WO2022079492A1

WO2022079492A1 - Methods and apparatuses for pneumatic liquid transfer

Info

Publication number: WO2022079492A1
Application number: PCT/IB2021/000696
Authority: WO
Inventors: Ryan Fobel; Hamed TINAFAR; Alexander KLENOV
Original assignee: Liberum Biotech Inc.
Priority date: 2020-10-14
Filing date: 2021-10-14
Publication date: 2022-04-21

Abstract

Devices and methods for portable, modular production of a biological material comprising a device compatible with receiving one or more cartridges, wherein production and purification of the biological material are handled inside the cartridge or multiple cartridges using pneumatic control. These devices and methods allow for touch-free production and purification of the biological material such that at least one liquid transfer or mixing step within the cartridge is carried out without liquid coming in contact with a pump or valve within the device. These devices and methods can be used for efficient on-demand production of biomolecules including protein, RNA, DNA or any combination thereof, while minimizing cross-contamination between runs.

Description

METHODS AND APPARATUSES FOR PNEUMATIC LIQUID TRANSFER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application no. 63/091,719, titled “PNEUMATIC LIQUID TRANSFER IN A MULTI-CHAMBER SET-UP AND RELATED METHODS” and filed on October 14, 2020, herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

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

BACKGROUND

[0003] The methods and apparatuses described herein generally relates to lab automation, and more particularly production or purification of biomolecular products using cell-free systems.

[0004] Cell-free protein synthesis (CFPS) systems may provide simple and sensitive tools for both biological research and therapeutic treatments. CFPS systems, also known as in vitro protein synthesis systems, have emerged as potent technology for high-throughput production of biomolecules for research and therapeutic applications. To date, lab automation has been hampered by bulky equipment that not only takes up a lot of space, but also require lengthy setup times. Current systems also typically require highly skilled operators and are prone to crosscontamination across runs. It would be beneficial to provide method and apparatuses that allow liquid handling inside of disposable cartridges that do not require a great deal of space, capital equipment or training to operate.

SUMMARY OF THE DISCLOSURE

[0005] Described herein are methods and apparatuses (e.g., systems, devices, cartridges, etc.) that may provide a novel pneumatic liquid handling device for biomolecular production and purification. Described herein are touch-free methods and apparatuses wherein at least one liquid transfer or mixing step within the cartridge is carried out without liquid coming in contact with a pump or valve within the device. Such a system minimizes cross-contamination across multiple runs. [0006] For example, described herein are apparatuses (e.g., devices and systems, including cartridges) for producing biomolecules that may be configured to be portable and modular. These apparatuses may include a device configured to receive one or more cartridges and to operate on each of the one or more cartridges to generate a biomolecule (e.g., a polypeptide, a polynucleotide, etc.) in a way that does not transfer liquid material from the cartridge into the device. These devices may pneumatically drive material within the cartridge, for example, by applying air or other fluid to generate positive pressure and and/or by applying vacuum to generate negative pressure. Thus, these devices may be isolated from the contents of the cartridge, preventing or minimizing contamination. The device described herein may be configured for use with a variety of differently configured cartridges. In some examples the device may be configured for use with a single chamber cartridge, or a cartridge having a single reaction chamber and a single collection chamber which may be part of the same cartridge or separate. In some examples the device may be configured for use with cartridges having multiple chambers, and one or two chamber cartridge or for use with a cartridge including a plurality of chambers that may be connected by channels within the cartridge itself. The device may control operation on the cartridge by pneumatically driving material (e.g., liquid, including buffers, etc.) between the chambers.

[0007] Any of the apparatuses described herein may be configured to controllably apply sparging, e.g., by bubbling air or other gas through one or more chambers of a cartridge. Sparging may be particularly beneficial for increasing reaction surface area available for in vitro synthesis of the biomolecule and in some cases for providing oxygen to the reaction, but must be controlled in order to be effective. Specifically, the rate of sparging must be controlled; excessive bubbling can cause protein denaturation, while optimal sparging may greatly enhance the yield of the biomolecules in these apparatuses. Sparging may also be very useful for mixing materials within the cartridge to avoid formation of concentration gradients and to overcome diffusion limitations. However, it may be particularly difficult to manage sparging in the context of a removable cartridge in order to prevent leakage and/or contamination of the device controlling operations on the cartridge.

