the apparatus in which a second container for receiving the third liquid is present. Regarding the method features, this means that step (ee) may comprise collecting at least a further portion of the third liquid in a second container for receiving the third liquid, said second container being arranged in fluid connection with the outlet of the static mixer. Collecting one or more portions of the third liquid in the second container can be performed by interrupting the fluid connection between the outlet of the static mixer and the first container for receiving the third liquid.

[0132] For example, the first portion of the third liquid may be directed into a second vessel that can function as a waste vessel when it leaves the static mixer through its outlet, followed by subsequent portions of the third liquid directed into the first vessel that can function as a product vessel. Optionally, further subsequent portions of the third liquid may be directed back into the second vessel, e.g., toward the end of batch production. In this manner, it is possible to eliminate material produced during the beginning or subsequent stages of the process and thus selectively collect the portion of the third liquid that best represents the target product quality.

[0133] In another preferred embodiment, the apparatus features both an in-line particle size measurement means and a first and second container for receiving a third liquid, and also exhibits means (such as pinch valves) for reversibly blocking the fluid connection between the outlet of the static mixer and each of the first and second containers. Furthermore, this embodiment also provides means for controlling, for example, the pinch valves to block or open the respective fluid connections depending on the particle size measurement.

[0134] This arrangement and configuration can be used for the method of the present invention such that in-line particle size measurements are made during steps (dd) and (ee) when the mixing process is ongoing and a third liquid is produced and discharged from the static mixing device through its outlet. Depending on the measured particle size, the pinch valve is operated to open the fluid connection between the outlet and a first container used as a product container while blocking the fluid connection to the second container, or to open the fluid connection between the outlet and a second container used as a waste container while blocking the fluid connection to the first container.

[0135] In other words, according to this embodiment, the method includes the steps of performing inline particle size measurement on the third liquid during steps (dd) and (ee), and if the inline particle size measurement gives an undesirable result, collecting the third liquid in a second container for receiving the third liquid by blocking the fluid connection between the outlet of the static mixer and the first container for receiving the third liquid, and if the inline particle size measurement gives a desirable result, collecting the third liquid in the first container for receiving the third liquid by blocking the fluid connection between the outlet of the static mixer and the second container for receiving the third liquid.

[0136] For example, in the initial stages of steps (dd) and (ee), the particle size measured in the third liquid may not yet meet a preset target criterion, such that a first quantity of the third liquid should be directed to a waste container, and this target criterion is achieved by the respective settings of the pinch valves. Once the desired particle size characteristics are reached, the valve settings are changed to direct the third liquid into a product container.

[0137] The method of the present invention may be used to prepare particles, such as microparticles or nanoparticles, of poorly water-soluble compounds, for example, by flash precipitation. For this purpose, one of the liquid substrates, e.g., the first liquid, may comprise an organic solution of the poorly soluble compound in a water-miscible solvent or solvent mixture, and the second liquid may represent an aqueous solution, such that mixing of the two liquids according to the present invention results in precipitation of the poorly soluble compound in the form of microparticles or, preferably, nanoparticles. Similarly, ionizable poorly soluble compounds can be precipitated as microparticles or nanoparticles using the method of the present invention by providing a first liquid that is an aqueous solution of the poorly soluble compound with a pH at which the compound is primarily ionized and soluble, and a second liquid that represents an aqueous solution that acts as a poor solvent for the poorly soluble compound due to its pH.

[0138] In some embodiments, the poorly soluble compound is a biologically active agent, such as a drug substance, a vaccine, or a diagnostic compound. Optionally, the first and / or second liquid may comprise one or more polymers, and the process may be carried out to produce polymer particles comprising the biologically active component.

[0139] In a further preferred embodiment of the method of the present invention, the first liquid comprises an organic solution of one or more lipids and the second liquid comprises an aqueous solution of a biologically active agent, such that lipid nanoparticles are formed upon mixing of the first and second liquids and the biologically active agent is associated with and / or encapsulated within the lipid nanoparticles.

[0140] Preferably, two or more lipids are used in the first liquid.For example, an organic (e.g., ethanol) solution of a combination of lipids known to be useful for producing lipid nanoparticles (LNPs) can be used.These lipids can include cationic or cationizable lipids, PEGylated lipids, structural lipids, and cholesterol.

[0141] As used herein, unless otherwise specified by the context, cationic lipid refers to the lipid that contains positive charge in aqueous environment at any pH, for example, the lipid that has quaternary nitrogen atom (i.e., ammonium moiety), while cationizable lipid refers to the lipid that contains positive charge only in aqueous environment at neutral or acidic pH, for example, the lipid that shows primary, secondary or tertiary amine.As used herein, PEG refers to polyethylene glycol, and PEGylated lipid refers to the lipid that is combined with PEG moiety.Structural lipid is preferably non-PEGylated zwitterionic lipid.

[0142] More preferably, the first liquid comprises the water-miscible solvent that lipid is dissolved in.The water-miscible solvent is preferably selected from ethanol, methanol, acetone, acetonitrile, acetic acid, formic acid, trifluoroacetic acid, acetaldehyde, butanol, ethylamine, and any combination thereof.A particularly preferred water-miscible solvent is ethanol.

[0143] In a more preferred embodiment, the first liquid composition essentially consists of a solution of one or more lipids in a water-miscible solvent selected from ethanol, methanol, acetone, acetonitrile, acetic acid, formic acid, trifluoroacetic acid, acetaldehyde, butanol, ethylamine, and any combination thereof. In certain embodiments, the first liquid composition essentially consists of a solution of one or more lipids in ethanol.

[0144] In some embodiments, the first liquid composition comprises: 40 to 60 mol % of a cationic lipid or a cationizable lipid, -0.5 to 2 mol% PEGylated lipid, 5 to 20 mol% of a non-PEGylated zwitterionic lipid, and - 30 to 50 mol % cholesterol, Here, the percentages are based on the total lipid content in the first liquid.

[0145] When this method is used to prepare lipid nanoparticles, it is more preferred that biologically active agent is oligonucleotide or polynucleotide.According to another preferred embodiment, oligonucleotide or polynucleotide is optionally modified mRNA molecule, and comprises the nucleic acid sequence that codes antigen, particularly tumor antigen, virus antigen, bacterial antigen, fungus antigen or protozoan antigen.

