Microfluidic devices and methods of use thereof

Abstract

Microfluidic devices for manufacturing therapeutics, including polynucleotide therapeutics, particularly mRNA therapeutics, are provided. An apparatus for processing polynucleotides in a sealed pathway environment is provided that includes an optical sensor to monitor operation and verify material usage for good manufacturing practice. The apparatus may be used for the manufacture and formulation of biomolecule-containing products, including therapeutic mRNA, particularly therapeutics for personalized care.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 62 / 885,159, entitled "MICROFLUIDIC APPARATUS AND METHODS OF USE THEREOF," filed August 9, 2019; U.S. Provisional Patent Application No. 62 / 885,170, entitled "METHODS AND APPARATUSES FOR MANUFACTURING THERAPEUTIC COMPOSITIONS," filed August 9, 2019; and U.S. Provisional Patent Application No. 62 / 914,374, entitled "METHODS AND APPARATUSES FOR MANUFACTURING FOR REMOVING MATERIAL FROM A THERAPEUTIC COMPOSITION," filed October 11, 2019, each of which is incorporated herein by reference in its entirety.

[0002] INCORPORATION BY REFERENCE All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0003] FIELD The devices and methods described herein may be used for the manufacture and formulation of biomolecule-containing products (including therapeutic mRNA), particularly therapeutics for personalized care. In particular, this document describes closed-circuit methods and devices for processing therapeutic polynucleotides, including at the point of care. [Background technology]

[0004] Currently available technologies for manufacturing and compounding polynucleotide therapeutics, particularly mRNA therapeutics, often expose the product to contamination and degradation. Currently available centralized production can be too costly, time-consuming, and susceptible to contamination for use in compounding therapeutics that may contain large numbers of polynucleotide species. The development of scalable polynucleotide manufacturing, producing single-patient doses, limiting contamination by eliminating touchpoints, input and process tracking to meet clinical manufacturing requirements, and use in point-of-care operations can expedite the use of these promising therapeutics. The use of microfluidic devices and processes can provide significant advantages toward these goals. Summary of the Invention [Problem to be solved by the invention]

[0005] Described herein are microfluidic devices for manufacturing therapeutics, including, in particular, mRNA therapeutics. For example, described herein are systems that can form therapeutic substances (including both drug substances and pharmaceutical agents) within dedicated, disposable, easily disposable microfluidic pathway devices (e.g., microfluidic pathway plate devices, chips, biochips, plates, etc.). Described herein are microfluidic pathway devices and control systems for operating the microfluidic pathway devices. [Means for solving the problem]

[0006] For example, described herein are control systems ("devices") for operating microfluidic pathway devices. These devices may be referred to herein as microfluidic devices, microfluidic control devices, microfluidic control systems, or microfluidic systems.

[0007] The microfluidic device may include a seat mount (e.g., a seat) for a microfluidic pathway device; a plurality of fluid vials, each of which either includes a fluid line or is configured to interface with a fluid line to form a closed fluid pathway; and a controller configured to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted within the seat mount. In any of these microfluidic devices, each fluid line may be configured to seal against the microfluidic pathway device mounted within the seat mount to form a closed fluid pathway. The microfluidic devices described herein may advantageously include any of the features described herein (or any combination thereof).

[0008] For example, a microfluidic apparatus may include a seat mount for a microfluidic pathway device; a reagent storage frame including a plurality of holders each configured to hold a fluid vial, each fluid vial either including a fluid line or configured to interface with a fluid line, and each fluid line configured to be biased against a microfluidic pathway device mounted in the seat mount by a biasing force; and a controller configured to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0009] The microfluidic apparatus may include a seat mount for the microfluidic pathway device, a fluid interface assembly including a plurality of fluid lines, each fluid line configured to be separately biased by a biasing force to the microfluidic pathway device mounted in the seat mount, a reagent storage frame including a plurality of fluid sample holders, each configured to hold a fluid vial and each configured to connect to the fluid interface assembly through one of the fluid lines of the fluid interface assembly, and a controller configured to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0010] The microfluidic device may include a reagent storage frame comprising a seat mount for the microfluidic pathway device, a plurality of pressure lines, and a plurality of holders each configured to hold a fluid vial, each fluid vial either comprising a fluid line or configured to interface with a fluid line, and further wherein each fluid line and each pressure line is configured to be biased with a bias force relative to the microfluidic pathway device mounted in the seat mount; and a controller configured to control application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0011] The microfluidic apparatus may include a seat mount for the microfluidic pathway device; a fluid interface assembly including a plurality of fluid lines and pressure lines, each fluid line and each pressure line configured to be biased by a bias force against the microfluidic pathway device mounted in the seat mount; a reagent storage frame including a plurality of fluid sample holders, each configured to hold a fluid vial and each configured to connect to the fluid interface assembly through 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 within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0012] The microfluidic apparatus may include a seat mount for a microfluidic pathway device; a plurality of pressure lines; a plurality of fluid vials, each fluid vial either comprising a fluid line or configured to couple with a fluid line, and further configured such that each fluid line and each pressure line is sealed to the microfluidic pathway device mounted in the seat mount to form a closed fluid pathway; and a controller configured to control application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0013] The microfluidic device may include a seat mount for a microfluidic pathway device; a plurality of fluid vials, each fluid vial either comprising a fluid line or configured to couple with a fluid line, and each fluid line configured to be sealed to the microfluidic pathway device mounted in the seat mount to form a closed fluid pathway; at least one optical sensor configured to monitor fluid within the microfluidic pathway device mounted in the seat mount; and a controller configured to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount, and to record and / or transmit optical data indicative of fluid within the microfluidic pathway during operation of the device.

[0014] The microfluidic apparatus may include a seat mount for the microfluidic pathway device; a fluid interface assembly including a plurality of fluid lines and pressure lines, each fluid line and each pressure line configured to be biased (e.g., spring-loaded) against the microfluidic pathway device mounted in the seat mount; a reagent storage frame including a plurality of fluid sample holders, each configured to hold a fluid vial and each configured to connect to the fluid interface assembly through 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 within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0015] In some variations, a microfluidic device configured to process therapeutic polynucleotides at a therapeutic site and to operate as a closed circuit may include a seat mount for a microfluidic pathway device; a fluid interface assembly including a plurality of fluid lines and pressure lines, each fluid line and each pressure line independently configured to be actuated relative to and sealingly connected to a microfluidic pathway device mounted in the seat mount; a reagent storage frame including a plurality of fluid sample holders, each configured to hold a fluid and each configured to couple to the fluid interface assembly through 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 within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount, the fluid interface assembly including a plurality of spring biases configured to independently bias each fluid line relative to and sealingly connected to the microfluidic pathway device mounted in the seat mount.

[0016] For example, an apparatus (e.g., a microfluidic device for forming a therapeutic polynucleotide) may include a seat mount for removably holding a microfluidic pathway device, a plurality of pressure lines, and a plurality of fluid vials each pressurized by one or more of the plurality of pressure lines, wherein each fluid vial either comprises a fluid line or is configured to interface with a fluid line, and wherein each fluid line and at least a subset of the pressure lines are independently biased relative to the microfluidic pathway device mounted within the seat mount to form a sealed, closed fluid pathway. and a controller configured to control the application of pressure through the pressure lines when the microfluidic pathway device is seated in the seat mount to drive fluid movement within the microfluidic pathway device, and to apply pressure to one or more of the pressure lines to open or close valves within the microfluidic pathway device during operation.

[0017] As described above, generally, the controller may be configured to control the apparatus to perform in vitro transcription (IVT) reactions in the microfluidic pathway device.

[0018] For example, an apparatus (e.g., a microfluidic device for forming a therapeutic polynucleotide, such as a therapeutic mRNA) may include a reagent storage frame including a seat mount for a microfluidic pathway device, a plurality of pressure lines, a fluid interface assembly including a plurality of fluid lines, a plurality of fluid vials configured to be pressurized, and a plurality of holders each configured to hold a fluid vial of the plurality of fluid vials, each fluid vial either comprising a fluid line of the plurality of fluid lines or configured to interface with a fluid line of the plurality of fluid lines, and further wherein each fluid line, and at least some of the pressure lines, are configured to be separately biased with a biasing force relative to a microfluidic pathway device mounted in the seat mount, and a controller configured to control application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

[0019] In some variations, a microfluidic device (e.g., a microfluidic device for forming a therapeutic polynucleotide, such as a therapeutic mRNA) includes a seat mount for removably holding a microfluidic pathway device; a plurality of pressure lines, at least a subset of the pressure lines configured to be independently biased relative to a pressure input at a microfluidic pathway device mounted within the seat mount; a plurality of fluid vials configured to be pressurized, each fluid vial either having a fluid output configured to be sealed relative to an input at the microfluidic pathway device or configured to couple with a fluid line independently biased relative to the microfluidic pathway device and configured to form a sealed closed fluid pathway; a first optical detector configured to monitor fluid in the fluid vials; a second optical detector configured to monitor fluid in the microfluidic pathway device mounted within the seat mount; and an optical detector configured to receive inputs from the first and second optical detectors and to control the application of pressure through the pressure lines. and a controller configured to apply pressure from the plurality of pressure lines and open and / or close valves; and drive fluid movement within the microfluidic pathway device based at least in part on the received input.

[0020] A microfluidic device configured for processing therapeutic polynucleotides at a therapeutic site and for operation as a closed circuit includes a seat mount for a microfluidic pathway device; a fluid interface assembly including a plurality of fluid lines and pressure lines, each fluid line and each pressure line configured to be independently actuated relative to and sealingly connected to a microfluidic pathway device mounted in the seat mount; a reagent storage frame including a plurality of pressurized fluid sample holders, each configured to hold a fluid vial and to couple to the fluid interface assembly through one of the fluid lines of the fluid interface assembly; a plurality of optical sensors disposed around the seat mount and the reagent storage frame for monitoring fluid levels in the fluid vials held by the reagent storage frame and fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount; and a controller in communication with the optical sensors and configured to control application of pressure through the pressure lines and drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount. and a collet configured to independently bias each fluid line to a microfluidic pathway device mounted in the seat mount and sealingly connect the fluid line to the microfluidic pathway device, and each of the seat mount, the fluid interface assembly, and the fluid sample holder is configured to be removable for sterilization.

[0021] In some variations, these microfluidic devices may be microfluidic devices for forming therapeutic polynucleotides (e.g., mRNA therapeutics) that may include a sheet mount for removably holding a microfluidic pathway plate device, a plurality of pressure lines, a plurality of fluid vials, each fluid vial either comprising a fluid line or configured to interface with a fluid line, and each fluid line, and at least a subset of the pressure lines, configured to be biased relative to the microfluidic pathway plate device held in the sheet mount to form a closed fluid pathway, and a controller configured to control the application of pressure through the pressure lines and facilitate fluid movement within the microfluidic pathway plate device when the microfluidic pathway plate device is held in the sheet mount, the controller configured to direct synthesis of synthetic templates, direct in vitro transcription (IVT) reactions using the templates to form therapeutic polynucleotides, and direct purification of the therapeutic polynucleotides within one or more microfluidic pathway plate devices held in the sheet mount.

[0022] A microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides, such as therapeutic mRNA) includes a sheet mount for removably holding a microfluidic path plate device; a plurality of pressure lines; a plurality of fluid vials, each of which either comprises a fluid line or is configured to couple with a fluid line, and each fluid line, and at least a subset of the pressure lines, are configured to be biased relative to the microfluidic path plate device held in the sheet mount to form closed fluid paths; and a controller configured to control the application of pressure through the pressure lines and drive fluid movement within the microfluidic path plate device when the microfluidic path plate device is held in the sheet mount, to determine the contents of the fluid vials, move sub-microliter volumes of material from the fluid vials to one or more reactors within the microfluidic path plate device held in the sheet mount, direct synthesis of synthetic templates, direct in vitro transcription (IVT) reactions using the templates to form therapeutic polynucleotides, and perform sequencing. and a controller configured to manage the purification of therapeutic polynucleotides in one or more microfluidic pathway devices held within the mount.

[0023] A controller configured to perform any of the methods described herein may be configured to, among other things, receive inputs (e.g., light input, pressure input, temperature / heat input, etc.) and process the inputs to control fluid movement within the microfluidic pathway device, temperature (including thermal cycling) of various regions of the microfluidic pathway device, flushing / bonding, opening / closing valves of the microfluidic device, sensing of the microfluidic device, etc. The controller may comprise one or more microprocessors, communication circuitry, memory, etc. The controller may include firmware, hardware and / or software.

[0024] Any of these devices may include one or more (e.g., multiple) optical sensors disposed around the seat mount and reagent storage frame that monitor fluid levels in the reagent storage frame and fluid movement within the microfluidic pathway device when the microfluidic pathway device is installed in the seat mount. Alternatively or additionally, the optical sensor may be at the bottom of the device (e.g., directly below the seat mount), facing upward, and may detect fluid volume, movement, etc. The device may include a seat mount release control configured to allow the seat mount to be removed from the device and sterilized separately. Any of these devices may include a fluid interface assembly release control configured to allow the fluid interface assembly to be removed from the device and sterilized separately, and / or a fluid sample holder release control configured to allow the fluid sample holder to be removed from the device and sterilized separately.

[0025] Any of these devices may include a thermal control device configured to regulate the temperature of at least one region of the microfluidic pathway device when the microfluidic pathway device is installed in the seat mount. Some devices may have two or more thermal control devices configured to regulate the temperature of different regions of the microfluidic pathway device. The thermal control device may include a Peltier element and / or may be configured to control the temperature of at least one region of the microfluidic pathway device between 4°C and 105°C (e.g., 4°C to 99°C, 4°C to 98°C, 4°C to 95°C, 4°C to 90°C, 4°C to 85°C, 4°C to 80°C, 4°C to 75°C, 4°C to 70°C, 4°C to 65°C, 4°C to 60°C, etc.).

[0026] Any of these apparatus may include a magnetic field applicator configured to apply a magnetic field to at least one region of the microfluidic pathway device when the microfluidic pathway device is seated in the seat mount. The magnetic field applicator may include a control arm attached to the reagent storage frame.

[0027] Generally, the controller may be configured to detect an identification code on a fluid vial held by the fluid sample holder, and in some variations, the identification code may include a bar code, RFID, or other method for identifying (particularly contactlessly) the contents of a component of the fluid sample holder. The controller may be configured to determine the level of a reagent held by the reagent storage frame.

[0028] Any of these devices may include an optical sensor drive configured to operate one or more of a plurality of optical sensors around the seat mount and reagent storage frame, and / or one or more electroluminescent panels or other backlighting devices configured to illuminate an area beneath a portion of the reagent storage frame.

[0029] The described methods and devices generally comprise one or more fluid power circuits to move substances (liquid substances) between fluid chambers (depots, fluid contacting sides, reactors, etc.) and channels of a microfluidic pathway device or channels within a microfluidic pathway device. The fluid power circuit may be a hydraulic or pneumatic circuit which may include one or more pressure channels and pressure receiving sides of chambers within a microfluidic device. The fluid power circuit may also be referred to as a microfluidic power circuit. A single microfluidic chip may contain multiple fluid power circuits. The fluid power circuit may comprise one or more pressure lines and an interface between the pressure lines of the microfluidic control device and one or more microfluidic chips in the microfluidic pathway device. One or more fluid power circuits may share components (valves, pressure lines, vacuum caps, etc.) with other overlapping fluid power circuits. Furthermore, for convenience, when the term "pneumatic" is used, it should be understood that general fluid power circuits (e.g., hydraulic and / or air pressure) may be used instead or additionally. The fluidic substance driven by the fluid power lines may be any suitable fluid (e.g., gas or liquid, such as air, water, oil, etc.).

[0030] Also described herein are microfluidic pathway devices (e.g., closed-path microfluidic pathway devices) for processing therapeutic polynucleotides in a closed pathway. As mentioned above, these microfluidic pathway devices may be referred to herein as microfluidic chips, microfluidic pathway plates, process chips, biochips, process plates, etc. Generally, microfluidic pathway devices may be microfluidic pathway plate devices that are substantially flat, plate-like structures, and these structures may be relatively thin (e.g., less than a few mm thick, e.g., 0.5-20 mm thick, 0.5-15 mm thick, 0.5-10 mm thick, etc.). The microfluidic pathway devices described herein may generally be at least partially transparent, particularly at the top of the microfluidic pathway device, to allow one or more optical sensors (cameras, CCDs, fiber optics, etc.) to be used to sense, detect, monitor, record, etc., activity, including fluid movement and / or movement of the elastic layer, such that the microfluidic pathway device is used with the microfluidic apparatus described herein.

[0031] Any of these microfluidic pathway devices, as described herein, may be configured to operate as closed-circuit devices, in which the chambers (and particularly the fluid contact chambers and fluid channels) are sealed to the fluid input / output lines (e.g., fluid lines) with sealed connections that prevent exposure to the environment (e.g., air). This may be particularly critical in the production of therapeutic mRNA, which may be degraded by exposure to RNases and other contaminants in the environment.

[0032] For example, a microfluidic pathway device may comprise 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-contacting side at the second surface and a pressure-receiving side at the first surface; a plurality of fluid channels extending from a fluid port, through the elastic membrane and the first surface, to the second surface, each fluid channel fluidly connecting with the fluid-contacting sides of the plurality of chambers; and a plurality of pressure channels extending from one or more pressure ports, through the first surface and the elastic layer, to the second surface, each pressure channel returning through the elastic layer to the first surface, each pressure channel of the plurality of pressure channels fluidly connecting with the pressure-receiving sides of one or more of the plurality of chambers, and further, the volume of the fluid-contacting side of each chamber may be adjusted by applying pressure from one or more of the pressure ports.

[0033] A microfluidic pathway device (e.g., for forming therapeutic polynucleotides, such as therapeutic mRNA) may comprise 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-contacting side at the second surface and a pressure-receiving side at the first surface; a plurality of fluid channels each extending from a fluid port through the first plate region to the second plate region and fluidly connecting with the fluid-contacting side of one or more chambers of the plurality of chambers; and a plurality of pressure channels each extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region and back through the elastic layer to the first plate region, wherein each pressure channel of the plurality of pressure channels extends within the first plate region and fluidly connects with one or more pressure-receiving sides of one or more of the plurality of chambers, Further, the fluid-contacting side of each chamber is concave so that when positive pressure on the pressure-receiving side drives the elastic layer against the fluid-contacting side, the elastic layer rests flat and tightly against the fluid-contacting side at the second surface.