[0008] The apparatuses described herein may also control the temperature of one or more of the chambers of the cartridge in order to control the cell-free synthesis of biomolecules. For example, the devices described herein may control the temperature of a reaction mixture within the cartridge. Temperature control is particularly challenging when applying sparging, as bubbling air or other gas through the solution may result in rapidly changing the temperature (e.g., cooling) the solution. [0009] For example, described herein are methods of cell-free, in vitro synthesis of a biomolecule using any of these systems. The method may include adding a nucleic acid substrate to a chamber of a disposable cartridge in pneumatic communication with a device for in vitro synthesis of biomolecules comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; sparging a gas through the reaction mixture within the chamber; and combining, in the cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, flow-through, washing and eluting the synthesized biomolecule material within the cartridge, wherein the one or more processors pneumatically controls the transfer of liquid within the cartridge so that liquid from the cartridge does not contact a pump or valve within the device for in vitro synthesis of biomolecules.

[0010] The substrate for capturing the synthesized biomolecule may be any appropriate capture substrate, such as a substrate for chromatography (e.g., a metal-charged affinity resin such as Ni- NTA agarose, calmodulin-sepharose, protein A agarose, glutathione agarose, cellulose, chitin, anti-Flag tag agarose, anti-V5 tag agarose, amylose resin or a custom substrate made by attaching a molecule for specific binding to a resin or matrix) or ion-exchange purification, a silica gel, etc.

[0011] The synthesis of the biomolecule may be, in particular, protein synthesis, e.g., synthesis of a polypeptide. Thus, the biomolecule may be polypeptide. The polypeptide may be a full-length protein or portion of a protein. In some examples the biomolecule may be a polynucleotide (e.g., DNA, RNA, etc.). In other examples, the bimolecular product may be a complex between two or more biomolecules (e.g., protein and RNA).

[0012] The nucleic acid substrate may be, for example, a DNA or mRNA template. In some examples the nucleic acid substrate may be configured for synthesis, e.g., by including appropriate upstream and/or downstream promotors, recognition sites, etc. In general, the nucleic acid substrate may be combined with all of the components (enzymatic components, reagents, etc.) to perform in vitro synthesis under the control of the device. The nucleic acid substrate may be combined with these components either before adding the nucleic acid substrate to the cartridge or after adding the nucleic acid substrate to the cartridge. For example, the nucleic acid substrate may form a reaction mixture after combining with all of the synthesis components once it is added to the cartridge (e.g., to a chamber of the cartridge). In some examples, the synthesis components may include a cell extract together comprising biological extracts and/or defined reagents. The synthesis components may include one or more enzymes for synthesizing the biomolecule, such as enzymes for transcription and/or translation. In some examples (when the biomolecule includes DNA, for example) the synthesis components may include a DNA polymerase. Other synthesis components that may be included are amino acids, nucleotides, cofactors, enzymes and other reagents that are necessary or helpful for synthesis, e.g., ribosomes, tRNA, polymerases, transcriptional factors, etc. When the nucleic acid substrate is a DNA template, the DNA may be first transcribed to mRNA. Alternatively the nucleic acid substrate may be mRNA, which could be used directly for translation. The nucleic acid substrate for cell- free protein synthesis can be either DNA or mRNA. DNA or mRNA can be in either linear or circular formats. Translation of mRNA or combined transcription and translation converts stored information into a desired protein. In some examples the methods and apparatuses described herein may continuously generate mRNA from a DNA template with a recognizable promoter. For example, an endogenous RNA polymerase may be used, or an exogenous RNA polymerase (e.g., a phage polymerase such as that from T7 or SP6) and may be included in the reaction mixture. Alternatively, mRNA can be continually amplified by inserting the message into a template for QB replicase. The nucleic acid substrate (e.g., mRNA) may be stabilized by chemical modification before it is added to the reaction mixture. Nucleases can be removed to help stabilize mRNA levels. The nucleic acid substrate can encode for any particular gene of interest.

