Apparatus and method for mixing small liquid volumes and use of the apparatus - Patents.com

Abstract

An apparatus (1) for mixing two liquids is provided. The apparatus comprises a static mixer (2) and a first feed module (6) and a second feed module (14) for feeding the two liquids to the mixer. The feed modules comprise a pressurizable chamber (7, 15) for housing a flexible container (8, 16) holding the liquids to be mixed. When the chamber containing the flexible container is pressurized, the liquids are forced through the static mixer. Pressurization is achieved by pressurized gas stored in a pressure reservoir chamber (11, 19) connectable to the chamber holding the flexible container. An associated method of mixing two liquids is also provided.

Description

[Background technology]

[0001] Process automation and upscaling and downscaling of mixing processes are routine tasks 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, a feed line, a chamber in which the mixing or reaction takes place, and an outlet to harvest the product. The chamber often represents the heart of such a system and can be bulky, for example a vessel, or miniaturized, as in microfluidic approaches. However, the configuration of the system, the conditions, and the quality of the raw materials also play a decisive role in these processes.

[0003] An example of a discontinuous mixing system is the device for mixing, storing and homogenizing liquids as disclosed in US Pat. 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 lower surface used as an outlet for the liquid and with further orifices at the top of the bag for adding various liquids to produce the mixture. One of the upper orifices is used for the liquid to return to the inside of the bag (with the help of a pump), allowing a closed circuit circulation. This system is intended for disposable use, preventing the cleaning and sterilization steps necessary when a rigid tank is used for mixing. It can handle bags with a volume of 25 to 3000 l, but does not seem suitable for the production of small amounts, for example μl or ml quantities.

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

[0005] Despite these and other solutions to mixing systems and methods, the need for methods and apparatus for producing small quantities of products that are fast, reliable, and can be performed under standardized conditions has not been fully met, particularly for applications in the field of personalized medicine.

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

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

[0008] According to a first aspect of the invention, an apparatus for mixing a first liquid and a second liquid is provided. The apparatus comprises a static mixer and first and second supply modules adapted to provide the first and second liquids to the static mixer. The supply modules are characterized in that each of them comprises 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 the first or second inlet of the static mixer, respectively, a pressure reservoir chamber for holding a pressurized gas, and a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber. The connector comprises a means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber, the means having an open state and a closed state, said fluid communication being for allowing a flow of pressurized gas from the pressure reservoir chamber to the substrate chamber so as to apply pressure to the outer surface of the flexible container and to push the first or second liquid from the container into the conduit. Furthermore, the connector and said means are arranged to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the substrate chamber upon changing the state of said means from closed to open.

[0009] Further features of the device are provided below in the detailed description of the invention.

[0010] In a further 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 comprises: (aa) providing a first flexible container holding a first liquid, the first flexible container being contained in a first pressurizable chamber; (bb) providing a second flexible container holding a second liquid, the second flexible container being contained in a second pressurizable chamber; (cc) providing a static mixer, the static mixer comprising: a first inlet for receiving a first liquid; a second inlet for receiving a second liquid; and an outlet for discharging a third liquid resulting from the mixing of the first liquid with the second liquid; (dd) pressurizing the first and second pressurizable substrate chambers with a pressurized gas that applies pressure to an exterior surface of each of the first and second pressurizable substrate chambers to force the first and second liquids through a static mixer to mix the first and second liquids; (ee) collecting the third liquid.

[0011] Again, further features and embodiments of the method are disclosed below in the detailed description of the invention. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing an example of a general configuration of an apparatus according to the present invention. [Diagram 2] FIG. 1 illustrates an apparatus having a valve between the interior space of a flexible container and the inlet of a static mixer. [Diagram 3] FIG. 1 shows a device having means for indicating the pressure and inlet of pressurized gas. [Figure 4] FIG. 1 shows, not to scale, an example of a jet impingement reactor useful as a static mixing device and for carrying out the method of the present invention. [Diagram 5] Figure 1 shows an example of a flexible container for holding a liquid substrate, e.g. a first or second liquid In principle such a flexible container can also be used as a product container. [Figure 6] FIG. 13 illustrates an alternative embodiment of the device. [Figure 7] 1 shows an alternative version of the device, in which the orientation of the device is generally designed for an upward direction of liquid flow, i.e. against gravity or from a lower to a higher position. Additionally, two containers for receiving a third liquid are arranged, one of which can be used as a product container and the other as a waste container. [Figure 8] FIG. 13 shows another version of the device comprising a means for in-line particle size measurement having a sensing window, an analyzer, and a controller in communication with a means, such as a pinch valve, for blocking the fluid connection to a container for receiving a third liquid. [Figure 9] 1 is a schematic diagram of the overall relationship between the pressure in a pressure reservoir chamber (P), the pressure in a corresponding substrate chamber (P), and the flow rate (F) of liquid as it is pushed out of a flexible container contained within the substrate chamber over time (t) when performing the method of the present invention. The diagram is not to scale and uses arbitrary units only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In a first aspect, the present invention provides an apparatus for mixing a first liquid with a second liquid, the apparatus comprising (a) a static mixer, (b) a first feed module for providing the first liquid, and (c) a second feed module for providing the second liquid to the static mixer. The static mixer itself is characterized in that it presents 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 with the second liquid. Further, each of the first and second supply modules comprises: (i) a substrate chamber for holding a flexible container, the 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 a first or second inlet of the static mixer, respectively; (iii) a pressure reservoir chamber for holding a pressurized gas; and (iv) a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber, the connector comprising a means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber, the fluid communication being for allowing a flow of the 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 to push 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. Furthermore, the connector and said means are arranged to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the substrate chamber upon changing the state of said means from closed to open.

[0014] The inventors have found that the apparatus defined herein is surprisingly accurate in controlling the flow of the first and second liquids into the static mixer at a predetermined flow rate, allowing for highly reproducible processing of the two liquid substrates into a product, i.e., a third liquid composition. Moreover, it allows for aseptic processing of very small batches, avoiding the dead volumes and ramp-up losses of larger scale equipment to generate liquid streams, especially equipment that uses pumps and flow or pressure control systems. The use of gas pressure applied to the exterior surface of such a flexible container as the driving force to achieve a controlled flow of liquid from the flexible container to the static mixer also avoids the initial fluid pressure oscillations associated with the use of pumps. This effect makes the claimed apparatus and process particularly useful for the preparation of small batches and short processing times. This is especially true for gas pressure provided suddenly by a pre-pressurized pressure reservoir chamber, rather than a continuous flow of pressurized gas. Moreover, the pressurized gas does not come into contact with the liquid, but rather with the exterior surface of the flexible container, and therefore cannot contaminate the liquid.

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

[0016] In the context of the present 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 mixing device is a T-piece. Since static mixing devices are intended to mix two liquids to produce a liquid mixture, i.e. a third liquid, they typically comprise at least two substrate inlets and a product outlet. These substrate inlets are referred to in the context of the present invention as a first inlet for receiving the first liquid and a second inlet for receiving the second liquid.

[0017] A feed module as used herein should be understood as a group of equipment components designed, adapted and / or configured to feed a substrate to a static mixer. According to the invention, the equipment comprises at least two feed modules, namely a first feed module for providing a first liquid to a first inlet of the static mixer and a second feed module for providing a second liquid to a second inlet. The main aspect that characterizes the invention is the design and configuration of these feed modules.

[0018] More specifically, the supply module is adapted to accommodate the flexible container in which the liquid substrates are initially provided and from which they are supplied 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 can significantly reduce the internal volume or space of the container, as in the case of collapsible containers, where the collapsibility arises from the flexibility of the container walls, similar to infusion bags.

[0019] Each of the two supply modules comprises a substrate chamber, which is a pressurizable chamber for holding such a flexible container. To operate the apparatus and mix the two liquids, the substrate chamber is pressurized to expel, e.g. squeeze, the liquid from the flexible container and to feed the liquid to the mixing device. Pressurization is achieved by a pressurized gas that is initially contained within a pressure reservoir chamber of each supply module and that can flow into the substrate chamber, e.g. to apply pressure to the exterior surface of the flexible container to start the mixing process.

