Method and apparatus for removing material from therapeutic compositions

Abstract

Methods and devices for the manufacture and use of therapeutics, particularly including mRNA therapeutics, that separate double-stranded RNA from single-stranded RNA as part of a continuous flow. These methods and devices can include formulating the RNA therapeutic using a permeable insert integrated into a microfluidic device. In particular, these methods and devices can include formulating the RNA therapeutic by removing double-stranded RNA from a solution of RNA within a microfluidic device that includes a cellulose material.

Description

Cross - References to Related Applications

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

[0002] [Incorporation by Reference] All publications and patent applications mentioned in this specification are hereby 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.

Technical Field

[0003] The present disclosure generally relates to methods and systems for manufacturing polynucleotide - based therapeutic agents quickly and in high yield. The present disclosure particularly relates to the automated manufacture of therapeutic mRNAs, including vaccines, and can be carried out quickly and efficiently. Such therapeutic agents may take into account patient - specific information and may be manufactured on - demand, either fully or partially, at the point of care (e.g., hospitals, clinics, etc.).

Background Art

[0004] Currently, manufacturing and formulation technologies for polynucleotide therapeutics, particularly mRNA therapeutics, often expose products to contamination and degradation. Currently available centralized production methods are too costly, too time-consuming, and too susceptible to contamination for use in therapeutic formulations that may contain multiple polynucleotide species. Developing scalable polynucleotide manufacturing, generating single-patient doses, eliminating touchpoints to prevent contamination, implementing input and process tracking to meet clinical manufacturing requirements, and enabling use in clinical settings can facilitate the availability of these promising therapeutics. Microfluidic equipment and processes can offer significant advantages in achieving these goals.

[0005] This specification describes methods and apparatus (e.g., systems) for manufacturing various therapeutic agents that can address the above-mentioned needs. [Overview of the Initiative] [Means for solving the problem]

[0006] This specification describes apparatus and methods useful for manufacturing a wide variety of vaccines and therapeutic agents. For example, described herein are methods and apparatus (e.g., systems, devices, etc.) for manufacturing personalized therapeutic agents, including vaccines. In one non-limiting example, the methods and apparatus described herein may be used to manufacture a therapeutic mRNA vaccine against cancer-specific antigens active in cutaneous T-cell lymphoma.

[0007] Therefore, generally speaking, what is described herein is an automated, high-yield method for producing mRNA therapeutics, which can be optionally implemented in clinical settings.

[0008] For example, this specification describes a method for forming (e.g., producing, creating, synthesizing) therapeutic polynucleotides using a system comprising a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising transporting reagents between one or more fluid depots and a plurality of reactors on one or more microfluidic devices in a sealed closed channel protected from contact with the atmosphere, in order to perform the steps of forming a synthesis template, producing therapeutic polynucleotides by in vitro transfer from the template, and purifying the therapeutic polynucleotides.

[0009] A method for producing therapeutic mRNA using a system comprising a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic plate devices, wherein the one or more microfluidic plate devices comprise a plurality of reactors, and the method may include the steps of: delivering template precursor material from one or more fluid depots to one or more first reactor regions of the plurality of reactors; processing the template precursor material to prepare a template from the template precursor material; transferring the template to one or more second reactor regions of the plurality of reactors; processing the template by in vitro transcription to form therapeutic mRNA; and transferring the therapeutic mRNA to one or more third reactor regions of the plurality of reactors; and purifying the therapeutic mRNA by two-dimensional (2D) purification within one or more third reactor regions, all of which are performed without exposing the template and therapeutic mRNA to contact with the atmosphere.

[0010] A method for producing therapeutic mRNA using a system comprising one or more microfluidic plate devices and multiple fluid depots in sealed fluid communication, wherein the one or more microfluidic plate devices comprises multiple reactors, the method comprising the steps of: using fluid power to deliver template precursor material from one or more fluid depots to one or more first reactor regions of the multiple reactors; processing the template precursor material to prepare a template from the template precursor material; using fluid power to transfer the template to one or more second reactor regions of the multiple reactors; and transferring the template to The procedure may include the steps of: processing the target to form therapeutic mRNA by in vitro transcription; using fluid power to transfer the therapeutic mRNA to one or more third reactor regions of a plurality of reactors and purifying the therapeutic mRNA by two-dimensional (2D) purification within one or more third reactor regions; using fluid power to transfer the therapeutic mRNA to one or more fourth reactor regions of a plurality of reactors and encapsulating the therapeutic mRNA with a delivery vehicle to form a therapeutic mRNA composition; and using fluid power to concentrate the therapeutic mRNA composition in one or more fifth fluid depots, all of which are performed without exposing the template and therapeutic mRNA to contact with the atmosphere.

[0011] The purification of therapeutic polynucleotides typically involves two-dimensional (2D) purification of the therapeutic polynucleotides in one or more reactors of a plurality of reactors. 2D purification may be performed in a substantially flat microfluidic device (e.g., a microfluidic plate device) as described herein and may involve the use of a material to remove material (e.g., double-stranded RNA) from the therapeutic polynucleotides. 2D purification of polynucleotides in a microfluidic device may require the use of a column and may be particularly advantageous compared to the techniques of the prior art, which may involve steps that are difficult or impossible to perform in a closed environment and / or in small quantities, as described herein. In one variation, the purification of therapeutic polynucleotides involves removing double-stranded mRNA using cellulose material in one or more reactors.

[0012] Any of these methods may further include formulating a therapeutic polynucleotide together with a delivery vehicle in one or more reactors on one or more microfluidic devices to form a therapeutic polynucleotide composition. The therapeutic polynucleotide (e.g., mRNA) may be encapsulated in a delivery vehicle as described herein, and in some modifications, may include additional mRNA in addition to the therapeutic mRNA, which may include adjuvant mRNA (e.g., mRNA encapsulating a protein that enhances the immune response). The delivery vehicle may include amphiphilic nanoparticles, such as aminolipidized peptoids.

[0013] In one modification, the therapeutic composition may be stored for later conjugation with a delivery vehicle (e.g., stored in a sealed enclosure, container, or vial at 4°C). The therapeutic composition may be transferred from a microfluidic device to a fluid depot in a microfluidic control device and used in the same microfluidic control device, or provided to a different microfluidic control device (e.g., geographically separated, including proximity to the hospital or clinic where it is to be used) for conjugation with a delivery vehicle. Conjugation of the therapeutic polynucleotide (e.g., therapeutic mRNA) with the delivery vehicle forms the therapeutic composition. The therapeutic composition may be further modified before use (e.g., injection into a patient).

[0014] For example, any of these methods may include dialysis and / or concentration of a therapeutic polynucleotide composition in one or more microfluidic devices. As used herein, dialysis may refer to the separation of particles in a liquid based on the difference in the ability of particles to pass through a membrane (dialysis membrane). In some modifications, dialysis includes countercurrent exchange.

[0015] The system may automatically and continuously perform the steps of forming a synthetic template, performing in vitro transfer from the template, and purifying therapeutic polynucleotides based on optical feedback from one or more sensors in the system.

[0016] In general, therapeutic polynucleotides may be mRNA. For example, therapeutic polynucleotides may be mRNA, circular RNA, or self-replicating RNA.

[0017] Generally, the system may transport reagents between one or more fluid depots and multiple reactors by fluid power, for example, using one or more fluid circuits. Fluid power generally refers to both pneumatic and hydraulic power. A fluid power circuit may include multiple elements (e.g., fluid lines, valves, etc.), and components of different fluid power circuits (also referred to herein as fluid circuits) may be shared by multiple fluid power circuits and switched via one or more valves under the control of a controller. For example, the system may transport reagents pneumatically between one or more fluid depots and multiple reactors. The use of fluid power controlled by a controller can advantageously enable non-contact control of processes used to form templates, therapeutic mRNA, and / or therapeutic compositions. For example, the system may transport reagents between one or more fluid depots and multiple reactors by deflecting one or more elastic layers within one or more microfluidic devices.

[0018] The methods described herein can be carried out entirely or partially in-situ (e.g., in a medical setting), as described above. Advantageously, the methods described herein may enable on-demand production of therapeutic mRNA without the use of preservatives or additives to therapeutic mRNA, which could reduce efficacy and / or cause complications. Since therapeutic compositions comprising therapeutic mRNA and a delivery vehicle may aggregate and cluster over time, in-situ formulation of the delivery vehicle into therapeutic polynucleotides (e.g., therapeutic mRNA) may be particularly beneficial. Furthermore, these methods can be carried out more rapidly than existing methods. For example, the system described herein may perform the steps of forming a synthetic template, performing in vitro transcription from the template to produce therapeutic polynucleotides, and purifying the therapeutic polynucleotides automatically and continuously in less than 5 days (e.g., less than 4 days, less than 3 days, etc.).

[0019] Any of these methods may further include sealing the fluid depot to one or more microfluidic devices and pressurizing the fluid depot before transporting reagents between the fluid depot and multiple reactors on one or more microfluidic devices. A controller may control the pressurization of the fluid depot.

[0020] In general, these methods and apparatus may record part or all of the manufacturing process, and this record may be optical (e.g., including video, etc., showing the movement of fluids within a microfluidic device) and / or non-optical sensor data (e.g., pressure measurements, temperature measurements). This manufacturing data may be stored, preserved, and / or transmitted for later review, including quality control and testing. Accordingly, any of these methods may include recording the movement of fluids within one or more microfluidic devices in a file related to the therapeutic polynucleotide being manufactured during the execution of the steps.

[0021] Any of the methods described herein can be performed automatically or semi-automatically by a system (e.g., a non-transient computer-readable medium encoding these instructions) including a computer (e.g., a processor) running software configured to perform all or part of these methods. For example, a non-transient computer-readable medium embodying instructions for producing therapeutic polynucleotides, when executed by a controller of a system including a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic devices, causes the controller to perform a method including pressurizing the plurality of fluid depots that are in fluid communication with one or more microfluidic devices, and transporting reagents between one or more fluid depots and a plurality of reactors on one or more microfluidic devices in a sealed closed channel protected from contact with the atmosphere, in order to perform the steps of forming a synthetic template, performing in vitro transfer from the template to produce therapeutic polynucleotides, and purifying the therapeutic polynucleotides (e.g., all within one or more microfluidic devices).

[0022] The command may further cause the controller to automatically and sequentially perform the steps of forming a synthetic template, performing in vitro transfer from the template, and purifying the therapeutic polynucleotide based on optical feedback from one or more optical sensors of the system. The command may further control the controller to purify the therapeutic polynucleotide by two-dimensional (2D) purification in one or more of the reactors, and / or to formulate the therapeutic polynucleotide with a delivery vehicle to form a therapeutic polynucleotide composition in one or more reactors on one or more microfluidic devices, and / or to dialyze and / or concentrate the therapeutic polynucleotide composition in one or more microfluidic devices.

[0023] Also described herein is an automated method of creating a synthetic double-stranded DNA template for mRNA synthesis using any of the closed-loop systems described herein. For example, an automated method of creating a synthetic double-stranded DNA template for mRNA synthesis using a closed-loop system comprising a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising, in a closed fluid path protected from contact with the atmosphere, transporting reagents between one or more of the plurality of fluid depots and a plurality of reactors on one or more microfluidic devices to bind the reagents, and forming a synthetic double-stranded DNA template for in vitro transcription of therapeutic mRNA.

[0024] The synthetic template (synthetic double-stranded DNA template) formed may contain no bacterial DNA and no endotoxin.

[0025] These methods may include receiving optical sensor data from one or more sensors of the closed-loop system in a controller of the closed-loop system, the controller controlling the operation of the closed-loop system based on the optical sensor data.

[0026] The method may include pressurizing the fluid depot, and / or transporting the reagents may include transporting a target synthetic gene and a synthetic in vitro transcription promoter cassette from one or more of the plurality of fluid depots to one or more first reactors within the microfluidic device, binding the target synthetic gene to the synthetic in vitro transcription promoter cassette to create a synthetic product, removing unreacted materials from the synthetic product, and amplifying the synthetic product to generate a synthetic double-stranded DNA template. The one or more microfluidic devices include a microfluidic plate device seated in the closed-loop system.

[0027] Advantageously, these methods involve creating a significant amount of template (mM amounts) compared to other systems which typically generate only femtomole amounts. The methods and apparatuses described herein can produce large amounts of template.

[0028] An automated method of creating a synthetic double-stranded DNA template for in vitro transcription using a closed-loop system that includes a plurality of fluid depots in sealed fluid communication with a microfluidic device includes transporting reagents including a synthetic gene of interest and a synthetic in vitro transcription promoter cassette from one or more of the plurality of fluid depots to one or more first reactors of the microfluidic device in a closed fluid path protected from contact with the atmosphere, combining the synthetic gene of interest and the synthetic in vitro transcription promoter cassette to generate a synthetic product, transporting the synthetic product within the microfluidic device to remove unreacted synthetic gene of interest and unreacted synthetic in vitro transcription promoter cassette from the synthetic product, and transporting the synthetic product within the microfluidic device and amplifying the synthetic product to generate a double-stranded DNA template. As described above, the step of amplifying the synthetic product may include generating an amplified DNA template that exceeds 1 mM.

[0029] Also described herein is an automated method for creating a synthetic double-stranded DNA template for in vitro transcription using a closed-loop system comprising a plurality of fluid depots in sealed fluid communication with a microfluidic device, the method comprising the steps of: transporting reagents, including a synthetic gene of interest and a synthetic in vitro transcription (IVT) accelerator cassette, from one or more fluid depots of the plurality to one or more first reactors of the microfluidic device in a closed-loop system protected from contact with the atmosphere; conjugating the synthetic gene of interest and the synthetic IVT accelerator cassette in one or more first reactors to produce a synthetic product; transporting the synthetic product to one or more second reactors of the microfluidic device to remove unreacted synthetic gene of interest and unreacted synthetic IVT accelerator cassette from the synthetic product; transporting the synthetic product to one or more third reactors of the microfluidic device to amplify the synthetic product to produce an amplified DNA template of more than 1 mM; and receiving optical sensor data from one or more sensors of the closed-loop system in a controller of the closed-loop system, the controller controlling the operation of the closed-loop system based on the optical sensor data.

[0030] For example, an automated method for creating a synthetic double-stranded DNA template for in vitro transcription using a closed-loop system including a microfluidic device and multiple fluid depots in sealed fluid communication includes the steps of: transporting the target synthetic gene and synthetic in vitro transcription (IVT) accelerator cassette from one or more fluid depots to one or more binding reactors of the microfluidic device using a first fluid-powered circuit in a closed-loop system protected from contact with the atmosphere; and binding the target synthetic gene to the IVT accelerator cassette to produce a synthetic product; and in the microfluidic device, using a second fluid-powered circuit, transporting the unreacted target synthetic gene and unreacted synthetic in vitro The process may include the steps of: removing a vitro transcription accelerator cassette from the synthetic product; using a third fluid-power circuit to transfer the synthetic product to one or more amplification reactors of a microfluidic device to amplify the synthetic product and generate an amplified DNA template of greater than 1 mM; and in a controller of the closed-loop system, receiving photosensor data from one or more photosensors of the closed-loop system, the controller controlling the first, second, and third fluid-power circuits based on the photosensor data to maintain the multiple fluid depots and microfluidic devices in a closed-loop sealed environment.

[0031] In all of these methods, the synthetic double-stranded DNA template does not contain bacterial DNA and does not contain endotoxins.

[0032] Any of these methods may include a step in the controller of the closed system receiving optical sensor data from one or more sensors of the closed system, and the controller controls the operation of the closed system based on the optical sensor data. The optical sensor data may be data from a camera or other imaging sensor. The methods described herein may use one or more fluid power circuits to move material between or within a plurality of fluid depots and microfluidic devices. The controller may adjust the operation of the fluid power circuits, or may adjust it using optical information. For example, the controller may determine that a fluid is in one or more parts of the closed system (e.g., depots, fluid lines, and / or areas of microfluidic sensors).

[0033] Any of these methods may include the step of transferring an amplified DNA template to one or more digestion reactors in a microfluidic device and enzymatically modifying the amplified synthetic product to generate a double-stranded DNA template.

[0034] As used herein, the step of conjugating a synthetic gene of interest with a synthetic in vitro transcription promoter cassette to produce a synthetic product may include producing a synthetic linear or circular ligation product. The conjugation step may be by ligation and / or by hybridization and / or annealing and / or primer extension. In one variation, the step of amplifying the synthetic product includes generating a linear, branched or circular amplified DNA product, and further including linearizing the amplified DNA product to generate a double-stranded DNA template. Ligation may include ligation by DNA ligase or ligation by primer extension. In one variation, the amplification step includes multiple substitution amplification (MDA). Alternatively, the amplification step may include polymerase chain reaction (PCR) amplification.

[0035] In one modification, the synthetic in vitro transcription accelerator cassette may consist of a double-stranded DNA template comprising a promoter, a 5'UTR, a cleavable linker, a 3'UTR, and a region encoding a polyA region consisting of at least 200 adenine residues or 200 thymidine residues. The double-stranded DNA template may include a polyA region of at least 300 bps in length at the 3' end of the synthetic gene of interest. Generally, the in vitro transcription accelerator cassette may be less than 1 kb in length. In one modification, the synthetic in vitro transcription accelerator cassette does not encode an antibiotic resistance gene and / or does not have an origin of replication (ORI).

[0036] Also described herein are automated methods and apparatus for performing in vitro transcription (IVT) using template materials (including, but not limited to, the above-described template materials) to form therapeutic mRNA. For example, described herein is a method and apparatus for automating an in vitro transcription (IVT) reaction using a system comprising a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising transporting reagents between one or more fluid depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere in order to perform in vitro transcription of therapeutic mRNA from a template in one or more microfluidic devices and to purify therapeutic polynucleotides.

[0037] For example, an automated method for performing an in vitro transcription (IVT) reaction using a system comprising a plurality of fluid depots configured to ensure sealed fluid communication with a microfluidic device, the method comprising the steps of: delivering a DNA template, polymerase, and nucleotides from one of the plurality of fluid depots and / or a portion of the microfluidic device to one or more IVT reactors of the microfluidic device; processing the DNA template and nucleotides in one or more IVT reactors to form therapeutic mRNA; and transferring the therapeutic mRNA to one or more purification reactor regions of the microfluidic device and purifying the therapeutic mRNA by two-dimensional (2D) purification within one or more purification reactor regions, wherein the microfluidic device and the plurality of fluid depots form a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0038] Generally, the system may include a controller for performing these methods, for example, the step of transporting a reagent by deflecting one or more elastic layers within a microfluidic device. These methods may include the controller of the system receiving optical sensor data from one or more sensors in the system, and the controller controls the operation of the system based on the optical sensor data. The controller may also control the pressurization of the fluid depot. In any of the methods described herein, the system may include a microfluidic device seated in the system.

[0039] Generally, a DNA template may consist of a double-stranded DNA template of the target synthetic gene and a synthetic in vitro transcription promoter cassette.

[0040] Any of these methods may include delivery and transport steps that use one or more fluid-powered circuits controlled by a controller to move DNA templates, polymerases, nucleotides, and therapeutic mRNA materials between or within multiple fluid depots and microfluidic devices. For example, the delivery and transport steps may be performed under the control of a controller, by deflecting one or more elastic layers within the microfluidic device to avoid contact with the atmosphere during the method.

[0041] In one modification, an IVT reactor is used, which may comprise a pair of connected chambers, each having a fluid-receiving portion and a pressure-receiving portion, the fluid-receiving portion being separated from the pressure-receiving portion by an elastic layer that can be deflected by the pressure-receiving portion to regulate the volume of the fluid-receiving portion. The DNA template may consist of a double-stranded DNA template of the synthetic gene of interest and a synthetic in vitro transcription accelerator cassette.

[0042] Any of these methods may include the step of sealing multiple fluid deposits by fluidizing them with multiple receiving ports on a microfluidic device.

[0043] For example, an automated method for performing an in vitro transcription (IVT) reaction using a system comprising multiple fluid depots configured to ensure sealed fluid communication with a microfluidic device may include the steps of pressurizing the multiple fluid depots, using one or more first fluid power circuits to deliver sub-microliter-precise amounts of DNA template, polymerase, and nucleotides from one of the multiple fluid depots and / or a part of the microfluidic device to one or more IVT reactors of the microfluidic device, processing the DNA template and nucleotides in one or more IVT reactors to form therapeutic mRNA, and using a second fluid power circuit to transfer the therapeutic mRNA to one or more purification reactor regions of the microfluidic device and purify the therapeutic mRNA by two-dimensional (2D) purification within one or more purification reactor regions, wherein the microfluidic device and the multiple fluid depots form a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0044] As described herein, what is described herein is software and / or firmware configured to perform any of the methods described herein. For example, what is described herein is a non-transient computer-readable medium embodying instructions for performing an in vitro transcription (IVT) reaction, which, when executed by a controller of a system including a plurality of fluid depots configured to ensure sealed fluid communication with one or more microfluidic devices, causes the controller to perform a method including, at any time during the reaction, by pneumatic pressure, delivering a metered amount of template material, polymerase, and nucleotides from the plurality of fluid depots to a first reactor of a microfluidic device, by pneumatic pressure; processing the template material and nucleotides in the first reactor to form therapeutic mRNA; and transferring the therapeutic mRNA from the first reactor through the microfluidic device by pneumatic pressure, such that the microfluidic device and the plurality of fluid depots form a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0045] As described above, in certain modifications, the methods and apparatus described herein may be used to formulate (e.g., compound) a therapeutic composition to encapsulate therapeutic mRNA in a delivery vehicle, for example, by automatically conjugating therapeutic polynucleotides (e.g., therapeutic mRNA) to a delivery vehicle. This may be done in an apparatus as described herein, and in certain modifications, may include forming a template and / or therapeutic polynucleotide. For example, a method for producing a therapeutic mRNA composition using a system comprising one or more microfluidic plate devices and a plurality of fluid depots in sealed fluid communication, wherein the one or more microfluidic plate devices comprises a plurality of reactors, and the method may include the steps of: delivering a template precursor material from one or more of the plurality of fluid depots to a first reactor among the plurality of reactors, processing the template precursor material to form a DNA template from the template precursor material; transferring the DNA template to a second reactor among the plurality of reactors, processing the DNA template by in vitro transcription to form therapeutic mRNA; transferring the therapeutic mRNA to a third reactor among the plurality of reactors, processing the therapeutic mRNA to bind it to a delivery vehicle to form a therapeutic mRNA composition; and transferring the therapeutic mRNA composition to a concentrator in fluid communication with the third reactor. The step of transferring the therapeutic mRNA to the third reactor may include transferring a plurality of different therapeutic mRNAs together with a delivery vehicle to form a therapeutic mRNA composition. The delivery and transfer steps may be performed by one or more fluid-power circuits in a system controlled by a controller. For example, the controller may control the fluid-power circuits by deflecting one or more elastic layers in one or more microfluidic plate devices. The method may be performed in a medical setting.

[0046] Any of these methods (and the systems that perform them) can be configured to automatically dialyze a therapeutic composition (e.g., mRNA encapsulated in a delivery vehicle) within the same microfluidic device, remove material, and / or concentrate the therapeutic composition. For example, any of these methods may include purifying the therapeutic mRNA composition by dialyzing it within one or more microfluidic pathway plate devices. Any suitable nanoparticles may be used (e.g., amphiphilic nanoparticles such as aminolipidized peptoids).

[0047] Furthermore, any of these methods may include two-dimensional (2D) purification in one or more reactors that are in fluid communication with the second reactor.

[0048] The method for producing the therapeutic mRNA composition may be particularly fast compared to known techniques and methods. For example, in the method described herein, the formation of the therapeutic composition (therapeutic mRNA and delivery vehicle), including the formation of a synthetic template (e.g., de novo synthesis without bacterial precursors), may be carried out in 5 days or less (e.g., 4 days or less, 72 hours or less, etc.).

[0049] For example, a method for producing a therapeutic mRNA composition using a system comprising one or more microfluidic plate devices and multiple fluid depots in sealed fluid communication, wherein the one or more microfluidic plate devices comprise multiple reactors, the method comprising the steps of: pressurizing the multiple fluid depots; controlling a first fluid power circuit to deliver template precursor material from one or more of the multiple fluid depots to a first reactor of the multiple reactors with sub-microliter precision and without contact with the atmosphere; processing the template precursor material to form a DNA template from the template precursor material; and controlling a second fluid power circuit to transfer the DNA template to a second reactor of the multiple reactors with sub-microliter precision and without contact with the atmosphere. The procedure may include the steps of: processing a DNA template by in vitro transcription to form therapeutic mRNA; controlling a third fluid power circuit to transfer the therapeutic mRNA to the third reactor among a plurality of reactors with sub-microliter precision and without contact with the atmosphere; processing the therapeutic mRNA to bind to a delivery vehicle to form a therapeutic mRNA composition; controlling the third fluid power circuit to transfer the therapeutic mRNA composition to a concentrator that is in fluid communication with the third reactor; and concentrating the therapeutic mRNA composition.

[0050] The methods and apparatus described herein may be used to provide on-demand synthesis of therapeutic polynucleotide compositions. In some modifications, these methods may include remotely synthesizing a portion of the components and using the apparatus to locally synthesize a therapeutic polynucleotide composition that can then be delivered to a patient. For example, a method for producing a therapeutic polynucleotide composition on demand, the method including receiving a therapeutic polynucleotide synthesized at a remote facility at a local facility, formulating the therapeutic polynucleotide composition at the local facility by performing the steps of forming the therapeutic polynucleotide composition by conjugating the therapeutic polynucleotide with a delivery vehicle in a microfluidic device held within the system in an automated system protected from contact with the atmosphere, and dialyzing the therapeutic polynucleotide composition within the microfluidic device, and providing the therapeutic polynucleotide composition.

