Microfluidic apparatus and method of use thereof

Abstract

To provide a microfluidic device for producing and formulating a polynucleotide therapeutic agent, which is less likely to cause contamination and deterioration of a product.SOLUTION: A microfluidic device comprises: a first fluidic input and a second fluidic input; a fluidic intersection channel to receive fluid from the first fluidic input and the second fluidic input, where the fluidic intersection channel opens into a first mixing chamber on an upper region of a first side of the first mixing chamber, where the first mixing chamber has length, width, and depth, where the depth is greater than about 1.5 times of depth of the fluidic intersection channel; an outlet channel on an upper region of a second side of the first mixing chamber, where the outlet channel has depth less than the depth of the first mixing chamber, and where an opening of the outlet channel is offset along width of the second side of the first mixing chamber relative to a fluidic intersection portion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to U.S. Provisional Patent Application No. 62 / 953,102, filed on 23 December 2019, entitled "MICROFLUIDIC APPARATUS AND METHODS OF USE THEREOF," which is incorporated herein by reference in its entirety.

[0002] [Incorporation by reference] All publications and patent applications referenced herein are incorporated herein by reference in whole to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Currently available technologies for manufacturing and formulating polynucleotide therapeutics, particularly mRNA therapeutics, often expose products to contamination and degradation. Currently available centralized production methods are too costly and time-consuming, and are susceptible to contamination, especially when used in therapeutic formulations that may contain multiple polynucleotide species. The development of scalable polynucleotides, production of single-dose patient doses, elimination of touchpoints to limit contamination, input and process tracking to meet clinical manufacturing requirements, and use in point-of-care operations can advance the use of these promising therapeutics. Microfluidic instruments and processes can offer significant advantages to these goals. [Overview of the project] [Means for solving the problem]

[0004] The apparatus and methods described herein may be used for the manufacture and formulation of biomolecular-containing products, particularly therapeutic agents for individualized care. In particular, this specification describes closed-channel methods and apparatus for point-of-care processing of therapeutic polynucleotides.

[0005] Generally, this specification describes apparatus and methods for compounding compositions using microfluidic devices. In particular, this specification describes methods and apparatus for compounding compositions using a microfluidic mixing chamber (or a series of interconnected microfluidic mixing chambers) configured to provide highly efficient mixing with a relatively small footprint. These mixing chambers may operate within specific flow rates to achieve a high degree of mixing. In some examples, the mixing chambers may be cooled to below room temperature to enhance mixing within the microfluidic mixing chambers described herein.

[0006] For example, the mixing chambers described herein may be called box mixing chambers and / or vortex mixing chambers. These mixing chambers may be part of a microfluidic device, e.g., a microfluidic device, which may be alternatively referred to herein as a microfluidic pathway device. These chambers may generally include a base having a base surface (e.g., bottom surface) enclosing the chamber, side walls, and a cover surface (e.g., top surface). These mixing chambers may also include inlets, e.g., mixing inlets, that receive input from two or more fluid pathways in (or into) the microfluidic device. The inlets supply fluid into the chamber (e.g., a box-shaped chamber) near a region on one side of the box chamber that is offset from the top and / or bottom, generally near the upper region on the first side of the chamber. The inlets are generally offset from the top and / or bottom of the chamber by a depth of more than about 1.5 times the depth of the inlet. The inlets may also be called fluid crossing channels. For example, a mixing inlet channel may include an opening to the mixing chamber in the first side wall of a vortex mixing chamber. These chambers may also include a mixing outlet channel. The mixing outlet channel may include an opening to the mixing chamber in the second side wall of the vortex mixing chamber. Generally, the vertical dimension of the vortex mixing chamber may be greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel.

[0007] The mixing channel described herein can receive two or more fluids from an inlet (e.g., a first fluid containing a first composition such as mRNA, and a second fluid containing a second composition such as a delivery vehicle). The fluids (which may be combined before being driven into the mixing chamber) may spread into the mixing chamber and be directed slightly downward against the wall opposite the inlet. This can form a curved or curled fluid path in which the fluids are mixed and combined with the new material entering the chamber from the inlet, directed downward and below across the inlet. The fluids continue to mix and can eventually be driven out of the outlet. In some examples, the first mixing chamber is coupled in series with one or more mixing chambers such that adjacent mixing chambers are supplied in series with each other. For example, the outlet of one chamber may be supplied to the inlet of another chamber. As described below, any of these methods and apparatus may include controlling the temperature of the mixing chamber to a temperature or temperature range (in some examples about 10 to about 20°C) that is calibrated to enhance mixing for mixing within the mixing chamber described herein. The enhanced mixing temperature may be based on the formulation being mixed within a particular geometric shape of the mixing chamber (in some examples, the sequence of mRNA and / or delivery vehicle). This optimal temperature may be determined experimentally and / or by simulation. As used herein, “delivery vehicle” refers to any substance that at least partially facilitates the in vivo, in vitro, or ex vivo delivery of polynucleotides to a targeted cell or tissue (e.g., a tumor). Referring to something as a delivery vehicle does not necessarily mean that it may not have a therapeutic effect. In one example, the delivery vehicle provides an additional therapeutic effect. In another example, the delivery vehicle does not provide an additional therapeutic effect. For example, the delivery vehicle may be an aminolipidized peptoid delivery vehicle that can at least partially encapsulate mRNA.

[0008] The apparatus and methods described herein may be used in conjunction with any suitable microfluidic apparatus (e.g., microfluidic devices, microfluidic systems, etc.), particularly in conjunction with enclosed (e.g., closed-path) microfluidic pathways where a high degree of mixing is desired, especially as part of a series, and in some examples, where space may be premium. Examples of microfluidic systems that may be used with any of the mixers and mixing techniques described herein can be found, for example, in U.S. Provisional Patent Application No. 62 / 885159, filed August 9, 2019, entitled “MICROFLUIDIC APPARATUS AND METHODS OF USE THEREOF” and U.S. Provisional Patent Application No. 62 / 885170, filed August 9, 2019, entitled “METHODS AND APPARATUSES FOR MANUFACTURING THERAPEUTIC COMPOSITION,” and filed August 10, 2020, entitled “METHODS AND APPARATUSES FOR MANUFACTURING FOR REMOVING MATERIAL FROM A THERAPEUTIC COMPOSITION,” each of which is incorporated herein by reference in its entirety.

[0009] Furthermore, this specification describes microfluidic devices comprising one or more of the described mixers. For example, a microfluidic device may include a first fluid input and a second fluid input, a fluid crossing channel configured to receive fluid from the first fluid input and the second fluid input, the fluid crossing channel opening into the first mixing chamber on the upper region of the first side of the first mixing chamber, the first mixing chamber having a depth of more than about 1.5 times the depth of the fluid crossing channel, and an outlet channel on the upper region of the second side of the first mixing chamber, the outlet channel having a depth less than the depth of the first mixing chamber, and further, the opening of the outlet channel being offset with respect to the fluid crossing along the width of the second side of the first mixing chamber.

[0010] The microfluidic device may include: a first fluid input channel and a second fluid input channel, wherein the first fluid inlet channel and the second fluid input channel converge to form a fluid crossing channel having depth and width extending from the top surface to the first bottom surface; a first mixing chamber having depth extending from the top surface to the second bottom surface, width extending from the first side to the second side, and length, wherein the depth of the first mixing chamber is greater than the depth of the fluid crossing channel, the width of the first mixing chamber is greater than the width of the fluid crossing channel, and furthermore, the first mixing chamber is fluidly connected to the fluid crossing channel at the top surface, adjacent to the first side; and an outlet channel, wherein the outlet channel is fluidly connected to the first mixing chamber at the top surface, adjacent to the second side of the mixing chamber.

[0011] The microfluidic device may include a first fluid input and a second fluid input, a fluid crossing channel configured to receive fluid from the first fluid input and the second fluid input, the fluid crossing channel opening into the first mixing chamber on the upper region of the first side of the first mixing chamber, the first mixing chamber having a depth of more than approximately 1.5 times the depth of the fluid crossing channel, a connecting channel on the upper region of the second side of the first mixing chamber, the connecting channel having a depth less than the depth of the first mixing chamber, and further, the opening of the connecting channel being offset from the fluid crossing along the width of the second side of the first mixing chamber, the connecting channel opening into the second mixing chamber, and an outlet channel extending from the second mixing chamber.

[0012] Any of these microfluidic devices may be configured as a single mixer where the outlet channel forms the mixer output (for example, without being connected in series with an additional mixing chamber). The outlet channel may be connected to the output of the microfluidic device, or it may provide an input for further processing, including combination with another fluid (and subsequent mixing). If the mixing chamber is configured as a single mixer, the outlet channel does not act as the sole input to a second mixing chamber connected in series with the first mixing chamber.

[0013] Any of these microfluidic devices may have a first mixing chamber depth that is approximately 2 to 4 times the depth of the fluid crossing channel. The depth of the first mixing chamber may be approximately 3 times the depth of the fluid crossing channel. In some examples, the width of the first mixing chamber is approximately 1.5 to 3 times the length of the box. For example, the width of the first mixing chamber may be approximately 2 times the length of the box.

[0014] The length of the first mixing chamber can be approximately 2 to 4 times the length of the fluid crossing channel. In some examples, the length of the first mixing chamber is approximately 3 times the length of the box.

[0015] In general, the mixers described herein can be integrated into microfluidic pathway devices. For example, the fluid crossing channel, the first mixing chamber, and the outlet channel may all be formed within a first layer, and furthermore, the upper surfaces of the fluid crossing channel, the first mixing chamber, and the outlet channel may be formed in a second layer.

[0016] In general, the inlets and outlets to and from the mixing chamber described herein may be separated from each other by offset. For example, the outlet channel may be in fluid communication with the first mixing chamber at a first length of the mixing chamber, and the fluid crossing channel may be in fluid communication with the mixing chamber at a second length of the mixing chamber.

[0017] The mixing chamber may be a box having square corners and / or rounded corners. For example, the first mixing chamber may have corner radii of about 65 to 85 μm at all or some of its corners. As used herein, “rounded” refers to a surface that transitions smoothly in a curve rather than abruptly at an angle. The rounded corner may have a non-zero radius of curvature, for example, 0.5 to 0.01 times the length of the shortest sidewall it connects to.

[0018] Any of the microfluidic devices in the examples described herein have a flow rate of about 0.25 mL / min to about 5 mL / min (e.g., about 0.25 mL / min to about 4 mL / min, about 0.25 mL / min to about 3 mL / min, about 0.25 mL / min to about 2 mL / min, about 0.25 mL / min to about 1.5 mL / min) and a pressure of about 1 psi (6.9 kPa) to about 30 psi (206.8 kPa), for example, about 1 psi to about 25 It may be configured to provide fluid pressure changes through the first mixing chamber, such as psi (approximately 6.9 kPa to 172.4 kPa), approximately 1 psi to approximately 22.5 psi (approximately 6.9 kPa to 155.1 kPa), approximately 1 psi to approximately 20 psi (approximately 6.9 kPa to 137.9 kPa), 1 to 10 psi (approximately 6.9 kPa to 68.9 kPa), and 1 to 5 psi (approximately 6.9 kPa to 34.5 kPa).

[0019] A microfluidic device can include any number of mixing chambers that can be connected in series. Accordingly, the microfluidic apparatus may include a plurality of fluidly connected mixing chambers. For example, the microfluidic path device may have a second mixing chamber, which has a depth extending from the top surface to the bottom surface of the second mixing chamber, a width extending from the first side to the second side of the second mixing chamber, and a length, wherein the depth of the second mixing chamber is greater than the depth of the outlet channel, the width of the second mixing chamber is greater than the width of the outlet channel, and the second mixing chamber is fluidly connected to the outlet channel on the top surface adjacent to the second side; and a second outlet channel having a depth and a width, which is fluidly connected to the second mixing chamber on the top surface adjacent to the first side of the second mixing chamber.

[0020] Any of these microfluidic devices may include one or more fluid pumps configured to pump fluid from the fluid intersection channel into the first mixing chamber by deflecting at least a portion of the elastic membrane within the microfluidic device. For example, the microfluidic device may include one or more fluid pumps between the plurality of blending chambers and the microfluidic mixer, and the fluid pumps are configured to pump fluid from the fluid intersection channel into the first mixing chamber by deflecting at least a portion of the elastic membrane within the microfluidic device. Alternatively or additionally, any of these apparatuses described herein (e.g., any of these microfluidic devices) can use a non-pulsating pressure source to drive fluid from the fluid intersection channel into one or more mixing chambers. Accordingly, the flow through the mixer can be continuous and non-pulsatile.

[0021] For example, any of these microfluidic devices may include a plurality of pressure ports configured to deflect the elastic layer within the microfluidic device to drive fluid through and between the first mixing chambers.

[0022] In some examples, the microfluidic device includes a flow restrictor in fluid communication with a first fluid input, and the flow restrictor includes a serpentine elongated fluid channel. In some examples, the outlet channel is in fluid communication with one or more final blending chambers.

[0023] In general, a microfluidic mixer (e.g., a mixing apparatus, mixing system, mixing device, microfluidic vortex mixing apparatus, etc.) may include a vortex mixing chamber that encloses the vortex mixing chamber and includes a base defining a bottom surface, side walls, and a top surface, a mixing inlet channel including an opening to the vortex mixing chamber at a first side wall of the vortex mixing chamber, and a mixing outlet channel including an opening to the vortex mixing chamber at a second side wall of the vortex mixing chamber, where the vertical dimension of the vortex mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel.

[0024] The first side wall and the second side wall may be on opposing side walls of the vortex mixing chamber. In some examples, the mixing inlet channel and the mixing outlet channel connect to the vortex mixing chamber at offset positions along the first side wall and the second side wall. The height of the opening of the mixing inlet channel and the height of the opening of the mixing outlet channel may be the same. The width of the opening of the mixing inlet channel and the width of the opening of the mixing outlet channel may be the same.

[0025] The opening of the mixing inlet channel and the opening of the mixing outlet channel may be arranged at the height of the respective first side wall and second side wall adjacent to the top surface of the vortex mixing chamber.

[0026] The mixing inlet channel may have a first end including a fluid intersection, and a second end including an opening to the vortex mixing chamber. In some examples, the fluid intersection may further include a first fluid input channel and a second fluid input channel configured to intersect the mixing inlet channel at the fluid intersection.

[0027] The first fluid channel and the second fluid channel may be configured to intersect each other at a fluid intersection at an angle of less than 180 degrees. The first fluid channel and the second fluid channel may be configured to intersect each other at a fluid intersection at an angle of more than 30 degrees.

[0028] In some examples, the vortex mixing chamber, mixing inlet channel, and mixing outlet channel may be a first vortex mixing chamber, a first mixing inlet channel, and a second mixing outlet channel, and the microfluidic device may further include a second vortex mixing chamber comprising a second vortex mixing chamber which includes a base defining a bottom surface, side walls, and a top surface surrounding the second vortex mixing chamber; a second mixing inlet channel which includes an opening to the second vortex mixing chamber in the first side wall of the second vortex mixing chamber; and a second mixing outlet channel which includes an opening to the second vortex mixing chamber in the second side wall of the second vortex mixing chamber, wherein the vertical dimension of the second vortex mixing chamber is greater than the vertical dimension of the second mixing inlet channel and greater than the vertical dimension of the second mixing outlet channel.

[0029] As used herein, a mixing apparatus may include a mixing system or a mixing device. A mixing apparatus may be referred to herein as a microfluidic mixer, or microfluidic mixing device, or microfluidic mixing system.

[0030] Any of the microfluidic devices described herein may include one or more of the following: the first and second side walls of the second vortex chamber are opposing side walls of the second vortex mixing chamber; the second mixing inlet channel and the second mixing outlet channel are connected to the second vortex mixing chamber at an offset position along the first and second side walls of the second vortex chamber; the height of the opening of the second mixing inlet channel is the same as the height of the opening of the second mixing outlet channel; the width of the opening of the second mixing inlet channel is the same as the width of the opening of the second mixing outlet channel; the openings of the second mixing inlet channel and the second mixing outlet channel are positioned at the height of the first and second side walls of the second vortex mixing chamber, respectively, adjacent to the upper surface of the second vortex mixing chamber; and any combination thereof.

[0031] Any of the microfluidic devices described herein may include a second mixing outlet channel having a first termination at an opening to a second vortex mixing chamber.

[0032] The microfluidic mixer described herein may be included as part of a microfluidic device (e.g., a microfluidic chip) formed between two layers, and may include one or more pumps, blend chambers, etc. For example, a microfluidic device includes a first plate and a second plate, an elastic layer disposed between the first plate and the second plate, and a microfluidic channel formed between the first plate and the second plate, wherein the microfluidic channel includes a plurality of blend chambers, each containing a fixed volume configured to drive fluid between the blend chambers by deflecting at least a portion of the elastic layer, and the microfluidic mixer includes a first fluid input and a second fluid input, and a fluid intersection configured to receive fluid from the first fluid input and the second fluid input, wherein the fluid intersection opens into the first blend chamber on the upper region of the first side of the first blend chamber, and the first blend chamber is deep within the fluid intersection. The system may include a fluid crossing having a depth of more than approximately 1.5 times the depth of the first mixing chamber, a connecting channel on the upper region of the second side of the first mixing chamber, wherein the connecting channel has a depth less than the depth of the first mixing chamber, and the opening of the connecting channel is offset from the fluid crossing along the width of the second side of the first mixing chamber, and the connecting channel opens into the second mixing chamber on the upper region of the first side of the second mixing chamber, and the depth of the second mixing chamber is more than approximately 1.5 times the depth of the connecting channel, and an output channel from the second mixing chamber on the upper region of the second side of the second mixing chamber, wherein the second side of the second mixing chamber is on the opposite side from the first side of the second mixing chamber.

[0033] The upper surface of the fluid intersection may be configured to be at the same height as the upper surface of the first mixing chamber. In some examples, the upper surface of the connecting channel may be configured to be at the same height as the upper surfaces of the first and second mixing chambers. In some examples, the microfluidic apparatus may also include one or more fluid pumps configured to pump fluid from the blend chamber into the microfluidic mixer by deflecting at least a portion of the elastic layer.

[0034] Any of these microfluidic devices may include one or more fluid pumps between a plurality of blend chambers and a microfluidic mixer, the fluid pumps configured to pump fluid from the blend chambers into the microfluidic mixer by deflecting at least a portion of an elastic layer. For example, a microfluidic device may include a plurality of microfluidic mixers. In some examples, the device may include a plurality of pressure ports to a first plate configured to deflect an elastic layer to drive fluid through the microfluidic mixer between the blend chambers.

[0035] In some examples, the microfluidic device includes a flow limiter in fluid communication with a first fluid input, the flow limiter including a meandering, elongated fluid channel. The output channel may be in fluid communication with one or more blend chambers. The blend chamber may be the final blend chamber, and / or each blend chamber may include a pair of blend chambers having a fixed volume formed between a first plate and a second plate, with a portion of the elastic layer dividing each chamber into a fluid-contact side in the second plate and a pressure-receiving side in the first plate.

[0036] Any of the methods and apparatus described herein, including mixing using one or more of the mixing chambers described herein, may include mixing at a lower temperature (e.g., mixing temperature) which is generally about 1 to about 30°C (e.g., about 2 to about 20°C, e.g., about 5 to about 18°C, about 5 to about 15°C, etc.). The mixing temperature may be enhanced with respect to the specific composition (e.g., therapeutic mRNA and / or delivery vehicle), and / or the geometric shape of the mixing chamber, and / or the flow rate (e.g., fluid pressure) of the fluid being mixed.