[0146] A particular advantage of the method according to the present invention is that it allows for the rapid and flexible production of small batches under aseptic conditions, preferably using the apparatus described herein. After the production of a batch, the apparatus is easily reconfigured and prepared for the production of another batch of the same or a different material. No pumps are involved in the flow and mixing of the first and second liquids to form the third liquid. There is no or very little structure in direct contact with the product, and these can be easily replaced. Parts that require sterilization before the production of a batch are easily sterilized.

[0147] The method of the present invention can further include a step of sterilizing the static mixer before step (dd) is performed, i.e., before pressurizing the first and second pressurizable chambers to force the first and second liquids into the static mixer and cause mixing of the two liquid substrates. Sterilization can be carried out by any sterilization method compatible with the material from which the mixing device is made. For example, sterilization can be carried out using steam. In one preferred embodiment, sterilization is carried out by gamma irradiation. This also applies when the mixing device is made of glass or plastic, especially when the glass or plastic mixing device is a jet impingement reactor as described in more detail above.

[0148] In a further aspect, the present invention provides the use of the above-described apparatus or method for the production of lipid nanoparticles.

[0149] Detailed Description of the Drawings The drawings included in this disclosure illustrate specific embodiments of devices provided in accordance with the present invention. None of the drawings are to scale.

[0150] FIG. 1 is a flow chart illustrating a pneumatic circuit of one embodiment of an apparatus for operating a static mixer for mixing a first liquid and a second liquid according to the present disclosure. The apparatus includes a first supply module (11) for supplying a first liquid to a first inlet of the static mixer (not shown), a second supply module (12) for supplying a second liquid to a second inlet of the static mixer, and a pressure supply module (2a) disposed upstream of the first supply module (11) and the second supply module (12). Solid arrows in the flow chart indicate the direction of flow of pressurized gas (e.g., pressurized air). Dashed lines enclose components included in and / or contributing to the function of the pressure supply module (2a) and components included in and / or contributing to the function of the first supply module (11) and the second supply module (12). Dotted lines, drawn as links between two features, indicate electrical signal communication (e.g., command, feedback, or both).

[0151] The pressure supply module (2a) includes a gas inlet (3) for receiving pressurized gas from an upstream gas source (not shown). The pressurized gas is directed at a specific pressure to a pressure amplifier (7), which outputs gas having a higher pressure than the gas received from the gas inlet (3). A pressure reservoir chamber (8) is located in the flow path downstream of the pressure amplifier (7). The pressurized gas held in this reservoir (8) can flow further downstream to the first supply module (11) and the second supply module (12) via a valve (21) and a flow path divider (6) that diverts the flow of pressurized gas to the first supply module (11) and the second supply module (12). Each of the two flow path portions downstream of the flow path divider (6) leading to the two supply modules (11, 12) is independently equipped with an electric pressure regulator (24a, 24b), also referred to as an electronic regulator, for regulating the pressure of the gas conducted to the pressure supply module via the valves (22a, 22b) and gas outlets (4a, 4b). As shown in the figure, the pressure supply module (2a) includes a central microcontroller (5) adapted to control the opening and closing of at least the valves (21, 22a, 22b) and the operation of the electric pressure regulators (24a, 24b). Furthermore, the microcontroller (5) is adapted to receive signals from pressure sensors (16a, 16b) communicating with the pressure reservoir chambers (13a, 13b) of the first supply module (11) and the second supply module (12), respectively. As shown, pressurized gas from a pressure supply module (2a) flows into a first supply module (11) and a second supply module (12) via a first gas outlet (4a) and a second gas outlet (4b), and then flows into pressure reservoir chambers (13a, 13b) located at the upstream ends of the respective supply modules (11, 12), as in this case. Valves (23a, 23b) located between the pressure reservoir chambers (13a, 13b) and the respective substrate chambers (14a, 14b) function as a means for reversibly blocking fluid communication between the respective reservoir chambers (13a, 13b) and the respective substrate chambers (14a, 14b). These may also be operated by a microcontroller (5).The pressure sensors (16a, 16b) of each pressure supply module (2a, 2b) are configured to sense the gas pressure in the corresponding pressure reservoir chamber (13a, 13b) and send an electrical signal thereto to the microcontroller (5), and the microcontroller (5) is configured to control the pressure provided by the pressure supply modules (2a, 2b) to the first supply module (11) and the second supply module (12).

[0152] 2 is a flow chart illustrating the pneumatic circuit and components of another embodiment of the apparatus (1). Again, a pressure supply module (2b) and first and second supply modules (11, 12) are provided. The pressure supply module (2b) is configured to deliver pressurized gas to the supply modules (11, 12) individually so that each supply module (11, 12) can operate at an individually selected operating pressure.

[0153] The pressure supply module (2b) comprises a gas inlet (3) reversibly connectable to a pressurized gas source (not shown), a first gas outlet (4a) for supplying pressurized gas to a pressure reservoir chamber (13a) of the first supply module (11), a second gas outlet (4b) for supplying pressurized gas to a pressure reservoir chamber (13b) of the second supply module (12), and a flow path (solid arrow) for the pressurized gas to flow from the gas inlet (3) to the first gas outlet (4a) and / or the second gas outlet (4b), the flow path comprising a flow path divider (6), and first and second pressure amplifiers (7a, 7b) arranged in the part of the flow path downstream of the flow path divider (6). Flow path branches are arranged to bypass each of the two pressure amplifiers (7a, 7b), and check valves (9a, 9b) are arranged in the flow path branches. The pressure in each downstream portion of the flow path supplying the first gas outlet (4a) and the second gas outlet (4b) is independently controllable by electrical pressure regulators, also referred to as electronic controllers (24a, 24b), and valves (21a, 22b) located upstream and downstream of the pressure amplifiers (7a, 7b), in combination with the microcontroller (5), which together form first and second pressure control circuits. Electrical signals representing input parameters of the control circuits are provided by first and second pressure sensors (16a, 16b), which are configured to detect the gas pressure in the first and second supply modules (11, 12), e.g., in their respective pressure reservoir chambers (13a, 13b).