[0051] The synthesis of therapeutic polynucleotides may be carried out using a microfluidic system at a remote facility, by performing the steps of forming a synthesis template in a closed-channel apparatus protected from contact with the atmosphere, forming therapeutic polynucleotides by in vitro transfer from the synthesis template, and purifying the therapeutic polynucleotides.

[0052] For example, a method for producing a therapeutic mRNA composition on demand, the method comprising synthesizing the therapeutic mRNA at a remote facility, transporting the therapeutic mRNA to a local facility, formulating the therapeutic mRNA composition at the local facility by performing the steps of forming the therapeutic mRNA composition by binding the therapeutic mRNA to a delivery vehicle in a microfluidic device in an automated closed-channel apparatus protected from contact with the atmosphere, and dialyzing the therapeutic mRNA composition within the microfluidic device, and providing the therapeutic mRNA composition. The local facility is a hospital or clinic and typically includes one or more microfluidic control systems as described herein. In one variation, the remote facility may be a manufacturing facility including one or more (e.g., multiple) microfluidic control systems.

[0053] The methods described herein may further include concentrating a therapeutic polynucleotide composition.

[0054] Any of these methods may include synthesizing therapeutic polynucleotides using the system described herein, then transporting them from a remote facility to a local facility (e.g., shipping) while storing them at low temperatures (e.g., cold and / or frozen), and receiving the therapeutic polynucleotides (e.g., mRNA) at the local facility. For example, the therapeutic polynucleotide composition may consist of an mRNA vaccine. Any of these methods may also include formulating the therapeutic polynucleotides using the system, which includes a number of fluid depots configured to ensure sealed fluid communication with a microfluidic device.

[0055] The first fluid-powered circuit may be used to deliver therapeutic polynucleotides and a delivery vehicle from multiple fluid depots to one or more reactors of a microfluidic device with sub-microliter precision and without contact with the atmosphere, in order to bind the therapeutic polynucleotides and the delivery vehicle.

[0056] As described above, any of these therapeutic compositions may contain multiple mRNAs (including, but not limited to, multiple therapeutic mRNAs) encapsulated in the same (or different) delivery vehicles. For example, forming a therapeutic polynucleotide composition at a local facility may further involve conjugating one or more additional therapeutic polynucleotides with the therapeutic polynucleotide and delivery vehicle. Any suitable delivery vehicle, including those described herein, may be used. The therapeutic polynucleotide may be mRNA such as linear mRNA, circular RNA, or self-replicating RNA.

[0057] Therapeutic polynucleotides may be stable at low temperatures (e.g., 4°C, 0°C, -10°C, etc.) for a certain period of time (e.g., more than 1 month, more than 2 months, more than 3 months, more than 6 months, more than 8 months, more than 9 months, more than 1 year, etc.) and may be stored remotely or locally. For example, these methods may include storing the therapeutic polynucleotides at a local facility before formulating the therapeutic composition.

[0058] For example, a method for producing a therapeutic mRNA composition using a closed-circuit system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed-circuit system protected from contact with the atmosphere, for the following steps: forming a DNA template in one or more microfluidic devices; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; and binding the mRNA to a delivery vehicle.

[0059] A method for producing a therapeutic mRNA composition using a closed-loop system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices (where the one or more microfluidic devices comprise a plurality of reactors) may include the steps of: delivering a template precursor material from one or more storage depots to a first reactor region of the plurality of reactors; processing the template precursor material to prepare a template from the template precursor material; transferring the template to a second reactor region of the plurality of reactors; processing the template by in vitro transcription to form therapeutic mRNA; and transferring the therapeutic mRNA to a third reactor region of the plurality of reactors; and processing the therapeutic mRNA to bind it to a delivery vehicle to form a therapeutic mRNA composition, wherein the material comprising the template precursor material and the delivery vehicle is delivered from the storage depots to the plurality of reactors without contact with the atmosphere.

[0060] A method for producing a therapeutic mRNA composition using a closed-circuit system comprising one or more microfluidic devices (for example, wherein one or more microfluidic devices comprise multiple reactors) and multiple storage depots in sealed fluid communication may include the steps of: inducing a fluid flow; delivering template precursor material from one or more storage depots to a first reactor region of the multiple reactors; processing the template precursor material to prepare a template from the template precursor material; transferring the template to a second reactor among the multiple reactors; processing the template by in vitro transcription to form mRNA; transferring the mRNA to a third reactor region among the multiple reactors; processing the mRNA to bind to a delivery vehicle to form a therapeutic mRNA composition; and transferring an mRNA product depot from one or more storage depots, wherein the material is delivered from the storage depots into the reactors of the microfluidic devices with sub-microliter precision and without contact with the atmosphere. In any of the methods described herein, any of the steps may be performed pneumatically, for example, the fluid flow may be induced pneumatically, and the fluid may be transferred pneumatically. Alternatively or additionally, the fluid may be driven mechanically, hydraulically, or otherwise.

[0061] In any of these methods (and apparatus for carrying them out), the closed-loop system may automatically and continuously perform the steps of forming a template, in vitro transcription of therapeutic mRNA from the template, purification of the therapeutic mRNA, and binding the mRNA to a delivery vehicle. The closed-loop system may pneumatically control the performance of the steps of forming a template, in vitro transcription of therapeutic mRNA from the template, purification of the therapeutic mRNA, and binding the mRNA to a delivery vehicle. In one modification, the closed-loop system pneumatically controls the performance of the steps of forming a template, in vitro transcription of therapeutic mRNA from the template, purification of the therapeutic mRNA, and binding the mRNA to a delivery vehicle by deflecting one or more membranes within one or more microfluidic devices.

[0062] Any of the methods and apparatus described herein may be configured to be set up and operated in a medical setting such as a hospital or clinic. This allows for the custom manufacturing of immediate / on-demand patient-specific therapeutics for specific patients. Alternatively or additionally, non-patient-specific therapeutic molecules may be formulated with a delivery vehicle in a "patient-personalized" manner. Due to the methods and apparatus described herein, any of these methods can be performed very quickly. For example, a closed-loop system may automatically and continuously perform the steps of forming a template, in vitro transcription of therapeutic mRNA from the template, purification of the therapeutic mRNA, and binding the mRNA to a delivery vehicle in less than five days. Alternatively, the system may use a pre-made template as input and perform the remaining steps in a shorter time.

[0063] The step of conjugating mRNA to a delivery vehicle (the step of formulating a therapeutic agent) may further include dialysis of the therapeutic mRNA composition using one or more microfluidic devices to purify the therapeutic mRNA composition.

[0064] Any of these methods may further include the steps of concentrating a therapeutic mRNA composition on one or more microfluidic devices and / or dialysis of the therapeutic agent.

[0065] For example, any suitable delivery vehicle containing amphiphilic nanoparticles may be used. For example, the amphiphilic nanoparticles may consist of aminolipidized peptoids.

[0066] Alternatively or additionally, in any of the methods and apparatus described herein, mRNA may be prepared in advance and stored for a period of time (e.g., at 10°C, 4°C, 0°C, -10°C, etc.). For example, any of these methods and apparatus for carrying them may include a library of therapeutic mRNA, and therapeutic mRNA may be combined individually or collectively (e.g., 2, 3, 4, 5, 6, etc., or more individual therapeutic mRNAs) to form a therapeutic mRNA composition. As described herein, therapeutic mRNA compositions may therefore be manufactured on demand and may be combined just-in-time into a single or more therapeutic mRNA composition "cocktail".

[0067] Also described herein are methods for forming templates (e.g., DNA templates). For example, a method for creating a synthetic double-stranded DNA template for in vitro transcription using a closed-loop system comprising a microfluidic device and a plurality of storage depots in communication with a sealed fluid may include the steps of: conjugating a synthetic gene of interest with a synthetic in vitro transcription promoter cassette to produce a synthetic linear or circular ligation product; removing unreacted synthetic gene of interest and unreacted synthetic in vitro transcription promoter cassette from the synthetic linear or circular ligation product; amplifying the circular ligation product to produce linear, branched or circular amplified DNA; and linearizing the amplified DNA ligation product to produce a double-stranded DNA template, wherein each of the conjugation, removal, amplification, and linearization steps is performed within the microfluidic device by the closed-loop system.

[0068] For example, a highly efficient automated method for creating a synthetic double-stranded DNA template for in vitro transcription involves the steps of: pneumatically delivering the target synthetic gene and a synthetic in vitro transcription promoter cassette, respectively, from one or more storage depots, which are in fluid communication with the microfluidic device, to the ligation reactor of the microfluidic device; creating a synthetic linear or cyclic ligate product by binding the target synthetic gene to the synthetic in vitro transcription promoter cassette; and pneumatically introducing one or more exonuclease agents from one or more storage depots to the ligation reactor, thereby combining the unreacted synthetic gene and the unreacted synthetic in vitro The process may include the steps of removing unreacted material by moving a vitro transcription accelerator cassette away from the synthetic linear or cyclic ligation product; pneumatically feeding the synthetic linear or cyclic ligation product to an amplification reactor of a microfluidic device and conjugating it with one or more amplifying agents to amplify the linear or cyclic ligation product to generate linear, branched, or cyclic amplified DNA; and pneumatically transferring the amplified DNA ligation product to a digestion reactor of a microfluidic device and linearizing the amplified DNA ligation product to generate a fully synthetic double-stranded DNA template free of unreacted input material, wherein the ligation reactor, amplification reactor, digestion reactor, and multiple storage depots form a closed-circuit sealed environment.

[0069] A method for creating a synthetic double-stranded DNA template for a therapeutic mRNA composition (using a closed-loop system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices) may include the step of transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, and forming a template for in vitro transcription of therapeutic mRNA in one or more microfluidic devices.

[0070] In general, the methods and apparatus described herein can produce double-stranded DNA templates that are free from bacterial DNA and / or endotoxins. The template production methods and apparatus described herein do not require bacterial culture. Furthermore, the therapeutic mRNA produced as described herein may be synthesized from the template without using bacterial polynucleotides. Therefore, any of the methods described herein may be for producing therapeutic mRNA that is free from bacterial DNA and / or isolated from endotoxins. In particular, the methods described herein are for producing double-stranded DNA templates that are free from bacterial DNA and / or endotoxins. Any of the methods described herein may be sterile production methods.

[0071] Any of these methods may include a step of digesting the synthetic in vitro transcription template with a type IIS restriction enzyme and / or a methylation-sensitive restriction enzyme. The binding step may include ligation by DNA ligase, ligation by DNA synthesis, or ligation by primer extension. The removal step may include digestion of linear DNA with an exonuclease or with a methylation-sensitive restriction enzyme. The exonuclease may include exonuclease V. The amplification step may include multiple substitution amplification (MDA). The amplification step may include amplification with Φ29 DNA polymerase. The amplification step may include generating branched amplified DNA. The amplification step may include polymerase chain amplification (PCR). The amplification step may include amplification with a thermostable DNA polymerase.

[0072] The linearization step may include digestion with a type IIs restriction enzyme. The linearization step may also include digestion with a BsaI restriction enzyme. The digestion step of the synthetic in vitro transcription template may include digestion with a methylation-sensitive restriction enzyme such as DpnI. The synthetic gene of interest may be linear. In one modification, the synthetic in vitro transcription accelerator cassette comprises a double-stranded DNA template including a promoter, a 5'UTR, a cleavable linker, a 3'UTR, and a portion encoding a polyA region consisting of at least 200 adenine residues or 200 thymidine residues. The synthetic in vitro transcription accelerator cassette may be delivered as a single unit or as two or more units. The portion encoding the polyA region may be at least 300 bps in length. In one modification, the portion encoding the polyA region may be at least 350 bps in length.

[0073] The target synthetic gene may include at least a portion of the T cell receptor. The target synthetic gene may also include a complementation-determining region (CDR).

[0074] The in vitro transcription facilitator cassette may be less than 2 kb in length. The in vitro transcription facilitator cassette may be less than 1 kb in length. The in vitro transcription facilitator cassette may be less than 700 base pairs in length. The synthetic in vitro transcription facilitator cassette does not need to encode an antibiotic resistance gene.

[0075] Synthetic linear or cyclic ligation products do not need to have origins of replication (ORIs). In vitro transcription accelerator cassettes do not need to have origins of replication (ORIs).

[0076] As described above, any step of the method described herein may be performed in a closed microfluidic device. The step may be performed in a closed microfluidic device, the coupling step may be performed in a different module (e.g., a different microfluidic device) than the amplification step, and the amplification step may be performed in a different module than the linearization step.

[0077] Any of these methods may include a step of purifying the template in the closed circuit of one or more microfluidic devices.

[0078] Furthermore, this specification describes methods for performing in vitro transcription using the closed-loop methods and apparatus described herein. For example, a method for performing an in vitro transcription (IVT) reaction using a closed-loop system (including, for example, a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices) may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform in vitro transcription of therapeutic mRNA from a template in one or more microfluidic devices.

[0079] A method for performing an in vitro transcription (IVT) reaction may include the steps of: automatically delivering DNA template, polymerase, and nucleotides in submicroliter-precise quantities by direct fluid flow from multiple storage depots to a first reactor of a microfluidic device; processing the template material and nucleotides in the first reactor to form therapeutic mRNA; and pneumatically transferring the therapeutic mRNA from the first reactor through the microfluidic device, wherein the first microfluidic device and the multiple storage depots may form a closed-circuit sealed environment to prevent exposure to the atmosphere.

[0080] The closed-loop system may operate automatically and continuously. The performance of the closed-loop system in in vitro transcription of therapeutic mRNA from a template may be controlled by pneumatic pressure.

[0081] Any of these methods may also include a step of purifying therapeutic mRNA in one or more microfluidic devices. The step of transporting reagents may include transporting reagents from multiple storage depots to the first reactor of the microfluidic device.

[0082] Furthermore, this specification describes methods for formulating therapeutic mRNA (e.g., by binding it to a delivery vehicle). For example, a method for producing a therapeutic mRNA composition (e.g., using a closed-loop system including a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices) may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed-loop system protected from contact with the atmosphere, in order to formulate a therapeutic mRNA composition by binding the therapeutic mRNA to a delivery vehicle in one or more microfluidic devices. The closed-loop system can automatically and continuously bind the mRNA to the delivery vehicle. The closed-loop system may control the binding of mRNA to the delivery vehicle by pneumatic pressure. For example, the closed-loop system may control the binding of mRNA to the delivery vehicle by deflecting one or more membranes in one or more microfluidic devices by pneumatic pressure.

[0083] The step of binding mRNA to a delivery vehicle may further include dialysis of the therapeutic mRNA composition in one or more microfluidic devices to purify the therapeutic mRNA composition, and / or enrichment of the therapeutic mRNA composition on one or more microfluidic devices.

[0084] For example, this specification describes a method for producing mRNA using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices. Any of these methods may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the steps of forming a template, performing in vitro transcription of mRNA from the template, and purifying the mRNA in one or more microfluidic devices.

[0085] A method for producing a therapeutic mRNA composition using a system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices may include transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the following steps in one or more microfluidic devices: forming a template; performing in vitro transcription of mRNA from the template; purifying the mRNA; and formulating the mRNA with a delivery vehicle.

[0086] A method for producing a therapeutic mRNA composition using a system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices may include transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the following steps in one or more microfluidic devices: forming a template; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; formulating the mRNA with a delivery vehicle; and dialysis and concentration of the formulated therapeutic mRNA.

[0087] A method for producing a therapeutic mRNA composition using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices comprises following a series of therapeutic mRNA composition formation steps encoded in a non-transient computer-readable medium, which may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the following steps in one or more microfluidic devices: forming a template; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; and binding the mRNA to a delivery vehicle.

[0088] Also described herein is a method for producing a therapeutic mRNA composition using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with a microfluidic device, the method comprising the steps of: performing in vitro transcription of therapeutic mRNA from a template on a microfluidic device; and purifying the therapeutic mRNA in one or more fluidly connected reactors on the microfluidic device.

[0089] Also described herein are therapeutic agents, particularly mRNA therapeutic agents, prepared by any of these methods. For example, described herein is therapeutic mRNA prepared using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, wherein the mRNA is prepared by transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the steps of forming a template, performing in vitro transcription of mRNA from the template, and purifying the mRNA in one or more microfluidic devices.

[0090] For example, described herein are therapeutic mRNAs prepared using a system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, wherein the mRNAs are prepared by transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the following steps in one or more microfluidic devices: forming a template; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; and formulating the mRNA with a delivery vehicle.

[0091] For example, described herein are therapeutic mRNAs prepared using a system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, wherein the mRNAs are prepared by transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform one or more of the following steps in one or more microfluidic devices: forming a template; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; formulating the mRNA with a delivery vehicle; and dialysis and concentration of the formulated therapeutic mRNA.

[0092] This specification describes a therapeutic mRNA composition formed according to a series of steps for forming a therapeutic mRNA composition encoded in a non-transient computer-readable medium using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the steps comprising transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere in order to perform one or more of the following steps: forming a template in one or more microfluidic devices; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; and binding the mRNA to a delivery vehicle. For example, the therapeutic mRNA may be a therapeutic mRNA composition formed using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising the steps of performing in vitro transcription of therapeutic mRNA from a template on a microfluidic device and purifying the therapeutic mRNA in one or more fluidly connected reactors on a microfluidic device.

[0093] Any of the systems described herein may include a controller configured to perform any of these methods. Accordingly, software, firmware, or hardware configured to perform any of the methods described herein is also described herein. For example, described herein is a non-transient computer-readable medium embodying instructions for producing mRNA, which, when performed by a controller of a system including a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, causes the controller to perform a method in which, in one or more microfluidic devices, one or more of the following steps are performed: forming a template; performing in vitro transcription of mRNA from the template; and purifying the mRNA, in a closed channel protected from contact with the atmosphere.

[0094] For example, described herein is a non-transient computer-readable medium embodying instructions for producing mRNA, including a therapeutic mRNA composition, which, when executed by a controller of a system including a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, causes the controller to perform one of the methods described herein.

[0095] Also described herein is a method for creating a synthetic double-stranded DNA template for mRNA using a closed-loop system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method which may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices for binding reagents in a sealed closed-loop system protected from contact with the atmosphere, and forming an in vitro transcription template for therapeutic mRNA.

[0096] For example, a method for creating a synthetic double-stranded DNA template for use as input to mRNA in an in vitro transcription reaction using a closed-loop system may include a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising transporting reagents between one or more of the plurality of storage depots and a plurality of reactors on one or more microfluidic devices in a closed-loop system protected from contact with the atmosphere, and forming an in vitro transcription template of therapeutic mRNA.

[0097] A method for producing an mRNA composition using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, wherein the one or more microfluidic devices comprise a plurality of reactors, and the method may include the steps of: delivering a template precursor material from one or more storage depots to a first reactor region of the plurality of reactors; processing the template precursor material to prepare a template from the template precursor material; transferring the template to a second reactor region of the plurality of reactors; processing the template by in vitro transcription to form mRNA; and transferring the mRNA to a third reactor region of the plurality of reactors and processing the mRNA to bind it to a delivery vehicle to form an mRNA composition, wherein the material comprising the template material and the delivery vehicle is delivered from the storage depots to the plurality of reactors without contact with the atmosphere.

[0098] A method for producing an mRNA composition using a system comprising one or more microfluidic devices and a plurality of storage depots in sealed fluid communication, wherein the one or more microfluidic devices comprise a plurality of reactors, and the method may include the steps of: pneumatically delivering a template precursor material from one or more storage depots to a first reactor region of the plurality of reactors; processing the template precursor material to prepare a template from the template precursor material; pneumatically transferring the template to a second reactor region of the plurality of reactors and processing the template by in vitro transcription to form mRNA; pneumatically transferring the mRNA to a third reactor region of the plurality of reactors and processing the mRNA to bind it to a delivery vehicle to form a therapeutic mRNA composition; and transferring the mRNA product to one or more storage depots, wherein the material is delivered from the storage depots to the reactors of the microfluidic devices with sub-microliter precision and without contact with the atmosphere.

[0099] A method for creating a synthetic double-stranded DNA template for in vitro transcription using a closed-loop system comprising a microfluidic device and a plurality of storage depots in communication with a sealed fluid, wherein one or more microfluidic plate devices comprise a plurality of reactors, and the method may include the steps of: conjugating a synthetic gene of interest with a synthetic in vitro transcription promoter cassette to produce a synthetic linear or circular ligation product; removing unreacted synthetic gene of interest and unreacted synthetic in vitro transcription promoter cassette from the synthetic linear or circular ligation product; amplifying the linear or circular ligation product to produce linear, branched, or circular amplified DNA; and linearizing the amplified DNA product to produce a double-stranded DNA template, wherein each of the conjugation, removal, amplification, and linearization steps is performed within the microfluidic device by the closed-loop system.

[0100] These methods may all be highly efficient automated methods, and may be highly efficient automated methods for creating synthetic double-stranded DNA templates for in vitro transcription. For example, the method may involve the steps of: pneumatically delivering the target synthetic gene and a synthetic in vitro transcription promoter cassette, respectively, from one or more storage depots of a plurality of storage depots that are in fluid communication with the microfluidic device to the ligation reactor of the microfluidic device, and generating a synthetic linear or cyclic ligation product by binding the target synthetic gene with the synthetic in vitro transcription promoter cassette; and pneumatically introducing one or more exonuclease agents from one or more storage depots to the ligation reactor, thereby combining the unreacted synthetic gene and the unreacted synthetic in vitro The steps may include removing unreacted material by removing a vitro transcription accelerator cassette from the synthetic linear or cyclic ligation product; pneumatically delivering the synthetic linear or cyclic ligation product to a multiple substitution amplification (MDA) or polymerase chain reaction (PCR) reactor in a microfluidic device and conjugating it with one or more amplifiers for amplifying the linear or cyclic ligation product to generate linear, branched, or cyclic amplified DNA; and pneumatically transferring the amplified DNA ligation product to a digestion reactor in a microfluidic device to linearize the amplified DNA ligation product and generate a double-stranded DNA template, wherein the ligation reactor, MDA or PCR reactor and digestion reactor, as well as multiple storage depots, form a closed-circuit sealed environment.

[0101] A method for preparing a synthetic double-stranded DNA template for in vitro transcription, comprising following a series of steps encoded in a non-transient computer-readable medium, the method comprising: preparing a synthetic linear or cyclic ligation product by delivering a synthetic gene of interest and a synthetic in vitro transcription promoter cassette, respectively, from one or more storage depots of a plurality of storage depots in fluid communication with the microfluidic device to the ligation reactor of the microfluidic device, thereby conjugating the synthetic gene of interest to the synthetic in vitro transcription promoter cassette; introducing one or more exonuclease agents from one or more storage depots of a plurality of storage depots to the ligation reactor, and unreacted synthetic gene and unreacted synthetic in vitro The process includes the steps of removing unreacted material by moving a vitro transcription accelerator cassette away from the synthetic linear or cyclic ligation product; sending the synthetic linear or cyclic ligation product to a multiple substitution amplification (MDA) or polymerase chain reaction (PCR) reactor in a microfluidic device and combining it with one or more amplifiers to amplify the linear or cyclic ligation product to generate linear, branched, or cyclic amplified DNA; and transferring the amplified DNA ligation product to a digestion reactor in a microfluidic device and linearizing the amplified DNA ligation product to generate a double-stranded DNA template, wherein the ligation reactor, MDA reactor and digestion reactor, as well as multiple storage depots, form a closed-circuit sealed environment.

[0102] A method for performing an in vitro transcription (IVT) reaction using a system comprising multiple storage depots configured to ensure sealed fluid communication with one or more microfluidic devices may include transporting reagents between one or more storage depots and multiple reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to perform in vitro transcription of therapeutic mRNA from a template in one or more microfluidic devices.

[0103] Also described herein is a method for performing an in vitro transcription (IVT) reaction, the method comprising following a series of steps coded in a non-transient computer-readable medium, the steps comprising, at any time during the reaction, delivering, by pneumatic means, a sub-microliter metered amount of template material, polymerase, and nucleotides from a plurality of storage depots to a first reactor of a microfluidic device; processing the template material and nucleotides in the first reactor to form therapeutic mRNA; and transferring, by pneumatic means, the therapeutic mRNA from the first reactor through the microfluidic device, the microfluidic device and the plurality of storage depots forming a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0104] Also described herein is a method for performing an in vitro transcription (IVT) reaction, the method comprising following a series of steps encoded in a non-transient computer-readable medium, the steps comprising delivering a controller-controlled amount of template material, polymerase, and nucleotides from a plurality of storage depots to a microfluidic device by an induced fluid flow, the controller following a series of steps including processing the template material and nucleotides in one or more reactors to form therapeutic mRNA, and transferring the therapeutic mRNA from one or more reactors through a microfluidic device, the microfluidic device and the plurality of storage depots forming a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0105] Furthermore, this specification describes a method for performing an in vitro transcription (IVT) reaction, the method comprising the steps of: delivering a pre-programmed amount of template material, polymerase, and nucleotides from a plurality of storage depots to a microfluidic device by an induced fluid flow; processing the template material and nucleotides in one or more first reactors within the microfluidic device to form therapeutic mRNA; and transferring the therapeutic mRNA from one or more first reactors to one or more second reactors suitable for mRNA purification through the microfluidic device, wherein the microfluidic device and the plurality of storage depots form a closed-circuit sealed environment that prevents exposure to the atmosphere.