[0037] For example, a method for compounding therapeutic mRNA with a delivery vehicle may include mixing the mRNA and the delivery vehicle in a microfluidic mixing chamber at a temperature of approximately 2 to approximately 20°C, the temperature of which is selected based on the composition of the mRNA and / or the composition of the delivery vehicle. The temperature may be selected based on one or more of the following: the polynucleotide sequence of the therapeutic mRNA, the sequence of the delivery vehicle, the molecular weight of the delivery vehicle, the molecular weight of the therapeutic mRNA, the charge of the delivery vehicle, the charge of the mRNA, the molecular weight of the delivery vehicle, the molecular weight of the mRNA, the flow rate of the mRNA and / or delivery vehicle in the microfluidic mixing chamber, and the dimensions of the microfluidic mixing chamber.

[0038] In any of these methods and apparatuses, mixing may include mixing within a microfluidic device including a microfluidic mixing chamber. Any of these methods may include maintaining the temperature of one or more mixing chambers separately from the rest of the microfluidic device. Mixing within a microfluidic mixing chamber may include passing mRNA and a delivery vehicle through a first opening into the mixing chamber of the microfluidic device, thereby driving the mRNA and delivery vehicle against the walls of the mixing chamber and to a depth greater than one-fold the depth of the first opening, to form a mixed fluid containing the therapeutic composition.

[0039] Passing through may involve driving the mRNA and delivery vehicle against the wall of the mixing chamber from a plane across the first opening to a depth of more than approximately 2.5 times the depth of the first opening. The mRNA and delivery vehicle may be driven against the wall of the mixing chamber from a plane across the first opening to a depth of more than approximately 3 times the depth of the first opening. The top of the first opening may be aligned with the top of the mixing chamber.

[0040] Furthermore, therapeutic compositions prepared using any of the methods described herein are also described. For example, therapeutic compositions comprising mRNA and a delivery vehicle, prepared by mixing mRNA and a delivery vehicle in a microfluidic mixing chamber at a temperature of about 2 to about 20°C, wherein the temperature is selected based on the composition of the mRNA and / or the composition of the delivery vehicle.

[0041] A method for mixing in a microfluidic device as described herein may include passing a first fluid and a second fluid through a first opening into a mixing chamber within the microfluidic device, thereby driving the first and second fluids against the wall of the mixing chamber to a depth greater than one-fold from the plane of the first opening to the depth of the first opening, thereby forming a mixed fluid, and discharging the mixed fluid from the mixing chamber through an outlet opening, wherein the mixing chamber is maintained at a temperature of about 2 to about 20°C.

[0042] A method for mixing in a microfluidic device may include passing a first fluid and a second fluid through at least one opening into a mixing chamber within the microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and from at least the plane of the first opening, and exiting the mixing chamber through an outlet opening, wherein the mixing chamber is maintained at a temperature of about 2 to about 20°C.

[0043] In some examples of the methods described herein, the method is a method for forming aggregated nanoparticles by mixing oligonucleotides and a delivery vehicle in a microfluidic device, which may include passing a first fluid containing oligonucleotide molecules and a second fluid containing a delivery vehicle chemical through at least one opening into a mixing chamber in the microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and from the plane of the opening, and exiting the mixing chamber through an outlet opening, wherein the mixing chamber is maintained at a temperature of about 2 to about 20°C.

[0044] A method of mixing within a microfluidic device may include passing a first fluid and a second fluid through a first opening into a mixing chamber within the microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber to a depth of more than 2.5 times the depth of the first opening from a plane intersecting the first opening to form a homogeneous mixed fluid, and exiting the mixing chamber through an outlet opening, wherein the outlet opening is on the opposite side of the first opening but offset from it, and the mixing chamber is maintained at a temperature of about 5 to about 20°C to uniformly mix the first and second fluids.

[0045] Passing the first fluid and the second fluid into the mixing chamber through the first opening may include driving the first fluid and the second fluid against the wall of the mixing chamber from a plane across the first opening to a depth of more than approximately 2.5 times the depth of the first opening.

[0046] As described above, in some examples, the mixing chamber may be maintained at a temperature of about 5 to about 15°C to uniformly mix the first and second fluids, and in some examples, the temperature of the mixture is maintained at about 5 to about 15°C (e.g., about 10°C). Any of these methods may involve passing the mixed fluid through the outlet opening to the second opening into the second mixing chamber, thereby driving the fluid against the wall of the second mixing chamber to a depth greater than one times the depth of the second opening from the plane of the second opening, thereby further mixing the mixed fluid. For example, the fluid may be driven against the wall of the mixing chamber to a depth of about three times or more the depth of the first opening from the plane across the first opening. As described above, the top of the first opening may be in line with the top of the first mixing chamber. In some examples, the outlet opening has a cross-sectional area equivalent to the cross-sectional area of ​​the first opening. The mixing chamber may be formed between the first and second layers of the microfluidic path device. The mixing chamber may have a length greater than its width, and furthermore, its length may be more than twice the width of the first opening.

[0047] Furthermore, this specification describes a therapeutic composition comprising mRNA and a delivery vehicle (DV), prepared as described herein, by passing a first fluid containing mRNA and a second fluid containing a delivery vehicle (DV) through a first opening into a mixing chamber in a microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber to a depth greater than one-fold from the plane of the first opening to a depth greater than one-fold from the depth of the first opening to form a mixed fluid, and then exiting the mixing chamber through an outlet opening, wherein the mixing chamber is maintained at a temperature of about 2 to about 20°C.

[0048] This specification also describes therapeutic methods using any of the compositions formed as described herein. In some cases, these compositions can be manufactured only to desired concentrations and volumes (and purities) when using the described methods. For example, a method for treating a disease may include synthesizing one or more therapeutic mRNAs in a microfluidic device, wherein one or more therapeutic mRNAs are in a first fluid and one or more delivery vehicles for the therapeutic mRNAs are in a second fluid; passing the first and second fluids through a first opening into a mixing chamber in the microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and to a depth greater than one-fold from the plane of the first opening to the depth of the first opening, to form a mixed fluid containing the therapeutic composition; discharging the mixed fluid through an outlet opening from the mixing chamber, such that the mixing chamber is maintained at a temperature of about 2 to about 20°C; and administering the therapeutic composition to a patient.

[0049] The mixing temperature, which is generally (but not necessarily) about 2 to about 20°C, may be selected (as an enhanced mixing temperature) based on the dimensions of the mixer (e.g., a box mixer), the composition of the mRNA (e.g., therapeutic mRNA), and / or the composition of the delivery vehicle (DV). Therefore, any of the methods described herein may involve calibrating or selecting the temperature of the mixing chamber to set an enhanced mixing temperature, and the temperature of the mixing chamber(s) may be controlled separately from the temperature(s) of other parts of the microfluidic device, including the mixer. In some examples, the mixing temperature may be calibrated or selected (for an enhanced mixing temperature) by modeling mixing in vitro or in vivo. For example, the optimal mixing temperature may be estimated and / or set based on the mRNA composition (e.g., as a function of one or more of the percentages or ratios of nucleotides that make up the mRNA(s), the length(s) of the mRNA(s), the concentration(s) of the mRNA(s). Additionally or alternatively, the optimal mixing temperature may be estimated based on the composition of the delivery vehicle, including but not limited to molecular weight, concentration, and charge. For example, in some cases, selecting and / or setting an enhanced mixing temperature involves selecting a temperature of about 2 to about 20°C based on the delivery vehicle and one or more therapeutic mRNAs. In some cases, the optimal temperature may be about 2 to 20°C (over about 2°C, over about 5°C, over about 10°C, over about 12.5°C, over about 15°C, over about 17.5°C, over about 20°C, etc.). For example, in some cases, the optimal temperature range may be about 2 to about 50°C. In some cases, the optimal mixing temperature may be calculated or estimated in part based on the geometric shape of the mixing chamber(s) and / or the fluid pressure and / or flow rate of the materials being mixed within the mixing chamber(s). The optimal mixing temperature may refer to the temperature at which the mixing chamber(s) can be maintained during mixing to result in a more uniform mixture after passing through the mixing chamber(s).

[0050] Passing the first and second fluids into the mixing chamber through the first opening may include driving the first and second fluids against the wall of the mixing chamber to a depth of more than approximately 2.5 times the depth of the first opening from a plane crossing the first opening. The fluids may be driven against the wall of the mixing chamber to a depth of more than approximately 3 times the depth of the first opening from a plane crossing the first opening. In some examples, the top of the first opening may be in line with the top of the mixing chamber. The outlet opening may have a cross-sectional area equivalent to the cross-sectional area of ​​the first opening. The mixing chamber may be formed between the first and second layers of the microfluidic path device. In some examples, the mixing chamber has a length greater than its width, for example, the length may be more than approximately twice the width of the first opening.

[0051] A method for forming a therapeutic composition for treating a disease is: The method may include passing one or more therapeutic mRNAs in a first fluid and one or more delivery vehicles for therapeutic mRNAs in a second fluid through a first opening into a mixing chamber in a microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber to a depth greater than one-fold the depth of the first opening from the plane of the first opening to form a mixed fluid containing the therapeutic composition; maintaining the temperature of the mixing chamber at a temperature determined by one or more therapeutic and / or delivery vehicles, the temperature being about 2 to about 20°C when forming the mixed fluid; and exiting the mixing chamber through an outlet opening.

[0052] As described above, any of these methods may include determining an optimal mixing temperature. For example, maintaining the temperature of the mixing chamber may further include selecting and / or setting the temperature of the mixing chamber to an enhanced mixing temperature. The enhanced mixing temperature may be determined by modeling the mixing in vitro and / or experimentally determining the enhanced mixing temperature in vivo. In any of these examples, the selection and / or setting of the enhanced mixing temperature may include determining a temperature or temperature range in which the yield of mRNA (e.g., mRNA expression) in vivo or in vitro is maximized for various temperatures in order to enhance (e.g., increase) the production of the active compound. Thus, the enhanced mixing temperature may include a temperature or temperature range in which mixing is performed for mRNA expression in the system(s) described herein. Determining and / or setting the enhanced mixing temperature may include selecting a temperature of about 2 to about 20°C based on the delivery vehicle and one or more therapeutic mRNAs.

[0053] For example, this specification describes a microfluidic device comprising: a first fluid input and a second fluid input; a fluid crossing channel for receiving fluids from the first fluid input and the second fluid input, the fluid crossing channel opening into the first mixing chamber on the upper region of the first side of the first mixing chamber, the first mixing chamber having length, width, and depth, the depth being more than about 1.5 times the depth of the fluid crossing channel; and an outlet channel on the upper region of the second side of the first mixing chamber, the outlet channel having a depth less than the depth of the first mixing chamber, the opening of the outlet channel being offset relative to the fluid crossing along the width of the second side of the first mixing chamber.

[0054] The microfluidic device may include: a first fluid input channel and a second fluid input channel, which converge into a fluid crossing channel having a width and a depth extending from the top surface to the first bottom surface; a first mixing chamber having a depth extending from the top surface to the second bottom surface, a width extending from the first side to the second side, and a length, wherein the depth of the first mixing chamber is greater than the depth of the fluid crossing channel, the width of the first mixing chamber is greater than the width of the fluid crossing channel, and the first mixing chamber is fluidly connected to the fluid crossing channel at the top surface, adjacent to the first side; and an outlet channel, which is fluidly connected to the first mixing chamber at the top surface, adjacent to the second side of the first mixing chamber.

[0055] The microfluidic device may include a first fluid input and a second fluid input, a fluid crossing channel for receiving fluid from the first fluid input and the second fluid input, the fluid crossing channel opening into the first mixing chamber on the upper region of the first side of the first mixing chamber, the first mixing chamber having width, length, and depth, the depth being more than approximately 1.5 times the depth of the fluid crossing channel, a connecting channel on the upper region of the second side of the first mixing chamber, the connecting channel having a depth less than the depth of the first mixing chamber, and further, the opening of the connecting channel being offset from the fluid crossing along the width of the second side of the first mixing chamber, the connecting channel opening into the second mixing chamber, and an outlet channel extending from the second mixing chamber.

[0056] In any of these microfluidic devices, the depth of the first mixing chamber may be approximately 2 to 4 times the depth of the fluid crossing channel. The depth of the first mixing chamber may be approximately 3 times the depth of the fluid crossing channel. The width of the first mixing chamber may be approximately 1.5 to 3 times the length of the box. The width of the first mixing chamber may be approximately 2 times the length of the box. The length of the first mixing chamber may be approximately 2 to 4 times the length of the fluid crossing channel. The length of the first mixing chamber may be approximately 3 times the length of the box.

[0057] The fluid crossing channel, the first mixing chamber, and the outlet channel may all be within the first layer, and the upper surfaces of the fluid crossing channel, the first mixing chamber, and the outlet channel include the second layer. The outlet channel may be in fluid communication with the first mixing chamber over a first length of the mixing chamber, and the fluid crossing channel may be in fluid communication with the mixing chamber over a second length of the mixing chamber.

[0058] The first mixing chamber may have rounded corners. The first mixing chamber may have a corner radius of about 65 to about 85 μm. The change in fluid pressure passing through the first mixing chamber at a flow rate of 0.25 to 5 mL / min may be about 6.9 kPa to about 206.8 kPa. The width of the first mixing chamber may be about 150 to about 600 μm, the depth of the first mixing chamber may be about 150 to about 500 μm, and the length of the first mixing chamber may be about 500 μm to about 1000 μm.

[0059] The connection channel may have a width of approximately 75 μm to approximately 225 μm, a depth of approximately 75 μm to approximately 225 μm, and a length of approximately 225 to approximately 550 μm.

[0060] Any of these microfluidic devices may include a second mixing chamber having a depth extending from the top surface to the bottom surface of the second mixing chamber, a width extending from the first side to the second side of the second mixing chamber, and a length wherein the depth of the second mixing chamber is greater than the depth of the outlet channel, the width of the second mixing chamber is greater than the width of the outlet channel, and further comprising a second mixing chamber that is fluidly connected to the outlet channel at the top surface, adjacent to the second side; and a second outlet channel having depth and width, which is fluidly connected to the second mixing chamber at the top surface, adjacent to the first side of the second mixing chamber.

[0061] Any of the microfluidic devices described herein may have one or more fluid pumps for pumping fluid from a fluid crossing channel into a first mixing chamber by deflecting at least a portion of an elastic membrane within the microfluidic device. Any of the microfluidic devices described herein may have one or more fluid pumps between a plurality of blending chambers and a microfluidic mixer, the fluid pumps for pumping fluid from a fluid crossing channel into a first mixing chamber by deflecting at least a portion of an elastic membrane within the microfluidic device.

[0062] The microfluidic device may include a plurality of fluid-connected mixing chambers, including a first mixing chamber.

[0063] Any of the microfluidic devices described herein may include a plurality of pressure ports configured to deflect an elastic layer within the microfluidic device to drive fluid between them through a first mixing chamber. Any of the microfluidic devices described herein may include a flow restrictor in fluid communication with the first fluid input, the flow restrictor comprising a meandering elongated fluid channel.

[0064] A microfluidic mixing apparatus may include a mixing chamber (e.g., including a base defining the bottom surface, side walls, and top surface surrounding the mixing chamber), a mixing inlet channel (e.g., having an opening to the mixing chamber in a first side wall of the mixing chamber), a mixing outlet channel (e.g., having an opening to the mixing chamber in a second side wall of the mixing chamber), or any combination thereof. The vertical dimension of the mixing chamber may be greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel.

[0065] The first and second side walls may be opposing side walls of the mixing chamber. The mixing inlet channel and mixing outlet channel may be connected to the mixing chamber at offset positions along the first and second side walls. The height of the opening of the mixing inlet channel may be the same as the height of the opening of the mixing outlet channel. The width of the opening of the mixing inlet channel may be the same as the width of the opening of the mixing outlet channel.

[0066] The openings of the mixing inlet channel and the mixing outlet channel may be positioned at the height of the first and second side walls, respectively, adjacent to the upper surface of the mixing chamber. The mixing inlet channel may have a first end including a fluid crossing and a second end including an opening to the mixing chamber.

[0067] The fluid intersection may further comprise a first fluid input channel and a second fluid input channel that intersect the mixing inlet channel at the fluid intersection. The first fluid channel and the second fluid channel may intersect each other at an angle of less than approximately 180 degrees at the fluid intersection. The first fluid channel and the second fluid channel may intersect each other at an angle of more than approximately 30 degrees at the fluid intersection.

[0068] The mixing chamber may be a first mixing chamber, the mixing inlet channel may be a first mixing inlet channel, and / or the mixing outlet channel may be a first mixing outlet channel. The microfluidic device further comprises a second mixing chamber, which includes a second mixing chamber surrounding the second mixing chamber, a base defining a bottom surface, side walls, and a top surface; a second mixing inlet channel including an opening to the second mixing chamber in a first side wall of the second mixing chamber; and a second mixing outlet channel including an opening to the second mixing chamber in a second side wall of the second mixing chamber, wherein the vertical dimension of the second mixing chamber is greater than the vertical dimension of the second mixing inlet channel and greater than the vertical dimension of the second mixing outlet channel.

[0069] The first and second side walls of the second chamber may be opposing side walls of the second mixing chamber, and the second mixing inlet channel and the second mixing outlet channel may be connected to the second mixing chamber at offset positions along the first and second side walls of the second chamber. The height of the opening of the second mixing inlet channel may be the same as the height of the opening of the second mixing outlet channel, and the width of the opening of the second mixing inlet channel may be the same as the width of the opening of the second mixing outlet channel.

[0070] The microfluidic apparatus according to any one of claims 23 to 34, wherein the second mixing outlet channel includes a first termination at an opening to a second mixing chamber.

[0071] Furthermore, this specification describes microfluidic apparatus comprising cascade microfluidic mixers, each of which may include a mixing chamber (including, for example, a base defining a bottom surface, side walls, and a top surface), a mixing inlet channel (for example, having an opening to the mixing chamber in a first side wall of the mixing chamber), a mixing outlet channel (for example, having an opening to the mixing chamber in a second side wall of the mixing chamber), or any combination thereof. The vertical dimension of the mixing chamber may be greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel. In addition, the cascade microfluidic mixers may be connected in series with each other, so that the mixing inlet channel of each cascade microfluidic mixer following the first microfluidic mixer in series may be connected to the mixing outlet of the earlier microfluidic mixer in series.

[0072] The microfluidic apparatus includes a first plate and a second plate, an elastic layer disposed between the first plate and the second plate, and a microfluidic path between the first plate and the second plate, wherein the microfluidic path comprises a plurality of blend chambers, each containing a fixed volume separated by a portion of the elastic layer, and the portion of the elastic layer is configured to deflect to drive fluid between the blend chambers of the plurality of blend chambers, and a first microfluidic mixer, wherein the first microfluidic mixer comprises a first fluid input and a second fluid input, and a fluid intersection that receives fluid from the first fluid input and the second fluid input, the fluid intersection opening into the first blend chamber on the upper region of the first side of the first blend chamber. The first mixing chamber may comprise a fluid crossing having a depth greater than 1.5 times the depth of the fluid crossing, and a connecting channel on the upper region of the second side of the first mixing chamber, wherein the connecting channel has a depth less than the depth of the first mixing chamber, and the opening of the connecting channel is offset from the fluid crossing along the width of the second side of the first mixing chamber, and the connecting channel opens into the second mixing chamber on the upper region of the first side of the second mixing chamber, and the second mixing chamber may comprise a connecting channel having a depth greater than 1.5 times the depth of the connecting channel, and an output channel from the second mixing chamber on the upper region of the second side of the second mixing chamber, wherein the second side of the second mixing chamber is on the opposite side from the first side of the second mixing chamber.

[0073] The upper surface of the fluid intersection may be substantially the same height as the upper surface of the first mixing chamber. The upper surface of the connecting channel may be configured to be the same height as the upper surfaces of the first and second mixing chambers.

[0074] Any of the microfluidic devices described herein may include one or more fluid pumps for pumping fluid from a blend chamber into a first microfluidic mixer by deflecting at least a portion of an elastic layer. The microfluidic devices described herein may include one or more fluid pumps between a plurality of blend chambers and a first microfluidic mixer, the fluid pumps for pumping fluid from a blend chamber into a first microfluidic mixer by deflecting at least a portion of an elastic layer.