[0034] Any of these microfluidic devices may be configured to form a static seal with one or more fluid and / or pressure lines. In some variations, ports (fluid ports, pressure ports, etc.) may be formed as channels that enter the body of the microfluidic device (e.g., cylindrical channels) and descend to openings through an elastic layer of the device, the bottom surface of which may have a passage to a second plate region having a diameter narrower than the diameter of the port channel and be supported by the second plate region (e.g., a second surface of the second plate region) such that input lines (fluid lines and / or pressure lines) are supported against the elastic layer when actuated against the elastic layer to form a seal.

[0035] For example, a microfluidic pathway device (eg, for forming a therapeutic polynucleotide) may be configured to operate in a generally closed circuit. The microfluidic pathway device may comprise 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, a portion of the elastic layer dividing each chamber into a fluid-contacting side at the second surface and a pressure-receiving side at the first surface; a plurality of fluid channels each extending from a fluid port through the first plate region to the second plate region and fluidly connecting with the fluid-contacting side of one or more of the plurality of chambers; and a plurality of pressure channels each extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region and back through the elastic layer to the first plate region, each pressure channel of the plurality of pressure channels extending within the first plate region and fluidly connecting with one or more pressure-receiving sides of one or more of the plurality of chambers, each fluid port extending laterally through the first plate region and a port channel opening onto an opening through the elastic layer having an opening diameter smaller than a diameter of the fluid channel second plate region, and further wherein the diameter of the fluid channel second plate region is smaller than the diameter of the port channel.

[0036] Any of these devices may be configured to reduce or eliminate air bubbles that may form in the fluid pathways, for example, by including one or more vacuum caps in the fluid circuitry of the device. For example, a microfluidic pathway device (e.g., for processing therapeutic polynucleotides in a closed circuit) 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, a portion of the elastic layer separating each chamber into a fluid-contacting side at the second surface and a pressure-receiving side at the first surface; a plurality of fluid channels each extending from a fluid port through the first plate region to the second plate region and fluidly connecting with the fluid-contacting side of one or more chambers of the plurality of chambers; a plurality of pressure channels each extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region and back through the elastic layer to the first plate region, each pressure channel of the plurality of pressure channels extending within the first plate region and fluidly connecting with one or more pressure-receiving sides of one or more of the plurality of chambers; and a small number of pressure channels between at least some of the plurality of chambers. and at least one vacuum cap, wherein the at least one vacuum cap comprises a bubble removal chamber formed between a first surface and a second surface, the resilient layer separating the bubble removal chamber into a fluid-contacting side of the bubble removal chamber at the second surface and a vacuum-receiving side at the first surface, the fluid-contacting side of the bubble removal chamber being in fluid communication with at least two of the fluid-contacting sides of the plurality of chambers, and at least a portion of the resilient layer between the fluid-contacting side and the vacuum-receiving side of the bubble removal chamber being gas-permeable.

[0037] Thus, any of these microfluidic pathway devices may include at least one vacuum cap between at least some of the plurality of chambers, the at least one vacuum cap comprising a bubble removal chamber formed between a first surface and a second surface, a gas permeable elastic layer separating the bubble removal chamber into a fluid contacting side of the bubble removal chamber at the second surface and a vacuum receiving side at the first surface, the fluid contacting side of the bubble removal chamber being in fluid communication with at least two of the fluid contacting sides of the plurality of chambers.

[0038] Any of these microfluidic pathway devices may be configured to prevent dead space areas even within the smallest chambers of the microfluidic pathway device, for example, the fluid-contacting side and the pressure-receiving side of the second surface may be concave so that when positive pressure on the pressure-receiving side drives the elastic layer against the fluid-contacting side, the elastic layer rests flat and tightly against the fluid-contacting side of the second surface.

[0039] Generally, these devices may be formed from one plate or multiple plates. For example, a single plate may include multiple surfaces, including interior surfaces. Alternatively, a device may include two or more plates stacked and / or nested together, which may include an elastic layer and / or membrane between them. In some variations of the microfluidic pathway 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 the first plate, and the second surface may be part of the second plate. Alternatively, the first surface may be part of the first plate region, and the second surface may be part of the second plate region; in some variations, the first and second plate regions may be part of the same plate; alternatively, the first and second plate regions may be part of different plates forming the microfluidic pathway device.

[0040] One or more pressure and fluid ports may be located adjacent to the exterior surface of the microfluidic pathway device. The pressure and fluid ports may be arranged in groups and / or spaced apart. Generally, the pressure and fluid ports may be located around the periphery of the exterior surface of the microfluidic pathway device along the top of the device and / or may be located such that a central region of the microfluidic pathway device is open and exposed for visualization (by one or more optical sensors) that may monitor fluid movement and / or processing of the microfluidic pathway device.

[0041] In some variations, the chambers of the microfluidic pathway device may be pairs of chambers, with the first chamber (e.g., fluid contacting portion) of each pair of chambers being fluidly connected to the second chamber (e.g., fluid contacting portion) of each pair of chambers. The pressure-receiving side of each chamber may be a separate (or separate) chamber. The chambers may be separately controlled by connection to pressure lines (separable and / or connectable) or by fluid power circuitry on the microfluidic pathway device. In some variations, a first chamber of a set of chambers may be connected to one of the chambers of the other set via a valved fluid connection. The valves may be part of the fluid power circuitry and may be opened / closed by a controller that applies fluid pressure (e.g., air pressure, hydraulic pressure, etc.) to deflect an elastic layer within a small chamber formed between the first and second surfaces.

[0042] As described above, the microfluidic pathway device may be a sealed pathway device. Operation of the device may be monitored and controlled by a controller device without contact with the liquid (e.g., containing the therapeutic polynucleotide, e.g., mRNA). In some variations, the microfluidic pathway device may be at least substantially translucent to visible or ultraviolet light. For example, the microfluidic pathway device is substantially transparent to visible or ultraviolet light.

[0043] Any of these methods and apparatus may be configured to purify polynucleotides (e.g., mRNA) within a microfluidic pathway device. For example, the microfluidic pathway device may include a material inserted into the fluid-contacting side of the channel, e.g., the material may include a cellulose material configured to selectively absorb double-stranded mRNA.

[0044] Any of these microfluidic pathway devices may be configured to remove impurities from therapeutic substances (e.g., "drug particles"), such as therapeutic mRNA substances (e.g., therapeutic mRNA encapsulated in a delivery vehicle). For example, any of the microfluidic pathway devices described herein may include one or more chambers configured for buffer preparation and / or drug particle concentration. In some cases, the device is configured to flow a drug particle solution tangentially through a chamber having one or more ultrafiltration membranes to separate the nanoparticles from the solvent, thereby purifying and / or concentrating the retentate drug particles. Smaller particles, such as solvent and ions, pass through the membrane as permeants and become waste, while drug particles (e.g., mRNA encapsulated in a delivery vehicle) in the same solvent may be collected downstream as retentate. In some variations, this may concentrate the drug particles. In some cases, biocompatible and stable buffers can be used for downstream processing for infusion into patients. Buffer adjustments can be made by adding diluents with the appropriate composition of water, salts, additives, and / or other ingredients. The concentration of a particular chemical can be increased by adding a buffer with a higher chemical concentration, and vice versa. For example, in some cases, the concentration of ethanol can be reduced by half by adding the same amount of water. The methods and devices described herein enable the formulation, buffer adjustment, and concentration of biomolecule-containing products to be performed in a single microfluidic pathway device. The formulation buffer can be adjusted to make it more biocompatible and stable for downstream processing and patient injection. The drug concentration can be adjusted after the formulation and buffer adjustment process to suit the drug's administration method. Accordingly, any of these devices can include one or more chambers with a membrane, such as an ultrafiltration membrane. The microfluidic pathway device can therefore include a concentrator. In some variations, the microfluidic pathway includes a dialysis chamber (e.g., within the thickness of the second surface, e.g., the second layer portion).

[0045] Any of these microfluidic pathway devices may include a delivery reservoir configured to deliver a preselected volume of fluid to at least one chamber, e.g., the preselected volume of the chamber may be, for example, between about 20 nanoliters and 5 mL (e.g., between 25 nL and 5 mL, between about 50 nL and 5 mL, between about 50 nL and 2 mL, about 25 nL, about 30 nL, about 50 nL, greater than about 75 nL, etc.).

[0046] The first and / or second layer portions may be formed from a hard material. Any of these microfluidic pathway devices may include a third layer portion (e.g., a third surface) that may be formed from a hard material, for example, overlaid on an elastic material. The hard material may be a polymer, for example, a cycloolefin copolymer, or glass.

[0047] For example, a microfluidic pathway device may include an elastic layer between (e.g., 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, with a portion of the elastic layer dividing each chamber into a fluid-contacting side and a pressure-receiving side; a plurality of fluid ports through the first plate, each fluid port having an exposed portion of the elastic layer supported by the second plate, each fluid port having an opening through the elastic layer to the second plate in fluid communication with the fluid-contacting side of one of the plurality of chambers; and a plurality of pressure ports through the first plate, each fluid port having an exposed portion of the elastic layer supported by the second plate, each fluid port having an opening through the elastic layer to the second plate in fluid communication with the pressure-receiving side of one of the plurality of chambers.

[0048] 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, formed between the first plate and the second plate, with a portion of the elastic layer dividing each chamber into a fluid-contacting side and a pressure-receiving side; a plurality of fluid ports, each of which passes through the first plate, through the elastic layer, and to the second plate, fluidly connecting with the fluid-contacting side of one of the plurality of chambers; and a plurality of pressure ports, each of which passes through the first plate, through the elastic layer, and to the second plate, then through the elastic layer back to the first plate, fluidly connecting with the pressure-receiving side of one of the plurality of chambers.

[0049] For example, a microfluidic pathway device may include a first plate having a first surface and a second surface and a thickness therebetween; a second plate having a first surface and a second surface and a thickness therebetween; 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 at the second surface of the second plate, the third plate having the first surface and the second surface; at least one chamber having a fixed volume, the at least one chamber being formed in the second surface of the first plate and the first surface of the second plate, a portion of the elastic layer dividing the at least one chamber into a fluid-contacting side and a pressure-receiving side; and a fluid channel extending from a fluid port through the thickness of the first plate, through the elastic layer, to a fluid channel opening through the thickness of the second plate, fluidly connecting with a connecting channel formed in the second surface of the second plate, the fluid channel connecting with the fluid-contacting side of the at least one chamber. The first plate may have a fluid port having a diameter through the thickness thereof that is larger than the diameter of the fluid channel opening through the elastic layer, the outlet channel extending from the fluid contacting side through the second surface of the second plate, and the port (e.g., valve) formed by the elastic layer being between the fluid contacting side and the outlet channel.

[0050] For example, a microfluidic pathway device for processing therapeutic polynucleotides in a closed channel may include a first plate having a first surface and a second surface and a thickness therebetween, a second plate having a first surface and a second surface and a thickness therebetween, 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 at the second surface of the second plate, at least one chamber having a fixed volume, the at least one chamber being formed at the second surface of the first plate and the first surface of the second plate, a portion of the elastic layer dividing the at least one chamber into a fluid-contacting side and a pressure-receiving side, and a fluid channel extending from a fluid port passing through the thickness of the first plate, through the elastic layer, to a fluid channel opening passing through the thickness of the second plate, fluidly connecting with a connecting channel formed at the second surface of the second plate and surrounded by the third plate, the fluid channel connecting with the fluid-contacting side of the at least one chamber. and a pressure channel extending from a pressure port through the thickness of the first plate, through the elastic layer, to a pressure channel opening through the thickness of the second plate, fluidly connecting with a connecting pressure channel formed in the second surface of the first plate and surrounded by the elastic layer, the pressure channel connecting with 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 fluid channel opening through the elastic layer, and further, the diameter of the pressure port through the thickness of the first plate is larger than the pressure channel opening through the elastic layer, an outlet channel extending from the fluid contacting side through the second surface of the second plate, and a valve (e.g., port) formed by the elastic layer is between the fluid contacting side and the outlet channel.

[0051] The microfluidic pathway device may include a pressure channel extending from a pressure port through the thickness of the first plate, through the elastic layer, to a pressure channel opening through the thickness of the second plate, in fluid communication with a connecting pressure channel formed in the second surface of the first plate and surrounded by the elastic layer, the pressure channel connecting to the pressure-receiving side of at least one chamber.

[0052] The microfluidic pathway devices described herein may include a plurality of pressure ports, the fluid ports being disposed adjacent an exterior surface of the microfluidic pathway device.

[0053] For example, a microfluidic pathway device for processing therapeutic polynucleotides in a closed channel may include a first plate having a first surface and a second surface and a thickness therebetween, the first surface forming an exposed outer surface; a second plate having the first surface and the second surface and a thickness therebetween; 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 at the second surface of the second plate, the third plate having the first surface and the second surface and a thickness therebetween, the second surface forming an exposed bottom surface at the bottom of the device; and at least one set of chambers, each having a fixed volume, the at least one set of chambers being formed on the second surface of the first plate and the first surface of the second plate, a portion of the elastic layer bifurcating each of the at least one set of chambers into a fluid contacting side and a pressure receiving side, each of the at least one set of chambers being fluidly connected therebetween. and a pressure channel extending from a pressure port through the thickness of the first plate, through the elastic layer, to a pressure channel opening through the thickness of the second plate, in fluid communication with a connecting pressure channel formed in the second surface of the first plate and surrounded by the elastic layer, the pressure channel connecting with a pressure receiving side of the at least one chamber; an outlet channel extending from the fluid contacting side through the second surface of the second plate, and a valve (e.g., port) formed by the elastic layer connecting the fluid contacting side and the outlet channel. It is between.

[0054] Also described herein are apparatuses (e.g., systems) that include both any of the microfluidic devices (e.g., microfluidic pathway device control systems) and one or more microfluidic pathway devices. For example, the system may include a microfluidic device, the apparatus including a seat mount for the microfluidic pathway device, a fluid interface assembly including a plurality of fluid lines and pressure lines, each configured to be actuated relative to the microfluidic pathway device mounted in the seat mount, and a reagent storage frame including a plurality of fluid sample holders, each configured to hold a fluid vial and connect to the fluid interface assembly through one of the fluid lines of the fluid interface assembly. As described in more detail below, in some variations, the fluid sample holders may be adapted to be actuated directly relative to the microfluidic pathway device without a separate fluid line, and the sample holders may form a fluid line. Fluid lines, or individually sample holders (eg, vials, containers, etc.), may be configured to be held within the microfluidic pathway device, for example, by pressure against an elastomeric sheet formed at a port.

[0055] The apparatus may include a plurality of optical sensors disposed around the seat mount and the reagent storage frame for monitoring fluid levels in the reagent storage frame and fluid movement within the microfluidic pathway device when the microfluidic pathway device is installed in the seat mount; a controller configured to control application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is installed in the seat mount; and a microfluidic pathway device, the microfluidic pathway device comprising: a first plate having a first surface and a second surface and a thickness therebetween, the first surface forming an exposed outer surface; a second plate having the first surface and the second surface and a thickness therebetween; an elastic layer sandwiched between the second surface of the first plate and the first surface of the second plate; and a third plate coupled to the second plate at the second surface of the second plate, the third plate having the first surface and the second surface and a thickness therebetween, the second surface forming an exposed bottom surface at the bottom of the device; and a microfluidic pathway device having a fixed volume. at least one chamber, the at least one chamber being formed on the second surface of the first plate and the first surface of the second plate, a portion of the elastic layer bifurcating the at least one chamber into a fluid contacting side and a pressure receiving side; a fluid channel extending from a fluid port passing through the thickness of the first plate, through the elastic layer, to a fluid channel opening passing through the thickness of the second plate, and fluidly connecting with a connecting channel formed on the second surface of the second plate and surrounded by a third plate, the fluid channel connecting with the fluid contacting side of the at least one chamber; and a pressure channel extending from a pressure port passing through the thickness of the first plate, through the elastic layer, to a pressure channel opening passing through the thickness of the second plate, and fluidly connecting with a connecting pressure channel formed on the second surface of the first plate and surrounded by the elastic layer, the pressure channel connecting with the pressure receiving side of the at least one chamber, the fluid port having a diameter larger than the fluid channel opening passing through the thickness of the first plate, and further having a diameter larger than the direct diameter of the pressure port passing through the thickness of the first plate.The outlet channel has a diameter larger than the pressure channel opening through the elastic layer and extends from the fluid contacting side through the second surface of the second plate, with a valve formed by the elastic layer between the fluid contacting side and the outlet channel.

[0056] Also described herein are methods of using any of these apparatus and devices. For example, a method of processing a fluid to form a therapeutic polynucleotide (e.g., a therapeutic mRNA) in a microfluidic pathway device may include sealingly and independently coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of the microfluidic pathway device, each distal end being biased to be driven against an elastic layer between a first surface and a second surface, the microfluidic pathway device comprising a plurality of chambers each divided into a fluid-contacting side formed on the second surface and a pressure-receiving side formed on the first surface, and applying positive and negative pressures within the pressure-receiving sides of the chambers to drive fluid through the fluid-contacting sides of the plurality of chambers and change the size of the plurality of fluid-contacting sides.

[0057] A method for processing fluid in a microfluidic pathway device may include sealingly and independently coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of a microfluidic pathway device, each distal end being biased to be driven against an elastic layer between a first plate and a second plate, the microfluidic pathway device comprising a plurality of chambers each separated into a fluid-contacting side formed in the second plate and a pressure-receiving side formed in the first plate, the fluid-contacting sides being interconnected; and driving fluid through the interconnected fluid-contacting sides and operating valves to measure fluid movement between the fluid-contacting sides of the plurality of chambers by application of positive and negative pressures in the pressure-receiving sides of the chambers and to change the size of the plurality of fluid-contacting sides.

[0058] Driving the fluid through the fluid contacting side may include deflecting a resilient layer sandwiched between the first surface and the second surface.

[0059] As mentioned above, any of these methods may involve optical feedback from the microfluidic pathway device to control the application of positive and negative pressure.