[0106] Also described herein is a method for performing an in vitro transcription (IVT) reaction, the method comprising following a series of steps encoded in a non-transient computer-readable medium, the steps comprising delivering, by an induced fluid flow, a quantity of template material, polymerase, and nucleotides controlled by a series of steps described below, from a plurality of storage depots to one or more first reactors of a first microfluidic device, the series of steps comprising processing the template material and nucleotides in one or more first reactors to form therapeutic mRNA, transferring the therapeutic mRNA from one or more first reactors through the first microfluidic device to one or more second reactors suitable for mRNA purification, and transferring the thus purified mRNA to complete the formulation of an mRNA therapeutic agent, wherein the first microfluidic device and the plurality of storage depots form a closed-circuit sealed environment to prevent exposure to the atmosphere.

[0107] Also described herein is a method for performing an in vitro transcription (IVT) reaction, the method comprising following a series of steps coded in a non-transient computer-readable medium, the steps of: pneumatically delivering a template material, polymerase, and nucleotides from a plurality of storage depots to one or more first reactors of a first microfluidic device; processing the template material and nucleotides in one or more first reactors to form therapeutic mRNA; transferring the therapeutic mRNA through the first microfluidic device from one or more first reactors to one or more second reactors suitable for mRNA purification; and transferring the purified mRNA to one or more third reactors to conjugate the purified mRNA with one or more delivery vehicles to form an mRNA therapeutic agent, wherein the first microfluidic device and the plurality of storage depots form a closed-circuit sealed environment to prevent exposure to the atmosphere.

[0108] For example, described herein is a method for performing an in vitro transcription (IVT) reaction, the method comprising following a series of steps encoded in a non-transient computer-readable medium, the steps of: pneumatically delivering a template material, polymerase, and nucleotides from a plurality of storage depots to one or more first reactors of a first microfluidic device; processing the template material and nucleotides in one or more first reactors to form therapeutic mRNA; transferring the therapeutic mRNA from one or more first reactors through the first microfluidic device to one or more second reactors made of cellulose and suitable for mRNA purification; and transferring the purified mRNA to one or more third reactors and conjugating the purified mRNA with one or more delivery vehicles to form an mRNA therapeutic agent, wherein the first microfluidic device and the plurality of storage depots form a closed-circuit sealed environment to prevent exposure to the atmosphere.

[0109] Also described herein is a method for producing a therapeutic mRNA composition using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method comprising transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere, in order to formulate the therapeutic mRNA composition by binding one or more therapeutic mRNAs to a delivery vehicle in one or more microfluidic devices.

[0110] A method for producing a therapeutic mRNA composition on demand using a system comprising a plurality of storage depots configured to ensure sealed fluid communication with one or more microfluidic devices, the method may include transporting reagents between one or more storage depots and a plurality of reactors on one or more microfluidic devices in a closed channel protected from contact with the atmosphere in order to perform one or more of the following steps: forming a template in one or more microfluidic devices; performing in vitro transcription of therapeutic mRNA from the template; purifying the therapeutic mRNA; and formulating the mRNA with a delivery vehicle.

[0111] Any of these methods and apparatus may be operated in a clinical setting (e.g., for the production of therapeutic mRNA). Any of these methods and apparatus may be performed rapidly and continuously, for example, for therapeutic drugs to be produced in less than 72 hours.

[0112] As described above, in any of these methods and apparatus, the polynucleotides formed (e.g., by IVT) within the microfluidic device may be purified before and after being bound to the delivery vehicle under the automatic control of a microfluidic control device.

[0113] In particular, the methods and apparatus described herein may include one or more microfluidic devices adapted for use with permeable insert materials, which, as part of the preparation process, may remove one or more target materials (e.g., double-stranded RNA) and / or add one or more additional materials (e.g., lyophilized materials) to the therapeutic material. The permeable insert material may be configured to be held within one or more fluid contact chambers of the microfluidic device and adapted so that a developing therapeutic solution passes through the permeable insert material. The permeable insert material may be compressible and / or deformable and / or elastic so that it can be manipulated by an elastic membrane within the chamber. The permeable insert may include a cover (e.g., an outer cover) that is permeable and contains a modifying material such as a solid, granular, or gel. In one modification, the permeable insert material is a cellulose material configured to selectively remove double-stranded RNA from a solution.

[0114] The permeable material may be porous (for example, it may contain pores). In one modification, the permeable material may be fibrous or layered. For example, the permeable material may be fibrous and may contain channels through which fluid can move. In one modification, channels may be formed in the permeable material to enable permeability to fluid. For example, the permeable material may have laser-formed channels or any other means by which the internal volume can be made accessible to fluid. In one modification, the permeable material may be formed from multiple layers arranged so that fluid can pass between the layers. Multiple thin layers of material accessible only from the surface may be stacked together. For example, functionalized graphene may be stacked (for example, to the extreme of a single atomic layer). As another example, aerogel slices may be processed to make them absorbent and then stacked to form a permeable material for an insert.

[0115] In any permeable material described herein, the material may be a preformed material configured to allow the passage of fluid at a predetermined flow rate and / or flow resistance, depending on the circumstances. The permeability of the material may be selected to allow flow through the permeable insert when held in the apparatus chamber or channel at the processing flow rates and fluid pressures described herein. As described above, the preformed permeable material may be porous, fibrous, or a stack of layers, and any of these materials may be functionalized to bond materials. As used herein, a functionalized material may include any material whose surface is modified by the addition of compounds, agents, or functional groups that specifically bond a target material. Materials may also be functionalized by surface treatments that attach specific atomic or molecular groups to alter certain properties of the material. Functionalization can be carried out by a variety of surface modification techniques, such as wet chemistry, or vapor, gas, and / or plasma chemistry and / or microwave-assisted chemistry techniques, including techniques that utilize surface chemistry to bond a desired material to the surface. Similar techniques can be used to modify materials, for example, to “activate” them.

[0116] The permeable material may be configured to maximize the total surface area of ​​the insert within the device and to allow selective binding of unwanted impurities and / or target products. Any of these permeable materials may be configured to provide sufficient permeability of the solution into the preform material for effective binding with the substance in the solution. In one modification, the permeable material consists of multiple preform materials, each larger than the channel or chamber within the microfluidic device, and each of these preform materials can still allow permeability of the solution into the interior to maximize the exposure of the solution to the functionally active substance.

[0117] As described above, the permeable implant inserts described herein may be configured to remove impurities (e.g., unwanted material) from the solution, and alternatively or additionally, the permeable implant may be configured to be releasably bonded to a desired material so that it can be eluted (e.g., after washing).

[0118] As described above, the methods and apparatus described herein may include the use of a microfluidic device comprising one or more permeable inserts configured to modify a solution forming a therapeutic agent. The permeable inserts may be adapted for use within the microfluidic devices described herein.

[0119] For example, as described herein, a permeable insert configured to fit within the fluid-contact side of a chamber. Therefore, the permeable insert may be sized and / or shaped to substantially fit within the volume of all or part (e.g., a cross-sectional area) of the fluid-contact side of the chamber. As described above, the permeable material may be a single pre-formed implant or a combination of multiple pre-formed materials forming the permeable implant. The permeable implant may generally allow the flow of solution into and through the material. In one modification, the permeable implant may also be compressible (e.g., "extractable") to allow the removal of fluid from within the permeable implant when a microfluidic device compresses the chamber into which the permeable implant is inserted. In one modification, the permeable implant has sufficient elasticity to return to its expanded shape after compression.

[0120] Permeable inserts and microfluidic devices may be configured such that a fluid, particularly a material used to generate a therapeutic composition, necessarily passes through the permeable insert during processing. In one modification, the permeable insert is a compressible and / or elastically deformable material (e.g., a stretchable material) that can deform as the volume of the fluid-contact chamber changes by deflecting an elastic material (e.g., an elastic layer) that separates the fluid-contact side of the chamber from the pressure-receiving side of the chamber. In another modification, the permeable insert is compressible but does not necessarily have to be elastically deformable. In another modification, the permeable insert is a swellable material that can swell within the fluid-contact side of the chamber when activated (e.g., by the addition of a fluid such as a buffer or water). The permeable insert may be compressed by deflecting an elastic material (layer) between the pressure-receiving side and the fluid-contact side of the chamber. In another modification, processing a therapeutic agent may involve permeating a solution used to formulate the therapeutic material between a number of chambers (e.g., 2, 3, 4, etc.) containing the permeable insert material. In one modification, the method may include adding and removing the solution from a chamber containing a permeable insert material.

[0121] In any of the methods and apparatus described herein, the therapeutic insert material can be compressed by deflecting an elastic membrane that separates the chamber into a fluid contact side and a pressure-receiving side, for example, by adjusting the pressure on the pressure-receiving side of the chamber, in order to expel the solution containing the therapeutic material (or the solution on which the therapeutic material is formed) from the fluid contact side of the chamber.

[0122] The permeable insert may be any suitable material that can be used to modify, further process, and / or alter the therapeutic material. For example, in one modification, the permeable insert may include a cellulose material configured to retain double-stranded RNA in order to remove double-stranded RNA from the solution as it passes through the permeable material. Alternatively or additionally, the permeable insert may include one or more materials that can be added from the permeable insert to the solution.

[0123] For example, any of the permeable inserts described herein may include one or more additional materials adsorbed to or on the permeable insert. In any of these modifications, the permeable insert may include release material. In one modification, for example, the permeable insert may include a cellulose insert that has been pretreated with DNAse to encapsulate DNAse in cellulose. This allows the insert to simultaneously allow for the removal of double-stranded RNA and the digestion of DNA material, such as a DNA template from an in vitro transcription step.

[0124] Any of the permeable inserts described herein may be configured as surface-functionalized inserts containing one or more additional agents attached, adsorbed, or otherwise included on or within the permeable insert. For example, in one modification, additional materials included in or on the permeable insert may be covalently bonded materials (e.g., antibodies or aptamers), electrostatically bonded materials, adsorbed enzymes (e.g., capable of selectively degrading impurities such as the DNAse described above), or covalently or non-covalently attached sensors (e.g., for detecting materials to be removed, such as double-stranded RNA or impurities). In one modification, the additional material or material may include, for example, a poly(dT) sequence for capturing polyadenylated RNA molecules bound to the surface(s) of the permeable implant (e.g., a poly(dT) sequence may be used within the permeable implant to separate mRNA). In one modification, one or more additional materials may include small molecules to enhance binding properties (e.g., a double-stranded RNA intercalator such as ethidium bromide may selectively bind double-stranded RNA material without binding single-stranded RNA). In one modification, the permeable insert may be at least partially coated with the material. For example, in one modification, the coating may be a carboxylate coating.

[0125] In some modifications, the permeable insert may contain a lyophilized material that can be released into the solution immediately or after a time delay. For example, in some modifications, the lyophilized material may dissolve in the solution when the solution comes into contact with the permeable insert. Examples of lyophilized materials may include one or more buffering materials (e.g., salts, chelating agents, detergents, polynucleotides, enzymes, proteins, etc.). In some modifications, the permeable insert may contain agents such as binders for binding to one or more materials in the solution. For example, the permeable insert may contain a portion of a bound immunotherapy agent, such as an antibody, or a FAB fragment, which can selectively remove substances from the solution.

[0126] Generally, a permeable insert may be configured to straddle the fluid-contact side of the chamber so that fluid passes over and / or through the permeable insert. The permeable insert may be paper, e.g., a sheet-like material. The permeable insert may be folded to straddle and / or at least partially fill the fluid-contact side of the chamber. The folded shape can therefore straddle the fluid-contact portion of the chamber while being configured to deflect (including elastically deflect). The folds may include simple folds (e.g., fan-shaped folds) or more complex folds, and generally, the folds may include one or more curved regions that can act as hinge (e.g., living hinge) regions and / or may be biased to return to an expanded shape after being compressed or otherwise deflected by the movement of an elastic membrane that divides the chamber into a fluid-contact chamber and a pressure-receiving chamber. In one modification, the permeable insert may form a sponge. The permeable insert may be formed as a foamed or inflated material.

[0127] In any variation of the permeable insert described herein, the size of the passages (e.g., pores, channels, chambers, etc.) within the permeable insert may be configured to allow or exclude substances based on their size. Thus, the permeable inserts described herein may be configured to perform size exclusion (e.g., size exclusion chromatography). For example, a large mRNA molecule having unreacted mononucleotides may be passed through a chamber containing a nanoporous insert, where the unreacted dNTPs diffuse into the insert and are physically contained therein, while the size of the passages (e.g., pores) can exclude the large molecule.

[0128] For example, in a modification in which the permeable insert contains cellulose (e.g., for double-stranded RNA removal), the cellulose may be in the form of paper (e.g., filter paper), which is folded or layered and includes folds configured to be retained within the fluid contact portion of the chamber. In one modification, the cellulose may be expanded or foamed. In another modification, the cellulose may be in the form of a sponge.

[0129] Permeable inserts may generally have pores of any suitable size. The pore sizes may be uniform or non-uniform, and in some modifications, the pore sizes may be distributed within a size range. For example, the pore sizes may be between approximately 1 μm and approximately 200 μm (e.g., 1 μm to 3 μm, 1 μm to 5 μm, 2 μm to 4 μm, 2 μm to 5 μm, 3 μm to 6 μm, 5 μm to 10 μm, 6 μm to 8 μm, 6 μm to 10 μm, 7 μm to 10 μm, 8 μm to 10 μm, 9 μm to 11 μm, 9 μm to 12 μm) m, 10μm to 12μm, 10μm to 15μm, 12μm to 15μm, 14μm to 16μm, 15μm to 18μm, 17μm to 19μm) and 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm From the lower limit sizes of 17μm, 18μm, 19μm, 20μm, 22μm, 25μm, 27μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 110μm, 120μm, to 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12 The pore size range is from μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 22μm, 25μm, 27μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 110μm, 120μm, etc., up to the upper limit, and the lower limit of pore size is smaller than the upper limit. The size range may also be a distribution, for example, more than 90% of the pores (e.g., more than 90%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%, more than 99.9%, etc.) are within the size distribution.

[0130] In any of the methods and apparatus described herein, the temperature of the microfluidic device may be controlled as described herein. In particular, the temperature of the chamber containing the permeable insert may be controlled. For example, when a solution containing a therapeutic material (or a solution on which a therapeutic material is formed) comes into contact with the permeable insert, the chamber containing the permeable insert of the microfluidic device may be maintained at a target temperature. The temperatures are, for example, between 2°C and 20°C, between 2°C and 5°C, between 2°C and 10°C, between 2°C and 15°C, between 5°C and 10°C, between 5°C and 15°C, between 5°C and 20°C, between 10°C and 15°C, between 10°C and 20°C, between 10°C and 30°C, between 10°C and 25°C, between 15°C and 25°C, between 15°C and 30°C, between 20°C and 25°C, between 20°C and 30°C, between 25°C and 30°C, between 25°C and 40°C, between 25°C and 35°C, between 30°C and 35°C, between 30°C and 40°C, between 35°C and 40°C, between 30°C and 50°C, between 30°C and 45°C, between 35°C and 45°C, between 35°C and 50°C, between 40°C and 45°C, The temperature may be maintained between 40°C and 50°C, between 45°C and 50°C, between 40°C and 60°C, between 40°C and 55°C, between 45°C and 55°C, between 45°C and 60°C, between 50°C and 55°C, between 50°C and 60°C, between 55°C and 60°C, between 50°C and 70°C, between 50°C and 65°C, between 55°C and 65°C, between 55°C and 70°C, between 60°C and 70°C, between 60°C and 75°C, between 65°C and 70°C, between 65°C and 80°C, between 70°C and 80°C, between 75°C and 80°C, between 60°C and 80°C, between 70°C and 90°C, between 75°C and 90°C, between 80°C and 90°C, between 85°C and 85°C, between 85°C and 90°C, etc. The temperature may be constant, or it may be changed (e.g., increased, decreased, etc.) before, during, and / or after exposure to the permeable insert.

[0131] In one modification, the permeable insert may be called a solid permeable insert, and the permeable (e.g., solid permeable) insert may be configured to remain completely contained within the fluid-contact side of the chamber. As described above, in one modification, the permeable insert may be configured as a permeable package surrounded by an outer part, e.g., a permeable cover, which surrounds the material and confines the material within the permeable cover. For example, the permeable cover may contain granular material or a gel (e.g., a hydrogel). The permeable cover may be formed of a material such as a membrane material that has sufficient permeability to allow fluid to pass through the cover and enter the volume contained within the cover. Thus, the permeable insert may have a compressible and / or elastically deformable pillow shape.

[0132] In general, the permeable insert may be inserted into the fluid contact chamber of a microfluidic device and may be configured to fit within the fluid contact chamber as described above. In one modification, the permeable insert may be configured to fit snugly into the fluid contact side of the chamber, and for example, the permeable insert may have a shape complementary to the shape of the fluid contact side of the chamber (e.g., ellipse, circle, square, rounded square, etc.). As described above, the permeable insert may be configured to span and / or fill a volume, in particular, to span a volume perpendicular to the direction of flow through the volume on the fluid contact side so that the fluid passes through the permeable insert.

[0133] For example, described herein is a microfluidic device which may include means for guiding a fluid flow of a solution within the microfluidic device, a plurality of chambers, and a permeable insert located in a first chamber of the plurality of chambers, the insert being configured to be compressible. The means for guiding the fluid flow of the solution may include any suitable means, in particular a plurality of pressure ports on the microfluidic device configured to receive positive or negative pressure for deflecting a membrane within the microfluidic device. For example, described herein is a microfluidic device which includes an elastic material sandwiched between a first plate and a second plate, and a plurality of chambers formed between the first plate and the second plate, the elastic material dividing each chamber into a fluid contact side and a pressure receiving side. Any of these microfluidic devices may include a solid permeable insert in the fluid contact side of the first chamber.

[0134] For example, this specification describes a microfluidic device comprising an elastic material sandwiched between a first plate and a second plate, a plurality of chambers formed between the first plate and the second plate, wherein a portion of the elastic material divides each chamber into a fluid contact side and a pressure receiving side, and a solid permeable insert within the fluid contact side of the first chamber, wherein when pressure is applied to the pressure receiving side of the first chamber, the insert is compressed by the deflection of the elastic material.

[0135] In one variation, the microfluidic device includes an elastic material sandwiched between a first plate and a second plate, a plurality of chambers formed between the first plate and the second plate, wherein a portion of the elastic material divides each chamber into a fluid contact side and a pressure receiving side, and a solid-permeable insert within the fluid contact side of the first chamber, the insert being made of a cellulose material configured to purify RNA, and is configured such that by applying pressure to the pressure receiving side, the elastic material dividing the first chamber flexes, allowing fluid to move in or out of the first chamber.

[0136] The microfluidic device may include an elastic material sandwiched between a first plate and a second plate, a plurality of chambers formed between the first plate and the second plate, wherein a portion of the elastic material divides each chamber into a fluid contact side and a pressure receiving side, a plurality of fluid ports configured to fluidly communicate with the fluid contact sides of the plurality of chambers, a plurality of pressure ports fluidly communicating with the pressure receiving sides of the plurality of chambers, and a solid permeable insert in the fluid contact side of the first chamber among the plurality of chambers, wherein when pressure is applied to the pressure receiving side of the first chamber from one or more pressure ports, the insert is compressed by the deflection of the elastic material.

[0137] In some modifications, particularly those in which a permeable insert is configured to remove undesirable substances (e.g., double-stranded RNA) from solution, such as modifications containing cellulose, the chamber may be called a separation chamber.

[0138] As described above, in any of these devices (e.g., systems, devices, etc.), the solid-permeable insert may include a cellulose material configured to purify RNA. For example, the solid-permeable insert may consist of a sheet of cellulose material. Alternatively or additionally, the solid-permeable insert may consist of a lyophilized material.

[0139] The solid permeable insert may have a profile that matches the profile of the first chamber. As mentioned above, the solid permeable insert may be elastic.

[0140] The solid permeable insert may consist of a permeable outer cover containing granular material. In one variation, the solid permeable insert includes a folded structure.

[0141] In one modification, the microfluidic device may include a second chamber fluidically connected to a first chamber. The device may be configured to transfer fluid between the first and second chambers by deflecting an elastic material. In one modification, the fluid may reciprocate between the first and second chambers.

[0142] The microfluidic device may include a plurality of individually addressable pressure ports extending through a first plate and configured to supply pressure to the pressure-receiving side of a plurality of chambers to move fluid on the fluid-receiving side.

[0143] This specification also describes methods for using any of the apparatuses described herein. For example, a method for processing therapeutic material in a fluid (e.g., an RNA sample) may include the steps of connecting a microfluidic device to a pressure source, applying pressure to transport the sample to the fluid contact side of the separation chamber of the microfluidic device, passing the sample through a solid-permeable insert in the fluid contact side of the separation chamber so that the sample is corrected by the solid-permeable insert, and applying pressure to transport the sample out of the fluid contact side of the separation chamber.

[0144] For example, a method for removing dsRNA from an RNA sample containing both double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) may include the steps of: connecting a microfluidic device to a pressure source; applying pressure to transfer the RNA sample to the fluid contact side of the separation chamber of the microfluidic device; passing the RNA sample through a solid-permeable insert in the fluid contact side of the separation chamber, wherein the solid-permeable insert is made of cellulose and the dsRNA is held by the insert; and applying pressure to transfer the RNA sample out of the fluid contact side of the separation chamber.

[0145] A method for removing dsRNA from an RNA sample containing both double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA) may include the steps of: connecting a microfluidic device to a pressure source; applying pressure to transport the RNA sample to the fluid contact side of the separation chamber of the microfluidic device, such that the RNA sample passes through a solid permeable insert containing cellulose within the fluid contact side of the separation chamber, so that the dsRNA is retained by the insert; and applying pressure to the pressure-receiving side of the separation chamber to transport the RNA sample out of the fluid contact side of the separation chamber.

[0146] This method may include the step of synthesizing an RNA sample by in vitro transcription within a microfluidic device. In one modification, the method may include the step of connecting the microfluidic device to a source of RNA sample.

[0147] The step of applying pressure to transport the RNA sample out of the fluid contact side may include applying pressure to the pressure-receiving side of the separation chamber to deflect the elastic material that separates the pressure-receiving side of the separation chamber from the fluid contact side of the separation chamber. Applying pressure to the pressure-receiving side of the separation chamber may include transporting the RNA sample from the fluid contact side of the separation chamber to the fluid contact side of the mixing chamber, and further applying pressure to the pressure-receiving side of the mixing chamber to transport the RNA sample back to the fluid contact side of the separation chamber. Transporting the RNA sample from the fluid contact side of the separation chamber may include compressing the solid-permeable insert by the elastic material that separates the pressure-receiving side of the separation chamber from the fluid contact side of the separation chamber.

[0148] Any of these methods may include a step of pre-moistening the solid-permeable insert. [Brief explanation of the drawing]

[0149] A patent file or application file must include at least one color drawing. Copies of the color drawing of this patent or patent application publication will be provided by the Patent Office upon request and payment of the required fees.

[0150] Novel features of the present invention are specifically described in the following claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and the accompanying drawings.

[0151] [Figure 1] Figure 1A is a schematic diagram illustrating one modified method for manufacturing mRNA therapeutics. Figure 1B is a schematic diagram illustrating one modified example of a process for manufacturing a patient-specific T-cell lymphoma vaccine formulation. [Figure 2A] This figure shows an example of a microfluidic device control system described herein. [Figure 2B]This figure schematically illustrates one modified example of a microfluidic device control system that may be used as described herein. [Figure 3] Figures 3A to 3C show modified examples of the microfluidic devices described herein. [Figure 4] This is a cross-sectional view through a portion of one modified example of the microfluidic device described herein. [Figure 5] This figure shows an example of a peptoid delivery vehicle that may be used in any of the methods described herein. [Figure 6] This figure shows an example of an in vitro transcription accelerator cassette useful for creating double-stranded DNA templates. [Figure 7] This figure shows an example of a double-stranded DNA template generated as described herein. [Figure 8] This figure shows a region of an example of a T cell receptor useful for creating double-stranded DNA for use in vaccines or therapeutics. [Figure 9] This figure shows an overview of one modified architecture for a microfluidic device reactor for generating double-stranded DNA. [Figure 10] This figure schematically illustrates an example of a codon optimization process that may be used in any of the methods and apparatus described herein. [Figure 11] This figure schematically shows an example of a functional diagram of a microfluidic device configured to perform IVT as described herein. [Figure 12] This figure schematically shows an example of a functional diagram of a microfluidic device configured as a pharmaceutical microfluidic device as described herein. [Figure 13] This figure schematically shows another example of a functional diagram of a microfluidic device configured as a pharmaceutical microfluidic device as described herein. [Figure 14] This figure schematically illustrates another example of a functional diagram of a microfluidic device for pharmaceutical formulations described herein. [Figure 15]This figure illustrates an example of an experiment to investigate in vivo mRNA expression and in vivo distribution using an exemplary model of mRNA therapeutic agent as described herein. [Figure 16] Figure 16A is a graph showing the therapeutic effect of an exemplary therapeutic mRNA vaccine described herein. Figure 16B is a graph showing the therapeutic effect of an exemplary therapeutic mRNA vaccine described herein. Figure 16C is a graph showing the therapeutic effect of an exemplary therapeutic mRNA vaccine described in the specification. Figure 16D is a graph showing the therapeutic effect of an exemplary therapeutic mRNA vaccine described herein. [Figure 17] Figure 17A shows systemic luciferase expression after injection of a conserved model mRNA therapeutic prepared as described herein. Figure 17B shows an example of quantitative expression of the model mRNA therapeutic from Figure 17A. [Figure 18] This figure schematically illustrates various time intervals over which filtration may be applied in the methods and apparatus described herein. [Figure 19A] This is a top view showing an example of a microfluidic device that includes a permeable insert within the fluid contact side of the chamber. [Figure 19B] This figure shows an example of a cross-section passing through a region of an example of a microfluidic device that includes a permeable insert on one side of a chamber. [Figure 19C] This figure shows an example of a microfluidic device, schematically illustrating a vacuum cap for bubble removal. [Figure 20A] This figure schematically illustrates a method for processing therapeutic material in a fluid (e.g., an RNA sample) using a microfluidic device including a permeable insert as described herein. [Figure 20B] This figure schematically illustrates a method for removing double-stranded RNA from therapeutic material using a microfluidic device including a permeable insert as described herein. [Figure 21]Figure 21A shows an example of a system including a microfluidic device in a Class 5 isolation cabinet within a Class 7 space. This system may be configured as a mini-factory. Figure 21B shows a microfluidic device in a Class 5 cabinet. [Modes for carrying out the invention]

[0152] This specification describes methods and apparatus for manufacturing therapeutics, which may include the use of fully automated, software-controlled microfluidic apparatus. These methods and apparatus may be used for personalized or individualized therapy. This specification also describes apparatus (e.g., systems, devices, etc.) and methods that include software control of any of the manufacturing steps described herein, including template formation, in vitro transcription, purification of therapeutic mRNA, mRNA enrichment, and compounding of mRNA(s) with one or more delivery vehicles. Software control may enable the automation of these methods so that any, some, or all of these steps for manufacturing one or more therapeutic mRNAs are performed accurately, precisely, and rapidly. Software control and precise delivery and transfer of reactive components by microfluidics offer opportunities to significantly reduce or eliminate manual operations, lower the need for equipment, shorten production cycle times, and ultimately lead to low-cost therapeutics produced just-in-time as needed, while increasing process control, efficiency, and reproducibility.