[0020] As mentioned above, the connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber and the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber, having an open state and a closed state, are arranged to achieve instantaneous pressure equilibration between the pressure reservoir chamber and the substrate chamber upon changing the state of said means from closed to open. When used in this context, the expression "instantaneous" means very rapid, such as less than one or two seconds, in particular very rapid compared to the duration of the flow of the respective liquids from the flexible container to the static mixer, which is brought about by such rapid pressure equilibration between the pressure reservoir chamber and the substrate chamber. In other words, the initial pressure equilibration occurs abruptly. Typically, the pressure equilibration requires only a fraction of a second and is already completed or nearly completed when the respective liquids initially reach the static mixer.

[0021] Instantaneous pressure equilibration between the pressure reservoir chamber and the substrate chamber involves a rapid and significant pressure increase in the substrate chamber where the pressurized gas exerts pressure on the outer surface of the flexible container rather abruptly, and a corresponding rapid pressure drop in the pressure reservoir chamber. As will be understood by those skilled in the art, the pressure equilibration achieved by instantaneous pressure equilibration is a dynamic equilibration in that the pressures in the pressure reservoir chamber and the substrate chamber, which are essentially the same after equilibration, decrease slightly over time as the respective liquids flow out of the flexible container, thus decreasing the overall volume of the flexible container and increasing the gas space in the substrate chamber. In one of the preferred embodiments, no additional pressurized gas is supplied to the pressure reservoir chamber or the device during pressure equilibration or during the subsequent flow of liquid from the flexible container to the static mixer, so that the equilibration pressures in the pressure reservoir chamber and the substrate chamber change only in response to the flow of liquid from the flexible container. In an alternative embodiment, an additional amount of pressurized gas is supplied at a time after the flow of liquid has begun.

[0022] In some preferred embodiments, the total equilibrium pressure drop caused by the flow of liquid from the flexible container to the static mixer is about 10% or less of the initial equilibrium pressure. In this way, the pressure drop during the mixing process cannot 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 to flow to the static mixer. For example, the internal volume of the pressure reservoir chamber can be at least about 10 times the volume of the liquid initially held by the flexible container, or the internal volume of the pressure reservoir chamber can be at least about 15 times, or even about 20 times or more, the volume of the liquid initially held by the flexible container.

[0023] Instantaneous equilibrium can be achieved by changing the state of the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber from its closed state to its open state. As understood in this context, the closed state means a completely closed state, such as a state of a fully closed valve that substantially prevents fluid flow, while the open state means a fully open state that allows substantially unrestricted fluid flow. In one of the preferred embodiments, the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber only has these two states, namely a fully closed state and a fully open state. In other words, this embodiment does not have an intermediate state, such as a pressure adjustment function, or if it does, such an intermediate state is not used when performing the process described herein. Therefore, in one of the preferred embodiments, neither the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber nor the connector generally comprises a pressure adjustment member.

[0024] Optionally, the open state also comprises a means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber in the open state, the means being at least about 1 mm apart from each other. 2 , or at least about 2 mm 2, 3mm 2 , 4mm 2 , or even 5mm 2 The pressure vessel may be characterized as having a fluid path for pressurized gas having a relatively large cross-sectional area, such as a pressure vessel.

[0025] The means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber may be integrated within the connector or may be located as part of the outlet of the pressure reservoir chamber or the inlet of the substrate chamber, where the connector provides fluid communication between the pressure reservoir chamber and the substrate chamber.

[0026] In one of the preferred embodiments, the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber are the only means for controlling the flow of pressurized gas between the pressure reservoir chamber and the respective substrate chamber.

[0027] A particular advantage of the present invention is that the pressure in the pressure reservoir chamber and the volume ratio of the pressure reservoir chamber to the substrate chamber of each supply module may be preselected to achieve a desired pressure in the substrate chamber and thereby a desired flow rate at which a given liquid is squeezed 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 may 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 upon opening the valve of the connector between these chambers) will be identical, and the resulting pressure in the chamber may 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. The actual flow rate of the liquid discharged from the flexible container contained in the substrate chamber will of course also 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 obtain a particular flow rate can be easily determined experimentally. Once that is known, the feed module can be easily configured to achieve such target pressure, i.e., by calculating and selecting a particular chamber volume and starting pressure of the pressure reservoir chamber. If the batch size changes and the volume of the flexible container and substrate chamber changes, the feed module can be easily reconfigured to achieve the same target pressure and the same 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.

[0028] Particularly useful embodiments are based on the selection of particular static mixing devices, chambers, valves, vessels, pressure ranges, and other features, as described below. In general, it should be understood that these preferences can be combined with each other, unless the context dictates otherwise.

[0029] In one of the preferred embodiments, the static mixer comprises or represents a T-piece mixer, a Y-piece mixer, a vortex mixer, a baffle-based static mixer, a microfluidic mixing device, a multi-inlet vortex mixer (MIVM), or a jet impingement reactor. As used herein, a T-piece mixer and a Y-piece mixer are mixing devices that comprise a T-piece or a Y-piece, respectively, and function to mix two liquids together within such a T-piece or Y-piece. A static vortex mixer is a precisely designed device for continuous mixing of liquids, typically based on a baffle-like structure shaped to create a vortex, which is a region within the liquid mixture where the flow rotates around an axis parallel to the overall flow direction. Thus, such a vortex mixer can also be understood as a special type of baffle-based 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 may 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 contacting the substrates with each other. A multi-inlet vortex mixer (MIVM) is a special type of static vortex mixer with more than two fluid inlets.

[0030] The function of a jet impingement reactor involves the injection of two fluid streams, e.g., a first stream of a first liquid and a second stream of a second liquid to be mixed, into a reactor cavity through a nozzle such that the streams collide in a turbulent mixing zone. Preferably, the first and second streams are injected from diametrically opposed positions in the reactor such that the streams collide substantially forward, i.e., substantially at an angle of about 180°. Examples of jet impingement reactors include confined impingement jet (CIJ) reactors and microjet reactors (MJR).

[0031] In one of the preferred embodiments (see also FIG. 4 ), the static mixer is a jet impingement reactor (31) having a mixing chamber (36) defined by an inner surface (32) of a mixing chamber wall (33), the mixing chamber (36) having a substantially spheroidal overall shape, the mixing chamber (36) preferably being - first and second fluid inlets (34), the first and second fluid inlets (34) being arranged at opposing positions facing each other on a first central axis (x) of the reaction mixing chamber (36), each of the first and second fluid inlets (34) comprising a nozzle (35); a fluid outlet (37) arranged at a third location, said third location being located on a second central axis (y) of said chamber (36), said second central axis (y) being perpendicular to the first central axis (x), The distance (d) between the nozzle (35) of the first fluid inlet (34) and the nozzle (35) of the second fluid inlet (34) is equal to or smaller than the diameter of the mixing chamber (36) along the first central axis (x). Furthermore, such a jet impingement reactor may have further features as described in co-pending European Patent Application No. 21192535.9, which is incorporated herein by reference.

[0032] As mentioned above, the apparatus comprises conduits for providing fluid communication between the interior space of the flexible container and the respective inlets of the static mixer. Specifically, the conduits of the first supply module provide a fluid connection between the interior space of the flexible container holding the first liquid and the first inlet, and the conduits of the second supply module provide a fluid connection between the interior space of the flexible container holding the second liquid and the second inlet. The conduits may comprise or represent, for example, tubes that may be flexible, such as tubes made of elastomeric (polymeric) material, or may be rigid, such as metal tubes.

[0033] In one embodiment, the conduits of the first and / or second supply modules comprise means for preferably reversibly blocking the fluid communication between the interior space of the flexible container and the respective inlet of the static mixer. Optionally, the means are valves. If the conduits are flexible tubes such as plastic tubing, the fluid communication may alternatively be initially blocked by a tube clamp, in which case the respective fluid connection is created by opening or removing the clamp, allowing fluid to flow from the flexible container to the static mixer when the substrate chamber is pressurized.

[0034] In one embodiment, there are no means for blocking the fluid connections arranged in the conduits of the first and / or second supply modules. The conduits may comprise a connection achieved, for example, by engagement of a connecting piece initially introduced as part of the outlet of the respective flexible container with a complementary connecting piece in the downstream part of the conduit leading to the respective inlet of the static mixing device.