[0075] The microfluidic apparatus may include a plurality of microfluidic mixers, including a first microfluidic mixer, and / or a plurality of pressure ports to a first plate configured to deflect an elastic layer to drive fluid through the first microfluidic mixer between blend chambers. Any of the microfluidic apparatuses described herein may include a flow restrictor in fluid communication with a first fluid input, the flow restrictor comprising a meandering elongated fluid channel.

[0076] The final blend chamber may include a pair of blend chambers having a fixed volume, each blend chamber positioned between a first plate and a second plate, with a portion of the elastic layer dividing each chamber into a fluid-contact side within the second plate and a pressure-receiving side within the first plate.

[0077] Furthermore, this specification describes a method for compounding therapeutic mRNA with a delivery vehicle, which comprises mixing the mRNA and the delivery vehicle in a microfluidic mixing chamber at a temperature of approximately 2 to approximately 20°C, the temperature being selected using at least the composition of the mRNA, the composition of the delivery vehicle, or a combination thereof.

[0078] One of these methods may include selecting a temperature using, at least, the polynucleotide sequence of the therapeutic mRNA, the sequence of the delivery vehicle, the molecular weight of the delivery vehicle, the molecular weight of the therapeutic mRNA, the charge of the delivery vehicle, the charge of the mRNA, the molecular weight of the delivery vehicle, the molecular weight of the mRNA, the flow rate of the mRNA and / or delivery vehicle in the microfluidic mixing chamber, and the dimensions of the microfluidic mixing chamber, or any combination thereof.

[0079] Mixing may include mixing within a microfluidic device, such as a microfluidic mixing chamber.

[0080] One of these methods may include maintaining the temperature of the mixing chamber separately from the rest of the microfluidic device at about 2 to about 20°C. Mixing within the microfluidic mixing chamber may include passing the mRNA and delivery vehicle through a first opening into the mixing chamber of the microfluidic device, thereby driving the mRNA and delivery vehicle against the walls of the mixing chamber to a depth greater than one-fold the depth of the first opening from the plane of the first opening. The methods described herein may include passing the mRNA and delivery vehicle against the walls of the mixing chamber, including driving them from a plane across the first opening to a depth greater than about 2.5-fold the depth of the first opening.

[0081] The mRNA and delivery vehicle can be driven against the wall of the mixing chamber from a plane across the first opening to a depth of at least three times the depth of the first opening. The top of the first opening may coincide with the top of the mixing chamber.

[0082] Furthermore, this specification describes a method of mixing, which includes passing a first fluid and a second fluid through a first opening into a mixing chamber of a microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber and to a depth greater than one times the depth of the first opening from the plane of the first opening to form a mixed fluid, and discharging the mixed fluid through an outlet opening from the mixing chamber, the mixing chamber being maintained at a temperature of about 2 to about 20°C.

[0083] The mixing method may include passing a first fluid and a second fluid through at least one opening into a mixing chamber within a microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and from at least the plane of the first opening, and exiting the mixing chamber through an outlet opening, wherein the mixing chamber is maintained at a temperature of 2 to 20°C.

[0084] Furthermore, this specification describes a method for mixing, which includes passing a first fluid containing oligonucleotide molecules and a second fluid containing a delivery vehicle chemical into a mixing chamber of a microfluidic device through at least one opening, thereby driving the first and second fluids against the walls of the mixing chamber and from the plane of the opening, and discharging the mixed fluid from the mixing chamber through an outlet opening, the mixing chamber being maintained at a temperature of about 2 to about 20°C.

[0085] A method of mixing in a microfluidic device may include passing a first fluid and a second fluid through a first opening into a mixing chamber within the microfluidic device, thereby driving the first and second fluids against the wall of the mixing chamber to a depth of more than 2.5 times the depth of the first opening from a plane across the first opening to form a substantial mixed fluid, and exiting the mixing chamber through an outlet opening, the outlet opening being on the opposite side of the first opening but offset from it, and the mixing chamber being maintained at a temperature of about 5 to about 20°C.

[0086] Passing the first fluid and the second fluid into the mixing chamber through the first opening may include passing the first fluid and the second fluid such that they are driven against the wall of the mixing chamber to a depth greater than approximately 2.5 times the depth of the first opening from a plane crossing the first opening.

[0087] Any of the methods described herein may include maintaining the temperature of the mixing chamber at about 5 to about 15°C. The methods described herein may include maintaining the temperature of the mixed fluid at about 5 to about 15°C. The methods described herein may include maintaining the temperature of the mixed fluid at about 10°C. The methods described herein may include passing the mixed fluid from the outlet opening through a second opening into a second mixing chamber, thereby driving the fluid against the wall of the second mixing chamber to a depth greater than one times the depth of the second opening from the plane of the second opening, thereby further mixing the mixed fluid. The fluid may be driven against the wall of the mixing chamber to a depth greater than about three times the depth of the first opening from the plane across the first opening.

[0088] In any of these methods, the upper part of the first opening may coincide with the upper part of the first mixing chamber. The outlet opening may have a cross-sectional area equal to the cross-sectional area of ​​the first opening. The mixing chamber may be between the first and second layers of the microfluidic device. The mixing chamber may have a length greater than its width, and the length may be more than about twice the width of the first opening.

[0089] This specification also describes a method for forming a composition, the method for synthesizing one or more therapeutic mRNAs in a microfluidic device, wherein one or more therapeutic mRNAs are in a first fluid and one or more delivery vehicles for the therapeutic mRNAs are in a second fluid, and the first and second fluids are passed through a first opening into a mixing chamber of the microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and to a depth greater than one-fold the depth of the first opening from the plane of the first opening to form a mixed fluid, wherein the mixing chamber is maintained at a temperature selected to enhance the mixing of the therapeutic mRNAs and delivery vehicles, and the mixed fluid is exited from the mixing chamber through an outlet opening. The temperature may be selected to enhance (e.g., increase) the mixing compared to mixing with all other parameters (except temperature), and is kept substantially constant, so that mixing at lower temperatures can be at or above the same level (e.g., temperatures between 2°C and 20°C), as described herein.

[0090] For example, the mixing chamber may be selected to enhance the mixing of therapeutic mRNA and the delivery vehicle and may be maintained at a temperature of 2–20°C.

[0091] One of these methods may include selecting an enhanced mixing temperature for the mixing chamber. Selecting an enhanced mixing temperature may include modeling in vitro or in vivo mixing. Selecting an enhanced mixing temperature may include selecting a temperature of about 2 to about 20°C based on the delivery vehicle and one or more therapeutic mRNAs. Passing the first and second fluids into the mixing chamber through the first opening may include passing the first and second fluids, thereby driving them against the wall of the mixing chamber to a depth of more than about 2.5 times the depth of the first opening from a plane across the first opening. The fluids may be driven against the wall of the mixing chamber to a depth of more than about 3 times the depth of the first opening from a plane across the first opening. The top of the first opening may coincide with the top of the mixing chamber. The outlet opening may have a cross-sectional area equal to the cross-sectional area of ​​the first opening. The mixing chamber may be between the first and second layers of the microfluidic device. The mixing chamber may have a length greater than its width, and furthermore, its length may be more than twice the width of the first opening.

[0092] This specification also describes a method for forming a therapeutic composition, which includes passing one or more therapeutic mRNAs in a first fluid and one or more delivery vehicles for therapeutic mRNAs in a second fluid through a first opening into a mixing chamber of a microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and to a depth greater than one-fold the depth of the first opening from the plane of the first opening to form a mixed fluid containing the therapeutic composition; maintaining the temperature of the mixing chamber at an enhanced mixing temperature determined to enhance mixing; and exiting the mixing chamber through an outlet opening.

[0093] The enhanced mixing temperature may be approximately 2 to 20°C. Maintaining this temperature may involve determining the enhanced mixing temperature for one or more therapeutic mRNAs and / or delivery vehicles.

[0094] Any of the methods described herein may include determining an enhanced mixing temperature by modeling mixing in vivo or in vitro. Any of these methods may include determining an enhanced mixing temperature by selecting a temperature of about 2 to about 20°C that produces better mixing than mixing at other temperatures of about 2 to about 20°C. Thus, an enhanced mixing temperature may be a temperature in the range of about 2 to 20°C at which mixing is maximum or near maximum compared to other temperatures in this temperature range. The enhanced mixing temperature may not be a peak (maximum) value, but may be within a temperature range corresponding to the peak mixing value (e.g., within 2°C, within 1.5°C, within 1°C, within 0.5°C, within 0.2°C, within 0.2°C, etc.).

[0095] Passing the first fluid and the second fluid into the mixing chamber through the first opening may include driving the first fluid and the second fluid against the wall of the mixing chamber to a depth of more than approximately 2.5 times the depth of the first opening from a plane crossing the first opening. The fluid may be driven against the wall of the mixing chamber to a depth of more than approximately 3 times the depth of the first opening from a plane crossing the first opening. The top of the first opening may coincide with the top of the mixing chamber. The outlet opening may have a cross-sectional area equivalent to the cross-sectional area of ​​the first opening. The mixing chamber may be formed between the first and second layers of the microfluidic device. The mixing chamber may have a length greater than its width, and the length may be more than twice the width of the first opening.

[0096] This specification also describes a method of mixing, which includes passing a first fluid and a second fluid through a first opening into a mixing chamber of a microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber to a depth greater than one times the depth of the first opening from the plane of the first opening to form a mixed fluid, and discharging the mixed fluid through an outlet opening from the mixing chamber.

[0097] The mixing method described herein may include passing a first fluid and a second fluid through a first opening into a mixing chamber, thereby driving the first fluid and the second fluid against the wall of the mixing chamber to a depth greater than approximately 2.5 times the depth of the first opening, from a plane across the first opening.

[0098] Any of the mixing methods described herein may be single-mixing-chamber mixers in which only a single mixing chamber (e.g., a box mixer) is used and substantially complete mixing can be achieved. Thus, these single-mixing-chamber mixers can achieve a high level of mixing with a very small footprint in microfluidic devices. The mixed fluids may be substantially mixed by the mixing chamber, which may be configured as a single mixer not connected to a second mixing chamber.

[0099] For example, a method of mixing in a microfluidic device may include passing a first fluid and a second fluid through a first opening into a mixing chamber within the microfluidic device, thereby driving the first and second fluids against the wall of the mixing chamber to a depth of more than 2.5 times the depth of the first opening from a plane across the first opening to form a substantially mixed fluid, and exiting the mixing chamber through an outlet opening, the outlet opening being on the opposite side of the first opening but offset from it, wherein the mixed fluid is substantially mixed by the mixing chamber, and the mixing chamber is configured as a single mixer not connected to a second mixing chamber.

[0100] Alternatively, these methods may be configured such that mixing is carried out by connecting two or more (e.g., three, four, five, six, etc.) mixing chambers in series. For example, the method may include passing the mixed fluid from an outlet opening through a second opening into a second mixing chamber, thereby driving the fluid against the wall of the second mixing chamber and to a depth greater than one times the depth of the second opening from the plane of the second opening, thereby further mixing the mixed fluid.

[0101] The fluid can be driven from a plane across the first opening to a depth of approximately three times or more the depth of the first opening, relative to the wall of the mixing chamber. The outlet opening may have a cross-sectional area equal to the cross-sectional area of ​​the first opening. The mixing chamber may have a length greater than its width, and the length is approximately twice the width of the first opening. The mixing chamber may have rounded corners. The change in fluid pressure through the mixing chamber at a flow rate of 0.25 to 5 mL / min may be approximately 6.9 kPa to approximately 206.8 kPa. The width of the mixing chamber may be approximately 150 to approximately 600 μm, the depth of the mixing chamber may be approximately 150 to approximately 500 μm, and the length of the mixing chamber may be approximately 500 μm to approximately 1000 μm.

[0102] All of the methods and apparatus described herein, in any combination, may be used to achieve the interests contemplated herein and described herein.

[0103] Novel features of the present invention are described in particular in the following claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the following accompanying drawings. [Brief explanation of the drawing]

[0104] [Figure 1A] These are schematic diagrams of mixing apparatuses, some examples of which are provided in this disclosure. [Figure 1B] These are schematic diagrams of mixing apparatuses, some examples of which are provided in this disclosure. [Figure 2A]The mixing behavior of an example of a mixing apparatus is schematically shown. [Figure 2B] The mixing behavior of an example of a mixing apparatus is schematically shown. [Figure 2C] The mixing behavior of an example of a mixing apparatus is schematically shown. [Figure 2D] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2E] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2F] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2G] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2H] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2I] Another example of the mixing apparatus described herein, demonstrating the mixing of ethanol and water, is shown. [Figure 2J] This shows mixing in another example of the mixing apparatus described herein. [Figure 3A] A schematic example of an apparatus (e.g., a microfluidic apparatus) including a mixer (e.g., a mixing device) as described herein is shown below. [Figure 3B] An example of a microfluidic apparatus including a mixer as described herein is shown. [Figure 3C] An example of a microfluidic apparatus including a mixer described herein is shown, and an enlarged view of the mixer (mixing apparatus) in Figure 3B is shown. [Figure 3D] Another example of a mixer in a microfluidic apparatus is shown, where there is a short distance between the mixer output and the junction with the dilution buffer. [Figure 3E] This is an example of a device with a longer distance between the mixer output and the junction with the dilution buffer. [Figure 4] This diagram shows selected characteristics of an exemplary apparatus, including an exemplary mixer, as illustrated by some examples of the present disclosure. [Figure 5]This is a schematic diagram of an exemplary apparatus, including exemplary mixers and processing components, as shown in some examples of the present disclosure. [Figure 6] This graph shows, as an example, the effect of temperature (e.g., cooling) on ​​mixing using the mixing apparatus described herein. [Figure 7A] Examples of mixing apparatuses with different configurations are shown. Figure 7A shows a single-chamber mixing apparatus. [Figure 7B] Examples of mixing apparatuses with different configurations are shown. Figure 7B shows three mixers connected in series as described herein. [Figure 7C] Examples of mixing apparatuses with different configurations are shown. Figure 7C shows an example of six mixers connected in series as described herein. [Figure 7D] Examples of mixing apparatuses with different configurations are shown. Figure 7D shows an example of 12 mixers connected in series as described herein. [Figure 8] This figure shows a mixing apparatus including three mixers connected in series, illustrating the deposits within the mixer. [Figure 9A] This figure shows a first example of a mixing apparatus having three mixers of different sizes connected in series ("3-box" mixer). In Figure 9A, each mixer in the mixing apparatus forms a box measuring 250 μm × 200 μm × 500 μm. [Figure 9B] This figure shows a first example of a mixing apparatus having three mixers of different sizes connected in series ("3-box" mixers). In Figure 9B, each mixer in the mixing apparatus is scaled up twice as much as the apparatus shown in Figure 9A, with each box forming each mixer measuring 500 μm × 400 μm × 1000 μm. [Figure 9C] Figure 9B is a graph comparing the mixing effects of a larger (scaled-up) mixing apparatus ("3-box L") and the mixing apparatus ("3-box") shown in Figure 9A. The bar graph shows the Z-mean value on the left scale, and the dots show the polydispersity index (PDI) on the right scale. [Figure 10A]This specification shows a mixing apparatus including three mixers connected in series. [Figure 10B] The mixing apparatus shown is similar to that shown in Figure 10A, but the corners are rounded, which reduces, and in some cases eliminates, the dead zone (areas with little to no fluid flow) within the mixer. [Figure 11A] An example of a mixing apparatus having three mixers connected in series at an angle greater than 90 degrees but less than 180 degrees is shown. Figure 11A shows a top perspective view of the mixing apparatus. The angle between adjacent mixers is 135 degrees. [Figure 11B] An example of a mixing apparatus having three mixers connected in series at an angle greater than 90 degrees but less than 180 degrees is shown. Figure 11B shows a cross-sectional view passing through the upper region of the mixing apparatus. The angle between adjacent mixers is 135 degrees. [Figure 11C] This graph shows the mixing effects of different configurations of mixing devices, including a mixing device with a 135-degree angle (the "3-box angle" shown in Figures 11A and 11B) and a curved mixing device (the "3-box curve" shown in Figures 10A and 10B), compared to a mixing device with three mixers connected in series ("3-box") as shown in Figure 7B, or a mixing device with a single mixer ("1-box") as shown in Figure 7A. In Figure 11C, the bar graph shows the Z-mean value on the left scale, and the dots show the polydispersity index (PDI) on the right scale. [Figure 12A] Another example of a mixing apparatus including three mixers connected in series, as described herein, is shown. [Figure 12B] This example shows a setup that does not involve a step from a narrower height channel to a deeper box in the mixer. [Figure 12C] This graph shows the particle size (due to mixing) after passing through a mixing device ("3-box") as shown in Figure 12A or a linear mixing channel ("3-box plane") as shown in Figure 12B multiple times. Compared to Figure 12B, the mixing device in Figure 12A shows a visibly smaller particle size (and therefore more efficient mixing). [Figure 13] Figures 7B, 9A, and 10A show graphs illustrating the effect of flow rates through a mixing apparatus with three mixers ("3-box" mixers) similar to those shown, demonstrating that higher flow rates result in smaller particle sizes and visibly better mixing compared to manual mixing. In Figure 13, the bar graph shows the volume-mean value on the left scale, and the dots show the polydispersity index (PDI) on the right scale. Four examples of 1 mL / min, four examples of 2 mL / min, and two examples of 4 mL / min are shown, along with a sample mixed by hand in a single sample. [Figure 14A] An example of a dialyzer is shown in both an oblique view and a cross-sectional view. [Figure 14B] An example of a dialyzer is shown in both an oblique view and a cross-sectional view. [Figure 15] This is a cross-sectional view of an example of the edge region of a dialyzer, similar to those shown in Figures 14A and 14B. [Figure 16] An example of a dialyzer described herein is shown, illustrating the exemplary flow rate (velocity) through the device. [Figure 17A] This is a diagram showing the flow rate through another example of a dialyzer apparatus shown herein, illustrating the flow rate through the device. [Figure 17B] This is a diagram showing the flow rate through another example of a dialyzer apparatus shown herein, illustrating the flow rate through the device. [Figure 17C] This is a diagram showing the flow rate through another example of a dialyzer apparatus shown herein, illustrating the flow rate through the device. [Figure 18] This figure shows an example of a concentrator described in this specification. [Figure 19] A schematic example of a microfluidic pathway device described herein, which includes mixing (using one or more vortex mixing chambers), dialysis, and concentration, is shown herein. [Figure 20] This is a schematic diagram of the pressure characteristics of a microfluidic apparatus, including a microfluidic mixer (e.g., a box mixer), compounding components, and a concentrator, according to some embodiments of the present disclosure. [Modes for carrying out the invention]

[0105] In general, this specification describes apparatuses (e.g., systems, devices, etc.) and methods for processing fluid mixtures containing but not limited to therapeutic polynucleotides. In particular, this specification describes mixing apparatuses that can mix materials in a microfluidic apparatus more rapidly and efficiently using less microfluidic space than previously described microfluidic mixers. Any of the mixing apparatuses described herein may include one or more mixers, which may be called box mixers or vortex mixers, which may be connected in series and may be included as part of a microfluidic apparatus. For example, any of these apparatuses and methods may be used as part of a closed-loop microfluidic apparatus and method, may be configured to be minimized, and in some cases may even be configured to eliminate manual handling. Closed-loop apparatuses and methods may provide a substantially sterile environment and may form a sterile pathway for processing from initial input (e.g., template) to output (formulated therapeutic agent). As used herein, the term “substantially” may mean almost or essentially all / essentially completely (e.g., more than 90%, more than 95%, more than 97.5%, more than 99%, more than 99.5%, more than 99.9%, etc.), and may include all or completely. For example, “substantially mixed” may mean a mixture that is almost or completely mixed, i.e., a homogeneously mixed mixture. Material inputs to the apparatus (nucleotides and any chemical components) may be sterile and may be introduced into the system without substantially any manual interaction. Mixing apparatuses described herein may enable complete and thorough mixing of these components for forming and / or compounding compositions using microfluidic apparatuses.