[0153] In some of the apparatuses (e.g., systems, devices, etc.) described herein, each batch of therapeutic material may be manufactured in a dedicated, single-use, disposable microfluidic device (also referred to herein as a biochip), which may be housed within a microfluidic device control system (also referred to herein as a control system). The entire production process may proceed as a closed-loop process with an aseptic design that does not come into contact with the atmosphere. The entire production process is automated and controlled by the control system, and a copy-exact process can be achieved regardless of the attributes of the facility housing the system. Production parameters, raw materials, and environmental data (including complete visual records) may be protected in the cloud and become part of an extensive, encrypted electronic file associated with each production run. Furthermore, purification processes and many QC assays can be performed in-line in a single fluid flow during the production process, and anomalies can be detected early using process control concepts developed in the semiconductor industry. By leveraging fully automated, software-controlled manufacturing methods, personalized and individualized mRNA therapeutics can be manufactured for patients in a cost-effective manner.

[0154] In particular, these methods and apparatus can synthesize mRNA therapeutics in vitro by a synthetic technique known as in vitro transcription (IVT). Typically, naked mRNA molecules are large polyanionic molecules that do not cross the cell membrane and are rapidly degraded by extracellular nucleases in vivo. The methods and apparatus described herein can produce formulations of mRNA molecules having one or more delivery vehicles designed to transport mRNA to a target (such as a tissue, body, or tissue region). For example, in one variation, the delivery vehicle may be a lipid-containing amphiphilic delivery vehicle that can provide packaging and protection of the mRNA cargo during circulation, evade immune recognition, and facilitate cellular uptake and release.

[0155] In general, as will be described in more detail herein, in certain modifications, all or part of the manufacturing process, including template synthesis, IVT, purification, and formulation by delivery vehicle, can be carried out in a highly controlled environment of one or more microfluidic devices, enabling the optimization of a robust, high-quality, and reproducible manufacturing process. definition

[0156] As used herein, the delivery vehicle can refer to any suitable nanoparticles. Examples of such nanoparticles may include, but are not limited to, amphiphilic nanoparticles such as aminolipidized peptoids.

[0157] As used herein, “amplification” may refer to polynucleotide (e.g., DNA) amplification. For example, amplification may be carried out entirely within a microfluidic plate device as described herein. Amplification may include, but is not limited to, multiple substitution amplification (MDA), polymerase chain reaction (PCR) amplification, loop-mediated isothermal amplification, LAMP, nucleic acid sequence-based amplification, strand displacement amplification, rolling circle amplification, ligase chain reaction, and the like.

[0158] As used herein, automation and semi-automation refer primarily to methods and processes that are performed without human intervention, and may be under the control of one or more computer processes. The automated methods may be supervised and / or directed by human input.

[0159] As used herein, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are interchangeable and refer to deoxyribonucleotides (DNA), ribonucleotides (RNA), and their functional analogs, such as complementary DNA (cDNA) of linear or cyclic conformations. The nucleic acid molecules provided herein may be single-stranded or double-stranded. Nucleic acid molecules consist of the nucleotide bases adenine (A), guanine (G), thymine (T), and cytosine (C). In RNA molecules, uracil (U) is present instead of thymine. Analogues of natural nucleotide bases, and nucleotide bases modified with bases, sugars, and / or phosphate moieties are also provided herein. The symbol “N” can be used to represent any nucleotide base (e.g., A, G, C, T, or U).

[0160] As used herein, “cassette” (e.g., synthetic in vitro transcription promoter cassette) means a polynucleotide sequence that includes, or can be operably ligated to, one or more expression elements, such as an enhancer, promoter, leader, intron, 5' untranslated region (UTR), 3'UTR, or transcription termination sequence. In some embodiments, the cassette comprises at least a first polynucleotide sequence capable of initiating transcription of a second polynucleotide sequence to which it is operably ligated, and optionally a transcription termination sequence operably ligated to the second polynucleotide sequence. The cassette may be provided as a single element or as two or more unligated elements.

[0161] As used herein, "polynucleotide" refers to a nucleic acid molecule containing multiple nucleotides, and generally refers to both "oligonucleotides" (polynucleotide molecules with a length of 18 to 25 nucleotides) and polynucleotides with a length of 26 nucleotides or more. Aspects of this disclosure refer to oligonucleotides having a length of 18 to 25 nucleotides (e.g., 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer), or medium-length polynucleotides having a length of 26 nucleotides or more (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 250, approximately 260, approximately 270, approximately 28 This includes polynucleotides of 0, approximately 290, or approximately 300 nucleotides, or longer polynucleotides having a length greater than approximately 300 nucleotides (e.g., between approximately 300 and approximately 400 nucleotides, between approximately 400 and approximately 500 nucleotides, between approximately 500 and approximately 600 nucleotides, between approximately 600 and approximately 700 nucleotides, between approximately 700 and approximately 800 nucleotides, between approximately 800 and approximately 900 nucleotides, between approximately 900 and approximately 1000 nucleotides, between approximately 300 and approximately 500 nucleotides, between approximately 300 and approximately 600 nucleotides, between approximately 300 and approximately 700 nucleotides, between approximately 300 and approximately 800 nucleotides, between approximately 300 and approximately 900 nucleotides, or a length of approximately 1000 nucleotides, or even greater than approximately 1000 nucleotides). If a polynucleotide is double-stranded, its length can similarly be described in terms of base pairs.

[0162] In this specification, “in vitro transcription” or “IVT” refers to the process of performing transcription in a non-cellular system to produce synthetic RNA molecules (synthetic mRNA) for use in a variety of applications, including therapeutic delivery to subjects. The resulting synthetic RNA molecules (transcripts) can be combined with a delivery vehicle. Synthetic transcripts include mRNA, antisense RNA molecules, shRNA, circular RNA molecules, ribozymes, etc. The IVT reaction can use a purified linear DNA template containing a promoter sequence and the sequence of the desired open reading frame, ribonucleotide triphosphates or modified ribonucleotide triphosphates, a buffer system containing DTT and magnesium ions, and an appropriate phage RNA polymerase.

[0163] A "template" or "double-stranded DNA template" is an isolated nucleic acid sequence containing the minimum constituent sequences necessary for in vitro transcription of the target open reading frame into which it was inserted.

[0164] As used herein, “fluid depot” may refer to a storage space for holding fluids, and may include vials, bottles, bags, tubes, etc. A fluid depot may also include fluid lines as part of the fluid line (e.g., passages or channels leading out of the body or chamber within the fluid depot).

[0165] The fluid power systems used in this book include both pneumatic and hydraulic systems. For convenience, the term "pneumatic" may sometimes include "hydraulic," and these terms may be used interchangeably.

[0166] As used herein, “therapeutic polynucleotide” means a polynucleotide (e.g., therapeutic mRNA) that may be part of a therapeutic polynucleotide composition for delivery to a subject to treat, prevent, improve or otherwise modify the subject’s health.

[0167] As used herein, “therapeutic polynucleotide composition” may refer to a composition comprising one or more polynucleotides (e.g., mRNA) encapsulated by a delivery vehicle administered to a subject. mRNA vaccines are merely one example of therapeutic polynucleotide compositions.

[0168] As used herein, "bacterial DNA-free" means the absence of bacterial DNA. Materials that are substantially free of bacterial DNA may contain bacterial DNA in amounts less than 0.1%, less than 0.01%, less than 0.001%, etc. As used herein, "endotoxin-free" means the absence of endotoxins. "Substantially free of endotoxins" means that the endotoxin content is less than 0.1%, less than 0.01%, less than 0.001%, etc.

[0169] In this specification, “binding” means a method for binding one component to another, such as ligation, synthesis, primer extension, annealing, recombination, or hybridization.

[0170] Microfluidic devices or microfluidic plate devices used herein are equivalently referred to as chips, cartridges, biochips, microfluidic plates, etc., and may include a plurality of fluidically interconnected chambers. These chambers may be divided into a fluid contact side and a pressure receiving side. The pressure receiving side may be part of a fluid power circuit, while the fluid contact side may be isolated from the external atmosphere and used for processing materials within the microfluidic device. Microfluidic devices described herein may generally be flat (e.g., having a thickness of less than about 4 cm, less than about 2 cm, less than about 1.5 cm, less than about 1 cm, etc.) and may include a plurality of pressure ports for communicating with one or more pressure lines for driving and / or controlling a fluid power circuit within the microfluidic plate device.

[0171] As used in this book, "on-demand" is intended to define when a method or service is performed, and is used in contrast to being stored, scheduled, ordered, or prepared in advance.

[0172] As used herein, the term "optical sensor" typically refers to a light sensing device, which may include one or more imaging devices. The optical sensor may include single lenses, cameras, stereo cameras, multi-lens cameras, digital still cameras, thermographic cameras, CCDs, optical fibers, and the like.

[0173] As used herein, “purification” means the physical and / or chemical separation of an ingredient (e.g., particles) from other unwanted ingredients (e.g., contaminants, fragments, etc.).

[0174] As used herein, "sealed fluid communication" and "sealed closed channel" may both refer to isolating a material (a fluid containing, for example, a template, a therapeutic polynucleotide and / or a solution of a therapeutic polynucleotide composition, but not limited to such material) from the surrounding atmosphere.

[0175] In this specification, “template precursor material” means the material necessary to form a template (e.g., a DNA double-strand template), and may include the synthetic gene of the target and an in vitro transcription accelerator cassette provided as one or more independent elements.

[0176] In this specification, 2D purification refers to purification performed in a substantially flat microfluidic device (e.g., a microfluidic plate device) as described herein, and includes the use of two or more adsorbents to remove material (e.g., double-stranded RNA, unreacted nucleotides, unreacted capping reagents, buffer components, etc.).

[0177] mRNA-based therapies can be used in multiple treatment approaches, including vaccination, immunotherapy, protein replacement therapy, tissue regeneration, and gene editing for the treatment of genetic diseases. In addition to their high efficacy, mRNA-based therapies also offer important advantages such as rapid development cycles, standardized manufacturing, transient expression, and a low risk of genome integration.

[0178] In some modifications, the mRNA therapeutics described herein may include mRNA encoding the target antigen or protein as the active ingredient in the final drug. Robust translation of mRNA requires a functional 5' cap structure. The 5' cap (or 7-methylguanosine cap) consists of a terminal 7-methylguanosine residue linked to the first transcription nucleotide via a 5'-5'-triphosphate bond. This cap is crucial for ribosome recognition of mRNA and protection from RNAs. The poly(A) tail binds to poly(A)-binding protein (PABP), interacts with the eukaryotic translation initiation factor eIF4G, and further forms a complex with eIF4E to synergistically regulate mRNA stability and translation initiation with the m7G cap. The length of the poly(A) tail is thought to influence the efficiency of the mRNA-to-protein translation process.

[0179] mRNA therapeutics can be broadly classified into at least five categories, used for (i) protein substitution, (ii) vaccines, (iii) effector protein expression, (iv) loss of function induction by dominant-negative protein expression, and (v) gene / genome editing. The methods and apparatus described herein may provide mRNA therapeutics for any (or more) of these categories.

[0180] The methods and apparatus described herein provide packaging and protection of circulating mRNA cargo, avoiding toxicity or immunogenicity concerns that may limit repeated administration, and can be used to formulate mRNA therapeutics to avoid immune recognition, localize the drug to desired tissue, and facilitate cellular uptake and release.

[0181] Generally, a method for producing mRNA therapeutics (including, but not limited to, patient-specific T-cell lymphoma vaccine formulations) may include any or all of the steps schematically shown in Figure 1A, and include the identification of a target protein and design of an mRNA sequence 101, and the preparation of a double-stranded DNA template for the target sequence 103. Using this sequence, mRNA may be generated for an in vitro transcription (IVT) reaction 105, and mRNA may be synthesized. This therapeutic mRNA may then be purified to remove process impurities and filtered to produce a pharmaceutical substance 107. The therapeutic mRNA may then be formulated together with a delivery vehicle (including, in variations, an adjuvant and delivery vehicle components for forming amphiphilic nanoparticles) 109. The formulation may then be processed and purified to produce a pharmaceutical product to be delivered to a patient 111. As described above, in some modifications, some of these steps may be performed remotely (e.g., 101-107) and some on-site (e.g., 109, 111), and in some modifications, all of them (e.g., 103, 105, 107, 109, 111) may be performed on-site.

[0182] As a specific example of a modification, Figure 1B shows an exemplary process for manufacturing a patient-specific T-cell lymphoma vaccine formulation. In Figure 1B, the process may include the identification of a clonally expanded TCR sequence (idiotype) expressed by lymphoma cells 121. The process may also include designing an mRNA vaccine sequence 123 and preparing a double-stranded DNA template for use in the IVT reaction 125. This template may be used in the IVT reaction to synthesize mRNA 127, and the therapeutic mRNA may be purified to remove process impurities and filtered to prepare the therapeutic mRNA as a pharmaceutical substance 129. Subsequently, the therapeutic mRNA may be formulated with an adjuvant and a delivery vehicle component to form amphiphilic nanoparticles 131. Next, post-formulation treatment 133 may be performed to produce a pharmaceutical product such as a therapeutic mRNA vaccine 135.

[0183] Any of these manufacturing steps can be optimized to be performed using an automated microfluidic device control system as described herein. For example, the production of a DNA template may be carried out in one or more microfluidic devices, and in the example shown in Figure 1B, a template microfluidic device (e.g., a template biochip) may be used. In this same example, the steps of transcribing mRNA in vitro and purifying the material to produce a pharmaceutical substance may be carried out in an IVT microfluidic device (e.g., an IVT biochip), and the pharmaceutical formulation step may be carried out in a formulation microfluidic device (e.g., a formulation biochip). These microfluidic devices may include input ports, metering valves, reaction chambers, and purification structures necessary to perform each step of the manufacturing process. Device

[0184] The methods described herein may generally be carried out using apparatus that may be used with and / or include one or more microfluidic devices (e.g., biochips), and systems configured to control the operation in the microfluidic devices (e.g., microfluidic control systems). These apparatuses may be referred to herein as microfluidic apparatuses, microfluidic control devices, microfluidic device control systems, microfluidic control systems, or microfluidic systems. Microfluidic devices (also called microfluidic plate devices) may be located within a microfluidic control system and may operate in a closed-loop manner to prevent some, more preferably almost all or all, of the manufactured components in the system from being exposed to the atmosphere. In particular, parts of the apparatus that come into contact with the fluid(s) in the system are prevented from being exposed to the atmosphere. Figure 2A shows an example of a microfluidic device control system including a microfluidic device management system 203 (hardware for holding microfluidic devices, applying positive / negative pressure to manipulate microfluidic operations within microfluidic devices, heating / cooling all or some areas of microfluidic devices, detecting one or more features from microfluidic devices and / or recording operations performed on one or more microfluidic devices), a controller (not shown), and a refrigerated container 205 (e.g., an ISO Class 5 cabinet). This system may use or include one or more microfluidic devices 201.

[0185] The microfluidic apparatus may be a microfluidic apparatus for forming therapeutic polynucleotides (e.g., mRNA therapeutics). The apparatus may include a seating mount for removably holding a microfluidic plate device, a plurality of pressure lines, a plurality of fluid vials, and a controller. Each fluid vial contains or is configured to bind to a fluid line, and each fluid line and at least a subset of pressure lines may be configured to be biased to the microfluidic plate device held in the seating mount to form a closed channel. The controller may be configured to control the application of pressure through the pressure lines to drive fluid motion within the microfluidic plate device when the microfluidic plate device is held in the seating mount. The controller may be configured to direct the synthesis of a synthetic template, direct an in vitro transcription (IVT) reaction using the template to form therapeutic polynucleotides, and direct the purification of therapeutic polynucleotides in one or more microfluidic plate devices held in the seating mount.

[0186] A microfluidic device (e.g., a microfluidic device for forming therapeutic polynucleotides such as therapeutic mRNA) may include a seating mount for removably holding a microfluidic plate device, a plurality of pressure lines, a plurality of fluid vials, and a controller. Each fluid vial contains or is configured to bind to a fluid line, and each fluid line and at least a subset of pressure lines may be configured to be biased to the microfluidic plate device held in the seating mount to form a closed channel. The controller may be configured to control the application of pressure through the pressure lines to drive fluid motion within the microfluidic plate device when the microfluidic plate device is held in the seating mount. The controller may be configured to determine the contents of the fluid vials, transfer a microliter or less of material from the vials to one or more reactors in the microfluidic plate device held in the seating mount, instruct the synthesis of a synthesis template, instruct an in vitro transcription (IVT) reaction using the template to form therapeutic polynucleotides, and instruct the purification of therapeutic polynucleotides in one or more microfluidic devices held in the seating mount.

[0187] The controller may be configured to perform any of the methods described herein, in particular, to receive and process inputs (e.g., optical input, pressure input, temperature / thermal input, etc.) to control the movement of fluid within a microfluidic device, the temperature (including thermal cycling) of various regions of the microfluidic device, cleaning / coupling, opening / closing valves in the microfluidic device, detection of the microfluidic device, etc. The controller may include one or more microprocessors, communication circuits, memory, etc. The controller may consist of firmware, hardware and / or software.

[0188] Any of these devices may include one or more optical sensors positioned around the seating mount and reagent storage frame to monitor the fluid level in the reagent storage frame and the fluid movement within the microfluidic device when the microfluidic device is seated on the seating mount. Alternatively or additionally, the optical sensor(s) may be positioned on the bottom of the device (e.g., below the seating mount) and oriented upward to detect fluid volume, movement, etc.

[0189] The methods and apparatus described generally include one or more fluid power circuits for moving material (liquid material) between the fluid chamber (depot, fluid contact side, reactor, etc.) and channels of a microfluidic device, or within the microfluidic device, and possibly between the microfluidic device and fluid depots (bale, bottle, container, etc.) within the apparatus. A fluid power circuit may be a hydraulic or pneumatic circuit that includes one or more pressure channels within the microfluidic device and the pressure-receiving side of the chamber. A fluid power circuit is sometimes referred to as a microfluidic power circuit. A single microfluidic chip may include multiple fluid power circuits, and a fluid power circuit may also include one or more pressure lines and interfaces between the pressure lines of a microfluidic control device and one or more microfluidic chips within the microfluidic device. One or more fluid power circuits may share components (valves, pressure lines, vacuum caps, etc.) with other overlapping fluid power circuits. Furthermore, it should be understood that where the term "pneumatic" is used for convenience, general fluid power circuits (e.g., hydraulic and / or pneumatic) may be used instead or additionally. The fluid material driven by the fluid power line may be any suitable fluid (e.g., gas or liquid such as air, water, or oil).

[0190] Furthermore, this specification describes microfluidic devices for processing therapeutic polynucleotides in a closed circuit (e.g., closed-circuit microfluidic devices). As mentioned above, these microfluidic devices may be referred to herein as microfluidic chips, microfluidic plates, process chips, biochips, process plates, etc. Generally, microfluidic devices may also be microfluidic plate devices, which may be substantially flat plate-like structures, and these structures may be relatively thin (e.g., less than a few millimeters thick, e.g., 0.5 to 20 mm thick, 0.5 to 15 mm thick, 0.5 to 10 mm thick, etc.). The microfluidic devices described herein may generally be at least partially transparent, in particular the upper part of the microfluidic device may be transparent, so that one or more optical sensors (cameras, CCDs, optical fibers, etc.) can be used together with the microfluidic device to sense, detect, monitor, record, etc., actions including fluid movement and / or movement of elastic layers, as used by the microfluidic apparatus described herein.

[0191] Figure 2B is a schematic diagram showing one modification of a microfluidic device control system that may be used as described herein. In this example, the apparatus includes a housing 233 surrounding a seating mount 215 that can hold one or more microfluidic devices 211, which may be single-use devices. The housing may be a chamber, enclosure, etc., which may include a lid or opening, and may be sealed when closed. The housing may surround a thermal regulator and / or may be configured to surround a thermally regulated environment (such as a refrigeration unit). The housing may form a sterile barrier. In one modification, the housing may form a humidified or humidity-controlled environment.

[0192] The seating mount 215 may be configured to secure the microfluidic device using one or more pins or other components configured to hold the microfluidic device in a predetermined orientation.

[0193] In one modification, the thermal control unit 213 may be positioned adjacent to the seated mount 215 to regulate the temperature of one or more microfluidic devices 211. The thermal control unit may include a thermoelectric component (e.g., a Peltier device) and / or one or more heat sinks to control the temperature of all or part of the microfluidic device. In one modification, one or more thermal control units may be included to regulate the temperature of one or more regions of the microfluidic device separately. The thermal control unit may include one or more thermal sensors (e.g., thermocouples) that can be used for feedback control of the microfluidic device and / or the thermal control unit.

[0194] In Figure 2B, the fluid interface assembly 209 may connect a liquid reagent and / or pressure (e.g., gas) to a microfluidic device 211 held in a seated mount 215, and assist in the delivery of fluid material from a pressure source 217 to the interior of the microfluidic device 211 according to positive / negative gaseous pressure. The fluid interface assembly may optionally assist in the fixation of the microfluidic device(s), as will be described in more detail below. The fluid interface assembly may be detachably coupled to the apparatus (and may be detached, or partially detached) for sterilization between uses.

[0195] The reagent storage frame 207 may be configured to include a plurality of fluid sample holders, each of which may hold a fluid vial configured to hold a reagent (e.g., nucleotide, solvent, water, etc.) for delivery to the microfluidic device 211, or the fluid vials may be configured to receive products from inside the microfluidic device 211. The reagent storage frame may be referred to as a reagent rack. In one modification, the reagent rack may include a plurality of pressure lines and / or manifolds configured to divide one or more pressure sources 217 into a plurality of pressure lines that can be applied to the microfluidic device, and which may be controlled independently or collectively (in a subcombination). Alternatively, the fluid depot (e.g., vials) may be configured to be directly fixed and sealed to the microfluidic device(s).

[0196] The fluid interface assembly may include a plurality of fluid lines and / or pressure lines and may include bias (e.g., spring-loaded) holders or tips that drive each fluid and / or pressure line individually and independently to the microfluidic device when held in the seating mount 215 (or, as previously stated, the device may be directly spring-mounted). Tubes, e.g., fluid lines and / or pressure lines, may be part of the fluid interface assembly or connected to the fluid interface assembly. In one modification, the fluid line consists of a flexible tube connecting between the reagent housing frame and the microfluidic device via a connector that locks the vial to the tube with a locking engagement (e.g., a ferrule). The ends of the channels, in one modification, the ends of the fluid lines / pressure lines, may be configured to be sealed to the microfluidic device, for example, by a sealing port formed in the microfluidic device, as described herein. For example, the ends of the fluid lines may be cut or formed to be flat (vertical in side view). The vials may be pressurized (e.g., to a pressure greater than 1 atmosphere, such as 2 atmospheres, 3 atmospheres, 5 atmospheres, etc.) via a connector which may also be connected to a pressure source. For example, the fluid vial may be pressurized to between 1 and 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, thereby preventing or reducing leakage. In one modification, the pressure difference between the fluid valve and the pneumatic valve may be about 5 psi (e.g., about 7 psi, 10 psi, 12 psi, 15 psi, 20 psi, etc.).

[0197] Each vial may be coded (for example, by an identifier that can be read by one or more sensors, as described later). The controller may monitor fluid levels, i.e., the amount of each material in the fluid interface assembly.

[0198] The apparatus may also include a magnetic field applicator 219, which may be configured to form a magnetic field in the area of ​​the microfluidic device 211. One or more sensors 205, which may be optical sensors, may be part of the apparatus and may sense one or more of the following: a barcode, the fluid level in a fluid vial held in a reagent storage frame, and the fluid movement in the microfluidic device 211 when the device is mounted in a seating mount 215.