[0035] To initiate pressurization of the substrate chamber, the means for reversibly blocking fluid communication between the pressure reservoir chamber and the substrate chamber is opened or removed, thereby opening or freeing the connector to create said fluid connection. As used herein, a connector is any piece, conduit, pipe, or tube that can allow pressurized gas to flow from the pressure reservoir chamber to the substrate chamber or between these chambers. The means may be a clamp or a valve if the connector material is flexible. In one of the preferred embodiments, the means is a valve.

[0036] The pressure reservoir chamber may be designed to be relatively large in order to achieve sufficient pressure in the substrate chamber without the need to pressurize the pressure reservoir chamber very highly before fluidly connecting the two chambers, for example by opening a valve of the connector. For example, the pressure reservoir chamber may have a larger volume than the substrate chamber with which it is in fluid communication. 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 one embodiment, the ratio of the volume of the pressure reservoir chamber to the volume of the substrate chamber is at least about 10, and optionally ranges from about 10 to about 100, for example from about 20 to about 50. For example, the pressure reservoir chamber may have an internal volume of 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.

[0037] In a further embodiment, the pressure reservoir chamber has a substantially cylindrical or columnar overall shape.

[0038] As will be appreciated by those skilled in the art, preparation of the apparatus for producing a batch of a liquid mixture (i.e., a third liquid) by mixing two liquid substrates (i.e., a first 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 that the pressure reservoir chambers should have at the start of the batch production, is selected with reference to the volume of that chamber and the corresponding substrate chamber, also taking into account the viscosity of the respective liquids, the flow resistance of the flow paths, and the desired flow rates. A typical initial pressure in the pressure reservoir chamber may be, for example, in the range of about 2 bar to 40 bar. Other pressures may be used depending on the choice of pressurized gas used.

[0039] Further, the pressure may be selected to force the first and second liquids out of the containers into their respective conduits and direct them to the static mixer at a flow rate in the range of about 10 to about 200 ml / min.

[0040] 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. Among the preferred gases are nitrogen, air, and carbon dioxide.

[0041] The (pressurized) gas may be filled into the pressure reservoir chamber through its connector, which is then fixed or connected to the respective substrate chamber. In one of the preferred embodiments, the pressure reservoir chamber comprises an inlet for the pressurized gas. This inlet may optionally comprise a means for closing the inlet, such as a valve. Furthermore, the inlet may optionally be independent of the connector. In this context, independent means functionally independent, which may be attached to or associated with the connector. For example, the connector may have a three-way valve connected to the inlet for the pressurized gas. The valve may have a first position in which there is no fluid communication between any of the gas inlet, the pressure reservoir chamber, and the substrate chamber, a second valve position that opens the fluid communication only between the gas inlet and the pressure reservoir chamber, and a third position that opens the fluid communication only between the pressure reservoir chamber and the substrate chamber. In an alternative preferred embodiment, the inlet is positioned independently of the connector and is equipped with a separate valve.

[0042] The inlet is preferably connectable to a pressurized gas source, such as by a pressure-resistant fitting. In a further embodiment, the inlet for pressurized gas is connected to a pressurized gas source. Optionally, the connection to the pressurized gas source is equipped with a pressure reducing means, such as a pressure regulator, e.g. a gas pressure regulating valve.

[0043] The pressure reservoir chamber may comprise a means for indicating pressure. This may be, for example, a pressure gauge or other means for measuring and indicating pressure as known in the art. Alternatively, the means for indicating pressure may be attached to a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber.

[0044] All of the embodiments and optional features relating to the gas inlets described above may be used for one or both of the supply modules. The device may optionally have an asymmetric configuration of the gas inlets, for example the gas inlet of the first or second supply module may be attached to the reservoir chamber and the gas inlet of the second or first supply module may be attached to the connector.

[0045] As mentioned above, the substrate chamber is adapted to hold a flexible container containing the first or second liquid. To facilitate the insertion and / or removal of such a flexible container, the substrate chamber of the first or second supply module may be provided with a means for opening and closing it. Preferably, both substrate chambers exhibit this feature. For example, the substrate chamber may comprise a two-piece wall or housing, e.g. a main part and a lid-like part, and a means for hermetically fixing the lid to the main part.

[0046] To ensure correct insertion of the container into the substrate chamber, the flexible containers holding the first and / or second liquids may be tagged with an identification means. This identification means may for example be an RFID tag. In this case, it is advantageous to equip each substrate chamber with a sensor adapted to recognize the identification means, such as an RFID reader. Optionally, the RFID reader may communicate with a controller that enables or disables the operation of the device, such that the device can be operated to perform the batch process only upon correct insertion of the designated flexible container.

[0047] According to further preferred embodiments, the dimensions of each substrate chamber are selected such 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 may be filled 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 substrate chamber, with at least about 30%, or at least about 50% of the total internal volume of the respective substrate chamber. As used herein, the total internal volume of the chamber, which is the basis of 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 substrate chamber.

[0048] At the same time, the dimensions of the two flexible containers holding the first and second liquids, respectively, may be different from each other. This results from a somewhat typical situation where one of the two liquids must be provided at a larger volume or flow rate than the other liquid in order to obtain the desired product. It is therefore another preferred embodiment that 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 be different from each other. In one embodiment, the difference between the internal volume of the substrate chamber of the first feed module and the internal volume of the substrate chamber of the second feed module is a factor ranging from about 1.5 to about 10, or from about 2 to about 5. In this context, the internal volume means the total internal volume of the respective chamber, i.e. when empty.

[0049] As mentioned above, the flexible container is intended to hold the liquid substrate, i.e. the first and second liquids, from which the third liquid is formed by mixing, using the device of the invention. Preferably, the containers are flexible to the extent that they are at least partially collapsible. In other words, their internal volume can vary substantially depending on the shape of the container walls at a given moment.

[0050] 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 mentioned above, one of the advantages of the present invention is that it allows for 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.

[0051] In one of the further preferred embodiments, the components of the device are set and / or oriented such that the liquids processed in the device or obtained by use of the device have an overall flow direction that is in an upward direction, i.e. against gravity. For example, the 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 at a higher position than their upstream ends. For the avoidance of doubt, the upstream ends of the conduits are the ends connected to the flexible container and the downstream ends of the conduits are connected to the first or second inlets of the static mixer. In this context, a higher position means a position above a relatively lower position with respect to the horizontal axis of the device in its normal operating orientation.

[0052] Similarly, the static mixing device or static mixer may be oriented such that its outlet is at a higher position compared to its inlet. This orientation of the static mixer in combination 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 in an upward direction. Such a 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 invention.

[0053] Optionally, the inlet of the products vessel may also be located higher relative to the outlet of the static mixer.

[0054] According to a further preferred embodiment, the device is adapted for a completely aseptic process. 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 in the device, i.e. the third liquid. Thus, the outlet of the static mixer is preferably fluidly connectable or actually connected to a container for receiving the third liquid, said container being optionally a flexible container. The connection may involve a sterile tube between the outlet of the static mixer and the container. This product container is also called the first container for receiving the third liquid. To avoid any doubt, the expression "first container" does not imply any processing sequence, for example, if the device is arranged with a first and a second container for receiving the third liquid, the process may be carried out such that the second container receives an amount of the third liquid before the first container.

[0055] Thus, the product container or the first container may be a sterile flexible bag that is 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, especially microbiological contaminants. Those skilled in the art will understand 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 tube to seal the product container and then disconnecting the container upstream of the clamp.

[0056] Optionally, the connection between the outlet of the mixing device and the product container may be equipped with 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 optionally used in other locations in the device, such as in the inlet for pressurized gas, or in a conduit for 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 at the inlet of the flexible container and provide for the aseptic filling of the secondary state 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.

[0057] In a further preferred embodiment, the device comprises a 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 a means for interrupting this fluid connection. Both electrically and pneumatically actuated pinch valves are suitable in the context of implementing the invention according to this embodiment.

