Microfluidic devices and methods of using the same

Abstract

Microfluidic devices for processing polynucleotides in a sealed path environment and methods of using the same are described herein. These devices include optical sensors to monitor operations and track material usage in order to enable good manufacturing practices.

Description

[0001] Cross-reference to related applications

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 62885159, filed August 9, 2019, entitled “Microfluidic Device and Method of Use Thereof”; U.S. Provisional Patent Application No. 62885170, filed August 9, 2019, entitled “Method and Apparatus for Manufacturing a Therapeutic Composition”; and U.S. Provisional Patent Application No. 62 / 914,374, filed October 11, 2019, entitled “Method and Apparatus for Manufacturing and Removing Material from a Therapeutic Composition”, which are incorporated herein by reference in their entirety.

[0003] By invoking

[0004] All publications and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent application were expressly and individually indicated to be incorporated by reference. Technical Field

[0005] The devices and methods described herein can be used for the manufacture and formulation of products containing biomolecules (including therapeutic mRNA), particularly for therapies intended for personalized care. Specifically, this document describes closed-pathway methods and devices for processing therapeutic polynucleotides, including at the point of care. Background Technology

[0006] Current technologies for manufacturing and formulating polynucleotide therapies (especially mRNA therapies) often result in product contamination and degradation. Centralized production currently available is prohibitively expensive, slow, and susceptible to contamination for the formulation of therapies that may involve multiple polynucleotide species. Developing scalable polynucleotide manufacturing, single-patient-dose production, eliminating contact points to limit contamination, providing input and process traceability to meet clinical manufacturing requirements, and enabling point-of-care operations can facilitate the use of these promising therapeutic modalities. Microfluidic instruments and processes can offer key advantages in achieving these goals. Summary of the Invention

[0007] This article describes microfluidic devices for manufacturing therapeutics, particularly mRNA therapy. For example, the systems described herein can form therapeutic materials (including drugs and pharmaceuticals) within dedicated, single-use, disposable microfluidic pathway devices (e.g., microfluidic pathway plate devices, chips, biochips, plates, etc.). Microfluidic pathway devices and control systems for operating these devices are described.

[0008] For example, what is described herein is a control system (“device”) for operating a microfluidic path device. These devices may be referred to herein as microfluidic devices, microfluidic control devices, microfluidic control systems, or microfluidic systems.

[0009] Microfluidic devices may include: a holder (e.g., a base) for a microfluidic path device; a plurality of fluid bottles, each fluid bottle including or configured to be coupled to a fluid line to form a closed fluid path; and a controller configured to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the holder. In any of these microfluidic devices, each fluid line may be configured to seal against the microfluidic path device located in the holder to form a closed fluid path. The microfluidic devices described herein may advantageously include any features (or any combination thereof) described herein.

[0010] For example, a microfluidic device may include: a holder for a microfluidic path device; a reagent storage frame including a plurality of supports, each support configured to hold a fluid bottle, wherein each fluid bottle includes or is configured to be connected to a fluid line, and wherein each fluid line is configured to bias against the microfluidic path device located in the holder; and a controller configured to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the holder.

[0011] The microfluidic device may include: a mount for a microfluidic path device; a fluid interface assembly including multiple fluid lines, each fluid line being configured to bias against the microfluidic path device located in the mount with a bias force; a reagent storage frame including multiple fluid sample holders, each fluid sample holder being configured to hold a fluid bottle and each fluid sample holder being configured to be connected to the fluid interface assembly via one of the fluid lines of the fluid interface assembly; and a controller configured to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the mount.

[0012] The microfluidic device may include: a mount for a microfluidic path device; multiple pressure lines; a reagent storage frame including multiple supports, each support configured to hold a fluid bottle, wherein each fluid bottle includes or is configured to be connected to a fluid line, and wherein each fluid line and each pressure line are configured to bias against the microfluidic path device located in the mount; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the mount.

[0013] The microfluidic device may include: a mount for a microfluidic path device; a fluid interface assembly including multiple fluid lines and pressure lines, wherein each fluid line and each pressure line is configured to bias against the microfluidic path device located in the mount; a reagent storage frame including multiple fluid sample holders, each fluid sample holder being configured to hold a fluid bottle, and each fluid sample holder being configured to be connected to the fluid interface assembly via one of the fluid lines of the fluid interface assembly; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the mount.

[0014] The microfluidic device may include: a mount for a microfluidic path device; multiple pressure lines; multiple fluid bottles, each fluid bottle including or configured to be connected to a fluid line, and wherein each fluid line and each pressure line are configured to seal against the microfluidic path device located in the mount to form a closed fluid path; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the mount.

[0015] The microfluidic device may include: a mount for a microfluidic path device; a plurality of fluid bottles, each fluid bottle including or configured to be connected to a fluid line, and wherein each fluid line is configured to seal against the microfluidic path device located in the mount to form a closed fluid path; at least one optical sensor configured to monitor fluid within the microfluidic path device located in the mount; and a controller configured to drive fluid movement within the microfluidic path device when the microfluidic path device is located in the mount, and to record and / or transmit optical data displaying the fluid in the microfluidic path during device operation.

[0016] The microfluidic device may include: a mount for a microfluidic path device; a fluid interface assembly including multiple fluid lines and pressure lines, wherein each fluid line and each pressure line is configured to be biased (e.g., spring-loaded) against the microfluidic path device located in the mount; a reagent storage frame including multiple fluid sample holders, each fluid sample holder configured to hold a fluid bottle, and each fluid sample holder configured to be coupled to the fluid interface assembly via one of the fluid lines of the fluid interface assembly; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the mount.

[0017] In some variations, a microfluidic device for processing therapeutic polynucleotides at the point of care and configured to operate as a closed pathway may include: a holder for the microfluidic pathway device; a fluid interface assembly including multiple fluid lines and pressure lines, wherein each fluid line and each pressure line is configured to be independently driven against the microfluidic pathway device located in the holder to form a sealed connection therewith; a reagent storage frame including multiple fluid sample holders, each fluid sample holder being configured to hold fluid, and each fluid sample holder being configured to be connected to the fluid interface assembly via one of the fluid lines of the fluid interface assembly; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic pathway device when the microfluidic pathway device is located in the holder; wherein the fluid interface assembly includes multiple spring biasing elements configured to independently push each fluid line against the microfluidic pathway device located in the holder to form a sealed connection therewith.

[0018] For example, an apparatus (e.g., a microfluidic device for forming therapeutic polynucleotides) may include: a holder for removably holding a microfluidic path device; multiple pressure lines; multiple fluid bottles, each pressurized by one or more pressure lines from the multiple pressure lines, wherein each fluid bottle includes or is configured to be coupled to a fluid line, wherein each fluid line and at least a portion of the pressure lines are configured to independently bias against the microfluidic path device located in the holder to form a sealed, closed fluid path; and a controller configured to control the pressure applied through the pressure lines to drive fluid movement in the microfluidic path device when the microfluidic path device is located in the holder, and to apply pressure to one or more pressure lines during operation to open or close valves in the microfluidic path device.

[0019] As described above, in general, the controller can be configured to control the device to perform in vitro transcription (IVT) reactions in a microfluidic pathway device.

[0020] For example, a device (e.g., a microfluidic device for forming therapeutic polynucleotides, such as therapeutic mRNA) may include: a frame for a microfluidic pathway device; multiple pressure lines; a fluid interface assembly including multiple fluid lines; multiple fluid bottles configured to be pressurized; a reagent storage frame including multiple supports, each support configured to hold a fluid bottle among multiple fluid bottles, wherein each fluid bottle either includes a fluid line among multiple fluid lines or is configured to be coupled to a fluid line among multiple fluid lines, and wherein each fluid line and at least some pressure lines are configured to individually bias against the microfluidic pathway device located in the frame; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement in the microfluidic pathway device when the microfluidic pathway device is located in the frame.

[0021] In some variations, a microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides, such as therapeutic mRNA) may include: a holder for removably holding the microfluidic path device; a plurality of pressure lines, at least a portion of which are configured to independently bias against a pressure input on the microfluidic path device located in the holder; a plurality of fluid bottles configured to be pressurized, each fluid bottle either including a fluid output configured to seal the input on the microfluidic path device, or configured to be coupled to a fluid line configured to independently bias the microfluidic path device to form a sealed closed fluid path; a first optical detector configured to monitor fluid within the fluid bottle; a second optical detector configured to monitor fluid within the microfluidic path device located in the holder; and a controller configured to receive input from the first and second optical detectors and control the pressure applied through the pressure lines to apply pressure from the plurality of pressure lines to open and / or close valves, and to drive fluid movement in the microfluidic path device at least in part based on the received input.

[0022] A microfluidic device for processing therapeutic polynucleotides at the point of care and configured to operate as a closed pathway may include: a holder for the microfluidic pathway device; a fluid interface assembly including multiple fluid lines and pressure lines, wherein each fluid line and each pressure line is configured to be independently driven abut against the microfluidic pathway device located in the holder to form a sealed connection thereto; a reagent storage frame including multiple pressurized fluid sample holders, each pressurized fluid sample holder being configured to hold a fluid bottle, and each pressurized fluid sample holder being configured to be coupled to the fluid interface assembly via one of the fluid lines of the fluid interface assembly; multiple An optical sensor, arranged around a holder and a reagent storage frame, monitors the fluid level within a fluid vial held by the reagent storage frame and fluid movement within the microfluidic path device when the microfluidic path device is located in the holder; and a controller, communicating with the optical sensors and configured to control pressure applied through the pressure lines to drive fluid movement within the microfluidic path device when the microfluidic path device is located in the holder; wherein the fluid interface assembly includes a plurality of clamps configured to independently press each fluid line against the microfluidic path device located in the holder to form a sealing connection thereto, and wherein each of the holder, fluid interface assembly, and fluid sample holder is configured to be removable for sterilization.

[0023] In some variations, these microfluidic devices can be microfluidic devices for forming therapeutic polynucleotides (e.g., mRNA therapeutic agents). The device may include: a holder for removably holding the microfluidic pathplate device; multiple pressure lines; multiple fluid bottles, each fluid bottle including or configured to be coupled to a fluid line, wherein each fluid line and at least a portion of the pressure line are configured to be biased against the microfluidic pathplate device held in the holder to form a closed fluid path; and a controller configured to control pressure applied through the pressure lines to drive fluid movement within the microfluidic pathplate device when the microfluidic pathplate device is held in the holder, wherein the controller is configured to direct the synthesis of a synthetic template, use the template to direct an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, and direct the purification of the therapeutic polynucleotide in one or more microfluidic pathplate devices held in the holder.

[0024] A microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides, such as therapeutic mRNA) may include: a holder for removably holding a microfluidic path plate device; multiple pressure lines; multiple fluid bottles, each fluid bottle including or configured to be coupled to a fluid line, wherein each fluid line and at least a portion of the pressure line are configured to be biased against the microfluidic path plate device held in the holder to form a closed fluid path; and a controller configured to control pressure applied through the pressure lines to drive fluid movement in the microfluidic path plate device when the microfluidic path plate device is held in the holder, wherein the controller is configured to determine the contents of the fluid bottles, transfer submicroliter amounts of material from the fluid bottles to one or more reactors in the microfluidic path plate device held in the holder, guide the synthesis of a synthetic template, use the template to guide an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, and guide the purification of the therapeutic polynucleotide in one or more microfluidic path plates held in the holder.

[0025] The controller is configured to perform any of the methods described herein, and in particular, can be configured to receive inputs (e.g., optical inputs, pressure inputs, temperature / heat inputs, etc.) and process inputs to control fluid movement in a microfluidic path device, temperature (including thermal cycling) in various regions of the microfluidic path device, flushing / engaging, opening / closing of valves in the microfluidic device, detection of the microfluidic device, etc. The controller may include one or more microprocessors, communication circuitry, memory, etc. The controller may include firmware, hardware, and / or software.

[0026] Any of these devices may include one or more (e.g., multiple) optical sensors arranged around the holder and reagent storage frame to monitor fluid levels within the reagent storage frame and fluid movement within the microfluidic path device when the microfluidic path device is located in the holder. Optionally or additionally, the optical sensors may be located on the bottom of the device (e.g., below the holder and may be directed upwards to detect fluid volume, movement, etc.). The device may include a holder release control configured to release the holder from the device for individual sterilization. Any of these devices may include a fluid interface assembly release control configured to release a fluid interface assembly from the device for individual sterilization, and / or a fluid sample holder release control configured to release a fluid sample holder from the device for individual sterilization.

[0027] Any of these devices may include a thermal control device configured to regulate the temperature of at least one region of the microfluidic path device when it is located in the pedestal. In some devices, there may be more than one thermal control device configured to regulate the temperature of different regions of the microfluidic path device. The thermal control device may include a Peltier device and / or may be configured to control the temperature of at least one region of the microfluidic path device between 4°C and 65°C.

[0028] Any of these devices may include a magnetic field applicator configured to apply a magnetic field to at least one region of the microfluidic path device when the microfluidic path device is in the pedestal. The magnetic field applicator may include a control arm mounted on a reagent storage frame.

[0029] Generally, the controller can be configured to detect an identification code on a fluid vial held by a fluid sample holder; in some variations, the identification code includes a barcode, RFID, or other means of identifying (particularly in a non-contact manner) the contents of the fluid sample holder component. The controller can also be configured to determine the level of reagent held by a reagent storage frame.

[0030] Any of these devices may include an optical sensor driver and / or one or more electroluminescent panels or other backlighting devices, the optical sensor driver being configured to move one or more of a plurality of optical sensors around a mount and a reagent storage frame, and the backlighting devices being configured to provide illumination to an area below a portion of the reagent storage frame.

[0031] The described methods and apparatus generally include one or more fluid power loops to move material (liquid material) between or within a fluid chamber (reservoir, fluid contact side, reactor, etc.) and channel of a microfluidic path device. The fluid power loop can be a hydraulic or pneumatic loop comprising one or more pressure channels and pressure receiving sides of a chamber within a microfluidic device, particularly a microfluidic device. The fluid power loop may also be referred to as a microfluidic power loop. A single microfluidic chip may include multiple fluid power loops; the fluid power loop may also include one or more pressure lines and interfaces between the pressure lines of the microfluidic control device and one or more microfluidic chips within the microfluidic path device. One or more fluid power loops may share components (valves, pressure lines, vacuum caps, etc.) with other overlapping fluid power loops. Furthermore, for convenience, it should be understood that when the term "pneumatic" is used, a general fluid power loop (e.g., hydraulic and / or pneumatic) may be used alternatively or additionally. The fluid material driven by the fluid power lines can be any suitable fluid (e.g., gas or liquid, such as air, water, oil, etc.).

[0032] This document also describes microfluidic pathway devices (e.g., closed-path microfluidic pathway devices) for processing therapeutic polynucleotides in closed pathways. As mentioned above, these microfluidic pathway devices may be referred to herein as microfluidic chips, microfluidic pathway plates, processing chips, biochips, processing plates, etc. Typically, microfluidic pathway devices can be microfluidic pathway plate devices, which can be generally flat structures; these structures can be relatively thin (e.g., less than a few millimeters thick, such as 0.5-20 mm, 0.5-15 mm, 0.5-10 mm, etc.). The microfluidic pathway devices described herein are typically at least partially transparent, particularly the top of the microfluidic pathway device, so that one or more optical sensors (cameras, CCDs, optical fibers, etc.) can be used to sense, detect, monitor, and record the movement of the microfluidic pathway device, including fluid movement and / or movement of the elastic layer, when the microfluidic pathway device is used by the microfluidic device described herein.

[0033] Any of these microfluidic pathway devices can be configured to operate as a closed-path device as described herein, wherein the chamber (especially the fluid contact chamber) and the fluid channel are sealed to the fluid inlet / outlet lines (e.g., fluid lines) via a hermetically sealed connection that prevents exposure to the environment (e.g., air). This is particularly important when manufacturing therapeutic mRNAs that can be degraded by exposure to RNA and other contaminants in the environment.