[0154] Each of the first supply module (11) and the second supply module (12) independently comprises a pressure reservoir chamber (13a, 13b) for holding pressurized gas received from the respective gas outlets (4a, 4b) of the pressure supply module (2b), a pressurizable substrate chamber (14a, 14b) for holding a flexible container having an interior space for holding a liquid substrate (not shown), connectors (not specifically shown but functionally indicated by solid arrows) for providing fluid communication between the pressure reservoir chambers (13a, 13b) and the pressurizable substrate chambers (14a, 14b) and allowing the flow of pressurized gas, and valves (23a, 23b) as means for reversibly blocking fluid communication between the pressure reservoir chambers (13a, 13b) and the respective pressurizable substrate chambers (14a, 14b). The already mentioned pressure sensors (16a, 16b) are adapted to sense the gas pressure in the first supply module (11) and the second supply module (12) in their respective pressure reservoir chambers (13a, 13b) and send an electrical signal to the microcontroller (5).

[0155] FIG. 3 is a front view, or user-facing side view, of a device (1) according to the present disclosure. The device (1) is shown in its operational orientation vertical, i.e., parallel to the vertical axis (y). As shown, the device (1) comprises a first opposing part (51), characterized as part of the body of the device. The first opposing part (51) includes a first cavity (53a) and a second cavity (53b), which respectively contain the first gas outlet (60a) and second gas outlet (60b) of a second pressure supply module as described and illustrated in FIGS. 1 and 2. Other features of the pressure supply module are located on the rear of the device (not shown).

[0156] The first and second cavities (53a, 53b) are configured to correspond to respective first and second cavities (not shown, see FIG. 4) of the second opposing part (52). Furthermore, the first and second cavities (53a, 53b) are adapted to be independently sealed by a circumferential gasket (not shown) that may surround or enclose the cavities or, as described with respect to FIG. 6, may optionally be attached to a frame as described further herein. The first and second cavities (53a, 53b) are further independently adapted and shaped to at least partially hold or accommodate any one or combination of a flexible substrate container and its associated features (e.g., ports), at least one conduit, a portion of a frame adapted to hold the aforementioned components, a portion of a static mixer, or a connecting piece associated with a static mixer, or an insert (not shown, see FIGS. 5 and 6), as described in FIG. 5. Regarding the location of the first and second gas outlets (60a, 60b), they are characterized in that, as shown in this embodiment, they are located near the top of the cavities (53a) and (53b) in the context of the vertical operating orientation of the device (1). Regarding the positioning and accommodation of the flexible container (see FIG. 6), it is generally preferred that the gas outlets (60a, 60b) provided for delivering pressurized gas are provided by the device (1) and are located on the first counter part (51) at positions selected so that the first contact point of the pressurized gas flow leaving the outlets during operation of the device is not the outer surface of the flexible substrate bag.

[0157] As further shown in this figure, the second counter part (52) is hingedly connected to the first counter part (51) of the device (1). The device (1) also comprises a series of fastening means (56a), such as in the form of rotatable clamps, arranged on the first counter part (51) circumferentially around the first cavity (53a) and the second cavity (53b). In some embodiments, such as the currently shown embodiment, at least five fastening means (56a), i.e., a total of ten clamps, are arranged circumferentially around each of the first and second cavities: four arranged in a row near the top of the cavities (53a, 53b) along an axis perpendicular to the vertical operating orientation (y) of the device; and four arranged parallel below the two cavities (53a, 53b), one located to the left of the first cavity (53a) and one located to the right of the second cavity (53b). These clamps correspond to complementary fastening means (not shown, see FIG. 4) featured on the second counter part (52). The rotating clamps can provide a securing means for locking the first and second counter parts together during operation of the apparatus and under high pressure conditions. The apparatus (1) further includes two valve actuators (57), as shown, that can be fitted with and actuated by valves (e.g., one-way checkcock valves) that may be useful in the mixing process or in the process of collecting the resulting mixed or reaction products.

[0158] Figure 4 shows a perspective view of the device (1) shown in Figure 3, not drawn to scale, and also shown in its operational orientation parallel to the vertical axis (y). As shown, the second counter part (52) of the device is rotatably and hingedly connected to a first counter part (51) that is part of the body of the device (1) via a pivotable hinge (55) and is configured to complementarily attach to the first counter part (51) that includes a first cavity (53a) and a second cavity (53b). The second counter part further comprises complementary fastening means (56b) in the form of a groove that is complementary to fastening means (56a) located on the first counter part (51), which is in the form of a rotatable clamp. The second counter part (52) further comprises a first cavity (54a) and a second cavity (54b) that match and correspond respectively to the first cavity (53a) and the second cavity (53b) of the first counter part (51). The cavities (54a, 54b) are adapted to be independently sealed by circumferential gaskets (not shown), which may optionally surround the cavities or alternatively be secured to a frame as described with respect to FIG. 6 and as further described herein. In addition to being complementary to the cavities (53a, 53b) of the first opposing part (51), the first and second cavities (54a, 54b) of the second opposing part (52) are further adapted and shaped to at least partially hold or accommodate, respectively, a flexible substrate container and its associated components (ports), at least one conduit, a portion of a frame adapted to hold the aforementioned components, a portion of a static mixer, or a connection component associated with a static mixer, or any one or combination of inserts such as those described in FIG. 5 (not shown, but see FIGS. 5 and 6). Other features of the device (1) such as those described in FIG. 1 or 2 (not shown) are located behind the first opposing part within the device housing or body.

[0159] Figure 5 shows a perspective view of the same device (1) as shown in Figure 4, with a first insert (58a) and a second insert (58b) fitted into the first and second cavities, respectively, of the first counter part (51), and a first insert (59a) and a second insert (59b) fitted into the first and second cavities, respectively, of the second counter part (52) of the device (1). The inserts (58a, 58b, 59a, 59b) shape the cavities to fit a shaped or sized flexible substrate container (not shown, but see Figure 6), providing a means not only to ensure that the container is contained and held in place during pressurization, but also to reduce the volume of the cavity and therefore the overall volume to which the pressure supply module must supply pressurized gas. In some embodiments, the insert is not sealably fitted into the cavity, but is only fitted in a manner that allows the insert to be secured; in other words, when the insert is placed in the cavity, pressurized gas can still diffuse through or between the contact surfaces of the cavity and the insert.