[0013] The synthesis components forming part of the reaction mixture may also include salts, particularly those that are biologically relevant, such as those of magnesium, manganese, potassium or ammonium. The pH of the reaction may be between pH 5.5-9. As mentioned, the apparatuses and methods using them described herein may include regulation of the temperature of the reaction (generally between 16° C and 40° C, but may be outside of these ranges). In some examples the temperature may be tightly regulated to particular ranges of temperatures during the various steps of the method. For example, the temperature of the reaction mixture when synthesizing the biomolecule (such as, but not limited to a polypeptide) may be regulated within +/- 0.1 degree, 0.2 degrees, 0.5 degrees, 1.0 degrees, 1.5 degrees, 2 degrees, 3 degrees, etc. The apparatus (e.g., device) may hold the temperature at this set temperature for all or a portion of the duration of a particular step (e.g., synthesis), e.g., for 0-16 hours, 1-20 hours, 2-18 hours, 2-16 hours, etc. The temperature may be monitored, and used to control (feedback) the temperature of the chamber and/or reaction mixture. Other steps in the method may be held to different temperatures, e.g., binding, flow-through, washing and eluting the synthesized biomolecule may each be performed at a predetermined temperature or within a predetermined temperature range. As mentioned, it may be particularly advantageous to monitor the temperature through the cartridge without contacting the liquid(s) within the cartridge. It may also be surprisingly advantageous to apply thermal energy (e.g., heat) to the liquid within the cartridge (including the reaction mixture within a chamber of the cartridge) without contacting the liquid within the cartridge. For example, infrared sensing has been found to be surprisingly effective in noncontact monitoring of the temperature of the liquid within the cartridge and infrared energy may also or alternatively be applied to control the temperature of a fluid within the cartridge (e.g., within a chamber of the cartridge). Thus, as will be described in greater detail below, the apparatuses descried herein, including the devices for in vitro synthesis, may control the temperature. For example, the synthesis reaction may be carried out at temperatures within the 22-28 °C range.

[0014] The use of pneumatic control, e.g., by one or more processors of the device pneumatically controlling the transfer of liquid within the cartridge is particularly helpful so that liquid from the cartridge does not contact a pump or valve within the device for in vitro synthesis of biomolecules. The apparatus may be configured to prevent, limit or prohibit fluid from the cartridge coming into contact with the device, including internal components of the device. For example, in some cases the cartridge may be configured so that the pneumatic line(s) in the cartridge to/from the device each include one or more traps (fluid or liquid traps) having a sufficient capacity and orientation so that fluid is not passed from the cartridge into the device, or even into a line (e.g., tubing) or manifold of the device.

[0015] Examples of cartridges are provided in detail below, but in general the cartridge may include: one or more chambers for storing or receiving reagents for synthesis and/or purification of a biomolecule (such as a protein, RNA, DNA or any combination thereof). The one or more chambers may include at least one reactor module (“reaction chamber”). The reactor module may facilitate production or processing of the biological material via a process selected from: continuous exchange cell-free synthesis, fed-batch cell-free synthesis, and/or batch cell-free synthesis. In some examples the same chamber may be used for the synthesis reaction as for the purification (e.g., by adding the purification material/substrate into the reaction chamber). In some examples the cartridge may include multiple chambers, including a dedicated purification module that may include one or more chambers for carrying out chromatography or ionexchange purification for separating the biological material from one or more components of a reaction solution.