[0106] Therefore, the mixing methods and apparatus described herein can be used to generate therapeutic agents with high reproducibility and rapid cycle times. Accordingly, the mixing methods and apparatus described herein may enable the synthesis, purification, and compounding of one or more therapeutic compositions (including, but not limited to, therapeutic polynucleotides) in a single, integrated microfluidic apparatus. All or some of these processing steps may be carried out in an undamaged fluid processing pathway that can be configured as one or more disposable microfluidic devices, also known as a microfluidic pathway chip, microfluidic pathway plate, process chip, biochip, or process plate. This may enable the synthesis of patient-specific therapeutic agents, including compounding at point of care (e.g., hospitals, clinics, pharmacies, etc.).

[0107] During the operation of the device, the fluid pathways may remain undamaged, and contamination can be substantially eliminated by non-contact monitoring (e.g., optical monitoring), including fluid flow measurement and mixing monitoring, as well as by manipulating (metering, mixing, etc.) the precise amount of microfluidic fluid using pressure applied from a deflectable membrane on the opposite side of the fluid chamber and channel.

[0108] These apparatuses and methods may be configured for use in point-of-care settings. For example, the methods and apparatus described herein may be configured to produce customized therapeutic compositions comprising one or more therapeutic polynucleotides (e.g., mRNA, microRNA, DNA, etc.).

[0109] Therefore, the methods and apparatus described herein can provide for therapeutic drugs scalable polynucleotide production, production of single-dose patient doses, elimination of touchpoints to limit contamination, input and process tracking to meet clinical manufacturing requirements, and use in point-of-care operations. The microfluidic apparatus and processes described herein can offer key advantages.

[0110] In general, the apparatus described herein may be a microfluidic apparatus. In some examples, these microfluidic apparatuses may include, but are not limited to, closed-path microfluidic apparatuses for processing biomolecular products such as therapeutic polynucleotides, which may include mixing and compounding the biomolecular products (e.g., in combination with a selected carrier or vehicle). These apparatuses may be configured to operate on one or more microfluidic devices. A microfluidic apparatus may include one or more microfluidic devices (e.g., blending chips, compounding chips, etc.).

[0111] The mixing devices described herein are remarkably effective and can be implemented along microfluidic channels. These mixing devices may be part of a microfluidic path device located in or between a pair of layers of material separated by a deflectable membrane. The fluid path containing the mixing device may be formed on one side of the deflectable membrane (for example, by forming a first layer(s) on one side of the membrane), the membrane may form the top of the mixing device, and the plate on the opposite side of the membrane may be flat. In some examples, the plate on the opposite side of the deflectable membrane may be notched to form a chamber opposite the mixer, which may be connected to a pressure channel to allow the membrane to be deflected into the mixer and / or the upper chamber, for example, to drive the fluid.

[0112] The mixing apparatus described herein may include one or more individual mixers arranged in series and connected by channels having smaller heights and / or widths. The mixers may be referred to as mixing components, box mixers, or vortex mixers. Examples of these mixers are described in more detail herein. Thus, a mixing apparatus including one or more mixers may collectively be referred to as a “microfluidic mixing apparatus,” a “box mixing apparatus,” or a “vortex mixing apparatus,” or simply a “mixing apparatus.” The mixers described herein, using vortex mixing within a vortex mixing chamber (e.g., a “box” or a series of fluid connection boxes), can efficiently mix non-uniform inflow streams or pulses of fluid material to obtain a more uniform mixed fluid mixture over very short distances along a microfluidic channel. This can be achieved over a wide range of fluid mixtures. One or more of the fluids being mixed may further include particles, such as drug substances containing encapsulated nanoparticles. Efficient mixing can also be obtained over a wide variety of ratios of components encombined in the fluids introduced for mixing, as will be discussed in detail below. In some examples of mixing apparatuses described herein, the fluid is considered to form a vortex within the chamber of the mixing apparatus, and as a result, the direction of the fluid flow may reverse itself in a circular vortex pattern, as shown in the fluid model presented below. Therefore, these mixing apparatuses may be called vortex mixers or vortex mixing chambers. It should be understood that references to mixing and mixing apparatuses described herein as vortex mixing or vortex mixing chambers are not intended to limit these methods and apparatuses to a specific operating theory.

[0113] Figure 1A shows a microfluidic mixer 100 having two fluid inlet channels 103 and 105 that are offset from each other and configured to transport one or more substances (e.g., biomolecular products (one or more), buffers, carriers, minor components) that can be combined together. Although two inlet channels are shown, three or more (four, five, six, etc.) may be used and converge on the same mixer. The fluid to be mixed may pass through the inlet channels under positive pressure. This pressure may be constant, variable, increasing, decreasing, and / or pulsatile. The mixer is configured to be positioned along a microfluidic flow path between an input terminal and an output terminal, and the mixed and compounded biomolecular products may be discharged from the microfluidic flow path and the device. The microfluidic device may have a first or upper plate and a second or lower plate. Microfluidic flow paths and microfluidic components (mixers, pumps, etc.) may be formed between them and may be machined, molded, or manufactured in any preferred manner. A microfluidic channel may have one or more widths along its length and one or more vertical dimensions. Generally, the top surface defining the microfluidic path may be at the same height throughout the entire microfluidic channel of the device.

[0114] In Figure 1A, the microfluidic mixer receives fluid from two fluid channels 103 and 105, each of which may have a width of approximately 50 to 500 μm (e.g., approximately 50 to 400 μm, 50 to 300 μm, 50 to 200 μm, 170 μm, 150 μm, 110 μm, 100 μm, 80 μm, 60 μm, 50 μm, etc.). The channels may have the same width (and / or cross-sectional diameter) or different widths (and / or cross-sectional diameters). Channels 103 and 105 may have vertical dimensions of approximately 20 to 200 μm (e.g., approximately 200 μm, 175 μm, 150 μm, 125 μm, 100 μm, 75 μm, 60 μm, 50 μm, 40 μm, or any value in between). The two channels 103 and 105 intersect at the fluid intersection 109, merging the two fluid streams together. The channels intersect at an angle 107. Angle 107 can be less than approximately 180 degrees (e.g., 5 to 179 degrees, 10 to 160 degrees, 15 to 145 degrees, 30 to 120 degrees, etc.). In some examples, angle 107 can be greater than approximately 30 degrees (e.g., angle 107 can be approximately 35 degrees, 40 degrees, 50 degrees, 60 degrees, 75 degrees, 90 degrees, 100 degrees, 120 degrees, 145 degrees, 160 degrees, or any value in between).

[0115] The merged channel exiting the fluid crossing 109 may have a width of, for example, about 50 μm to about 200 μm (e.g., about 50 to about 180, about 50 to 150, about 50 to 140, about 50 to 130, about 50 to 120 μm, about 110 μm, about 100 μm, about 80 μm, about 60 μm, about 50 μm, etc.). The merged channel is a mixing inlet channel and may have a vertical dimension that matches the inlet vertical dimension (one or more), for example, about 100 μm, about 75 μm, about 60 μm, about 50 μm, about 40 μm, or one or more of any values ​​in between (e.g., all of them). The mixing inlet channel has a first end including the fluid crossing and a second end including an opening to the vortex mixing chamber. The mixing inlet channel enters the vortex mixing chamber 115, and the channel has a mixing inlet opening 111 into the vortex mixing chamber 115, passing through the side wall of the vortex mixing chamber 115.

[0116] By increasing the vertical dimension 121 of the vortex mixing chamber 115 so that it is larger than the vertical dimension 119 of the mixing inlet channel opening 111, and in some examples so that it is larger than the vertical dimension 123 of the mixing outlet channel opening 113, remarkably enhanced mixing is achieved. Figure 1B shows an example of a microfluidic mixing unit 130. An example of a vortex mixing chamber provides a common top surface for both the mixing chamber 115 and the inlet channel opening, thereby causing the incoming fluid to descend vertically toward the bottom surface. Furthermore, the vortex mixing chamber 115 may be configured such that the fluid exits the vortex mixing chamber 115 at the outlet opening 113 of the mixing outlet channel. The outlet opening may be configured similarly to the opening 111 to the mixing chamber, for example, the vertical dimension of the opening 113 in this example may be smaller than the vertical dimension of the vortex mixing chamber 115 and may share the same top surface as the vortex mixing chamber, and the height and / or cross-sectional area of ​​the outlet may be the same as the height and / or cross-sectional area of ​​the inlet opening 111. To exit the vortex mixing chamber 115, the fluid is moved upward and laterally, and the inlet opening 111 and outlet opening 113 are also located on both sides of the vortex mixing chamber 115 (and on the opposing walls in Figure 1B). In some examples, the openings 111, 113 are positioned vertically through the side walls of the vortex mixing chamber 115 such that the upper limits of the openings 111, 113 (e.g., the top surfaces defining the mixing inlet and mixing outlet channels) are the same vertical dimension as the top surface of the vortex mixing chamber 115. The openings 111, 113 are further positioned on the opposing side walls of the vortex mixing chamber 115 and are horizontally offset from each other. The fluid forms a vortex around the horizontal axis of the box and mixes strongly as it moves from the opening 111 to the opening 113 of the mixing outlet channel. Generally, the outlet opening is offset from the inlet opening so that the fluid is deflected away from the side wall of the chamber opposite the inlet and deflected "downward" toward the bottom as part of the mixing.

[0117] Generally, the vortex mixing chamber 115 has a base defining the bottom surface, one or more side walls, and a top surface surrounding the vortex mixing chamber. The vortex chamber may have shapes such as rectangular, elliptical, circular, or hexagonal, and the side walls may be curved (for example, having a radius of curvature 127 which is 0.5 to 0.01 times the length of the shortest side wall it connects to). As described above, the mixing inlet channel and the mixing outlet channel open into the vortex mixing chamber at the first and second side walls of the vortex mixing chamber, respectively. The sizes of the vortex mixing chambers 115, 120 and the mixing inlet / outlet channels may be selected to provide efficient mixing for a particular overall flow rate or range of flow rates.

[0118] In some examples, the mixing inlet channel and / or mixing outlet channel may each have a width of approximately 50 to 200 μm (e.g., approximately 50 to 170 μm, approximately 170 μm, approximately 110 μm, approximately 100 μm, approximately 80 μm, approximately 60 μm, approximately 50 μm, etc.). The mixing inlet channel and / or mixing outlet channel may each have a vertical dimension of approximately 75 μm, approximately 60 μm, approximately 50 μm, approximately 40 μm, or any value in between. In some examples, the mixing inlet channel and the mixing outlet channel may have the same width and height as each other.

[0119] As shown in Figure 1A, the mixing assembly may include a plurality of vortex mixing chambers connected together such that the inlet of a subsequent vortex mixing chamber is connected to the outlet of a preceding vortex mixing chamber, and connecting channels 132 may be connected between each vortex mixing chamber. The microfluidic vortex mixing units may have the same and / or dimensions, or they may have different shapes and / or dimensions.

[0120] In some examples, the microfluidic vortex mixing unit(s) 130 may have a length of approximately 250 μm to approximately 1100 μm (e.g., approximately 300 μm, approximately 350 μm, approximately 400 μm, approximately 500 μm, approximately 600 μm, approximately 1000 μm, approximately 1100 μm, or any length in between). The vortex mixing chamber may have a width of approximately 175 μm to approximately 600 μm (e.g., approximately 200 μm, approximately 250 μm, approximately 275 μm, approximately 400 μm, approximately 500 μm, approximately 600 μm, or any width in between). In some examples, the chamber may have a vertical height of approximately 125 μm to 500 μm (e.g., approximately 125 μm, 150 μm, 170 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, or any height in between). As described above, in some examples, the corners of the vortex mixing chamber may be rounded, and the corners may have a radius of curvature of approximately 50 μm to 90 μm, or approximately 50 μm, 60 μm, 75 μm, 80 μm, 90 μm, or any radius in between. The dimensions of the vortex mixing chamber and mixing inlet / outlet channels may be selected to allow for easy machining and to minimize changes in fluid pressure (delta P) while maximizing mixing over a small distance. Efficient use of the microfluidic tip surface area is also included as a design consideration. For example, the mixing / size selection factor may include the height of the vortex mixing chamber, which is approximately 2 to 5 times the height of the mixing inlet / outlet channel (e.g., approximately 2, 3, 4, 5, approximately 2 to 4, etc.). In some examples, the height of the vortex mixing chamber is approximately 3 times the height of the mixing inlet / outlet channel. The length of the sidewalls of the vortex mixing chamber may be approximately 2 or more times the width of the mixing inlet / outlet channel (e.g., approximately 2, 3, 4, approximately 2 to 5, approximately 2 to 4, etc.).

[0121] In general, the dimensional specifications for mixing chambers provided herein are illustrative, and for example, the dimensions provided above may be used in mixers having flow rates of about 0.1 to 10 mL / min (e.g., about 0.2 mL / min to about 5 mL / min, about 0.5 mL / min to about 4 mL / min, etc.). The dimensions described herein may be scaled up or down to provide larger or smaller flow rates (e.g., at different dimensional values) to achieve the same vortex flow (e.g., equivalent mixing) for a given applied pressure (e.g., about 6.99 kPa to about 206.8 kPa). Thus, the dimensions provided herein may be scaled (e.g., scaled of these structures) to enable different flow rates.

[0122] For example, a microfluidic mixing apparatus may include a mixing inlet channel, a microfluidic vortex mixing chamber, and a mixing outlet channel, the mixing inlet and outlet channels having a width of approximately 100 μm and a vertical dimension of approximately 50 μm from the base of the mixing inlet / outlet channel(s), one or more, and the vortex mixing chamber having a length of 350 μm, a width of 250 μm, a height of 150 μm, and a radius of curvature of approximately 75 μm at the rounded corners of the chamber. Thus, the mixing inlet opening 111 is horizontally offset along a 350 μm long sidewall, and along an opposing 350 μm long sidewall, from the mixing outlet opening 113 by a maximum of approximately 150 μm.

[0123] In another example, the microfluidic mixer may include a mixing inlet channel, a microfluidic vortex mixing unit, and a mixing outlet channel, the mixing inlet channel and mixing outlet channel may have a width of about 150 μm and a vertical dimension of about 50 μm from the base of the mixing inlet / outlet channel(s), one or more, and the vortex mixing chamber may have a length of 500 μm, a width of 250 μm, a height of 150 μm, and a radius of curvature of about 75 μm at the rounded corners of the chamber. Thus, the mixing inlet opening 111 is horizontally offset along a 500 μm long sidewall, and along an opposing 500 μm long sidewall, from the mixing outlet opening 113 by up to about 200 μm.

[0124] As described above, the vortex mixing unit 130 may be arranged along a microfluidic channel paired with a second (or more) vortex mixing unit, similar to the pair of vortex mixing units shown in Figure 1A. The second vortex mixing unit may be configured similarly to the first vortex mixing unit. That is, the vortex mixing chambers 115 and 120 may have the same dimensions to provide similar vortex and mixing characteristics as described above. For example, the respective mixing inlet and outlet channels may relate as follows: the first mixing outlet channel of the first vortex mixing unit may have a first end including an opening 113 to the first vortex mixing chamber 115, and the second end including a second mixing inlet channel of the second vortex mixing unit including an opening 131 to the second vortex mixing chamber 120. For example, the second mixing outlet channel of the second vortex mixing unit has a first end at an opening 133 to the second vortex mixing chamber 120 and a second end at an outlet 117 from the mixing device, along a microfluidic path. A pair of two microfluidic vortex mixing units can be designed to minimize the pressure drop (delta P), and as described above, for a smaller pair of mixing units having a vortex chamber with a length of 350 μm, a width of 250 μm, and a height of 150 μm, the delta P per pair at a flow rate of 0.5 mL / min is approximately 10.3 kPa (e.g., approximately 6.9 kPa to approximately 206.8 kPa, approximately 6.9 kPa to approximately 172.4 kPa, approximately 6.9 kPa to approximately 106.0 kPa, approximately 6.9 kPa to approximately 103.4 kPa, approximately 6.9 kPa to approximately 68.9 kPa, approximately 6.9 kPa to approximately 34.5 kPa, etc.). The delta P at a flow rate of 1 mL / min for a pair of vortex mixing units with larger chamber dimensions of 500 μm in length, 250 μm in width, and 150 μm in depth is approximately 2.4 psi (16.5 kPa).

[0125] In some examples, as shown in Figure 3A, a third or fourth microfluidic vortex mixing unit may be included along the microfluidic channel to provide a two-stage mixing apparatus. The first chain of mixing units 330 constitutes the first stage, and this mixing product may then be combined with the second group of mixing units 331 in the second stage. Additional microfluidic vortex mixing units may be configured as described above. Generally, each individual vortex mixing unit may have the same features and dimensions, or different dimensions. In Figure 3A, the first stage 330 includes four mixing units (e.g., two pairs of mixing units) which may be connected as shown. For example, the second end of the second mixing outlet channel is continuous with the opening to the third mixing inlet channel and the third vortex mixing chamber. The third mixing outlet channel may have a first end including an opening to the third vortex mixing chamber and a second end which may be an opening to the fourth mixing inlet channel and the fourth vortex chamber. The fourth mixing outlet channel may have a first end which may be an opening to the fourth vortex mixing chamber and a second end which may be an output from the microfluidic mixer.

[0126] Returning to Figures 2A and 2C, Figure 2A shows a flow trajectory model for a microfluidic mixer 200 having a pair of microfluidic vortex mixing units 130, similar to the mixer 100 in Figure 1A, and represents the mixing capacity of the microfluidic mixer (showing the mixing of equal volumes of ethanol (arrow 255) and water (arrow 257) by mixing ratio). The shading of the arrows indicates the mixing ratio. Figure 2A is a transparent flow model of a 1:1 mixture of ethanol introduced into the first inlet 203 and water introduced into the second inlet 205. The volume ratio of ethanol to water along the microfluidic flow path, indicated by the arrows, shows the substantial mixing in the vortex mixer. Inlet 203 is labeled to represent a volume ethanol ratio of 100%, while the second inlet 205 is labeled to indicate the absence of ethanol. At the fluid intersection 209, the flow moves laminarily with little mixing, as indicated by region 204 showing separate, unmixed ethanol and water flows. However, region 206, enclosed by the white ellipse, shows a rapid change in ethanol concentration as the fluid flow cascades down into the vortex mixing chamber 215 (which may be similar to regions 115 and 120 of a typical microfluidic vortex mixing unit 130), at which point the water edge of the flow contains approximately 0.1667 (v / v) ethanol. When the flow reaches the mixing outlet opening region 208, the flow now contains approximately the same volumetric proportion of ethanol, and the remainder of the flow in region 212 (arrow 259) is a 0.5000 (v / v) mixture of ethanol. Figure 2B shows a representation of a similar experiment, with side views of both the first and second vortex mixing units (215, 220) along the microfluidic path, both of which may be similar to 115, 120 of a typical microfluidic vortex mixing unit 130. The side view in Figure 2B more clearly shows the movement of the vortex that causes the fluid to flow out of the vortex mixing chamber 215.A 1:1 ethanol:water v / v flow can be shown to be substantially mixed at the opening 211 of the mixing outlet channel from the vortex mixing chamber 215 to somewhere between 0.4167 and 0.5833 ethanol / water v / v. In this example, there is a small area at 214 where a volume ratio of 0.667 is still present, but the ethanol / water v / v ratio of 0.4167 to 0.5833 is dominant throughout most of the area 216. A second vortex mixing unit 226 can be used for final mixing in the vortex mixing chamber 220 to produce a fully mixed ethanol / water from the pair of microfluidic vortex mixing units at a second output 217. Within the vortex mixing chamber, the arrangement of the chamber allows for mixing in which the flow is directed downward toward the bottom of the chamber, as shown in Figure 2B. As will be explained in more detail below, the enhanced mixing temperature can be determined by calibrating the geometric shape, particularly the depth and flow rate of the chamber, which allows for near-perfect mixing within a single vortex mixing chamber.