[0160] The inserts (58a, 58b, 59a, 59b), although not shown to scale, occupy a substantial portion of the space or volume of the respective cavities into which they are inserted and may include at least one recess adapted to hold at least a portion of the flexible substrate container (and its associated port), and optionally at least one conduit. As shown, the inserts (58a, 58b) fitted into the first counter part (51) cover the first and second gas outlets (60a, 60b) of the pressure supply module, while means for diffusing the pressurized gas (61a, 61b) are simply provided in the form of cutouts or recesses at the top of the inserts (relative to the operating orientation of the device), resulting in uncovered portions of the cavities through which the pressurized gas can diffuse. The inserts (58a, 58b) may provide additional technical functionality with respect to reducing or avoiding the generation of undesirable pressurized gas affecting potentially sensitive connection points between the ports and conduits of the external flexible substrate container and / or bag by providing covers over the first and second gas outlets (60a, 60b), respectively. In other embodiments, the inserts may not include means for diffusing the pressurized gas and still cover the respective gas outlets (60a, 60b), but as noted above, because the inserts are not sealably secured or fitted to the cavities, the pressurized gas can still flow or diffuse into the negative space between the fitting of the insert and its respective cavity.

[0161] A pressurizable substrate chamber can be formed by fastening a second opposing part (52) to a first opposing part (51), such that the first cavity of the first opposing part (51) is aligned with the first cavity of the second opposing part (52), and the second cavity of the first opposing part (51) is aligned with the second cavity of the opposing part (52). The insert functions to reduce the volume within the chamber that needs to be filled with pressurized gas introduced from a pressure supply module. This improves the efficiency of the pressurization process with respect to the performance of the method according to the present disclosure. In some embodiments, the insert simply fits into the cavity, while in other embodiments, the insert is fixed or fastened (e.g., screwed) to the cavity by a fastening means (not shown). For example, depending on the desired production scale, it is conceivable that alternative inserts different from those illustrated can be utilized to accommodate different sizes or shapes of flexible substrate containers used in the method according to the present disclosure for mixing two fluid substrates. In some embodiments, the first insert (58a) of the first counter part (51) and the first insert (59a) of the second counter part (52) may be the same, i.e., identical inserts, as well as for the second inserts (58b, 59b). In other embodiments, the first and second inserts (58a, 58b) used in the cavities of the first counter part (51) may be different from their counterparts in the second counter part.

[0162] FIG. 6 shows a front or user-facing side elevational view of a frame (80), not drawn to scale, configured and adapted for use with a device (1) according to the present disclosure, such as a device similar to the device (1) described in FIGS. 3-5.

[0163] The frame (80) is shown in a vertical, operational orientation parallel to the vertical axis (y). The frame (80) is assembled with and holds at least the following components: flexible containers (81, 82, 83), a conduit (84), and a static mixing device (85). The frame (80) includes a first sealable area (231) and a second sealable area (232) for holding or accommodating the first and second flexible substrate containers (81, 82). The sealable areas (231, 232) are surrounded by a circumferential gasket (86). The first sealable area (231) and the second sealable area (232) are positioned adjacent to each other, and the dimensions and arrangement of these areas, as well as the circumferential gasket (86), correspond to the respective first and second cavities of the second opposing part of the apparatus (1) according to the present disclosure. On the back side of the frame (80), not shown, sealable areas (231, 232) similarly correspond to the first and second cavities of the first opposing part of the device. Within the sealable areas (231, 232), the first and second flexible substrate containers (81, 82) are held by means (226) for fixing them in their designated positions. The means (226) are connectable with corresponding through-holes (see 228) in the peripheral zones or sealing edges (227) of the respective flexible containers. Each flexible substrate container (81, 82) has three ports, including an outlet port (91) and a sealed inlet port (92). The outlet port (91) is fluidly connected to a static mixer (85) via a conduit (84), which is held in place by the means (223) for holding the static mixer. Additionally, outlet ports (91) are located at the top of the substrate containers (81, 82) to allow the fluid substrate or liquid (not shown) held therein to be discharged from the substrate containers (81, 82) in a direction against gravity so as to flow into the static mixer (85).

[0164] Also shown is a flexible product container (83), which is positioned toward the front and top of the frame (80) and is held by three fastening means (226) located in positions corresponding to three through holes (228) in the peripheral region of the product container (83). Note that a flexible waste container may also be assembled to the frame (80), but is not visible as it is attached to the back side of the frame. The product container (83) has three ports located on its bottom surface (in its operational orientation), including an inlet port (94) for receiving a third fluid from the static mixer (85) via conduit (84), and two outlet ports (95). One of the outlet ports (95) is fluidly connected via flexible tubing to a sampling tube (241), the downstream end of which is fluidly connected to a sterile filter (242), with a pinch valve (246) and a sterile disconnect (247) located upstream of the sampling tube (241) to facilitate withdrawal of a product sample. The other outlet port (95) is fluidly connected via flexible tubing to a sterile connector (245). Also shown is an additional sterile filter (249) in fluid communication with the inlet port of the product container via a Y-fitting (248). This configuration can be used to add a diluent to the product container (83) before, during, or after the mixing process, such as to dilute or alter the composition of the third fluid received from the static mixer (85). The frame (80) further comprises four through holes (251) in the central region, which allow the frame (80) to be fixed to a device according to the present disclosure and / or allow protruding fastening or locking means featured on a first opposing part of the device to access corresponding fastening or locking means on a second opposing part of the device when the device is in operation.

[0165] When frame (80) is secured or held in place on an apparatus according to the present disclosure by a securing or retaining means present on the apparatus, such as one or more hooks, and all of the described components are assembled as shown in this figure, a second opposing part, which may be rotatably and hingedly connected to a first opposing part, is secured to and sealably joined with the first opposing part, and frame (80) includes at least one circumferential gasket arranged to seal the respective first and second cavities to both the first and second opposing parts. Thus, the first and second cavities of the two opposing parts can be separately sealed to form first and second pressurizable substrate chambers of each supply module of the apparatus.

[0166] Although the particular embodiment shown in the present drawing of frame (80) has a slightly different shape for the first sealable area (231) compared to the shape of the corresponding first cavities (53a, 54b) of the first and second opposing parts (51, 52) of device (1) as shown in Figures 3-5, those skilled in the art will understand that frames provided with or adapted with correspondingly shaped sealable areas can be devised for use in combination with the embodiment of device as shown in Figures 3-5.

[0167] FIG. 7 shows a perspective view of an exemplary static mixing device (70) that can be used in combination with a frame (80) and adapted to operate an apparatus according to the present disclosure for mixing a first liquid and a second liquid.