[0016] In any of these cartridges, the cartridge may include a connection (e.g., a first connection) between two or more of the chambers of the cartridge, wherein fluid transfer via the first connection is controlled by a flow controller (such as a valve or a hydrophobic frit), e.g., at a bottom of the chamber. For example, in cartridges having multiple chambers the chamber may include multiple connections individually between some chambers directly or collectively by connecting to a single channel. Each of the chambers may be coupled to the device via a pneumatic channel that can apply positive or negative pressure (e.g., by applying air or vacuum) or simply block the channel or open it to the atmosphere. In some examples the chamber(s) may be connected to the device by a second connection, wherein the second connection is established between the device and the cartridge chamber(s) via pneumatic ports. The cartridge may therefore include a pneumatic connection to the device at both a top and a bottom of the chamber. Each of these channels into and/or out of the chambers (from the top or bottom) may be valved by one or more pneumatically controlled valves. The pneumatically controlled valves may also be connected via a line that provides pneumatic (positive or negative pressure) to open/close the valve. As mentioned, in some cases the bottom of the chamber may be instead or additionally connected to a porous matrix (e.g., a hydrophobic matrix, which may be configured as a frit) for applying sparging and/or preventing fluid or a substrate (e.g., a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material) from out of the bottom of the cartridge. In general, the device controller may use multiple pneumatic lines to control valves and to control the pressure within the chamber(s) of the cartridge to control the cell-free, in vitro synthesis of the biomolecule.

[0017] Thus, the system may be configured for liquid transfer or mixing within the cartridge carried out without liquid coming in contact with a pump or valve within the device. For example, the arrangement of the pneumatic valves, channels and liquid traps, as well as in some examples the porous matrix, in the one or more channels of the cartridge may be arranged so that cell-free, in vitro synthesis may be performed within the cartridge without the risk of fluid from the cartridge contacting the device (e.g., contacting a pump and/or valve within the device). [0018] Also described herein are methods of cell-free production of a biological material using a device compatible with receiving one or more cartridges such as the cartridges described above. For example, a cartridge may include one or more chambers for storing or receiving reagents for synthesis or purification of a biomolecule (e.g., protein, RNA, DNA or any combination thereof); at least one reactor module, wherein the reactor module facilitates production of the biological material via a process (e.g., a process selected from: continuous exchange cell-free synthesis; fed-batch cell-free synthesis; or batch cell-free synthesis); a purification module comprising one or more chambers for carrying out chromatography or ionexchange for separating the biological material from one or more components of a reaction solution; a first connection between two or more of cartridge chambers, wherein fluid transfer via the first connection is controlled by at least one flow controller; and a second connection, wherein the second connection is established between the device and the cartridge via pneumatic ports; wherein the platform is capable of at least one liquid transfer or mixing step within the cartridge that is carried out without liquid coming in contact with a pump or valve within the device. In any of these apparatuses (e.g., the portable, modular apparatuses) or methods described herein, the reaction and purification modules may share some or all of their chambers. The synthesis of the biological material may include transcription, translation, amplification or a combination thereof.

[0019] The at least one flow controller may be contained within the cartridge. The flow controller may be a porous matrix (e.g., configured as a hydrophobic frit) or pressure-responsive valve. As will be described in detail below it may be particularly helpful to include a porous matrix (e.g., a hydrophobic porous matrix) at the bottom of one or more of the chambers of the cartridge. The porous matrix may be configured to provide for both sparging (applying bubbles) and for removing liquid, by applying negative pressure (e.g., suction) through the porous matrix and/or providing positive pressure (e.g., air pressure) into the chamber, e.g., from the top, so that fluid may pass through the porous matrix.

[0020] The description of provided herein typically refers to the “top” and “bottom” of the chambers and/or cartridges described herein. The term top and bottom may refer to the orientation of the cartridge when the cartridge is configured to be operated in a particular (e.g., upright, vertical) position. In some examples the cartridge may be oriented in the device at an angle relative to vertical or even horizontally. Although bubbling is typically performed up/down relative to gravity, in some examples, sparging may be performed at an angle relative to the cartridge or horizontally.