[0127] Another example of flow modeling is shown in Figure 2C, which shows a 1:10 ratio of ethanol (input at inlet 203) to water (input at inlet 205). Shading indicates the ratio of ethanol to water (per scale). Region 218 shows the flow alongside ethanol / water, where the volume ratio of ethanol has already decreased to 0.7500–0.4167 as the flow begins to cascade down into the vortex mixing chamber 215. Region 220 shows a portion of the flow where the volume ratio of ethanol 263 is fully equilibrated at the point where it exits the second vortex mixing chamber 220, with a slightly increased volume ratio of ethanol at 0.1667–0.2500, and is output from the microfluidic mixer at 217. The arrangement of the mixing inlet opening, mixing outlet opening, and vortex mixing chamber as shown provides a mixing apparatus that is substantially insensitive to the ratio of the fluids being mixed, such that both 1:1 and 1:10 ratios of fluids in the two inlet channels are brought to the equilibrated mixture as it exits the mixing apparatus at 217.

[0128] Figures 2D to 2J show examples of other vortex mixing apparatuses illustrating the effect of a vortex mixing chamber configuration on total mixing at exemplary pressures and flow rates.

[0129] For example, Figures 2D–2E show an example of a apparatus including a series of vortex mixing chambers, each of which has a channel inlet 211 with a width of 100 μm and a depth of 50 μm. The depth is measured from the top surface (e.g., top plate), and the vortex mixing chamber is approximately square (with rounded sides) and has a length of 250 μm, a width of 250 μm, and a depth of 100 μm. Thus, in this example, the depth of the vortex mixing chamber is twice the depth of the inlet, and the inlet opening and the chamber have a common top surface, and as a result, the maximum drop from the inlet opening of the vortex mixing chamber to the top (or bottom, depending on the reference frame) is approximately the same as the depth of the inlet. In this case, for a 1:10 ethanol:water mixture, as indicated by the shaded arrow, mixing is not completed after the second continuous vortex mixing chamber 226 (connected in series). Figure 2E shows the pressure drop of the same example (showing six continuously connected vortex mixing chambers). Mixing is substantially completed by the third mixing chamber. The pressure drop from each of the water 205 and ethanol 203 is approximately 20.3 lbf / in. 2 The channel supplies approximately 0.76 lbf / in between each continuous vortex mixing chamber. 2 It only descends.

[0130] Figures 2F and 2G show another example of an apparatus including a series of vortex mixing chambers, each of which is similarly (as in Figures 2D-2E) 100 μm wide × 50 μm deep at the channel inlet 211. The vortex mixing chambers in this example are approximately rectangular (with rounded sides) and have a length of 350 μm, a width of 250 μm, and a depth of 100 μm. Thus, this example has vortex mixing chambers that are 1.4 times longer but otherwise similarly sized, as shown in Figures 2D and 2E above. In Figures 2F and 2G, the depth of the vortex mixing chambers is also twice the depth of the inlets, and the inlet openings and chambers have a common top surface, so that the maximum drop from the inlet opening of the vortex mixing chambers to the top (or bottom, depending on the reference frame) is approximately the same as the depth of the inlets. The 1:10 ethanol:water mixing profile (indicated by shaded arrows) is approximately the same as in the examples in Figures 2D and 2E. Figure 2G shows the pressure drop for the same example (also showing six sequentially connected vortex mixing chambers). Mixing is substantially completed after exiting the second mixing chamber. 20.19 (e.g., 20.19 lbf / in) 2 and 20.19 lbf / in 2 The pressure drop from each of the water 205 and ethanol 203 supply channels is approximately 0.75 lbf / in between each continuous vortex mixing chamber. 2 It only descends.

[0131] Figures 2H and 2I show an example of an apparatus including a series of vortex mixing chambers, each of which has a channel inlet 211 (as shown in Figures 2D-2G) with a width of 100 μm and a depth of 50 μm. The vortex mixing chambers in this example are approximately rectangular (with rounded sides) and have a length of 500 μm, a width of 250 μm, and a depth of 100 μm. Thus, this example has vortex mixing chambers that are twice as long but otherwise similarly sized, as shown in Figures 2D and 2E above. In Figures 2H and 2I, the depth of the vortex mixing chambers is also twice the depth of the inlets, and the inlet openings and chambers have a common top surface, so that the maximum drop from the inlet opening of the vortex mixing chambers to the top (or bottom, depending on the reference frame) is approximately the same as the depth of the inlets. The 1:10 ethanol:water mixing profile (indicated by shaded arrows) is approximately the same as in the examples in Figures 2D and 2E. Figure 2I shows the pressure drop for the same example (also showing six sequentially connected vortex mixing chambers). Mixing is substantially complete after exiting the second mixing chamber. Approximately 20 (e.g., 20.33 lbf / in) 2 and 20.37 lbf / in 2 The pressure drop from each of the water 205 and ethanol 203 supply channels is approximately 0.75 lbf / in between each continuous vortex mixing chamber. 2 It only descends.

[0132] Figure 2J shows an example of a device including a series of vortex mixing chambers, each of which has a channel inlet 211 with a width of 100 μm and a depth of 50 μm, as shown in Figures 2D to 2I. The vortex mixing chambers in this example are approximately rectangular (with rounded sides) but are approximately three times the depth of the channel inlet openings. In Figure 2J, the vortex mixing chambers have a length of 350 μm, a width of 250 μm, and a depth of 150 μm. Thus, this example has vortex mixing chambers with a similar shape to those shown in Figures 2F to 2G, but with a depth 50% greater. Therefore, in Figure 2J, the depth of the vortex mixing chambers is three times the depth of the inlets, and the inlet openings and chambers have a common top surface, resulting in a maximum drop from the inlet opening of the vortex mixing chambers to the top (or bottom, depending on the reference frame) being approximately twice the depth of the inlets. The 1:10 ethanol:water mixing profile (indicated by the shaded arrow) shows that mixing is very efficient in this example at these pressures and flows, and shows almost complete mixing after the first vortex mixing chamber, as indicated by the arrow showing the volume percentage of ethanol. The pressure drop between the vortex mixing chambers is approximately the same as that shown in Figures 2D to 2I. Thus, at these pressures and flows, the relative drop from the inlet to the vortex mixing chamber appears to correlate strongly with the mixing efficiency, for example, compared to the length of the chamber. In the mixing model in Figure 2J, vortex and complete mixing are achieved in one stage.

[0133] Any of the vortex mixing chambers described herein may be part of a microfluidic mixing apparatus, which may comprise one or more vortex mixing chambers. A microfluidic mixing apparatus may be implemented as part of a microfluidic device. For example, a microfluidic mixing apparatus used as part of a microfluidic apparatus for mixing and compounding biomolecular products is shown in Figures 3A-3E and 4. As described above, a microfluidic apparatus may be formed from a first plate and a second plate, and microfluidic channels may be formed in portions of one or both plates. In Figure 3A, the microfluidic apparatus 300 may comprise an elastic layer positioned between the first plate and the second plate. This apparatus also comprises two mixing regions 330, 331 and is configured to mix materials from three separate inputs 303, 305, 335. Similar to the mixing apparatus described above, the first fluid is introduced into the first fluid input 303, and the second fluid is introduced into the second fluid input 305, and these intersect at a fluid intersection 309, which may be configured as a fluid intersection 109. The fluids may be configured to be driven at a pressure higher than atmospheric pressure and supported by an inlet valve 332 (for example, positive or negative pressure may be applied). The merged flow continues into four vortex mixing chambers 330 arranged in series along the microfluidic flow path. Each of the individual vortex mixing chambers of the mixing apparatus 330 may be configured as the microfluidic vortex mixing unit 130 described above and may have any of the dimensions described above. The two-stage mixing apparatus may be configured to output the mixed fluid through a single output from the final microfluidic vortex mixing chamber (the fourth in series).

[0134] The apparatus 300 is further configured to mix a third fluid component. After outputting the mixed fluid from the first-stage mixing apparatus 330, the output channel becomes the third fluid inlet 333, intersecting with the fourth fluid inlet 335, and introducing the third fluid component at the second fluid intersection 319 as described above. The merged fluid flow is then fed into the vortex mixing chambers of the second mixing stage 331, which are arranged continuously along the microfluidic flow path. Each of these vortex mixing chambers of the second stage 331 may be configured as any of the microfluidic vortex mixing chambers described above. Complete mixing can be achieved using a single vortex mixing chamber in either the first or second-stage mixing path, but in some examples, additional mixing chambers may allow for further mixing, for example, by providing a buffer in flow rate. The mixed fluid traversing through the vortex mixing chambers may be output from the mixing path (e.g., from the second stage) in a single channel and may continue along the microfluidic flow path for further processing in other areas of the microfluidic apparatus.

[0135] The microfluidic pathway apparatus 300 in Figure 3A also includes a vacuum cap 334 which can be held under negative pressure to remove gas from the liquid (fluid) line by drawing it through a membrane over the fluid pathway, provided that the cap is gas-permeable. For example, a polydimethylsilicone (PDMS) elastomer film is sufficiently gas-permeable to enable this. In the case of the cascade mixer shown in this example, there are three fluid-driven chambers configured to drive a first fluid component, a second fluid component, and a third fluid component, each into their respective inlet channels. Each fluid-driven chamber has a fixed volume and is formed between a first plate and a second plate. A portion of the elastic layer positioned between the first and second plates divides each fluid-driven chamber into a fluid-contact side in the second plate and a pressure-receiving side in the first plate. The pressure-receiving side can be pressurized to drive the fluid through the chamber into the mixers 330, 335. Each fluid-driven chamber includes a fluid port (from a microfluidic channel) that fluidly connects to the respective fluid-contact side of each first fluid and each fluid-contact side of the second fluid-driven chamber via respective fluid channels in the second plate, and a pressure port that extends through the first plate into the second plate and fluidly connects to the pressure-receiving side of the fluid-driven chamber via respective pressure channels that extend through the second plate along the first plate. The volume of the fluid-contact side of the fluid-driven chamber can be adjusted by applying pressure or vacuum from the respective pressure ports. The fluid ports of the fluid-driven chamber may further include flow restrictors 336. In some examples, the flow restrictors may include meandering elongated fluid channels.

[0136] In general, the methods and apparatus described herein may involve the use of multiple fluids (e.g., materials in the fluids, including mRNA, buffers, salts, and delivery vehicles) that can be supplied from an external reservoir. Any of these methods and apparatus may include one or more vacuum cap structures and valves for releasing the fluids in a controlled manner so that all fluids advance to the starting point without bubbles and the mixing result is stable throughout the mixing process. As described above, the vacuum cap may be configured to reduce or remove bubbles from the fluid(s). The apparatus and methods described herein may also include valves connected to one or more waste collection areas. In some examples, initial results may be sent to the waste output as needed to maintain the overall quality of the output.

[0137] The microfluidic mixing apparatus 300 may further include a fourth fluid-driven chamber, which may be positioned along the microfluidic channel following the mixing apparatus. In Figure 3A, a vacuum cap 338 may be included. Although two cascade-type mixers are shown, additional mixers may be included as part of the fluid channel. In this way, the step of forming a nanoparticle-based therapeutic agent can be broken down into steps achieved in a very timely and controlled manner along the cascade. For example, in the first mixer, polynucleotides such as mRNA in water can be mixed with one or more delivery vehicle molecules in ethanol to form complex nanoparticles. Using the second mixer, a diluent such as a citrate-based buffer solution for pH adjustment can be added. If more mixers are used, additional steps may be included. For example, it may be desirable to add a surface layer to the nanoparticles formed in the first mixing step to enhance the bioactivity of the nanoparticles. This can be done by combining the output fluid stream of the first mixer with a solution containing the desired overcoating material in the second mixed structure. This can then be combined with a pH-adjusting buffer solution in a third mixer. It may also be useful to prepare a mixture of polynucleotides and water in an upstream mixer structure where the polynucleotides and delivery vehicle molecules are combined. In this way, the more concentrated polynucleotides, typically produced in the mRNA synthesis process, can be uniformly diluted with water before the nanoparticle formation step. Similarly, the upstream mixing of ethanol and delivery vehicle molecules can be performed before the mixer combining the polynucleotide solution and the delivery vehicle solution.

[0138] Figures 3B and 3C show examples of microfluidic devices configured as continuous mixers. In Figure 3B, the microfluidic device 350 includes multiple mixers arranged in parallel. As described above, the microfluidic device may include two or more plates separated by a deflectable membrane, and chambers and channels are formed on the upper and / or lower surfaces of the plates which can be divided by the membrane. In this example, the device may be configured to receive multiple reagents, e.g., mRNA, delivery vehicles, diluents, etc., which can be pumped directly from reagent containers (e.g., vials, tubes, not shown) located outside the microfluidic device (e.g., “tip”). Mixers may be used to mix reagents for distribution from the microfluidic device into, for example, a collection container (not shown). The microfluidic device may include ports for coupling to one or more pressure lines 352 which can be used to selectively apply pressure (e.g., positive and / or negative air pressure) to control one or more valves (e.g., allowing the flow of reagents to turn the tip on / off). Reagents can be pressurized in reagent containers and driven onto the microfluidic device when valves that allow them to flow are opened.

[0139] Figure 3C shows a magnified view of area D of the apparatus 300 in Figure 3B. In this example, the mixer 369 may be configured as a single mixer as described herein. Three inputs are shown for each of the three reagents, including an mRNA input 355, a delivery vehicle input 357, and a diluent input 359. Valves 363 may be opened / closed by selectively applying positive and / or negative pressure (e.g., by a controller) to allow fluid to flow. In the example shown in Figure 3C, each reagent is also coupled to a vacuum cap 361 which can be used to remove air (e.g., bubbles) from the fluid before it passes through and enters the mixer 369. For example, a vacuum cap may have negative pressure applied to draw air through a membrane that allows air to pass through but not fluid.

[0140] In Figure 3C, the mixer 369 includes a first fluid input 365 and a second fluid input 367 that intersect in a fluid cross channel leading into the mixer 369. In this example, the mRNA reagent is mixed with the delivery vehicle in the mixer as described above. The output of the mixer forms a cross with an input 371 for the dilution buffer just upstream of the output 354 of the microfluidic device ("tip").

[0141] In this example, the mixer can operate continuously or nearly continuously, as the material of its volume arrives from an off-tip container and the output from the tip can be stored in an off-tip storage container. Therefore, in this example, the fluid can be driven through the mixer directly by applying pneumatic pressure. In some cases, which may be used for smaller volumes or more discrete (including metered) volumes of material, the fluid can be driven through the channel and / or mixer by deflecting a film between the plates of the microfluidic device.

[0142] The example shown in Figure 3C may be configured to prevent clogging or accumulation of material in the mixer, which will be described in more detail below with reference to Figure 8. For example, Figures 3D and 3E show examples of microfluidic apparatus similar to those shown in Figures 3B and 3C, in which the dilution buffer is added (with or without mixing using the mixer) to a mixed solution of reagents, e.g., mRNA and delivery vehicle, after being mixed in the mixing chamber. In Figure 3D, the output channels 373 of mixers 369, 369', and 369'' extend only a very short distance (e.g., less than about 100 μm, less than about 150 μm, less than about 200 μm, less than about 400 μm, less than about 500 μm, etc.) before crossing the dilution buffer input 371. In contrast, in Figure 3E, the microfluidic apparatus is configured such that, in the longest case, the output channels 373' of three mixers 369, 369', and 369'' arranged in series have widths of, for example, over 600 μm, over 700 μm, over 800 μm, over 900 μm, over 1000 μm, and so on.

[0143] Devices with output channels shorter than, for example, 500 μm (such as about 400 μm, about 300 μm, etc.) can generally be more compact than other designs while still providing enhanced mixing. Furthermore, mixing with the dilution buffer at a very short distance from the input may result in less material deposition. Alternatively or additionally, shortening the distance between the first 369'' and the second 369' mixer (or the second and third 369) can also reduce or eliminate deposition. For example, the mixing devices described herein may include less than about 500 μm (e.g., less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm) between mixing chambers arranged in series. In some examples, these devices may include a dilution buffer input at or near the output of the mixing device.

[0144] Figure 4 shows an example of a portion of another microfluidic device that also experiences a pressure drop across device 400, including a mixing subassembly 433 configured like device 300. A first fluid component may be introduced into the microfluidic device at input 403'. In this example, the fluid flow is 23.28 lbf / in 2 starts at a pressure of (160.5 kPa), traverses through flow restrictor 434 and vacuum cap 435, and reaches the first fluid inlet 403 at a pressure of 20.15 lbf / in 2 (138.9 kPa). A second fluid component is at input 405' at 23.30 lbf / in 2 at a pressure of (160.6 kPa), through its respective flow restrictor and fluid drive chamber, at 20.17 lbf / in 2 at a pressure of (139.0 kPa) is introduced into the second fluid inlet 405. The two fluids intersect at equalized pressure, are mixed in the first vortex mixing chamber, for example, at 16.73 lbf / in 2The mixture can then pass through the subsequently sequentially arranged chambers of the mixing subassembly 433 until it exits output 417 at (115.3 kPa). The mixture then enters the second stage 445 of the cascade mixer, where it can cross with a third fluid component at the second fluid crossing. In this example, the third fluid component is, for example, 23.30 lbf / in at input 407. 2 It is entered into the microfluidic path device 400 at a pressure of (160.6 kPa), crosses the flow limiter 457, and flows at 16.73 lbf / in. 2 The fluid reaches the fourth fluid inlet 435 at (115.3 kPa), where the pressure is equalized with the fluid arriving from the third fluid inlet 417. The merged flow passes through the last pair of vortex mixing chambers of the second stage of the mixing subassembly, at 14.36 lbf / in. 2 Entering 438 at (99.0 kPa). The pressure can be further reduced within 438, and the fluid at output 414 is, for example, 7.60 lbf / in. 2 It can be output at a pressure of (52.4 kPa). In some examples, this mixing subassembly can be fluidly connected in line with additional processing components on either the same microfluidic device (microfluidic pathway device) or a separate microfluidic device.

[0145] In general, the mixers described herein can be cascaded together. Cascaded mixers may provide additional mixing and may enable a higher degree of mixing at increased flow rates. For example, any of the microfluidic devices described herein may be a cascade of microfluidic vortex mixers, each of which may include a vortex mixing chamber comprising a base defining a bottom surface, side walls, and a top surface surrounding the vortex mixing chamber; a mixing inlet channel having an opening to the vortex mixing chamber in a first side wall of the vortex mixing chamber; and a mixing outlet channel having an opening to the vortex mixing chamber in a second side wall of the vortex mixing chamber, wherein the vertical dimension of the vortex mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel, and furthermore, the microfluidic vortex mixers are connected in series, so that the mixing inlet channel of each microfluidic vortex mixer following the first microfluidic vortex mixer in series is connected to the mixing outlet of the preceding microfluidic vortex mixer in series.

[0146] For example, Figure 5 shows a microfluidic device configured as a microfluidic path compounding apparatus 500, which includes a cascade mixing subassembly 510 similar to that shown in Figure 4. This mixing subassembly may include multiple vortex mixing chambers configured in series. The apparatus 500 also includes pumps 520 and 550, as well as fluid-driven chambers 530, 540, and 560 (which may act as blending chambers).

[0147] temperature In any of the mixing apparatuses described herein, the applicants have surprisingly found that it is beneficial to mix certain materials, such as mRNA in aqueous solution and a delivery vehicle (e.g., in ethanol), at temperatures below room temperature (e.g., below approximately 25°C), such as below 20°C, below 18°C, below 15°C, below 12.5°C, below 10°C, below 8°C, below 7°C, etc.