[0058] 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 said 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 change the composition of the third liquid after discharge from the static mixer by adding one or more further components. Typically, these one or more further components are added in liquid form, i.e. in the form of a liquid diluent. The second fluid inlet allows the introduction of such a liquid diluent either before or after the first container receives the third liquid.

[0059] It should be noted that the liquid diluent may have other functions, or even a completely different important function than diluting the third liquid. For example, the liquid diluent may serve to adjust the pH of the product to a particular value, and for this purpose may contain a pH adjuster, such as an acid, a base, a buffer salt, or a buffer system. Alternatively or additionally, the liquid diluent may contain a tonicity agent, such as sodium chloride, a sugar, a sugar alcohol, or any other osmotically active compound. It may also contain a lyophilization aid, such as a sugar or a 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 agents, and / or lyophilization aids may also be used.

[0060] To facilitate aseptic introduction of a liquid diluent into the container through one of the fluid inlets, an in-line sterile filtration means may be disposed upstream of the respective fluid inlet. Such a sterile filtration means typically comprises a filter housing and a filter, such as a filter membrane having an effective pore size of, for example, 0.2 or 0.22 μm. After aseptic introduction of the liquid into the container, the sterile filtration means may be aseptically removed by melt clamping, or the inlet upstream of the filtration means may be melt sealed while simultaneously removing the filtration means.

[0061] In addition to the product container or the first container, there may also be a second container for receiving a third liquid, i.e., its amount. In other words, the outlet of the static mixer may be fluidly connectable to a 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-piece. An exemplary arrangement with two containers downstream of the static mixer is shown in FIG. 7.

[0062] One particular advantage of such an arrangement is that the second vessel may be used for product waste, such as a product obtained at the very beginning of the operation of the apparatus or at the end of a batch process. This may be particularly useful where there is a risk that the product will only achieve its target specifications after the beginning stage or before the beginning stage of a batch process.

[0063] In order to use both the first and second containers without interrupting the mixing process, means may 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 of the preferred embodiments, the device is configured to have both the first and second containers in fluid connection with the outlet of the static mixer for receiving the third liquid, and the means for reversibly interrupting the fluid connection are arranged upstream of each of the two containers and at a location where the fluid paths to the two containers split, for example downstream of a T-piece if used.

[0064] Again, a pinch valve is also suitable as a means for blocking the fluid connection between the static mixer outlet and a 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 invention according to this embodiment.

[0065] 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. Thus, the device may comprise 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 liquids, and without the need to withdraw a sample of the third liquid for measurement purposes.

[0066] In one embodiment, the means for in-line particle size measurement is located at the downstream portion of the static mixer, at the outlet of the static mixer, or downstream of and in fluid connection with the outlet of the static mixer, which should be understood such that at least a sensor that detects a signal from which particle size is determined is located at one of the specified locations.

[0067] The means for in-line particle size measurement may comprise a transparent sensing or measurement 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 measurement window may be provided in the form of a capillary tube made of glass or plastic.

[0068] These features allow for in-line particle size measurement of the third liquid by optical methods such as light scattering or laser diffraction. To detect the signal emitted or returned from the third liquid during its flow in the device, a transparent portion or window must be present in one of the walls of the structure through which the third liquid flows. Such a transparent portion or window may be downstream of and in fluid connection with the outlet of the static mixer, such as in the downstream portion of the static mixer, at the outlet of the static mixer, or near the outlet of the static mixer, and, if two containers for receiving the third liquid are used, preferably upstream of the location where the fluid paths to these two containers separate. For example, a capillary tube made of glass or transparent plastic may form part of the structure through which the third liquid passes from the static mixer to the product container. Such a capillary tube may be part of the mixer itself. Indeed, according to one of the embodiments of the present invention, the static mixing device is made of glass or plastic. Also preferred is a static mixing device made of transparent glass or plastic. In this context, transparent means sufficiently transparent to allow the transmission of an optical signal for particle size measurement.

[0069] Those skilled in the art will understand that the guidance provided herein referring to the location of the means for in-line particle size measurement refers primarily 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 a specific distance to the specified location.

[0070] In one embodiment, a transparent region, e.g., a capillary made of glass or plastic, has an inner surface coated with a layer of a material that exhibits low protein or nucleic acid binding. Alternatively, the capillary may be made of a type or grade of glass or plastic that exhibits low protein or nucleic acid binding.

[0071] As mentioned above, it may be desirable to position and orient the device components so that the main 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 container 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 inlet of the first and / or second container 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 container.

[0072] Since 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 further comprising an outlet for withdrawing the liquid. This is particularly true for the first container.

[0073] In a further aspect, the invention provides a method for mixing a first liquid and a second liquid, such as to obtain a third liquid, the method being essentially characterised in that an apparatus as described above is used for mixing the liquids.

[0074] 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 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; and (cc) providing (i) a first inlet for receiving the first liquid, (ii) a second inlet for receiving the second liquid. and (iii) an outlet for discharging a third liquid resulting from mixing of the first and second liquids, (dd) independently pressurizing the first and second pressurizable substrate chambers with a pressurized gas that applies pressure to an exterior surface of each of the first and second pressurizable substrate chambers to force the first and second liquids through 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 by a pressurized gas.

[0075] As one skilled in the art will appreciate, steps (aa), (bb), and (cc) may be performed in any order, and optionally simultaneously. Steps (aa), (bb), and (cc) followed by steps (dd) and (ee) may be performed essentially simultaneously or with a substantial overlap in time, in that step (ee) may be started and ended with a slight delay after the start and end of step (dd).

[0076] 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.

[0077] As noted above, step (dd) may include a sudden or rapid increase in pressure in each of the first and second pressurizable substrate chambers. In a preferred embodiment, the pressure in the substrate chambers increases from the initial pressure to the maximum process pressure in less than about 2 seconds, preferably less than about 1 second.

[0078] The abrupt nature of this pressure rise may also be described in relation to the duration of the subsequent step (ee) of collecting the third liquid. In a preferred embodiment, the duration of the abrupt pressure rise in each of the first and second pressurizable substrate chambers to the initial equilibrium pressure is less than about 10 percent of the duration of step (ee), or less than about 5 percent, less than about 2 percent, or even less than about 1 percent of the duration of step (ee). As will be appreciated, the duration of step (ee) substantially reflects the period during which the third liquid is actually generated in the static mixing device from the mixing of the first liquid with the second liquid.

[0079] In further related embodiments, the initial pressure is about ambient pressure and the maximum process pressure is in the range of about 1 to 16 bar. Maximum 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, are also preferred.

[0080] As mentioned above, pressurization of the substrate chambers is achieved by pressurized gas exerting pressure on the outer surface of each substrate chamber. In a preferred embodiment, this pressurized gas is provided by a pressure reservoir chamber, as already explained. In this case, the maximum process pressure in the substrate chamber corresponds to the initial equilibrium pressure reached upon pressure equilibrium between the pressure reservoir chamber and the respective substrate chamber.

[0081] Thus, in a related embodiment, each of the first and second pressurizable substrate chambers are connected to a first and second pressure reservoir chamber for holding pressurized gas by a connector for providing fluid communication between the pressure reservoir chambers and the respective pressurizable substrate chamber, and means are arranged for reversibly blocking fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber, said means having an open state and a closed state, and prior to carrying out the pressurizing step (dd), each pressure reservoir chamber is in a pressurized state such that its pressure is about 2-20 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. For the avoidance of doubt, it is to be understood that 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. The first means is arranged to reversibly block fluid communication between the first pressure reservoir chamber and the first pressurizable substrate chamber, which may be associated with the first connector, and the second means is arranged to reversibly block fluid communication between the second pressure reservoir chamber and the second pressurizable substrate chamber, which 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".

[0082] In a further related embodiment, the pressurizing step (dd) comprises the substeps of (i) changing the state of the means for reversibly blocking fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber from closed to open to generate a pressure equilibrium between each pressure reservoir chamber and the respective pressurizable substrate chamber within a period of about 2 seconds or less, and (ii) forcing essentially the entire amount of said first and second liquids from their respective flexible containers to flow through a static mixer to generate a third liquid over a period of at least about 5 seconds, preferably at least 10 seconds, for example from about 10 seconds to about 15 minutes. Those skilled in the art will appreciate that the duration of the liquid flow will vary depending on the batch size as well as the flow rate.