[0060] These methods may include controlling a valve by deflecting an elastic layer between a first surface and a second surface. For example, the controller may control fluid power (e.g., air pressure, hydraulic pressure) through fluid power circuits (e.g., fluid lines, valves, etc.) to control processing of the microfluidic pathway device.

[0061] Generally, the actuating step may include driving fluid through interconnected fluid contacting sides and manipulating valves to measure fluid movement between the fluid contacting sides of the plurality of chambers through application of positive and negative pressure. The fluid contacting sides may be interconnected.

[0062] For example, described herein is a method for processing fluid in a microfluidic pathway device, the method including: sealingly and independently coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of the microfluidic pathway device, each distal end being biased (e.g., spring-loaded) to drive the distal end against an elastic layer between a first plate and a second plate, the microfluidic pathway device comprising a plurality of chambers, each chamber being divided into a fluid-contacting side formed in the second plate and a pressure-receiving side formed in the first plate; and pneumatically driving fluid through a plurality of fluid-contacting sides of the plurality of chambers by adjusting the application of positive and negative pressures within the pressure-receiving sides of the chambers to vary the size of the plurality of fluid-contacting sides.

[0063] In some variations, a method for processing fluid within a microfluidic pathway device may include sealingly (and independently) coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of a microfluidic pathway device, each distal end being biased to drive the distal end against an elastic layer between a first plate and a second plate of the microfluidic pathway device, the microfluidic pathway device comprising a plurality of chambers, each chamber being divided into a fluid-contacting side formed in the second plate and a pressure-receiving side formed in the first plate, and driving fluid through the plurality of fluid-contacting sides of the plurality of chambers.

[0064] The novel features of the invention are set forth with particularity in the following claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0065] [Figure 1] 1 is a schematic diagram of one variation of a system comprising a microfluidic device and a microfluidic pathway device as described herein. [Figure 2A] FIG. 1 shows an example of the front view of a closed-circuit microfluidic device for processing therapeutic polynucleotides at the point of care. [Figure 2B] FIG. 1 shows an example of the backside of a closed-circuit microfluidic device for processing therapeutic polynucleotides at the point of care. [Figure 2C] 1 is an example of an apparatus as described herein. [Figure 3] FIG. 1 is a partially exploded view of an example of a closed-circuit microfluidic device for processing therapeutic polynucleotides at a point of care, as described herein, including a reagent storage frame, a fluid interface assembly, a thermal control and sensor assembly, and a microfluidic pathway device. [Figure 4A]FIG. 1 is an isometric view of an example reagent storage frame. [Figure 4B] FIG. 4B is a side view of the reagent storage frame of FIG. 4A. [Figure 5A] FIG. 1 is an isometric view of an example of the top surface (eg, top) of a reagent storage frame. [Figure 5B] FIG. 5B is an isometric view of the underside (eg, bottom) of the reagent storage frame of FIG. 5A. [Figure 6A] FIG. 1 is a top view of an example fluid interface assembly. [Figure 6B] FIG. 6B is a bottom view of the fluid interface assembly of FIG. 6A. [Figure 7A] FIG. 1 is a perspective view of a tubing compression connector assembly configured as a bias (eg, spring bias) configured to bias a fluid line against a microfluidic pathway device and make a sealing connection to the microfluidic pathway device. [Figure 7B] FIG. 1 is a side view of a tubing compression connector assembly configured as a bias (eg, spring bias) configured to bias a fluid line against a microfluidic pathway device and make a sealing connection to the microfluidic pathway device. [Figure 7C] FIG. 7C is a graphic depiction of a tubing retainer engaged within a tubing compression connector (e.g., a collet) similar to that shown in FIGS. 7A-7B. [Figure 7D] 1A-1C show examples of fluidic cartridges held in spring contact with ports (e.g., fluidic ports) of a microfluidic pathway device. [Figure 7E] 1A-1C show an example of a fluidic cartridge held in spring contact with a port (e.g., a fluidic port) of a microfluidic pathway device. [Figure 8A] FIG. 1 is an isometric top view of an example of a seat mount, multiple optical sensors arranged around the seat mount on a gantry, and a thermal control device directly below the seat mount, as described herein. [Figure 8B]8B is a side view of the subassembly (including the seat mount, optical sensor, and thermal control device) of FIG. 8A. FIG. [Figure 8C] FIG. 8B is a detailed view of the seat mount according to FIG. 8A. [Figure 9A] FIG. 1 is a top view of an example of a microfluidic pathway device. [Figure 9B] FIG. 9B is an isometric view of the microfluidic pathway device of FIG. 9A. [Figure 9C] 9A-9B, showing an enlarged example of a portion of a microfluidic path device such as that shown in FIGS. 9A-9B, including a vacuum cap for air bubble removal. [Figure 9D] FIG. 1 illustrates an example of a vacuum cap as is described herein. [Figure 9E] FIG. 1 illustrates an example of a vacuum cap as is described herein. [Figure 9F] FIG. 1 shows another example of a microfluidic pathway device configured as a large capacity device (eg, a "10X device"). [Figure 10A] FIG. 1 is a cross-sectional side view of an example of a microfluidic pathway device. [Figure 10B] 1 is an example cross-sectional view of a portion of an example microfluidic pathway device as described herein. [Figure 10C] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10D] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10E]1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10F] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10G] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10H] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10I] 1 is a side cross-sectional view illustrating the operation of a portion of an example microfluidic pathway device that interacts with a closed-circuit microfluidic device as described herein, in which fluid and pressure lines engage the microfluidic pathway device to precisely control fluid movement in the microfluidic pathway device. [Figure 10J] FIG. 10 is a cross-sectional view of another example of a portion of a microfluidic pathway device including a priming valve or priming cap as described herein. [Figure 10K] FIG. 10 is a cross-sectional view of another example of a portion of a microfluidic pathway device including a priming valve or priming cap as described herein. [Figure 11]FIG. 1 is an exploded view of the layers comprising a microfluidic pathway device according to one embodiment of the present disclosure. [Figure 12] FIG. 12A illustrates various exemplary microfluidic pathway devices as described herein. FIG. 12B illustrates various exemplary microfluidic pathway devices as described herein. FIG. 12C illustrates various exemplary microfluidic pathway devices as described herein. FIG. 12D illustrates a variation of a microfluidic pathway device similar to that shown in FIGS. 12A-12C. FIG. 12E illustrates a variation of a microfluidic pathway device similar to that shown in FIGS. 12A-12C. FIG. 12F illustrates a variation of a microfluidic pathway device similar to that shown in FIGS. 12A-12C. [Figure 13] 1A-1C are cross-sectional views of examples of microfluidic pathway devices showing closed fluid pathways. [Figure 14] FIG. 1 is a schematic diagram illustrating some of the features of a microfluidic pathway device controller as described herein. [Figure 15A] 1A and 1B are top views of an example of a microfluidic pathway device with a heat spreader. [Figure 15B] FIG. 1B is a bottom view of an example microfluidic pathway device with a heat spreader. [Figure 16A] 1 shows an example of a system with microfluidic devices in a class 5 isolation cabinet in a class 7 space. The system may be configured as a mini-factory. [Figure 16B] FIG. 1 shows a microfluidic device in a class 5 cabinet. DETAILED DESCRIPTION OF THE INVENTION

[0066] Generally, described herein are apparatus (e.g., systems, devices, etc.) and methods for processing therapeutic polynucleotides. In particular, these apparatus and methods may be closed-circuit apparatus and methods configured to minimize or eliminate manual handling during operation. Closed-circuit apparatus and methods provide a nearly completely sterile environment, and components may provide a sterile pathway for processing from initial input (e.g., template) to output (formulated therapeutic). Input of materials (nucleotides, and any chemical components) into the apparatus is sterile and may be input into the system without requiring virtually any manual interaction.

[0067] The methods and devices described herein can produce therapeutics with very fast cycle times and a very high degree of reproducibility. The devices described herein are configured to provide synthesis, purification, dialysis, formulation, and concentration of one or more therapeutic compositions (including, but not limited to, therapeutic polynucleotides) in a single, integrated device. All or some of these processing steps are performed in a continuous fluid processing pathway, which may be configured as one or a series of consumable microfluidic devices (which may also be referred to as microfluidic channel chips, microfluidic channel plates, process chips, biochips, or process plates). This enables the synthesis, including formulation, of patient-specific therapeutics at the point of care (e.g., hospitals, clinics, pharmacies, etc.).

[0068] During operation of the device, the fluid pathways remain substantially continuous, and contamination can be substantially eliminated by non-contact monitoring (e.g., optical monitoring), including fluid flow measurement, mixing monitoring, etc., and by using pressure applied from deflectable membranes on opposite sides of the fluid chambers and channels to manipulate precise microfluidic volumes (metering, mixing, etc.).

[0069] These devices and methods may be configured for use in therapeutic settings. For example, the methods and devices described herein may be configured for producing customized therapeutic compositions containing one or more therapeutic polynucleotides (e.g., mRNA, microRNA, DNA, etc.).

[0070] Thus, the methods and devices described herein may lead to the development of scalable polynucleotide manufacturing, single-patient dose production, limiting contamination by eliminating touch points, input and process tracking to meet clinical manufacturing requirements, and use in therapeutic point-of-care operations. The use of microfluidic devices and processes described herein can provide significant advantages.

[0071] Generally, the devices described herein may be microfluidic devices (e.g., microfluidic control devices). In some variations, these microfluidic devices may comprise closed-circuit microfluidic devices for processing therapeutic polynucleotides at the point of care. These devices may be configured to operate in one or more microfluidic pathway devices. A microfluidic device may comprise one or more microfluidic pathway devices (e.g., process chips, formulation chips, etc.) or may be configured for use with a microfluidic pathway device; thus, a microfluidic device may not comprise a microfluidic pathway device. In some variations, a microfluidic device (either with or without a microfluidic pathway device) may be referred to as a system.

[0072] Generally, a microfluidic device as described herein may include a seat mount for the microfluidic device, a fluid interface assembly including a plurality of fluid lines and pressure lines, a reagent storage frame including a plurality of fluid sample holders each configured to hold a fluid vial and each configured to connect to the fluid interface assembly through one of the fluid lines of the fluid interface assembly, a plurality of optical sensors disposed around the seat mount and reagent storage frame for monitoring fluid levels in the reagent storage frame and fluid movement within the microfluidic pathway device, and a controller configured to control the application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device. In any of these devices, each fluid line and each pressure line may be configured to be driven relative to the microfluidic pathway device mounted within the seat mount.

[0073] The controller may coordinate processes on and in the microfluidic pathway device, including moving one or more fluids in the microfluidic pathway device, mixing one or more fluids in 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 a magnetic field (e.g., when using magnetic beads), etc. The controller may comprise software, firmware, and / or hardware. In some variations, the controller may receive input from a user and provide output (e.g., via a monitor, touchscreen, etc.). The controller may communicate with a remote server, for example, to confirm operation of the device, reorder materials (e.g., components such as nucleotides, microfluidic pathway devices, etc.), and / or download protocols.

[0074] FIG. 1 shows a diagram of one example of a system for processing polynucleotides, including an apparatus having a housing 103 enclosing a seat mount 115 that can hold one or more microfluidic pathway devices 111, which can be disposable devices. The housing can be a chamber, enclosure, etc., and can include a lid or opening and be sealed when closed. The housing can enclose a temperature regulator and / or be configured to be enclosed in a temperature-regulated environment (such as a cooling unit). The housing can form a sterility barrier. In some variations, the housing can form a humidified or humidity-controlled environment.

[0075] The seat mount 115 may be configured to secure the microfluidic pathway device using one or more pins or other components configured to hold the microfluidic pathway device in a fixed and predetermined orientation.

[0076] In some variations, a thermal controller 113 may be positioned adjacent to the seat mount 115 to regulate the temperature of one or more microfluidic pathway devices 111. The thermal controller may include a thermoelectric component (e.g., a Peltier element) and / or one or more heat sinks to control the temperature of all or a portion of the microfluidic pathway device. In some variations, two or more thermal controllers may be included to separately regulate the temperature of different regions of one or more regions of the microfluidic pathway device. The thermal controller may include one or more thermal sensors (e.g., thermocouples, etc.) that may be used for feedback control of the microfluidic pathway device and / or the thermal controller.

[0077] In FIG. 1, the fluid interface assembly 109 couples liquid reagents and / or pressure (e.g., gas) to the microfluidic pathway device 111 held within the seat mount 115 and also couples pressure from a pressure source 117 to the microfluidic pathway device 111. The fluid interface assembly may aid in the delivery of fluidic materials and positive / negative gas pressure to the interior. The fluid interface assembly may optionally aid in securing the microfluidic pathway device, as described in more detail below. The fluid interface assembly may be removably coupled to the device (and may be removed or portions thereof removed) for sterilization between uses.

[0078] The reagent storage frame 107 is configured to include a plurality of fluid sample holders, each of which may hold a fluid vial configured to hold a reagent (e.g., nucleotides, solvents, water, etc.) for delivery to the microfluidic device 111, or alternatively, the fluid vials may 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 a manifold configured to split one or more pressure sources 117 into multiple pressure lines applied to the microfluidic pathway device that are controlled independently or collectively (in subcombination).

[0079] The fluid interface assembly may comprise multiple fluid and / or pressure lines and may comprise bias (e.g., spring-loaded) holders or chips that individually and independently actuate each fluid and / or pressure line into the microfluidic pathway device when the microfluidic pathway device is held within the seat mount 115. Tubing (e.g., fluid and / or pressure lines) may be part of and / or connected to the fluid interface assembly. In some variations, the fluid lines comprise flexible tubing connecting between the reagent storage frame and the microfluidic pathway device via connectors that couple vials to tubing in locking engagements (e.g., ferrules). The ends of the fluid pathways, and in some variations, the ends of the fluid / pressure lines, may be configured to seal against the microfluidic pathway device, for example, at sealing ports 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 vial may be pressurized (e.g., pressures >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, the fluid vial may be pressurized to 1-20 psig (e.g., 5 psig / 20 psia, 10 psig, etc.). Negative or positive pressure may be applied, for example, a vacuum (e.g., -7 psig or 7 psia) may be applied to draw the fluid back into the vial (e.g., depot) at the end of the process. Generally, the fluid vial may be driven at a lower pressure than the pneumatic valve to prevent or reduce leakage. In some variations, the pressure differential between the fluid and the pneumatic valve may be from about 1 psi to about 25 psi (e.g., about 3 psi, about 5 psi, 7 psi, 10 psi, 12 psi, 15 psi, 20 psi, etc.).

[0080] As described in more detail below, the fluid lines (or, in some variations, directly the fluid vials) and pressure lines may be actuated against ports (pressure ports or fluid ports) formed in the microfluidic pathway device to form seals. Each pressure line and / or fluid line (or fluid vial) may be individually actuated against a valve seat in the microfluidic pathway with a biasing force that may form a seal at the port. The biasing force (which may be pressure from a spring or other force-applying element) may be greater than the pressure in the fluid vial (and / or fluid line) and pressure line and configured to maintain a leak-free seal. For example, the pressure differential between the fluid vial and the biasing force may 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.), which may be referred to as the valve closing pressure. Generally, this bias force (valve closing pressure) may exceed the fluid driving pressure by, for example, an amount (e.g., 3 psi, 5 psi, 7 psi, 10 psi, etc.) that may be design-dependent. The bias force may be constant or adjustable. The bias force may be applied to maintain a seal with a port on the microfluidic pathway assembly. In some variations, the bias force may be adjusted based on the pressure in a fluid line (e.g., fluid vial) or pressure line. The bias force for each fluid line (or fluid vial) and pressure line may be individually adjustable.

[0081] Each vial may be coded (e.g., with an identifier that can be read by one or more sensors, as described below), and the controller may monitor the fluid levels and, therefore, the amount of each substance within the fluid interface assembly.

[0082] The apparatus may include a magnetic field applicator 119 and may be configured to create a magnetic field in the region of the microfluidic pathway device 111. One or more sensors 105, which may be optical sensors, may be part of the apparatus and may sense one or more of a barcode, fluid levels in fluid vials held in a reagent storage frame, and fluid movement within the microfluidic pathway device 111 when the device is mounted within the mounting sheet 115.

[0083] The sensor may measure the process in the device, for example, by measuring a light indicator. In some variations, visual / optical markers may be used to estimate yield. For example, fluorescence emission may be used to detect process yield or residue by tagging with a fluorophore. Alternatively or additionally, dynamic light scattering may be used to measure particle size distribution within a portion of the microfluidic pathway device (e.g., a mixing section). In some variations, sensor measurements may be performed using one or two optical fibers to transmit light (e.g., laser light) and detect the emerging light signal. An instrument package may be attached remotely from the device. Such contactless sensing may be recommended.

[0084] In any of the methods and apparatus described herein, a sensor (e.g., a video sensor) may record all activity in a microfluidic pathway device (e.g., a chip or cartridge). For example, the entire run for synthesizing and / or processing a substance (such as a therapeutic RNA) may be recorded by one or more video sensors, including a video sensor that may visualize the microfluidic pathway device, e.g., from above. Processing in the microfluidic pathway device may be visually confirmed, and this record may be kept for later quality control and / or processing. Accordingly, video recordings of processing may be saved, archived, and / or transmitted for later review and / or analysis.

[0085] The interior of the device, for example, within the housing 103, may further be configured to be sterilizable. In particular, portions of the device may be removed and individually sterilized. Sterilization may be performed, for example, by UV irradiation or any other sterilization method necessary to limit contamination or meet regulatory requirements. The device, including the housing, may be stored in a High Efficiency Particulate Air (HEPA) filtered environment. The device, including the housing, may be stored in a temperature-controlled enclosure.

[0086] As described above, the apparatus may include, among other tasks, applying pressure and at least driving fluid movement through the microfluidic pathway device 111 and may be controlled by a controller 121. The controller may be completely or partially external to the housing. The controller may be configured to include user input / output. For example, a system user interface 123 may allow for easy operation and management of the apparatus and microfluidic pathway device.

[0087] Any of the devices described herein may include some or all of the components shown in Figure 1, but not all components are required. Although only some of the connections between components are shown in Figure 1, additional (or alternative) connections may be used.