[0199] The sensor can perform process measurements on the device, for example, by measuring optical indicators. In one variation, visual / optical markers may be used to estimate yield. For example, fluorescence may be used to detect process yield or residual material by tagging with a fluorescent substance. Alternatively or additionally, dynamic light scattering may be used to measure the particle size distribution within a portion of the microfluidic device (e.g., a mixing portion). In one variation, the sensor measurement may be performed by detecting the outgoing optical signal using one or two optical fibers that transmit light (e.g., laser light) through them. The instrument package may be mounted remotely from the device. Such non-contact sensing may be preferred in some cases.

[0200] In any of the methods and apparatus described herein, a sensor (e.g., a video sensor) may record all activity on a microfluidic device (e.g., a chip or cartridge). For example, the entire operation for synthesizing and / or processing a material (e.g., therapeutic RNA) may be recorded by one or more video sensors, including, for example, a video sensor capable of visualizing the microfluidic device from above. The processing on the microfluidic device may be visually tracked, and this recording may be retained for later quality control and / or processing. Thus, video recordings of processing may be stored, kept and / or transmitted for subsequent review and / or analysis.

[0201] The internal parts of the instrument, for example, within the housing 233, may be configured to be further sterilizable. In particular, parts of the instrument may be removed and sterilized individually. Sterilization may be performed, for example, by UV irradiation or any other sterilization method that may be required to limit contamination or to meet regulatory requirements. The apparatus, including the housing, may be housed in a high-efficiency particulate air (HEPA) filtration environment. The apparatus, including the housing, may be housed in a temperature-controlled enclosure. Furthermore, the apparatus itself may include one or more temperature-controlled areas. In any of the apparatuses described herein, the apparatus may include a temperature-controlled area (for example, within the housing) for storing reagents and / or mRNA (e.g., therapeutic mRNA) at a storage temperature (e.g., -10°C to 20°C, e.g., 10°C, 4°C, -10°C, etc.). Any of these apparatuses may include a library of manufactured mRNA that can be formulated individually or in combination with one or more additional mRNAs and delivery vehicles.

[0202] As described above, the microfluidic device control system may be controlled by a controller 221, including applying pressure through the microfluidic device 211 to drive at least fluid movement. The controller may be located entirely or partially outside the housing. The controller may be configured to include user inputs / outputs. For example, the system's user interface 223 may allow for easy operation and instruction of the apparatus and microfluidic device(s).

[0203] Any of the devices described herein may include all or some of the components shown in Figure 2B, and not all components are necessarily required. Figure 2B shows only some of the connections between the components, and additional (or alternative) connections may be used.

[0204] A microfluidic device control system can support all production activities within a microfluidic device, including reagent supply, fluid control, temperature control, mixing, purification, and process monitoring. Production activities on the microfluidic device control system can be accessed and controlled through application software.

[0205] A microfluidic device may be configured to include one or more reactors for manufacturing steps performed to precisely prepare therapeutic (e.g., therapeutic mRNA) material. The same microfluidic device may operate on one or more microfluidic devices in series and / or parallel without interrupting the continuity of the microfluidic device control system. For example, when manufacturing a therapeutic agent using multiple processing steps performed in multiple reactors using multiple microfluidic devices, a fluid product (possibly multiple) containing partial products from one microfluidic device may be transferred by the device, in a closed-loop manner, to one or more additional microfluidic devices, which may include moving the fluid containing the microfluidic device product to a storage depot of the microfluidic device control system.

[0206] Each microfluidic device may be configured to include one or more reactors for processing during the manufacturing process. For example, Figures 3A–3C show three examples of microfluidic devices. These examples illustrate three different types of microfluidic devices: a template microfluidic device (Figure 3A), an in vitro transfer (IVT) microfluidic device (Figure 3B), and a formulation microfluidic device (Figure 3C). Each of these examples of microfluidic devices may be configured to include functions for performing a series of unit operations in a highly reproducible controlled manner.

[0207] In one modification, the microfluidic device may be configured as a multilayer structure including two rigid layers with a flexible membrane sandwiched between two ridged layers. Figure 4 is a cross-sectional view (lateral to the plane of the microfluidic device) through an example of a microfluidic device having multiple layers forming a reactor for processing therapeutic agents as described herein. The reactor may include a chamber containing seals, channels, valves, and a pumping chamber formed from multiple layers. For example, the microfluidic device may be formed from two or more rigid or semi-rigid plates 403, 405 and at least one elastic layer 407. The elastic layer 407 may be a sheet of liquid-impermeable elastic material. The elastic layer may be gas-permeable or may be treated to be gas-permeable, including in various regions. A single continuous sheet of elastic material may be used, but in one modification, multiple sheets of elastic material may be used, or the “sheet” may be formed from portions of multiple sheets. Alternatively, layers and elastic sheets may be laminated. Generally, chambers for holding, valvering, and / or pumping fluid may be formed on plates on both sides of the elastic layer, such that the elastic layer divides the chamber into a liquid-containing side and a pressure-applied (e.g., gas) side. The overall volume of the chamber(s) may be constant and may be formed on both the first (e.g., upper) plate and the second (e.g., lower) plate, 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 to reduce (to zero, closing the chamber) the volume on the liquid-containing side or increase (to a predetermined maximum value) the volume on the liquid-containing side. Also, to apply negative or positive pressure to the pressure-receiving side 419 of one or more chambers, the pressure-applying side may be connected to a pressure port 443 on the upper plate 403, which is connected to a pressure channel 447. The liquid-containing side 417 of each chamber, opposite the pressure-applying side, may be connected to a fluid port 423 via a fluid channel 421.Both the fluid port and the pressure port may be formed by openings to the upper plate 403 and the elastic layer 407, and the pressure line is pressed into the elastic layer 407, which is supported on the underside of the port by the opposite rigid or semi-rigid layers, 405, 409, thus enabling a sealed connection isolated from the atmosphere even when there are multiple different input lines.

[0208] In Figure 4, the microfluidic device 400 includes a first (e.g., upper) plate 403 having a first (e.g., upper) surface 411, a second (e.g., bottom) surface 429, and a thickness between them. The first surface 411 may form an exposed outer surface. The microfluidic device also includes a second plate 405 having a first (e.g., upper) surface 431, a second (e.g., bottom) surface 433, and a thickness between them. An elastic layer 407 is sandwiched between the second surface 429 of the first plate 403 and the first surface 431 of the second plate 405. A third plate 409 is bonded to the second plate directly or indirectly on the second surface 433 of the second plate. The third plate 409 also has a first (e.g., upper) surface, a second (bottom) surface, and a thickness between them. The second surface of the third plate may form the bottom surface of the microfluidic device. Each plate may be formed of multiple layers, which may be stacked or otherwise connected. For example, in Figure 4, the third plate 409 includes an optional second elastic layer 413 that can help bond the third plate to the second plate, and in this example, the second elastic layer 413 forms the first surface 435 of the third plate 409. The layers and plates shown in Figure 4 may not be to scale (for example, the elastic layer 407 may be thinner than the plate).

[0209] Furthermore, the microfluidic device 400 shown in Figure 4 may include a plurality of chambers 415, 416, 418, 420, each having a certain volume. These chambers are formed by cut-out regions (e.g., rounded / curved cuts) into the second (bottom) surface 429 of the first plate 403 and the first (top) surface 431 of the second plate 405, and the elastic layer 407 branches these chambers so that each includes a liquid-containing side 417 and a pressure (e.g., gas-containing) side 419. The microfluidic device 400 may also include a plurality of liquid (e.g., fluid) channels. In Figure 4, a single fluid channel 421 extends from a fluid port 423 that penetrates the thickness of the first plate 403 to a liquid channel opening 425 through the elastic layer 407, and substantially penetrates the thickness of the second plate 405 to the bottom surface 433 of the second plate. The liquid channel 421, which extends parallel to the lower surface of the third plate, has its length formed on the lower surface 433 of the second plate, with the upper surface of the third plate 409 as the boundary.

[0210] With respect to the fluid port 423, the diameter of the opening in the first plate 403 that forms the fluid port 423, extending through the thickness of the first plate, may be larger than the diameter of the fluid channel opening 425 that extends through the elastic layer 407 into the liquid (e.g., fluid) channel 421. The fluid channel opening 425 may be centrally located with respect to the bottom of the fluid port opening, and may be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line coupling interface that will be connected to the fluid port.

[0211] The fluid channel 421 is connected to the liquid-containing side 417 of the first chamber 415. This first chamber has a relatively low holding volume (fixed volume), but may be configured as a valve that can be fully opened and closed by the movement of the elastic layer 407.

[0212] The microfluidic device 400 also includes a plurality of pressure channels that can be independently controlled to apply positive and / or negative pressure. In Figure 4, a single pressure port 443 connected to the fourth chamber 420 is shown, but each of the chambers 415, 416, and 418 may independently operate and control the movement of the portion of the elastic layer 407 that branches these chambers and connect each chamber independently to separate pressure ports and pressure channels for valves and / or pumps. In one modification, pressure ports may be shared among the plurality of chambers. In Figure 4, the pressure (e.g., gas) port 443 is similar to the fluid (e.g., liquid) port 423 and includes an opening that penetrates the first plate 403 completely to the exposed elastic layer 407, and through the elastic layer that forms the pressure (e.g., gas) channel opening 445 to the opening. The pressure channel opening 445 extends from a pressure port 443 that substantially penetrates the thickness of the first plate 403 and is a cut-out channel along the bottom of the second plate (or alternatively, a cut-out region at the top of the third plate), and is continuous with a pressure channel 447 that passes through the second plate and the elastic layer 407 and returns to the region of the pressure channel in the first plate that connects to the pressure (e.g., gas) containing portion 419 of the fourth chamber 420. As described for a similar fluid (e.g., liquid) port, the diameter of the pressure port 443 that penetrates the thickness of the first plate 403 may be larger than the diameter of the pressure channel opening 445 that penetrates the elastic layer 407, and may be centrally located or offset by a greater margin than the wall thickness of the pressure line or pressure line coupling interface that will connect to the pressure port.

[0213] In the cross-section of the microfluidic device 400 shown in Figure 4, there are other fluid (e.g., liquid) lines, fluid ports, pressure lines, and multiple connections to pressure ports, but these are not shown because they are outside the shown plane. For example, in Figure 4, the liquid-containing side / part 417 of the fourth chamber may be connected to an additional valve (chamber) and / or channel, including an outlet channel extending from the liquid-containing side 417, for example. Additional chambers (e.g., configured as valves), not shown, may be formed as described above. In one modification, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid receiving depot, such as a vial or tube. This receiving depot may be held in a reagent storage frame.

[0214] Generally, such microfluidic devices and microfluidic apparatuses are configured to perform multiple complex steps without human intervention, in a completely sealed (sealed and protected from the atmosphere) state by the apparatus on the microfluidic device. Fluids may be metered using a fixed-volume chamber and moved, mixed, filtered, etc., by applying air pressure to flex the area of ​​an elastic layer.

[0215] In one modification, a chamber within a microfluidic device may be configured as a mixing chamber for mixing fluids within the microfluidic device. In another modification, a chamber(s) may be configured as a dialysis chamber, which may include a dialysis material and one or more countercurrent channels for dialysis of substances in the fluid. In yet another modification, one or more chambers may be configured as a concentrator for concentrating therapeutic material.

[0216] While various microfluidic devices may differ in the arrangement of channels, ports, and chambers, their basic architecture is the same, and many functional elements are shared that can be used in different configurations to perform different protocols. These functional elements include, as mentioned above, input ports, metering valves, pumps, reaction chambers, mixing structures, and purification structures.

[0217] Any of these microfluidic devices may include one or more bubble removal chambers, or one of the chambers on the fluid contact side may be configured as a bubble removal chamber to remove bubbles in the fluid on the fluid-containing side. The bubble removal chamber may also be called a vacuum cap and may generally be configured to apply negative pressure on the opposite side of the membrane while the fluid is held within the fluid contact side of the chamber. The membrane may be at least partially gas permeable, as described above. Figures 19A-19C show an example of a bubble removal chamber. All, or more preferably, part 1988 (e.g., only the cap area) of the membrane partitioning the chamber may be in contact with the vacuum, for example, via a vacuum line 1987 on the top surface or top plate of the device, as shown in Figure 19C. In operation, the vacuum cap 1938 may remove or reduce bubbles in the line by holding the fluid within the fluid contact side of the chamber and applying negative pressure on the upper side (pressure-receiving side) of the chamber. The membrane separating the chamber into a fluid contact side and a pressure receiving side may be gas permeable, allowing gas to be removed from the liquid (fluid) side by negative pressure drawing gas (e.g., air, nitrogen, etc.) through the membrane above the flow path. For example, the membrane (or region of membrane within the vacuum cap) may be, for example, a polydimethylsilicone (PDMS) elastomer film, which is sufficiently gas permeable to remove gas from the liquid side of the membrane. A fluid chamber having a fixed volume as described herein (e.g., formed between a first plate and a second plate) may include or be coupled to one or more bubble removal chambers (vacuum caps) and / or be configured as a bubble removal chamber. In one modification, the portion of the elastic layer positioned between the first and second surfaces forming the chamber, for example, the portion separating the fluid contact side on the second surface (and / or second plate) from the pressure receiving side on the first surface (and / or first plate), may have minimal deflection (or no deflection at all). For example, the upper, pressure-receiving side may be minimally spaced and / or nearly coplanar (e.g., flat) with the relaxed membrane, while the fluid-contacting side is concave and extends into the second surface (second plate).The controller may, for example, retain fluid within the vacuum cap region by applying positive pressure to the pressure-receiving side of a valve to close the valve on either or both sides (inlet and outlet) of the vacuum cap, or by applying negative pressure to the pressure-receiving side of the pressure-receiving vacuum cap. The absolute amount of negative pressure applied (e.g., magnitude of negative pressure) may be less than that applied to deflect the membrane (e.g., less than the absolute value of the positive pressure applied to close the valve and / or pump). Alternatively, in one modification, the membrane may be configured to be deflected (e.g., deflected upward) relative to a first surface and / or plate to draw fluid into the enlarged fluid-contact side of the chamber. The membrane may be held by negative pressure applied to the first upper surface, allowing bubbles (e.g., air bubbles) to be removed. The controller may retain the fluid in the vacuum chamber for a period 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 minute and a half 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 Figure 19C, pressure may be applied via a pressure line 1987 communicating with the pressure-receiving surface of the chamber formed between the first and second surfaces of the apparatus (e.g., the first plate and the second plate). The vacuum cap 1938 may be valved by one or more valves 1992. The fluid may exit the fluid contact side through a fluid line 1989 on the opposite side of the vacuum cap.

[0218] The fluid contact side of the chamber of the pressure cap may be in fluid communication with a fluid port that is fluidly connected to the fluid contact side of each chamber via one or more fluid channels (similar to the valves and reactors described herein), and this fluid port may be located on the second surface and / or plate. The pressure receiving side of the vacuum cap may be in fluid communication with a pressure port extending through the first surface / plate (e.g., within the surface / plate) so as to be in fluid communication with the pressure receiving port / side via a pressure channel extending through the second plate and along the first plate, as described herein.

[0219] Any microfluidic device described herein may be a substantially thin microfluidic plate device, as described above. Therefore, in-plate / on-plate processing may be carried out substantially two-dimensionally (2D), including the purification of any polynucleotide (e.g., mRNA). 2D purification of polynucleotides is particularly advantageous compared to prior art, as described herein, which may involve steps requiring the use of columns and which may be difficult or impossible to perform in a closed environment and / or in small quantities.

[0220] Furthermore, as shown in Figure 4, the fluid-contact side (and / or pressure-receiving side) of each chamber may be configured such that the elastic layer seats flush and gapless with the fluid-contact side on the second surface when the positive pressure on the pressure-receiving side drives the elastic layer against the fluid-contact side. In one modification, the fluid-contact side and / or pressure-receiving side may be concave. This concave may have a shallow elliptical cross-section to allow the elastic layer to easily sit coplanar with the wall of the fluid-contact side (and / or pressure-receiving side). The elastic layer is pressed against (e.g., seated) against the wall of the chamber (e.g., the fluid-contact side) so that there are no dead retention portions of the chamber.

[0221] The microfluidic device can be connected to a microfluidic device control system via a series of spring-loaded connections for both reagents and pneumatic lines used to manage fluid movement and valve control. The reagent and gas lines may be sealed by pressure against an elastomer layer embedded in the microfluidic device, forming a completely sealed pathway from the reagent vial to the microfluidic device and from the microfluidic device to the transfer vial. This sealed pathway is maintained for all reactions within the microfluidic device, effectively eliminating contact with the atmosphere and minimizing the risk of contamination.

[0222] The microfluidic device control systems described herein may provide a sterile controlled environment and may include interfaces for loading reagents and taking output. In any of the devices (e.g., systems) described herein, the device may include a housing that provides a controlled environment, and this housing may also be located within a controlled environment. For example, a sealed device may be in a Class 5 environment located within a Class 7 environment.

[0223] Microfluidic device control systems may be designed to connect to all actuators in a single step. These control systems may also scan identifiers (e.g., barcodes) for all reagents and microfluidic devices and monitor fluid levels. Generally, these microfluidic device control systems may automate all or part of the functions of the microfluidic devices and generate visual records of all process steps that can be monitored, stored, transmitted, or reviewed later (e.g., for optical quality control analysis of intermediate process outputs).

[0224] As described above, the microfluidic device management system may include hardware such as nests (microfluidic device holders) that can be designed to ensure that microfluidic devices are properly aligned and can be inserted in only one direction. This may be managed, for example, via two pins and / or notches in the nest that conform to the shape of the microfluidic device. The microfluidic device management system (control system) may also include a vial rack for holding reagents and transfer vials, a downward-facing camera for recording all liquid and valve movements and product transfers, a side camera on a rail for capturing barcodes and detecting liquid levels, and a robotic arm with a magnet for bead manipulation. The microfluidic devices are secured with vacuum chucks and are in good contact with a Peltier element for temperature control. After the microfluidic devices are installed, mating with all connectors is achieved in one step by lowering the top of the microfluidic device management system with a dowel pin guide system.

[0225] As mentioned above, the microfluidic device control system may include a control panel that interfaces with all electronic devices (CPU, Ethernet RIO device controller, etc.), as well as valves, manifolds, and pressure regulators for pneumatic control. Any of these systems may also include a refrigerated cabinet or chamber (e.g., an ISO Class 5 safety cabinet) that acts like a biosafety hood, providing a microbiologically safe enclosure through HEPA air filters and airflow control. Furthermore, this cabinet may also ensure that all reagents are kept at the appropriate temperature throughout the manufacturing process. The cabinet may also be equipped with UV lamps for sterilizing the microfluidic devices and all internal microfluidic device management system elements. The microfluidic device management system itself may reside in an environment that is an ISO Class 7 room (e.g., a 6-foot x 6-foot ISO Class 5 small environment). All interaction between the operator and the system, including the loading of reagent vials and microfluidic devices, may be performed according to best aseptic techniques. Delivery vehicle

[0226] The methods and apparatus described herein can be adapted to a wide range of mRNA delivery vehicles. For example, delivery vehicles can be adapted to electroporation and gene guns, viral delivery by adenovirus (AV) or adeno-associated virus (AAV), exosomes and liposomes, cationic polymer encapsulation, and lipid nanoparticle (LNP) formulations. Manufacturing of therapeutic drugs

[0227] This specification describes methods and apparatus (e.g., devices and systems) for manufacturing therapeutic drugs. These methods and apparatus may be used to manufacture patient-specific therapeutic drugs in a very rapid period of time. In particular, these methods and apparatus may be used to manufacture therapeutic drugs based on polynucleotides such as mRNA, as described above. As part of this process, the methods and apparatus may perform some or all of the steps described above, including the manufacture of an IVT DNA template, the execution of an IVT reaction to produce therapeutic mRNA, the purification of therapeutic mRNA, the formulation of the mRNA with a delivery vehicle to form a therapeutic composition, and post-formulation processing of the final drug. IVT mold creation

[0228] The methods for producing DNA templates described herein, particularly for producing synthetic DNA templates, are considered particularly useful for producing better, more scalable, faster, and safer vaccines and therapeutic agents. The use of synthetic templates for mRNA synthesis by IVT is beneficial in many ways, including preventing potential microbial contamination. Solutions containing synthetic DNA templates generally do not contain contaminating cells, cell extracts, and endotoxins from cells. These solutions may be particularly suitable for being part of vaccines to be injected into patients, where there is substantially no risk of toxicity from contaminating cells, cell extracts, or endotoxins.

[0229] In one modification, an in vitro transcription-promoting cassette (IFC) as described herein is an in vitro transcribed double-stranded DNA. Figure 6 shows an example of an in vitro transcription-promoting cassette useful for preparing a double-stranded DNA template. The in vitro transcription-promoting cassette includes functional elements configured to effectively promote in vitro transcription (e.g., from the target insertion gene), such as a promoter, a portion encoding the 5' untranslated region (5'UTR), a portion encoding the 3' untranslated region (3'UTR), and a portion encoding the poly-A tail. The in vitro transcription-promoting cassette also includes one or more linkers useful for cloning the target gene into the in vitro transcription-promoting cassette for gene expression, and restriction sites to ensure linearization of the template.

[0230] In vitro transcription-promoting cassettes can be manufactured synthetically or non-synthetically, but are generally manufactured synthetically. In one variation, the method for manufacturing a synthetic in vitro transcription-promoting cassette involves using a commercially available DNA synthesizer, such as those available from Twist Bioscience (San Francisco, California) or Thermo Fisher Scientific (Waltham, Massachusetts). Furthermore, an in vitro transcription-promoting cassette can be assembled from separate parts of DNA, or it can be synthesized as a single part. In one embodiment, the in vitro transcription-promoting cassette is linear and includes interchangeable ends that can be ligated together. In one embodiment, the in vitro transcription-promoting cassette is circular. In one embodiment, the circular in vitro transcription-promoting cassette includes a site (e.g., a restriction endonuclease site) between the polyA region encoding part and the promoter, configured to produce linear DNA containing, in order, a portion encoding the promoter, 5'UTR, linker region, 3'UTR, and polyA region upon application of a suitable restriction endonuclease. Generally, in vitro transcription-promoting cassettes do not encode antibiotic resistance genes. For example, a synthesized in vitro transcription-promoting cassette does not grow in biological (e.g., bacterial) cells, and therefore does not require antibiotic resistance genes and does not require antibiotic selection. Generally, in vitro transcription-promoting cassettes do not have origins of replication (ori) or associated regulatory elements to promote DNA replication. For example, a synthesized in vitro transcription-promoting cassette does not grow in biological (e.g., bacterial) cells and does not require ori for replication, so ori is unnecessary. The total length of an in vitro transcription-promoting cassette can be smaller than that of many plasmids. In vitro transcription-promoting cassettes can be less than 2 kb, less than 1.5 kb, less than 1.0 kb, less than 900 bps, less than 800 bps, less than 700 bps, or less than 600 bps in length.

[0231] As described above, in vitro transcription-promoting cassettes contain promoters. The enzyme RNA polymerase binds to the promoter and initiates transcription of RNA from the target gene (for example, after a double-stranded DNA template has been assembled from the cassette and the target gene). Examples of promoters useful for transcription within the cassette include the natural or modified T7 promoter, the natural or modified T3 promoter, or the natural or modified SP6 promoter.

[0232] Furthermore, the in vitro transcription accelerator cassette contains regions encoding interchangeable 5' untranslated region (5'UTR) and interchangeable 3' untranslated region (3'UTR). These regions themselves are not translated into proteins or peptides, but they help regulate the translation of mRNA into proteins and peptides. The in vitro transcription accelerator cassette also contains a region encoding the poly-A tail. The poly-A tail in mRNA is a long chain consisting of tens to hundreds of repeated adenine residues. The poly-A tail on mRNA is thought to perform several functions, including increasing the stability of mRNA in the cytoplasm and facilitating translation from mRNA to protein. Unlike the rest of the mRNA sequence, which is directly encoded by the template DNA in mRNA, the poly-A tail is not usually (e.g., in nature) directly encoded by DNA. Rather, naturally occurring DNA contains an abbreviation called a polyadenylation signal (e.g., AATAAA), which, along with other DNA sequences, signals the cell's transcription mechanism to add the poly-A tail to the mRNA being synthesized. In other words, the length of the poly(A) tail of naturally occurring mRNA is determined by the cell producing the mRNA. As shown in Figure 6, the in vitro transcription accelerator cassette described herein contains a region of DNA that directly codes for the poly(A) tail (e.g., the entire tail). The length of the poly(A) tail is determined by the length of the region of DNA that directly codes for the poly(A) tail (e.g., the number of adenines or poly(A) or thymidine or poly(T)). The region of DNA that directly codes for the poly(A) tail may be at least 100 bp long, at least 200 bp long, at least 300 bp long, at least 400 bp long, or at least 500 bp long, and may be between these sizes (e.g., 350 base pairs long). The poly(A) tail can be added to mRNA prepared using the cassette as a template using the same process used to prepare the rest of the mRNA. This has the advantage of significantly simplifying the process of preparing the entire mRNA including the poly(A) tail. mRNA production does not require living cells or complex extracts from cells containing cellular DNA, cellular RNA, cell membrane proteins, etc.Instead, as described herein, a well-defined transcription mixture can be used to generate the entire mRNA, including the poly(A) tail, from a double-stranded DNA template, and a transcription mixture that generally does not contain toxic byproducts, such as those found in transcription mixtures made using cells or cell extracts, can be produced. Such a well-defined mixture can be safely delivered to the patient with minimal cleanup. If the double-stranded DNA template is also produced from a well-defined mixture that is substantially free of toxic byproducts, the transcript produced from the double-stranded DNA template is suitable for direct injection into the patient with only the minimum necessary washing. Described herein is a double-stranded DNA template produced from a well-defined mixture that is substantially free of toxic byproducts.