[0021] As mentioned, flow out of (or in some cases into) a chamber of the cartridge may be regulated by a flow controller. In some examples, the flow controller may a membrane, MEMS device or rotary pump and/or valve. The flow controller may contain an elastomeric membrane. One particular example of a flow controller is a porous matrix, and in particular, a hydrophobic porous matrix, which may be configured to act in three modes of operation. The first mode is that of sparging, in which air or other gas is applied (by positive pressure) into the porous matrix, and allowed to form bubbles that are released up and pass through the chamber at a rate that is controlled by the one or more processors of the device, for example, by controlling the pressure applied by from, e.g., the air pump, of the pressure manifold. The second mode may be to block or prevent fluid and/or material (e.g., the reaction mixture) within the chamber of the cartridge from flowing or leaking out, e.g., from the bottom of the chamber, at low pressure. The third mode may be to allow flow of fluid (but not material such as the substrate for capturing the synthesized biomolecule) out through the porous matrix. The threshold at which the porous matrix switches between the second and third modes may depend on the material properties of the porous matrix, the size of the pores, and the fluids being blocked/passed. In some cases, the porous matrix may be a hydrophobic matrix material, e.g., a hydrophobic substrate. In some examples the porous matrix may be one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE).

[0022] For example, a method of cell-free, in vitro synthesis of biomolecules may include: adding a nucleic acid substrate to a chamber of a disposable cartridge held within a device for in vitro synthesis comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; applying a gas, under the control of the one or more processors, through a porous matrix at a bottom of the reaction chamber to sparge the gas through the reaction mixture within the chamber; and combining, in the cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by removing unbound components, washing, and eluting or synthesized biomolecule material within the cartridge, wherein the one or more processors applies pressure to drive liquid through the porous matrix for one or more of removing unbound components, washing and eluting.

[0023] As mentioned, in any of these methods and apparatuses, the purification module may include a chromatography or an ion-exchange chamber or column. Alternatively or additionally, a chromatography or an ion-exchange chamber or column can be separately attached to the cartridge.

[0024] In any of these methods and apparatuses the cartridge may be connected to at least one other removable cartridge(s) or chamber(s) for the collection of the product and/or waste. [0025] In any of the portable, modular platforms described herein, the cartridge may be supplied with at least one twist cap lid or screw for providing a sealable entry port for one or more reagents. In general, the device may be connected to at least one removable cartridge for the formation of the biomolecule. The cartridge may include a collection chamber which may be integrated into or separatable from the rest of the cartridge. For example, the cartridge may include a chamber (subcartridge) for collecting product biomolecule material and a twist cap lid attached to the cartridge collection chamber (subcartridge). The product, once prepared, can be sealed within the removable subcartridge using the twist cap lid.

[0026] As will be described in greater detail below, in any of these apparatuses the cartridge may be pneumatically sealed to the cartridge. Force may be applied by the device to seal the device to the pneumatic inputs on the cartridge, or the seal may be simply made using a magnetic connection. Either or both the cartridge or the device may include sealing connectors (e.g., including a gasket or gaskets, oil/gel, or the like) to maintain the seal between the cartridge and the manifold of the device. In some examples the device may include a clamp and/or lock for securing the cartridge in sealing communication with the cartridge. In some examples the connection between the device and the cartridge may be include a magnetic connector, mechanical clamp, luer lock and/or a lid.

[0027] The devices described herein may include non-contact monitoring of one or more properties (e.g., production rate, temperature, flow rate, pressure, pH, etc.) within the cartridge. For example, any of these apparatuses may include or be configured for optical tracking. Optical tracking may monitor the production of the raw biomolecular product via the production (e.g., of GFP protein) or activation of a measurable molecular reporter (e.g., of GFP1-10 by binding to synthesized GFP11) to track the reaction progress, the optical tracking directly taking place through the body of the cartridge or part thereof without having to move the sample outside the cartridge for measurement purposes. Other optical (non-contact) monitoring may include temperature sensing (e.g., infrared temperature sensing). Any of the apparatuses and methods described herein may include an optical system to measure product yield; the optical measurement may be done through the body of the cartridge or part thereof without having to move the sample outside the cartridge for measurement purposes.