[0088] FIG. 2A shows the front of device 200, which may include any of the features of the devices in system 100. In FIG. 2A, housing 103 (e.g., cover) is shown in an open position showing reagent storage frame 107, sensor 105 (configured as a camera or optical sensor in this example) that monitors fill levels and detects barcodes in the area below the horizontal surface of reagent storage frame 107, and control arm 219 for magnetic field applicator 119. Another sensor (camera) 105' is configured to be positioned above the top surface of the microfluidic pathway device (not visible in FIG. 2A). In FIG. 2A, the magnet and control arm for the upper sensor, each of which may separately comprise one or more actuators that may be connected and controlled by a controller (not shown), may be attached to the reagent storage frame. The device also includes a gantry on which side sensors (cameras) move to visualize reagent containers and / or other components of the device. The upper sensor may be configured to interrogate the microfluidic pathway device when installed within the device, and this information may be used, for example, by a controller to operate the device.

[0089] Figure 2B shows the back side of the apparatus 200, where power and pressure are connected through the access area 203. Figure 2C shows another example of a system including a microfluidic pathway device.

[0090] The housing 103 may be made from any suitable material, such as a polymer, metal, or composite. The housing is moisture resistant and protects the sterility of the enclosed components during operation of the device. The housing may be included within a cooling device and designed to keep reagents cool when stored in a reagent storage frame during the course of a batch or continuous run of the device 200.

[0091] In Figure 3, an exploded three-dimensional view of a portion of the apparatus is provided, including the seat mount 115, microfluidic pathway device 111, fluid interface assembly 109, and reagent storage frame 107, showing how these parts fit together and coordinate to provide functionality for the microfluidic pathway device 111 in apparatus 300. Apparatus 300 may be similar to the apparatus of Figures 1 or 2 and may have any of its features. In Figure 3, the reagent storage frame 107 is shown with several reagent vials and connectors (similar to Figure 2A). Rods 303 are shown connecting the reagent frame 107 to the fluid interface assembly 109, and can be attached (e.g., by screws) to the frame 107 and assembly 109 at receptacles adjacent to the seat mount 115. The seat mount 115 itself provides a frame on which the microfluidic pathway device 111 may be placed, and alignment pins (not visible in FIG. 3) may tightly align each of these vertically arranged components to the microfluidic pathway device 111.

[0092] Two or more sets of pins may be used for alignment. For example, the seat mount 115 (also called the lower nest) may have two or more short (e.g., 1.5 mm) pins that protrude above the device to align the microfluidic pathway device 111 when placed in the lower nest. There may be two long (e.g., 6 mm) pins that protrude downwards and mate with (visible) holes in the lower nest. These act to guide the upper nest into position, followed by smaller (e.g., 1.5 mm) pins that may function to provide a pin and slot mechanism to allow final adjustments, also in the fluid interface assembly 109.

[0093] The seat mount 115 is secured to the base 305 and may permit or restrict (e.g., limit) adjustment of the horizontal placement of the microfluidic pathway device 111. In some variations, the microfluidic pathway device 111 may be supported in a substantially horizontal plane, which may be beneficial for minimizing the pressure required to drive fluid movement throughout the microfluidic pathway device 111. In some other variations, the microfluidic pathway device may be supported at an orientation within about 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or about 15 degrees from the horizontal. Small deviations from the horizontal orientation may help remove air bubbles from the fluid in the chambers and lines passing through the microfluidic pathway device 111. In still other variations, the microfluidic pathway device may be supported in a substantially vertical orientation relative to the base 305 or may be supported at an orientation within about 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or about 15 degrees from the vertical.

[0094] The seat mount 115, the fluid interface assembly 109, and / or the fluid sample holder may be formed from any suitable material, such as, for example, a polymer, glass, metal, or composite. The seat mount 115, the fluid interface assembly 109, and / or the fluid sample holder may be configured to be sterilized, such as by autoclaving or exposure to gamma radiation. The device 300 may further include one or more of a seat mount 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, the fluid interface assembly, and / or the fluid sample holder can be removed from the device and sterilized separately. These components may be removed and / or reinserted separately and / or collectively.

[0095] The thermal control device 113 may be positioned adjacent to the microfluidic pathway device 111 and below the sheet mount 115. The thermal control device 113 may be configured to control the temperature of at least one region of the microfluidic pathway device 111 to between about 4°C and about 105°C, or any selected temperature (or range of temperatures) therebetween. The thermal control device may be any suitable temperature control device, such as, in one non-limiting example, a Peltier element and / or multiple Peltier elements. Generally, the thermal control device may be configured to allow for independent temperature control in different thermal regions simultaneously.

[0096] The optical sensor 105 may be disposed on the base 305 and oriented to sense fluid fill levels in fluid vials disposed within the reagent storage frame and reduce the possibility of interrupting process execution within the microfluidic pathway device 111. The optical sensor 105 may sense the barcode on the fluid vial to positively identify the identity and / or lot number of the reagent or product vial. The optical sensor transmits information about the fill level or barcode to the controller 121, where the information may be stored or acted upon. This can help provide chain of custody data essential for the regulatory management of individual therapeutic drugs.

[0097] The optical sensors 105 may be movably disposed within rails 307 on the base 305 (e.g., as part of a gantry) and may further be operably connected to an optical sensor drive. The optical sensor drive may be configured to actuate one or more of the multiple optical sensors 105 around the seat mount 115 and / or the reagent storage frame 107. The multiple optical sensors 105 may be actuated in unison by coordinating movement with an optical drive belt (e.g., drive chain) 309.

[0098] 4A shows a top view of a reagent storage frame 107, which may be used in any of the apparatus described herein. The reagent storage frame 107 may support a magnetic field applicator 119, which, in use, may further include a control arm 219 capable of controlling the positioning of a magnet 414 adjacent at least one region of the microfluidic pathway device 111. 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 be the same size or may have one or more different sizes. The fluid sample holders 416 include caps that can be pressurized above ambient atmospheric pressure (e.g., to approximately 1 PSI, 2 PSI, 3 PSI, 4 PSI, 5 PSI, or more) to allow fluid from within a fluid vial contained by the fluid sample holder to be driven into the microfluidic pathway device 111. Alternatively, the fluid sample holder may not be pressurized at all and may operate at ambient atmospheric pressure. In yet another variation, the fluid sample holder cap may allow a reduction in the applied pressure to draw or accept fluid driven from the microfluidic pathway device 111. Multiple fluid sample holders may distribute the applied pressure to multiple fluid vials. The applied pressure may be distributed all the way to the fluid interface assembly. The fluid sample holder cap, in one non-limiting example, may include a Luer connection or the like that can provide a leak-tight connection under pressure. The fluid sample holder 416 may be connected to the fluid interface assembly 109 via fluid lines for delivery to the microfluidic pathway device 111. The fluid lines connecting the fluid sample holder (and the fluid vials held therein) to the fluid interface assembly may have the shortest possible length and be configured to prevent wear and lag.

[0099] The connections between the fluid sample holder, fluid lines, and microfluidic pathway device can form a sealed, closed pathway that is isolated when the microfluidic pathway device is installed in the seat mount, which is beneficial for protection from contamination when processing therapeutic polynucleotides.

[0100] The reagent storage frame may provide a support to which a sensor / camera support arm 418 is connected. The support arm 418 supports an overhead sensor / camera 412 configured to image and detect signals from the microfluidic pathway device 111. The sensor / camera 412 may be a camera configured to record fluid movement within the microfluidic pathway device 111 and / or detect signals emitted from within one or more chambers of the microfluidic pathway device.

[0101] The signal detected by the sensor operating as a signal detector may be a visible signal, a fluorescent signal, a UV absorbance signal, or an IR absorbance signal. The signal detector is a non-contact signal detector, e.g., does not directly contact the substance emitting the signal. In some variations, the signal detector is configured to measure nanoparticle size distribution. The signal detector may be configured to measure dynamic light scattering (DLS).

[0102] Figure 4B shows a side view of the reagent storage frame 107 having a horizontal surface 421 on which a fluid vial 422 contained by a fluid sample holder 416 may be placed. The reservoir portion of the fluid vial 422 protrudes below the horizontal surface 421, allowing an optical sensor to visualize an identification code 425, such as a bar code or RFID tag on the fluid vial, and to sense a fluid level 427 of the fluid within the fluid vial (e.g., the meniscus). In some variations, an electroluminescent panel 429 may be positioned below the horizontal surface 421 of the reagent storage frame 107 to provide additional illumination and assist in sensing. Also visible in Figure 4B is a magnetic arm controller 432 for the magnetic field applicator 119.

[0103] Figure 5A shows a perspective view of the top of the fluid interface assembly 109 of the apparatus 100, 200, 300. 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 actuated and sealingly connected to a microfluidic pathway device 111 mounted within a seat mount 115. For the fluid lines 534, a bias spring within a fitting 538 may apply a force to maintain the fluid line engaged with the microfluidic pathway device 111. Figure 5B is a perspective view of the bottom of the fluid interface assembly 109, showing the fluid lines 534 extending from the bottom of the horizontal surface 421 of the fluid interface assembly 109 and engaging the microfluidic pathway device 111. The fluid and pressure lines may have distal ends for engaging a substantially planar microfluidic path device, allowing for a good seal to be formed with the microfluidic path device.

[0104] Figure 6A shows the top surface of the fluid interface assembly 109. The fluid interface assembly has a central opening 602 through which the microfluidic pathway device 111 may be imaged by one or more of a plurality of optical sensors. Pressure lines may pass through 642 which may be located on the outer surface of the fluid interface assembly (pressure lines not shown), with fluid line 534 and pressure line 641 disposed around the outer surface of the central opening 602 for input to the microfluidic pathway device 111. Figure 6B shows the bottom surface of the fluid interface assembly and shows that a sealed end exit point 643 for fluid line 534 and a sealed end 645 for pressure line 536 are disposed around the outer surface of the central opening 602.

[0105] FIG. 7A shows a fitting 746 that can be used to engage a fluid line 534 to a microfluidic pathway device 111 mounted in a seat mount for a sealed connection within the microfluidic pathway device 111. The fitting 746 includes two mechanisms to provide flexibility for the fluid line 534 secured therein while providing a robust sealed connection that minimizes leakage and minimizes fluid line stress during assembly and operation of the device. A spring bias 748 can be used to bias the fluid line against the microfluidic pathway device. Additionally, a collet 749 can be used to bias the fluid line 534 against the microfluidic pathway device 111. The end of the microfluidic line 534 is cut flat to smoothly engage the microfluidic pathway device 111. In some variations, both of these mechanisms can be used to secure the fluid line. In another variation, a spring bias 748 can be used. Collet 749 may be used to enhance grip (e.g., in one direction) so that as fluid and / or pressure lines are driven into the microfluidic pathway device, they cannot back out of the microfluidic pathway device and break the seal with the microfluidic pathway device. In yet other variations, other suitable connectors, such as gaskets or other types of compression seals, may be used to secure the microfluidic pathway device. 7B shows a side view of the fitting with a spring bias 748 and collet 749. FIG. 7C shows a graphic representation of the fitting 746 with an engaged fluid line 534. The spring bias 748 is engaged against the fitting base, and the collet 749 grips the fluid line 534. The flat cut end 735 is pushed beyond the collet 749 to engage the microfluidic routing device 111.

[0106] In some variations, all or some of the fluid lines and fluid vials may alternatively or additionally be configured as fluid cassettes that connect to the microfluidic pathway device 111. Any of these fluid vials (e.g., fluid depots) may be configured with fluid lines integrated as part of the fluid depot. One example is shown in Figures 7D-7E, where a fluid cassette 750 may have a pressure port 753 and a flat-cut or flat-molded fluid input port 755. This fluid input port is configured as a channel with a fluid line 755 integrated into the rest of the fluid depot / fluid vial 750. The fluid input port 755 (shown as an integrated fluid line in Figures 7D and 7E) may be biased (e.g., spring-loaded in some variations) against the microfluidic pathway device 111 and contact and seal against an elastomeric layer (e.g., elastic layer) within the microfluidic pathway device 111 at the port. As described above, the bias force (also referred to as valve closing pressure) may be greater than the pressure in the fluid vial / fluid line and pressure line to prevent leakage (e.g., greater than 2 psi, 5 psi, 7 psi, 10 psi, etc.) than the pressure in the fluid line / fluid vial or pressure line. For example, the ports of the microfluidic pathway device may be configured to receive pressures up to about 5 psig (e.g., 5 psig, 7 psig, 10 psig, 12 psig, 15 psig, etc.), which is slightly higher than the pressurization of the fluid vial 422 as described herein. In some variations, the fluid vial (e.g., fluid cassette) 750 need not be mounted on a separate reagent storage frame, but can be spring mounted directly on the device 111 or to the fluid interface assembly 109, which may reduce or eliminate tubing. Bias contact with the elastic layer of the microfluidic pathway device 111 may initiate a sealed opening of the fluid cassette for use. The fluid cassette 750 may facilitate barcoding and identification. The use of multiple fluid cassettes 750 may simplify the structure required to supply reagents within the microfluidic pathway device 111.This design may enable the use of an isolating sterile inner liner within the fluidic cassette 750, eliminating exposure of reagents stored therein to the gas used to pressurize the fluidic cassette. Alternatively, other fluidic cassette designs may achieve isolation from exposure to the same gas by using a low-sliding-force piston or other structural mechanism that can isolate reagents held within the fluidic cassette 750 from the gas used to drive fluid to the microfluidic pathway device. Figure 7E shows a close-up of the fluidic input port 755, which directly engages the microfluidic pathway device. In any of the variations, unless otherwise clear from the context, the phrase "fluid line" may include either or both the fluidic cassette (also referred to as a fluid vial) and / or tubing connected to a fluid source (e.g., a depot, such as a bottle, vial, tubing, etc.).

[0107] FIG. 8A is a perspective view of the bottom substrate 800 of the device of FIG. 1, 2, or 3, showing the base 305 to which the seat mount 115 and multiple optical sensors 105 are connected. In this view, four optical sensors 105 are positioned around each edge of the base and move along a gantry comprising one or more rails 307, which, when in position, may be configured to image along the area surrounding the seat mount and reagent storage frame. The optical sensors 105 may be driven along the rails 307 by optical sensor drives 851. The optical sensors may be driven in unison by optical sensor drive belts 309. Each of the optical sensors 105 has an angled mirror assembly 853, which allows imaging of a field of view 855 while minimizing the space occupied by the optical sensors 105 themselves. The bottom substrate 800 in this example may include a second set of alignment pins 857 that may help align the fluid interface assembly and reagent storage frame to the seat mount. Figure 8B is a side view showing the arrangement of the base 305, optical sensors 105 (three visible in this view), optical sensor drive belt 309, and optical sensor drive 851. The second set of alignment pins 857 form an area around the seat mount 115, and the cooling fan 859 of the thermal control device 113 is visible directly below the seat mount 115. Figure 8C shows a detailed view of the components located on the bottom substrate 800. The seat mount 115 is secured by mounting springs 865, supporting it in the desired orientation and providing an opening for the thermal control device, e.g., a microfluidic pathway device, at the location of the Peltier surface (thermal control surface) 863. The seat mount 115 is releasably secured to the mounting spring 865 and has a release lever or connector (not shown) that allows the seat mount 115 and fluid interface assembly 109 (e.g., upper nest) to be removed and sterilized between uses, for example, by autoclaving. Adjustment holes 861 may be present in the seat mount 115 for adjusting the microfluidic pathway device 111 to properly seat within the seat mount 115. The adjustment holes may be near the outer edges of the seat mount 115.Alignment dowel pins may be provided. Thermal control surface 863 may include or be configured to include a vacuum chuck, and vacuum grooves may be present on the top surface to allow a vacuum to be applied and to suck the microfluidic pathway device down in good thermal contact with thermal control surface 863.

[0108] Generally, the seat mount may be simply referred to as a "seat" and is configured to be mounted to one or more microfluidic pathway devices, either fixed or loose within the device.

[0109] The microfluidic pathway device 111 may, in some variations, be supported in a substantially horizontal orientation or at an orientation within about 1, 2, 3, 4, 5, 7, 9, 10, 11, 13, or about 15 degrees from horizontal to aid in bubble control. A fan 859 is visible below the seat mount 115.

[0110] As described above, the apparatus (e.g., system, apparatus, or device) may include a controller. The controller may be configured to control the application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is installed in the seat mount. The controller may be configured to communicate with the optical sensor and may sense the identity of a code on a fluid vial or may sense the identity of a code on the microfluidic pathway device. The code on the fluid vial and / or the microfluidic device may be an optical code or an RFID code. The controller may be configured to communicate with the optical sensor and may sense the level of a reagent within a fluid vial. The controller may be configured to send instructions to the optical sensor drive to selectively position the optical sensor to sense the code or to sense the fluid level within a fluid vial disposed in the reagent storage frame. The controller may be configured to control the time of introduction of a reagent within the microfluidic pathway device. The controller may be configured to control the amount of reagent driven within the microfluidic pathway device. The controller may be configured to control positive gas pressure to at least one fluid input of the microfluidic pathway device. The controller may be configured to isolate at least a portion of the product within a subregion of the microfluidic device for export. In some variations, the controller may be configured to perform an in vitro transcription (IVT) reaction within the microfluidic pathway device. The controller may comprise a memory, one or more data stores.

[0111] The apparatus may include one or more processors configured to direct and / or control the apparatus, and the one or more processors may analyze information from the apparatus and / or the microfluidic pathway device.

[0112] The device may include a user interface for inputting / exporting instructions and at least one of information about the status of the device, the identity of reagents within the device, and the workflow being performed. In some variations, the device may include a graphical user interface configured to provide input to the processor.

[0113] The device may include a remote database for data and image storage and retrieval. Identification codes, visual logs, and other information may be stored in any format suitable for operation of the device and / or to meet regulatory requirements for manufacturing and dispensing individual therapeutic drugs.