[0034] For example, a microfluidic pathway device may include: an elastic layer sandwiched between a first surface and a second surface; a plurality of chambers formed between the first surface and the second surface, wherein a portion of the elastic layer divides each chamber into a fluid contact side in the second surface and a pressure receiving side in the first surface; a plurality of fluid channels, each fluid channel extending from a fluid port, the elastic membrane, through the first surface, and into the second surface to be in fluid communication with the fluid contact side of the plurality of chambers; and a plurality of pressure channels, each pressure channel extending from one or more pressure ports, through the first surface and the elastic layer, into the second surface, and returning through the elastic layer into the first surface, wherein each of the plurality of pressure channels is in fluid communication with one or more pressure receiving sides of the plurality of chambers, and wherein the volume of the fluid contact side of each chamber can be adjusted by applying pressure from one or more pressure ports.

[0035] A microfluidic pathway device (e.g., for forming a therapeutic polynucleotide, such as therapeutic mRNA) may include: an elastic layer sandwiched between a first plate region having a first surface and a second plate region having a second surface; a plurality of chambers, each having a fixed volume and formed between the first and second surfaces, wherein a portion of the elastic layer divides each chamber into a fluid contact side in the second surface and a pressure receiving side in the first surface; a plurality of fluid channels, each fluid channel extending from a fluid port through the first plate region and into the second plate region to be in fluid communication with the fluid contact side of one or more of the plurality of chambers; and a plurality of pressure channels, each pressure channel extending from one or more pressure ports through the first plate region and the elastic layer, into the second plate region, and returning through the elastic layer and into the first plate region, wherein each pressure channel of the plurality of pressure channels extends within the first plate region and is in fluid communication with one or more pressure receiving sides of one or more of the plurality of chambers, wherein the plurality of fluid contact sides of the plurality of chambers are interconnected, and wherein the fluid contact side of each chamber is recessed such that when a positive pressure on the pressure receiving side drives the elastic layer against the fluid contact side, the elastic layer is flush with and without gaps to the fluid contact side of the second surface.

[0036] Any of these microfluidic devices can be configured to form a secure seal with one or more fluid and / or pressure lines. In some variations, the ports (fluid ports, pressure ports, etc.) can be formed as channels (e.g., cylindrical channels) leading into the body of the microfluidic device, extending through an opening in the device's elastic layer; the underside of this elastic layer can be supported by a second plate region (e.g., a second surface of the second plate region) having a channel leading into the second plate region, the diameter of which is narrower than the diameter of the port channel, so that when the input line (fluid line and / or pressure line) is driven against the elastic layer, the input line can be supported against the elastic layer to form a seal.

[0037] For example, microfluidic pathway devices (e.g., for forming therapeutic polynucleotides) are typically configured to operate within a closed pathway. A microfluidic pathway device may include: an elastic layer sandwiched between a first plate region having a first surface and a second plate region having a second surface; a plurality of chambers, each having a fixed volume and formed between the first and second surfaces, wherein a portion of the elastic layer divides each chamber into a fluid contact side in the second surface and a pressure receiving side in the first surface; a plurality of fluid channels, each extending from a fluid port through the first plate region and into the second plate region to fluidly communicate with the fluid contact side of one or more of the chambers; and a plurality of pressure channels, each extending from one or more pressure ports, through the first plate region and the elastic layer, into the second plate region, and returning through the elastic layer and into the first plate region, wherein each of the plurality of pressure channels extends within the first plate region and is in fluid communication with one or more pressure receiving sides of one or more of the chambers, wherein each fluid port includes a port channel extending laterally through the first plate region and leading to an opening through the elastic layer, the diameter of which is smaller than the diameter of the port channel, and wherein the diameter of the fluid channel in the second plate region is smaller than the diameter of the port channel.

[0038] Any of these devices can be configured to reduce or eliminate bubbles that may form within the fluid path, for example by including one or more vacuum caps within the fluid loop of the device. For example, a microfluidic pathway device (e.g., for processing therapeutic polynucleotides in a closed path) may include: an elastic layer sandwiched between a first plate region having a first surface and a second plate region having a second surface; a plurality of chambers, each having a fixed volume and formed between the first and second surfaces, wherein a portion of the elastic layer divides each chamber into a fluid contact side in the second surface and a pressure receiving side in the first surface; a plurality of fluid channels, each extending from a fluid port through the first plate region and into the second plate region to fluidly communicate with the fluid contact side of one or more of the chambers; and a plurality of pressure channels, each extending from one or more pressure ports through the first plate region and the elastic layer, into… The second plate region extends through the elastic layer into the first plate region, wherein each of the plurality of pressure channels extends within the first plate region and is in fluid communication with one or more pressure receiving sides of one or more of the plurality of chambers, and at least one vacuum cap is located between at least some of the plurality of chambers, wherein the at least one vacuum cap includes a debubbling chamber formed between a first surface and a second surface, wherein the elastic layer divides the debubbling chamber into a fluid contact side of the debubbling chamber in the second surface and a vacuum receiving side in the first surface, further wherein the fluid contact side of the debubbling chamber is in fluid communication with at least two fluid contact sides of the plurality of chambers, and wherein at least a portion of the elastic layer located between the fluid contact side and the vacuum receiving side of the debubbling chamber is gas permeable.

[0039] Therefore, any of these microfluidic pathway devices may include at least one vacuum cap between at least some of the multiple chambers, wherein the at least one vacuum cap includes a defoaming chamber formed between a first surface and a second surface, wherein a breathable elastic layer divides the defoaming chamber into a fluid contact side of the defoaming chamber in the second surface and a vacuum receiving side in the first surface, and wherein the fluid contact side of the defoaming chamber is in fluid communication with at least two fluid contact sides of the multiple chambers.

[0040] Any of these microfluidic pathway devices can be configured to prevent dead space regions even within the smallest compartment of the microfluidic pathway device. For example, the fluid contact side and pressure receiving side in the second surface are recessed and configured such that when the positive pressure in the pressure receiving side drives the elastic layer against the fluid contact side, the elastic layer is flush with and without gaps to the fluid contact side in the second surface.

[0041] In general, these devices can be formed from one or more plates. For example, a single plate may include multiple surfaces, including an inner surface. Alternatively, the device may include two or more plates that can be stacked and / or laminated together, including an elastic layer and / or membrane between them. In some variations of the microfluidic path device, the first and second surfaces may be part of at least one plate or plate region. For example, the first surface may be part of a first plate, and the second surface may be part of a second plate. Alternatively, the first surface may be part of a first plate region, and the second surface may be part of a second plate region; in some variations, the first and second plate regions may be part of the same plate; or the first and second plate regions may be part of different plates forming the microfluidic path device.

[0042] One or more pressure ports and fluid ports may be located near the periphery of the microfluidic path device. The pressure ports and fluid ports may be arranged in groups and / or spaced apart. Generally, the pressure ports and fluid ports may be arranged along the top of the device around the periphery of the microfluidic path device, and / or may be arranged such that the central region of the microfluidic path device is open and exposed for visualization (via one or more optical sensors) that can monitor the fluid movement and / or handling of the microfluidic path device.

[0043] In some variations, the chambers of the microfluidic pathway device can be paired chambers, wherein the first chamber (e.g., the fluid contact portion) of each pair of chambers is fluidly connected to the second chamber (e.g., the fluid contact portion) of each pair of chambers. The pressure receiving side of each chamber can be individually controlled by connection to a separate (or separable and / or connectable) pressure line or fluid power loop on the microfluidic pathway device. In some variations, the first chamber of a pair of chambers can be connected to any other pair of chambers via a fluid connection with a valve. This valve can be part of a fluid power loop and can be opened / closed by a controller that applies fluid pressure (e.g., pneumatic, hydraulic, etc.) to deflect an elastic layer in the small chamber formed between the first and second surfaces.

[0044] As described above, the microfluidic pathway device can be a hermetically sealed pathway device. The operation of this device can be monitored and controlled by a controller device without contact with the liquid (e.g., containing therapeutic polynucleotides, such as mRNA). In some variations, the microfluidic pathway device can be at least substantially transparent to visible or ultraviolet light. For example, the microfluidic pathway device is substantially transparent to visible or ultraviolet light.

[0045] Any of these methods and devices can be configured to purify polynucleotides (e.g., mRNA) on / in a microfluidic pathway device. For example, a microfluidic pathway device may include material inserted into the fluid contact side of a channel; for example, the material may include cellulose material configured to selectively absorb double-stranded mRNA.

[0046] Any of these microfluidic pathway devices can be configured to remove impurities from therapeutic materials (e.g., “drug particles”), such as therapeutic mRNA materials (e.g., therapeutic mRNA encapsulated in a delivery carrier). For example, any microfluidic pathway device described herein may include one or more chambers configured for buffer adjustment and / or drug particle concentration. In some cases, the device is configured to allow a drug particle solution to flow tangentially through a chamber having one or more ultrafiltration membranes to separate the nanoparticles from the solvent, thereby purifying and / or concentrating the drug particles in the retentate. Smaller particles (e.g., solvent and ions) may be wasted as permeation material through the membrane, while drug particles (e.g., mRNA encapsulated in a delivery carrier) in the same solvent may be collected downstream as retentate. In some variations, this may cause drug particle concentration. In some cases, a biocompatible and stable buffer may be used for downstream processing for injection into a patient. Buffer adjustment can be achieved by adding a diluent containing water, salts, excipients, and / or other components of appropriate composition. The concentration of certain chemicals can be increased by adding a buffer with a higher chemical concentration, and vice versa. For example, in some cases, adding the same volume of water can reduce the ethanol concentration by half. The methods and apparatus described herein allow for the formulation, buffer conditioning, and concentration of biomolecular products within a microfluidic pathway device. The formulation buffer can be adjusted to a more biocompatible and stable buffer for downstream processing and patient injection. Drug concentrations can also be adjusted to a volume acceptable for the administration method after the formulation and buffer conditioning processes. Therefore, any of these devices can include one or more membrane-containing chambers, such as ultrafiltration membranes. Thus, the microfluidic pathway device can include a concentrator. In some variations, the microfluidic pathway includes a dialysis chamber (e.g., within the thickness of a second surface, such as a second layer portion).

[0047] Any of these microfluidic pathway devices may include a delivery reservoir configured to deliver a preselected volume of fluid to at least one chamber; for example, the preselected volume of the chamber may be between, for example, about 20 nanoliters and 5 mL (e.g., 25 nL to 5 mL, about 50 nL to 5 mL, about 50 nL to 2 mL, greater than about 25 nL, about 30 nL, about 50 nL, about 75 nL, etc.).

[0048] The first and / or second layer portions may be formed of a rigid material. Any of these microfluidic pathway devices may include a third layer portion (e.g., a third surface) that may be formed of a rigid material, such as laminated onto an elastic material. The rigid material may be a polymer, such as a cycloalkene copolymer or glass.

[0049] For example, a microfluidic pathway device may include: an elastic layer located between a first plate and a second plate (e.g., sandwiched between the first plate and the second plate); a plurality of chambers, each having a fixed volume, each chamber formed between the first plate and the second plate, wherein a portion of the elastic layer divides each chamber into a fluid contact side and a pressure receiving side; a plurality of fluid ports through the first plate, each fluid port including an exposed portion of the elastic layer supported by the second plate, wherein each fluid port includes an opening through the elastic layer and into the second plate, the opening being in fluid communication with the fluid contact side of one of the plurality of chambers; and a plurality of pressure ports through the first plate, each pressure port including an exposed portion of the elastic layer supported by the second plate, wherein each fluid port includes an opening through the elastic layer and into the second plate, the opening being in fluid communication with the pressure receiving side of one of the plurality of chambers.

[0050] The microfluidic pathway device may include: an elastic layer sandwiched between a first plate and a second plate; a plurality of chambers, each having a fixed volume, each chamber formed between the first plate and the second plate, wherein a portion of the elastic layer divides each chamber into a fluid contact side and a pressure receiving side; a plurality of fluid ports, each fluid port passing through the first plate and the elastic layer and entering the second plate to be in fluid communication with the fluid contact side of one of the plurality of chambers; and a plurality of pressure ports, each pressure port passing through the first plate and through the elastic layer into the second plate, then returning through the elastic layer and into the first plate to be in fluid communication with the pressure receiving side of one of the plurality of chambers.

[0051] For example, a microfluidic pathway device may include: a first plate having a first surface and a second surface and a thickness between them; a second plate having a first surface and a second surface and a thickness between them; an elastic layer sandwiched between the second surface of the first plate and the first surface of the second plate; a third plate coupled to the second plate on a second surface of the second plate, the third plate having a first surface and a second surface; at least one chamber having a fixed volume formed in the second surface of the first plate and the first surface of the second plate, wherein a portion of the elastic layer divides the at least one chamber into a fluid contact side and a pressure receiving side; a fluid channel extending from a fluid port through the thickness of the first plate to a fluid channel opening through the elastic layer and through the thickness of the second plate for fluid connection with a connection channel formed in the second surface of the second plate, wherein the fluid channel is connected to the fluid contact side of the at least one chamber; wherein the diameter of the fluid port through the thickness of the first plate is larger than the diameter of the fluid channel opening through the elastic layer; and an outlet channel extending from the fluid contact side through the second surface of the second plate, wherein a port (e.g., a valve) formed by the elastic layer is located between the fluid contact side and the outlet channel.

[0052] For example, a microfluidic pathway device for processing therapeutic polynucleotides in a closed pathway may include: a first plate having a first surface and a second surface and a thickness between them; a second plate having a first surface and a second surface and a thickness between them; an elastic layer sandwiched between the second surface of the first plate and the first surface of the second plate; a third plate coupled to the second plate on the second surface of the second plate; at least one chamber having a fixed volume formed in the second surface of the first plate and the first surface of the second plate, wherein a portion of the elastic layer divides the at least one chamber into a fluid contact side and a pressure receiving side; a fluid channel extending from a fluid port through the thickness of the first plate to a fluid channel opening through the elastic layer and through the thickness of the second plate, to interact with the fluid channel formed in the second surface of the second plate and formed by… A fluid connection is defined by a third plate, wherein the fluid connection is connected to the fluid contact side of at least one chamber; a pressure channel extends from a pressure port through the thickness of the first plate to a pressure channel opening through the elastic layer and into the thickness of the second plate, to be fluidly connected to a connecting pressure channel formed in the second surface of the first plate and defined by the elastic layer, wherein the pressure channel is connected to the pressure receiving side of at least one chamber; wherein the diameter of the fluid port through the thickness of the first plate is larger than the diameter of the fluid channel opening through the elastic layer, and wherein the diameter of the pressure port through the thickness of the first plate is larger than the diameter of the pressure channel opening through the elastic layer; and an outlet channel extends from the fluid contact side through the second surface of the second plate, wherein a valve (e.g., a port) formed by the elastic layer is located between the fluid contact side and the outlet channel.

[0053] The microfluidic pathway device may include a pressure channel extending from a pressure port through a first plate thickness to a pressure channel opening through an elastic layer and into a second plate thickness, to be fluidly connected to a connecting pressure channel formed in a second surface of the first plate and defined by the elastic layer, wherein the pressure channel is connected to the pressure receiving side of at least one chamber.

[0054] The microfluidic path device described herein may include multiple pressure ports, and the fluid ports are arranged in the periphery of the microfluidic path device.