[0168] According to some preferred embodiments, the static mixer (70) may represent a jet impingement reactor. The illustrated mixer (70) comprises a main housing (71) having an outlet port (72) and first and second inlet ports (73, 74). In this case, the static mixer (70) is shown in an operational orientation with the outlet port (72) facing upward so that the third fluid formed from the mixture of the first and second liquids and exiting the outlet port (72) flows against gravity. First and second inlet connection pieces (75, 76) are fitted to the first and second inlet ports (73, 74), respectively. Barbed connectors (77) are provided at the upstream ends of the connection pieces (75, 76) and at the downstream end of the outlet port (72).

[0169] FIG. 8 shows a schematic diagram of a typical overall relationship between the pressure (P) in a pressure reservoir chamber, the pressure (P) in a corresponding pressurizable substrate chamber, and the flow rate (F) of liquid as it is forced out of a flexible container housed in the substrate chamber when carrying out a method of the present invention according to some embodiments. The diagram is not to scale and uses arbitrary units only. Initially, i.e., before step (dd) of the method, the pressure (101) in the pressure reservoir chamber containing the pressurized gas is substantially higher than the pressure (102) in the corresponding pressurizable substrate chamber holding a flexible container filled with a liquid substrate. During this stage, the respective means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber are in a closed state, and the pressure (102) in the substrate chamber may be at or near ambient pressure. Step (dd) may be initiated by changing the state of the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber from a closed state to an open state, thereby causing a sudden or rapid pressure equilibrium (103) between the pressure reservoir chamber and the substrate chamber, e.g., within less than two or three seconds. Note that this pressure equilibrium involves a sudden drop in pressure in the pressure reservoir chamber and a corresponding sudden increase in pressure in the substrate chamber, where this pressure increase is above ambient pressure levels. The now increased pressure (104) in the pressurizable substrate chamber, i.e., the equilibrium pressure or target process pressure, acting on the exterior surface of the flexible container holding the liquid, now causes the liquid to flow out of the flexible container toward the static mixer at a substantially constant flow rate (105). During the flow of liquid, additional pressurized gas may be introduced into the supply module containing the pressure reservoir chamber and the pressurizable substrate chamber so that the target process pressure is maintained until the mixing process is substantially complete. However, even without supplying additional pressurized gas during the mixing process, the pressure (104) in the pressurizable substrate chamber after equilibration remains approximately constant because the volume of liquid flowing out of the flexible container is relatively small compared to the total volume of the pressure reservoir chamber and the pressurizable substrate chamber.

[0170] Depending on the specific optional process parameters or the type and sensitivity of the pressure or flow sensors for measuring pressure (P) and flow rate (F), slight deviations from the overall shape of the graph shown in FIG. 9 may be observed when using the apparatus or performing the process according to the present invention. For example, during rapid pressure equilibration (103), a very short initial peak may be observed for the pressure (102) or flow rate (105) in the substrate chamber if, for example, the fluid path upstream of the static mixing device is pre-filled with liquid. Such a short peak may be interpreted as an initial pulse or "shock wave" traveling through the liquid. This short peak does not change the overall characteristics of the process. Furthermore, a very slight decrease or increase in the pressure (104) in the substrate chamber after rapid equilibration over time may be observed. A slight decrease may result, for example, from a slight increase in the volume of pressurized gas in the substrate chamber, while a slight increase may result from a slight temperature increase of the pressurized gas during the process. However, the inventors have found that when using an apparatus as described herein, such as an apparatus having a pressure supply module as outlined in FIG. 1 or FIG. 2, a process can be performed with very fast initiation of liquid flow and highly controlled pressure. [Explanation of symbols]

[0171] 1 device 2a, 2b Pressure supply module 3 Gas inlet of pressure supply module 11 First supply module 12 Second Supply Module 13a Pressure reservoir chamber of first supply module 13b Pressure reservoir chamber of second supply module 14a Pressure substrate chamber of first supply module 14b Pressure substrate chamber of second supply module 15a, 15b Valves as means for reversibly blocking fluid communication 16a Pressure sensor for first supply module 16b Pressure sensor for second supply module 4a, 60a First gas outlet of pressure supply module 4b, 60b Second gas outlet of pressure supply module 5 Microcontrollers 6 Flow Divider 7,7a,7b Pressure amplifier 8 Pressure Supply Module Pressure Reservoir Chamber 21, 21a, 21b valves 22a, 22b valves 9a, 9b Check valve 23a, 23b valves 24a,24b,25a,25b Electronic regulator 51 first opposing part 52 Second opposing part 53a First cavity of first opposing part 53b Second cavity of first opposing part 54a First cavity of second opposing part 54b Second cavity of second opposing part 55 Hinge 56a,56b Fastening means 57 Valve Actuator 58a Insert for the first cavity of the first counter part 58b Insert for the second cavity of the first counter part 59a Insert for the first cavity of the second counter part 59b Insert for the second cavity of the second counter part 61a, 62b Pressurized gas diffusion means 80 frames 81,82,83 Flexible containers 84 Conduit 70,85 Static Mixer 86 Circumferential gasket 91 outlet port of first or second flexible substrate container 92 inlet port of first or second flexible substrate container 93 Flexible waste container inlet port 94 Flexible product container inlet port 95 Resealable outlet port of flexible product container 96 First inlet port of static mixer 97 Second inlet port of static mixer 98 Static mixer outlet port 231 first sealable area 232 Second sealable area 223 Means for retaining static mixing devices 226 Means for securing flexible substrates, product or waste containers 228 Perforations in the peripheral area of ​​flexible containers 227 Sealing edges of flexible substrates, product or waste containers 241 Sampling tube 242 Sterile Filter 245 Sterile Connector 246 Pinch valve 247 Sterile Disconnector 248 Y-joint 249 Sterile Filter 251 Through holes for matching hooks or fastening means of devices 71 Main housing of static mixer 72 Static mixer outlet port 73 First inlet port of static mixer 74 Second inlet port of static mixer 75 First inlet connection part 76 Second inlet connection piece 77 Barbed Connector 101 Pressure in the pressure reservoir chamber before equilibration 102 Pressure in the pressurizable substrate chamber before equilibration 103 Rapid Pressure Equalization 104 Pressure in the pressurizable substrate chamber and pressure reservoir chamber after equilibration 105 Constant flow rate phase P pressure F flow rate t time