[0083] In further embodiments, no additional pressurized gas is supplied to any of the pressure reservoir chambers during steps (dd) and (ee). Thus, the equilibrium pressure is the main driving force for the first and second liquids to flow through the static mixer, depending on the geometric orientation of the device, particularly in the absence of any contribution of gravity to the flow of the liquids. As mentioned above, in some of the preferred embodiments, the device comprises 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 such that it is at a higher position than the upstream end. In other words, at least the flexible container and the static mixer are arranged such that the flow of the liquid occurs in an upward direction.

[0084] With regard to optional or preferred features of the static mixer, chamber and other device features, reference is made to the disclosure above. In other words, the embodiments and preferences provided for the apparatus provided according to the present invention are also applicable to the method, so that preferred embodiments of the method are characterized by the use of preferred apparatus features to perform the mixing of two fluids. Furthermore, optionally preferred process features are specifically described in the context of the apparatus, as necessary to explain its function.

[0085] For example, in one of the preferred embodiments, as described above, the first container for receiving the third liquid, which may also be called the product container, is configured to receive a liquid diluent either before or after receiving the third liquid. To fill the container with the liquid diluent, the container may at least initially comprise an additional inlet and a means for in-line sterile filtration associated with this inlet. Thus, the method of the present invention may comprise a step of filling the first container with the liquid diluent, preferably through the additional inlet of the container and a sterile filter associated with this inlet, before or after the third liquid is received in said first container. The liquid diluent is preferably an aqueous liquid composition. As mentioned, it may comprise a pH adjuster, a tonicity agent, a lyophilization aid, or any combination thereof.

[0086] Whether or not the third liquid collected in the first (or product) vessel downstream of the static mixer is combined with a liquid diluent, it may continue with further processing steps, for example to further purify or characterize the product, or to make the product more stable and easier to handle, store, and transport. Preferably, such further processing is also carried out under sterile conditions.

[0087] For example, further processing may include steps of 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 may be useful for the purpose of concentrating the product, 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 to remove or reduce the concentration of certain solutes, such as free molecules of biologically active components, i.e. molecules that are not incorporated in liposomes or lipid nanoparticles. Freezing or lyophilization may be useful to convert 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. As mentioned above, the device may comprise a means for in-line particle size measurement of the third liquid. Thus, one of the preferred embodiments of the method of the present invention is also to take advantage of this device feature to perform a step of performing an in-line particle size measurement on the third liquid before said third liquid is received by the first container. In this way, it can be ensured that the key product parameter, i.e. the target particle size, is actually achieved by the product in the third liquid form as it is collected in the product container. This embodiment is particularly relevant for the production of small batches of liquid products that include liposomes or lipid nanoparticles.

[0088] The in-line particle size measurement step may also be advantageous when operating in a further preferred embodiment of the device in which there is a second container for receiving the third liquid. In terms of 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. The collection of the part or parts of the third liquid in the second container may be brought about by interrupting the fluid connection between the outlet of the static mixer and the first container for receiving the third liquid.

[0089] For example, an initial portion of the third liquid as it exits the static mixer through its outlet may be directed into a second vessel that may function as a waste vessel, followed by a subsequent portion of the third liquid into the first vessel that may function as a product vessel. Optionally, yet another subsequent portion of the third liquid may be directed again into the second vessel, for example toward the end of the batch production. In this manner, it is possible to selectively collect a portion of the third liquid, which eliminates material generated during the beginning or subsequent stages of the process and thus best represents the target product quality.

[0090] In another preferred embodiment, the apparatus features both an arrangement with means for in-line particle size measurement, as well as an arrangement with first and second containers for receiving a third liquid, and also presents means, e.g., 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, e.g., for controlling the pinch valves to block or open the respective fluid connections in response to the particle size measurement.

[0091] With respect to the method of the present invention, this apparatus arrangement and configuration may be used such that an in-line particle size measurement is performed 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 operates such that there is an open fluid connection between the outlet and a first container used as a product container while the fluid connection to the second container is blocked, or such that there is an open fluid connection between the outlet and a second container used as a waste container while the fluid connection to the first container is blocked.

[0092] In other words, according to this embodiment, the method includes performing an in-line particle size measurement on the third liquid during steps (dd) and (ee), and if the in-line 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 in-line 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.

[0093] For example, in the initial stages of steps (dd) and (ee), the particle size measured in the third liquid may not yet meet the pre-set target criteria, so a first amount of the third liquid should be directed to a waste container, which is achieved by the respective settings of the pinch valves. Once the desired particle size characteristics are reached, the valve settings are changed, whereby the third liquid is now directed to a product container.

[0094] The method of the present invention may be used to create particles, such as microparticles or nanoparticles, of poorly water-soluble compounds, for example by flash precipitation. For this purpose, one of the liquid substrates, for example 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, so that the mixing of the two liquids according to the present invention results in the precipitation of the poorly soluble compound in the form of microparticles or preferably nanoparticles. Similarly, ionizable poorly soluble compounds may 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 mainly 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.

[0095] 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 method may be performed such that polymer particles comprising the biologically active component are produced.

[0096] 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 by mixing the first and second liquids, and the biologically active agent is associated with and / or encapsulated within the lipid nanoparticles.

[0097] Preferably, two or more lipids are used in the first liquid.For example, an organic (e.g. ethanolic) solution of a combination of lipids known to be useful for making lipid nanoparticles (LNPs) may be used.These lipids may include cationic or cationizable lipids, PEGylated lipids, structured lipids, and cholesterol.

[0098] As used herein, unless the context dictates otherwise, a cationic lipid is a lipid that contains a positive charge in an aqueous environment of any pH, e.g., a lipid having a quaternary nitrogen atom (i.e., an ammonium moiety), whereas a cationizable lipid is a lipid that contains a positive charge only in an aqueous environment of neutral or acidic pH, e.g., a lipid that displays a primary, secondary, or tertiary amine.

[0099] In specific embodiments, the cationic or cationizable lipid is N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), N4-cholesteryl-spermine HCl salt (GL67), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-distearoyl-3-dimethylammonium propane (18:0DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0DAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0DAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODA P) (18:1 DAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), 1,2-dioleoyloxy-3-trimethylammonium-propane (chloride salt) (18:1 TAP or DOTAP), 1,2-stearoyl-3-trimethylammonium propane (chloride salt) (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride salt) (16:0 TAP), 1,2-dimyristoyl-3-trimethylammonium propane (chloride salt) (14:0 TAP), dimethyldioctadecylammonium (bromide salt) (18:0 DDAB), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt) (14:1 EPC Tf salt), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (16:0-18:1 EPC Cl salt), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:1 EPC Cl salt), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:0 EPC Cl salt), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (16:0 EPC Cl salt), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (14:0 EPC Cl salt), 1,2-Dilauryl-sn-glycero-3-ethylphosphocholine (chloride salt) (12:0EPC Cl salt), O,O'-Ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol·HCl), 4-(Dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl yl ester (D-Lin-MC3-DMA), ([(4-hydroxybutyl))azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)) (ALC-0315), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200 ), cetyl-trimethylammonium bromide (CTAB), penta-amine N15-cholesteryloxycarbonyl-3,712-triazapentadecane-115-diamine (CTAP), bis-guanidinium-spermidine-chol (BGSC), bis-guanidinium-trenic (BGTC), N,N-distearyl-N-methyl-N-2[N'-(N2-guanidino-L-lysinyl)]aminoethyl ammonium chloride ( DSGLA), O-(2R-1,2-di-O-(1Z,9Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)carbamate) (BTCA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), N-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N'-{2-[N,N-bis(2-aminoethyl)-amino]ethyl}-2,2-ditetradecylpropanediamide (DiTT4), cholesteryloxypropan-1-amine (COPA), and cholesteryl-2-aminoethylcarbamate (CAEC). Optionally, the first liquid comprises a mixture of two or more of the above cationic or cationizable lipids. The cationic or cationizable lipids can interact with negatively charged oligonucleotides and polynucleotides, aiding in the association and / or encapsulation of such nucleotides within or with the lipid nanoparticles.