[0055] For example, a microfluidic pathway device for processing therapeutic polynucleotides in a closed pathway may include: a first plate having a first surface and a second surface and a thickness between the first and second surfaces, the first surface forming an exposed outer surface; a second plate having a first surface and a second surface and a thickness between them; an elastic layer sandwiched between the second surface of the first plate and the first surface of the second plate; a third plate connected to the second plate on the second surface of the second plate, the third plate having a first surface and a second surface and a thickness between them, the second surface forming an exposed bottom surface at the bottom of the device; at least one pair of chambers, each pair of chambers having a fixed volume, the at least one pair of chambers being formed in the second surface of the first plate and the first surface of the second plate, wherein a portion of the elastic layer divides each of the at least one pair of chambers into A fluid contact side and a pressure receiving side, wherein each pair of chambers is in fluid communication; a fluid channel extending from a fluid port through the thickness of the first plate to a fluid channel opening through the elastic layer and through the thickness of the second plate, to be fluidly connected to a connecting channel formed in the second surface of the second plate and defined by a third plate, wherein the fluid channel is connected to the fluid contact side of at least one chamber; a pressure channel extending from a pressure port through the thickness of the first plate to a pressure channel opening through the elastic layer and into the thickness of the second plate, to be fluidly connected to a connecting pressure channel formed in the second surface of the first plate and defined by the elastic layer, wherein the pressure channel is connected to the pressure receiving side of at least one chamber; and an outlet channel extending from the fluid contact side through the second surface of the second plate, wherein a valve (e.g., a port) formed by the elastic layer is located between the fluid contact side and the outlet channel.

[0056] This document also describes devices (e.g., systems) including any microfluidic device (e.g., a microfluidic path device control system) and one or more microfluidic path devices. For example, a system may include: a microfluidic device comprising: a holder for the microfluidic path device; a fluid interface assembly including multiple fluid lines and pressure lines, wherein each fluid line and each pressure line is configured to be actuated against the microfluidic path device located in the holder; and a reagent storage frame including multiple fluid sample holders, each fluid sample holder configured to hold a fluid bottle, and each fluid sample holder configured to be coupled to the fluid interface assembly via one of the fluid lines of the fluid interface assembly. As described in more detail below, in some variations, even without separate fluid lines, the fluid sample holders may be adapted to be directly actuated against the microfluidic path device; the sample holders may form fluid lines. Fluid lines, or separate sample holders (e.g., bottles, containers, etc.), may be configured to be held against a resilient seat formed in the microfluidic path device by pressure, for example, at a port.

[0057] The device may also include a plurality of optical sensors arranged around a frame and a reagent storage frame to monitor fluid levels within the reagent storage frame and fluid movement within the microfluidic path device when the microfluidic path device is in the frame; and a controller configured to control pressure applied via a pressure line to drive fluid movement within the microfluidic path device when the microfluidic path device is in the frame; and the microfluidic path device comprising: a first plate having a first surface and a second surface and a thickness between the first and second surfaces, the first surface forming an exposed outer surface; a second plate having a first surface and a second surface and a thickness between them; an elastic layer sandwiched between the second surface of the first plate and the first surface of the second plate; and a third plate connected to the second plate on a second surface of the second plate, the third plate having a first surface and a second surface and a thickness between them, the second surface forming an exposed bottom surface at the bottom of the device; At least one chamber having a fixed volume is formed in a second surface of a first plate and a first surface of a second plate, wherein a portion of an elastic layer divides the at least one chamber into a fluid contact side and a pressure receiving side; a fluid channel extending from a fluid port through the thickness of the first plate to a fluid channel opening through the elastic layer and through the thickness of the second plate, to be fluidly connected to a connecting channel formed in the second surface of the second plate and defined by a third plate, wherein the fluid channel is connected to the fluid contact side of the at least one chamber; a pressure channel extending from a pressure port through the thickness of the first plate to a pressure channel opening through the elastic layer and into the thickness of the second plate, to be fluidly connected to a connecting pressure channel formed in the second surface of the first plate and defined by the elastic layer, wherein the pressure channel is connected to the pressure receiving side of the at least one chamber; wherein the diameter of the fluid port through the thickness of the first plate is larger than the diameter of the fluid channel opening through the elastic layer, and wherein the diameter of the pressure port through the thickness of the first plate is larger than the diameter of the pressure channel opening through the elastic layer; and an outlet channel extending from the fluid contact side through the second surface of the second plate, wherein a valve formed by the elastic layer is located between the fluid contact side and the outlet channel.

[0058] This document also describes methods for using any of these devices and apparatuses. For example, a method of processing fluids in a microfluidic pathway device to form a therapeutic polynucleotide (e.g., therapeutic mRNA) may include: sealing and independently coupling the distal ends of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid ports or pressure ports on a surface of the microfluidic pathway device, wherein each distal end is biased to be driven against an elastic layer between a first surface and a second surface, wherein the microfluidic pathway device includes a plurality of chambers, each chamber being divided into a fluid contact side formed in the second surface and a pressure receiving side formed in the first surface; and driving fluid through the fluid contact sides of the plurality of chambers by applying positive and negative pressures within the pressure receiving sides of the plurality of chambers to change the dimensions of the plurality of fluid contact sides.

[0059] A method for handling fluid in a microfluidic pathway device may include: sealing and independently connecting the distal ends of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid ports or pressure ports on a surface of the microfluidic pathway device, wherein each distal end is biased to be driven against an elastic layer between a first plate and a second plate, wherein the microfluidic pathway device includes a plurality of chambers, each chamber being divided into a fluid contact side formed in the second plate and a pressure receiving side formed in the first plate, wherein the fluid contact sides are interconnected; and driving fluid through the interconnected fluid contact sides, and operating valves to meter fluid movement between the fluid contact sides of the plurality of chambers by applying positive and negative pressures to the pressure receiving sides of the plurality of chambers to change the size of the plurality of fluid contact sides.

[0060] Driving fluid through the fluid contact side may include deflecting an elastic layer sandwiched between a first surface and a second surface.

[0061] As mentioned above, any of these methods may include optical feedback from the microfluidic path device to control the application of positive and negative pressure.

[0062] These methods may include controlling the valve by deflecting an elastic layer between the first and second surfaces. For example, a controller may control fluid power (e.g., pneumatic, hydraulic) via a fluid power loop (e.g., fluid line, valve, etc.) to control the handling of a microfluidic path device.

[0063] In general, the actuation may include driving fluid through interconnected fluid contact sides and operating valves by applying positive and negative pressures to meter fluid movement between multiple chambers of fluid contact sides. The fluid contact sides may be interconnected.

[0064] For example, the method described herein is a method of handling fluid in a microfluidic pathway device, the method comprising: sealing and independently coupling the distal ends of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid ports or pressure ports on the surface of the microfluidic pathway device, wherein each distal end is biased (e.g., spring-loaded) to drive the distal end against an elastic layer between a first plate and a second plate, wherein the microfluidic pathway device comprises a plurality of chambers, each chamber being divided into a fluid contact side formed in the second plate and a pressure receiving side formed in the first plate; and pneumatically driving fluid through the plurality of fluid contact sides of the plurality of chambers by coordinating the application of positive and negative pressures on the pressure receiving sides of the plurality of chambers to change the size of the plurality of fluid contact sides.

[0065] In some variations, the method of handling fluid in a microfluidic pathway device may include: sealingly (and independently) connecting the distal ends of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid ports or pressure ports on the surface of the microfluidic pathway device, wherein each distal end is biased to drive the distal end against an elastic layer between a first plate and a second plate of the microfluidic pathway device, wherein the microfluidic pathway device includes a plurality of chambers, each chamber being divided into a fluid contact side formed in the second plate and a pressure receiving side formed in the first plate; and driving fluid through the plurality of fluid contact sides of the plurality of chambers. Attached Figure Description

[0066] The novel features of the invention are set forth in the following claims. The features and advantages of the invention will be better understood by referring to the following detailed description and accompanying drawings, which illustrate exemplary embodiments using the principles of the invention, wherein:

[0067] Figure 1 This is a schematic diagram of a variation of a system that includes microfluidic devices and microfluidic pathways as described herein.

[0068] Figure 2A-2B An example of a closed-path microfluidic device for treating therapeutic polynucleotides at the point of care is shown, both front and back.

[0069] Figure 2C Examples of devices as described in this article.

[0070] Figure 3 This is a partially exploded view of an example of a closed-path microfluidic device for processing therapeutic polynucleotides at the point of care, as described herein, including a reagent storage frame, fluid interface assembly, thermal control device and sensor assembly, and microfluidic path device.

[0071] Figure 4A This is an isometric view of an example of a reagent storage frame.

[0072] Figure 4B yes Figure 4A Side view of the reagent storage frame.

[0073] Figure 5A This is an example isometric view of the upper surface (e.g., top) of the reagent storage frame.

[0074] Figure 5B yes Figure 5A An isometric view of the lower surface (e.g., bottom) of the reagent storage frame.

[0075] Figure 6A This is a top view of an example fluid interface component.

[0076] Figure 6B yes Figure 6A A bottom view of the fluid interface component.

[0077] Figures 7A-7B These are perspective and side views of a pipe compression connector assembly configured to be biased (e.g., spring-biased) to push a fluid line against a microfluidic path device to form a sealed connection thereto.

[0078] Figure 7C It is similar to Figures 7A-7B The illustration shows a pipe that remains engaged within a pipe compression connector (e.g., a clamp).

[0079] Figures 7D-7E An example of a fluid cartridge that can maintain spring contact with a port (e.g., a fluid port) of a microfluidic path device is shown. Figure 7E An enlarged view of the port interface area is shown.

[0080] Figure 8A This is an isometric top view of an example of the mount as described herein, the multiple optical sensors arranged around the mount on the rack, and the thermal control device below the mount.

[0081] Figure 8B yes Figure 8A Side view of the sub-components (including the mount, optical sensor, and thermal control device).

[0082] Figure 8C It is based on Figure 8A Detailed view of the frame.

[0083] Figure 9A This is a top view of an example of a microfluidic pathway device.

[0084] Figure 9B yes Figure 9A An isometric view of a microfluidic path device.

[0085] Figure 9C It shows Figures 9A-9B An enlarged example of a portion of the microfluidic path device shown includes a vacuum cap for removing air bubbles.

[0086] Figure 10A This is a side sectional view of an example of a microfluidic pathway device.

[0087] Figure 10B This is a cross-sectional example of a portion of a microfluidic path device as described herein.

[0088] Figure 10C-10I Operation of a portion of an example of a microfluidic path device interacting with a closed-path microfluidic device as described herein is illustrated in a side cross-sectional view. In this example, fluid lines and pressure lines engage with the microfluidic path device, allowing for precise control of fluid movement within the device.

[0089] Figure 11 This is an exploded view of the layers including a microfluidic pathway device according to an embodiment of the present disclosure.

[0090] Figures 12A-12C Examples of various microfluidic pathway devices as described herein are shown.

[0091] Figure 12D-12F It shows something similar to Figures 12A-12C Examples of variations of the microfluidic path device are shown.

[0092] Figure 13 A cross-section of an example of a microfluidic path device is shown, illustrating a closed fluid path.

[0093] Figure 14 Some of the functions of the microfluidic path device controller described herein are illustrated schematically.

[0094] Figures 15A-15B Top and bottom views of an example of a microfluidic path device including a heat sink are shown respectively.

[0095] Figure 16A An example of a system incorporating microfluidic devices within a Level 5 isolation cabinet within a Level 7 space is shown. The system can be configured for small-scale manufacturing.

[0096] Figure 16B The microfluidic device is shown in a Level 5 cabinet. Detailed Implementation

[0097] In general, this document describes devices (e.g., systems, apparatuses, etc.) and methods for processing therapeutic polynucleotides. Specifically, these devices and methods can be closed-path devices and methods configured to minimize or eliminate manual operation during operation. Closed-path devices and methods provide a nearly completely sterile environment, and components provide a sterile path of processing from the initial input (e.g., template) to the output (compound therapy). The material inputs to the device (nucleotides and any chemical components) can be sterile and can be introduced into the system with virtually no human interaction.

[0098] The methods and apparatus described herein can produce treatments with very high reproducibility and very fast cycle times. The apparatus described herein is configured to provide the synthesis, purification, dialysis, compounding, and concentration of one or more therapeutic compositions (including, but not limited to, therapeutic polynucleotides) in a single integrated device. All or part of these processing steps can be performed in an uninterrupted fluid processing pathway, which can be configured as one or a series of consumable microfluidic pathway devices (also referred to as microfluidic pathway chips, microfluidic pathway plates, processing chips, biochips, or processing plates). This allows for the synthesis of patient-specific treatments, including compounding, at the point of care (e.g., hospitals, clinics, pharmacies, etc.).

[0099] During equipment operation, the fluid path can remain essentially uninterrupted, and contamination can be largely eliminated through non-contact monitoring (e.g., optical monitoring), including fluid flow measurement, mixing monitoring, etc., and by manipulating precise microfluidic quantities (metering, mixing, etc.) using pressure applied from deflectable membranes on opposite sides of the fluid chamber and channel.

[0100] These devices and methods can be configured for use at the point of care. For example, the methods and devices described herein can be configured to manufacture custom therapeutic compositions comprising one or more therapeutic polynucleotides (e.g., mRNA, microRNA, DNA, etc.).

[0101] Therefore, the methods and apparatus described herein provide scalable polynucleotide manufacturing, production of single-patient doses, elimination of contact points to limit contamination, input and process traceability to meet clinical manufacturing requirements, and use in point-of-care operations for therapeutic purposes. The microfluidic instruments and processes described herein offer key advantages.

[0102] equipment

[0103] In general, the devices described herein can be microfluidic devices (e.g., microfluidic control devices). In some variations, these microfluidic devices may include closed-path microfluidic devices for processing therapeutic polynucleotides at the point of care. These devices may be configured to operate on one or more microfluidic path devices. Microfluidic devices may include one or more microfluidic path devices (e.g., process chips, formulation chips, etc.) or may be configured to be used with microfluidic path devices; therefore, microfluidic devices may not include microfluidic path devices. In some variations, microfluidic devices (with or without microfluidic path devices) may be referred to as systems.

[0104] In general, the microfluidic devices described herein may include: a mount for the microfluidic device; a fluid interface assembly including multiple fluid lines and pressure lines; a reagent storage frame including multiple fluid sample holders, each fluid sample holder configured to hold a fluid bottle, and each fluid sample holder configured to be connected to the fluid interface assembly via a fluid line; multiple optical sensors arranged around the mount and reagent storage frame for monitoring fluid levels within the reagent storage frame and fluid movement within the microfluidic path device; and a controller configured to control pressure applied via the pressure lines to drive fluid movement within the microfluidic path device. In any of these devices, each fluid line and each pressure line may be configured to be driven against the microfluidic path device located in the mount.

[0105] The controller can coordinate processes including moving one or more fluids onto a microfluidic pathway device, mixing one or more fluids on the microfluidic pathway device, adding one or more components to the microfluidic pathway device, metering fluids in the microfluidic pathway device, regulating the temperature of the microfluidic pathway device, applying magnetic fields (e.g., when using magnetic beads), etc. The controller may include software, firmware, and / or hardware. In some variations, the controller may receive input from a user and may provide output (e.g., via a monitor, touchscreen, etc.). The controller may communicate with a remote server, for example, to track device operation, reorder materials (e.g., components such as nucleotides, microfluidic pathway devices, etc.), and / or download protocols, etc.

[0106] Figure 1A schematic diagram of an example system for processing polynucleotides is shown, comprising a device having a housing 103 that encloses a holder 115 for holding one or more microfluidic pathway devices 111, which may be disposable devices. The housing may be a chamber, shell, etc., and may include a lid or opening; it may be sealable when closed. The housing may enclose a thermal regulator and / or may be configured to be enclosed in a thermally regulated environment (e.g., a refrigeration unit). The housing may form a sterile barrier. In some variations, the housing may form a humid or humidity-controlled environment.

[0107] The bracket 115 can be configured to secure the microfluidic path device using one or more pins or other components configured to hold the microfluidic path device in a fixed and predefined orientation.

[0108] In some variations, a thermal control device 113 may be located near the mount 115 to regulate the temperature of one or more microfluidic path devices 111. The thermal control device may include thermoelectric components (e.g., Peltier devices) and / or one or more heat sinks for controlling all or part of the temperature of the microfluidic path device. In some variations, more than one thermal control device may be included for individually regulating the temperature of different regions within one or more areas of the microfluidic path device. The thermal control device may include one or more thermal sensors (e.g., thermocouples, etc.) which can be used for feedback control and / or thermal control of the microfluidic path device.