[0028] In some examples, the apparatuses described herein may include a valve that is pneumatically operated to open, close, or open and close. In some cases the valve may be default open, default closed, or neither default open nor closed (e.g., floating). For example, the flow controller may be a valve having at least one elastomeric membrane that is pneumatically, mechanically and/or electronically controlled to open, close, or open and close. In some cases, as mentioned above, the flow controller contains at least one MEMS device that is pneumatically, mechanically or electronically controlled. The flow controller may contain at least one rotary valve that is pneumatically, mechanically or electronically controlled.

[0029] In any of the apparatuses or methods described herein, one or more sensors may be used to evaluate proper loading of the cartridge when connected to the device. For example, a pressure sensor may be used to track pressure data or to guide control of fluids within the cartridge. Pressure may be sensed one or more of the inputs (e.g., inlets), and/or outputs (e.g., outlets) for pneumatic pressure into/out of the cartridge. The sensor(s), including pressure sensors, may be integrated into the device (the device for in vitro synthesis) holding the cartridge. In some examples the pressure sensor may be part of the cartridge, or both part of the cartridge and part of the device. Multiple pressure sensors may be used, or a single pressure sensor may be multiplexed. In some cases the pressure sensor may be coupled to a pressure manifold, which may distribute pressure (positive and/or negative pressure) to one or more inlets/outlets of the cartridge. [0030] In any of the apparatuses and methods described herein the cartridge may be configured to increase the surface-to-volume ratio of one or more chambers of the cartridge. For example, one or more chambers in which cell-free synthesis takes place may be configured to increase surface-to-volume ratio. In some examples the surface-to-volume ratio of a chamber may be increased by including a serpentine structure. Alternatively or additionally, the one or more chambers in which cell-free synthesis takes place may include beads or bead-like structures to increase surface-to-volume ratio. Alternatively or additionally, the one or more chambers in which cell-free synthesis takes place may be configured to apply sparging (e.g., bubbling of gases) through the reaction chamber. As will be described in greater detail below, in some examples it is advantageous to use a porous matrix that is configured to both apply sparging (forming bubbles), and to act as a passive threshold valve, preventing liquid (and in particular, aqueous liquid) from passing into/out of the chamber below a pressure threshold, but allowing passing of the liquid (but not solid material such as a substrate for capturing the synthesized biomolecule) to pass when the pressure across the porous matrix exceeds a threshold.

[0031] Any of the cartridges described herein may include a purification module, which may include a substrate for capturing the synthesized biomolecule. The purification module may be separately attached to the device or the cartridge, or it may be integrated into the cartridge. The substrate for capturing the synthesized biomolecule may be, for example, beads coated with a charged functional groups, a gel including beads coated with a charged functional group, beads coated with an affinity group, etc. In some examples the substrate for capturing the synthesized biomolecule may be configured to bind to the biomolecule under a first set of purification conditions and the biomolecule may be released from the substate under a second set of purification conditions.