[0172] The following examples serve to illustrate the present invention but should not be understood as limiting the scope of the invention. [Example]

[0173] Example 1 A prototype device according to the present invention was constructed and tested. A non-optimized device (e.g., not optimized for minimum dead volume) included a jet impingement reactor, such as that described in co-pending European Patent Application No. 21192535.9 or International Patent Application Publication No. 2023 / 025736, as a static mixer. The substantially spherical reaction chamber had a diameter of 5 mm, and the first and second inlets were each provided by a simple orifice nozzle with a pinhole diameter of 200 μm. Water was used as a substitute for both the first and second liquids. Water was provided in two flexible containers similar to infusion bags and placed in the first and second substrate chambers, respectively. The internal volume of the substrate chambers was approximately 10 L, and the volume of water in each flexible container was approximately 500 mL. The first and second pressure reservoir chambers each had a volume of approximately 20 L and were filled with pressurized air at a pressure of 15 bar. Furthermore, the jet impingement reactor and the conduits for providing fluid communication between each of the two flexible containers and the respective inlets of the jet impingement reactor were pre-filled with water. The apparatus further included a flow meter (Cori-Flow™) and various pressure sensors disposed in the fluid conduits between the interior space of the flexible containers and the first or second inlets of the jet impingement reactor, respectively.

[0174] Instantaneous pressure equilibrium between the pressure reservoir chambers and the corresponding substrate chambers was achieved by simultaneously opening the magnetic valves located on the connectors between them. Pressure readings were recorded once per second, and it was observed that an initial equilibrium pressure of approximately 9 bar was achieved in each substrate chamber just 1 second after the valves were opened. By the end of the trial run, which lasted 43 seconds, the equilibrium pressure rose slightly to approximately 10 bar in each substrate chamber, likely due to a slight temperature increase during this phase. At each point, the pressure in the first substrate chamber was virtually identical to the pressure in the second substrate chamber.

[0175] Pressure equilibration immediately induced a flow of water downstream from the flexible container in the substrate chamber, toward and through the jet impingement reactor. The flow rates remained fairly constant starting approximately 2 seconds after initial pressure equilibration. The flow rate of the first feed module increased very slightly and uniformly from approximately 53 to 54 mL / min, while the flow rate of the second feed module increased from approximately 54 to 55 mL / min. A flow rate peak was observed within the first 1 or 2 seconds, which was considered an artifact caused by the initial pressure impulse traveling through the water-filled system. While the very slight difference between the two feed modules is likely caused by minimal pinhole differences due to manufacturing tolerances, the slight and technically quite negligible increase in flow rate over time could result from the corresponding pressure increase.

[0176] In summary, the experiments demonstrate that the device is suitable for achieving near-instantaneous initiation of flow of two liquids from a flexible substrate container into a static mixing device, followed by highly controlled flow rates and flow ratios between the flow rates. It also demonstrates that the device can be used to perform small-scale mixing of two liquids without a pump.

[0177] Example 2 A prototype apparatus similar to that described in Example 1, except that the reaction chamber of the jet impingement reactor had a diameter of 3 mm, was used to mix two model liquids that would form solid barium sulfate particles upon mixing. The first liquid consisted of approximately 500 mL of an aqueous barium chloride solution, and the second liquid consisted of approximately 500 mL of an aqueous barium sulfate solution. The two liquids were contained in flexible bags placed in first and second substrate chambers, each with a volume of approximately 10 L. The first pressure reservoir chamber had a volume of 20 L and was filled with pressurized air to a pressure of 10.9 bar. The second pressure reservoir chamber also had a volume of 20 L and was pressurized with air to 6.1 bar. The connectors between the first pressure reservoir chamber and the first substrate chamber and between the second pressure reservoir chamber and the second substrate chamber were equipped with solenoid valves. When the valves were opened simultaneously, essentially instantaneous pressure equilibration between each pressure reservoir chamber and its associated substrate chamber was observed. A pressure of 7.2 bar was recorded for the first supply module (i.e., the first pressure reservoir chamber and the first substrate chamber), and a pressure of 4.6 bar was recorded for the second supply module (i.e., the second pressure reservoir chamber and the second substrate chamber). The overpressure caused the two liquids to flow from their respective flexible containers at a total flow rate (i.e., the sum of the flow rates of the first and second liquid streams) of 75.5 mL / min, with a flow rate ratio between the first and second liquids of 0.73. The liquid product (i.e., the third liquid) resulting from mixing the first and second liquids in the jet impingement reactor was an aqueous dispersion of barium sulfate nanoparticles with a z-average particle size of 79.4 nm and a polydispersity index of 0.14, as measured by dynamic light scattering (DLS) using an Anton Paar™ Litesizer 500.

[0178] To assess the reproducibility of the mixing process, the experiment was repeated twice. In both additional runs, the instantaneous pressure equilibration resulted in similar pressures as in the first experiment, as shown in Table 1, and in comparable products with respect to nanoparticle properties, thus demonstrating a high degree of process robustness and reproducibility.

[0179]

Table 1

Claims

1. 1. An apparatus for operating a static mixer for mixing a first liquid and a second liquid, the static mixer comprising a first inlet for receiving the first liquid, a second inlet for receiving the second liquid, and an outlet for discharging a third liquid resulting from mixing the first liquid and the second liquid, the apparatus comprising: (a) a first supply module for supplying the first liquid to the first inlet; (b) a second supply module for supplying the second liquid to the second inlet; and (c) a pressure supply module disposed upstream of the first and second supply modules; Equipped with Each of the first and second supply modules independently: (i) a pressure reservoir chamber for holding pressurized gas, said pressure reservoir chamber having an inlet and / or an outlet for the pressurized gas; (ii) a pressurizable substrate chamber for holding a flexible container, the flexible container having an interior space for holding the first liquid or the second liquid, respectively; (iii) a connector for providing fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the fluid communication being for allowing a flow of pressurized gas from the pressure reservoir chamber to the pressurizable substrate chamber; and (iv) a means for reversibly blocking the fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber, the means having an open state and a closed state, a means having an open state and a closed state, the connector and the means being arranged to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the pressurizable substrate chamber upon changing the state of the means from a closed state to an open state; (v) a pressure sensor for measuring the pressure of pressurized gas in the supply module at or downstream of the pressure reservoir chamber; Equipped with The pressure supply module includes: (i) a gas inlet reversibly connectable to a source of pressurized gas; (ii) a first gas outlet for supplying pressurized gas to the pressure reservoir chamber of the first supply module; (iii) a second gas outlet for supplying pressurized gas to the pressure reservoir chamber of the second supply module; (iv) a flow path comprising a flow path divider for pressurized gas to flow from the gas inlet to the first gas outlet and / or the second gas outlet; (v) at least one pressure amplifier disposed in the flow path; (vi) a first pressure control circuit adapted to control the pressure of pressurized gas delivered to the pressure reservoir chamber of the first supply module; (vii) a second pressure control circuit adapted to control the pressure of pressurized gas delivered to the pressure reservoir chamber of the second supply module; and An apparatus comprising:

2. 2. The apparatus of claim 1, wherein each of the first and second pressure control circuits of the pressure supply module comprises a valve and / or an electrical pressure regulator disposed between the flow path divider and the respective first or second gas outlet.