[0100] As used herein, PEG means polyethylene glycol, and a PEGylated lipid is a lipid conjugated with a PEG moiety.In a specific embodiment, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), distearoyl-rac-glycerol-PEG2K (DSG-PEG2000), 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-2000-DMPE Na), N-(carbonyl-methoxypolyethylene glycol 750)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-750-DSPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](ammonium salt) (DSPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](ammonium salt) (16:0 PEG2000 PE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000](ammonium salt) (14:0 PEG2000 PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000](ammonium salt) (18:1 PEG2000 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000](ammonium salt) (18:0 PEG2000 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000](ammonium salt) (18:0 PEG5000 PE), N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-2000-DPPE Na). Optionally, the first liquid composition comprises a mixture of two or more of the above PEGylated lipids.In one of the preferred embodiments, the first liquid comprises only one PEGylated lipid.Optionally, the PEGylated lipid is the same as the cationic or cationizable lipid.In an alternative and more preferred embodiment, the first liquid comprises one PEGylated lipid that is not cationic or cationizable and one PEGylated lipid that is not PEGylated and one cationic or cationizable PEGylated lipid.

[0101] The structured lipid is preferably a non-PEGylated zwitterionic lipid. In a specific embodiment, the non-PEGylated zwitterionic lipid is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (18:1 (Δ9-cis)PE or DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA, Na), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. (16:0PC or DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (1,2-POPE), 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC), and trans-2-aminoacyclohexanol (TACH). Optionally, the first liquid comprises a mixture of two or more of the non-PEGylated zwitterionic lipids described above. In one of the preferred embodiments, it comprises only one non-PEGylated zwitterionic lipid.

[0102] More preferably, the first liquid comprises a water-miscible solvent in which lipid is dissolved.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.One of the particularly preferred water-miscible solvents is ethanol.

[0103] In a more preferred embodiment, the first liquid consists essentially 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 a specific embodiment, the first liquid composition consists essentially of a solution of one or more lipids in ethanol.

[0104] In some embodiments, the first liquid composition comprises: - 40 to 60 mol % of a cationic or cationizable lipid; -0.5 to 2 mol% PEGylated lipid; -5 to 20 mol % of a non-PEGylated zwitterionic lipid; -30 to 50 mol% cholesterol, wherein the percentage is based on the total lipid content in the first liquid.

[0105] When this method is used to prepare lipid nanoparticles, it is more preferred that the biologically active agent is an oligonucleotide or polynucleotide.According to another preferred embodiment, the oligonucleotide or polynucleotide is an optionally modified mRNA molecule that comprises the nucleic acid sequence that codes for an antigen, in particular a tumor antigen, a virus antigen, a bacterial antigen, a fungus antigen or a protozoan antigen.

[0106] A particular advantage of the device according to the invention is that it allows for rapid and flexible production of small batches under sterile conditions. After production of a batch, the device is easily reconfigured and prepared for production of another batch of the same or different material. No pumps are involved in flowing and mixing the first and second liquids to form the third liquid. Few structures are in direct product contact and these may be easily replaced. Parts that require sterilization prior to production of a batch are easily sterilized.

[0107] For example, the method of the invention may comprise a step of sterilizing the static mixer before step (dd) is performed, i.e. before pressurizing the first and second pressurizable chambers to force said first and second liquids through the static mixer and cause mixing of the two liquid substrates. Sterilization may be carried out by any sterilization method compatible with the material from which the mixing device is made. For example, sterilization may be carried out using steam. In one of the preferred embodiments, sterilization is carried out by gamma irradiation. This is also true when the mixing device is made of glass or plastic, in particular when the glass or plastic mixing device is a jet impingement reactor as described in more detail above.

[0108] According to a further aspect, the present invention provides the use of a pressurized gas for forcing a first liquid to flow from a flexible container to a static mixer, said liquid being mixed with a second liquid to form a third liquid, said pressurized gas being provided to suddenly apply a pressure of 1 bar to 16 bar to the outer surface of said flexible container without being in fluid communication with said first liquid. Preferably, the pressurized gas is also used in the same way to force a second liquid to flow from a (second) flexible container to a static mixer. In one of the specific embodiments, the pressurized gas is provided to suddenly apply a pressure of 2 bar to 12 bar to the outer surface of said flexible container.

[0109] In some preferred embodiments, the static mixer is part of the apparatus described above, the use further involving one or more of the features described in the context of the methods disclosed herein.

[0110] In a further aspect, the present invention provides the use of the above-mentioned device or method for the production of lipid nanoparticles.

[0111] The drawings included in this disclosure illustrate certain embodiments of devices provided in accordance with the present invention. None of the drawings are to scale.

[0112] Figure 1 shows the general configuration of the apparatus according to the invention. In detail, an apparatus (1) for mixing two liquids is shown using a static mixing device (2) having a first inlet (3) for receiving a first liquid (9), a second inlet (4) for receiving a second liquid (17), an outlet (5) for discharging the liquid mixture (product) and a first supply module (6) for providing the first liquid (9) to the first inlet (3). The first supply module (6) comprises a substrate chamber (7) for holding a flexible container (8), the flexible container (8) having an interior space for holding a first liquid (9), a conduit (10) for providing fluid communication between the interior space of the flexible container (8) and the first inlet (3), a first pressure reservoir chamber (11) for holding a pressurized gas, and a connector (12) with a valve (13) for providing and reversibly blocking fluid communication between the pressure reservoir chamber (11) and the substrate chamber (7). The apparatus further comprises a second supply module (14) for providing a second liquid (17) to the second inlet (4). The second supply module (14) comprises a second substrate chamber (15) for holding a second flexible container (16), the second flexible container (16) having an interior space for holding a second liquid (17), a conduit (18) for providing fluid communication between the interior space of the second flexible container (16) and the second inlet (4), a second pressure reservoir chamber (19) for holding pressurized gas, and a connector (20) providing fluid communication between the second pressure reservoir chamber (19) and the second substrate chamber (15) and with means (21) for reversibly blocking the fluid communication.

[0113] Figure 2 shows an apparatus similar to that described in Figure 1, further comprising valves (26, 27) between the interior space of the flexible container (8, 16) and the inlets (3, 4) of the static mixer (2). In this embodiment, the outlet (5) is provided with a means (28) for reversibly blocking the discharge of the liquid mixture (product) from the static mixing device (2).

[0114] Figure 3 shows a similar device further comprising means for indicating pressure (24, 25). Furthermore, the pressure reservoir chambers (11, 19) comprise an inlet (29) for pressurized gas with means (22, 23) for closing the inlet (29). In this embodiment, the means (22) for reversibly blocking fluid communication between the second pressure reservoir chamber (19) and the second substrate chamber (15) is a three-way valve.

[0115] Figure 4 shows, not to scale, an example of a jet impingement reactor (31). It comprises a reaction chamber (36) defined by an inner surface (32) of a chamber wall (33) and is substantially spherical except for two fluid inlets (34) and a fluid outlet (37). The fluid inlets (34) are arranged at opposite positions on a first central axis (x) of the reaction chamber (36) and face each other. Each of the fluid inlets (34) comprises a nozzle (35), which in this embodiment is a plain orifice nozzle. The fluid outlet (37) is located on a second central axis (y) perpendicular to the first central axis (x). The distance (d) between the two nozzles (35) is substantially the same as the diameter of the spherical reaction chamber (36).

[0116] FIG. 5 shows an optional type of flexible container (40) holding a first or second liquid (41). In principle, however, a flexible container (40) having the same or a very similar design may also be useful as a container for receiving a third liquid. In this case, the flexible container (40) comprises an inlet (42) having a filtering means (43), such as a sterile filter, to allow the aseptic introduction of the liquid (41). Optionally, a part of the inlet (42) may be removed by a heat clamp, which closes and seals the remaining part of the inlet (42) and at the same time allows the filtering means (43) to be removed, before introducing the pre-filled flexible container (40) into the pressurizable substrate chamber. Furthermore, an outlet (44) is provided through which the liquid (41) may flow from the flexible container (40) to the inlet of a static mixing device. The downstream end of the outlet (44) presents a connecting piece (45) which may engage with a complementary connecting piece, for example to establish a fluid connection with a static mixing device. The container further comprises an identification means (46), such as an RFID code.