[0114] Generally, the devices described herein may include one or more disposable microfluidic pathway devices and reusable components or subsystems, and selected portions of these subsystems may be sterilized. For example, one or more of the fluid sample holder (all or a portion thereof, such as a fluid container holder, tubing, etc.), the fluid interface assembly (all or a portion thereof), and / or the seat mount (all or a portion thereof, such as a seat portion) for the microfluidic pathway device may be removable, sterilizable, and replaceable. The device may be configured to allow one or more of these regions to be detached and removed from the device. For example, the device may include a seat mount release control configured to detach the seat mount from the device so that they can be sterilized separately, and / or a fluid interface assembly release control configured to detach the fluid interface assembly from the device so that they can be sterilized separately, and / or a fluid sample holder release control configured to detach the fluid sample holder from the device so that they can be sterilized separately. The release mechanism may be a lock release, one or more screws, pins, hinges, etc. Any of these devices may include integrated guides or mounts that support various portions of the device and may be configured to lift (either automatically, manually, or semi-manually) portions of the device away from other areas, allowing access and removal / replacement of certain components, such as all or part of the mount, fluidic interface assembly, and / or fluidic sample holder.

[0115] (Microfluidic Pathway Device) The apparatus described above is configured to support and control operations in a microfluidic pathway device and to perform polynucleotide processing. The polynucleotide may be any type of polynucleotide, including but not limited to ribonucleic acid, deoxyribonucleic acid, etc. The polynucleotide may comprise only natural nucleotide units or any type of synthetic or semi-synthetic nucleotide units. Processing may include, but is not limited to, in-vitro synthesis, purification, concentration, preparation, and analysis. stomach.

[0116] An example of a microfluidic pathway device for synthesizing therapeutic polynucleotides in a closed channel is shown in Figures 9A-9B, 9F, 10A-E, and 11, where Figure 9A is a view of the upper surface 911 of device 900 looking down through the multiple layers that form device 900, and Figure 9B is a perspective view of device 900. Figures 10A and 10C-10H are side views through the layers of device 900, showing an example arrangement of layers and channels, chambers, and ports formed therethrough.

[0117] FIG. 10A shows an example of the arrangement of layers in a microfluidic pathway to form chambers, including seals, channels, valves, and pump chambers. Generally, these devices may be advantageously formed from a rigid or semi-rigid plate and at least one elastic layer. The elastic layer may be a sheet of elastic material that is liquid-impermeable. The elastic layer may be slightly gas-permeable or may be treated as being more or less gas-permeable in various regions. A single continuous sheet of elastic material may be used, although in variations, multiple sheets of elastic material may be used, or a "sheet" may be formed from sections of multiple sheets. The layers and elastic sheets may be superimposed together. Generally, chambers for holding, valving, and / or pumping fluids may be formed in the plate on either side of the elastic layer, allowing the elastic layer to bisect the chamber into a liquid-containing side and a pressure (e.g., gas) application side. The total volume of the chamber may be constant and may be formed in both the first (e.g., upper) and second (e.g., lower) plates, but this volume may be divided into a pressure side and a liquid side. By applying positive or negative pressure to the pressure side, the elastic sheet may be deformed, decreasing the volume of the liquid-containing side (to zero, closing the chamber) or increasing the volume of the liquid-containing side (to a predetermined maximum value). The pressure-application side of the chamber may be connected to a pressure port, e.g., via a pressure channel in the upper plate (or between the upper plate and the elastic layer), for applying negative or positive pressure. The liquid-containing side may be connected to a fluid port via a fluid channel, as described herein. As described in more detail herein, both the fluid port and the pressure port may be formed by openings in the upper plate and the elastic layer, allowing for sealed connections that are isolated from the atmosphere, even with multiple different input lines.

[0118] In FIG. 10A, the microfluidic pathway device 900 includes a first (e.g., upper) surface formed on one side of a first plate 903. The first plate includes a first (e.g., top or upper) surface 911 and a second (e.g., bottom or lower) surface 929, with a thickness between the two. 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, with a thickness therebetween. 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 coupled, either directly or indirectly, to the second plate at 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 (lower or bottom) surface and a thickness therebetween. The second surface of the third plate may form the bottom surface of the microfluidic pathway device. Either of the plates may be formed from a number of layers that may be stacked or otherwise connected together. For example, in FIG. 10A, the third plate 909 includes an optional second elastic layer 913 that may serve to connect the third plate with the second plate, the second elastic layer 913 in this example forming the first surface 935 of the third plate 909. The layers and plates shown in FIG. 10A may not be to scale (e.g., the elastic layer 907 may be thinner than the plates).

[0119] 10A may include multiple chambers 915, 916, 918, 920, each having a fixed volume, formed by cutout areas (e.g., circular / curved cuts) in the second (bottom) surface 929 of the first plate 903 and the first (top) surface 931 of the second plate 905, and the elastic layer 907 branches these chambers 915, each having a liquid-containing side 917 and a pressure (e.g., gas-containing) side 919. The microfluidic pathway device 900 may include multiple liquid (e.g., fluid) channels. In FIG. 10A, a single fluid channel 921 is shown extending from a fluid port 923 through the thickness of the first plate 903, through the elastic layer 907, through most of the thickness of the second plate 905, and down to a fluid channel opening 925 at the bottom surface 933 of the second plate, where the entire length of the fluid channel 921 running parallel to the bottom surface of the third plate is formed within the bottom surface 933 of the second plate and is surrounded by the top surface of the third plate 909.

[0120] With respect to fluid port 923, the diameter of the opening into first plate 903 forming fluid port 923 extending through the thickness of the first plate may be larger than the diameter of fluid channel opening 925 extending through elastic layer 907 to liquid (e.g., fluid) channel 921. 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 a fluid line or fluid line connection interface connecting to the fluid port.

[0121] Fluid channel 921 connects to the liquid-containing side 917 of first chamber 915. This first chamber may be configured as a valve, having a relatively small holding volume (a fixed volume) but which can be fully opened or closed by movement of elastic layer 907.

[0122] The microfluidic pathway device 900 also includes multiple pressure channels that can be independently controlled to apply positive and / or negative pressure. While a single pressure port 943 is shown in FIG. 10A and connected to the fourth chamber 920, each of chambers 915, 916, and 918 may be connected to a separate pressure port and pressure channel to independently manipulate and control the movement of the portion of the elastic layer 907 that bisects those chambers, independently adjust the valves of each chamber, and / or pump each chamber. In some variations, pressure ports may be shared among multiple chambers. In FIG. 10A, the pressure (e.g., gas) port 943 is similar to the fluid (e.g., liquid) port 925, with an opening that penetrates the first plate 903, down to the exposed elastic layer 907, and to an opening through the elastic layer that forms the pressure (e.g., gas) channel opening 945. Pressure channel opening 945 connects to pressure (e.g., gas) channel 947 that extends from pressure port 943 through most of the thickness of first plate 903, into a cutout channel along the bottom of the second plate (or alternatively into the top cutout region of the third plate), back through the second plate and elastic layer 907, and to the region of the pressure channel in the first plate that connects to pressure (e.g., gas) containing portion 919 of fourth chamber 920. As described for the analogous fluid (e.g., liquid) port, the diameter of pressure port 943 through the thickness of first plate 903 may be larger than the diameter of pressure channel opening 945 through elastic layer 907 and may be centered or offset beyond the wall thickness of the pressure line or pressure line connecting interface that connects to the pressure port.

[0123] In the cross-section of microfluidic pathway device 900 shown in FIG. 10A, there are numerous connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports that are not shown because they are outside the plane of the drawing. For example, in FIG. 10A, the liquid-containing side or portion 917 of the fourth chamber may be connected to additional valves (chambers) and / or channels, including, for example, outlet channels extending from the liquid-containing side 917. Additional chambers not shown (e.g., configured as valves) may be formed as described above. In some variations, the outlet channels may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid-receiving depot, e.g., a vial, tube, or the like. This receiving depot may be held within a reagent storage frame.

[0124] Generally, this microfluidic pathway device and arrangement of microfluidic devices are configured such that numerous complex steps can be performed by devices on the microfluidic pathway device in a completely closed (sealed and protected from the atmosphere) manner without the need for manual manipulation. Fluids may be metered using fixed volume chambers, moved by applying air pressure to deflect regions of an elastic layer, mixed, filtered, etc.

[0125] Returning to FIG. 9A, the microfluidic pathway device 900 may include at least one set of chambers 953, each of which may be like the chambers 920 described above, with a liquid (fluid) side 917, a pressure (e.g., gas) side 919, fluid connections, pressure connections, and fluid / pressure lines. Furthermore, each of the set may be connected to one another by a fluidic connector 955. The fluidic connector 955 may be used in conjunction with positive and / or negative pressure applied to the pressure sides of the chambers to drive liquid on the liquid side between the two chambers and mix this liquid within each of the chambers. Any liquid within the liquid between the two chambers may be driven by deflecting the elastic layer between the fixed volumes of the chambers bifurcated by the elastic layer.

[0126] Any of the microfluidic pathway devices described herein may include one or more connections for electronics, including electrical sensors, on the device. For example, in FIG. 9A , the microfluidic pathway device may include a region 982 configured to include one or more electrical contacts for communication with one or more sensors (or other electronics) on the microfluidic pathway device. In some variations, the electronics may include electrical circuitry, etc. The electrically active region 982 may include one or more conductors for making electrical contact with a device, and the electrical contact may provide power, data, etc. For example, the electrically active region may be configured as one or more conductor pads for connecting to one or more connector pins (e.g., spring-loaded or otherwise biased connector pins) that may be attached to or part of the upper nest (e.g., fluidic interface assembly).

[0127] The microfluidic pathway device 900 may include two or more sets of chambers, each set of chambers being used for a different process applied to polynucleotides. For example, a first set of chambers 953 may be used for polynucleotide synthesis. A second set of chambers 955 may be used for purification of the synthesized polynucleotides. Fluid in the first set of chambers 953 may be driven into the second set of chambers when pressure is applied to the pressure-receiving side 919 of each chamber and a valve 959 between the first set of chambers 953 and the second set of chambers 955 is opened. The valve chamber 959 may be formed by an elastic layer 907 within the connector channel between the two sets of chambers.

[0128] 9A and 9B, the microfluidic pathway device 900 may have a plurality of pressure ports 943 and fluid ports 923. The plurality of pressure ports and fluid ports may , may be disposed adjacent to the exterior surface of the microfluidic path device and configured to connect to the fluid interface assembly 109 as described above.

[0129] Ports (e.g., sealing valves) may be formed from an elastic layer as described above along the entire length of connecting channel 939 (either a pressure channel or a fluidic channel) as shown in FIG. 9A for valve 961, which may control the timing of delivery of reagents actuated from fluidic port 923, but which, when placed in series with one or more similarly configured valves, may enable metering into the chambers of the device. For example, in FIG. 9A, three valve chambers are shown (described in more detail below); the first of these three valves functions as a peristaltic pump, while the middle valve may be a metering chamber that meters small volumes (e.g., having a metering volume of approximately 10 nL, 20 nL, 25 nL, 50 nL, 75 nL, 100 nL, etc.). The port and channel structure is shown in FIG. 10A, described above. The size of the channels, and particularly the size of the chambers connected to the channels, allows for metered volumes dispensed along the fluidic connecting channels 939, 921 and delivered to the chambers 953 connected to the fluidic connecting channels 939, 921. In some variations, the metered volumes may be as small as 50 nL. Metered volumes of approximately 100 nL, 1 microliter, 5 microliters, or more may be imported. Various valve sizes may be preselected for incorporation into the microfluidic pathway device 900, and reagents may be connected to the appropriate metered size at the user's choice.

[0130] Additionally, two or more valve bodies 961 may be provided in series along the fluid connecting channel 939. A series of valves 961 may function as a peristaltic pump to move fluids, including (but not limited to) viscous fluids. The ability to function as a peristaltic pump for fluids in general may be particularly advantageous in moving fluids that may be viscous or that may contain suspended particles, such as purification or capture beads.

[0131] As mentioned above, the microfluidic pathway device 900 may include a delivery or export reservoir or depot 963. In FIG. 9A, a preselected volume may be formed similarly to the chamber configuration described above, or may include only a metering side, as desired. In either case, a valve may be used to meter the desired volume into the reservoir 963. Valve 965 can control the delivery of fluid from the reservoir 963. If a larger volume is desired, the delivery may be repeated. Alternatively, if the reservoir 963 is preselected to be an export reservoir, valve 965 may be opened to deliver fluid from chamber 957, while retention valve 967 may be closed to export only a measured volume of fluid to the reservoir 963. This fluid may then be exported to a fluid vial in a reagent storage frame for further processing or testing. In some variations, the chamber, reservoir, or depot (e.g., 963) may be configured as the metering portion of a 1 μL pump formed, for example, by a three valve structure (967, 965, 967). The chamber may be configured, for example, for waste export from the mixing chamber 957.

[0132] An advantage of the microfluidic pathway device 900 may be the closed pathway nature of its configuration. While fluid vials, fluid lines, and microfluidic pathway devices are connected, operation of the apparatus may be performed without exchanging materials in or out of the system, particularly in or out of the fluid pathways of the microfluidic pathway device, for processes including synthesis and formulation of polynucleotides for biological delivery (as therapeutics such as drugs, vaccines, etc.). Thus, the entire system may operate as a closed pathway, and / or individual microfluidic pathway devices may operate within the system as closed pathways (protected from the atmosphere).

[0133] Some variations of processing that can be performed within the microfluidic pathway device 900 may include purification. One variation of purification may involve incorporating a substance into the fluid side 917 of a chamber or channel. The substance may be configured to absorb a selected portion from the fluid mixture within the chamber or channel. In one variation, the substance may include a cellulose material capable of selectively absorbing double-stranded mRNA from the mixture. The cellulose material may be inserted into only one chamber of a set of chambers, such that mixing of fluids from the first chamber to the second chamber of the set effectively removes the double-stranded mRNA from the fluid mixture and subsequently transfers it to another set of chambers further downstream for further processing or export.

[0134] Some variations of microfluidic device 900 may further comprise a concentrator within a chamber disposed within the thickness of the second plate and that may be in fluid communication with an outlet channel such as 949. The polynucleotides may be concentrated by draining the excess fluid medium, and the concentrated polynucleotide mixture may be exported out of microfluidic pathway device 900 for further handling 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.

[0135] The microfluidic pathway device 900 may be formed from a material that is at least substantially translucent to visible and / or ultraviolet light. By substantially translucent, it is meant that at least 90% of the light is transmitted through the material, as compared to a translucent material. In some variations, the microfluidic pathway device 900 may be formed from a material that is substantially transparent to visible and / or ultraviolet light. By substantially translucent, it is meant that at least 90% of the light is transmitted through the material, as compared to a completely transparent material.

[0136] As described above, the first plate and / or the second plate may be formed from a rigid material. The third plate may also be formed from a rigid material. In some variations, the third plate may be formed from a rigid material overlaid on a resilient material. The plates may be formed from the same or different materials. For example, the rigid material may be a polymer or glass. The polymer or glass may be biocompatible, e.g., it may not filter any monomers or soluble small molecules that are toxic to living cells. Polynucleotide products processed within the microfluidic pathway device may be administered to animals, so toxic contaminants are preferably reduced or eliminated by material selection. Suitable biocompatible polymers may be used, including medical-grade polycarbonate urethane, silicone polycarbonate urethane, and polyether urethane, among others. In some variations, the polymer may be a cycloolefin copolymer.

[0137] FIG. 9F shows another example of a microfluidic pathway device 900 similar to that shown in FIG. 9A, but configured to process larger volumes. For example, while the device shown in FIGS. 9A-9B is configured to process a specific volume per device cycle (e.g., 500 μg per IVT cycle), the variation shown in FIG. 9F may be configured to process up to five times this volume (e.g., 5 mg per IVT cycle). The high-volume microfluidic pathway device shown in FIG. 9F may have chambers 953, 915 that extend slightly outside the device, allowing for significantly larger chambers that are further divided into fluid-contacting and pressure-receiving sides by an elastic layer. In FIG. 9F, the area is roughly the same footprint and therefore may be held within the same sheet mount as the device in FIGS. 9A-9B, but may hold and process significantly larger fluid volumes. The controller may be adapted to automatically determine the size of the microfluidic pathway device chamber and / or the type of microfluidic pathway device (eg, template formation, IVT processing, etc.).

[0138] As noted above, a microfluidic pathway device may be configured such that the chambers are formed from the upper and lower surfaces of one or more plates that extend slightly outside the plane of the microfluidic pathway device, as compared to the variations shown in Figures 9A-9B. Any of the microfluidic pathway devices may be referred to herein as a microfluidic pathway plate device, as noted above. A plate, as used herein, may be generally planar, but may include one or more regions that extend above or outside the device, as shown in Figure 9F.

[0139] 10B shows an example of a portion of a microfluidic pathway device in a partially transparent perspective view. In this example, the device comprises a first plate 903 having first and second surfaces and a thickness therebetween, a second plate 905 having first and second surfaces 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 coupled to the second plate at the second surface of the second plate.

[0140] The portion of the microfluidic pathway device shown in Figure 10B also includes a section through three chambers 915, 914, and 918, each having a fixed volume. The chambers are formed within the second surface of the first plate and the first surface of the second plate, respectively, and a portion of the elastic layer bifurcates each chamber into a fluid-retaining side and a pressure-applying side. Fluid channels extend from fluid ports (not visible in Figure 10B) through the first plate to the elastic layer 907, through the elastic layer, to fluid channel openings through most of the thickness of the second plate, where they fluidly connect with connecting channels formed within the second surface of the second plate (or between the second and third plates). This connecting channel may be surrounded by the third plate. The fluid channels then extend back through the second plate to connect to the fluid (e.g., liquid)-retaining portion of one of the chambers, preferably configured as a valve. In Figure 10B, three chambers configured as valves are connected together, allowing fluid to be pumped between them in small, metered volumes (e.g., 10 nL). Negative pressure (or, in some variations, no pressure) may be applied to the pressure-retaining side 919 of chamber 915 to open the valve, pulling the elastic layer up and into the upper (pressure-retaining) side of the chamber. Positive pressure may be applied to close the valve, deflecting the elastic layer bifurcating the chambers against the curved wall below the fluid-retaining side of the chamber.