[0233] Furthermore, the in vitro transcription accelerator cassette contains one or more linker regions. The linker region is located between the 5'UTR and 3'UTR. The linker region contains at least one cleavable site, and generally two. If there are two or more cleavable sites, they may have the same sequence or different sequences. One or more cleavable restriction sites are useful for inserting the gene of interest (GOI) into the in vitro transcription accelerator cassette to produce a synthetic linear or cyclic ligation product. The gene of interest is generally inserted between the 5'UTR and 3'UTR of the in vitro transcription accelerator cassette, but in some cases, the 5'UTR or 3'UTR sequence may be included with the gene of interest and inserted into the in vitro transcription accelerator cassette together with the gene of interest. The cleavable sites(s) are restriction endonuclease sites, such as type II (type IIG, type IIS) restriction endonucleases, e.g., BsaI, BbsI, AarI, HhaI, HindIII, NotI, BbvCI, EcoRI, BglII, FokI, AlwI, AcuI, or BcgI, which are available from New England Biolabs (NEB, Ipswich, Massachusetts), Promega Corporation (Madison, Wisconsin), or Thermo Fisher Scientific (Waltham, Massachusetts).

[0234] The gene of interest (GOI) described herein is generally a short fragment of DNA that codes for a functional product molecule (RNA or protein). The gene of interest may code for a specific protein, a part of a protein, or a specific function. In some cases, it may also contain instructions for producing RNA that does not code for a specific protein or part of a protein (for example, it may code for untranslated functional RNA).

[0235] The target gene, useful for insertion into an in vitro transcription accelerator cassette, can be produced synthetically or non-synthetically, but will generally be produced synthetically. The target synthetic gene can be produced using commercially available DNA synthesizers and methods, such as those available from Twist Bioscience (San Francisco, California) or Thermo Fisher Scientific (Waltham, Massachusetts). Furthermore, the target gene can be assembled from separate DNA molecules, but is generally synthesized as a single piece. The target gene may be produced as a linear or circular fragment of DNA. A circular target gene may be digested with restriction enzymes to produce a linear target gene. The produced target gene may be purified (e.g., by column chromatography or electrophoresis).

[0236] Generally, the gene of interest is cleaved before binding to the in vitro transcription accelerator cassette. In particular, the gene of interest may be cleaved with the same restriction endonuclease(s) used to cleave the in vitro transcription accelerator cassette, or it may be generated by enzymatic amplification. Generally, the gene of interest does not encode an antibiotic resistance gene. For example, the synthesized gene of interest does not grow in biological (e.g., bacterial) cells and does not require antibiotic selection, and therefore does not require an antibiotic resistance gene. Generally, the gene of interest does not have an origin of replication (ori) or associated regulatory elements to facilitate DNA replication. For example, the synthesized gene of interest does not require an ori because it does not grow in biological (e.g., bacterial) cells and therefore does not require an ori for replication. In some embodiments, the full length of the gene of interest can be made smaller than that of many plasmids. The target gene may be less than 2kb, less than 1.5kb, less than 1.0kb, less than 900bps, less than 800bps, less than 700bps, or less than 600bps, less than 500bps, less than 400bps, or less than 300bps, less than 200bps, or less than 100bps.

[0237] In some cases, the target gene is a T cell receptor (TCR) or a part of a T cell receptor, and is intended to treat, for example, CTCL or other diseases or conditions mediated by a T cell receptor (TCR) or part of a T cell receptor. For example, during T cell development, cells must rearrange the T cell receptor (TCR) gene to create and express a novel TCR molecule. Since TCR rearrangement occurs early in T cell development and before the development of mature T-cell lymphomas such as CTCL, all malignant CTCL cells express identical clonal TCRs containing unique TCRα and TCRβ subunits. Because this TCR is specific to lymphoma cells and foreign to the immune system, it is an excellent target for treatment.

[0238] The target gene may be part or all of the complementarity-determining region (CDR). The CDR is a highly variable portion of the TCR sequence that mediates the binding of T cells to the antigen-major histocompatibility complex (MHC). Figure 8 shows one region of the T cell receptor useful for creating double-stranded DNA for use in vaccines or therapeutics. In particular, the CDR3 region, spanning the junction of the V(D)J and C regions, has the highest variability and represents a truly unique protein fragment that should be found only in lymphoma cells. Therefore, a CDR3 region extended by 10 amino acids at both the C-terminus and N-terminus constitutes the vaccine peptide fragment. In some methods, the unique sequence of a gene, such as the T cell receptor (TCR) or a portion of the T cell receptor, is determined from the individual, and the target gene is manufactured to be identical to the T cell receptor (TCR) or portion of the T cell receptor from the individual. In some cases, the sequence may be controlled by certain known methods, such as codon optimization or optimized RNA stability or expression, but the sequence of the target gene is still based on the sequence obtained from the individual. In one embodiment, the gene sequence of interest includes a T cell receptor having a DNA sequence identical to the DNA sequence from the patient, or a DNA sequence that has been controlled in a known manner relative to the DNA sequence from the patient.

[0239] This specification describes methods for preparing double-stranded DNA templates, particularly synthetic double-stranded DNA templates. Double-stranded DNA templates may be particularly useful for in vitro transcription to generate mRNA for use in vaccines or other therapeutic agents delivered by injection or other means to patients.

[0240] Existing DNA templates and mixtures for in vitro transcription generally contain crude or semi-purified cell extracts (e.g., bacteria, other microorganisms, or other extracts) and may be complex and undefined. Such extracts may contain bacteria, other microorganisms, or other DNA, endotoxins, and / or other undesirable components. When used for generating DNA templates or performing in vitro transcription as part of a vaccine or therapeutic process, undesirable components can increase the risk of serious side effects. For example, endotoxins are large molecules of lipopolysaccharide found in the outer wall of Gram-negative bacteria and are commonly used as sources for cell extracts used in in vitro transcription reactions. Endotoxins entering the bloodstream by injection, etc., can cause various problems in humans and other animals, such as inflammation and sepsis, posing a significant health risk. The methods described herein may be particularly useful for creating double-stranded DNA templates that are free from biological contaminants (bacteria, other microorganisms, or other contaminants), free from bacterial (or other microorganisms or unwanted) DNA, and / or free from endotoxins. The methods described herein may include the use of defined or synthesized components for creating a target gene, for creating an in vitro transcription accelerator cassette, and / or for creating a double-stranded DNA template (or any intermediate used to create these materials). Defined or synthesized components may be made from defined or synthesized components such as DNA synthesizers, purified nucleotides, and purified enzymes. Defined or synthesized components may be essentially free of bacteria, other microorganisms, or other DNA, endotoxins, and / or other undesirable components. By avoiding the use of biological components, biological contaminants such as DNA and endotoxins do not contaminate the DNA template (or other components) in the first place. Double-stranded DNA templates and downstream materials are safer as they do not require difficult or cumbersome purification steps.The methods and other methods described herein may include the steps of: conjugating a synthetic gene of interest with a synthetic in vitro transcription facilitator cassette to produce a synthetic linear or circular ligation product; removing unreacted synthetic gene of interest and unreacted synthetic in vitro transcription facilitator cassette; amplifying the circular ligation product to produce linear, circular, or branched amplified DNA; and linearizing the amplified DNA ligation product to produce a double-stranded DNA template.

[0241] As described above, the production of a synthetic linear or cyclic ligation product by ligating a target gene with an in vitro transcription facilitator cassette may include inserting the target gene into the in vitro transcription facilitator cassette. Figure 7 shows the double-stranded DNA template produced as described herein. The target gene and the in vitro transcription facilitator cassette may have the same restriction endonuclease site(s) as described elsewhere herein, and the method may include digesting the target gene and the in vitro transcription facilitator cassette with a restriction endonuclease targeting the restriction endonuclease site to create a suitable end, and ligating the target gene into the cassette. The method may include combining the target gene, the in vitro transcription facilitator cassette, restriction enzyme buffer, energy source, one or more restriction enzymes, ligase buffer, and ligase, and incubating the mixture for an appropriate time. The buffer(s) may be suitable for a particular restriction endonuclease and / or ligase, and may be one buffer or two (or more) buffers. The endonuclease and / or ligase buffer may be a commercially available buffer (e.g., NEB, Promega) and / or a buffer containing tris, potassium, magnesium, sodium chloride, and dithiothreitol at a pH of about 7.4 to about 9.0, such as tris acetate (e.g., 6 mM to 90 mM), potassium acetate (50 mM to 100 mM), magnesium acetate (5 mM to 10 mM), bovine serum albumin (BSA, 50 ug / ml to 200 ug / ml), and dithiothreitol (1 mM). The ligase may be a commercially available one (e.g., NEB, Promega, Thermo Fisher Scientific) or a ligase such as T3 DNA ligase, T4 DNA ligase, or T7 DNA ligase. Digest may be performed for 10 minutes to 4 hours, or any time in between (e.g., 30 minutes, 1 hour, 2 hours, etc.). The ligation step may be performed for 10 minutes to 4 hours, or any time in between (e.g., 30 minutes, 1 hour, 2 hours, etc.). The digestion step and the ligation step may be performed simultaneously or sequentially.The energy source may be adenosine 5'-triphosphate (ATP) (e.g., 0.1 mM to 5 mM). Incubation may be continued by adding any of the components, such as restriction enzymes or ligases, over time. In some embodiments, only materials proven to be non-animal-derived (AOF) will be used for therapeutic manufacturing to reduce the risk of transmitting infectious agents. Some of these methods, where the in vitro transcription facilitator cassette is not cyclic, include a step of ligating the ends of the in vitro transcription facilitator cassette to produce a cyclic in vitro transcription facilitator cassette. Alternatively, other methods for ligating the gene of interest with the in vitro transcription facilitator cassette, such as the Chubak method, may be used. Alternatively or additionally, prior to exponential amplification, primer extension can be used to ligate the in vitro transcription facilitator cassette with the gene of interest to produce a linear molecule.

[0242] The method or other methods described herein may include the step of removing the unreacted synthetic gene of interest and the unreacted synthetic in vitro transcription facilitator cassette separated from the synthetic linear or cyclic ligation product, or the step of purifying double-stranded DNA separated from the unreacted synthetic gene of interest and the unreacted synthetic in vitro transcription facilitator. Removal of the unreacted synthetic gene of interest and the unreacted synthetic in vitro transcription facilitator cassette separated from the synthetic cyclic ligation product can be carried out using an enzyme such as an exonuclease (exonuclease V, etc.) in a suitable exonuclease buffer (NEB, Promega, Thermo Fisher). The method may also include digesting the synthetic gene of interest and the unreacted synthetic in vitro transcription facilitator. This method may include passing the digested mixture through a resin or column such as an ion exchange resin or a size exclusion resin to retain either the unreacted synthetic gene of interest and / or the unreacted in vitro transcription accelerator cassette in the column, or retaining a double-stranded DNA template in the column, and passing the double-stranded DNA template or the synthetic gene of interest and / or the unreacted in vitro transcription accelerator cassette through the resin or column. Some embodiments may also include retaining the digested nucleotides in the resin or column, or passing the digested nucleotides through the resin or column. Some embodiments may include washing and / or eluting the resin or column. Some embodiments may also include retaining the digested nucleotides in the resin or column. Some embodiments may include binding the unreacted synthetic gene of interest and / or the unreacted synthetic in vitro transcription accelerator cassette to beads, or binding double-stranded DNA to beads, holding the beads with a magnet, and removing either the double-stranded DNA or the unreacted synthetic gene of interest and / or the unreacted synthetic in vitro transcription accelerator cassette from the double-stranded DNA. Resins, columns, and magnetic beads can be obtained from companies such as Bangs Laboratories (Fischer's, Indiana), Beckman Coulter (Blair, California), Millipore (Burlington, Massachusetts), and Thermo Fisher, VWR (Radnoll, Philadelphia).One embodiment may include the use of a methylation-sensitive restriction enzyme.

[0243] The methods and other methods described herein may include amplifying linear or circular ligation products to produce amplified DNA. Some methods include amplifying linear or circular ligation products to produce linear amplified DNA. Other methods include amplifying linear or circular ligation products to produce linear, branched, or circular amplified DNA. The amplified products may be amplified by helicase-dependent amplification (HAD), loop-mediated isothermal amplification (LAMP), multiple substitution amplification (MDA), nucleic acid sequence-based amplification (NASBA), polymerase chain reaction (PCR), rolling circle amplification (RCA), self-sustaining sequence replication (3SR), or strand substitution amplification (SDA). A buffer suitable for the reaction, deoxyribonucleotide triphosphate (dNTPs), enzymes (DNA polymerase), and primers are added as needed. The amplification temperature and timing are controlled. This method may include the step of heating a linear or circular ligation product (e.g., between 70°C and 100°C) to denature the DNA, and then cooling it. This method may also include the step of adding a denaturation buffer configured to denature DNA to the linear or circular ligation product to denature the DNA, and then adding a neutralization buffer to the denatured DNA mixture to neutralize the denaturation buffer, leaving the denatured DNA. This method may also include the step of adding an enzyme such as a DNA polymerase enzyme (e.g., Bst DNA polymerase, Φ29 DNA (Phi29) polymerase, Taq DNA polymerase) to amplify or extend the denatured DNA, and amplifying or extending the DNA with the enzyme to produce amplified DNA (e.g., branched, circulating, or linear amplified DNA).

[0244] One method includes the step of purifying amplified or extended DNA by separating it from buffers, enzymes, nucleotides, and other unwanted components. This method may include passing the amplified or extended DNA through beads, resins, or columns, such as ion exchange resins, magnetic beads, or size exclusion resins, to retain the amplified or extended DNA, or passing the amplified or extended DNA through beads, resins, or columns to retain unwanted enzymes and other components on the beads, resins, or columns. One embodiment includes washing and / or eluting and / or drying and / or rehydrating the resin or column. One embodiment includes repeating one or more of these steps. One embodiment includes repeating two or more depots of beads, resins, or columns and one or more of the washing / eluting / drying / and / or rehydration steps. One embodiment includes binding DNA to beads, holding the beads with a magnet, and removing unwanted components and contaminants from the DNA and beads (washing). Suitable resins, columns, and magnetic beads are available from Bangs Laboratories (Fischer's, Indiana), Beckman Coulter (Blair, California), Millipore (Burlington, Massachusetts), and Thermo Fisher, VWR (Randall, Philadelphia).

[0245] In some embodiments, the amplified DNA may be branched or circular, rather than linear. One method includes the step of linearizing the DNA to produce linearized template DNA. Another method may include the step of adding restriction endonuclease (in appropriate buffer) to purified amplified or extended DNA, incubating the DNA with the restriction endonuclease, and linearizing the DNA. The restriction enzyme is selected to cleave outside the 5'UTR, the gene of interest, the 3'UTR, and the poly-A region. In some embodiments, the restriction enzyme cleaves between the 3'UTR of one extended or amplified DNA and the 5'UTR of an adjacent (and downstream) extended or amplified DNA. The restriction enzyme may be any restriction enzyme, e.g., a type IIs restriction enzyme as described above with respect to restriction enzyme digestion for conjugating the synthetic gene of interest with a synthetic in vitro transcription accelerator cassette to create a linear or circular synthetic ligation product. In one example, the restriction enzyme is at least one of BsaI, BbsI, AarI, HhaI, HindIII, NotI, BbvCI, BglII, FokI, AlwI, AcuI, or BcgI, available from New England Biolabs (NEB; Ipswich, Massachusetts), Promega Corporation (Madison, Wisconsin), or ThermoFisher Scientific (Waltham, Massachusetts). In one embodiment, the restriction endonuclease is the same(s) used to insert the synthetic gene of interest into the in vitro transcription accelerator cassette. In another embodiment, the restriction endonuclease is different from the(s) used to insert the synthetic gene of interest into the in vitro transcription accelerator cassette. Also described herein are microfluidic device reactors for producing the double-stranded DNA described herein.

[0246] Figure 9 shows a modified architecture of a microfluidic biochip reactor for generating double-stranded DNA. The method and other methods described herein may involve generating double-stranded DNA from a gene of interest and an in vitro transcription facilitator cassette in a sterile, closed biochip where all components are maintained sterile during generation. The sterile, closed biochip is sealed off from the atmosphere. Figure 9 shows a microfluidic biochip reactor having four interconnection reactors (e.g., modules or chambers) on which DNA precursors at different stages along the pathway to double-stranded DNA template travel. For example, Figure 9 may include a ligation reactor (ligation reaction chamber 901), a pre-mixing chamber 903, an amplification reactor (amplification reaction chamber 905), and a digestion reactor (digestion reaction chamber) 907 (connectors and valves are not shown in this example). Different steps of the method described herein are carried out in different modules or chambers. The gene of interest and the in vitro transcription facilitator cassette are mixed together in the pre-mixing chamber. In the ligation reaction chamber, the target gene is bound to an in vitro transcription promoter cassette to produce a ligation product. In the amplification reaction chamber, the ligation product is amplified, generating amplified DNA. The amplified DNA is further processed. For example, it may be digested in a digestion reaction chamber to remove unwanted DNA or to separate different copies of the amplified target gene. IVT response

[0247] The next step may be an IVT reaction to produce mRNA. This step may be carried out within the same or a different microfluidic device (e.g., a certain modification of the IVT microfluidic device) that can be housed in the microfluidic device control system described earlier. A high-level mRNA production process showing the main steps and subcompartments within the IVT microfluidic device is shown in Figure 11.

[0248] An IVT reaction may involve combining a DNA template with a T7 polymerase enzyme, nucleotides, and a capping reagent, and incubating the reaction under controlled conditions to produce capped mRNA molecules. The IVT reaction may be carried out in a reaction chamber of a microfluidic device (e.g., an IVT microfluidic device), and process parameters such as temperature, mixing, and reagent addition (both at the start of the reaction and during the reaction) may be controlled to an optimized level. The process may be driven by a controller as described above. Buffers and solutions may be supplied via an array of microvalves, and the volume may be controlled using a pre-set program specific to the protocol optimized for each mRNA drug substance.

[0249] After the IVT reaction, DNAse treatment may be performed to degrade the template DNA. This step may be carried out within the IVT reaction chamber (part of the IVT reactor), and parameters such as dilution ratio, enzyme / buffer concentration, temperature, and mixing may be controlled to optimal levels. This procedure may be performed autonomously and recorded by a monitoring camera. Purification of IVT

[0250] DNAse-treated mRNA may be purified to remove impurities and by-products. In particular, degraded templates, unreacted nucleotides, enzymes (T7 polymerase and DNAse), and double-stranded RNA can affect the quality and immunogenicity of the drug substance. A two-step solid-phase reversible immobilization method using carriers with different surface chemistry can be used for purification. In the first step, a cellulose membrane can be used to selectively capture double-stranded RNA under precisely controlled binding conditions, and the unbound fraction can be eluted into a second purification chamber. The second purification step may use 1-2 m carboxyl-coated paramagnetic beads that selectively capture mRNA longer than 500 bp. Several washes can then be performed to remove unbound material, including nucleotides, enzymes, and degraded templates. The pure mRNA can then be eluted with USP-grade water. In-line microfluidics-based purification allows for a fully integrated workflow without exposing the material to air, avoids the use of toxic mobile phases used in conventional HPLC-based methods, and significantly reduces manual labor.

[0251] As described above, these methods and apparatuses are generally sterile methods and apparatuses that can produce therapeutic mRNA, or any or all of the components for producing therapeutic mRNA, without exposure to an external atmosphere and / or to sources of RNAse and / or contaminants that may otherwise be required. For example, as described herein, these methods can be carried out without adding a bacterial source of polynucleotides (e.g., in template DNA) and / or without adding components such as plasticizers that may be present during purification via HPLC or other conventional techniques. Apparatus and methods for purification within a microfluidic device (e.g., using pure cellulose) are described herein.

[0252] The purified mRNA can be quantified by UV absorption at 260 nm or by fluorescence using an mRNA-specific fluorescent dye. Additional mRNA QC steps may be performed to confirm purity and identity. The entire mRNA production process may be carried out within a microfluidic device control system, and the addition and transfer of reagents may be performed via the above-mentioned closed-loop microfluidic device control system, for example, using sterile techniques. Finally, filtration may be performed, for example, through a 0.22 m filter. If the final product meets acceptance criteria such as yield (e.g., more than 6.5 ug of mRNA per 1 ul of starting IVT by UV vis / fluorometry), identity (e.g., 100% consensus homology to the target by sequencing), integrity (e.g., mutation rate of less than 1% by sequencing), purity (e.g., CE, more than 95% of the single-band product), capping efficiency (HPLC, more than 95% capped mRNA), residual double-stranded RNA (e.g., FRET / Immunoblot, less than 0.02% (1 ng)), bacterial components (e.g., HCP ELISA (for DNA and protein), less than X), bacterial components (e.g., HC-DNA, less than X), endotoxin (e.g., LAL test, less than 0.2 EU / ml), and bioburden (e.g., microbial limit test (MLT)), it may be considered a low bioload pharmaceutical substance and released for use in pharmaceutical formulations. Purification of mRNA into ANP

[0253] The purified mRNA can be combined with a delivery component to form a nanoparticle formulation. This process is illustrated in Figure 12. For example, an aqueous solution of mRNA cargo (therapeutic mRNA, also referred to herein as the pharmaceutical substance) may be combined with an ethanol solution of the delivery vehicle in a microfluidic mixing structure within a formulation microfluidic device. The substance can then undergo two post-formulation processing steps, first an on-chip dialysis step to replace buffering components in the formulated product, and then a concentration step to reduce the volume of the drug to meet specifications. By implementing these steps in a microfluidic device-based manufacturing apparatus, a highly controlled formulation process can be achieved that requires no human intervention and minimizes the possibility of human error.

[0254] In general, components of the manufacturing method described herein, including, for example, synthesizing a template, performing IVT to generate mRNA, purifying the mRNA, binding the mRNA to a delivery vehicle to form a therapeutic composition, dialysis of the therapeutic composition, and / or concentration of the therapeutic composition, may be carried out in a single microfluidic device and / or multiple microfluidic devices as shown in Figures 3A-3C above. Thus, the channels may be continuous or partially continuous (e.g., continuous across components of the manufacturing process, such as one or more of template formation, IVT, mRNA purification, binding of mRNA to a delivery vehicle to form a therapeutic composition, dialysis of the therapeutic composition, and / or concentration of the therapeutic composition). In all cases, the same controller device may be used, or different controller devices may be used. The product of each of these components may be stored in a fluid vial (e.g., a depot) in the controller device and transferred to a new or subsequent microfluidic device. Thus, in any of these methods and apparatuses, the product may be protected from exposure to the atmosphere.

[0255] As described above in one modification, a peptoid-based lipid formulation may be used as the drug vehicle, which may incorporate both a cationic group and a lipid moiety into the N-substituted peptide (i.e., peptoid) backbone. The delivery vehicle component may be a monodisperse, fully characterizable chemical substance that can be procured by conventional means.

[0256] Maintaining small, uniform particle size in mRNA ANP formulations may require a controlled and consistent formulation process. The delivery vehicle component is rapidly mixed with mRNA in a controlled ratio using the methods and apparatus described herein. Exposure of the DV component to an aqueous solution and the interaction between cationic (+) lipids and anionic (-) mRNA may trigger particle formation. This process can be carried out by using the microfluidic devices described herein (to control particle size and uniformity). The mRNA may be dissolved in an acidic buffer (pH 3-5) which can help ensure complete protonation of the basic functional groups (such as amines) on the delivery vehicle that are responsible for its cationic charge. The delivery vehicle may also be dissolved in a water-miscible organic solvent (typically ethanol), which promotes the formation of nano-sized particles when exposed to an aqueous cargo solution. Immediately after mixing, the pH of the solution may be stabilized with a neutral buffer. The resulting formulation can be stored at 4°C for several weeks without significant degradation of function. Alternatively, the formulation process can be carried out just-in-time in a clinical setting.

[0257] Pharmaceutical microfluidic devices as described herein may be designed to accomplish these pharmaceutical operations. Figure 13 shows a schematic diagram of a typical architecture of such a microfluidic device that may be used. The first part of the pharmaceutical microfluidic device may include pre-diluting both mRNA and DV components into separate staging chambers. Input materials may be advanced from sterile barcoded vials into these pre-mixing chambers. The mRNA material is pre-diluted with acidic pharmaceutical buffer, and the delivery vehicle component is diluted with ethanol. At this stage, the concentrations of both materials can be adjusted to a volume ratio that matches the required specification of the target DV / mRNA ratio and, for example, a 3:1 aqueous:ethanol ratio, which has been previously shown to achieve good mixing behavior.

[0258] Microfluidic devices containing a mixing structure can precisely control the mixing rate of materials. Faster or slower mixing may be provided and controlled (e.g., by a controller). For example, a microfluidic device containing a mixing structure may provide a significantly increased DV / mRNA mixing rate. At the start of the mixing process, equal pressure may be applied to both mixing chambers, thereby forcing the fluid through the microfluidic structure at, for example, 0.5 mL / min. The shape of this structure may be determined by a rapid mixing time of approximately 3 ms. Under these conditions, water-insoluble lipid domains on peptoid molecules are exposed to the mRNA aqueous solution, which may lead to the formation of amphiphilic nanoparticles (ANPs).