[0109] exist Figure 1 In this device, the fluid interface assembly 109 connects liquid reagents and / or pressure (e.g., gas) to the microfluidic path device 111 held in the holder 115, and can assist in the delivery of fluid materials and positive / negative gas pressure from the pressure source 117 to the interior of the microfluidic path device 111. As described in more detail below, the fluid interface assembly can optionally help secure the microfluidic path device. The fluid interface assembly can be detachably connected to the device (and can be detached or partially detachable) for sterilization between uses.

[0110] The reagent storage frame 107 is configured to include multiple fluid sample holders, each of which can hold a fluid bottle configured to hold reagents (e.g., nucleotides, solvents, water, etc.) for delivery to the microfluidic device 111, or alternatively, the fluid bottle can be configured to receive product from within the microfluidic pathway device 111. The reagent storage frame may be referred to as a reagent rack. In some variations, the reagent rack includes multiple pressure lines and / or manifolds configured to branch one or more pressure sources 117 into multiple pressure lines that can be applied to the microfluidic pathway device and can be controlled independently or collectively (in a sub-combination manner).

[0111] The fluid interface assembly may include multiple fluid lines and / or pressure lines, and may include biased (e.g., spring-loaded) supports or tips that, when held in the holder 115, individually and independently drive each fluid and / or pressure line to the microfluidic pathway device. Conduits (e.g., fluid lines and / or pressure lines) may be part of the fluid interface assembly and may be connected to the fluid interface assembly. In some variations, the fluid lines include flexible tubing connected between the reagent storage frame and the microfluidic pathway device via a connector that locks the bottle to the tubing (e.g., a sleeve). The ends of the fluid pathway (in some variations, the ends of the fluid lines / pressure lines) may be configured to seal against the microfluidic pathway device, for example at a sealed port formed in the microfluidic pathway device, as described herein. For example, the ends of the fluid lines may be cut or formed flat (vertical in side view). The bottle may be pressurized (e.g., greater than 1 atm, such as 2 atm, 3 atm, 5 atm, etc.) via a connector that may also be connected to a pressure source. For example, fluid bottles can be pressurized to 1-20 psig (e.g., 5 psig / 20 psia, 10 psig, etc.). Negative or positive pressure can be applied; for example, at the end of the process, a vacuum (e.g., -7 psig or 7 psia) can be applied to pump the fluid back into the bottle (e.g., storage chamber). Generally, the driving pressure of the fluid bottle may be lower than that of the pneumatic valve, which can prevent or reduce leakage. In some variations, the pressure difference between the fluid and the pneumatic valve can be between about 1 psi and about 25 psi (e.g., about 3 psi, about 5 psi, 7 psi, 10 psi, 12 psi, 15 psi, 20 psi, etc.).

[0112] As described in more detail below, fluid lines (or in some variations, fluid bottles) and pressure lines can be driven against ports (pressure ports or fluid ports) formed in the microfluidic path device to form a seal. Each pressure line and / or fluid line (or fluid bottle) can be individually driven against a valve seat in the microfluidic path by a bias pressure, which forms a seal at the port. The bias pressure (which can be generated by a spring or other force-applying element) can be configured to be greater than the pressure within the fluid bottle (and / or fluid line) and the pressure line to maintain a leak-proof seal. For example, the pressure difference between the fluid bottle and the bias pressure can be greater than about 5 psi (e.g., greater than about 2 psi, greater than about 3 psi, greater than about 5 psi, greater than about 7 psi, greater than about 10 psi, etc.) and can be referred to as the valve closing pressure. In general, this bias pressure (valve closing pressure) may exceed the fluid drive pressure, for example, by a design-related amount (e.g., 3 psi, 5 psi, 7 psi, 10 psi, etc.). The bias pressure can be constant or adjustable. Bias pressure can be applied to maintain a seal with the ports on the microfluidic path assembly. In some variations, the bias pressure can be adjusted based on the pressure within the fluid line (e.g., fluid bottle) or pressure line. The bias pressure for each fluid line (or fluid bottle) and pressure line can be adjusted individually.

[0113] Each bottle can be coded (e.g., by an identifier that can be read by one or more sensors, as described below). The controller can monitor the fluid level, thereby monitoring the amount of each material in the fluid interface assembly.

[0114] The device may also include a magnetic field applicator 119, which can be configured to generate a magnetic field in the region of the microfluidic path device 111. One or more sensors 105 (which may be optical sensors) may be part of the device and, when the device is mounted within the holder 115, may sense one or more of the following: a barcode, the fluid level in a fluid bottle held within a reagent storage frame, and fluid movement within the microfluidic path device 111.

[0115] Sensors can measure processes on equipment, for example, by measuring optical indicators. In some variations, visual / optical markings can be used to estimate yield. For example, fluorescence can be used to detect process yield or residual material by marking with fluorophores. Optionally or additionally, dynamic light scattering can be used to measure particle size distribution within a portion of a microfluidic path device, such as a mixing section. In some variations, sensor measurements can be accomplished using one or two optical fibers to transmit light (e.g., a laser) and detect the light signal output. The instrument package can be mounted remotely to the equipment. This non-contact sensing may be preferred.

[0116] In any of the methods and devices described herein, sensors (e.g., video sensors) can record all activities on a microfluidic pathway device (e.g., a chip or cartridge). For example, the entire process for synthesizing and / or processing materials (e.g., therapeutic RNA) can be recorded by one or more video sensors, including those that can visualize the microfluidic pathway device, for example, from above. Processing on the microfluidic pathway device can be visually tracked, and this recording can be retained for subsequent quality control and / or processing. Thus, video recordings of the processing can be saved, stored, and / or transmitted for subsequent review and / or analysis.

[0117] The internal parts of the device, such as within housing 103, can be further configured to be sterilizable. Specifically, portions of the device can be removed and sterilized individually. Sterilization can be performed by, for example, ultraviolet radiation or any other sterilization method required to limit contamination or meet regulatory requirements. The device, including the housing, can be housed in a high-efficiency particulate air (HEPA) filtered environment. The device, including the housing, can be housed within a temperature-controlled enclosure.

[0118] As described above, the device can be controlled by controller 121, including applying pressure through microfluidic path device 111 to at least drive fluid movement, and other tasks. The controller may be wholly or partially located outside the housing. The controller may be configured to include user inputs / outputs. For example, the system's user interface 123 may allow for simple operation and guidance of the device and microfluidic path device.

[0119] Any device described herein may include Figure 1 All or some of the components shown; not all components are necessary. Figure 1 The diagram only shows some of the connections between components; additional (or alternative) connections may be used.

[0120] Figure 2A The front side of device 200 is shown; device 200 may have any of the features of the device in system 100. Figure 2A In the diagram, the housing 103 (e.g., a cover) is shown in the open position, revealing the reagent storage frame 107, sensor 105 (configured as a camera or optical sensor in this example), and control arm 219 for the magnetic field applicator 119. The sensor can monitor the fill level and detect barcodes in the area below the horizontal plane of the reagent storage frame 107. Another sensor (camera) 105' is configured to be positioned above the upper surface of the microfluidic path device (in...). Figure 2A (Not visible in the middle). Figure 2AIn this device, a control arm for the magnet and an upper sensor can be mounted on a reagent storage frame. The control arm and upper sensor may each include one or more actuators that can be connected to and controlled by a controller (not shown). The device also includes a gantry on which a side sensor (camera) can move to visualize reagent containers and / or other components of the device. The upper sensor can be configured to observe the microfluidic path device when mounted in the device, and this information can be used to operate the device, for example, via the controller.

[0121] Figure 2B The back of device 200 is shown, where electrical power and pressure can be connected via access area 203. Figure 2C Another example of a system including a microfluidic path device is shown.

[0122] The housing 103 can be made of any suitable material, such as polymers, metals, or composite materials. The housing is moisture-proof and protects the sterility of the enclosed components during equipment operation. The housing can be designed to be contained within a refrigerator to preserve reagents at low temperatures while they are stored on reagent storage frames for batch or continuous operation of the equipment 200.

[0123] exist Figure 3 The diagram provides an exploded view of a portion of the device, including the frame 115, microfluidic pathway 111, fluid interface assembly 109, and reagent storage frame 107, illustrating how these components are assembled and aligned to provide functionality to the microfluidic pathway 111 in the device 300, which may be similar to... Figure 1 Or a device of type 2, and having any of its characteristics. Figure 3 In the image, reagent storage frame 107 is shown to have several reagent bottles and connectors (similar to...). Figure 2A Rod 303 is shown as connecting reagent frame 107 to fluid interface assembly 109, and can be connected (e.g., by screw) to frame 107 and assembly 109 at a receiving port adjacent to holder 115. Holder 115 itself may include a frame in which microfluidic pathway device 111 can be placed, and locating pins (in...) Figure 3 (Not visible in the middle) Each of these vertically arranged components can be firmly oriented to the microfluidic path device 111.

[0124] Two or more sets of pins can be used for calibration. For example, the mount 115 (also called the lower nest) may have two or more short (e.g., 1.5 mm) pins that align with the microfluidic path device 111 when placed in the lower nest and may protrude upwards on the device. Alternatively, there may be two long pins (e.g., 6 mm) that press into the upper nest (e.g., the fluid interface assembly) and protrude downwards to engage with orifices (visible) in the lower nest. These can be used to guide the upper nest into place so that the smaller (e.g., 1.5 mm) pins can then be found in the fluid interface assembly 109 to produce pin and slot features that result in final alignment.

[0125] The bracket 115 can be secured to the base 305 and can allow or restrict (e.g., constrain) adjustments to the horizontal arrangement of the microfluidic path device 111. In some variations, the microfluidic path device 111 can be supported in a substantially horizontal plane, which is useful for minimizing the pressure required to drive the fluid movement throughout the microfluidic path device 111. In some other variations, the microfluidic path device can be supported at approximately 1, 2, 3, 4, 5, 7, 9, 10, 11, 13 degrees or approximately 15 degrees to the horizontal plane. Small deviations from the horizontal direction can help remove air bubbles from the fluid within the chamber and the tubing passing through the microfluidic path device 111. In other variations, the microfluidic path device can be supported in a substantially perpendicular direction to the base 305, or at approximately 1, 2, 3, 4, 5, 7, 9, 10, 11, 13 or approximately 15 degrees to the vertical plane.

[0126] The holder 115, fluid interface assembly 109, and / or fluid sample holder may be formed of any suitable material, such as polymer, glass, metal, or composite material. The holder 115, fluid interface assembly 109, and / or fluid sample holder may be configured to be sterilized, for example, by autoclaving or gamma radiation exposure. The device 300 may further include one or more of the following: a holder release control configured to release the fluid interface assembly from the device, a fluid assembly release control configured to release the fluid interface assembly from the device, and / or a fluid sample holder release control configured to release the fluid sample holder from the device. These release controls may be engaged such that each of the reagent storage frame, fluid interface assembly, and / or fluid sample holder can be released from the device for individual sterilization. These components may be released individually and / or jointly and / or reinserted.

[0127] A thermal control device 113 may be disposed below the mounting bracket 115, adjacent to the microfluidic path device 111. The thermal control device 113 may be configured to control the temperature in at least one region of the microfluidic path device 111 between about 4 degrees Celsius and about 65 degrees Celsius, or any selected temperature within this range. The thermal control device may be any suitable temperature control device, for example, in a non-limiting example, a Peltier device and / or multiple Peltier devices. Typically, the thermal control device may be configured to allow simultaneous and independent control of the temperature of different thermal regions.

[0128] An optical sensor 105 can be mounted on a base 305 and can be oriented to sense the fluid fill level within a fluid vial positioned within a reagent storage frame, thereby reducing the likelihood of process interruptions within the microfluidic pathway device 111. The optical sensor 105 can also sense barcodes on the fluid vials to definitively identify the reagent or product vial's identity and / or batch number. The optical sensor can send information about the fill level or barcode to a controller 121, where this information can be stored or action can be taken. This helps provide chain-of-custody data crucial for regulatory control of personalized treatment.

[0129] Optical sensor 105 may be movably disposed within track 307 on base 305 (e.g., as part of a hanger) and may be further operably connected to an optical sensor driver. The optical sensor driver may be configured to move one or more of a plurality of optical sensors 105 around base 115 and / or reagent storage frame 107. The plurality of optical sensors 105 may move in unison via coordinated movement with optical drive belts (e.g., drive chains) 309.

[0130] Figure 4AThe upper surface of a reagent storage frame 107 is shown, which can be used in any of the devices described herein. The reagent storage frame 107 may support a magnetic field applicator 119, which may further include a control arm 219 for controlling the positioning of a magnet 414 in at least one region adjacent to the microfluidic pathway device 111 during use. One or more fluid sample holders 416 are disposed on the reagent storage frame 107. In some variations, the reagent storage frame 107 may include multiple fluid sample holders 416, each of which may have the same size or may have one or more different sizes. Each fluid sample holder 416 includes a cap that can be pressurized above ambient atmospheric pressure (e.g., to about 1 PSI, 2 PSI, 3 PSI, 4 PSI, 5 PSI or higher) such that fluid from a fluid vial contained in the fluid sample holder can be driven into the microfluidic pathway device 111. Alternatively, the fluid sample holder may be completely unpressurized and can be operated at ambient atmospheric pressure. In another variation, the fluid sample holder cap allows for the application of reduced pressure to draw fluid or receive fluid driven from the microfluidic path device 111. Multiple fluid sample holders can distribute the applied pressure to multiple fluid bottles. The applied pressure can be distributed down to the fluid interface assembly. As a non-limiting example, the fluid sample holder cap may include a Luer connection or the like, which provides a leak-free connection under pressure. The fluid sample holder 416 can be connected to the fluid interface assembly 109 via a fluid line for delivery to the microfluidic path device 111. The fluid line connecting the fluid sample holder (and the fluid bottle held therein) and the fluid interface assembly can be configured to have the shortest possible length to prevent waste and lag.

[0131] The connections between the fluid sample holder, fluid lines, and microfluidic pathway devices can form a sealed and enclosed pathway that is isolated when the microfluidic pathway device is located in the holder. This enclosed pathway provides effective protection against contamination when handling therapeutic polynucleotides.

[0132] The reagent storage frame may also provide a support to which a sensor / camera support arm 418 is attached. The support arm 418 supports an overhead sensor / camera 412 configured to image and detect signals from the microfluidic path device 111. The sensor / camera 412 may be a camera configured to record fluid movement within the microfluidic path device 111 and / or detect signals emanating from one or more chambers of the microfluidic path device.

[0133] The signal detected by the sensor, which acts as a signal detector, can be a visible signal, a fluorescent signal, an ultraviolet absorption signal, or an infrared absorption signal. The signal detector is a non-contact signal detector; for example, it does not directly contact the material emitting the signal. In some variations, the signal detector is configured to measure the size distribution of nanoparticles. The signal detector can also be configured to measure dynamic light scattering (DLS).

[0134] Figure 4B A side view of a reagent storage frame 107 is shown, having a horizontal surface 421 in which a fluid bottle 422, accommodated by a fluid sample holder 416, can be disposed. The reservoir portion of the fluid bottle 422 protrudes below the horizontal surface 421, allowing optical sensors to visualize identification codes 425, such as barcodes or RFID tags on the fluid bottle, and to sense the fluid level 427 (e.g., meniscus) within the fluid bottle. In some variations, an electroluminescent panel 429 may be located below the horizontal surface 421 of the reagent storage frame 107 to provide additional illumination to aid sensing. Figure 4B The magnetic arm controller 432 of the magnetic field applicator 119 can also be seen.