[0141] Pressure channels 947 may extend from pressure ports (not visible in FIG. 10B) formed as channels through the thickness of the first plate down into the elastic layer 907. Pressure channel openings may be formed through the elastic layer into the thickness of the second plate and fluidly connect with connecting pressure channels formed in the second surface of the first plate (not visible in FIG. 10B) or between the second and third plates, which may then extend back up (in a U-shaped path) through the second plate to channels formed in the first plate (or between the first and second plates) and connect to the pressure-retaining side of one or more chambers. The diameter of the fluid ports through the thickness of the first plate is typically larger than the fluid channel openings through the elastic layer, and the diameter of the pressure ports through the thickness of the first plate may be larger than the pressure channel openings through the elastic layer. Any of the microfluidic pathway devices may be configured to transport fluid from the fluid-retaining side of a chamber or channel through a second surface of a second plate and / or to a depot (e.g., holder, bottle, container, vial) located, for example, in a rack of a reagent storage frame. The chamber may also include an outlet channel extending through the chamber configured as a valve that can be opened to allow pumping into a fluid supply (such as a valve, tube, etc.).

[0142] In any of the microfluidic pathway devices described herein, fluid may travel from the top, through the first plate, through the seal formed by the elastic layer, through the second plate, and then back up along the second plate to a chamber (e.g., a chamber possibly configured as a valve) bifurcated by the elastic material. Similarly, pressure flow (positive or negative) may travel from the top, through the first plate, through the seal formed by the elastic layer, through the second plate, along the bottom of the second plate, and then back up, through the second plate and the elastic layer, and then along the bottom of the first plate, connecting to the pressure-retaining side of the chamber bifurcated by the elastic layer. Generally, the elastic layer may bifurcate the chambers by actuating them uniformly or non-uniformly; for example, the upper (pressure) chamber may be larger or smaller than the lower (liquid-retaining) chamber. The application of positive or negative pressure to control a valve and / or pump or meter a fluid within a chamber may be referred to herein as pneumatic or pneumatic barrier deflective ("pneumodeflective").

[0143] 10C-10H illustrate the operation of a microfluidic pathway device (similar to the exemplary device shown in cross section in FIG. 10A) engaging a microfluidic device, and in particular, having multiple fluid 1033 and / or pressure 1043 lines. In FIG. 10C, the microfluidic pathway device 900 is shown in cross section, and multiple fluid 1033 and / or pressure 1043 lines (two visible in FIG. 10B) are shown adjacent to the microfluidic pathway device, each independently aligned to a set of ports, including fluid port 923 (to which fluid line 1033 is aligned) and pressure port 943 (to which pressure line 1043 is aligned). In any of the variations described herein, the pressure and fluid lines may extend from the fluid interface assembly, as described above. Each pressure and fluid line may be coupled to an independent bias (e.g., biased toward the microfluidic pathway device) by a compression connector, as described above and shown in Figures 7A-7C. Because each fluid and pressure connection can be independently biased (e.g., separately driven toward the microfluidic pathway device) and deflected up / down to press and seal against an exposed elastic layer supported on one side by a rigid second plate, the pressure connection may form a highly tolerant seal with the pressure or fluid line, as shown in Figure 10C. Thus, the device has high tolerance for orientation adjustment, allowing the device to be slightly misaligned and / or angled relative to the fluid and / or pressure lines. The compression connector may exert a force against the supported elastic layer to maintain the seal and prevent contamination or exposure to the outside environment. A biasing element (e.g., a spring) may press the end of the fluid or pressure line against the microfluidic pathway device. The closer the pressure or fluid lines are to the microfluidic pathway device, the stronger the force applied, but each fluid or pressure line may be pushed back, for example, at least slightly upward.Finally, the apparatus may be configured to connect to the microfluidic pathway device around the periphery of the microfluidic pathway device, thereby distributing forces and allowing for balanced and supported contact with the microfluidic pathway device all from the same side of the device.

[0144] Fluid line 1033 and pressure line 1043 shown in FIG. 10D are spring-biased against fluid and pressure ports of a microfluidic pathway device similar to those shown in FIGS. 7A-7D, allowing their distal open ends to be driven against the plane of a first elastic layer 907 of the microfluidic pathway device, which is supported beneath the elastic layer by a second layer. Each pressure and fluid line may then be separately driven (as indicated by the arrows) against the elastic layer, e.g., by a spring or other biasing element, to form a seal against elastic layer 907. The separate pressure and fluid lines may then be independently controlled to direct fluid into fluid line 939 to valve and / or meter fluid within the microfluidic pathway device. For example, in FIG. 10E, fluid 1044 may be driven through fluid line 1033, through first plate 903, through an opening in elastic layer 907, into a channel through second plate 905, and until it reaches first chamber 915, configured as valve 919. First chamber 915 comprises 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 may be circular in shape as described herein, or may have cylindrical or straight walls. In this example, the valve is opened by applying negative pressure through a pressure line (not shown). First chamber 915 is fluidly connected to second chamber 916, configured as a metering chamber. In FIG. 10E, metering chamber 916 is opened by applying negative pressure to the upper pressure-receiving portion of the bifurcated chamber. In this manner, the chamber is maximally opened and has a known volume (e.g., 50 nL, 100 nL, 150 nL, 200 nL, 250 nL, 300 nL, 500 nL, etc.). The adjacent chamber 918 may be configured as a valve similar to 915 and may remain closed to allow the measurement chamber to fill completely. In some variations, the elastic layer may be partially permeable to air, eliminating air bubbles and allowing the fluid 1044 to fill completely.

[0145] As mentioned above, in some variations, the microfluidic pathway device may include one or more bubble removal chambers in which air bubbles within the fluid on the fluid-containing side may be removed, and / or any of the chambers on the fluid-contacting side of the chamber may be configured as a bubble removal chamber. A bubble removal chamber may be referred to as a vacuum cap and is generally configured to retain fluid within the fluid-contacting side of the chamber while applying negative pressure to the other side of the membrane. The membrane may be at least partially gas-permeable, as described above. Any of the pressure-receiving sides of the chambers in the microfluidic pathway devices described herein may be configured with one or more protrusions 988 on the upper side (pressure-receiving side) of the chamber that prevent the elastic layer separating the pressure-receiving side of the chamber from the fluid-receiving side of the chamber from seating against the top of the pressure-receiving side. In FIG. 9C, the pressure-receiving side includes elongated protrusions 988 to the pressure-receiving side, which prevent the elastic layer from sealing against the pressure-receiving side, thereby maximizing the surface area over which a vacuum applied to the pressure-receiving side will draw gas (e.g., air) through the gas-permeable elastic layer, removing air bubbles from the liquid.

[0146] The protrusion may extend any suitable depth into the pressure-receiving side. For example, the protrusion may be referred to as a spacer and may extend to the full depth of the pressure-receiving side, or to about 0.3 to 1 times (e.g., 0.4 to 1, about 0.5 to 1, about 0.6 to 1, etc.) the depth of the pressure-receiving side. In some variations, two or more protrusions may be used. The protrusion may be cylindrical and have multiple arms (e.g., extending from an apex) to maximize the amount of membrane separated from the wall of the pressure-receiving side, even when a vacuum is applied to the pressure-receiving side.

[0147] In some variations, the chamber formed by the pressure-receiving side and the fluid-containing side may therefore be slightly uneven in volume, as protrusions into the pressure-receiving side take up some of the volume. Accordingly, the elastic layer dividing the chamber may be in contact with vacuum through a vacuum line 987 separate from the top surface of the pressure-receiving side, as shown in Figures 9C, 10B, and 10J-10K (described below). In operation, the vacuum cap 938 may retain fluid within the fluid-contacting side of the chamber and remove or reduce air bubbles within the line by applying negative pressure to the upper (pressure-receiving) side of the chamber. As noted above, the elastic layer dividing the chamber into the fluid-contacting and pressure-receiving sides may be gas-permeable, allowing negative pressure to remove gas (e.g., air, nitrogen, etc.) from the liquid (fluid) side by drawing the gas through a membrane covering the fluid path. For example, the elastic layer (which may be a membrane) in a vacuum cap may be, for example, a PolyDiMethylSilicone (PDMS) elastomer membrane that is sufficiently gas permeable to allow gas removal from the liquid side of the membrane. A fluid chamber having a fixed volume (e.g., formed between a first plate and a second plate) as described herein may be coupled to and / or configured as one or more bubble removal chambers (e.g., a vacuum cap, a priming cap, a priming valve, etc.). In some variations, for example, a portion of the elastic layer disposed between the first and second surfaces that forms a chamber separating the fluid-contacting side of the second surface (and / or second plate) from the pressure-receiving side of the first surface (and / or first plate) is only minimally deflected (or not deflected at all) so as not to rest flat against the surface or wall of the pressure-receiving side. For example, the upper pressure-receiving side may be configured with corners and / or one or more protrusions so that the resilient layer remains exposed rather than resting flat against the upper pressure-receiving side.However, the fluid receiving side may also be curved (e.g., concave) so that the elastic layer is driven flat against the fluid receiving side without a hold-up region, allowing all or substantially all of the fluid on the fluid receiving side to be expelled when a positive pressure is applied to the opposing pressure receiving side.

[0148] To remove air (e.g., air bubbles), the controller may apply negative pressure to the pressure-receiving side of the vacuum cap, e.g., by closing valves on one or both sides (inlet and outlet) of the vacuum cap, e.g., by applying positive pressure to the pressure-receiving side of the valve, retaining the fluid within the vacuum cap region. The absolute amount (e.g., magnitude) of the applied negative pressure may be the same as or different from (e.g., less than) the negative pressure applied to deflect membranes in other chambers and / or may be the same as or different from (e.g., less than) the absolute value of positive pressure applied to close valves and / or pumps. Alternatively, in some variations, the membrane may be configured to deflect (e.g., deflect upward) against the first surface and / or plate, e.g., to draw fluid into the enlarged fluid-contacting side of the chamber. As described above, negative pressure on the pressure-receiving side of the elastic layer may be maintained to remove gas (e.g., air bubbles) through the membrane. The controller may receive input (e.g., from one or more optical sensors) detecting air on the fluid-contacting side, e.g., by detecting one or more air bubbles, and may apply a vacuum in the vacuum cap until the air is removed. In some variations, the controller may hold the fluid in the vacuum chamber for a period of time sufficient to remove all or some 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, 1 second to 5 minutes, 2 seconds to 5 minutes, 5 seconds to 5 minutes, etc.). In FIG. 9C, pressure is applied to the first and second surfaces (e.g., 9C, the fluid may exit the fluid contacting side from fluid line 989 on the opposite side of the vacuum cap.

[0149] The fluid-contacting sides of the chambers of the pressure cap (similar to the valves and reactors described herein) may be in fluid communication with a fluid port that fluidly connects the fluid-contacting sides of each of the chambers via one or more fluid channels that may be in a second surface and / or plate. The pressure-receiving side of the vacuum cap may be in fluid communication with a pressure-receiving port or pressure ports that fluidly connect with the pressure-receiving side via pressure channels that extend through (e.g., and into) a first surface / plate and through a second plate as described herein and along the first plate.

[0150] 9D-9E show another variation of a vacuum cap 938'. In this example, the vacuum cap may be configured so that the pressure-receiving side 944 of the chamber has a corner, as shown. A resilient layer 948 separates the chamber into a pressure-receiving side 944 and a fluid-contacting side 946. In FIG. 9B, the resilient layer is in a neutral position. Fluid may be driven into the fluid-contacting side, as shown in FIG. 9E, and air may be removed by applying a vacuum (negative pressure) to the pressure-receiving side 944. The pressure-receiving side in this example also includes a protrusion 988, as shown in FIG. 9E, that prevents the resilient layer 948 from pressing against the wall of the pressure-receiving side, leaving a larger surface area of ​​the resilient layer exposed and allowing more air to be removed through the resilient layer. In some variations, the device may include a variation of the vacuum cap configured as a priming valve or priming chamber. In this variation, fluid may be drawn from one or more fluid depots into the chambers of the microfluidic pathway device by drawing the fluid into channels and one or more chambers and / or priming chambers or valves. Figure 10B shows an example of a priming valve, and Figures 10J-10K show the operation of a priming chamber or valve.

[0151] In FIG. 10F, the first chamber (valve 915) may be closed by applying positive pressure at the pressure-receiving portion above the first chamber. This limits the amount of fluid in the metering chamber 916 to a precise volume, and this metered fluid may then be released into another chamber (e.g., a fourth chamber configured as a mixing chamber, etc.) by opening the valve and holding / mixing chamber (FIG. 10G) and applying positive pressure to drive the fluid from the metering chamber 916 into the holding / mixing chamber 920 (FIG. 10H), as shown in FIGS. 10G-10H. Valve 918 may then be closed, as shown in FIG. 10I. The fluid in the mixing chamber may be combined with additional fluids (e.g., from other portions of the microfluidic device or other metered volumes in the same fluid path as shown) and mixed or otherwise processed within the chamber.

[0152] Any of the microfluidic pathway devices described herein may be a microfluidic pathway plate device, as described above, in which the device is sufficiently thin. Thus, processing in / on the plate, including purification of any polynucleotide (e.g., mRNA), may be performed substantially in two dimensions (2D). Purification of polynucleotides in 2D is particularly advantageous compared to prior art techniques that require the use of columns and may involve steps that are difficult or impossible to perform in a closed-circuit environment and / or in the small volumes described herein.

[0153] Additionally, as shown in the figures (e.g., FIGS. 10A-10I), the fluid-contacting side (and / or pressure-receiving side) of each chamber may be configured such that when positive pressure on the pressure-receiving side drives the elastic layer against the fluid-contacting side, the elastic layer rests flat against the fluid-contacting side at the second surface without any gaps. In some variations, the fluid-contacting side and / or pressure-receiving side may be concave. The concave surface may have a somewhat shallow elliptical cross-section, allowing the elastic layer to easily rest flat against the wall of the fluid-contacting side (and / or pressure-receiving side). The elastic layer may be pressed (e.g., rests) against the wall of the chamber, eliminating any deadholds (e.g., on the fluid-contacting side) of the chamber.

[0154] In addition to valves that open and / or close channels, the first elastic layer may be used to pump fluid in and out of the chamber, as described above. For example, in some variations, a chamber (e.g., accessible through a fluid channel with one or both valves open) may be provided and filled with fluid from a fluid port. Negative pressure may be applied from a pressure port connected to the upper half of the bisected chamber (divided by the elastic layer). Application of negative pressure may assist in priming the device by drawing fluid into the channel and removing air through the elastic layer. Thus, in any of the variations described herein, the elastic layer may be gas-permeable. Once primed, fluid may be expelled from the chamber by opening a distal valve and applying positive pressure to the other side of the elastic layer, driving the fluid out of the chamber.

[0155] Any of the chambers 915, 916, 918, 920 in the examples shown in Figures 10A-10I may be configured as a priming chamber or priming valve (typically, the priming chamber is larger than the priming valve, and in some variations, the priming chamber may be scaled). The priming valve may be used to draw fluid into the microfluidic pathway device and remove flowing air by introducing fluid (e.g., driven by positive pressure from one or more fluid depots attached to the microfluidic pathway device) and / or by applying pumping force from one or more chambers (e.g., pump chambers) in the plate. In some variations, the fluid is driven primarily or initially by the application of positive pressure from the depots.

[0156] 10J-10K illustrate the operation of a microfluidic device with a priming valve 938″. In FIG. 10J, the microfluidic device is similar to that shown in FIGS. 10A-10I, but with the addition of a priming valve in fluid communication with the input (fluid port 923) of fluid line 1033. Positive pressure applied at a fluid depot connected to the fluid line can drive fluid into the microfluidic pathway device, but air in the line may prevent or make driving fluid into the device difficult. As shown in FIG. 10K, once the microfluidic device is sealed to fluid line 1033 and pressure line 1043 (as described above), fluid can be driven into the channel connected to the first priming valve 938″. Chambers or valves 915 downstream of the priming valve may be closed by application of positive pressure in a pressure line connected to the chamber / valve 915, while negative pressure (vacuum) may be applied to the priming valve to remove air flowing ahead of the liquid in the channel. The controller may then (e.g., after allowing sufficient time based on a predetermined time and / or after sensing, e.g., optically, that the air has been removed) open the closed chambers or valves 915, as described above with respect to Figures 10B-10I, to allow the primed fluid to move into the device. In some variations, particularly when the elastic layer is permeable to air, air may be removed from each of the chambers / valves, including using negative pressure in a pressure-receiving valve.

[0157] Any of the devices described herein may be used as described and illustrated above. For example, the methods and devices described herein may be particularly useful for use in producing mRNA therapeutics using in vitro transcription (IVT), as described above. For example, the methods and devices may synthesize therapeutics containing one or more mRNAs in a single, continuous fluid pathway that provides an RNase-free environment. These mRNAs may be customized for individual patients.

[0158] [Example]

[0159] Any of the devices described herein may be used, for example, to manufacture therapeutics, including, in particular, mRNA therapeutics. For example, a system as described herein may comprise an integrated hardware-software system in which each batch of therapeutic (including both drug substance and drug product) may be produced within a dedicated, disposable, easily disposable microfluidic pathway device (which may be referred to as a chip or biochip). Therapeutic production may proceed in a sterile, closed-circuit system, with all production steps automated, enabling a copy-exact process. This may result in rapid improvement of "individual" production batches while providing the high levels of reproducibility, control, and quality required for the release of therapeutics for clinical use.

[0160] Any of the apparatuses described herein may be used with one or more microfluidic pathway devices, and in some variations, different microfluidic pathway devices may be used in series or in parallel by the same apparatus to perform different portions of a procedure. For example, in one variation in which therapeutic mRNA is produced, a first microfluidic pathway device may be used for DNA template production as part of a template microfluidic pathway device ("template biochip"). The resulting template may be transferred in a closed loop by the system to a second microfluidic pathway device (e.g., from the first microfluidic pathway device to a depot within the system and / or directly to the second microfluidic pathway device). In some variations, the second microfluidic pathway device may be configured to perform in vitro transcription of mRNA and purification of material to produce a drug substance (e.g., in an "IVT biochip" or IVT microfluidic pathway device). The product from this second microfluidic pathway device may then be transferred (either directly or via an intermediate depot, for example, in a reagent storage frame) to a third microfluidic pathway device, such as a compounding microfluidic pathway device (e.g., a "compounding biochip"). Formulation may then be performed in the compounding microfluidic pathway device.

[0161] 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 in the manufacturing process in a continuous, closed circuit.