3. 3. The apparatus of claim 2, wherein the valve and / or the electrical pressure regulator are configured to be operated by a microcontroller.

4. 4. The apparatus of claim 3, wherein the microcontroller is configured to receive a signal from the pressure sensor positioned to measure gas pressure within the respective first or second supply module.

5. 5. The apparatus of claim 1, wherein the pressure supply module further comprises a pressure reservoir chamber disposed in the flow path for holding pressurized gas.

6. The apparatus of claim 5 , wherein the pressure reservoir chamber is located upstream of the flow path divider.

7. 7. The apparatus of claim 5 or 6, wherein the pressure supply module comprises another valve disposed in the flow path between the pressure reservoir chamber and the flow path divider, the valve optionally configured to be operated by the microcontroller.

8. 5. The apparatus of claim 1, wherein the pressure supply module comprises two pressure amplifiers disposed downstream of the flow path divider, a first pressure amplifier fluidly connected to the first gas outlet and a second pressure amplifier fluidly connected to the second gas outlet.

9. 9. The apparatus of claim 1, wherein the pressure supply module further comprises a flow path branch arranged to bypass the at least one pressure amplifier, and wherein a check valve is arranged in the flow path branch.

10. 10. The apparatus of claim 1, wherein the static mixer is selected from the group consisting of a T-mixer, a Y-mixer, a vortex mixer, a baffled static mixer, a microfluidic mixing device, a multi-inlet vortex mixer (MIVM), and a jet impingement reactor.

11. 11. The device of claim 1, wherein the pressure reservoir chamber has a larger volume than the pressurizable substrate chamber with which it is in fluid communication.

12. 12. The device of claim 11, wherein the ratio of the volume of the pressure reservoir chamber to the volume of the pressurizable substrate chamber is at least about 5, and optionally at least about 10.

13. 13. The device of claim 1, wherein the open state and the closed state are the only states of the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber.

14. The means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber has a width of at least about 1 mm when the means is in an open state. ２ 14. The apparatus of claim 1, further comprising a fluid path for pressurized gas having a cross-sectional area of

15. 15. The device of claim 1, wherein the connector and the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber do not include a pressure adjusting member.

16. 16. The device of claim 1, wherein the means for reversibly blocking fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber is the only means for controlling the flow of pressurized gas between the pressure reservoir chamber and each of the pressurizable substrate chambers.

17. 17. The apparatus of claim 1, wherein the gas inlet of the pressure supply module is connected to the source of pressurized gas.

18. 18. The apparatus of claim 17, wherein the source provides gas at a pressure of about 6 to 10 bar.

19. 19. Apparatus according to any one of claims 1 to 18, wherein the pressure amplifier is adapted to increase the pressure of pressurized gas received from the pressurized gas source by at least 50%.

20. The apparatus of claim 7 , wherein the pressure sensor is configured to sense pressure within the pressure reservoir chamber of the first or second supply module.

21. 21. The apparatus of any one of claims 1 to 20, wherein the pressure supply module is adapted to maintain a pressure in the pressure reservoir chamber of the first and / or second supply module of about 2 to 20 bar when the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the pressurizable substrate chamber is in the open state.

22. 22. An apparatus according to any one of claims 1 to 21, wherein the pressurizable substrate chamber of the first and / or second supply module comprises means for opening and closing to allow insertion or removal of the respective flexible container.

23. 23. The apparatus of any one of claims 1 to 22, wherein the internal volume of the pressurizable substrate chamber of the first supply module is different from the internal volume of the pressurizable substrate chamber of the second supply module.

24. The static mixer is a jet impingement reactor having a mixing chamber defined by the inner surfaces of a mixing chamber wall, the mixing chamber having a substantially spheroidal overall shape, and the mixing chamber preferably comprises: a first fluid inlet and a second fluid inlet, the first fluid inlet and the second fluid inlet being arranged facing each other at opposite positions on a first central axis of the reaction chamber, each of the first fluid inlet and the second fluid inlet comprising a nozzle; a fluid outlet arranged at a third location, said third location being located on a second central axis of said chamber, said second central axis being perpendicular to said first central axis; Equipped with 24. The apparatus of any one of claims 1 to 23, wherein a distance between the nozzle of the first fluid inlet and the nozzle of the second fluid inlet is equal to or less than a diameter of the mixing chamber along the first central axis.

25. 25. The apparatus of any one of claims 1 to 24, wherein the static mixer is oriented such that an outlet of the static mixer is higher than first and second inlets of the static mixer.

26. 26. The apparatus of any one of claims 1 to 25, wherein the outlet of the static mixer is fluidly connectable to a first container for receiving the third liquid, the container being optionally a flexible container.

27. 27. The apparatus of claim 26, wherein the outlet of the static mixer is fluidly connectable to a second container for receiving the third liquid, the second container optionally being a flexible container.

28. 28. The apparatus of any one of claims 1 to 27, wherein the static mixer is made of plastic.

29. 29. The device of any one of claims 1 to 28, comprising first and second opposing parts, at least one of the first or second opposing parts comprising a first cavity and a second cavity, the first and second opposing parts being configured to be fixed to one another so that one of the pressurizable substrate chambers is formed to include the first cavity and the other of the pressurizable substrate chambers is formed to include the second cavity.

30. 30. The device of claim 29, wherein the second opposing part is rotatable and hingedly connected to the first opposing part.