[0117] Figure 6 shows another version of the device (1) similar to that of figure 2, but with some further modifications. In this version, the flexible containers (8, 16) for the first and second liquids (9, 17) have an identification tag (46), such as an RFID tag, and each of the first and second substrate chambers (7, 15) has a sensor (47), such as an RFID reader, configured to read the identification tag. The flexible containers (8, 16) further comprise a closed or sealed inlet (48), i.e. a structure that remains after the fusion sealing of the original inlet. The conduit between the flexible containers (8, 16) and the inlets (3, 4) of the static mixer (2) is arranged without a valve. An engaging connection piece (45) is shown, which serves to ensure that a fluid connection between the flexible containers (8, 16) and the inlets (3, 4) is established or maintained. The container (30) for receiving the third fluid is shown as a flexible container. The remaining features are similar to figure 2.

[0118] Figure 7 shows a further alternative version of the apparatus (1). In this version, the general direction of flow of liquid from the flexible container (8, 16) in the pressurizable substrate chamber (7, 15) through the static mixing device (2) to the container (30, 49) for receiving the third liquid is upwards, i.e. against gravity or from a lower to a higher position. The inlets (3, 4) of the static mixer (2) are higher than the outlets (10, 18) of the flexible containers (8, 16) holding the first and second liquids (9, 17). The orientation of the static mixer (2) itself is such that the outlet (5) is higher than the inlets (3, 4) and the inlet of the container (30, 49) for receiving the third liquid is higher than the outlet (5) of the static mixer. The first container (30) for the third liquid, which may serve as a product container, is pre-filled with a certain amount of liquid diluent. A second container (49) for a third liquid, which may serve as a waste container, is also fluidly connected to the outlet (5) of the static mixer (2) as is the first container (30). Means (28) for reversibly blocking the fluid connection to the outlet (5) of the static mixer (2) are arranged in both the first container (30) and the second container (49). Other features shown here have already been described.

[0119] FIG. 8 shows another version of the device (1). In this case, the second container (49) for receiving the third liquid, which may serve as a waste container, already contains a portion of the third liquid, indicating that the mixing process, i.e. steps (dd) and (ee) according to the method of the invention, has already started. In this embodiment, a means for in-line particle size measurement of the third liquid is provided, which comprises a sensing window (50) arranged downstream of and in fluid connection with the static mixing device (2), allowing the transmission of an optical signal between the third liquid and an analyzer (51) by means of a sensor capable of receiving such an optical signal. The analyzer (51), typically adapted for in-line particle size measurement, may be in (e.g., electrical or wireless) communication with a controller (52), which also communicates (e.g., electrical or wireless) with two means (28), such as pinch valves, for blocking the fluid connection between the outlet (5) of the static mixing device (2) and the first container (30) or the second container (49) for receiving the third liquid, respectively. By operation of the controller (52), the portion of the third liquid whose particle size measured by the analyzer (51) through the sensing window (50) does not match the desired particle size can be directed to the second container (49), i.e., waste container, in that the controller (52) operates the valve (28) (for example) to cut off the fluid connection of the static mixer (2) to the first container (30), but not to the second container (49). Once the third liquid has the desired particle size as measured by the analyzer (51), the controller (52) can direct the third liquid to the first container (30), i.e., product container, by operating the valve (28) (for example) to cut off the fluid connection of the static mixer (2) to the second container (49), but not to the first container (30). The other features shown in FIG. 8 are the same as in the previous figures and have already been described.

[0120] FIG. 9 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 pushed over time (t) from a flexible container housed in the substrate chamber when performing a method of the invention according to one embodiment. The diagram is not to scale and uses arbitrary units only. Initially, i.e. before performing 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 the flexible container with the liquid. During this stage, the respective means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber are in their closed state, and the pressure (102) in the substrate chamber can be at or close to the ambient pressure. Step (dd) may be initiated by changing the state of the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber from closed to open, which causes a sudden pressure equilibrium (103) between the pressure reservoir chamber and the substrate chamber. It is noted that this pressure equilibrium is accompanied by a sudden drop in the pressure in the pressure reservoir chamber and a corresponding sudden increase in the pressure in the substrate chamber, which is now above the ambient pressure level. The now increased pressure (104) in the substrate chamber, i.e. the equilibrium pressure acting on the outer surface of the flexible container holding the liquid, now pushes the liquid to flow out of the flexible container towards the static mixer at a substantially constant flow rate (105). During the flow of the liquid, no further pressurized gas is introduced into the pressure reservoir chamber or the substrate chamber. The pressure in the substrate chamber after equilibrium (104) remains approximately constant, since the volume of liquid flowing out of the flexible container is very small compared to the total volume of the pressure reservoir chamber and the substrate chamber.

[0121] Depending on the particular optional process parameters or depending on the type and sensitivity of the pressure or flow sensors for measuring the 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 device or performing the process according to the invention. For example, during the 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 short peaks may be interpreted as an initial pulse or "shock wave" moving through the liquid. They do not change the overall characteristics of the process. Moreover, instead of a very slight decrease in the pressure (104) in the substrate chamber over time after the rapid equilibration, a slight increase in the pressure (104) over time may be observed. The slight decrease may result, for example, from a slight increase in the volume of the pressurized gas in the substrate chamber, while the slight increase may result from a slight temperature increase of the pressurized gas during the process. Small pressure changes over time may also result in very small changes in flow rate, even though the flow rate ratio between the first and second liquids remains substantially unaffected. Factors that may result in a small increase in pressure (104) and flow rate (105) over time may also substantially compensate for the effects of factors that may result in a small decrease in pressure (104) and flow rate (105) over time, such that pressure (104) and flow rate (105) remain substantially constant after the sudden pressure equilibration. [Explanation of symbols]

[0122] 1 device 2. Static Mixing Device 3,4 entrance 5 exit 6,14 Supply module 7,15 Substrate chamber 8,16,40 Flexible container 9,17,41 liquid 10,18 Conduit 11,19 Pressure Reservoir Chamber 12,20 Connector 13 Valve 21 Means for reversibly interrupting fluid communication 22, 23, 53 Means for closing the inlet for pressurized gas 24,25 Means for indicating pressure 26,27 Valve 28 Means for reversibly blocking the flow of liquids 29 Inlet for pressurized gas 30 Product container 31 Jet Impingement Reactor 32 Inner surface of chamber wall 33 Chamber wall 34 Fluid inlet 35 Nozzle 36 Reaction Chamber 37 Fluid outlet 42 Entrance 43 Filtration means 44 Exit 45 Connection piece 46 Means of Identification 47 Sensors 48 Sealed Entrance 49 Waste Container 50 Detection window 51 Analyzer 52 Controller 101 Pressure in the pressure reservoir chamber 102 Pressure in the substrate chamber 103 Sudden Equilibrium Phase 104 Pressure in the substrate chamber after equilibration 105 Liquid Flow Rate

[0123] The following examples serve to illustrate the invention but should not be understood as limiting its scope. EXAMPLES

[0124] Example 1 A prototype device according to the invention was constructed and tested. The non-optimized device (e.g. not optimized with respect to minimum dead volume) comprised as a static mixer a jet impingement reactor as described in co-pending European patent application No. 21192535.9. 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 liquid. Water was provided in two flexible containers similar to the infusion bags and placed in the first and second substrate chambers, respectively. The internal volume of the substrate chambers was about 10 L and the volume of water in each flexible container was about 500 mL. The first and second pressure reservoir chambers each had a volume of about 20 L and were filled with pressurized air at a pressure of 15 bar. Furthermore, the jet impingement reactors and conduits for providing fluid communication between each of the two flexible vessels and the respective inlets of the jet impingement reactors were pre-filled with water. The apparatus was further equipped with a flow meter (Cori-Flow™), and various pressure sensors, each disposed in a fluid conduit between the interior space of the flexible vessel and the first or second inlet of the jet impingement reactor.