[0162] As illustrated above, the microfluidic pathway device may be installed within an apparatus (e.g., a system) that may include any of the elements described above. For example, returning to FIG. 2C, the apparatus (e.g., a system) may include a microfluidic pathway device 250, a microfluidic pathway device management system 260 or device, a control panel (e.g., user input, monitoring, and analysis), and, in some variations, a temperature-regulated (e.g., refrigerated) environment such as a cabinet 270. The system may support all production activities within the microfluidic pathway device, such as reagent supply, fluid control, temperature control, mixing, purification, and process monitoring. Manufacturing activities in the system may be accessed and controlled through application software.

[0163] The microfluidic pathway devices and apparatus (e.g., systems) for operating them described herein may serve as reactors for the manufacturing steps performed in the three different microfluidic pathway device types described above. For example, the template microfluidic pathway device, the IVT microfluidic pathway device, and the compounding microfluidic pathway device may be configured with mechanisms for performing a series of unit operations in a controlled and highly reproducible manner. As described above, microfluidic pathway devices are typically multilayer structures.

[0164] For example, a microfluidic pathway device may include cyclic olefin copolymer (COC) and silicone. The COC layer may be made of TOPAS 5013L-10, and the silicone layer may be made of Wacker Silpuran medical-grade silicone. Features for each layer may be created by machining (prototype stage) or injection molding (production stage). Fabrication of the microfluidic pathway device may include sterilizing the layers with 100% isopropanol, silicon oxide sputtering, oxygen plasma activation, vacuum bonding, marking the microfluidic pathway device (e.g., barcoding and / or RFID labeling), sterilizing the assembled microfluidic pathway device (e.g., by UV-C or Gamma Ray sterilization), and storing the microfluidic pathway device in a sterile wafer mask handling box. Oxygen plasma exposure sterilizes the individual layers prior to assembly, but subsequent sterilization may add an additional level of sterility assurance. Different microfluidic pathway device types may have different designs as shown, for example, Figures 12A-12C show schematic examples of template (Figure 12A), IVT (Figure 12B), and compounding (Figure 12C) microfluidic pathway devices. All of these example microfluidic pathway devices may share a similar basic architecture and number of functional elements that can be used in different configurations to perform different protocols. Functional elements may include input ports, metering valves, pumps, reaction chambers, mixing structures, and purification structures as described above.

[0165] Figures 12D-12E show alternative embodiments of the microfluidic pathway devices shown in Figures 12A-12C. Figure 12D shows a diagram of another example of a template microfluidic pathway device configured to form templates in a closed-path system as described herein; specifically, the microfluidic pathway device shown in Figure 12D may be used to form synthetic (e.g., non-bacterial) templates. Figure 12E is another example of an mRNA in vitro transcription (IVT) microfluidic pathway device similar to that shown in Figure 12B. Figure 12F is another example of a formulation microfluidic pathway device similar to that shown in Figure 12C, which may be used to encapsulate polynucleotides (e.g., mRNA) in a delivery vehicle as described herein. Any of these microfluidic pathway devices may be sterilized using, for example, gamma irradiation and packaged aseptically. The microfluidic pathway device may be configured to process batches of a predetermined size (e.g., about 5 mg of mRNA / 2-4 days, about 50 mg of mRNA / 2-4 days, about 100 mg of mRNA / 2-4 days, etc., or in some variations, 2 g / week).

[0166] The microfluidic pathway device may interface with a control system through a series of spring-loaded connections for both reagents and pneumatic lines used to manage fluid movement and valve control. Reagent and gas lines may be sealed by pressure against the elastomeric layer of the microfluidic pathway device, creating a completely sealed pathway from the reagent vial to the biochip and from the biochip to the export vial. Figure 13 shows an example of a sealed pathway that may be maintained throughout all reactions within the microfluidic pathway device, effectively preventing any contact with the atmosphere and minimizing the risk of contamination. Figure 13 shows a cross-sectional view of an example microfluidic pathway device installed within a control system, in which the microfluidic pathway device 1301 is mounted within a seat mount 1305 and a fluid interface assembly 1307 connects fluid and pressure lines from a reagent storage frame 1309. The closed fluid pathway 1315 originates from a reagent storage depot in the reagent storage frame 1309 and enters the microfluidic pathway device 1301 for processing (via fluid lines held by the fluid interface assembly to the microfluidic pathway device 1301). The product is subsequently exported from the microfluidic pathway device back to the depot 1313 in the reagent storage frame.

[0167] The microfluidic pathway device control system (e.g., controller hardware / software, seat mount, fluid interface assembly, reagent storage frame, sensors, etc.) may provide the backbone for all electronic and hardware components. The microfluidic pathway device control system may be sterile and maintain a controlled environment. The system may provide interfaces for loading reagents and retrieving outputs, hold the microfluidic pathway device, and provide single-step connections to all actuators.

[0168] The microfluidic pathway device control system may monitor and control the operation of the device via one or more sensors, as described above. For example, the microfluidic pathway device control system may scan all reagent and microfluidic pathway device barcodes and monitor fluid levels. The microfluidic pathway device control system may automate all microfluidic pathway device functions. As described above, these microfluidic pathway device control systems may generate visual records of all process steps and / or provide optical quality control (QC) analysis of intermediate process outputs.

[0169] The microfluidic pathway device control system (which may also be referred to herein as a management system) may include the components described above, such as a seat mount ("nest" or "holder") that can be configured to precisely align the microfluidic pathway device during use, e.g., allowing the microfluidic pathway device to be inserted only in a single orientation. For example, pins (e.g., two dowel pins) and / or notches in the nest may be aligned with the shape of the microfluidic pathway device. The microfluidic pathway device management system may include a vial rack to hold reagent and export vials, a downward-facing camera to record all fluid and valve movements, and product export. A side camera on a rail may capture barcodes and detect fluid levels, and a robotic arm with a magnet, for example, may be controlled for bead manipulation. The microfluidic pathway device may be held in place by a vacuum chuck that ensures good contact with a thermal control device (e.g., a Peltier element) for temperature control. Once the microfluidic pathway device is in place, in some variations, connections to all connectors may be achieved in a single step by lowering the top of the microfluidic pathway device management system through a dowel-guided system.

[0170] The control panel may be configured as the main interface for all electronic devices (e.g., CPU, Ethernet RIO device controller) and pneumatic control valves and manifolds and pressure regulators. In some variations, the microfluidic pathway device control system may be kept in a refrigerated container or cabinet (e.g., an ISO Class 5 safety cabinet) that provides a microbiologically safe enclosure through HEPA air filtering and airflow management, ensuring all reagents are maintained at the correct temperature throughout the manufacturing process. The cabinet may also include UV lamps for sterilization of the microfluidic pathway device and all internal microfluidic pathway device management system components. In some variations, the microfluidic pathway device control system may reside in a mini-environment (e.g., a 6-foot by 6-foot ISO Class 5 mini-environment) that may itself be within a clean room (e.g., an ISO Class 7 room). All operator interactions with the system, including loading reagent vials and biochips, may be performed following aseptic methods. All reagents and consumables may be placed in a double-bagged area, wiped clean, and opened in a sterile environment to control contamination risk.

[0171] The microfluidic pathway device operating system described herein may be automated by a controller. The controller may load a process protocol that defines the type of microfluidic pathway device and the reagents to be used and ensure the correct microfluidic pathway device type is used. The controller may 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 may execute a series of steps defined in the protocol, control automated valves, pumps, and blender actuators, temperature controllers, cameras, magnetic arms, and other necessary controllers. The controller may create a batch log of events and process parameters and record measurements from peripheral devices and in-line measurements involving light sources and detection systems. In some variations, this log may be stored as a full digital batch record in the cloud.

[0172] In use, an operator may select a protocol to run, for example, from a library of preset protocols, or a user may input a new protocol (or modify an existing protocol). From the protocol, the controller tells the operator which microfluidic pathway device type to use, what to do with the vial contents, and where to place the vial within the nest. The operator may load the microfluidic pathway device, necessary reagents, and export vials into the system. The application may verify the presence of required peripherals, identify the microfluidic pathway device, scan identifiers (e.g., barcodes) for each reagent and product vial, and ensure that the vials match the bill-of-reagents for the selected protocol. After verifying the starting materials and necessary equipment, the controller may execute the protocol. During execution, valves and pumps operate to deliver reagents, reagents are mixed, temperatures are controlled, reactions occur, measurements are performed, and products are pumped into destination vials. At the end of the protocol, a production batch record is created in the cloud. The batch record is encrypted and the system measurements are uploaded to the cloud. An example data flow map is shown in Figure 14, which illustrates some of the controller's functions.

[0173] As used herein, the term "processing polynucleotides" may include many types of operations, including, but not limited to, synthesizing polynucleotides, purifying polynucleotides, concentrating solutions containing polynucleotides, preparing polynucleotides, and any combination thereof. As used herein, the term "substantially horizontal" refers to a surface that is substantially horizontal. When defined as horizontal, it means that the surface is within + / - X degrees of horizontal to the ground (e.g., X may be, for example, 0.1 degrees, 0.5 degrees, 1 degree, 2 degrees, 3 degrees, 5 degrees, 10 degrees, etc.).

[0174] Any of the microfluidic pathway devices described herein may include a heat spreader in the microfluidic pathway device or in a portion of the microfluidic pathway device to provide consistent heating in this portion of the device. For example, Figures 15A-15B show another example of a microfluidic pathway device 1501 (shown here as an IVT microfluidic pathway device similar to that shown in Figures 12B and 12E above), but with a large heat spreader 1507 on the bottom side. In FIG. 15B, a microfluidic pathway device is shown comprising a plurality of reactors 1503, 1505 (as described above) fluidically coupled to the fluid-contacting sides of chambers formed between two surfaces of the microfluidic pathway device. The top of the device (as shown in FIG. 15A) comprises peripherally arranged fluid ports 1509 and pressure ports 1511. The microfluidic pathway device exhibits a plurality of fluid-power circuits between pressure ports 1511, actuated valves 1512, metering chambers 1513, and reactors 1505, 1503. All of these chambers, valves, and reactors may be formed as part of a fixed-volume chamber formed between a first surface and a second surface, in which an elastic layer may divide each chamber into a fluid-contacting side (reactor, metering chamber, etc.) at the second surface and a pressure-receiving side at the first surface (which forms part of the fluid-power circuit).

[0175] In the bottom view of the microfluidic pathway device 1501 shown in FIG. 15B, the device includes a heat spreader (e.g., copper or other highly thermally conductive material attached to the bottom of the microfluidic pathway device). The highly thermally conductive material can be, for example, copper, aluminum, silver, or a material such as highly thermally conductive pyrolytic graphite. The heat spreader 1507 can be mechanically attached to the microfluidic pathway device with fasteners and / or can be held in place with an adhesive. In some variations, the microfluidic pathway device heat spreader can include or be formed from a thermally conductive adhesive. The microfluidic pathway device can be thinner beneath the heat spreader, improving heat transfer to materials within the microfluidic pathway device. In some variations, the heat spreader can increase the rigidity of the microfluidic pathway device.

[0176] Although Figure 15B shows a single heat spreader 1507 at the bottom of the microfluidic pathway device, two or more heat spreaders may be used. For example, multiple heat spreaders may be used to create different temperature zones. The microfluidic pathway device may comprise a plastic material as part of its body (e.g., a plate), and plastics are generally poor thermal conductors, so lateral temperature differences between different zones of the microfluidic pathway device may be maintained. In some variations, the microfluidic pathway device may be just below the reactor region.

[0177] In some variations, the heat transfer area may be attached to the flat bottom and / or placed within a pocket in that part.

[0178] The devices described herein may comprise and / or be used with one or more isolation chambers. For example, in some variations, the devices described herein may be part of a therapeutic polynucleotide manufacturing "factory" that may produce, for example, therapeutic polynucleotides for delivery to a subject. The therapeutic polynucleotide may be, for example, therapeutic mRNA. Figures 16A-16B show an example of a device that may be used by itself as a factory device or as part of a parallel manufacturing unit. In Figure 16A, devices 1601, 1601' may comprise or be held within a Class 5 isolation cabinet 1603, which may itself be held within a Class 7 isolation space. In Figure 16A, the cabinet comprises two microfluidic control devices 1601, 1601'. The devices may be part of an assembly factory providing copy-exact GMP units that can automatically manufacture therapeutic polynucleotides, such as therapeutic mRNA, for rapid patient use. These devices may be highly reconfigurable, allowing for rapid deployment and low-cost production. In some variations, these devices may be deployed in on-demand manufacturing "factory" units. In some variations, these devices may be set up as part of a mobile unit that can be deployed to remote sites temporarily or for longer periods.

[0179] As used herein, when a feature or element is referred to as being "on" another feature or element, the feature or element is directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. When a feature or element is referred to as being "connected," "attached," or "coupled" to another feature or element, it should also be understood that the feature or element may be directly "connected," "attached," or "coupled" to the other feature or element, or that intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, features and elements so described or illustrated may be applicable to other embodiments. It will also be understood by those skilled in the art that a reference to a structure or feature being located "adjacent" to another feature may have a portion that overlaps the adjacent feature or a portion that is underlying the adjacent feature.

[0180] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention. For example, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates. It should be further understood that as used herein, the terms "comprises" and / or "comprising" specify the presence of 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 groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ."

[0181] Spatially relative terms, such as "under," "below," "lower," "over," and "upper," may be used herein to facilitate describing the relationship of one element or feature to another, as illustrated in the figures. It should be understood that the spatially relative terms are intended to encompass different orientations of the device during use or operation, in addition to the orientation depicted in the figures. For example, if the device in the figures were inverted, an element described as being "under" or "beneath" another element or feature would then be oriented "over" the other element or feature. Thus, the exemplary term "under" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. Similarly, the terms "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only, unless otherwise specified.

[0182] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element described below may be referred to as a second feature / element, and similarly, a second feature / element described below may be referred to as a first feature / element, without departing from the teachings of the present invention.

[0183] Throughout this specification and the claims that follow, unless the context dictates otherwise, the word "comprise," and variations such as "comprises" and "comprising," mean that various components may be used in combination in methods and articles (e.g., compositions and apparatuses, including devices and methods). For example, the term "comprising" should be understood to imply the inclusion of any stated elements or steps, but not the exclusion of any other elements or steps.

[0184] Generally, any of the apparatus and methods described herein should be understood to be inclusive, although all or a subset of the components and / or steps may alternatively be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" various components, steps, subcomponents, or substeps.

[0185] As used in this specification and claims, including when used in the examples, unless otherwise stated, all numbers may be read as if preceded by the word "about" or "approximately," even if the term does not explicitly appear. The terms "about" or "approximately," when used when describing a size and / or location, may indicate that the described value and / or location is within a reasonably expected range of values ​​and / or locations. For example, a numerical value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. It should also be understood that any numerical value given herein includes about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges subsumed therein. As will be appreciated by those of skill in the art, when a value is disclosed, it is understood that "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed. For example, if a value "X" is disclosed, "less than or equal to X" and "greater than or equal to X" (e.g., where X is a number) are also disclosed. Throughout the application, data is provided in a number of different formats, and it is understood that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, a particular data point "10" " and a specific data point "15" is disclosed, it is understood that numbers greater than, greater than, less than, less than, less than, or equal to 10 and 15 are also considered disclosed, as are numbers between 10 and 15. It is understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0186] While various exemplary embodiments have been described above, any of numerous modifications may be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, the order in which various described method steps are performed is often changed in alternative embodiments, and in other alternative embodiments, one or more method steps may be skipped entirely. Optional features of various device and system embodiments may be included in some embodiments but not in others. As such, 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.

[0187] The examples and figures included herein show, for purposes of illustration and not limitation, specific embodiments in which the subject matter may be practiced. As noted above, other embodiments may be used and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such embodiments of the inventive subject matter may be referred to herein, individually or collectively, by the term "invention," merely for convenience, and are not intended to spontaneously limit the scope of this application to any single invention or inventive concept, if in fact more than one is disclosed. Thus, while specific embodiments have been illustrated and described herein, any arrangements designed to achieve the same purpose may be substituted for the specific embodiments illustrated. The present disclosure is intended to cover any and all adaptations or modifications of the various embodiments. Combinations of the above embodiments, as well as other embodiments not specifically described herein, will be apparent to those skilled in the art upon reviewing the above description.

Claims

1. 1. A microfluidic apparatus for forming therapeutic polynucleotides, the apparatus comprising: a sheet mount for removably holding a microfluidic pathway device; a plurality of pressure lines; a plurality of fluid vials, each fluid vial either comprising a fluid line or configured to couple with a fluid line, wherein each fluid line, and at least a subset of the pressure lines, are configured to be biased relative to the microfluidic pathway device held in the sheet 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 within the microfluidic pathway device when the microfluidic pathway device is held in the sheet mount, the controller being configured to manage synthesis of synthetic templates, manage in vitro transcription (IVT) reactions using the templates to form therapeutic polynucleotides, and manage purification of the therapeutic polynucleotides within one or more microfluidic pathway devices held in the sheet mount.

2. a plurality of fluid vials, each fluid vial either comprising a fluid line or configured to couple with a fluid line, and each fluid line, and at least a subset of the pressure lines, configured to be biased relative to the microfluidic pathway device held within the seat mount to form a closed fluid path; and a controller configured to control the application of pressure through the pressure lines and drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is held within the seat mount, to determine the contents of the fluid vials, move sub-microliter quantities of material from the fluid vials to one or more reactors within the microfluidic pathway device held within the seat mount, direct synthesis of synthetic templates, and direct in vitro transcription (IVT) reactions using the templates to form therapeutic polynucleotides, and to control the flow of the therapeutic polynucleotides through one or more reactors held within the seat mount. and a controller configured to manage the purification of said therapeutic polynucleotide within a number of microfluidic pathway devices.

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

4. 4. The apparatus of claim 1, further comprising a plurality of optical sensors positioned to monitor fluid levels in the plurality of fluid vials and to monitor fluid movement within the microfluidic pathway device when the microfluidic pathway device is held within the seat mount.

5. The apparatus of any one of claims 1 to 4, further comprising a fluid interface assembly configured to hold the fluid line.

6. 6. The device of any one of claims 1-5, further comprising a fluid interface assembly release control configured to release the fluid interface assembly from the device.

7. 7. The apparatus of any one of claims 1 to 6, further comprising a reagent storage frame release control configured to release the reagent storage frame from the apparatus.