[0259] Immediately after mixing, ANP can be diluted by adding 1:1 neutral PBS in-line. This neutralizes the acidic formulation buffer, which may prepare the formulation for dialysis and concentration. All of these steps are controlled by a microfluidic device control system, ensuring reproducible particle size and formulation characteristics.

[0260] This microfluidic device enables the formulation of personalized therapeutics in a clinical setting. For example, a therapeutic could be for treating CTCL or other diseases or conditions mediated by T cell receptors (TCRs) or parts of T cell receptors. Personalized therapeutics can be based on a specific patient's genetic characteristics (e.g., genotype), including generating a specific mRNA composition based on the patient's own sequence. The methods and apparatus described herein can also, or alternatively, enable personalized therapies. Personalized therapeutics may be based on a patient's phenotype, such as a category to which the patient belongs, such as a risk factor category. Therefore, personalized therapeutics may be adapted to a patient based on their category. For example, a microfluidic formulation device could, for instance, enable the mixing of multiple mRNAs to generate a patient-personalized therapeutic composition from a subset of mRNAs from a larger library, based on the components and proportions (amounts) of each component, which may be determined from the patient's phenotypic data. Any of these compositions can then be formulated in a clinical setting to produce a personally optimized therapeutic. Processes to produce pharmaceuticals after formulation

[0261] Once ANP is formed during the formulation process, several post-processing steps can be completed using a formulation microfluidic device. These may include buffer exchange and dialysis for ethanol removal, followed by evaporative concentration to reduce the volume for drug delivery. See, for example, Figure 14.

[0262] The obtained nanoparticles may be analyzed on a microfluidic device (e.g., by a microfluidic device control system) for purposes such as size distribution using dynamic light scattering (DLS) and % mRNA encapsulation using fluorescence. The analysis can be completed with a small amount of final formulation material flowing from the main channel into an optically transparent sampling chamber. In this chamber, light scattering measurements may be performed using a fiber optic light source to determine particle size and dispersion. Next, RNA concentration is measured before and after particle disruption by adding a washing agent using a fluorescent mRNA-specific probe. This assay may elucidate mRNA concentration for administration information and the ratio of mRNA encapsulated in ANP to free mRNA in solution. For example, analytical methods that may be used to test formulated mRNA drugs include optical clarity (e.g., visible, no aggregates, clear solution by visual inspection), lipid composition characterization (e.g., by HPLC), size (e.g., DLS, 80-300 nm), % encapsulation (e.g., over 95% encapsulation by fluorescence measurement), dispersibility (e.g., DLS, PDI < 0.25), endotoxin (e.g., LAL test, < 0.2 EU / ml), sterility (e.g., culture (USP), < X cfu), pH (e.g., USP, pH 7.4 + / - 0.2), and potency (e.g., bioassay / ELISA, X EC). 50 ) may be included. Examples

[0263] As described above, the methods and apparatus described herein can be used, for example, to manufacture mRNA therapies, including therapies for cutaneous T-cell lymphoma (CTCL). Mature T cells express a unique TCR formed by a combination of two proteins: alpha and beta chains in αβ T cells, or delta and gamma chains in δγ T cells. Each TCR chain is formed by a unique recombination event in which one of a number of exons encoding the V, (D), and J regions of a gene is joined by a process called V(D)J recombination. This V(D)J recombination event is quasi-random and can produce a large number of combinations, thus creating TCR diversity. Furthermore, random addition or deletion of nucleotides occurs at the exon junctions during the V(D)J recombination process, resulting in even greater TCR diversity, which forms an individual's TCR repertoire. Healthy individuals deeply sequenced by next-generation technology have 1–5 × 10¹⁶ T 6 It is estimated that individuals possess multiple TCRs, and without infection, a single TCR would not typically account for more than 5% of the total population. T-cell lymphoma arises from the clonal expansion of a single malignant T cell and forms tumors in lymphoid tissue (spleen and lymph nodes) or other tissues such as the skin, liver, and gastrointestinal tract.

[0264] Another method has been developed to sequence an individual's TCR repertoire and to diagnose and identify clonally expanded TCRs in T-cell lymphoma patients. One commonly used method involves sequencing TCRβ or γ genome rearrangements using a panel of carefully developed PCR primers to control amplification bias. Thus, multiplex PCR can perform target enrichment followed by next-generation sequencing. Such methods have been validated, for example, by Adaptive Biotechnologies' Immunoseq assay, and are used clinically as a diagnostic tool and for quantifying minimal residual disease. Another method involves directly deep sequencing cDNA without target enrichment to identify significantly overexpressed TCR strands. The identification of lymphoma TCRs is usually referred to as lymphoma idiotype or chronotype.

[0265] Idiotype determination can be performed by biopsy and sequencing of the sample to identify the lymphoma idiotype. Digital data on patient-specific idiotypes may be used for patient-specific vaccine design. Design of the Http vaccine

[0266] The production of mRNA-based patient-specific cancer vaccines may begin with the design of DNA sequences corresponding to personalized target peptides that can generate specific and immunologically effective epitope presentation by antigen-presenting cells (APCs). To achieve this, the first step may involve extracting complementarity-determining regions (CDRs) from idiotype TCR chains (αβ or γδ). The CDR3 region can be extracted by performing sequence alignment with respect to the canonical TCR. CDRs are highly variable portions of the TCR sequence that mediate binding to the antigen-MHC complex. In particular, the CDR3 region spanning the junction of the V(D)J and C regions is the most variable and represents a truly unique protein fragment that should be present only in lymphoma cells. Thus, for example, a CDR3 region extended by 10 amino acids each at both the C-terminus and N-terminus constitutes a vaccine peptide fragment, as described above with respect to Figure 8.

[0267] Since the TCR has two chains (α and β), there are two CDR3s per patient, although in a small number of cases only one CDR3 may be identified. As an example, assuming the presence of CDR3α and CDR3β, the vaccine peptide can be designed with the following structure: CDR3α-linker-CDR3β, where the linker is the standard GSGGGSGGGSGGGS sequence commonly used in the design of single-chain variable fragment (ScFv) molecules.

[0268] Once the final amino acid sequence of the vaccine peptide is determined, the design process may include codon optimization, which can lead to a DNA sequence that (i) is highly transcribed and translated, resulting in good protein expression; (ii) is suitable for DNA synthesis; (iii) contains the adapter sequences required for the template generation step; and (iv) excludes sequence motifs that interfere with the template generation process, such as restriction enzymes. Codon optimization may be performed, for example, to balance the sequence GC and to remove sequence repeats, internal promoter sequences, stop sequences, splice sequences, recombinant sequences, and internal ribosome entry sites (IRESs). Furthermore, codon usage can be adapted to those observed in highly expressed human genes. A schematic diagram of an example of a codon optimization step that may be used is shown in Figure 10.

[0269] Once the sequence design is complete, the optimized sequence can be synthesized as a linear DNA molecule. The template for IVT may be prepared as described above. For example, an IVT-compatible double-stranded DNA template may be generated prior to mRNA synthesis by IVT. The DNA template may consist of (i) a protein-coding sequence (or CDS) defined as a set of codons corresponding to the target patient-specific peptide to be produced, (ii) a non-coding sequence including the 5' untranslated region (5'UTR) and 3'UTR, (iii) a poly(A) sequence that protects the mRNA from exonuclease activity, and (iv) a promoter sequence that invites the RNA polymerase enzyme to transcribe the DNA template into mRNA.

[0270] Therefore, as a template generation process, it is also possible to pair a patient-specific peptide coding sequence, which is synthetic linear DNA (e.g., from a DNA synthesizer), with a general functional element required for IVT.

[0271] Methods based on microfluidic devices and those described herein may be far more rapid and efficient than currently practiced bacterial culture-based methods, which involve template generation, take ~4 days or more, result in a variable-length poly-A tail (due to the bacterial recombination process), and carry the risk of carryover of bacterial proteins, bacterial DNA, and endotoxins. In contrast, methods and apparatus described herein can be carried out in 1 day (~12 hours), result in a consistent poly-A tail (greater than 300 bp), and may not involve any contact with host nucleic acids or host cell proteins. The final double-stranded DNA may be made from chemically produced nucleotides and can therefore be considered of synthetic origin.

[0272] As described above, these methods may include four steps: (i) ligation of sGOI and TIFC, and removal of non-ligated material by exonuclease treatment; (ii) cyclic amplification of the ligated product by a technique called multi-substitution amplification (MDA); (iii) linearization of the amplified product by digestion with IIs restriction enzyme; and (iv) purification on the chip by impurity removal. As a final step, the purified template may be filtered through a 0.22 μm filter. To ensure the quality of the material obtained before use in the IVT reaction, many analytical tests may be performed, including tests for yield (e.g., >50 ug at 1 ug / ul, 260 / 280 nm ratio >1.8), identity (e.g., 100% consensus homology), integrity (e.g., mutation rate <1%), purity (e.g., product in a single band by CE >95%), and endotoxin (e.g., <0.2 EU / ml).

[0273] Thus, the pharmaceutical can include a mixture of mRNA encoding a patient-specific TCR peptide and CpG as an adjuvant at a ratio of 1:1. The nucleic acid mix can be encapsulated in 200 nm ANPs that protect the mRNA from degradation by RNase and also play a role in promoting cellular uptake and release into the cytoplasm. The ability to utilize the mRNA directly in the cytoplasm where the translation process occurs may be necessary for the mechanism of action of the active ingredient. ANPs consist of a nucleic acid component, the cationic amine-functionalized peptide NTX-DV-0024, and 2 wt% PEG-lipid, and the overall ratio is mRNA:DV = 5:1 w / w. The size distribution of the ANPs may be unimodal with a Z-average particle diameter of about 200 nm. The ANPs can be suspended in phosphate-buffered saline (0.144 mg / mL potassium phosphate monobasic, 9.0 mg / mL sodium chloride, 0.795 mg / mL disodium phosphate) with the target pH set to 7.4. All excipients for the formulation may generally be recognized as safe. The final product is sterile, physiologically isotonic, and has an osmotic pressure of 295 ± 20 mOsm / kg.

[0274] mRNA itself has an attractive safety profile, but for example, levels of particles below visible light, host cell proteins (HCPs), host cell DNA, process-related impurities, bioburden, bacterial endotoxin levels, sterility, levels of eluates and extracts are mainly related to safety and must be minimized and controlled according to established safety profiles and industry standards. Furthermore, by eliminating or minimizing the presence of residual template DNA, double-stranded RNA, and enzymes used in the production of IVT mRNA, a safe and effective product can be ensured.

[0275] As described above, the methods and apparatuses described herein can include quantitative analysis of particulate matter by one or more procedures such as, for example, light obscuration particle counting tests, and / or microscopic particle counting tests. In order to reach a final conclusion regarding compliance with requirements, it may be necessary to test some preparations with both light obscuration particle counting tests and microscopic particle counting tests. Since nanoparticle preparations are essentially opaque due to light scattering by droplets and / or particle aggregates present in the injectant, filtration and subsequent microscopic analysis of the filter may be used for particulate matter analysis. An optical microscope adjusted to a magnification of 100±10 may be used to visualize particles smaller than about 1 μm, and the nominal pore size of the filter used in this method is at most 1.0 μm, and pharmaceutical nanoparticles in the range of 100 nm to 250 nm will not interfere with particulate matter detection.

[0276] The template generation method described herein does not use live microorganisms such as bacteria and relies on enzymatic reactions and the use of chemically synthesized nucleotides, so the template and mRNA products are fully synthetic.

[0277] Regarding residual host cell DNA in the final pharmaceutical product, the methods and apparatuses described herein may be less than 10 ng / dose and less than 200 base pairs at the dose of the final formulation. In addition to minimizing the presence of process-related impurities, product-related impurities can be controlled through the manufacturing process, formulation development and optimization, and identification of appropriate storage conditions described herein. Although IVT mRNA products are intended to be manufactured and administered as soon as possible, the stability profile can meet defined acceptance criteria at least until administration. The period for maintaining appropriate stability of the therapeutic agents described herein may be at least 30 days under refrigerated conditions.

[0278] Once IVT mRNA enters the cytoplasm, its pharmacological effects are governed by the same cellular mechanisms that control the stability and translation of native mRNA. Therefore, the potency of IVT mRNA is highly dependent on its bioavailability in the cytoplasm, and the focus should be on developing products that maximize cellular uptake.

[0279] The storage containers (e.g., depots) described herein may generally protect the product from the external environment (including protection from oxygen ingress and, where applicable, photodegradation), be sterilizable, ensure that sterility is maintained throughout the storage period, be compatible with the product formulation, and contribute little to no leaching chemicals to the product during storage. For example, a Type I borosilicate glass vial with a halobutyl rubber stopper adequately protects the product and ensures that sterility, safety, and efficacy are maintained throughout the storage period.

[0280] A preliminary screening was conducted to maximize mRNA expression, minimize its impact on cell viability, and obtain a favorable in vivo distribution profile. The results showed that mRNA expression was maximized, the impact on cell viability was minimized, and a favorable in vivo distribution profile was obtained. For this experiment, a bioluminescence assay based on firefly luciferase (Fluc) expression was selected. This assay allows for high-throughput quantitative measurement of gene expression by uptake of mRNA from each candidate delivery vehicle. For initial evaluation, 36 aminolipidized peptoids were synthesized by solid-phase peptoid synthesis and isolated by lyophilization and / or precipitation. These candidate materials exhibited structural variations in both cationic and lipid domains. These 36 materials were conjugated with Fluc mRNA in different ratios along with 2% (w / w) of lipid-immobilized PEG (1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000). Processing several cell lines, including HeLa, HepG2, and JAWSII dendritic cells, allowed for the selection of six candidate leads.

[0281] The in vivo mRNA expression and in vivo distribution of candidate delivery vehicles were quantified by Fluc expression after intravenous, subcutaneous, and intramuscular injection into Balb / c mice. Based on expression levels (in vitro and in vivo) and in vivo distribution (in vivo), NTX-DV-0024 was selected as a candidate, and mRNA encoding ovabumin was synthesized and evaluated as a model vaccine. Ovabumin is an ideal candidate for proof-of-concept studies because it has been very well studied from the perspective of vaccination, and reagents for tracking epitope presentation and T cell response are commercially available. Initial evaluation of the OVA mRNA produced as described herein was performed in an in vitro model using JAWSII mouse (C57BL / 6) dendritic cells. Briefly, JAWSII cells were transfected with candidate OVA mRNA for 24 hours using a commercially available transfection reagent (e.g., Lipofectamine2000™), and then the cells were stained with a fluorescent antibody for MHC-I conjugated to the SIINFEKL epitope. The mean fluorescence intensity (MFI) of the stained population represents an indicator of overall antigen presentation. This is schematically illustrated in Figure 15.

[0282] Using this assay, mRNA produced by the method described herein was compared and evaluated with commercially available material. In this case, the produced mRNA showed a 42% higher level of SIINFEKL presentation on MHC-I compared to the commercially available control. Furthermore, the reproducibility of mRNA synthesis on the device was demonstrated, and SIINFEKL+ JAWSII cells with the same level were obtained using 5 batches of OVA mRNA (NTX-RNA-0184).

[0283] mRNA candidates were similarly evaluated as vaccine candidates in mouse in vivo experiments. C57BL / 6 mice were injected (IV) with commercially available or manufactured mRNA (produced using the microfluidic devices described herein) and a delivery vehicle. Peripheral blood was isolated 7 days post-injection and stained with a fluorescent MHC-I tetramer specific to T cells that recognize the OVA epitope. Subsequently, the percentage of OVA-specific CD8+ T cells was quantified by flow cytometry. In this experiment, as in the previous study, the produced mRNA increased the percentage of OVA-specific T cells in peripheral blood by 50% compared to the commercially available control, demonstrating the strength of these molecules as vaccine candidates.

[0284] The first demonstration of the in vivo efficacy of an mRNA-based vaccine (manufactured by the method described herein) was in a mouse, OVA-expressing, EG.7, syngenic T-cell lymphoma model. This model is a physiologically relevant animal model of adaptive lymphoma and is related to the immunotherapeutic mechanism of action of NTX-0565. The syngeneic mouse model is created by transplanting an immortalized mouse cancer cell line into a mouse host of the same inbred background strain (homograft). The syngeneic host mouse is capable of functionally inducing the host antitumor immune response necessary for immunotherapy studies. The syngeneic model is characterized by its complete mouse immune capacity, diversity of immune cell infiltration into tumors, comprehensive mouse tumor, immune cell-stromal interface, and ease of tumor synchronization for pharmacological studies, including gene and protein expression history.

[0285] A mouse E.G7-OVA lymphoma tumor model was used. The E.G7-OVA tumor cell line is an EL-4 lymphoma derivative cell line engineered to possess one genome copy of the OVA antigen, which is stably and robustly expressed. This method enables a highly specific immune response to exogenous OVA antigens, making these tumor cells ideal for cancer vaccine research. Tumor growth inhibition has been demonstrated in the literature using DNA vaccines, cell-based vaccines, and siRNA vaccines. In the initial efficacy trial, the vaccine component was mRNA encoding the OVA antigen, and the trial design followed previous literature on this animal model using mRNA-based vaccines. Randomized concurrent negative controls were conducted in parallel using phosphate-buffered saline, the delivery vehicle for the test substance. Animals were matched for age and sex, and group assignment was randomized to ensure a mean distribution of tumor size and body weight. The investigational drug was blinded to prevent bias from the person responsible for the in vivo trial. Sample analysis was again blinded to eliminate analyst bias. Inclusion and exclusion criteria for acceptance into the trial were defined in the proposal before commencement. Endpoints, observation frequency, and schedule were predefined in the study protocol before the start of the study. Euthanasia criteria and animal nursing interventions were also predefined.

[0286] This initial study demonstrated that the mRNA vaccine described herein had a statistically very significant therapeutic effect (Figures 16A-D). When the OVA mRNA vaccine was administered intravenously to Group 2, this group began to show statistically very significant results on day 14 post-tumor transplantation (***, p<0.0005, compared to Negative Control by Multiple Dunnett's Comparison Test on day 14). The tumor volume on day 21 was 797 mm² in Group 2. 3 In contrast, the control group for Group 1 was 2000mm 3This was the case (**, p<0.005 as measured by the Multiple Dunnett's Comparisons Test on day 21). This translates to 61.42% tumor growth inhibition when animals receive mRNA vaccination manufactured as described herein (IV). Tumor growth inhibition (TGI%) is measured in the negative control group (group 1) compared to the specified endpoint tumor volume (2000 mm). 3 Tumor growth suppression rate (%) was calculated on day 21 post-transplant when terminal tumor volume was reached. The tumor growth suppression rate (%) was defined by the following formula: TGI (%) = (TV control group - TV treated group) / TV control × 100, and is relative to the negative control on day 21 post-transplant when all control animals reached terminal tumor volume. This suppression of tumor growth resulted in a very statistically significant increase in survival in the mRNA-vaccinated group, with a median time to endpoint of 25 days for group 2, mRNA-vaccinated animals, and a median time to endpoint of 23 days for group 1, vehicle-treated animals (*measured by log-rank test compared to control, p<0.01). No observable toxicity based on body weight and laboratory test results was observed at the given dose. In Figures 16A-16C, the manufactured mRNA-based vaccine demonstrates in vivo efficacy in the mouse lymphoma E.G7 sibling model. As seen in the individual animal curves (Figures 16A, 16B) and the mean tumor volume for each group (Figure 16C), tumor growth was inhibited by 61.4% (*p<0.01). This inhibition of tumor growth was linked to a statistically significant increase in survival for the mRNA-vaccinated group (Figure 16D) (**, p<0.01).

[0287] In addition, to supplement the above physical measurements, in vivo testing of the stored drug using reporter gene expression was conducted over a week. In this experiment, preparations of firefly luciferase mRNA and NTX-DV-0028 were prepared 1) 7 days before injection, 2) 3 days before injection, and 3) 1 hour before injection. After preparation, the preparations were stored at 4°C until administration. Subsequently, Balb / c mice were administered the three materials by tail vein injection at a dose of 0.25 mg / kg. Eight hours after injection, whole-body bioluminescence was measured, and the resulting images are shown in Figure 17A, with the quantified results shown in Figure 17B (showing the quantified luminous flux of whole-body luciferase expression after injection of the stored mRNA preparation). There was no substantial loss of measured bioluminescence during this one-week storage experiment. The stored materials for 3 days and 7 days were all within the margin of error compared to the materials prepared immediately before injection. This functional stability data supports the particle size stability data described above and strongly indicates that the mRNA formulations described herein are stable for storage at 4°C for at least one week.

[0288] Generally, mRNA pharmaceutical materials formulated with ANP together with the delivery vehicle molecule as described herein may have a size of approximately 200 nm, making it possible to eliminate the need to use a 0.2-micron sterile filter at the end of the final formulation step to prevent loss. Therefore, multiple methodological filtration steps can be incorporated throughout the manufacturing process to mitigate sterility risks while avoiding the collapse of the final ANP pharmaceutical. Figure 18 schematically illustrates the various times at which filtration may be applied. Before the IVT reaction, both the purified template and individual IVT reagents (including dNTPs, enzymes, etc.) may be filtered through a 0.22 μm filter (Figures 18A and B). After mRNA production is completed in the IVT microfluidic device, all input materials will be filtered before the final formulation step (where ANP is formed). These include medicinal drug substances (mRNA, e.g., C in Figure 18), adjuvants (CpG), amphiphilic peptoid delivery vehicle components and buffers (D in Figure 18), DMG-PEG2000 delivery vehicle components (D in Figure 18), and dialysis buffers (E in Figure 18). In addition to 0.22 micron filtration of the input materials, the final amphiphilic nanoparticle medicinal product may be filtered through a 0.45 micron filter to remove particulate or aggregated material (F in Figure 18). This larger filtration step may help prevent the breakdown of ANP and maintain the efficacy of the final medicinal product.

[0289] To complement the inconspicuous filtration step described above, the microfluidic device control system described herein can be designed to ensure the safety and sterility of the final pharmaceutical product using one or more sealed microfluidic devices that perform operations necessary for pharmaceutical manufacturing, such as template preparation, in vitro transfer (IVT), compounding with a delivery vehicle to amphiphilic nanoparticles (ANPs), and buffer exchange and concentration steps, as described herein. These microfluidic devices are located within a temperature-controlled Class 5 laminar hood and may further be housed in, for example, a Class 7 6x6 cleanroom. The mRNA reactor(s) may be automated devices protected from humans and the external environment. Reagent and product channels may be delivered from pressurized sterile containers to the reactor core using single-use sterile nuclease-free tubing. The final product formulations and pharmaceuticals can be manufactured in a fully closed system within the multilayer microfluidic device(s) described above. Transmissive insert

[0290] Any of the microfluidic devices described herein may include one or more permeable inserts for processing a solution of therapeutic material (or a solution in which the therapeutic material is formed). The permeable inserts may be inserted into the fluid-contact side of a chamber within the microfluidic device. Fluid entering or passing through the fluid-contact side of the chamber must pass through the permeable inserts and may be configured to be modified by the permeable inserts. Any suitable permeable insert may be used. For example, the permeable inserts may include materials configured to remove undesirable substances, and in one example, the permeable inserts include cellulose material configured to remove double-stranded RNA (dsRNA) from a therapeutic solution of single-stranded RNA (ssRNA).

[0291] Figure 19A shows an example of a microfluidic device 1900 including a permeable insert 1969 within the fluid contact surface of a chamber 1957. In Figure 19A, the microfluidic device 1900 may include at least one pair of chambers 1953, 1957, 1957', each of which may include a fluid contact side 1917, a pressure (e.g., gas) side 1919, a fluid connection, a pressure connection, and a fluid / pressure line, which may be formed to the thickness of the microfluidic device. In one modification, the chambers are paired, and each chamber of the pair may be connected to each other by a fluid connector 1955. The fluid connector 1955 may be used in conjunction with positive and / or negative pressure applied to the pressure side of the chamber(s) to drive a liquid on the liquid side between the two chambers and to mix this liquid within each of the chambers. The chamber may be branched by an elastic material (e.g., an elastic layer or membrane), and by deflecting the elastic material within a fixed volume of the chamber, any liquid in the liquid can be driven in / out of the liquid-contact side of the chamber (e.g., between two chambers).

[0292] The microfluidic device 1900 may include a plurality of pairs of chambers, one of which may include a permeable insert. Each pair of chambers may be used for different processes. For example, the first pair of chambers 1953 may be used for RNA synthesis. The second pair of chambers 1957, 1957' may be used for the purification of the synthesized polynucleotides. By applying pressure to the pressure-receiving side 1919 of each chamber and opening the valve 1959 between the first pair of chambers 1953 and the second pair of chambers 1957, fluid from the first pair of chambers 1953 may be driven into the second pair of chambers. The valve chamber 1959 may be formed by an elastic layer 1907 in a connector channel between the two pairs of chambers.

[0293] The microfluidic device 1900, as shown in Figures 19A and 19B, may have a plurality of pressure ports 1943 and a plurality of fluid ports 1923. The plurality of pressure ports and the plurality of fluid ports may be arranged adjacent to the periphery of the microfluidic device and are configured to be connected to the fluid interface assembly 209 as described above.