[0148] Any of the microfluidic path devices described herein may operate as part of a system that includes temperature control, including temperature control of the mixing portion (mixing subassembly) of the microfluidic device. Thus, a mixing subassembly including one or more vortex mixing chambers may be cooled to a temperature of about 20°C to 5°C during the operation of the mixing subassembly, for example, about 18°C ​​to 5°C, about 15°C to 5°C, or about 15°C to 8°C.

[0149] In some examples, the entire microfluidic device, including the mixing chamber, may be temperature-controlled to the mixing temperature. Alternatively, only a portion of the microfluidic device may be temperature-controlled as described herein. For example, only the mixing chamber(s) may be temperature-controlled to the mixing temperature, while other parts of the microfluidic device may be temperature-controlled to one or more different temperatures. In some examples, the microfluidic device (or any of its sub-regions, such as the mixing chamber(s)) may be temperature-controlled to the mixing temperature only during mixing, and maintained at a different temperature at other times.

[0150] Figure 6 is a graph showing the effect of temperature on mixing using the mixing apparatus described herein. Figure 6 shows mixing using the same mixing apparatus as shown in Figures 1A and 1B above (corresponding to “8 / 28 Box” mixing) and compares it to the use of unmixed (“Cell Only”) and hand-mixed (“8 / 28 Hand”) samples. The samples include cells transfected with a fluorescent agent, allowing for quantification of the effectiveness of the mixing, with larger fluorescence signals indicating higher mixing efficiency, which is measured as corrected bioluminescence (RLU). Hand-mixed reagents (“8 / 28 Hand Master,” “8 / 28 Hand Aliquot 1,” and “8 / 28 Hand Aliquot 2”) had corrected bioluminescence that was nearly equivalent to that mixed at 21°C in the above-described mixing apparatus (e.g., “8 / 28 Box 21C1,” “8 / 28 Box 21C2,” “8 / 28 Box 21C3,” and “8 / 28 Box 21C4”). Surprisingly, mixtures prepared at lower temperatures, e.g., 10°C, exhibited significantly higher bioluminescence (compared to "8 / 28 Boxrun 7 10C" and "8 / 28 Boxrun 8 10C"). In Figure 6, mixtures prepared at lower temperatures, e.g., 10°C, using the same mixing apparatus, had almost twice the bioluminescence compared to mixtures prepared using the same mixing apparatus or by hand at 21°C. For the reagent combinations shown, at higher temperatures (e.g., 40°C and 60°C), bioluminescence was approximately the same as that at 21°C.

[0151] The mixing temperature can be set manually or automatically. In some cases, the mixing temperature, which is typically around 20°C to 5°C but is not exclusive, can be determined based on the mRNA being mixed (e.g., therapeutic mRNA) and / or the delivery vehicle. For example, the enhanced mixing temperature can be determined using a combination of mRNA and delivery vehicle, as described above. The enhanced mixing temperature can be determined empirically (e.g., experimentally) and / or by calculation based on, for example, the size, molecular weight, sequence, etc., of the mRNA and / or delivery vehicle.

[0152] As described above, the mixing apparatus described herein may be part of a microfluidic apparatus (e.g., a microfluidic device) and may include a first fluid input and a second fluid input, and a fluid crossing channel configured to receive fluid from the first fluid input and the second fluid input, wherein the fluid crossing channel opens into the first mixing chamber on the upper region of the first side of the first mixing chamber. The first mixing chamber may have a depth greater than about 1.5 times the depth of the fluid crossing channel. The device may also include an outlet channel on the upper region of the second side of the first mixing chamber, wherein the outlet channel has a depth less than the depth of the first mixing chamber, and further, the opening of the outlet channel is offset with respect to the fluid crossing along the width of the second side of the first mixing chamber.

[0153] In some examples, a microfluidic mixing apparatus may include multiple mixers (e.g., multiple mixing chambers) as part of the apparatus, and these may be connected in series. For example, Figures 7A–7D show examples of mixing apparatuses with one (Figure 7A), three (Figure 7B), six (Figure 7C), and twelve (Figure 7D) mixing chambers. As mentioned above, surprisingly, nearly uniform mixing can be achieved using a single mixing chamber (see, for example, Figures 2A–2E). In some cases, especially when the mixture contains particles suspended in the fluid being mixed, complete or near-complete mixing can be achieved using two or more, for example, two or three mixing chambers. This is shown below and is particularly surprising given the relatively small dimensions (e.g., footprint) of the mixing apparatus, even when relatively high flow rates and low pressures (e.g., pressures of about 6.9 kPa to about 206.8 kPa, and flow rates of 1 mL / min to about 10 mL / min) are used. The mixing apparatus described herein may have a total length of approximately 2 mm or less from input to output (for example, approximately 1.75 mm or less, approximately 1.7 mm or less, approximately 1.6 mm or less, approximately 1.5 mm or less, approximately 1.4 mm or less, approximately 1.2 mm or less, approximately 1 mm or less, approximately 0.8 mm or less, approximately 0.7 mm or less, etc.). Even with these relatively short lengths, nearly uniform mixing can be achieved.

[0154] All apparatuses and methods described herein provide superior mixing to manual mixing, including greater uniformity and, as a result, smaller particle size for mixtures containing particles (which may otherwise cluster). However, mixtures containing particles may present certain challenges to microfluidic mixing. For example, repeated and / or continuous use of a microfluidic mixer may result in particle deposition within the channels of the microfluidic mixer. Figure 8 illustrates this potential problem. Figure 8 shows an image of a mixer. The mixer comprises three series-connected mixers (mixing chambers) described herein, having, for example, widths / depths / lengths of approximately 250 / 200 / 500 μm. In this figure, the mixer mixes a fluid containing material (e.g., mRNA and molecules of delivery vehicles containing or not containing mRNA, such as molecules of aminolipidized peptoid delivery vehicles) for continuous operation to form a therapeutic agent (e.g., mRNA encapsulated in a delivery vehicle), but over time this has resulted in material deposition within the mixer.807 During operation, such deposition can lead to clogging. The apparatus and methods described herein may be configured to reduce or prevent clogging and / or buildup of materials.

[0155] For example, in some cases, the number of mixing chambers may be limited. Therefore, in some cases, three or fewer mixing chambers may be used. As shown above and illustrated above, in some cases, two mixing chambers may be connected in series for mixing within the mixing apparatus. In some cases, three mixing chambers may be connected in series for mixing within the mixing apparatus. Alternatively, in some cases, only a single mixing chamber may be included. These configurations may have the additional advantage of having a substantially smaller footprint compared to other mixers.

[0156] In some examples, the size of the chambers and / or channels of the mixing apparatus can increase proportionally. Larger mixing chambers can reduce particle deposition in the channels. For example, in some examples, the dimensions of one or more mixing chambers may be approximately 225 to 600 μm in width (e.g., approximately 250 to 600 μm, approximately 300 to 550 μm, etc.), approximately 175 to 425 μm in depth (e.g., approximately 200 to 400 μm, approximately 300 to 425 μm, etc.), and approximately 450 to 1050 μm in length (e.g., approximately 500 μm to 1000 μm, etc.). For example, one or more mixing chambers may have a width / depth / length of approximately 500 / 400 / 1000 μm. Similarly, the connecting channel(s) may have a width of approximately 75 μm to approximately 225 μm (e.g., approximately 100 μm to approximately 200 μm), a depth of approximately 75 μm to approximately 225 μm (e.g., approximately 100 μm to approximately 200 μm), and a length of approximately 225 μm to approximately 525 μm (e.g., approximately 250 μm to approximately 500 μm).

[0157] Figures 9A and 9B show examples of similar but scaled mixing apparatuses. The mixing apparatus in Figure 9A shows a mixing chamber 905 and a connecting channel 903, which are a first set of dimensions (e.g., the width / depth / length of the mixing chamber is approximately 250 / 200 / 500 μm and the width / depth / length of the connecting channel is approximately 100 / 100 / 250 μm). In Figure 9B, the same shape is scaled up by a factor of two (e.g., twice), resulting in the mixing chamber and connecting channel having twice the width, depth, and length (e.g., the mixing chamber has a width / depth / length of approximately 500 / 400 / 1000 μm and the connecting channel has a width / depth / length of approximately 200 / 200 / 500 μm).

[0158] As shown in Figure 9C, the overall mixing effect of the mixing apparatus was comparable for both smaller (e.g., Figure 9A) and larger (e.g., Figure 9B) dimensions, and both the average particle size and particle dispersion were tested for both. Dispersion is a measure of the heterogeneity of molecular or particle size in a mixture. A collection of objects is called homogeneous if the objects have the same size, shape, or mass. A sample of objects with inconsistent size, shape, and mass distribution is called heterogeneous. The polydispersity index (PDI) is used as a measure of the spread of molecular weight distribution, and therefore an indicator of size distribution. A larger PDI indicates a wider molecular weight distribution. The PDI of a polymer is calculated as the ratio of the weight average to the number average molecular weight. The degree of dispersion (e.g., PDI) can be measured by light scattering measurements such as dynamic light scattering and / or direct measurement, for example, via mass spectrometry, using matrix-assisted laser desorption / ionization (MALDI) or electrospray ionization with tandem mass spectrometry (ESI-MS). The polydispersity index is dimensionless and is scaled so that values ​​less than 0.05 are rarely seen except as a high monodispersity standard. Values ​​greater than 0.7 may indicate that the sample has a very wide size distribution and is therefore not uniform in size. The Z-mean size or Z-mean value can be used in dynamic light scattering as a parameter (also known as the cumulant mean) to provide a hydrodynamic parameter applicable to particles in dispersions or molecules in solutions.

[0159] As shown in Figure 9C, the PDIs for both the smaller (Figure 9A) and larger (Figure 9B) mixers were reasonably similar; however, the Z-mean was slightly lower in the smaller mixer compared to the larger mixer.

[0160] Any of the mixing apparatuses described herein may have rounded or curved corners and / or edges. For example, Figure 10A shows a mixing apparatus similar to those shown above (e.g., Figures 7B, 9A, etc.), and Figure 10B shows an example of a mixing apparatus having rounded edges 1015 and / or corners at the bottom and / or top of the mixing apparatus. Rounded (e.g., curved, curved, etc.) edges / corners can prevent dead or stagnant areas in the mixer where particles may accumulate. Furthermore, rounded edges can also amplify the mixing in the mixing chamber (as described above, since the fluid is driven against the walls and rotates in the chamber, thereby enhancing the mixing). In some examples, the opening from the mixing chamber to the outlet (e.g., into a connecting channel) may include a sloping or funnel shape, such as a funnel shape with a sloping diameter (or width and depth) of the opening, which can provide a gentler transition between the mixing chamber and the channel(s).

[0161] Any of the apparatuses described herein may be configured such that the mixers are at an angle to each other, as described above. In some examples, the angle is about 90 degrees (as shown in Figures 7B–7D), and the mixing chamber is positioned perpendicular to the connecting channel. Figures 11A–11B show another example of a mixing apparatus where the angle between the mixing chamber and the connecting channel is about 135 degrees (see Figure 11B) when observed from above. Thus, in some examples, the angle between the mixing chamber and the connecting channel (which may be called the box angle or mixing chamber angle) can be between 90 and 180 degrees, such as about 100 degrees, about 110 degrees, about 120 degrees, about 130 degrees, about 135 degrees, about 140 degrees, about 150 degrees, and about 160 degrees. Increasing this angle beyond 90 degrees can reduce sedimentation and / or increase the flow rate (for lower pressures). Conversely, in some cases, it is preferable to reduce the angle to less than 90 degrees, which can increase mixing efficiency.

[0162] As shown in Figure 11C, there was no significant change in the Z-mean (e.g., particle size) or PDI between angled and curved configurations (e.g., 135 degrees vs. 90 degrees). As shown in Figure 11C, in general, a single-stage mixer (e.g., a mixer with only a single mixing chamber) can mix even particles sufficiently. Compared to a three-stage device of similar dimensions, the final particle size and PDI values ​​were found to be equivalent to, or in some cases even better than, those of a single-stage mixer. Therefore, a highly compact single-stage device may be used, and flow limitations can be reduced.

[0163] In some examples, the mixing apparatus described herein may result in substantially less sedimentation over time. For example, apparatus with curved side walls and / or bottom and / or top may result in sedimentation of less than 25% per hour and / or flow velocity (e.g., less than 20%, less than 15%, less than 10%, less than 5%, etc.).

[0164] As described above, the flow rate can be controlled. The flow rate can also affect mixing. Generally, faster flow rates through these devices can result in smaller particle sizes, which can reflect enhanced mixing. This is shown in Figure 13. In Figure 13, several examples of similar microfluidic mixing devices on the same microfluidic device substrate (e.g., "chip") are provided in parallel repeats of 1 mL / min (1-4), 2 mL / min (1-4), and 4 mL / min (1-2). The flow rate can be adjusted to the particle size and / or dimensions of the microfluidic device. In Figure 13, both the volume-averaged size (size in nm) and PDI are reasonably equivalent, and the particle size decreases as the flow rate increases.

[0165] As described above, mixing can be enhanced by a “step” or transition from a smaller opening at the input of the mixing chamber to the mixing chamber, and then back to a smaller diameter in the output (or connection) channel(s). However, in some examples, as shown in Figure 12B, the mixing apparatus may have the same height between the input, output, and mixing chamber. Figure 12A shows another example of a mixing apparatus, similar to Figure 7B, including three mixers connected in series. For comparison, Figure 12B shows an example of an apparatus that does not include a step from a narrower-height channel to a deeper box of mixers. In general, these mixers may not yield the high level of mixing shown for other embodiments, as shown in Figure 12C. Figure 12C is a graph showing particle size (to be mixed) after multiple runs through a mixing apparatus as shown in Figure 12A ("3 boxes") or a linear mixing channel as shown in Figure 12B ("3 box plane"), and compared to Figure 12B, the mixing apparatus in Figure 12A shows substantially smaller particle size (and therefore more efficient mixing).

[0166] Optional embodiments Furthermore, this specification describes additional examples of microfluidic devices. These devices may include the mixers described herein, each comprising one or more additional, optional microfluidic components. For example, the outlet channel of the mixer may be in fluid communication with one or more of a pair of final blend chambers, dialysis chambers, or evaporation chambers. Microfluidic pathway devices (e.g., microfluidic chips) may include microfluidic dialysis chambers and / or microfluidic concentrators. Dialysis chambers and / or concentrators may be very compact and efficient and may operate on or within the boundaries of a highly efficient and precise microfluidic device. The mixing methods and devices described herein may also enable the microfluidic device to purify, dialysis, and concentrate one or more therapeutic compositions (including, but not limited to, therapeutic polynucleotides) in a single, integrated device.

[0167] For example, a microfluidic path device includes a first plate and a second plate, a fluid contact chamber having a fixed volume formed in the first plate, a dialysis buffer chamber having a fixed volume formed in the second plate, wherein the fluid contact chamber is separated from the dialysis buffer chamber by a dialysis membrane positioned between the first plate and the second plate, and a plurality of pressure ports passing through the first plate, the fluid contact chamber comprising a plurality of channels partitioning the fluid contact chamber.

[0168] The microfluidic dialysis chamber may have a fluid contact chamber formed on a first plate and a dialysis buffer chamber formed on a second plate, wherein the fluid contact chamber is separated from the dialysis buffer chamber by a dialysis membrane positioned between the first plate and the second plate, and a plurality of pressure ports passing through the first plate, and the fluid contact chamber comprises a plurality of channels that partition the fluid contact chamber.

[0169] Any of these microfluidic dialysis chamber devices may include an inlet to a fluid contact chamber and an outlet from the fluid contact chamber, with the inlet located on the opposite side of the length and the opposite side of the width of the fluid contact chamber. The inlet may be offset from the side of the fluid contact chamber by approximately 15% to 35% of the width of the fluid contact chamber. Any of these dialysis devices may include an elastic membrane sandwiched between a first plate and a second plate. The periphery of the dialysis membrane may be sealed by an elastic membrane.

[0170] For example, a microfluidic pathway device may include a first plate and a second plate, a fluid contact chamber having a fixed volume formed in the first plate, and a concentration chamber having a fixed volume formed in the second plate, wherein the fluid contact chamber is separated from the concentration chamber by a hydrophobic membrane disposed between the first plate and the second plate, a plurality of pressure ports passing through the first plate, and a plurality of individually addressable membrane-driven pumps controlled by the pressure ports, and is configured to drive fluid through the fluid contact chamber and drive dry air through the concentration chamber.

[0171] The microfluidic pathway device may include a mixer, a dialysis subassembly, and a concentrator subassembly, wherein the mixer-dialysis subassembly and the concentrator are formed between a first plate and a second plate.

[0172] A dialyzer may be formed as part of a microfluidic pathway device and may include a first chamber separated from a second chamber by a dialyzing membrane, the first and / or second chambers may be divided into a plurality of channels. The first channel is configured to allow the fluid to be dialyzed to pass through, and the second channel is configured to allow the dialyzing solution to pass through. The dialyzing solution may pass through the second channel in a reverse flow direction (for example, opposite to the direction of the fluid flowing through the first channel).

[0173] In some examples, a dialyzer is formed between a first plate and a second plate (e.g., a first layer and a second layer) of a microfluidic pathway device. A first channel may be formed within the first plate, a second channel within the second plate, and a dialyzer membrane may be sealed between the first and second plates. In some examples, an elastic membrane may be sandwiched between the first and second plates, and the dialyzer membrane may be sandwiched between the first and second plates across an opening in the elastic membrane and sealed by the elastic membrane (e.g., around its periphery). The first chamber of the dialyzer may include an inlet on one end of the first chamber and an outlet on the opposite end. The inlet and outlet may be offset from the side edges of the first chamber, for example, at a position between 15 and 45 percent of the width of the chamber from the first side edge, with a width formed between the side edges. Similarly, the outlet may be on the opposite side of the chamber (separated by most of the chamber's length), and the inlet is offset from the second side (opposite the first side's edge) by the same or approximately the same amount from the first side's edge (e.g., 15-45% of the chamber's width).

[0174] Alternatively, in some examples, the elastic membrane is not used to seal the dialysis membrane. Therefore, the dialysis membrane can be reliably held in place by the engagement of the first plate with the second plate. In some examples, an additional (e.g., a third plate) and / or elastic membrane may be included, for example, below or above the presumed first and second plates.

[0175] The first and / or second chambers of the dialyzer may be divided into a plurality of channels as described above. In some examples, the channels may be parallel or extend in a straight line. In some examples, the channels may extend in a curved or zigzag line. The channels may have a uniform cross-sectional diameter, or they may have different diameters, and / or they may have the same cross-sectional diameter.

[0176] Figure 14A shows a perspective view of an example of a dialyzer described herein. In Figure 14A, the dialyzer is a subregion (or dialyzer module) of a microfluidic device, comprising a first plate 1401, a second plate 1403, and an elastic membrane 1405 sandwiched between the first and second plates. An opening (invisible) through the elastic membrane may be extended by a dialyzer membrane 1407. The first chamber is separated from the second chamber of the dialyzer by the dialyzer membrane 1407, and each illustrated chamber is divided into a plurality of parallel channels extending the length of the first and second chambers.

[0177] Figure 14B is an example of a cross-section through a dialyzer similar to that shown in Figure 14A. In Figure 14B, the dialyzer includes a first chamber 1411, a second chamber 1413, and a dialyzer membrane 1407 between the first and second chambers. The inlet 1422 to the second chamber is also shown as the outlet 1423 from the first chamber (the outlet of the second chamber and the inlet of the first chamber are not visible in Figure 14B). Channels within each chamber may be formed by the plate from which the chamber is formed. In some examples, the channels are on only one side (e.g., the first chamber side), while in some examples, the channels are on both sides, and may be on opposite sides of each other or offset from each other.