8. 8. The apparatus of any one of claims 1 to 7, further comprising a thermal control device configured to regulate a temperature of at least one region of the microfluidic pathway device when the microfluidic pathway device is held within the seat mount.

9. 9. The apparatus of any one of claims 1 to 8, further comprising a magnetic field applicator configured to apply a magnetic field to at least one region of the microfluidic pathway device when the microfluidic pathway device is mounted within the seat mount.

10. 10. The apparatus of claim 9, wherein the magnetic field applicator comprises a control arm attached to the reagent storage frame.

11. The apparatus of any one of claims 1 to 10, wherein the controller is configured to detect an identification code on the fluid vial.

12. 12. The apparatus of any one of claims 1 to 11, wherein the controller is configured to determine a level of reagent held by one or more of the plurality of fluid vials.

13. 13. The apparatus of claim 1, further comprising an optical sensor drive configured to operate one or more of a plurality of optical sensors around the seat mount or around the plurality of vials.

14. The apparatus of any one of claims 1 to 13, further comprising one or more adjustment pins configured to adjust the microfluidic pathway device within the seat mount.

15. 15. The device of any one of claims 1 to 14, further comprising an enclosure having a cover configured to maintain sterility of the device during operation of the device.

16. The apparatus of any one of claims 1 to 15, further comprising the microfluidic pathway device.

17. 17. The apparatus of any one of claims 1 to 16, further comprising a fluid interface assembly having a central opening through which the microfluidic pathway device is imaged by one or more of a plurality of optical sensors, and further wherein the sealed ends of each of the fluid and pressure lines are disposed around an outer surface of the central opening.

18. The apparatus of any one of claims 1 to 17, further comprising a signal detector configured to detect a signal from within the microfluidic pathway device.

19. 20. The apparatus of claim 18, wherein the signal is one or more of a visible signal, a fluorescent signal, a UV absorbance signal, or an IR absorbance signal.

20. 20. The apparatus of claim 18, wherein the signal detector is configured to measure a nanoparticle size distribution.

21. The apparatus of claim 18 , wherein the signal detector is configured to measure dynamic light scattering (DLS).

22. The apparatus of claim 1 , wherein the controller is further configured to isolate at least a portion of the product within a subregion of the microfluidic pathway device for export.

23. 23. The apparatus of any one of claims 1 to 22, wherein the seat mount secures the microfluidic pathway device so that the microfluidic pathway device is disposed in a substantially horizontal orientation.

24. 24. The apparatus of any one of claims 1 to 23, wherein the seat mount secures the microfluidic pathway device so that the microfluidic pathway device is oriented within 15 degrees of the horizontal.

25. 1. A microfluidic device for forming a therapeutic polynucleotide, comprising: a seat mount for removably holding a microfluidic pathway device; a plurality of pressure lines; and a plurality of fluid vials each pressurized by one or more pressure lines of the plurality of pressure lines, wherein each fluid vial either comprises a fluid line or is configured to couple with a fluid line, and wherein each fluid line, and at least a subset of the pressure lines, are configured to be independently biased relative to the microfluidic pathway device mounted in the seat mount to form a closed fluid pathway; and a controller configured to control the application of pressure through the pressure lines when the microfluidic pathway device is mounted in the seat mount to drive fluid movement within the microfluidic pathway device, and to apply pressure to one or more of the pressure lines to open or close valves within the microfluidic pathway device during operation.

26. 26. The apparatus of claim 25, wherein the controller is configured to perform an in vitro transcription (IVT) reaction within the microfluidic pathway device.

27. 26. The apparatus of claim 25, further comprising a reagent storage frame.

28. 26. The apparatus of claim 25, further comprising a plurality of optical sensors positioned to monitor fluid levels in the plurality of fluid vials and to monitor fluid movement within the microfluidic pathway device when the microfluidic pathway device is held within the seat mount.

29. 26. The apparatus of claim 25, further comprising a fluid interface assembly configured to hold the plurality of fluid vials.

30. 30. The device of claim 29, further comprising a fluid interface assembly release control configured to release the fluid interface assembly from the device.

31. 26. The apparatus of claim 25, further comprising a fluid sample holder release control configured to release the fluid sample holder from the apparatus.

32. 26. The apparatus of claim 25, further comprising a thermal control device configured to regulate a temperature of at least one region of the microfluidic pathway device when the microfluidic pathway device is installed within the seat mount.

33. 33. The apparatus of claim 32, wherein the thermal control device comprises a Peltier element.

34. a magnetic field adapter configured to apply a magnetic field to at least one region of the microfluidic pathway device when the microfluidic pathway device is mounted within the seat mount; 26. The apparatus of claim 25, further comprising a licator.

35. 35. The apparatus of claim 34, wherein the magnetic field applicator comprises a control arm attached to the reagent storage frame.

36. 26. The apparatus of claim 25, wherein the controller is configured to detect an identification code on the fluid vial held by the fluid sample holder.

37. 37. The apparatus of claim 36, wherein the identification code comprises a bar code.

38. 26. The apparatus of claim 25, wherein the controller is configured to determine a level of reagent held by one or more of the plurality of fluid vials.

39. 26. The apparatus of claim 25, further comprising an optical sensor drive configured to operate one or more of a plurality of optical sensors around the seat mount or around the plurality of vials.

40. 26. The apparatus of claim 25, further comprising one or more alignment pins configured to align the microfluidic pathway device within the seat mount.

41. 26. The device of claim 25, further comprising an enclosure having a cover configured to maintain sterility of the device during operation of the device.

42. 26. The apparatus of claim 25, further comprising the microfluidic pathway device.

43. 26. The apparatus of claim 25, further comprising a fluid interface assembly having a central opening through which the microfluidic pathway device is imaged by one or more of the plurality of optical sensors, and further wherein the sealed ends of each of the fluid and pressure lines are disposed around an outer surface of the central opening.

44. 26. The apparatus of claim 25, further comprising a signal detector configured to detect a signal from within the microfluidic pathway device.

45. 45. The apparatus of claim 44, wherein the signal is one or more of a visible signal, a fluorescent signal, a UV absorbance signal, or an IR absorbance signal.

46. 45. The apparatus of claim 44, wherein the signal detector is configured to measure a nanoparticle size distribution.

47. 45. The apparatus of claim 44, wherein the signal detector is configured to measure dynamic light scattering (DLS).

48. 26. The apparatus of claim 25, wherein the seat mount secures the microfluidic pathway device such that the microfluidic pathway device is disposed in a substantially horizontal orientation.

49. 26. The apparatus of claim 25, wherein the seat mount secures the microfluidic pathway device such that the microfluidic pathway device is oriented within 15 degrees of horizontal.

50. a fluid interface assembly including a plurality of fluid lines; a plurality of fluid vials configured to be pressurized; and a reagent storage frame including a plurality of holders each configured to hold a fluid vial of the plurality of fluid vials, each fluid vial either comprising one of the plurality of fluid lines or configured to connect to one of the plurality of fluid lines, and further configured such that each fluid line and at least some of the pressure lines are separately biased with a biasing force relative to the microfluidic pathway device mounted in the seat mount; and a controller configured to control the application of pressure through the pressure lines to drive fluid movement within the microfluidic pathway device when the microfluidic pathway device is mounted in the seat mount.

51. 51. The apparatus of claim 50, wherein each fluid line comprises a mechanical bias configured to drive the end of the fluid line against the microfluidic pathway device mounted within the seat mount with the biasing force.

52. 52. The apparatus of claim 51, wherein the mechanical bias comprises a spring.

53. 51. The apparatus of claim 50, wherein the fluid interface assembly is movably positioned on the seat mount.

54. 51. The apparatus of claim 50, wherein the fluid interface assembly is configured to move relative to the reagent storage frame.

55. 51. The apparatus of claim 50, wherein the fluid interface assembly has a central opening through which the microfluidic pathway device is imaged by one or more optical sensors.

56. 56. The apparatus of claim 55, wherein the sealed ends of each of the fluid and pressure lines are disposed around an outer surface of the central opening.

57. 51. The apparatus of claim 50, further comprising a signal detector configured to detect a signal from within the microfluidic pathway device.

58. 58. The apparatus of claim 57, wherein the signal is one or more of a visible signal, a fluorescent signal, a UV absorbance signal, or an IR absorbance signal.

59. 1. A microfluidic apparatus for forming a therapeutic polynucleotide, comprising: a seat mount for removably holding a microfluidic pathway device; a plurality of pressure lines, at least a subset of the pressure lines configured to be independently biased relative to a pressure input at the microfluidic pathway device mounted in the seat mount; a plurality of fluid vials configured to be pressurized, each fluid vial either including a fluid output configured to seal an input at the microfluidic pathway device or configured to couple with a fluid line configured to be independently biased relative to the microfluidic pathway device to form a sealed closed fluid pathway; a first optical detector configured to monitor fluid in the fluid vials; a second optical detector configured to monitor fluid in the microfluidic pathway device mounted in the seat mount; and an optical detector configured to receive inputs from the first and second optical detectors and to control the application of pressure through the pressure lines, apply pressure from the plurality of pressure lines, and activate valves. and a controller configured to open and / or close and facilitate fluid movement within the microfluidic pathway device based at least in part on the received input.

60. 60. The apparatus of claim 59, wherein the controller is configured to read an identification marker on each of the fluid vials.

61. 61. The device of claim 60, wherein the identification marker comprises one or more of a bar code, a QR code, and an alphanumeric code.

62. 60. The apparatus of claim 59, wherein the controller is configured to coordinate fluid movement through a plurality of chambers of the microfluidic pathway device.

63. 60. The apparatus of claim 59, wherein the controller is configured to simultaneously coordinate fluid movement through multiple chambers of the microfluidic pathway device.

64. 60. The apparatus of claim 59, wherein the controller is configured to store and / or transmit an optical record of the fluid movement within the microfluidic pathway device.

65. a plurality of fluid channels extending from a fluid port through the elastic membrane and the first surface to the second surface, each fluid channel fluidly connecting with the fluid contacting side of the plurality of chambers; and a plurality of pressure channels extending from one or more pressure ports through the first surface and the elastic layer to the second surface, each pressure channel fluidly connecting with the pressure receiving side of one or more of the plurality of chambers, and further wherein a volume of the fluid contacting side of each of the chambers can be adjusted by applying pressure from one or more of the pressure ports.

66. a plurality of fluid channels extending from a fluid port through the first plate region to the second plate region, the fluid channels fluidly connecting the fluid contacting side of one or more of the plurality of chambers; and a plurality of pressure channels extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region, the plurality of pressure channels extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region, the plurality of pressure channels extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region, the plurality of pressure channels extending from one or more pressure ports through the elastic layer to the first plate region, the plurality of pressure channels fluidly connecting one or more pressure receiving sides of one or more of the plurality of chambers. the fluid contacting side is concave such that when a positive pressure on the pressure receiving side drives the elastic layer against the fluid contacting side, the elastic layer rests flat and tightly against the fluid contacting side at the second surface.

67. a plurality of fluid channels extending from a fluid port through the first plate region to the second plate region, the plurality of fluid channels fluidly connecting the fluid contacting side of one or more of the plurality of chambers; and a plurality of pressure channels extending from one or more pressure ports through the first plate region and the elastic layer to the second plate region and back through the elastic layer to the first plate region, the plurality of pressure channels extending within the first plate region and fluidly connecting one or more pressure receiving sides of one or more of the plurality of chambers. a port channel opening onto an opening through said elastic layer having an opening diameter smaller than a diameter of said panel, and further wherein the diameter of said fluid channel second plate region is smaller than the diameter of said port channel.

68. A microfluidic pathway device for processing therapeutic polynucleotides in a closed path, comprising: 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 surface and the second surface, a portion of the elastic layer dividing each chamber into a fluid contacting side at the second surface and a pressure receiving side at the first surface; and a fluid port extending from the first plate region to the first plate region. a plurality of fluid channels extending from one or more pressure ports through the first plate region and elastic layer to the second plate region and fluidly connecting the fluid contacting side of one or more of the plurality of chambers; a plurality of pressure channels extending from one or more pressure ports through the first plate region and elastic layer to the second plate region and back through the elastic layer to the first plate region, each pressure channel of the plurality of pressure channels extending within the first plate region and fluidly connecting one or more pressure receiving sides of one or more of the plurality of chambers; and at least one vacuum cap between at least some of the plurality of chambers, the at least one vacuum cap comprising a bubble removal chamber formed between the first surface and the second surface, the elastic layer dividing the bubble removal chamber into a fluid contacting side of the bubble removal chamber at the second surface and a vacuum receiving side at the first surface, the fluid contacting side of the bubble removal chamber being in fluid communication with at least two of the fluid contacting sides of the plurality of chambers, and at least one vacuum cap, at least a portion of said elastic layer between said vacuum receiving side and said vacuum cap being gas permeable.

69. 68. The microfluidic pathway device of any one of claims 65 to 67, further comprising at least one vacuum cap between at least some of the plurality of chambers, the at least one vacuum cap comprising a bubble removal chamber formed between the first surface and the second surface, a gas permeable elastic layer separating the bubble removal chamber into a fluid contacting side of the bubble removal chamber at the second surface and a vacuum receiving side at the first surface, the fluid contacting side of the bubble removal chamber being in fluid communication with at least two of the fluid contacting sides of the plurality of chambers.

70. 70. The microfluidic pathway device of any one of claims 65 to 69, wherein the fluid contacting side and the pressure receiving side of the second surface are concave, and configured such that when a positive pressure on the pressure receiving side drives the elastic layer against the fluid contacting side, the elastic layer rests flat and tightly against the fluid contacting side of the second surface.

71. The microfluidic pathway device of any one of claims 65 to 70, wherein the first surface and the second surface are part of at least one plate.

72. 72. The microfluidic pathway device of any one of claims 65 to 71, wherein the first surface is part of a first plate and the second surface is part of a second plate.

73. 73. The microfluidic pathway device of any one of claims 65 to 72, wherein the one or more pressure ports and fluid ports are located adjacent an outer surface of the microfluidic pathway device.

74. 74. The microfluidic pathway device of any one of claims 65 to 73, wherein at least two of the chambers are fluidly connected.

75. 75. The microfluidic pathway device of claim 65, wherein the chambers comprise a plurality of sets of chambers, a first chamber of each set of chambers being fluidly connected to a second chamber of each set of chambers.

76. 76. The microfluidic routing device of claim 75, wherein a first set of chambers of said plurality of sets of chambers is connected to any of the other sets of chambers via a valved fluid connection.

77. The microfluidic pathway device according to any one of claims 65 to 76, wherein the microfluidic pathway device is a sealed pathway device.

78. 78. The microfluidic pathway device of any one of claims 65 to 77, wherein the microfluidic pathway device is at least substantially translucent to visible or ultraviolet light.

79. The microfluidic pathway device of any one of claims 65 to 78, wherein the microfluidic pathway device is substantially transparent to visible or ultraviolet light.

80. 80. The microfluidic pathway device of any one of claims 65 to 79, further comprising a substance inserted into the fluid contacting side of the channel.

81. 81. The microfluidic pathway device of any one of claims 65 to 80, wherein the material comprises a cellulose material configured to selectively absorb double-stranded mRNA.

82. 82. The microfluidic pathway device of any one of claims 65 to 81, further comprising a concentrator within the thickness of said second surface.

83. 83. The microfluidic pathway device of any one of claims 65 to 82, further comprising a dialysis chamber within the thickness of the second surface.

84. 84. The microfluidic pathway device of any one of claims 65 to 83, further comprising a delivery reservoir configured to deliver a preselected amount of fluid to said at least one chamber.

85. 85. The microfluidic pathway device of any one of claims 65 to 84, wherein the preselected volume of the chamber is between 50 nanoliters and 2 mL.

86. 86. The microfluidic pathway device of any one of claims 65 to 85, wherein the first surface and / or the second surface are formed from a hard material.

87. 87. The microfluidic pathway device of any one of claims 65 to 86, further comprising a third surface.

88. A microfluidic pathway device according to any one of claims 65 to 87, wherein the third surface is formed from a rigid material overlaid on an elastic material.

89. 89. The microfluidic channel device of claim 88, wherein the hard material is a polymer or glass.

90. 90. The microfluidic pathway device of claim 89, wherein the polymer is a cycloolefin copolymer.

91. 1. A method for processing fluids and forming therapeutic polynucleotides in a microfluidic pathway device, comprising: sealingly and independently coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of a microfluidic pathway device, each distal end being biased to be driven against an elastic layer between a first surface and a second surface, the microfluidic pathway device comprising a plurality of chambers each divided into a fluid contacting side formed on the second surface and a pressure receiving side formed on the first surface; and applying positive and negative pressures in the pressure receiving sides of the chambers to drive fluids through the fluid contacting sides of the plurality of chambers and change the size of the plurality of fluid contacting sides.

92. 92. The method of claim 91, wherein driving a fluid through the fluid contacting side comprises deflecting a resilient layer sandwiched between the first surface and the second surface.

93. 92. The method of claim 91, further comprising receiving optical feedback from the microfluidic pathway device to control the application of positive and negative pressure.

94. 92. The method of claim 91, further comprising controlling a valve by deflecting a resilient layer between the first surface and the second surface.

95. 92. The method of claim 91, wherein the driving step includes driving fluid through interconnected fluid contacting sides and manipulating valves to measure fluid movement between the fluid contacting sides of the plurality of chambers by application of positive and negative pressure.

96. 92. The method of claim 91, wherein the fluid contacting sides are interconnected.

97. 1. A method for processing fluid in a microfluidic pathway device, comprising: sealingly and independently coupling a distal end of each of a plurality of fluid lines and a plurality of pressure lines to a plurality of fluid or pressure ports on a surface of a microfluidic pathway device, each distal end being biased to be driven against an elastic layer between a first plate and a second plate; the microfluidic pathway device comprising a plurality of chambers each divided into a fluid contacting side formed in the second plate and a pressure receiving side formed in the first plate, the fluid contacting sides being interconnected; and driving fluid through the interconnected fluid contacting sides and operating valves to measure fluid movement between the fluid contacting sides of the plurality of chambers by application of positive and negative pressures within the pressure receiving sides of the chambers, and changing the size of the plurality of fluid contacting sides.

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