[0125] Instantaneous pressure equilibrium between the pressure reservoir chambers and the corresponding substrate chambers was achieved by simultaneously opening the magnetic valves located at the connectors between the pressure reservoir chambers and the corresponding substrate chambers. Pressure values ​​were recorded once per second and it was observed that already 1 second after opening the valves, an initial equilibrium pressure of about 9 bar was obtained in each substrate chamber. Until the end of the test run, which lasted for 43 seconds, the equilibrium pressure rose slightly to about 10 bar in each substrate chamber, probably due to a slight temperature increase during this phase. At each time point, the pressure in the first substrate chamber was practically identical to the pressure in the second substrate chamber.

[0126] Pressure equilibration immediately induced a flow of water from the flexible vessel in the substrate chamber in the downstream direction, i.e., towards and through the jet impingement reactor. The flow rates were fairly constant starting about 2 seconds after the initial pressure equilibration: the flow rate of the first feed module increased very slightly and uniformly from about 53 to about 54 mL / min, and the flow rate of the second feed module increased from about 54 to about 55 mL / min. During the first second or two, a flow rate peak was observed, which was considered an artifact caused by the initial pressure impulse traveling through the water-filled system. The very slight difference between the two feed modules is likely caused by minimal pinhole differences due to manufacturing tolerances, but the slight and technically quite negligible increase in flow rate over time could result from the corresponding increase in pressure.

[0127] In summary, the experiments demonstrate that the apparatus 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 small-scale mixing of two liquids can be performed without pumps using this apparatus.

[0128] Example 2 A prototype apparatus similar to that of Example 1 was used to mix two model liquids, which upon mixing would form solid barium sulfate particles. The first liquid consisted of approximately 500 mL of an aqueous solution of barium chloride, and the second liquid consisted of approximately 500 mL of an aqueous solution of barium sulfate. The two liquids were provided in flexible bags that were placed in first and second substrate chambers, each having 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. Upon simultaneous opening of the valves, a substantially instantaneous pressure equilibration between each pressure reservoir chamber and its connected substrate chamber was observed: for the first supply module (i.e., the first pressure reservoir chamber and the first substrate chamber), a pressure of 7.2 bar was recorded, and for the second supply module (i.e., the second pressure reservoir chamber and the second substrate chamber), a pressure of 4.6 bar was recorded. 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 of the first liquid with the second liquid in the jet impingement reactor was an aqueous dispersion of barium sulfate nanoparticles having 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.

[0129] To assess the reproducibility of the mixing process, the experiment was repeated twice. In both additional test runs, the instantaneous pressure equilibration resulted in similar pressures as the first experiment, as shown in Table 1, resulting in a comparable product in terms of nanoparticle properties, thus demonstrating a high degree of process robustness and reproducibility.

[0130]

Table 1

Claims

1. 1. An apparatus for mixing a first liquid and a second liquid, said apparatus comprising: (a) a static mixer, a first inlet for receiving said first liquid; a second inlet for receiving said second liquid, and a static mixer having an outlet for discharging a third liquid resulting from the mixing of said first liquid with said second liquid; (b) a first supply module for providing the first liquid to the first inlet; (c) a second supply module for providing the second liquid to the second inlet; Each of the first and second supply modules comprises: a substrate chamber for holding a flexible container, said flexible container having an interior space for holding said first liquid or said second liquid, respectively; 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; a pressure reservoir chamber for holding pressurized gas; a connector for providing fluid communication between the pressure reservoir chamber and the substrate chamber, the fluid communication being for allowing a 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 out of the container and into the conduit; - means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber, said means having an open state and a closed state; The device, wherein the connector and the means are arranged to achieve instantaneous pressure equilibrium between the pressure reservoir chamber and the substrate chamber upon changing the state of the means from closed to open.

2. 10. The apparatus of claim 1, wherein the static mixer comprises a T-piece mixer, a Y-piece mixer, a vortex mixer, a baffle-based static mixer, a microfluidic mixing device, a multi-inlet vortex mixer (MIVM), or a jet impingement reactor.

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

4. 4. The device of claim 3, wherein the ratio of the volume of the pressure reservoir chamber to the volume of the substrate chamber is at least about 10.

5. - the open state and the closed state are the only states of the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber; and / or - said means for reversibly blocking said fluid communication between said pressure reservoir chamber and said substrate chamber in its open state comprises a fluid path for said pressurized gas having a cross-sectional area of ​​at least about 1 mm 2 ; and / or - the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber are the only means for controlling the flow of pressurized gas between the pressure reservoir chamber and the respective substrate chamber; and / or - the connector and the means for reversibly blocking the fluid communication between the pressure reservoir chamber and the substrate chamber do not comprise a pressure adjusting member; 3. The device according to claim 1 or 2.

6. 3. The apparatus of claim 1 or 2, wherein the 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.

7. 3. The apparatus of claim 1, wherein the conduit for providing fluid communication between the interior space of the flexible container and the first or second inlet of the static mixer has 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 located higher than the upstream end, and / or the static mixer being oriented such that its outlet is located higher than its first and second inlets.

8. The apparatus of claim 1 , wherein the outlet of the static mixer is fluidly connectable to a first container and / or a second container for receiving the third liquid.

9. 9. The apparatus of claim 8, wherein the first and / or second containers for receiving the third liquid are located higher than the outlet of the static mixer and / or comprise an outlet for withdrawing the third liquid or a mixture of the third liquid and the liquid diluent.

10. 3. The apparatus of claim 1, wherein the internal volume of the substrate chamber of the first supply module differs from the internal volume of the substrate chamber of the second supply module, the difference in volume being a factor ranging from about 1.5 to about 10, or from about 2 to about 5.

11. 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: - first and second fluid inlets, the first and second fluid inlets being arranged at opposite positions on a first central axis of the reaction chamber so as to face each other, and each of the first and second fluid inlets 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; 3. The apparatus of claim 2, 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.

12. 1. A method of mixing a first liquid and a second liquid, the method 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, said static mixer comprising: a first inlet for receiving said first liquid; a second inlet for receiving said second liquid, and 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 that applies pressure to the exterior surfaces of the first and second pressurizable substrate chambers, respectively, to force the first and second liquids through the static mixer to mix the first and second liquids; (ee) collecting the third liquid.

13. 13. The method of claim 12, wherein said pressurizing step (dd) comprises a sudden increase in pressure in each of said first and second pressurizable substrate chambers from an initial pressure to a maximum process pressure within less than about 2 seconds, preferably less than about 1 second.

14. 14. The method of claim 13, wherein the initial pressure is about ambient pressure and the maximum process pressure is in the range of about 1 to 16 bar.

15. each of the first and second pressurizable substrate chambers is connected to a first and second pressure reservoir chamber for holding the pressurized gas by a connector for providing fluid communication between the respective pressure reservoir chamber and the respective pressurizable substrate chamber, and means are arranged for reversibly blocking the fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber, the means having an open state and a closed state, and before performing the pressurizing step (dd): - each said pressure reservoir chamber is under pressure such that its pressure is about 2 to 20 times higher than the pressure of each said pressurizable substrate chamber; The method according to claim 12 or 13, wherein the means for reversibly blocking the fluid communication between the respective pressure reservoir chamber and the respective pressurizable substrate chamber are in the closed state.

16. The pressurizing step (dd) (i) changing the state of the means for reversibly blocking the fluid communication between each pressure reservoir chamber and the respective pressurizable substrate chamber from closed to open to create pressure equilibrium between each pressure reservoir chamber and the respective pressurizable substrate chamber within a period of about 2 seconds or less; (ii) forcing essentially all of the first and second liquids from the respective flexible containers to flow through the static mixer to produce the third liquid over a period of at least about 5 seconds, preferably at least 10 seconds, for example from about 10 seconds to about 15 minutes.

17. 14. The method of claim 12 or 13, wherein no additional pressurized gas is supplied to either of the pressure reservoir chambers during steps (dd) and (ee).

18. - said first liquid comprises an organic solution of one or more lipids, - the second liquid comprises an aqueous solution of a biologically active agent; 14. The method of claim 12 or 13, 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.

19. 1. Use of pressurized gas to force a first liquid to flow from a flexible container into a static mixer, wherein the liquid is mixed with a second liquid to form a third liquid, and the pressurized gas is provided to suddenly apply a pressure of 1 to 16 bar to an outer surface of the flexible container without being in fluid communication with the first liquid.

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