[0178] Figure 15 shows an example of the edge region of a dialyzer, such as the examples shown in Figures 14A and 14B, illustrating the sealing between the upper and lower chambers and the dialysis membrane. In Figure 15, the first plate 1501 includes the first chamber 1511, which is divided into several connected channels. The second plate 1503 is attached to the bottom of the first plate and includes the second chamber 1505, which is also divided into channels. Channel dividers 1509, 1519 of the first and / or second plates form contact points between them that crimp the dialysis membrane 1515.

[0179] At the edge of the dialyzer 1500, the elastic membrane 1521 can be sandwiched between the first plate and the second plate. As shown in Figure 15, the edge of the elastic membrane (e.g., a silicone membrane) can also fix (e.g., seal) the dialyzer membrane between the first plate and the second plate.

[0180] During operation, the dialyzer portion of the microfluidic pathway device may include an inlet on the sample-processing side of the device for driving the solution to be dialyzed into the first chamber of the dialyzer (by applying pressure). Figure 16 shows the first chamber divided by multiple parallel channels. In this example, the inlet 1601 is located in the upper common region of the channel from which the fluid to be dialyzed can flow toward the outlet 1603. In this example, the inlet and outlet are located on both sides of the width and on both sides of the length of the chamber. Shading indicates the flow rate (velocity, Z, in cm / sec) through the chamber from the inlet to the outlet. This arrangement of the inlet and outlet results in non-uniform flow, as demonstrated by the shaded map, indicating that flow is slower through more peripheral channel regions.

[0181] Figure 17A shows an example where the inlet 1701 and outlet 1703 are positioned within a common region at the end of the chamber, slightly inward from the longer side of the chamber (e.g., 15% to 35% of the chamber width, e.g., about a quarter of the width) on both the width and length sides. (Indicated by heatmap key 17B) The resulting flow velocity is considerably more uniform, with slightly faster regions within the channels closest to the inlet and outlet. In the examples of Figures 17A–17C, the maximum flow rate may be, for example, about -1.1 cm / sec, while the minimum flow rate may be, for example, about -0.9 cm / sec. Figure 17C shows an upper common region 1707 supplying into a channel that extends the length of the first chamber of the dialyzer, which may have localized areas of higher flow rates 1711, 1709. In this example, the pressure between the inlet and outlet can drop to a delta of 0.22 psi (1.52 kPa), for example, from approximately 14.92 psi (102.87 kPa) to 14.70 psi (101.35 kPa) when the flow rate is 0.5 mL / min.

[0182] During use, a dialyzer can be used to dialyze a solution containing therapeutic materials, for example, to remove undesirable materials from a solution. As the solution flows through the first chamber, the dialysate can flow in the same or opposite direction to the second chamber, which is opposite the first chamber. The second chamber may have essentially the same structure as the first chamber described above.

[0183] Furthermore, this specification describes a concentrator. The concentrator may have the same structure as the dialyzer described above, but the membrane may be a membrane that allows water vapor to pass through (allowing evaporation through it) so that air can flow across the (hydrophobic membrane) in the second chamber as the fluid passes through the first chamber, thereby evaporating and concentrating the solution.

[0184] In some examples, a concentrator is configured to have one or more pathways (channels) through a first fluid passage chamber, and possibly a second chamber through which a gas (e.g., air) passes. Figure 18 shows an example of a concentrator device (e.g., a concentrator subassembly for a microfluidic pathway device). In Figure 18, the concentrator includes an elongated channel from inlet 1801 to outlet 1803 in the first chamber. A membrane (not shown in Figure 18) allows water vapor to spread between the first and second chambers. The gas can pass through the second chamber to remove water, and thus concentrate the solution as it passes through the first chamber. The evaporation rate may be related to the flow rate through the concentrator. In Figure 18, the shaded map shows the velocity (cm / sec) through the first chamber of the concentrator.

[0185] During use, the concentrator can be highly efficient and can concentrate a manufactured dose of therapeutic agent from a microfluidic pathway device to a concentration range that allows for dilution (e.g., 2 mL to 0.1 mL) into an injectable dose form.

[0186] The exemplary concentrator shown in Figure 18 is a 25.4 mm × 25.4 mm square. The membrane is a Sterlite PTFE membrane with a pore size of 0.22 μm and a thickness of 37 μm. In Figure 18, the input flow rate is approximately 0.5 mL / min. The dialysis membrane transport rate is 0.483 mL / min, and the resulting output flow rate is approximately 0.019 mL / min, or 1.1 mL / hour. In this example, for speeds of approximately 4.321 cm / sec to 0.160 cm / sec, the pressure drop between the inlet and outlet may be, for example, 14.96 psi (103.15 kPa) at the inlet and 14.70 psi (101.35 kPa) at the outlet (delta of 1.8 kPa).

[0187] As described above, any of the microfluidic pathway devices described herein may include one or more dialyzers and / or one or more concentrators (dialyzer subassemblies and / or concentrator subassemblies). Figure 19 schematically shows a microfluidic pathway device including both a series of mixers 1903 for compounding therapeutic agents (e.g., therapeutic RNA) formed on or added to the microfluidic pathway device, including, for example, the addition of a delivery vehicle, and a series dialyzer 1905 between the mixers (one or more) and the concentrator 1907. The first input 1911, the second input 1913, and the third input 1915 may be inserted as described above with reference to Figure 4. After compounding / mixing, dialysis, and concentration, the final product may be output from the concentrator 1931 and used, stored, or further processed. In this manner, the preparation of nanoparticle therapeutic agents, including dialysis and concentration to a final injectable form, may be carried out using a single continuous flow microfluidic device without intermediate storage of materials generated in the compounding process.

[0188] Figure 20 shows an example of the relationship between pressure and position on an exemplary microfluidic path device, such as the device schematically shown in Figure 19. In Figure 20, the flow resistance and pressure of the input pressures (V1-V4) can be regulated and / or monitored by the system to control the final concentration by adjusting the concentrator.

[0189] Where a feature or element is referred to herein as being "on top of" another feature or element, it may also be directly on top of or interposing to the other feature or element. In contrast, where a feature or element is referred to as being "directly on top of" another feature or element, there is no interposing feature or element. Also, where a feature or element is referred to as being "connected," "attached," or "joined" to another feature or element, it will be understood that it may be directly connected to, attached to, or joined to the other feature or element, or that there may be an interposing feature or element. In contrast, where a feature or element is referred to as being "directly connected," "directly attached," or "directly joined" to another feature or element, there is no interposing feature or element. Features and elements described or shown in part with respect to one example may be applicable to other embodiments. Also, references to structures or features located on another feature "adjacent" will be understood to include overlapping or underlying portions of the adjacent feature.

[0190] The terms used herein are for the purpose of illustrating only specific embodiments and are not intended to limit the invention. For example, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context specifically indicates otherwise. As used herein, the terms “comprises” and / or “comprising” specify the presence of the described features, steps, actions, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, actions, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the related enumerated items and may be abbreviated as “ / ”.

[0191] Spatially relative terms such as “under,” “below,” “lower,” “over,” and “upper” may be used herein to describe the relationship of one element or feature to another element or feature(single or multiple) as shown in the drawings. It will be understood that spatially relative terms are intended to encompass different orientations of a device in use or operation, in addition to the orientation shown in the drawings. For example, if the device in the drawing is inverted, an element described as “under” or “beneath” another element or feature will be oriented “above” the other element or feature. Thus, the exemplary term “under” can encompass both upward and downward orientations. The device may be oriented differently (by 90 degrees or in other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. Similarly, terms such as “upwardly,” “downwardly,” “vertical,” and “horizontal” are used herein for illustrative purposes only, unless otherwise specifically indicated.

[0192] The terms “first” and “second” may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context otherwise indicates. These terms may be used to distinguish one feature / element from another. Thus, the first feature / element discussed below may be called the second feature / element, and similarly, without departing from the teachings of the invention, the second feature / element discussed below may be called the first feature / element.

[0193] Throughout this specification and the claims thereafter, unless the context otherwise requires otherwise, the word “comprise,” and examples such as “comprises” and “comprising,” mean that various components may be used together in methods and articles (e.g., compositions and apparatus including devices and methods). For example, the term “comprising” would be understood to mean the inclusion of any described element or step, but not the exclusion of other elements or steps.

[0194] Where used herein and in the claims, including in the examples, all numbers, unless otherwise expressly specified, can be interpreted as if they were about or approximately, even if the words about or approximately do not appear expressly. In all cases where the phrase about or approximately is used, actual values ​​(e.g., quantity, distance, etc.) may be used. The phrase about or approximately may be used when describing size and / or location to indicate that the stated value and / or location is within a reasonable range of prediction for the value and / or location. For example, a numerical value may have an extent of + / -0.1% of the stated value (or range of values), + / -1% of the stated value (or range of values), + / -2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / -10% of the stated value (or range of values), and so on. Unless otherwise indicated, numerical values ​​given herein should be understood to include (about) or approximately the value. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range enumerated herein is intended to include all subranges contained therein. It should also be understood, as will be well understood by those skilled in the art, that a value is disclosed as also disclosing "less than or equal to," "greater than or equal to," and possible ranges between values. For example, if the value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" (for example, X is a numerical value) are also disclosed. Furthermore, throughout this application, the data is provided in several different formats, and it will be understood that this data represents end and start points, and also represents a range of any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it will be understood that the range is greater than 10 and 15, 10 and 15 or more, less than 10 and 15, 10 and 15 or less, and 10 and 15, and also 10 to 15.It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0195] While various exemplary examples have been described above, any of several modifications can be made to these examples without departing from the scope of the invention, as described in the claims. For example, the order in which various described method steps are performed may often be changed in alternative examples, and in other alternative examples, one or more method steps may be skipped entirely. Optional features of various device and system examples may be included in some examples and not in others. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as described in the claims.

[0196] The examples and figures included herein illustrate, not limiting, specific examples in which the subject matter may be implemented. As stated above, other examples can be used and derived therefrom so that structural and logical substitutions and modifications can be made without departing from the scope of this disclosure. Such examples of the subject matter of the present invention may be referenced individually or collectively herein by the term “invention” for convenience only, without intending to spontaneously limit the scope of this application to any single invention or inventive concept when two or more are actually disclosed. Thus, while specific examples have been illustrated and described herein, any arrangement calculated to achieve the same objective may be substituted for the specific examples shown. This disclosure is intended to cover any and all adaptations or examples of various embodiments. Combinations of the above embodiments, as well as other embodiments not specifically described herein, will be apparent to those skilled in the art upon consideration of the above description.

[0197] [Implementation Method] (1) A microfluidic device, A first fluid input and a second fluid input, A fluid crossing channel that receives fluid from the first fluid input and the second fluid input, wherein the fluid crossing channel opens into the first mixing chamber on the upper region of the first side of the first mixing chamber, and the first mixing chamber has length, width, and depth, the depth being more than 1.5 times the depth of the fluid crossing channel, An outlet channel on the upper region of the second side of the first mixing chamber, wherein the outlet channel has a depth less than the depth of the first mixing chamber, and the opening of the outlet channel is offset with respect to the fluid intersection along the width of the second side of the first mixing chamber, A microfluidic device equipped with the following features. (2) Microfluidic devices, A first fluid input channel and a second fluid input channel, the first fluid input channel and the second fluid input channel, which converge into a fluid cross channel having a width and a depth extending from the top surface to the first bottom surface, A first mixing chamber having a depth extending from the upper surface to the second bottom surface, a width extending from the first side to the second side, and a length, wherein the depth of the first mixing chamber is greater than the depth of the fluid crossing channel, the width of the first mixing chamber is greater than the width of the fluid crossing channel, and the first mixing chamber is fluidly connected to the fluid crossing channel at the upper surface, adjacent to the first side. An outlet channel, wherein the outlet channel is located adjacent to the second side of the first mixing chamber on its upper surface and is fluidly connected to the first mixing chamber, A microfluidic device equipped with the following features. (3) Microfluidic devices, A first fluid input and a second fluid input, A fluid crossing channel that receives fluid from the first fluid input and the second fluid input, wherein the fluid crossing channel opens into the first mixing chamber on the upper region of the first side of the first mixing chamber, and the first mixing chamber has width, length, and depth, the depth being more than approximately 1.5 times the depth of the fluid crossing channel, A connecting channel on the upper region of the second side of the first mixing chamber, wherein the connecting channel has a depth less than the depth of the first mixing chamber, and furthermore, the opening of the connecting channel is offset with respect to the fluid intersection along the width of the second side of the first mixing chamber, and the connecting channel opens into the second mixing chamber. An outlet channel extending from the second mixing chamber, A microfluidic device equipped with the following features. (4) The microfluidic device according to Embodiment 1 or 2, wherein the first mixing chamber is configured as a single mixer in which the outlet channel forms a mixer output, without being connected in series with any additional mixing chambers. (5) The microfluidic device according to any one of embodiments 1 to 4, wherein the depth of the first mixing chamber is about 2 to about 4 times the depth of the fluid crossing channel.

[0198] (6) The microfluidic device according to any one of embodiments 1 to 5, wherein the depth of the first mixing chamber is approximately three times the depth of the fluid crossing channel. (7) The microfluidic device according to any one of embodiments 1 to 6, wherein the width of the first mixing chamber is about 1.5 to about 3 times the length of the first mixing chamber. (8) The microfluidic device according to any one of embodiments 1 to 7, wherein the width of the first mixing chamber is approximately twice the length of the first mixing chamber. (9) The microfluidic device according to any one of embodiments 1 to 8, wherein the length of the first mixing chamber is about 2 to about 4 times the length of the fluid crossing channel. (10) The microfluidic device according to any one of embodiments 1 to 9, wherein the length of the first mixing chamber is approximately three times the length of the fluid crossing channel.

[0199] (11) A microfluidic device according to any one of embodiments 1 to 10, wherein the fluid crossing channel, the first mixing chamber, and the outlet channel are all located within a first layer, and the upper surfaces of the fluid crossing channel, the first mixing chamber, and the outlet channel are part of a second layer. (12) A microfluidic device according to any one of embodiments 1 to 11, wherein the outlet channel is in fluid communication with the first mixing chamber over a first length of the mixing chamber, and the fluid crossing channel is in fluid communication with the mixing chamber over a second length of the mixing chamber. (13) The microfluidic device according to any one of embodiments 1 to 12, wherein the first mixing chamber has rounded corners. (14) The microfluidic device according to any one of embodiments 1 to 13, wherein the first mixing chamber has an angular radius of about 65 to about 85 μm. (15) A microfluidic device according to any one of embodiments 1 to 14, wherein the change in fluid pressure passing through the first mixing chamber at a flow rate of 0.25 to 5 mL / min is approximately 6.9 kPa to approximately 206.8 kPa.

[0200] (16) A microfluidic device according to any one of embodiments 1 to 15, wherein the width of the first mixing chamber is about 150 to about 600 μm, the depth of the first mixing chamber is about 150 to about 500 μm, and the length of the first mixing chamber is about 500 μm to about 1000 μm. (17) The microfluidic device according to Embodiment 3, wherein the connection channel has a width of about 75 μm to about 225 μm, a depth of about 75 μm to about 225 μm, and a length of about 225 to about 550 μm. (18) A second mixing chamber having a depth extending from the top surface to the bottom surface of the second mixing chamber, a width extending from the first side to the second side of the second mixing chamber, and a length, wherein the depth of the second mixing chamber is greater than the depth of the outlet channel, and the width of the second mixing chamber is greater than the width of the outlet channel, and further, the second mixing chamber is fluidly connected to the outlet channel at the top surface, adjacent to the second side, A second outlet channel having depth and width, the second outlet channel being fluidly connected to the second mixing chamber, with its upper surface adjacent to the first side of the second mixing chamber, A microfluidic device according to embodiment 1 or 2, further comprising the above. (19) The microfluidic device according to any one of embodiments 1 to 18, further comprising one or more fluid pumps for pumping fluid from the fluid crossing channel into the first mixing chamber by deflecting at least a portion of the elastic membrane in the microfluidic device. (20) A microfluidic device according to any one of embodiments 1 to 18, further comprising one or more fluid pumps for pumping fluid from the fluid crossing channel into the first mixing chamber by deflecting at least a portion of the elastic membrane in the microfluidic device.

[0201] (21) The microfluidic device according to Embodiment 1 or 2, comprising a plurality of fluid-connected mixing chambers, including the first mixing chamber. (22) A microfluidic device according to any one of embodiments 1 to 21, further comprising a plurality of pressure ports for deflecting an elastic layer within the microfluidic device and driving a fluid through the first mixing chamber. (23) A microfluidic device according to any one of embodiments 1 to 22, further comprising a flow restrictor in fluid communication with the first fluid input, wherein the flow restrictor includes a meandering elongated fluid channel. (24) A microfluidic mixing apparatus, A mixing chamber comprising a base defining the bottom surface, side walls, and top surface surrounding the mixing chamber, The first side wall of the mixing chamber includes a mixing inlet channel with an opening to the mixing chamber, The second side wall of the mixing chamber includes a mixing outlet channel that includes an opening to the mixing chamber, The vertical dimension of the mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel. Microfluidic mixing device. (25) The microfluidic apparatus according to embodiment 24, wherein the first side wall and the second side wall are opposing side walls of the mixing chamber.

[0202] (26) The microfluidic apparatus according to embodiment 24 or 25, wherein the mixing inlet channel and the mixing outlet channel are connected to the mixing chamber at offset positions along the first side wall and the second side wall. (27) The microfluidic apparatus according to any one of embodiments 24 to 26, wherein the height of the opening of the mixing inlet channel is the same as the height of the opening of the mixing outlet channel. (28) A microfluidic apparatus according to any one of embodiments 24 to 27, wherein the width of the opening of the mixing inlet channel and the width of the opening of the mixing outlet channel are the same. (29) A microfluidic apparatus according to any one of embodiments 24 to 28, wherein the opening of the mixing inlet channel is located at the height of the first side wall adjacent to the upper surface of the mixing chamber, and the opening of the mixing outlet channel is located at the height of the second side wall adjacent to the upper surface of the mixing chamber. (30) The microfluidic apparatus according to any one of embodiments 24 to 29, wherein the mixing inlet channel has a first end including a fluid crossing and a second end including the opening to the mixing chamber.

[0203] (31) The microfluidic apparatus according to embodiment 30, wherein the fluid intersection further includes a first fluid input channel and a second fluid input channel that intersect with the mixing inlet channel at the fluid intersection. (32) A microfluidic apparatus according to any one of embodiments 24 to 31, wherein the first fluid channel and the second fluid channel intersect each other at the fluid intersection at an angle of less than approximately 180 degrees. (33) A microfluidic apparatus according to any one of embodiments 24 to 32, wherein the first fluid channel and the second fluid channel intersect each other at the fluid intersection at an angle of more than approximately 30 degrees. (34) The mixing chamber is a first mixing chamber, the mixing inlet channel is a first mixing inlet channel, the mixing outlet channel is a first mixing outlet channel, and the microfluidic device further comprises a second microfluidic mixing device, the second microfluidic mixing device is A second mixing chamber comprising a base defining the bottom surface, side walls, and top surface surrounding the second mixing chamber, The first side wall of the second mixing chamber includes a second mixing inlet channel with an opening to the second mixing chamber, The second mixing chamber includes a second mixing outlet channel in the second side wall of the second mixing chamber, which includes an opening to the second mixing chamber. The vertical dimension of the second mixing chamber is greater than the vertical dimension of the second mixing inlet channel and greater than the vertical dimension of the second mixing outlet channel. A microfluidic apparatus according to any of embodiments 24 to 33. (35) Furthermore, The first side wall and the second side wall of the second mixing chamber are opposing side walls of the second mixing chamber. The second mixing inlet channel and the second mixing outlet channel are connected to the second mixing chamber at offset positions along the first and second side walls of the second mixing chamber. The height of the opening of the second mixing inlet channel and the height of the opening of the second mixing outlet channel are the same. The width of the opening of the second mixing inlet channel and the width of the opening of the second mixing outlet channel are the same. The opening of the second mixing inlet channel and the opening of the second mixing outlet channel are positioned at the height of the first and second side walls of the second mixing chamber, respectively, adjacent to the upper surface of the second mixing chamber. or Any combination of those, A microfluidic apparatus according to any of embodiments 24 to 34.