[0294] For valve 1961, which can control the timing of reagent delivery driven from fluid port 1923, and which can also enable metering into the chamber of the apparatus when placed in series with one or more similarly configured valves, it is also possible to form the port (e.g., seal valve) from an elastic layer along the length of the connecting channel 1939 (either a pressure channel or a fluid channel), as shown in Figure 19A. For example, in Figure 19A, three valve chambers are shown (described in more detail below), where the first of these three valves may act as a peristaltic pump, while the middle valve may be a small metering chamber (e.g., having metering volumes of approximately 10 nL, 20 nL, 25 nL, 50 nL, 75 nL, 100 nL, etc.). The size of the channel (in particular the size of the chamber connected to the channel) allows for metering of the volume dispensed along the fluid connecting channels 1939, 1921 and delivered to the chamber 1953 connected to the fluid connecting channels 1939, 1921. In one variation, the metered volume may be approximately 50 nL. Metered volumes of approximately 100 nL, 1 microliter, 5 microliters, or more may be transferred. Various valve sizes may be pre-selected for integration into the microfluidic device 1900, and reagents may be connected to an appropriate metering size at the user's discretion.

[0295] Furthermore, a plurality of valve bodies 1961 may be included in a row along the fluid connection channel 1939. The series of valve bodies 1961 may act as a peristaltic pump for moving fluids including, but not limited to, viscous fluids. In general, the ability to function as a peristaltic pump for fluids may be particularly advantageous when moving fluids that are viscous or that may contain suspended particles such as purified or captured beads.

[0296] As described above, the microchannel device 1900 may also include a delivery or transfer reservoir or depot 1963. In FIG. 19A, the preselected volume may be formed similar to the chamber structure described above, or may optionally include only the metering side. In either case, valves may be used to meter the desired volume into the reservoir 1963. The valve 1965 may control the delivery of fluid from the reservoir 1963. If a larger volume is desired, the delivery may be repeated. Alternatively, if the reservoir 1963 has been preselected to be a transfer reservoir, the valve 1965 opens and pumps fluid out of the chamber 1957, while the valve 1967 remains closed, which allows only a measured volume of fluid to be transferred to the reservoir 1963. This fluid may then be transferred to a fluid vial on the reagent storage frame for further processing or testing. In one variation, the chamber, reservoir or depot (e.g., 1963) may be configured as the metering section of a 1 μL pump formed by, for example, three valve structures (1967, 1965, 1967). The chamber may be configured, for example, for the transfer of waste from the mixing chamber 1957.

[0297] The microfluidic device 1900 may be a sealed pathway structure. While the fluid vial, fluid line, and microfluidic device are connected, the operation of the apparatus can be carried out without exchange of substances inside and outside the system, particularly inside and outside the channels of the microfluidic device for processing including the synthesis of polynucleotides (RNA) and the preparation of biological delivery (as therapeutic drugs, vaccines, etc.). Thus, the entire system may operate as a closed pathway, and / or individual microfluidic devices may operate within the system as closed (air-protected) pathways.

[0298] Generally, these microfluidic devices may include incorporating one or more permeable inserts 1969 within the fluid side 1917 of a chamber or channel. The permeable inserts may be configured to absorb selected sites (e.g., selected material) from the fluid mixture in the chamber or channel. The absorbed material may be unwanted material to be purified from the solution, or it may be desired material to be removed from the solution, subsequently eluted, and further processed. In one modification, the permeable material of the insert may include a cellulose material capable of selectively absorbing double-stranded mRNA from the mixture. The cellulose material may be inserted into only one of a pair of chambers, so that when a fluid is mixed or passed through the permeable insert in the first chamber, the double-stranded RNA is effectively removed from the fluid mixture and then transferred to another pair of chambers further downstream for further processing or transfer.

[0299] In one variation of the microfluidic device 1900, a concentrator may further be included within the chamber, which may be located within the thickness of the second plate and be in fluid communication with an outlet channel such as 1949. The polynucleotides may be concentrated by expelling excess fluid medium, and the concentrated polynucleotide mixture is transferred outside the microfluidic device 1900 for further handling or use. In one variation, the concentrator may be a dialysis chamber. For example, a dialysis membrane may be located within or between the plates of the microfluidic device.

[0300] The microfluidic device 1900 may be formed of a material that is at least substantially translucent to visible light and / or ultraviolet light. Substantially translucent means that at least 90% of light is transmitted compared to a translucent material. In one variation, the microfluidic device 1900 may be formed of a material that is substantially translucent to visible light and / or ultraviolet light. Substantially translucent means that at least 90% of light is transmitted through the material compared to a completely translucent material.

[0301] The microfluidic device may be formed of two or more plates stacked on top of each other so that chambers and / or channels are formed between the plates, with an elastic material sandwiched between the first and second plates. The first and / or second plates may be formed of a rigid material. The plates may be formed of the same material or of different materials. For example, the rigid material may be a polymer or glass. The polymer or glass may be biocompatible and, for example, does not leach monomers or soluble small molecules toxic to living cells. Any suitable biocompatible polymer may be used, including medical-grade polycarbonate urethane, silicone polycarbonate urethane, polyether urethane, etc. In one modification, the polymer may be a cycloolefin copolymer.

[0302] Figure 19B shows a cross-section through a portion of a microfluidic device, showing a permeable insert 1969 in the fluid contact side 1917 of a chamber 1920, which is branched into a fluid contact side and a pressure receiving side 1919 by an elastic material 1907. Thus, the microfluidic device can be configured as a multilayer structure with a flexible membrane 1907 sandwiched between two more rigid layers 1903, 1905. Figure 19B shows a portion of a cross-sectional view (lateral to the plane of the microfluidic device) through an example of a microfluidic device having multiple layers forming a reactor for processing therapeutic agents, as described herein. The reactor may include a chamber containing seals, channels, valves, and a pumping chamber formed from multiple layers. For example, the microfluidic device may be formed from two or more rigid or semi-rigid plates 1903, 1905 and at least one elastic layer 1907. The elastic layer 1907 may be a sheet of an elastic material that is impermeable to liquids. The elastic layer is gas permeable, or may be treated to be gas permeable, including in various regions. A single continuous sheet may be used as the elastic material, but in some modifications, multiple sheets of elastic material may be used, or the “sheet” may be formed from portions of multiple sheets. The layer and the elastic sheet may also be laminated. Generally, chambers for holding, valvering, and / or pumping fluid may be formed in plates on both sides of the elastic layer, such that the elastic layer divides the chamber into a liquid-containing side and a pressure-applying side (e.g., gas). The overall volume of the chamber(s) may be constant and may be formed in both the first (e.g., upper) plate and the second (e.g., lower) plate, 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 to reduce (to zero, closing the chamber) the volume on the liquid-containing side or increase (to a predetermined maximum value) the volume on the liquid-containing side. Furthermore, to apply negative or positive pressure to the pressure-receiving side 1919 of one or more chambers, a pressure channel 1947 may be connected, for example, via a pressure port 1943 provided in the upper plate 1903.Furthermore, the liquid-containing side 1917 opposite the pressure-applied side of each chamber may be connected to a fluid port 1923 via a fluid passage 1921. Both the fluid port and the pressure port may be formed by openings to the upper plate 1903 and the elastic layer 1907, and since the pressure line is pushed into the elastic layer 1907 which is supported below the port by the opposite rigid or semi-rigid layer(s) 1905, 1909, a sealed connection isolated from the atmosphere is possible even when there are multiple different input lines.

[0303] In Figure 19B, the microfluidic device 1900 includes a first (e.g., upper) plate 1903 having a first (e.g., upper) surface 1911, a second (e.g., lower) surface 1929, and a thickness between them. The first surface 1911 may form an exposed outer surface. The microfluidic device also includes a second plate 1905 having a first (e.g., upper) surface 1931, a second (e.g., lower) surface 1933, and a thickness between them. An elastic layer 1907 is sandwiched between the second surface 1929 of the first plate 1903 and the first surface 1931 of the second plate 1905. In this example, a third plate 1909 is bonded to the second plate directly or indirectly on the second surface 1933 of the second plate. The third plate 1909 also has a first (e.g., upper) surface, a second (e.g., lower) surface, and a thickness between them. The second surface of the third plate may form the bottom surface of the microfluidic device. Each plate may be formed of multiple layers, which may be stacked or otherwise connected. For example, in Figure 19B, the third plate 1909 includes an optional second elastic layer 1913 that can help bond the third plate to the second plate, and in this example, the second elastic layer 1913 forms the first surface 1935 of the third plate 1909. The layers and plates shown in Figure 19B may not be to scale (for example, the elastic layer 1907 may be thinner than the plate).

[0304] Furthermore, the microfluidic device 1900 shown in Figure 19B may include a plurality of chambers 1915, 1916, 1918, and 1920, each having a certain volume. These chambers are formed by cut-out regions (e.g., rounded / curved cuts) into the second (bottom) surface 1929 of the first plate 1903 and the first (top) surface 1931 of the second plate 1905, and the elastic layer 1907 branches these chambers 1915 such that each includes a liquid-containing side 1917 and a pressure-receiving (e.g., gas-containing) side 1919. The microfluidic device 1900 may also include a plurality of liquid (e.g., fluid) channels. In Figure 19B, a single fluid channel 1921 extends from a liquid port 1923 that penetrates the thickness of the first plate 1903 to a liquid channel opening 1925 through the elastic layer 1907, and substantially penetrates the thickness of the second plate 1905 to the bottom surface 1933 of the second plate. The liquid channel 1921, which extends parallel to the lower surface of the third plate, has its length formed on the lower surface 1933 of the second plate, with the upper surface of the third plate as the boundary.

[0305] With respect to the fluid port 1923, the diameter of the opening to the first plate 1903 that forms the fluid port 1923, extending through the thickness of the first plate, may be larger than the diameter of the fluid channel opening 1925 that extends through the elastic layer 1907 into the liquid (e.g., fluid) channel 1921. The fluid channel opening 1925 may be centrally located relative to the bottom of the fluid port opening and may be offset from the wall of the fluid port opening by at least the expected wall thickness of the fluid line or fluid line coupling interface that will be connected to the fluid port.

[0306] The fluid channel 1921 is connected to the liquid-containing side 1917 of the first chamber 1915. This first chamber may be configured as a valve having a relatively low holding volume (fixed volume) but which can be completely opened and closed by the movement of the elastic layer 1907.

[0307] The microfluidic device 1900 also includes a plurality of pressure channels that can be independently controlled to apply positive and / or negative pressure. In Figure 19B, a single pressure port 1943 connected to the fourth chamber 1920 is shown, but each of the chambers 1915, 1916, and 1918 may independently operate and control the movement of the portion of the elastic layer 1907 that branches these chambers and connect each chamber independently to separate pressure ports and pressure channels for valves and / or pumps. In one modification, pressure ports may be shared among the plurality of chambers. In Figure 19B, the pressure (e.g., gas) port 1943 is similar to the fluid (e.g., liquid) port 1923 and includes an opening that penetrates the first plate 1903 completely and leads to the exposed elastic layer 1907, through the elastic layer to form the pressure (e.g., gas) channel opening 1945. The pressure channel opening 1945 extends from a pressure port 1943 that penetrates substantially the thickness of the first plate 1903 and is a cut-out channel along the bottom of the second plate (or alternatively, a cut-out region at the top of the third plate), and is continuous with a pressure channel 1947 that passes through the second plate and the elastic layer 1907 and returns to the region of the pressure channel in the first plate that connects to the pressure (e.g., gas) containing section 1919 of the fourth chamber 1920. As described for a similar fluid (e.g., liquid) port, the diameter of the pressure port 1943 that penetrates the thickness of the first plate 1903 may be larger than the diameter of the pressure channel opening 1945 that penetrates the elastic layer 1907, and may be centrally located or offset by a greater margin than the wall thickness of the pressure line or pressure line coupling interface that will connect to the pressure port.

[0308] In the cross-section of the microfluidic device 1900 shown in Figure 19B, there are multiple connections to other fluid (e.g., liquid) lines, fluid ports, pressure lines, and pressure ports, but these are not shown because they are outside the shown plane. For example, in Figure 19B, the liquid-containing side / part 1917 of the fourth chamber may be connected to an additional valve (chamber) and / or channel, for example, an outlet channel extending from the liquid-containing side 1917. Additional chambers (e.g., configured as valves) not shown may be formed as described above. In one modification, the outlet channel may deliver fluid from one or more chambers through another fluid port (not shown) to a fluid receiving depot, such as a vial or tube. This receiving depot may be held in a reagent storage frame.

[0309] As described above, the permeable insert 1969 may be inserted into the fluid contact side of the separation chamber and configured to be compressed by an elastic material that separates the fluid contact chamber from the pressure-receiving side of the chamber. In this example, a positive or negative pressure applied to the pressure-receiving side (e.g., through a pressure port directed to this chamber) can deflect the elastic material and change the volume on the fluid contact side. A fluid may be driven into the chamber 1920 having the permeable insert 1969, and the fluid may pass through the insert to modify the solution. In a modified example where the permeable insert is compressible, it may be compressed to remove and discharge the fluid from the chamber. In one modified example, the permeable insert may then expand (or allow expansion) back to an expanded configuration, allowing the fluid to pass through again or undergo further processing.

[0310] The permeable inserts described herein can generally be used to modify solutions containing (or on which therapeutic materials are formed). Figure 20A schematically shows an example of a method for processing therapeutic materials in a fluid (e.g., an RNA sample) using one of the apparatuses described herein. For example, the method may first include mounting a microfluidic device (or one or more microfluidic devices) to a microfluidic device control system 2001. This step may include coupling the microfluidic device to a pressure source. In some modifications, this step (or an additional step) may include coupling the microfluidic device to a source of therapeutic material, such as RNA. Optionally, in some modifications, the method may include synthesizing the therapeutic material in the microfluidic device, such as generating therapeutic RNA by in vitro transcription 2003.

[0311] This method may further include transporting a sample containing therapeutic material (e.g., RNA) to the fluid contact portion of a processing chamber containing a permeable insert. For example, this may include applying pressure to transfer the sample to the fluid contact side of the separation chamber of a microfluidic device 2005. In one modification, pressure can be applied by deflecting an elastic material (e.g., an elastic membrane) within the microfluidic device to drive a fluid containing the therapeutic substance (or putative therapeutic material) to the fluid contact side of the chamber. As part of this step, the fluid sample (including therapeutic / provisional therapeutic) can be passed through a permeable insert in the fluid contact side of the separation chamber to modify the sample 2007. For example, the therapeutic / provisional therapeutic substance may be added or removed by interacting with the permeable insert.

[0312] Finally, pressure may be applied to transfer the sample from the fluid contact side of the separation chamber by, for example, bending an elastic material such as an elastic membrane that separates the fluid contact side of the chamber from the pressure receiving side of the chamber.2009

[0313] Figure 20B shows a specific example of a method for processing therapeutic material (e.g., an RNA sample) in a fluid using any of the apparatus described herein. For example, in Figure 20B, the method may be a method for removing dsRNA from an RNA sample containing both double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA). In this variation, the method may include connecting a microfluidic device to a pressure source 2011. In one variation, this may include connecting a microfluidic device to a source of therapeutic RNA and / or performing in vitro transcription of therapeutic RNA in the microfluidic device, as described above 2013. The method then may include transferring the RNA sample to the fluid contact side of the separation chamber of the microfluidic device under pressure 2015. The RNA sample may then pass through a solid, permeable insert containing collagen located in the fluid contact side of the separation chamber 2017, so that the cellulose binds to the dsRNA and the dsRNA is retained by the insert. Subsequently, pressure is applied to transfer the RNA sample from the fluid contact side of the isolation chamber, leaving the ssRNA in the therapeutic solution. This step may be repeated as needed to remove all or substantially all dsRNA.

[0314] As mentioned above, further processing (such as binding with a delivery vehicle, dialysis, or concentration) may be performed thereafter.

[0315] The apparatus described herein may include one or more isolation chambers and / or be used in conjunction with one or more isolation chambers. For example, in one modification, the apparatus described herein may be part of a therapeutic polynucleotide manufacturing “factory” capable of producing therapeutic polynucleotides for delivery to a subject, for example. The therapeutic polynucleotides may be therapeutic mRNA, for example. Figures 21A-21B show an example of an apparatus that may be used as a factory apparatus by itself or as part of a parallel manufacturing apparatus. In Figure 21A, apparatus 2101, 2101' may include or be housed in a Class 5 isolation cabinet 2103, the isolation cabinet may itself be housed in a Class 7 isolation space. In Figure 21A, the cabinet includes two microfluidic control devices 2101, 2101'. The apparatus may be part of an assembly plant providing a copy-exact GMP unit capable of rapidly and automatically manufacturing therapeutic polynucleotides, such as therapeutic mRNA, for patient use. These apparatuses can be highly reconfigurable, enabling rapid deployment and low-cost production. In one variation, these devices may be deployed as on-demand manufacturing "factory" units. In another variation, these devices may be configured as part of mobile units deployed temporarily or permanently in remote locations.

[0316] In this specification, when a feature or element is referred to as being "on top of" another feature or element, it may be directly on top of the other feature or element, or there may be intervening features and / or elements. 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. Also, when a feature or element is referred to as being "connected," "attached," or "joined" to another feature or element, it will be understood that it may be directly connected to, attached to, or joined to the other feature or element, or there may be intervening features or elements. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly joined" to another feature or element, there are no intervening features or elements. Although a feature and element have been described or illustrated in relation to one embodiment, such features and elements may be applicable to other embodiments. Also, it will be understood by those skilled in the art that a reference to a structure or feature positioned "adjacent" to another feature may have a portion that overlaps with or lies beneath the adjacent feature.

[0317] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the invention. For example, as used herein, each component may be plural unless the context clearly indicates otherwise. As used herein, the terms “including” and / or “including” specify the presence of the described 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 terms “and / or” include any and all combinations of one or more of the items described herein and may be abbreviated as “ / ”.

[0318] In this specification, for the sake of ease of explanation, spatially relative terms such as “below,” “lower,” “below,” “above,” and “above” may be used to describe the relationship of one element or feature to another element or feature, as shown in the figures. It will be understood that spatially relative terms are intended to encompass different orientations of the device in use or operation, in addition to the orientation depicted in the figures. For example, if the device in the figure is inverted, an element described as “below” or “below” another element or feature would then be oriented “above” the other element or feature. Thus, the exemplary term “below” can encompass both up and down orientations. The device may also be in other orientations (rotated 90 degrees, or in other orientations), and the spatially relative descriptors used herein shall be interpreted accordingly. Similarly, terms such as “upward,” “downward,” “vertical,” and “horizontal” are used herein for illustrative purposes only, unless otherwise specified.

[0319] In this specification, the terms “first” and “second” may be used to describe various features / elements (including steps), but unless otherwise indicated by the context, these features / elements should not be limited by these terms. These terms may be used to distinguish one feature / element from another. Thus, the first feature / element described below may be referred to as the second feature / element, and similarly, the second feature / element described below may be referred to as the first feature / element without departing from the teachings of the present invention.

[0320] Throughout this specification and the subsequent claims, unless otherwise required by context, the word “contains” means that various components may be employed collaboratively in methods and articles (e.g., compositions and apparatus including devices and methods). For example, the term “contains” would be understood to mean that it includes any described element or step, but does not exclude other elements or steps.

[0321] Generally, any apparatus and method described herein should be understood to be comprehensive, however, all or a subset of components and / or steps may be alternatively exclusive and may be expressed as "consisting only of" or alternatively "essentially consisting only of" various components, steps, subcomponents or substeps.

[0322] Where used herein and in the claims, including where used in examples, unless otherwise expressly specified, all numerical values ​​can be read as if preceded by the words “about” or “approximately,” even if the term does not explicitly appear. The phrase “about” or “approximately” is used when describing magnitude and / or location and can indicate that the described value and / or location is within a reasonable expected range of the value and / or location. For example, a numerical value may have values ​​such as ±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. Furthermore, any numerical value shown herein should be understood to include 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 referred to herein is intended to include all subranges contained therein. Furthermore, where a value is disclosed, it is understood that, in order to be appropriately understood by those skilled in the art, "less than or equal to that value," "greater than or equal to that value," and possible ranges between values ​​are also disclosed. For example, if the value "X" is disclosed, not only "less than or equal to X" but also "greater than or equal to X" (e.g., X is a number) is disclosed. Also, throughout this application, it is understood that data is provided in many different formats, and this data represents the endpoints and starting points of any combination of data points, and ranges. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that not only the range between 10 and 15 is disclosed, but also the ranges greater than 10, 10 or greater, less than 10, 10 or less, and equivalent to 10 and 15 are disclosed. It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0323] While various exemplary embodiments have been described above, any of numerous modifications can be made to these embodiments without departing from the scope of the invention as described in the claims. For example, the order in which various described method steps are performed may often be changed in alternative 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 one embodiment but not in another. Therefore, the foregoing description is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention as defined in the claims.

[0324] The examples and illustrations included herein are illustrative and not limiting, illustrating specific embodiments in which the subject matter may be implemented. As noted, other embodiments may be utilized and derived therefrom, allowing for structural and logical substitutions and modifications without departing from the scope of this disclosure. Such embodiments of the subject matter of the present invention may be referred to individually or collectively in this specification merely for convenience, using the term “invention,” and where multiple inventions are actually disclosed, there is no intention to spontaneously limit the scope of this application to any single invention or inventive concept. Thus, while specific embodiments are illustrated and described herein, any arrangement calculated to achieve the same objective may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the embodiments described above, and other embodiments not specifically described herein, will be apparent to those skilled in the art upon consideration of the above description.

Claims

1. A method for producing a therapeutic single-stranded mRNA composition, The aforementioned method, A step of pressurizing one or more microfluidic plate devices containing multiple reactors and multiple fluid deposits that are in sealing fluid communication, A step of controlling a first fluid power circuit to deliver a template precursor material from one or more fluid depots among the plurality of fluid depots to a first reactor among the plurality of reactors with sub-microliter precision and without contact with the atmosphere. A step of processing the template precursor material to form a DNA template from the template precursor material, A step of controlling a second fluid power circuit to transfer the DNA template to the second reactor among the plurality of reactors with sub-microliter precision and without contact with the atmosphere, The step of processing the DNA template by in vitro transcription to form therapeutic single-stranded mRNA, A step of controlling a third fluid power circuit to transfer the therapeutic mRNA to the third reactor among the plurality of reactors with sub-microliter precision and without contact with the atmosphere. The steps include: forming the therapeutic mRNA composition by performing a process to bind the therapeutic mRNA to an mRNA delivery vehicle, and The step includes concentrating the therapeutic mRNA composition, The device described above, An elastic material sandwiched between the first plate and the second plate, A plurality of chambers formed between a first plate and a second plate, wherein a portion of the elastic material divides each chamber into a fluid contact side and a pressure receiving side, A permeable material in the fluid contact side of the first chamber among multiple chambers, Equipped with, The permeable material is configured to purify RNA, and the elastic material dividing the first chamber is configured to flex upon application of pressure to the pressure-receiving side, allowing fluid to move in and out of the first chamber. The method further comprises purifying a therapeutic single-stranded polynucleotide, and the method comprises removing double-stranded RNA. The aforementioned purification process Connecting the device to a pressure source, The RNA sample is transported under pressure to the fluid contact side of the separation chamber of a microfluidic plate device, allowing the RNA sample to pass through a permeable material within the fluid contact side of the separation chamber, and ensuring that the dsRNA is retained by the permeable material. Applying pressure to the pressure-receiving side of the separation chamber to transport the RNA sample from the fluid-contact side of the separation chamber, Further including, method.

2. The method according to claim 1, wherein the step of transferring the therapeutic single-stranded mRNA to a third reactor includes transferring a plurality of different therapeutic mRNAs together with the delivery vehicle to form a single-stranded mRNA composition.

3. The method according to claim 1, wherein the first fluid power circuit and the second fluid power circuit are controlled by a controller.

4. The method according to claim 3, wherein the controller controls the fluid power circuit by deflecting one or more elastic layers in the one or more microfluidic plate devices.

5. The method described above is the method according to claim 1, which is performed in a medical setting.

6. The method according to claim 1, wherein the step of binding the therapeutic single-stranded mRNA to the delivery vehicle further comprises dialysis of the therapeutic single-stranded mRNA composition in one or more microfluidic pathway plate devices to purify the therapeutic single-stranded mRNA composition.

7. The method according to claim 1, wherein the delivery vehicle comprises amphiphilic nanoparticles.

8. The method according to claim 7, wherein the amphiphilic nanoparticles include an aminolipidized peptoid.

9. The method according to claim 1, further comprising the step of purifying the therapeutic single-stranded mRNA by two-dimensional (2D) purification in one or more of the plurality of reactors that are in fluid communication with the second reactor.

10. The method for producing the therapeutic single-stranded mRNA composition, wherein the method is carried out for 72 hours or less, according to claim 1.

11. The method according to claim 1, wherein the first reactor among the plurality of reactors is located on the first microfluidic plate device among the one or more microfluidic plate devices, and the third reactor among the plurality of reactors is located on the second microfluidic plate device.

12. The method according to claim 1, further comprising the step of receiving optical sensor data from one or more sensors of a system, wherein the controller controls the operation of a closed system based on at least a portion of the optical sensor data, in a controller of a system including a plurality of fluid depots.

13. The method according to claim 1, further comprising the steps of transferring the therapeutic mRNA composition to a concentrator having fluid communication with the third reactor, and concentrating the therapeutic single-stranded mRNA composition.

14. The method according to claim 1, further comprising recording the movement of fluid in one or more microfluidic devices while the steps described above are being performed in a file related to the therapeutic polynucleotide being manufactured.

15. The method further includes the step of processing the DNA template by in vitro transcription, and then transferring the therapeutic mRNA into a fluid vial. The method according to claim 1, further comprising the step of controlling a third fluid-powered circuit to transfer therapeutic mRNA to a third reactor, the step of transferring therapeutic single-stranded mRNA to a subsequent microfluidic device including a third reactor among a plurality of reactors.

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