[0135] Figure 5A A perspective view of the top surface of the fluid interface assembly 109 of devices 100, 200, and 300 is shown. The fluid interface assembly 109 may include multiple fluid lines 534 and pressure lines 536, wherein each fluid line and each pressure line is configured to be independently driven to abut against a microfluidic path device 111 located in a mount 115 to form a sealing connection therewith. For the fluid lines 534, a biasing spring within a fitting 538 provides force to maintain the fluid line engagement with the microfluidic path device 111. Figure 5B This is a perspective view of the lower side of the fluid interface assembly 109, showing a fluid line 534 exiting from the lower side of the horizontal surface 421 of the fluid interface assembly 109 and engaging with the microfluidic path device 111. The fluid line and pressure line may have substantially flat distal ends for engagement with the microfluidic path device to form a good seal with it.

[0136] Figure 6A The top surface of the fluid interface assembly 109 is shown. The fluid interface assembly has a central opening 602 through which one or more of a plurality of optical sensors can image the microfluidic path device 111. A pressure line 642 may pass through and may be located around the periphery of the fluid interface assembly (pressure line not shown), and fluid lines 534 and pressure lines 641 are arranged for inputting the microfluidic path device 111 around the periphery of the central opening 602. Figure 6BThe bottom surface of the fluid interface assembly is shown, which shows the sealed end outlet point 643 of the fluid line 534 and the sealed end 645 of the pressure line 536 arranged around the central opening 602.

[0137] Figure 7A Fitting 746 is shown, which is used to engage a fluid line 534 against a microfluidic path device 111 located in a holder to form a sealed connection within the microfluidic path device 111. Fitting 746 includes two mechanisms to provide a robust sealed connection, minimizing leakage, while providing flexibility for the fluid line 534 held therein, thereby minimizing stress on the fluid line during device setup and operation. A spring-biased member 748 can be used to press the fluid line against the microfluidic path device. Additionally, a clamp 749 can be used to push the fluid line 534 against the microfluidic path device 111. The microfluidic line 534 can terminate at a flat cut for clean engagement with the microfluidic path device 111. In some variations, both mechanisms can be used to secure the fluid line. In other variations, the spring-biased member 748 can be used. The clamp 749 can be used to enhance gripping (e.g., in one direction) to prevent the fluid and / or pressure lines from exiting the microfluidic path device and disrupting the seal with the microfluidic path device, even when the fluid and / or pressure lines are driven against it. In other variations, other suitable connectors can be used to engage the fluid lines to the microfluidic path device, such as gaskets or other types of compression seals. Figure 7B A side view of the fitting, including spring biasing element 748 and chuck 749, is shown. Figure 7C A graphic representation of a fitting 746 with an engaged fluid line 534 is shown. A spring-biased member 748 engages against the fitting base, and a clamp 749 grips the fluid line 534. A cut-off end 735 is pushed past the clamp 749 to engage against the microfluidic path device 111.

[0138] In some variations, all or some of the fluid lines and fluid bottles may instead be additionally configured to connect to the fluid cartridge of the microfluidic path device 111. Figures 7D-7EAn example is shown in which the fluid cartridge 750 may have a pressure port 753 and a cut-out or flat-molded fluid inlet port 755. The fluid inlet port 755 may be held against the microfluidic path device 111 by biasing (e.g., spring-loaded in some variations) to contact and seal an elastic layer (e.g., a spring layer) within the microfluidic path device 111 at the port. As described above, the biasing pressure (also known as the valve closing pressure) may be greater than the pressure within the fluid bottle / fluid line and pressure line, greater than the pressure in the fluid line / fluid bottle or pressure line (e.g., greater than 2 psi, 5 psi, 7 psi, 10 psi, etc.) to prevent leakage. For example, the port of the microfluidic path device may be configured to receive a pressure of approximately 5 psig (e.g., 5 psig, 7 psig, 10 psig, 12 psig, 15 psig, etc.), which may be slightly higher than the pressurization of the fluid bottle 422 described herein. In some variations, the fluid bottle (e.g., fluid cartridge) 750 may not need to be mounted on a separate reagent storage frame, but can instead be spring-loaded directly onto the device 111 or the fluid interface assembly 109, which can reduce or eliminate tubing. Biased contact with the elastic layer of the microfluidic path device 111 actuates the sealed opening of the fluid cartridge for use. The fluid cartridge 750 is easily barcoded and identifiable. Using multiple fluid cartridges 750 simplifies the structure required for supplying reagents within the microfluidic path device 111. This design allows for the use of an isolating sterile liner within the fluid cartridge 750, eliminating contact between the reagents stored therein and the gas used to pressurize the fluid cartridge. Alternatively, other fluid cartridge designs can achieve the same isolation as exposure to gas by using a low-slip piston or other structural features that separate the reagents held within the fluid cartridge 750 from the gas used to drive the fluid into the microfluidic path device. Figure 7E An enlarged view of the fluid inlet port 755, which directly engages with the microfluidic path device, is shown. In any variation, unless the context clearly indicates otherwise, the phrase "fluid line" may include either or both of a fluid cartridge (also known as a fluid bottle) and / or a conduit connected to a fluid source (e.g., a reservoir, like a bottle, jar, tube, etc.).

[0139] Figure 8A yes Figure 1A perspective view of the bottom substrate 800 of device 1, 2, or 3 shows a mount 115 and a base 305 to which multiple optical sensors 105 are attached. In this view, four optical sensors 105 are positioned around each edge of the base and can be configured to move in place along a hanger including one or more tracks 307 to image an area around the mount and reagent storage frame. The optical sensors 105 can be driven along the tracks 307 by optical sensor drivers 851. The optical sensors can be uniformly driven by optical sensor drive belts 309. Each optical sensor 105 has an angled mirror assembly 853 that allows imaging of the field of view 855 while minimizing the space occupied by the optical sensor 105 itself. The bottom substrate 800 in this embodiment also includes a second set of positioning pins 857, which can facilitate alignment of the fluid interface assembly and reagent storage frame with the mount. Figure 8B This is a side view showing the arrangement of the base 305, optical sensors 105 (three are visible in this figure), optical sensor drive belt 309, and optical sensor drive device 851. A second set of locating pins 857 forms the area around the bracket 115, and the cooling fan 859 of the thermal control device 113 can be seen below the bracket 115. Figure 8C A detailed view of the components arranged on the bottom substrate 800 is shown. A holder 115 is secured by a mounting spring 865 to be supported in a desired orientation and to position the opening where the microfluidic path device will be placed at a thermally controlled level, such as a Peltier surface (thermal control surface) 863. The holder 115 may be releasably secured to the mounting spring 865 and has a release lever or connector (not shown) that allows the holder 115 and the fluid interface assembly 109 (e.g., the upper nest) to be removed and sterilized between uses, for example, by autoclaving. Alignment holes 861 may be present on the holder 115 for aligning the microfluidic path device 111 for proper placement within the holder 115. The alignment holes may be located near the outer edge of the holder 115. Alignment pins may also be included. The thermal control surface 863 may also include or be configured to include a vacuum chuck; a vacuum groove may be present in the upper surface, allowing a vacuum to be applied to draw the microfluidic path device down to maintain good thermal contact with the thermal control surface 863.

[0140] In general, the mount can be simply referred to as the "mount" and is configured to securely or loosely position one or more microfluidic path devices within the device.

[0141] In some variations, the microfluidic path device 111 may be supported in a substantially horizontal orientation, or in an orientation within a range of approximately 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or approximately 15 degrees above the horizontal plane, to aid in bubble control. A fan 859 is visible beneath the mount 115.

[0142] As described above, the device (e.g., system, apparatus, or device) may include a controller. The controller may be configured to control pressure applied via a pressure line to drive fluid movement within the microfluidic path device when the microfluidic path device is located in a holder. The controller may be configured to communicate with an optical sensor and to sense the identity of a code on a fluid bottle or a code on the microfluidic path device. The code on the fluid bottle and / or microfluidic device may be an optical code or an RFID code. The controller may be configured to communicate with an optical sensor and to sense the reagent level within the fluid bottle. The controller may be configured to send instructions to an optical sensor driver to selectively position the optical sensor for sensing a code or sensing the fluid level within a fluid bottle positioned on a reagent storage frame. The controller may be configured to control the timing of reagent introduction within the microfluidic path device. The controller may be configured to control the volume of reagent to be driven within the microfluidic path device. The controller may be configured to control the positive pressure of gas at at least one fluid inlet of the microfluidic path device. The controller may be configured to isolate at least a portion of the product within a sub-region of the microfluidic device for outlet. In some variations, the controller can be configured to perform in vitro transcription (IVT) reactions within a microfluidic pathway device. The controller may also include memory and one or more data stores.

[0143] The device may include one or more processors configured to instruct and / or control the device. The one or more processors may also analyze information from the device and / or the microfluidic pathway device.

[0144] The device may include a user interface for inputting / outputting at least one of instructions and information regarding device status, the identity of reagents within the device, and the ongoing workflow. In some variations, the device may include a graphical user interface configured to provide input to a processor.

[0145] The device may also include a remote database for storing and retrieving data and images. Identification codes, visual logs, and other information can be stored in any format suitable for device operation and / or to meet regulatory requirements for the manufacture and formulation of personalized treatments.

[0146] In general, the devices described herein may include one or more single-use microfluidic pathway devices, and reusable components or subsystems; selected portions of these subsystems may be sterilized. For example, one or more fluid sample holders (all or part, such as fluid container holders, tubing, etc.), fluid interface assemblies (all or part), and / or a holder for the microfluidic pathway device (all or part, such as a seat portion) may be removable, sterilizable, and replaceable. The device may be configured to allow the one or more areas to be released and removed from the device. For example, the device may include a holder release control configured to release a holder from the device for individual sterilization and / or a fluid interface assembly release control configured to release a fluid interface assembly from the device for individual sterilization, and / or a fluid sample holder release control configured to release a fluid sample holder from the device for individual sterilization. The release mechanism may be a locking release device, one or more screws, pins, hinges, etc. Any of these devices can be configured to allow parts to be lifted from other areas of the device (automatically, manually, or semi-manually), including integrated guides or mounts supporting the various parts of the device, thereby allowing access to and removal / replacement of certain components, such as the mount, fluid interface assembly, and / or all or part of the fluid sample holder.

[0147] Microfluidic pathway devices

[0148] The aforementioned device is configured to support and control operations within a microfluidic pathway to perform polynucleotide processing. The polynucleotide can be any type of polynucleotide, including but not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc. The polynucleotide may consist only of natural nucleotide units, or may include any type of synthetic or semi-synthetic nucleotide units. Processing may include, but is not limited to, in vitro synthesis, purification, concentration, formulation, and analysis.

[0149] Examples of microfluidic pathway devices for synthesizing therapeutic polynucleotides in closed pathways are shown in Figures 9A-9B As shown in 10A-E and 11, where Figure 9A This is a view of the upper surface 911 of the device 900, viewed downwards through the multiple layers forming the device 900. Figure 9B This is a perspective view of device 900. Figure 10A and 10C -10H is a side view of the layer through which the device 900 is constructed, showing an example of the arrangement of layers and passageways, rooms and ports formed therethrough.

[0150] Figure 10AAn example arrangement of layers forming microfluidic pathways such as seals, channels, valves, and chambers (including pumping chambers) is shown. Generally, these devices can be advantageously formed from a rigid or semi-rigid plate and at least one elastic layer. The elastic layer can be a sheet of impermeable elastic material. The elastic layer can have a certain degree of gas permeability, or can be treated to have more or less gas permeability in different areas. Although a single continuous elastic material sheet can be used, in some variations, multiple elastic material sheets can be used, or a “sheet” can be formed from fragments of multiple sheets. The layers and elastic sheets can be laminated together. Generally, a chamber for retaining, valve-controlling, and / or pumping fluid can be formed in a plate on either side of the elastic layer, such that the elastic layer equally divides the chamber into a liquid-containing side and a pressure (e.g., gas)-applying side. The total volume of the chamber can be constant, and a first (e.g., upper) plate and a second (e.g., lower) plate can be formed, but this volume can be divided into a pressure side and a liquid side. By applying positive or negative pressure to the pressure side, the elastic sheet can deform to reduce the volume of the liquid-containing side (to zero, closing the chamber) or increase the volume of the liquid-containing side (to a predetermined maximum value). The pressure application side of the chamber can be connected to a pressure port, for example, through a pressure channel in the upper plate (or between the upper plate and the elastic layer), for applying negative or positive pressure. As described herein, the liquid-containing side can be connected to a fluid port via a fluid channel. As will be described in more detail herein, both the fluid port and the pressure port can be formed through openings leading to the upper plate and the elastic layer, allowing for a sealed connection isolated from the atmosphere even in the presence of multiple different inlet lines.

[0151] exist Figure 10A In this microfluidic pathway device 900, a first (e.g., upper) plate 903 is included, having a first (e.g., top or upper) surface 911 and a second (bottom or lower) surface 929, and a thickness between them. The first surface 911 may form an exposed outer surface. The microfluidic pathway device also includes a second plate 905, having a first (e.g., upper or top) surface 931 and a second (e.g., lower or bottom) surface 933, and a thickness between them. An elastic layer 907 is sandwiched between the second surface 929 of the first plate 903 and the first surface 931 of the second plate 905. A third plate 909 is directly or indirectly coupled to the second plate on the second surface 933 of the second plate. The third plate 909 also has a first (e.g., upper or top) surface and a second (bottom or bottom) surface, and a thickness between them. The second surface of the third plate may form the bottom surface of the microfluidic pathway device. Any plate may be formed from multiple layers, which may be laminated or otherwise joined together. For example, in Figure 10A In this example, the third plate 909 includes an optional second elastic layer 913, which can help connect the third plate to the second plate; the second elastic layer 913 forms the first surface 935 of the third plate 909. Figure 10A The layers and plates shown may not be to scale (e.g., the elastic layer 907 may be thinner relative to the plate).

[0152] Figure 10A The illustrated microfluidic pathway device 900 may further include multiple chambers 915, 916, 918, 920, each with a fixed volume. These chambers are formed by cut-out regions (e.g., circular / curved cuts) in the second (bottom) surface 929 of the first plate 903 and the first (upper) surface 931 of the second plate 905; an elastic layer 907 branches these chambers 915 such that each chamber includes a liquid-containing side 917 and a pressure (e.g., gas-containing) side 919. The microfluidic pathway device 900 may also include multiple liquid (e.g., fluid) channels. Figure 10A The diagram shows a single fluid channel 921 extending from a fluid port 923 through the thickness of a first plate 903 to a fluid channel opening 925 through the elastic layer 907 and through most of the thickness of a second plate 905, up to the bottom surface 933 of the second plate. The length of the liquid channel 921 extending parallel to the bottom surface of the third plate is formed in the bottom surface 933 of the second plate and is defined by the upper surface of the third plate 909.

[0153] Regarding fluid port 923, the diameter of the opening leading to the first plate 903, thus forming a fluid port 923 extending through the thickness of the first plate, may be larger than the diameter of the fluid channel opening 925 extending through the elastic layer 907 and into the liquid (e.g., fluid) channel 921. The fluid channel opening 925 may be centered relative to the bottom of the fluid port opening and may be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line connection interface to which the fluid port will be connected.

[0154] Fluid passage 921 is connected to the liquid-containing side 917 of the first chamber 915. The first chamber can be configured as a valve having a relatively low holding volume (fixed volume) but which can be fully opened or closed by movement of the elastic layer 907.

[0155] The microfluidic pathway device 900 also includes multiple pressure channels, which can be independently controlled to apply positive and / or negative pressure. Figure 10A The diagram shows a single pressure port 943 connected to the fourth chamber 920, but each chamber 915, 916, 918 can be connected to separate pressure ports and pressure channels for independent operation and control of the movement of the portion of the elastic layer 907 that separates these chambers, to independently valve and / or pump each chamber. In some variations, the pressure port can be shared among multiple chambers. Figure 10AIn this design, a pressure (e.g., gas) port 943 is similar to a fluid (e.g., liquid) port 925 and includes an opening that extends completely through the first plate 903 down to the exposed elastic layer 907, and then through the opening in the elastic layer to form a pressure (e.g., gas) channel opening 945. The pressure channel opening 945 is continuous with a pressure (e.g., gas) channel 947 that extends from the pressure port 943, through a large portion of the thickness of the first plate 903, and in a cut-out channel along the bottom of the second plate (or optionally into the cut-out area at the top of the third plate), and upwards back through the second plate and elastic layer 907 to the pressure channel within the first plate, connecting to the pressure (e.g., gas) receiving portion 919 of the fourth chamber 929. As described for a similar fluid (e.g., liquid) port, the diameter of the pressure port 943 extending through the thickness of the first plate 903 can be larger than the diameter of the pressure channel opening 945 extending through the elastic layer 907, and can be centered or offset from a wall thickness greater than that of the pressure line or pressure line connection interface to which the pressure port will be connected.