[0204] (36) The microfluidic apparatus according to any one of embodiments 24 to 35, wherein the second mixing outlet channel includes a first termination at the opening to the second mixing chamber. (37) A microfluidic apparatus comprising a cascade microfluidic mixer, wherein each of the cascade microfluidic mixers is A mixing chamber including a base defining the bottom surface, side walls, and top surface, The first side wall of the mixing chamber includes a mixing inlet channel with an opening to the mixing chamber, The second side wall of the mixing chamber includes a mixing outlet channel that includes an opening to the mixing chamber, The vertical dimension of the mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel. The cascade microfluidic mixers are connected in series with respect to each other, so that the mixing inlet channel of each of the cascade microfluidic mixers that follows the first microfluidic mixer in the series is connected to the mixing outlet of the preceding microfluidic mixer in the series. Microfluidic devices. (38) Microfluidic devices, The first plate and the second plate, An elastic layer disposed between the first plate and the second plate, The microfluidic pathway between the first plate and the second plate, wherein the microfluidic pathway is A plurality of blend chambers, each containing a fixed volume separated by a portion of the elastic layer, wherein the portion of the elastic layer is for deflecting fluid to drive fluid between the blend chambers among the plurality of blend chambers, A first microfluidic mixer is provided, and the first microfluidic mixer is A first fluid input and a second fluid input, A fluid crossing portion that receives fluid from the first fluid input and the second fluid input, wherein the fluid crossing portion opens into the first mixing chamber on the upper region of the first side of the first mixing chamber, and the first mixing chamber has a depth of more than 1.5 times the depth of the fluid crossing portion. A connecting channel on the upper region of the second side of the first mixing chamber, wherein the connecting channel has a depth less than the depth of the first mixing chamber, and further, the opening of the connecting channel is offset with respect to the fluid intersection along the width of the second side of the first mixing chamber, and the connecting channel opens into the second mixing chamber on the upper region of the first side of the second mixing chamber, and further, the second mixing chamber has a depth greater than 1.5 times the depth of the connecting channel, An output channel from the second mixing chamber on the upper region of the second side of the second mixing chamber, wherein the second side of the second mixing chamber is on the opposite side from the first side of the second mixing chamber, Microfluidic devices. (39) The microfluidic apparatus according to embodiment 38, wherein the upper surface of the fluid intersection is substantially the same height as the upper surface of the first mixing chamber. (40) The microfluidic apparatus according to embodiment 38 or 39, wherein the upper surface of the connecting channel is configured to be at the same height as the upper surface of the first mixing chamber and the upper surface of the second mixing chamber.

[0205] (41) A microfluidic apparatus according to any one of embodiments 38 to 40, further comprising one or more fluid pumps for pumping fluid from the blend chamber into the first microfluidic mixer by deflecting at least a portion of the elastic layer. (42) A microfluidic apparatus according to any one of embodiments 38 to 41, further comprising one or more fluid pumps between the plurality of blend chambers and the first microfluidic mixer, wherein the fluid pumps pump fluid from the blend chambers into the first microfluidic mixer by deflecting at least a portion of the elastic layer. (43) The microfluidic apparatus according to any one of embodiments 38 to 42, wherein the microfluidic apparatus comprises a plurality of microfluidic mixers, including the first microfluidic mixer. (44) A microfluidic apparatus according to any one of embodiments 38 to 43, further comprising a plurality of pressure ports to the first plate, configured to deflect the elastic layer in order to drive a fluid through the first microfluidic mixer between the blend chambers. (45) A microfluidic device according to any one of embodiments 38 to 44, further comprising a flow restrictor in fluid communication with the first fluid input, wherein the flow restrictor includes a meandering elongated fluid channel.

[0206] (46) The microfluidic apparatus according to embodiment 45, further comprising a pair of blend chambers having a fixed volume, each blend chamber being positioned between the first plate and the second plate, and a portion of the elastic layer dividing each chamber into a fluid contact side within the second plate and a pressure receiving side within the first plate. (47) A method for compounding therapeutic mRNA with a delivery vehicle, comprising mixing the therapeutic mRNA and the delivery vehicle in a microfluidic mixing chamber of a microfluidic device at a temperature of about 2 to about 20°C, wherein the temperature is selected using at least the composition of the therapeutic mRNA, the composition of the delivery vehicle, or a combination thereof. (48) The method according to Embodiment 47, further comprising selecting the temperature using at least the polynucleotide sequence of the therapeutic mRNA, the sequence of the delivery vehicle, the molecular weight of the delivery vehicle, the molecular weight of the therapeutic mRNA, the charge of the delivery vehicle, the charge of the therapeutic mRNA, the molecular weight of the delivery vehicle, the molecular weight of the therapeutic mRNA, the flow rate of the therapeutic mRNA and / or the delivery vehicle in the microfluidic mixing chamber, and the dimensions of the microfluidic mixing chamber, or any combination thereof. (49) The method according to embodiment 47, wherein the mixing is performed in a microfluidic device comprising the microfluidic mixing chamber. (50) The method according to Embodiment 47, further comprising maintaining the temperature of the mixing chamber separately at about 2 to about 20°C for the rest of the microfluidic device.

[0207] (51) The method according to Embodiment 47, wherein mixing in the microfluidic mixing chamber is such that the therapeutic mRNA and the delivery vehicle pass through a first opening into the mixing chamber of the microfluidic device, thereby driving the therapeutic mRNA and the delivery vehicle against the wall of the mixing chamber to a depth greater than one times the depth of the first opening from the plane of the first opening. (52) The method according to Embodiment 51, wherein passing the therapeutic mRNA and the delivery vehicle against the wall of the mixing chamber from a plane across the first opening to a depth greater than approximately 2.5 times the depth of the first opening. (53) The method according to Embodiment 51, wherein the therapeutic mRNA and the delivery vehicle are driven against the wall of the mixing chamber from a plane crossing the first opening to a depth of three times or more the depth of the first opening. (54) The method according to embodiment 51, wherein the upper part of the first opening is aligned with the upper part of the mixing chamber. (55) A method of mixing, The first fluid and the second fluid are passed through the first opening into the mixing chamber of the microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber and from the plane of the first opening to a depth greater than one times the depth of the first opening, thereby forming a mixed fluid. The mixed fluid is to be discharged from the mixing chamber by passing it through the outlet opening. Includes, The mixing chamber is maintained at a temperature of approximately 2 to approximately 20°C. method.

[0208] (56) A method of mixing, The first fluid and the second fluid are passed through at least one opening into a mixing chamber within the microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber and driving them as a mixed fluid from at least the plane of the first opening. The mixed fluid is discharged from the mixing chamber by passing it through an outlet opening, and the mixing chamber is maintained at a temperature of 2 to 20°C. Methods that include... (57) A method of mixing, A first fluid containing oligonucleotide molecules and a second fluid containing a delivery vehicle chemical are passed through at least one opening into the mixing chamber of a microfluidic device, thereby driving the first and second fluids against the walls of the mixing chamber and from the plane of the opening. The mixed fluid is to be discharged from the mixing chamber by passing it through the outlet opening. The mixing chamber is maintained at a temperature of approximately 2 to approximately 20°C. method. (58) A method of mixing in a microfluidic device, The first fluid and the second fluid are passed through the first opening into the mixing chamber within the microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber from a plane crossing the first opening to a depth of approximately 2.5 times the depth of the first opening, thereby forming a substantial mixed fluid. The mixed fluid is discharged from the mixing chamber by passing it through an outlet opening, wherein the outlet opening is on the opposite side of the first opening but is offset from the first opening. The mixing chamber is maintained at a temperature of approximately 5 to approximately 20°C. method. (59) The method according to embodiment 58, wherein passing the first fluid and the second fluid into the mixing chamber through the first opening is such that the first fluid and the second fluid are driven against the wall of the mixing chamber from the plane across the first opening to a depth greater than approximately 2.5 times the depth of the first opening. (60) The method according to embodiment 58 or 59, further comprising maintaining the temperature of the mixing chamber at about 5 to about 15°C.

[0209] (61) The method according to any one of embodiments 58 to 60, further comprising maintaining the temperature of the mixed fluid at about 5 to about 15°C. (62) The method according to any one of embodiments 58 to 61, further comprising maintaining the temperature of the mixed fluid at about 10°C. (63) The method according to any one of embodiments 58 to 62, further comprising passing the mixed fluid from the outlet opening through a second opening into a second mixing chamber, thereby driving the fluid against the wall of the second mixing chamber and driving from the plane of the second opening to a depth greater than one times the depth of the second opening to further mix the mixed fluid. (64) The method according to any one of embodiments 58 to 63, wherein the fluid is driven against the wall of the mixing chamber from a plane crossing the first opening to a depth of about three times or more the depth of the first opening. (65) The method according to any one of embodiments 58 to 64, wherein the upper part of the first opening is aligned with the upper part of the first mixing chamber.

[0210] (66) The method according to any one of embodiments 58 to 65, wherein the outlet opening has a cross-sectional area equal to the cross-sectional area of ​​the first opening. (67) The method according to any one of embodiments 58 to 66, wherein the mixing chamber is located between the first layer and the second layer of the microfluidic device. (68) The method according to any one of embodiments 58 to 67, wherein the mixing chamber has a length greater than its width, and the length is more than about twice the width of the first opening. (69) A method for forming a composition, The synthesis of one or more therapeutic mRNAs in a microfluidic device, wherein the one or more therapeutic mRNAs are in a first fluid, and the delivery vehicles for the one or more therapeutic mRNAs are in a second fluid, The first fluid and the second fluid are passed through the first opening into the mixing chamber of the microfluidic device, thereby driving the first fluid and the second fluid against the walls of the mixing chamber and from the plane of the first opening to a depth greater than one times the depth of the first opening, to form a mixed fluid, wherein the mixing chamber is maintained at a temperature selected to enhance the mixing of the therapeutic mRNA and the delivery vehicle. The mixed fluid is to be discharged from the mixing chamber by passing it through the outlet opening. Methods that include... (70) The method according to Embodiment 69, wherein the mixing chamber is selected to enhance the mixing of the therapeutic mRNA and the delivery vehicle and is maintained at a temperature of 2 to 20°C.

[0211] (71) The method according to embodiment 69, further comprising selecting an enhanced mixing temperature for the mixing chamber. (72) The method according to embodiment 71, wherein selecting the enhanced mixing temperature is to model mixing in vivo or in vitro within a mixing chamber. (73) The method according to Embodiment 71, wherein the selection of the enhanced mixing temperature includes selecting a temperature of about 2 to about 20°C based on the delivery vehicle and the one or more therapeutic mRNAs. (74) The method according to embodiment 69, wherein passing the first fluid and the second fluid into the mixing chamber through the first opening is such that the first fluid and the second fluid are driven against the wall of the mixing chamber from a plane across the first opening to a depth greater than approximately 2.5 times the depth of the first opening. (75) The method according to embodiment 69, wherein the fluid is driven against the wall of the mixing chamber from a plane crossing the first opening to a depth of about three times or more the depth of the first opening.

[0212] (76) The method according to embodiment 69, wherein the upper part of the first opening is aligned with the upper part of the mixing chamber. (77) The method according to embodiment 69, wherein the outlet opening has a cross-sectional area equal to the cross-sectional area of ​​the first opening. (78) The method according to embodiment 69, wherein the mixing chamber is located between the first layer and the second layer of the microfluidic device. (79) The method according to embodiment 69, wherein the mixing chamber has a length greater than its width, and further wherein said length is more than twice the width of the first opening. (80) A method of forming a therapeutic composition, comprising: passing one or more therapeutic mRNAs in a first fluid and a delivery vehicle for said one or more therapeutic mRNAs in a second fluid through a first opening into a mixing chamber within a microfluidic device, whereby said first fluid and said second fluid are driven against the walls of said mixing chamber, and are driven from the plane of said first opening to a depth of more than 1 times the depth of said first opening to form a mixed fluid comprising said therapeutic composition; maintaining the temperature of said mixing chamber at an enhanced mixing temperature determined to enhance mixing; passing said mixed fluid out of said mixing chamber through an outlet opening; A method, comprising the above steps.

[0213] (81) The method according to embodiment 80, wherein said enhanced mixing temperature is from about 2 °C to about 20 °C. (82) The method according to embodiment 80, wherein said maintaining comprises determining said enhanced mixing temperature for said one or more therapeutic mRNAs and / or said delivery vehicle. (83) The method according to embodiment 82, further comprising determining said enhanced mixing temperature by modeling mixing within said mixing chamber in vivo or in vitro. (84) The method according to embodiment 82, further comprising determining said enhanced mixing temperature by selecting a temperature in the range of about 2 °C to about 20 °C that provides better mixing compared to mixing at other temperatures in the range of about 2 °C to about 20 °C. (85) The method according to Embodiment 80, wherein passing the first fluid and the second fluid into the mixing chamber through the first opening is such that the first fluid and the second fluid are driven against the wall of the mixing chamber from a plane across the first opening to a depth greater than approximately 2.5 times the depth of the first opening.

[0214] (86) The method according to embodiment 80, wherein the fluid is driven against the wall of the mixing chamber from a plane crossing the first opening to a depth of about three times or more the depth of the first opening. (87) The method according to embodiment 80, wherein the upper part of the first opening is aligned with the upper part of the mixing chamber. (88) The method according to embodiment 80, wherein the outlet opening has a cross-sectional area corresponding to the cross-sectional area of ​​the first opening. (89) The method according to embodiment 80, wherein the mixing chamber is formed between the first layer and the second layer of the microfluidic device. (90) The method according to embodiment 80, wherein the mixing chamber has a length greater than its width, and further, the length is more than twice the width of the first opening.

[0215] (91) A method of mixing, The first fluid and the second fluid are passed through the first opening into the mixing chamber of the microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber and from the plane of the first opening to a depth greater than one times the depth of the first opening, thereby forming a mixed fluid. The mixed fluid is to be discharged from the mixing chamber by passing it through the outlet opening. Methods that include... (92) The method according to embodiment 91, wherein passing the first fluid and the second fluid into the mixing chamber through the first opening is such that the first fluid and the second fluid are driven against the wall of the mixing chamber from the plane across the first opening to a depth greater than approximately 2.5 times the depth of the first opening. (93) The method according to embodiment 91 or 92, wherein the mixed fluid is substantially mixed by the mixing chamber, and the mixing chamber is configured as a single mixer not connected to a second mixing chamber. (94) The method according to embodiment 91 or 92, further comprising passing the mixed fluid from the outlet opening through a second opening into a second mixing chamber, thereby driving the fluid against the wall of the second mixing chamber and driving from the plane of the second opening to a depth greater than one times the depth of the second opening to further mix the mixed fluid. (95) The method according to embodiment 91 or 92, wherein the fluid is driven against the wall of the mixing chamber from a plane crossing the first opening to a depth of about three times or more the depth of the first opening.

[0216] (96) The method according to any one of embodiments 91 to 95, wherein the outlet opening has a cross-sectional area equal to the cross-sectional area of ​​the first opening. (97) The method according to any one of embodiments 91 to 96, wherein the mixing chamber has a length greater than its width, and the length is more than about twice the width of the first opening. (98) The method according to any one of embodiments 91 to 97, wherein the mixing chamber has rounded corners. (99) The method according to any one of embodiments 91 to 98, wherein the change in fluid pressure passing through the mixing chamber at a flow rate of 0.25 to 5 mL / min is approximately 6.9 kPa to approximately 206.8 kPa. (100) The method according to any one of embodiments 91 to 99, wherein the width of the mixing chamber is about 150 to about 600 μm, the depth of the mixing chamber is about 150 to about 500 μm, and the length of the mixing chamber is about 500 μm to about 1000 μm.

[0217] (101) A method of mixing in a microfluidic device, The first fluid and the second fluid are passed through the first opening into the mixing chamber within the microfluidic device, thereby driving the first fluid and the second fluid against the wall of the mixing chamber from a plane crossing the first opening to a depth of approximately 2.5 times the depth of the first opening, thereby forming a substantially mixed fluid. The mixed fluid is discharged from the mixing chamber by passing it through an outlet opening, wherein the outlet opening is on the opposite side of the first opening but is offset from the first opening. The mixture includes, wherein the mixed fluid is substantially mixed by the mixing chamber, and the mixing chamber is configured as a single mixer not connected to a second mixing chamber. method.

Claims

1. A microfluidic mixing device, A mixing chamber comprising a base defining the bottom surface, side walls, and top surface surrounding the mixing chamber, The first side wall of the mixing chamber includes a mixing inlet channel with an opening to the mixing chamber, The second side wall of the mixing chamber includes a mixing outlet channel that includes an opening to the mixing chamber, The vertical dimension of the mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel. The width of the mixing chamber is greater than the width of the mixing inlet channel and greater than the width of the mixing outlet channel. The first side wall and the second side wall are opposing side walls of the mixing chamber, The first side wall and the second side wall are elongated in the width direction of the mixing chamber, one end of the first side wall in the width direction of the mixing chamber faces one end of the second side wall in the width direction of the mixing chamber, and the other end of the first side wall in the width direction of the mixing chamber faces the other end of the second side wall in the width direction of the mixing chamber. The opening of the mixing inlet channel is located at one end of the first side wall, and the opening of the mixing outlet channel is located at the other end of the second side wall. The upper surface of the mixing chamber is at the same height as the upper surface of the mixing inlet channel and the upper surface of the mixing outlet channel, and the lower surface of the mixing chamber is located below the lower surface of the mixing inlet channel and the lower surface of the mixing outlet channel. Microfluidic mixing device.

2. The microfluidic mixing apparatus according to claim 1, wherein the mixing chamber has an elongated rectangular parallelepiped shape in the width direction of the mixing chamber.

3. A microfluidic apparatus comprising a cascade microfluidic mixer, wherein each of the cascade microfluidic mixers is A mixing chamber including a base defining the bottom surface, side walls, and top surface, The first side wall of the mixing chamber includes a mixing inlet channel with an opening to the mixing chamber, The second side wall of the mixing chamber includes a mixing outlet channel that includes an opening to the mixing chamber, The vertical dimension of the mixing chamber is greater than the vertical dimension of the mixing inlet channel and greater than the vertical dimension of the mixing outlet channel. The cascade microfluidic mixers are connected in series with respect to each other, so that the mixing inlet channel of each of the cascade microfluidic mixers located after the first microfluidic mixer in the series is connected to the mixing outlet of the earlier microfluidic mixer in the series. The width of the mixing chamber is greater than the width of the mixing inlet channel and greater than the width of the mixing outlet channel. The first side wall and the second side wall are opposing side walls of the mixing chamber, The first side wall and the second side wall are elongated in the width direction of the mixing chamber, one end of the first side wall in the width direction of the mixing chamber faces one end of the second side wall in the width direction of the mixing chamber, and the other end of the first side wall in the width direction of the mixing chamber faces the other end of the second side wall in the width direction of the mixing chamber. The opening of the mixing inlet channel is located at one end of the first side wall, and the opening of the mixing outlet channel is located at the other end of the second side wall. The upper surface of the mixing chamber is at the same height as the upper surface of the mixing inlet channel and the upper surface of the mixing outlet channel, and the lower surface of the mixing chamber is located below the lower surface of the mixing inlet channel and the lower surface of the mixing outlet channel. Microfluidic devices.

4. The microfluidic apparatus according to claim 3, wherein the mixing chamber has an elongated rectangular parallelepiped shape in the width direction of the mixing chamber.

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