31. 31. The device of claim 30, wherein the first and second opposing parts have a vertical operating orientation and the hinge connection is located on an underside or lower end of the second opposing part.

32. 32. The device of any one of claims 29 to 31, wherein at least one circumferential gasket is provided between the first and second opposing parts to separately seal each of the pressurizable substrate chambers.

33. 33. The device of claim 32, wherein at least two circumferential gaskets are provided between the first opposing part and the second opposing part to separately seal each of the pressurizable substrate chambers, and a frame for holding the flexible containers for holding the first liquid and the second liquid is sealed between the at least two circumferential gaskets.

34. 34. The apparatus of any one of claims 29 to 33, wherein the pressurizable substrate chamber and the pressure reservoir chamber of the first supply module are fluidly connected through an opening in the first opposing part, the opening being the first gas outlet of the pressure supply module, and the pressurizable substrate chamber and the pressure reservoir chamber of the second supply module are fluidly connected through an opening in the first opposing part, the opening being the second gas outlet of the pressure supply module.

35. Apparatus according to any one of claims 1 to 34, adapted to carry out a method according to any one of claims 36 to 53.

36. 1. A method for mixing a first liquid and a second liquid, comprising: (aa) providing a first flexible container for holding the first liquid, the first flexible container being contained within a first pressurizable substrate chamber; (bb) providing a second flexible container for holding the second liquid, the second flexible container being contained within a second pressurizable substrate chamber; (cc) providing a static mixer, the static mixer comprising: a first inlet for receiving said first liquid; a second inlet for receiving said second liquid, and - providing a static mixer having an outlet for discharging a third liquid resulting from the mixing of said first liquid with said second liquid; (dd) independently pressurizing the first and second pressurizable substrate chambers with pressurized gas exerting pressure on the exterior surfaces of the first and second flexible containers to force the first and second liquids into the static mixer and mix the first and second liquids; (ee) collecting the third liquid; A method comprising:

37. 37. The method of claim 36, wherein the pressurizing step (dd) comprises a first sub-step, characterized by abruptly increasing the pressure of each of the first and second pressurizable substrate chambers from an initial pressure to a target process pressure within less than about 2 seconds and / or within less than about 5% of the total time required to mix the first liquid and the second liquid, and wherein the target process pressure is independently selected for each pressurizable substrate chamber.

38. The first sub-step comprises: changing the state of the means for reversibly blocking the fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber from a closed state to an open state to create pressure equilibrium between each pressure reservoir chamber and the respective pressurizable substrate chamber within a period of about 2 seconds or less; 38. The method of claim 37, comprising:

39. 39. The method of claim 37 or 38, wherein the pressurizing step (dd) includes a second sub-step, wherein the target process pressure is maintained in each of the first and second pressurizable substrate chambers for at least 90% of the total time required to mix the first and second liquids.

40. 40. The method of any one of claims 37 to 39, wherein the initial pressure is about ambient pressure and the target process pressure is in the range of from above 1 bar to about 16 bar, preferably in the range of from about 2 bar to 12 bar.

41. Step (dd) is generating the third liquid over a period of at least about 10 seconds, e.g., from about 10 seconds to about 15 minutes, by flowing essentially all of the first and second liquids from their respective flexible containers through the static mixer; 41. The method of any one of claims 36 to 40, comprising:

42. each of the first and second pressurizable substrate chambers is connected to a first or second pressure reservoir chamber for holding pressurized gas by a connector for providing fluid communication between the respective pressure reservoir chamber and the respective pressurizable substrate chamber, and means having an open state and a closed state for reversibly blocking the fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber are arranged, and before performing the pressurizing step (dd): - each said pressure reservoir chamber is under pressure such that the pressure in each said pressure reservoir chamber is about 2 to 20 times higher than the pressure in each said pressurizable substrate chamber; - A method according to any one of claims 36 to 41, wherein the means for reversibly blocking the fluid communication between each said pressure reservoir chamber and each said pressurizable substrate chamber is in the closed state.

43. - said first liquid comprises an organic solution of one or more lipids, - the second liquid comprises an aqueous solution of a biologically active agent; 43. The method of any one of claims 36 to 42, wherein lipid nanoparticles are formed by mixing the first liquid with the second liquid, and the biologically active agent is associated with and / or encapsulated within the lipid nanoparticles.

44. 44. The method of claim 43, wherein the biologically active agent is an oligonucleotide or polynucleotide.

45. 45. The method of claim 44, wherein the oligonucleotide or polynucleotide is an optionally modified mRNA molecule comprising a nucleic acid sequence encoding an antigen, in particular a tumor, viral, bacterial, fungal or protozoal antigen.

46. 46. ​​The method of any one of claims 34 to 45, wherein step (ee) comprises collecting at least a portion of the third liquid in a first container for receiving the third liquid, the first container being disposed in fluid communication with the outlet of the static mixer.

47. 47. The method of claim 46, further comprising filling the first container with a liquid diluent before or after the third liquid is received in the first container.

48. 48. The method of claim 47, wherein the liquid diluent comprises a pH adjuster, a tonicity agent, a lyophilization aid, or any combination thereof.

49. 49. The method of any one of claims 46 to 48, further comprising the step of performing an in-line particle size measurement on the third liquid before the third liquid is received in the first container.

50. 50. The method of any one of claims 46 to 49, wherein step (ee) comprises collecting at least a further portion of the third liquid in a second container for receiving the third liquid, the second container being disposed in fluid connection with the outlet of the static mixer.

51. - performing an in-line particle size measurement on said third liquid between steps (dd) and (ee); - collecting the third liquid in the second container for receiving the third liquid by blocking the fluid connection between the outlet of the static mixer and the first container for receiving the third liquid if the in-line particle size measurement gives an undesired result; - if the in-line particle size measurement gives a desired result, collecting the third liquid in the first container for receiving the third liquid by blocking the fluid connection between the outlet of the static mixer and the second container for receiving the third liquid; 51. The method of claim 49 or 50, comprising:

52. 52. The method of any one of claims 36 to 51, further comprising further processing the third liquid or the mixture of the third liquid and the liquid diluent collected according to step (ee) by at least one of tangential flow filtration, chromatography, freezing, or lyophilization.

53. 53. The method of claim 52, wherein the further processing steps are performed under sterile conditions.

54. 54. A method according to any of claims 36 to 53, wherein for carrying out the method an apparatus according to any of claims 1 to 35 is used.

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