[0156] exist Figure 10A In the cross-section of the microfluidic path device 900 shown, there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports (not shown), as these may be outside the plane shown. For example, in Figure 10A In this configuration, the liquid-containing side or portion 917 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, an outlet channel extending from the liquid-containing side 917. As described above, additional chambers (e.g., configured as valves) may be formed. In some variations, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid receiving reservoir, such as a bottle, tube, etc. This receiving reservoir may be held within a reagent storage frame.

[0157] Overall, this configuration of the microfluidic path device and microfluidic apparatus is designed to enable the device to perform multiple complex steps in a completely closed (sealed and unaffected by the atmosphere) manner without any human intervention. A fixed volume of metered fluid can be used, and the fluid can be moved, mixed, and filtered by applying air pressure to deflect regions of the elastic layer.

[0158] Back Figure 9AThe microfluidic pathway device 900 may include at least a pair of chambers 953, each chamber being similar to chamber 920, including a liquid (fluid) side 917, a pressure (e.g., gas) side 919, a fluid connection, a pressure connection, and a fluid / pressure line as described above. Furthermore, each of the pairs may be interconnected via a fluid connector 955. The fluid connector 955 may be used in conjunction with positive and / or negative pressure applied to the pressure side of the chamber to drive liquid in the liquid side between the two chambers, thereby mixing the liquid in each chamber. Deflecting an elastic layer between fixed volumes of chambers separated by the elastic layer can drive any liquid between the two chambers.

[0159] Any microfluidic pathway device described herein may include one or more connections for electronic devices (including electrical sensors) on the device. For example, in Figure 9A In this device, the microfluidic path may include a region 982 configured to include one or more electrical contacts for communicating with one or more sensors (or other electronic devices) on the microfluidic path. In some variations, the electronic devices may include circuitry, etc. The electrically active region 982 may include one or more conductors for electrical contact with the device; the electrical contact may provide power, data, etc. For example, the electrically active region may be configured as one or more contact pads for connection to one or more connector pins (e.g., spring-loaded or otherwise biased connector pins) that may be attached to an upper housing or a portion thereof (e.g., a fluid interface assembly).

[0160] The microfluidic pathway device 900 may include more than one pair of chambers, each pair of chambers being usable for different processes applied to polynucleotides. For example, the first pair of chambers 953 may be used for the synthesis of polynucleotides. The second pair of chambers 955 may be used for the purification of the synthesized polynucleotides. When pressure is applied to the pressure receiving side 919 of the respective chamber and the valve 959 between the first pair of chambers 953 and the second pair of chambers 955 is opened, fluid from the first pair of chambers 953 may be driven to the second pair of chambers. The valve chamber 959 may be formed by an elastic layer 907 within a connector channel between the two pairs of chambers.

[0161] Figure 9A and 9B The microfluidic path device 900 shown may have multiple pressure ports 943 and fluid ports 923. The multiple pressure ports and fluid ports may be arranged adjacent to the periphery of the microfluidic path device and configured to connect to the fluid interface assembly 109 as described above.

[0162] The port (e.g., a sealing valve) can be formed by an elastic layer as described above along, for example... Figure 9AThe length of the connection channel 939 (pressure channel or fluid channel) of the valve 961 shown is formed, and the valve 961 can control the delivery time of the reagent driven from the fluid port 923, but when placed in series with one or more valves of similar construction, it can also allow metering of the chamber of the device. For example, in Figure 9A The diagram shows three valve chambers (described in more detail below); the first of these three valves can be used as a peristaltic pump, while the middle valve can be a small metering chamber (e.g., metering volumes of approximately 10 nL, 20 nL, 25 nL, 50 nL, 75 nL, 100 nL, etc.). As described above, the structure of the ports and channels is... Figure 10A As shown in the diagram, the dimensions of the channels, particularly the dimensions of the chambers connected to the channels, allow for the metering of the volume dispensed and delivered along the fluid connection channels 939, 921 to the chambers 953 connected to the fluid connection channels 939, 921. In some variations, the metered volume can be as small as 50 nL. Metering volumes of approximately 100 nL, 1 μL, 5 μL, or more can be input. Various valve sizes can be pre-selected for integration with the microfluidic path device 900, and reagents can be connected to the appropriate metering size by user selection.

[0163] Furthermore, along the fluid connection channel 939, a row may include more than one valve body 961. A series of valves 961 can function as a peristaltic pump to move fluids, including (but not limited to) viscous fluids. Generally, the ability of a peristaltic pump as a fluid may be particularly advantageous for moving fluids that may be viscous or contain suspended particles (e.g., for cleaning or trapping beads).

[0164] As described above, the microfluidic path device 900 may further include a delivery or output reservoir or tank 963. Figure 9A In this configuration, the preselected volume can be formed in a manner similar to the chamber structure described above, or it can consist only of the metering side. In either case, a valve can be used to meter the desired volume entering reservoir 963. Valve 965 can control the fluid delivery from reservoir 963. If a larger volume is required, delivery can be repeated. Alternatively, if reservoir 963 is preselected as an output reservoir, valve 965 can be opened to deliver fluid from chamber 957 while valve 967 remains closed, allowing only the measured fluid volume to be output to reservoir 963. This fluid can then be output to a fluid bottle on a reagent storage frame for further processing or testing. In some variations, the chamber, reservoir, or reservoir (e.g., 963) can be configured, for example, as the metering section of a 1L pump formed by three valve structures (967, 965, 967). The chamber can be configured to, for example, discharge waste from mixing chamber 957.

[0165] The advantage of the microfluidic pathway device 900 lies in the closed-path nature of its structure. When fluid bottles, fluid lines, and the microfluidic pathway device are connected, the device can be operated without exchanging any materials entering or leaving the system, particularly the fluid paths into / out of the microfluidic pathway device used for processing, including the synthesis of polynucleotides and their preparation for biodelivery (as therapeutic agents, such as drugs, vaccines, etc.). Therefore, the entire system can operate as a closed path, and / or individual microfluidic pathway devices can operate as closed paths within the system (unaffected by the atmosphere).

[0166] Some variations of the processing that can be performed within the microfluidic pathway device 900 may include purification. One variation of purification may include adding a material to the fluid side 917 of a chamber or channel. This material may be configured to absorb selected portions of the fluid mixture in the chamber or channel. In one variation, the material may include a cellulose material that selectively absorbs double-stranded mRNA from the mixture. The cellulose material may be inserted into only one chamber of a pair of chambers, such that after mixing fluid from the first chamber of the pair to the second chamber, the double-stranded mRNA can be effectively removed from the fluid mixture, which can then be transferred to another pair of downstream chambers for further processing or output.

[0167] Some variations of the microfluidic device 900 may further include a concentrator located within a chamber, which may be disposed within the thickness of the second plate and may be in fluid communication with an outlet channel, such as 949. Polynucleotides can be concentrated by discharging excess fluid medium, and the concentrated polynucleotide mixture can be output from the microfluidic pathway device 900 for further processing or use. In some variations, the concentrator may be a dialysis chamber. For example, a dialysis membrane may be present within or between the plates of the microfluidic pathway device.

[0168] The microfluidic path device 900 may be formed of a material that is at least substantially translucent to visible and / or ultraviolet light. "Substantially translucent" means that at least 90% of the light is transmitted through the material compared to a translucent material. In some variations, the microfluidic path device 900 may be formed of a material that is substantially transparent to visible and / or ultraviolet light. "Substantially transparent" means that at least 90% of the light is transmitted through the material compared to a completely transparent material.

[0169] As described above, the first and / or second plates can be formed of a rigid material. The third plate can also be formed of a rigid material. In some variations, the third plate can be formed of a rigid material laminated onto an elastic material. The plates can be formed of the same material or different materials. For example, the rigid material can be a polymer or glass. The polymer or glass can be biocompatible, for example, it will not leach any monomers or soluble small molecules that are toxic to living cells. Polynucleotide products processed in microfluidic pathway devices can be administered to animals, so it is preferable to reduce or eliminate toxic contaminants by selecting materials. Any suitable biocompatible polymer can be used, including medical-grade polycarbonate-urethane, siloxane polycarbonate-urethane, polyether urethane, etc. In some variations, the polymer can be a cyclic olefin copolymer.

[0170] Figure 10B An example of a portion of a microfluidic pathway device is shown, partially transparent and in perspective view. In this example, the device includes a first plate 903 having a first surface and a second surface and a thickness therebetween, a second plate 905 having a first surface and a second surface and a thickness therebetween, and an elastic layer 907 sandwiched between the second surface of the first plate and the first surface of the second plate. A third plate 909 is attached to the second plate on the second surface of the second plate.

[0171] Figure 10B The illustrated microfluidic pathway device also includes cross-sections through three chambers 915, 914, and 918, each chamber having a fixed volume. Each chamber is formed in the second surface of the first plate and the first surface of the second plate, and a portion of the elastic layer divides each chamber into a fluid retention side and a pressure application side. Fluid channels extend from the fluid port (in...) Figure 10B The fluid channel extends (not visible in the middle) through the first plate to the elastic layer 907, reaching a fluid channel opening that extends through the elastic layer and through most of the thickness of the second plate, to fluidly connect with a connecting channel formed in the second surface of the second plate (or between the second and third plates). This connecting channel may be defined by the third plate. The fluid channel then extends upward back through the second plate to connect to a fluid (e.g., liquid) holding portion of one of the chambers, and preferably to a chamber configured as a valve. Figure 10B In this design, three chambers configured as valves are connected together, allowing fluid to be pumped between them and metered in small volumes (e.g., 10 nL). A negative pressure (or, in some variations, zero pressure) can be applied to the pressure-holding side 919 of chamber 915 to open the valve, pulling the elastic layer upwards to the upper side of the chamber (pressure-holding side). A positive pressure can be applied to close the valve, deflecting the bifurcated elastic layer against the lower curved wall of the liquid-holding side of the chamber.

[0172] Pressure channel 947 can be formed from the pressure port (in) of the channel Figure 10B (Not visible in the middle) Extends downward through the thickness of the first plate to the elastic layer 907. Pressure channel openings may be formed through the elastic layer within the thickness of the second plate to interact with those formed in the second surface of the first plate (in the middle). Figure 10B The pressure channel between the first plate (not visible in the middle) or the second and third plates is in fluid communication; the pressure channel may then extend upwards back (in a U-shaped path) through the second plate to a channel formed in the first plate (or between the first and second plates) to connect to the pressure holding side of one or more chambers. The diameter of the fluid port through the thickness of the first plate is typically larger than the opening of the fluid channel through the elastic layer, and the diameter of the pressure port through the thickness of the first plate may be larger than the opening of the pressure channel through the elastic layer. Any microfluidic path device may also include an outlet channel extending from the fluid holding side of the chamber or channel through a second surface of the second plate and / or through a chamber configured as a valve that can be opened to allow fluid in the channel or chamber to be pumped into a reservoir (e.g., a holder, bottle, container, tube, etc.) that may be located, for example, in a rack of a reagent storage frame.

[0173] In any microfluidic pathway device described herein, fluid can flow from the top through a first plate, through a seal formed by an elastic layer, through a second plate, and then along the second plate and back up into a chamber bisected by an elastic material (e.g., in some cases, a chamber configured as a valve). Similarly, a pressure flow (positive or negative) can flow from the top through a first plate, through a seal formed by an elastic layer, through a second plate, along the bottom of the second plate, then back up through the second plate and the elastic layer, and then along the bottom of the first plate to connect to the pressure-holding side of a chamber bisected by an elastic layer. Generally, the elastic layer can bisecte the chambers by driving them equally or unequally; for example, the upper (pressure) chamber can be larger or smaller than the lower (liquid-holding) chamber. Applying positive or negative pressure to control the fluid within a valve and / or pump or metering chamber may be referred to herein as pneumatic or pneumatic barrier deflection (“pneumatic deflection”).

[0174] Figure 10C-10H A microfluidic pathway device (similar to) is shown that engages with a microfluidic device, particularly with multiple fluid lines 1033 and / or pressure lines 1043. Figure 10A The operation of the exemplary device shown in the cross-section. Figure 10C The image shows a cross-section of the microfluidic path device 900; multiple fluid lines 1033 and / or pressure lines 1043. Figure 10BTwo lines (as can be seen) are shown near the microfluidic path device, each line independently aligned with a pair of ports, including a fluid port 923 (to which fluid line 1033 is aligned) and a pressure port 943 (to which pressure line 1043 is aligned). In any variation described herein, as mentioned above, the pressure lines and fluid lines may extend from the fluid interface assembly. As mentioned above and Figures 7A-7C As shown, each pressure and fluid line can be connected to an independent bias (e.g., biased towards the microfluidic path device) via a compression connector. Because each fluid and pressure connection can be independently biased (e.g., driven individually towards the microfluidic path device), but can be biased up / down to push and seal against an exposed elastic layer supported on one side by a rigid second plate, the pressure connection can form a highly tolerant seal between the pressure or fluid lines, such as... Figure 10C As shown. Therefore, the device is highly tolerant of alignment and orientation, allowing for slight displacement and / or angulation relative to the fluid and / or pressure lines. Compression connectors can also apply force to the supported elastic layer to maintain a seal and prevent contamination or exposure to the external environment. Bias elements (e.g., springs) can push the ends of the fluid or pressure lines against the microfluidic path device. The closer the pressure or fluid line is to the microfluidic path device, the greater the applied force; however, each fluid or pressure line may be pushed back, for example, upwards, at least slightly. Finally, the device can be configured to mate with the microfluidic path device around its periphery, distributing force from the same side of the device and achieving a balanced and supported contact with the microfluidic path device.

[0175] exist Figure 10D In the diagram, fluid line 1033 and pressure line 1043 are driven by spring bias to abut against the fluid and pressure ports of the microfluidic path device, similar to... Figures 7A-7D The bias shown causes the distal opening end to be driven against the flat surface of the first elastic layer 907 of the microfluidic path device, which is supported by a second layer below. Therefore, each pressure line and fluid line can be individually driven (as indicated by the arrow), for example by a spring or other biasing element, against the elastic layer to form a seal against the elastic layer 907. Thereafter, the individual pressure lines and fluid lines can be independently controlled to apply fluid through the fluid line 939 and to valve regulate and / or meter the fluid within the microfluidic path device. For example, in Figure 10EIn this configuration, fluid 1044 is driven through fluid line 1033, through first plate 903, through an opening in elastic layer 907, and into a channel through second plate 905 until it reaches first chamber 915, configured as valve 919. First chamber 915 includes an upper circular portion (pressure portion) formed in the first plate and a lower circular portion (fluid portion) formed in the second plate, bifurcated by a portion of elastic layer 907. The upper and lower portions of any chamber can be circular, as described herein, or cylindrical or straight-walled. In this example, a negative pressure is applied via a pressure line (not shown) to open the valve. First chamber 915 is in fluid communication with second chamber 916, configured as metering chamber. Figure 10E In this configuration, metering chamber 916 is opened by applying negative pressure to the upper pressure receiving portion of the bifurcation chamber. Therefore, the chamber is opened to its maximum extent, and its volume is known (e.g., 50 nL, 100 nL, 150 nL, 200 nL, 250 nL, 300 nL, 500 nL, etc.). The adjacent chamber 918 can also be configured as a valve similar to 915 and can remain closed to allow the metering chamber to be completely filled. In some variations, an elastic layer may be partially permeable to air, thereby removing air bubbles and allowing fluid 1044 to be completely filled.

[0176] As described above, in some variations, the microfluidic path device includes one or more defoaming chambers, and / or any chamber on the fluid contact side of the chamber can be configured as a defoaming chamber, wherein air bubbles in the fluid containing the fluid side can be removed. The defoaming chamber may be referred to as a vacuum cap and is typically configured to apply a negative pressure on the opposite side of the membrane while the fluid is held on the fluid contact side of the chamber. As described above, the membrane may be at least partially permeable. All or more preferably a portion 988 of the membrane separating the chambers (e.g., cap region only) can be in contact with a vacuum via a vacuum line 987, for example, the vacuum line being in the upper surface or upper plate of the device, such as... Figure 9CAs shown. In operation, the vacuum cap 938 removes or reduces air bubbles in the pipeline by holding fluid on the fluid contact side of the chamber and applying negative pressure on the upper (pressure receiving) side of the chamber. The membrane dividing the chamber into the fluid contact side and the pressure receiving side can be gas-permeable, such that negative pressure can remove gas from the liquid (fluid) side by drawing gas (e.g., air, nitrogen, etc.) through the membrane covering the fluid path. For example, the membrane (or membrane region in the vacuum cap) can be, for example, a polydimethylsiloxane (PDMS) elastic membrane with sufficient gas permeability to allow gas removal from the liquid side of the membrane. As described herein, a fluid chamber having a fixed volume (e.g., formed between a first plate and a second plate) may include or be coupled to one or more defoaming chambers (vacuum caps) and / or may be configured as a defoaming chamber. In some variations, the elastic layer portion disposed between the first surface and the second surface to form a chamber divided, for example, into a fluid contact side in the second surface (and / or the second plate) and a pressure receiving side in the first surface (and / or the first plate) may experience only minimal (or no) deviation. For example, the upper pressure receiving side may be minimally spaced and / or nearly flush with the relaxed membrane (e.g., flat), while the fluid contact side is recessed and extends into the second surface (second plate). The controller can retain fluid within the vacuum cap region, for example by blocking valves on either side (inlet and outlet) of the vacuum cap, such as by applying positive pressure to the pressure receiving side of the valve, and can apply negative pressure to the pressure receiving side of the vacuum cap. The absolute amount of the applied negative pressure (e.g., the magnitude of the negative pressure) can be less than the negative pressure applied to deflect the membrane (e.g., less than the absolute value of the positive pressure used to close the valve and / or pump). Alternatively, in some variations, the membrane can be configured to deflect against the first surface and / or plate (e.g., upward deflection), for example, to draw fluid into the enlarged fluid contact side of the chamber. The membrane can be held in place by applying negative pressure to the first upper surface, thereby allowing the removal of air bubbles (e.g., air bubbles). The controller can maintain fluid within the vacuum chamber for a sufficient time to remove all or part of the gas (e.g., 1 second or more, 5 seconds or more, 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 1.5 minutes or more, 2 minutes or more, 5 minutes or more, between 1 second and 5 minutes, between 2 seconds and 5 minutes, between 5 seconds and 5 minutes, etc.). Figure 9C Pressure can be applied via pressure line 987, which communicates with the pressure receiving side of a chamber formed between the first and second surfaces (e.g., first and second plates) of the device. Vacuum cap 938 can be fitted with one or more valves 992. Fluid can exit from the fluid contact side via fluid line 989 located on the opposite side of the vacuum cap.

[0177] The fluid contact side of the chamber of the pressure cap (as with the valves and reactors described herein) may be in fluid communication with a fluid port, which is in fluid communication with the fluid contact side of each chamber via one or more fluid channels that may be located in the second surface and / or plate. The pressure receiving side of the vacuum cap may be in fluid communication with a pressure port that extends through the first surface / plate (e.g., and into the surface / plate) to be in fluid communication with the pressure receiving port or side via a pressure channel that extends through the second plate and along the first plate, as described herein.

[0178] exist Figure 10F In this configuration, the first chamber (valve 915) can be closed by applying positive pressure to the upper pressure receiving portion of the first chamber. This limits the amount of fluid within the metering chamber 916 to a precise quantity; the metered fluid can then be injected into another chamber (e.g., a fourth chamber, configured as a mixing chamber, etc.), such as... Figure 10G-10H As shown, by opening the valve and holding / mixing chamber (Figure 10G) and applying positive pressure, fluid is driven from metering chamber 916 into holding / mixing chamber 920. Figure 10H Then, valve 918 can be closed, as follows: Figure 10I As shown. The fluid in the mixing chamber can be combined, mixed, or otherwise processed in the mixing chamber with additional fluids (e.g., from other parts of the microfluidic path device, or another metering of the same fluid path shown).

[0179] Any microfluidic pathway device described herein can be a microfluidic pathway plate device, as described above, wherein the device is substantially thin. Therefore, in-plate / on-plate processing can be performed substantially in two dimensions (2D), including the purification of any polynucleotide (e.g., mRNA). Purifying polynucleotides in 2D is particularly advantageous compared to prior art, which may require the use of columns and may involve steps that are difficult or impossible to perform in a closed pathway environment and / or the small volumes described herein.

[0180] Additionally, as shown in the figure (for example, Figure 10A-10I As shown, the fluid contact side (and / or pressure receiving side) of each chamber can be configured such that when the positive pressure of the pressure receiving side drives the elastic layer against the fluid contact side, the elastic layer is flush with and without gaps to the fluid contact side in the second surface. In some variations, the fluid contact side and / or pressure receiving side can be concave. The concave surface may have a slightly shallow elliptical cross-section to allow the elastic layer to easily flush against the wall of the fluid contact side (and / or pressure receiving side). The elastic layer may push (e.g., sit) against the wall of the chamber such that there is no dead retention portion in the chamber (e.g., the fluid contact side).

[0181] In addition to valves that open and / or close the passage, the first resilient layer can also be used to pump fluid into / out of the chamber, as described above. For example, in some variations, a chamber (e.g., accessible via a fluid passage opened by valves on one or both sides) can be provided, allowing fluid to be filled from a fluid port. Negative pressure can be applied from a pressure port connected to the upper half of the bisected chamber (bisected by the resilient layer). Applying negative pressure can aid the infusion device by drawing fluid into the passage and removing air through the resilient layer. Thus, in any variation described herein, the resilient layer can be gas-permeable. Once infusion is complete, fluid can be expelled from the chamber by opening a distal valve and applying positive pressure to the opposite side of the resilient layer.

[0182] Any device described herein can be used as described and illustrated. For example, as described above, the methods and devices described herein may be particularly helpful in generating mRNA therapies using in vitro transcription (IVT). For example, the methods and devices can synthesize therapeutic agents containing one or more mRNAs in a single, uninterrupted fluid pathway that provides an RNase-free environment. These mRNAs can be tailored for individual patients.

[0183] Example

[0184] Any of the devices described herein can be used, for example, to manufacture therapeutic agents, particularly mRNA therapeutic agents. For instance, the systems described herein may include integrated hardware-software systems in which each batch of therapeutic material (including drugs and pharmaceuticals) can be produced within a dedicated, single-use, disposable microfluidic pathway device (which may be referred to as a chip or biochip). Therapeutic production can be carried out in a sterile, closed-path system, and all production steps can be automated to achieve a precisely replicated process. This can provide rapid turnaround for “personalized” production batches while providing the required high level of reproducibility, control, and quality for the release of therapeutic materials for clinical use.

[0185] Any device described herein can be used with one or more microfluidic pathway devices; in some variations, different microfluidic pathway devices can be used sequentially or in parallel by the same device to perform different parts of the procedure. For example, in one variation for producing therapeutic mRNA, a first microfluidic pathway device can be used as part of a template microfluidic pathway device (“template biochip”) for DNA template production. The resulting template can be transferred by the system in a closed pathway to a second microfluidic pathway device (e.g., from the first microfluidic pathway device to a reservoir in the system and / or directly to the second microfluidic pathway device). In some variations, the second microfluidic pathway device can be configured to perform in vitro transcription of mRNA and purification of the material to produce a drug (e.g., on an “IVT biochip” or an IVT microfluidic pathway device). The product from the second microfluidic pathway device can then be transferred (directly or via, for example, an intermediate reservoir on a reagent storage frame) to a third microfluidic pathway device, such as a formulation microfluidic pathway device (e.g., a “formulation biochip”). Drug product formulation can then be performed on the formulation microfluidic pathway device.

[0186] Each microfluidic pathway device may include input ports (fluid ports, pressure ports, etc.) and chambers (e.g., metering valves, reaction chambers, and purification structures) that can perform each step of the manufacturing process in a continuous and closed-loop manner.

[0187] As described above, microfluidic pathway devices can be placed within a device (e.g., a system) that may include any of the elements described above. For example, returning to... Figure 2C The equipment (e.g., the system) may include a microfluidic pathway device 250, a microfluidic pathway device management system 260 or device, control panels (e.g., for user input, monitoring, and analysis), and a temperature-controlled (e.g., refrigeration) environment, such as a cabinet 270, in some variable conditions. The system can support all production activities within the microfluidic pathway device, such as reagent supply, fluid control, temperature control, mixing, purification, and process monitoring. Manufacturing activities on the system can be accessed and controlled via application software.

[0188] The microfluidic pathway devices described herein and the equipment (e.g., systems) for operating them can be used as reactors for manufacturing steps performed on three different types of microfluidic pathway devices, as described above. For example, template microfluidic pathway devices, IVT microfluidic pathway devices, and formulation microfluidic pathway devices can be configured to include features that perform a set of unit operations in a controlled and highly reproducible manner. As described above, microfluidic pathway devices are typically multilayer structures.

[0189] For example, a microfluidic pathway device can be composed of a cyclic olefin copolymer (COC) and a siloxane. The COC layer can be made of TOPAS5013L-10, and the silicon layer can be made of Wacker Silpuran medical-grade silicon. Features of each layer can be produced by machining (prototyping stage) or injection molding (production stage). Fabrication of the microfluidic pathway device may include: sputtering with 100% isopropanol and silica, oxygen plasma activation, vacuum-bonded cleaning layers, marking the microfluidic pathway device (e.g., barcodes and / or RFID tags), sterilization of the assembled microfluidic pathway device (e.g., by UV-C or gamma rays), and storage of the microfluidic pathway device in a sterilized wafer mask processing cassette. Although oxygen plasma exposure can sterilize individual layers before assembly, subsequent sterilization can add additional sterility assurance. Different types of microfluidic pathway devices can have the different designs shown, for example, in Figures 12A-12C The template is shown in the image. Figure 12A ), IVT Figure 12B ) and preparation ( Figure 12C This is a schematic diagram of a microfluidic pathway device. All these exemplary microfluidic pathway devices can share a similar basic architecture and a number of functional elements that can be used in different configurations to perform different protocols. Functional elements may include, as described above, input ports, metering valves, pumps, reaction chambers, mixing structures, and purification structures.

[0190] Figure 12D-12E It shows Figures 12A-12C Alternative embodiments of the microfluidic path device shown. Figure 12D Another example diagram of a template microfluidic pathway device is shown, configured to form a template in a closed-path system as described herein; specifically... Figure 12D The microfluidic pathway device shown can be used to form synthetic (e.g., non-bacterial) templates. Figure 12E This is another example of a microfluidic pathway device for in vitro transcription (IVT) of mRNA, similar to that shown in Figure 12B. Figure 12F It is similar to Figure 12C Another example of a microfluidic pathway device is shown, which can be used to encapsulate polynucleotides (e.g., mRNA) in a delivery vector, as described herein. Any of these microfluidic pathway devices can be sterilized, for example using gamma radiation, and can be aseptically packaged. The microfluidic pathway devices can be configured to process batches of predetermined sizes (e.g., about 5 mg mRNA / 2–4 days, about 50 mg mRNA / 2–4 days, about 100 mg mRNA / 2–4 days, etc.; in some variations, 2 g / week).

[0191] The microfluidic pathway device interfaces with a control system via a set of spring-loaded connections for reagents and pneumatic lines for managing fluid movement and valve control. The reagent and gas lines can be sealed by applying pressure to the elastic layer (elastic layer) of the microfluidic pathway device, thus forming a completely sealed pathway from the reagent vial to the biochip and from the biochip to the outlet vial. Figure 13 An example of a sealed path is shown, which can be maintained by all reactions within the microfluidic path device, thereby effectively preventing any contact with the atmosphere and minimizing the risk of contamination. Figure 13 A cross-sectional view of an example microfluidic path device installed in a control system is shown, wherein the microfluidic path device 1301 is located in a mount 1305, and a fluid interface assembly 1307 is connected to fluid and pressure lines from a reagent storage frame 1309. A closed fluid path 1315 begins from a reagent storage container on the reagent storage frame 1309 and enters the microfluidic path device 1301 for processing (via the fluid lines held abutting against the microfluidic path device 1301 by the fluid interface assembly). The product is then output from the microfluidic path device back to the storage container 1313 in the reagent storage frame.

[0192] A microfluidic pathway device control system (e.g., controller hardware / software, mount, fluid interface assembly, reagent storage frame, sensors, etc.) can provide the backbone for all electronic and hardware components. The microfluidic pathway device control system can be sterile and maintain a controlled environment. The system can also provide interfaces for loading reagents and retrieving outputs, and can maintain the microfluidic pathway device while providing single-step connectivity to all actuators.

[0193] As described above, the microfluidic pathway device control system can also monitor and control the operation of the device via one or more sensors. For example, the microfluidic pathway device control system can scan all reagent and microfluidic pathway device barcodes and monitor fluid levels. The microfluidic pathway device control system can also automate all microfluidic pathway device functions. As mentioned above, these microfluidic pathway device control systems can also generate visual records of all process steps and / or provide optical quality control (QC) analysis that can provide intermediate process outputs.

[0194] The microfluidic path device control system (also referred to herein as a management system) may include the aforementioned components, such as a holder (“nest” or “support”), which may be configured to ensure proper alignment of the microfluidic path device during use, for example, to allow insertion in only a single direction. For example, pins (e.g., two interlocking pins) and / or notches in the nest may match the shape of the microfluidic path device. The microfluidic path device management system may also include bottle racks for holding reagents and outlet bottles, a downward-looking camera for recording all liquid and valve movements, and a product outlet. Side cameras on the track can capture barcodes and detect fluid levels, and can control robotic arms (e.g., robotic arms with magnets) to perform bead manipulation. The microfluidic path device may be held in place by a vacuum chuck that ensures good contact with a temperature control device (e.g., a Peltier device) for temperature management. Once the microfluidic path device is in place, in some variations, the top of the microfluidic path device management system is lowered via an interlocking pin guiding system, allowing engagement with all connectors in a single step.

[0195] The control panel can be configured as the main interface for all electronic equipment (e.g., CPU, Ethernet RIO device controllers) as well as pneumatically controlled valves and manifolds and pressure regulators. In some variations, the microfluidic path device control system can be housed in a refrigerated container or cabinet (e.g., an ISO Class 5 safety cabinet) that provides a microbiologically safe, enclosed space through HEPA air filtration and airflow management, ensuring all reagents are maintained at the correct temperature throughout the manufacturing process. The cabinet may also be equipped with UV lamps for sterilizing the microfluidic path device and all internal microfluidic path device management system components. In some variations, the microfluidic path device control system can be located in a microenvironment (e.g., a 6ft x 6ft ISO Class 5 microenvironment), which itself can be located in a clean room (e.g., an ISO Class 7 room). Interactions between operators and the system (including loading reagent vials and biochips) are performed aseptically. All reagents and consumables are double-bagled into the area and can be wiped clean and opened in an aseptic environment to control the risk of contamination.

[0196] The microfluidic pathway device operating system described herein can be automatically executed by a controller. The controller can load process protocols defining the microfluidic pathway device type and the reagents to be used, and can ensure the correct microfluidic pathway device type is used. The controller can also capture reagent and microfluidic pathway device identifiers (e.g., barcodes) and ensure that reagents are released for use, are not expired, and are loaded in the correct location. The controller can also execute a series of steps defined in the protocol to automate valves, pumps and stirrer actuators, temperature controllers, cameras, magnetic arms, and other necessary controllers. The controller can also create batch logs of events and process parameters and can record measurements from peripheral devices as well as online measurements involving light sources and detection systems. In some variants, this log can be stored in the cloud as a complete digital record.

[0197] In use, the operator can select a protocol from, for example, a pre-set protocol library, or the user can enter a new protocol (or modify an existing one). Based on the protocol, the controller informs the operator which type of microfluidic pathway device to use, what the bottle contents should be, and where to place the bottle in the nest. The operator can load the microfluidic pathway device, the required reagents, and the outlet bottle into the system. The application confirms the presence of the required peripherals, identifies the microfluidic pathway device, and scans the identifiers (e.g., barcodes) of each reagent and product bottle to ensure the bottles match the reagent list for the selected protocol. After confirming the starting materials and required equipment, the controller executes the protocol. During execution, valves and pumps are actuated to deliver reagents, mix reagents, control temperature, initiate reactions, take measurements, and pump the product to the destination bottle. At the end of the protocol, a production batch record is created in the cloud. The batch record is encrypted, and system measurement results are uploaded to the cloud. Figure 14 The example data flow diagram shown illustrates some of the controller's functions.

[0198] As used herein, the term "processing polynucleotides" can include a variety of operations, including but not limited to synthesizing polynucleotides, purifying polynucleotides, concentrating solutions containing polynucleotides, formulating polynucleotides, and any combination thereof. As used herein, when referring to a surface, the term "basic level" means that the surface is within a level range of + / - X degrees relative to the ground (e.g., X can be 0.1 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 5 degrees, 10 degrees, etc.).

[0199] Any microfluidic path device described herein may include a heat sink on or a portion thereof to balance (even out) the heat within that portion of the device. For example, Figures 15A-15B Another example of the microfluidic path device 1501 is shown (here shown as similar to the one described above). Figure 12B and12E The illustrated IVT microfluidic path device has a large heatsink 1507 on the bottom side. Figure 15B The microfluidic pathway device includes multiple reactors 1503 and 1505 (shown as fluid contact sides of chambers formed between two surfaces of the microfluidic pathway device as described above). The top of the device (shown in FIG. 15A) includes peripherally arranged fluid ports 1509 and pressure ports 1511. The microfluidic pathway device shows pressure port 1511, actuation valve 1512, metering chamber 1513, and multiple fluid power loops between reactors 1505 and 1503. All these chambers, valves, and reactors can be formed as part of a fixed-volume chamber formed between a first and a second surface, wherein an elastic layer divides each chamber into a fluid contact side (reactor, metering chamber, etc.) in the second surface and a pressure receiving side (forming part of the fluid power loop) in the first surface.

[0200] exist Figure 15B In the bottom view of the microfluidic path device 1501 shown, the device includes a heat sink (e.g., copper or other highly thermally conductive material attached to the bottom of the microfluidic path device). The highly thermally conductive material can be, for example, copper, aluminum, silver, or similar materials with high thermal conductivity, such as pyrolytic graphite. The heat sink 1507 can be mechanically attached to the microfluidic path device using fasteners and / or adhered to the appropriate location using adhesives. In some variations, the microfluidic path device heat sink may include or be formed from thermally conductive adhesives. The microfluidic path device may be thinner below the heat sink to improve heat transfer to the material within the microfluidic path device. In some variations, the heat sink may also increase the stiffness of the microfluidic path device.

[0201] although Figure 15B A single heat sink 1507 is shown located at the bottom of the microfluidic path device, but multiple heat sinks can be used. For example, multiple heat sinks can be used to create different temperature zones. The microfluidic path device may include a plastic material as part of the body (e.g., a plate); plastic is generally a poor thermal conductor, so it can maintain lateral temperature differences between different areas of the microfluidic path device. In some variations, the microfluidic path device may be located only below the reactor region.

[0202] In some variations, the heat transfer area is attached to a flat bottom and / or can be placed in a pouch for the component.

[0203] The devices described herein may include and / or may be used with one or more isolation chambers. For example, in some variations, the devices described herein may be part of a therapeutic polynucleotide production "factory" that can produce therapeutic polynucleotides, for example, for delivery to a subject. Therapeutic polynucleotides may be, for example, therapeutic mRNA. Figures 16A-16B An example of equipment is shown that can be used independently as factory equipment or as part of a parallel manufacturing unit. Figure 16A In this configuration, devices 1601 and 1601' may include or be housed within a Level 5 isolation cabinet 1603; the cabinet itself may be placed within a Level 7 isolation space. In Figure 16A, the cabinet includes two microfluidic control devices 1601 and 1601'. These devices may be part of an assembly plant providing precisely replicated GMP units capable of automated manufacturing of therapeutic polynucleotides, such as therapeutic mRNA for rapid patient delivery. These devices are highly reconfigurable, allowing for rapid deployment and low-cost production. In some variations, they may be manufacturing "factory" units deployed on demand. In some variations, these devices may be part of a mobile unit that can be deployed temporarily or for longer periods to remote locations.

[0204] When a feature or element is referred to herein as being “on” another feature or element, it may be directly on the other feature or element, or there may be intermediate features and / or elements present. Conversely, when a feature or element is referred to as being “directly located” on another feature or element, there are no intermediate features or elements. It should also be understood that when a feature or element is referred to as being “connected,” “attached,” or “joined” to another feature or element, it may be directly connected, attached, or joined to the other feature or element, or there may be intermediate features or elements present. Conversely, when a feature or element is referred to as being “directly connected,” “directly attached,” or “directly joined” to another feature or element, there are no intermediate features or elements. Although described or illustrated with respect to one embodiment, the features and elements thus described or illustrated may be applicable to other embodiments. Those skilled in the art will also understand that when referring to a structure or feature “adjacent” to another feature, it may have overlapping portions or be located below the adjacent feature.

[0205] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. For example, the singular forms “a,” “an,” and “the” used herein also include the plural forms unless the context clearly indicates otherwise. It should also be understood that the terms “comprising” and / or “including” as used in this specification specify the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items and may be abbreviated to “ / ”.

[0206] For ease of description, spatial relative terms such as “below,” “under,” “down,” “above,” “up,” etc., may be used herein to describe the relationship of one element or feature to another, as illustrated in the figure. It should be understood that spatial relative terms are intended to cover orientations in which the device differs from those shown in the figure during use or operation. For example, if the device in the figure were inverted, an element described as “below” or “below other elements or features” would be oriented “above other elements or features.” Thus, the exemplary term “below” can include both above and below orientations. The device may be oriented in other ways (rotated 90 degrees or otherwise), and the spatial relative descriptors used herein are interpreted accordingly. Similarly, unless explicitly stated otherwise, the terms “up,” “down,” “vertical,” “horizontal,” etc., are used herein for illustrative purposes only.

[0207] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited to these terms unless the context otherwise requires. These terms may be used to distinguish one feature / element from another. Thus, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element, without departing from the teachings of the invention.

[0208] In this specification and the following claims, unless the context otherwise requires, the word "comprising" and variations such as "comprising" and "including" refer to various components that can be used together in a method and article (e.g., compositions and devices that include both apparatus and method). For example, the term "comprising" will be understood to include any of the stated elements or steps, but does not exclude any other elements or steps.

[0209] In general, any device and method described herein should be understood as inclusive, but all components and / or steps or subsets thereof may alternatively be exclusive and may be described as “composed of” or “mainly composed of” various components, steps, subcomponents or substeps.

[0210] As used in this specification and claims, including in the embodiments, unless otherwise expressly stated, all numbers are to be understood to begin with "about" or "approximately," even if the term is not explicitly stated. When describing magnitude and / or location, the phrases "approximately" or "approximately" may be used to indicate that the described value and / or location is within a reasonable expected range of values ​​and / or locations. For example, a numerical value may be + / - 0.1% of a given value (or range), + / - 1% of a given value (or range), + / - 2% of a given value (or range), + / - 5% of a given value (or range), + / - 10% of a given value (or range), etc. Unless the context otherwise requires, any numerical value given in this agreement should also be understood to include approximately or close to that value. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical ranges described herein are intended to include all subranges contained therein. It should also be understood that when a value is disclosed, the terms "less than or equal to" that value, "greater than or equal to" that value, and the possible range between that value are also disclosed, as would be appropriately understood by someone skilled in the art. For example, if the value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" are also disclosed (e.g., where X is a numerical value). It should also be understood that throughout the application, data is provided in a variety of different formats, and that the data represents a range of endpoints and start points, as well as any combination of data points. For example, if specific data point "10" and specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to 10 and 15, and values ​​between 10 and 15 are considered disclosed. It should also be understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0211] Although various exemplary embodiments have been described above, numerous changes can be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, in alternative embodiments, the order in which the various method steps are performed may generally be changed, and in other alternative embodiments, one or more method steps may be omitted entirely. Optional features of the various apparatus and system embodiments may be included in some embodiments but 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 set forth in the claims.

[0212] The examples and illustrations included herein show specific embodiments in which the subject matter can be practiced by way of illustration and not limitation. As noted above, other embodiments can be utilized and derived therefrom, allowing for structural and logical substitutions and changes without departing from the scope of this disclosure. Such embodiments of the subject matter of the invention may be referred to herein individually or collectively as the term "invention," and are intended only for convenience and not to voluntarily limit the scope of this application to any single invention or inventive concept (if more than one invention or inventive concept is disclosed). Thus, although specific embodiments have been shown and described herein, any arrangement intended to achieve the same purpose may replace the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of the various embodiments. Combinations of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art upon reading the foregoing description.

Claims

1. A microfluidic device for forming therapeutic polynucleotides, the device comprising: A holder for removably holding a microfluidic path plate device; Multiple pressure lines; Multiple fluid bottles, wherein each fluid bottle either includes a fluid line or is configured to be connected to a fluid line. Each fluid line and at least a portion of the pressure lines are configured to be biased against a microfluidic path plate device held in the holder to form a closed fluid path. An optical sensor driving device configured to move one or more of a plurality of optical sensors around the mount or around the plurality of fluid bottles; and A controller, configured to control the application of pressure via a pressure line to drive fluid movement within the microfluidic pathplate device when the microfluidic pathplate device is held in a holder, wherein the controller is configured to, within one or more microfluidic pathplate devices held in a holder, guide the synthesis of a synthetic template, use the template to guide an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide, and guide the purification of the therapeutic polynucleotide.

2. A microfluidic device for forming therapeutic polynucleotides, the device comprising: A holder for removably holding a microfluidic path plate device; Multiple pressure lines; Multiple fluid bottles, wherein each fluid bottle either includes a fluid line or is configured to be connected to a fluid line. Each fluid line and at least a portion of the pressure lines are configured to be biased against a microfluidic path plate device held in the holder to form a closed fluid path. An optical sensor driving device configured to move one or more of a plurality of optical sensors around the mount or around the plurality of fluid bottles; and A controller, configured to control the application of pressure via a pressure line to drive fluid movement within the microfluidic pathplate device when the microfluidic pathplate device is held in a holder, wherein the controller is configured to: determine the contents of a fluid bottle within one or more microfluidic pathplate devices held in a holder; transfer submicroliter amounts of material from the fluid bottle to one or more reactors within the microfluidic pathplate device held in a holder; guide the synthesis of a synthetic template; use the template to guide an in vitro transcription (IVT) reaction to form a therapeutic polynucleotide; and guide the purification of the therapeutic polynucleotide.

3. The apparatus according to claim 1 or 2, further comprising a reagent storage frame for holding a plurality of fluid bottles.

4. The device according to claim 1 or 2, further comprising a plurality of optical sensors arranged to monitor fluid levels in the plurality of fluid bottles and to monitor fluid movement in the microfluidic pathplate device when the microfluidic pathplate device is held in the holder.

5. The device according to claim 1 or 2, further comprising a seat release control device configured to release the seat from the device.

6. The device according to claim 1 or 2, further comprising a thermal control device configured to regulate the temperature of at least one region of the microfluidic path plate device when the microfluidic path plate device is held in the holder.

7. The device of claim 3, further comprising a magnetic field applicator configured to apply a magnetic field to at least one region of the microfluidic pathplate device when the microfluidic pathplate device is located in the pedestal.

8. The device according to claim 7, wherein, The magnetic field applicator includes a control arm mounted to the reagent storage frame.

9. The device according to claim 1 or 2, wherein, The controller is configured to detect the identification code on the fluid bottle.

10. The device according to claim 1 or 2, wherein, The controller is configured to determine the level of reagent held by one or more of the plurality of fluid bottles.

11. The device of claim 1 or 2, further comprising a fluid interface assembly having a central opening through which one or more of a plurality of optical sensors are capable of imaging the microfluidic path plate device, wherein the sealed end of each of the fluid lines and pressure lines is arranged around the periphery of the central opening.

12. The device of claim 1 or 2, further comprising a signal detector configured to detect a signal from within the microfluidic path plate device, wherein the signal is one or more of a visible signal, a fluorescence signal, an ultraviolet absorption signal, or an IR absorption signal.

13. The device according to claim 12, wherein, The signal detector is configured to measure the nanoparticle size distribution via dynamic light scattering (DLS).

14. A microfluidic device for forming therapeutic polynucleotides, the device comprising: A holder for removably holding a microfluidic path device; Multiple pressure lines; Multiple fluid bottles, each pressurized by one or more pressure lines from the multiple pressure lines, wherein each fluid bottle either includes a fluid line or is configured to be connected to a fluid line. Each fluid line and at least a portion of the pressure lines are configured to be independently biased against the microfluidic path device located in the mount to form a sealed, closed fluid path. An optical sensor driving device configured to move one or more of a plurality of optical sensors around the mount or around the plurality of fluid bottles; and The controller is configured to control the application of pressure through pressure lines to drive fluid movement in the microfluidic path device when the device is in a pedestal, and to apply pressure to one or more pressure lines during operation to open or close valves in the microfluidic path device.

15. A microfluidic device for forming therapeutic polynucleotides, the device comprising: A mount for microfluidic pathway devices; Multiple pressure lines; Includes fluid interface assemblies for multiple fluid lines; Multiple fluid bottles are configured to be pressurized; A reagent storage frame includes multiple supports, each support configured to hold one of a plurality of fluid bottles, wherein each fluid bottle either includes one of the plurality of fluid lines or is configured to be connected to one of the plurality of fluid lines, wherein each fluid line and at least some pressure lines are configured to individually bias against a microfluidic pathway device located in the frame with bias pressure. An optical sensor drive device configured to move one or more of a plurality of optical sensors around the mount or around the plurality of fluid bottles; and The controller is configured to control the application of pressure through the pressure line to drive fluid movement in the microfluidic path device when the microfluidic path device is in the pedestal.

16. A microfluidic device for forming therapeutic polynucleotides, the device comprising: A holder for removably holding a microfluidic path device; Multiple pressure lines, at least a portion of which are configured to independently bias against a pressure input on a microfluidic path device located in a mount; Multiple fluid bottles are configured to be pressurized, wherein each fluid bottle either includes a fluid output end configured to seal an input end on a microfluidic path device, or is configured to be connected to a fluid line configured to be independently biased against the microfluidic path device to form a sealed closed fluid path. The first optical sensor is configured to monitor the fluid inside the fluid bottle; A second optical sensor is configured to monitor fluid within a microfluidic path device located in the pedestal; An optical sensor driving device configured to move the first optical sensor and / or the second optical sensor around the mount or around the plurality of fluid bottles; The controller is configured to receive inputs from the first optical sensor and the second optical sensor, and to control the application of pressure through the pressure lines to apply pressure from the plurality of pressure lines to open and / or close valves, and to drive fluid movement in the microfluidic path device based at least in part on the received inputs.

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