[0032] In general, the cartridge may be configured to seal to a pneumatic interface in the device so that one or more pneumatic inlets/outlets (ports, also referred to as pneumatic ports) of the cartridge may be sealingly coupled to the pneumatic manifold within the device. In some cases the sealing of the pneumatic port connection between the cartridge and the device is enhanced by a soft gasket material such as rubber, silicone or an elastomeric polymer. The sealing of the pneumatic port connection may be established or improved by one or several magnets. The sealing of the pneumatic port connection may include one or more mechanical systems such as a push-release latch, lid or motorized mechanism. Sealing of the pneumatic port connection may include the use of a vacuum seal. In some examples sealing of the pneumatic port connection may be maintained (at least in part) by at least one of the pneumatic signals from the device being exerted in the form of negative pressure. [0033] In some examples, described herein are systems that are configured as portable, modular systems for synthesizing (and purifying) a biomolecule. These systems may include: a device compatible with receiving one or more cartridges; wherein each cartridge or combination of cartridges comprise: (a) at least one input chamber for storing or receiving reagents for synthesis or purification of a biomolecule, wherein the biomolecule comprises protein, RNA, DNA or any combination thereof; at least one receiving chamber for collecting product, waste or product intermediates; at least one porous matrix (e.g., a hydrophobic frit), valve or combination thereof separating the input chamber from a receiving chamber, wherein the cartridge facilitates production of the biomolecule via a process selected from: continuous exchange cell-free synthesis; fed-batch cell-free synthesis; or batch cell-free synthesis; wherein the system (e.g., device and cartridge) is configured to perform liquid transfer or mixing within the cartridge without liquid from the cartridge coming in contact with a pump or valve within the device. [0034] A method of cell-free production of a biomolecule may include: a device compatible with receiving one or more cartridges; wherein each cartridge or combination of cartridges comprises: at least one input chamber for storing or receiving reagents for synthesis or purification of biological material, wherein the biomolecule comprises protein, RNA, DNA or any combination thereof; at least one receiving chamber for collecting product, waste or product intermediates; at least one porous matrix (e.g., hydrophobic frit), valve or combination thereof separating the input chamber from a receiving chamber, wherein the cartridge facilitates production of the biomolecule via a process selected from: continuous exchange cell-free synthesis; fed-batch cell-free synthesis; or batch cell-free synthesis; wherein the device is configured to transfer liquid (and/or mixing) within the cartridge without liquid from the cartridge coming in contact with the device (e.g., a pump or valve within the device).

[0035] The one or more enzymes for synthesizing the biomolecule may include enzymes for one or more of transcription, translation, amplification or a combination thereof.

[0036] As mentioned, any of the cartridges described herein may be configured to increase a surface-to-volume ratio within, e.g., a reaction chamber and/or purification chamber of the cartridge. For example, described herein are methods of cell-free, in vitro synthesis of biomolecules that include: adding a nucleic acid substrate to a chamber of a disposable cartridge held within a device for in vitro synthesis comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; introducing the reaction mixture into a porous matrix or micro-scale channels within the cartridge to increase surface interactions; controlling the temperature of the reaction mixture within the cartridge; combining, in the cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to enable purification; and pneumatically controlling, by the one or more controllers of device binding, flow-through, washing or elution to purify a synthesized biomolecule material.

[0037] A micro-scale channel may be configured as a channel having a relatively narrow diameter, and a much longer length. As used herein the term “microscale” may refer to dimensions having a scale on the order of microns. In reference to microscale channels, the diameter of the channel may be within the range of microns (e.g., 1 pm to 1 mm or more), while the length is much longer (typically mm or cm). For example a micro-scale channel may have a diameter of between 1 pm and 5 mm (between 1 pm and 3 mm, between 1 pm mm and 2 mm, between 1 pm mm and 1 mm, between 10 pm and 5 mm, between 10 pm and 4 mm, between 1 pm and 3 mm, between 10 pm and 2 mm, etc.) and a length that is typically more than 10 fold (lOx) greater, or more than 20 fold (20x), more than 30 fold (30x) more than 40 fold (40x) more than 50 fold (50x) more than 75 fold (75x), more than 100 fold (lOOx), more than 500 fold (500x), more than 1000 fold (lOOOx), etc. longer than it is wide. The micro-scale channel may be serpentine, including numerous turns that may double-back. In some examples the micro-scale channel may extend in a helical direction, and not limited to a single plane. This may permit a higher radius of turning while still providing a significant increase in length compared to diameter.

[0038] As mentioned, the methods and apparatuses described herein may be configured to sparge (bubble) gas, such as air, into the cartridge in a controlled manner to enhance synthesis of one or more biomolecule while preventing foaming and/or protein denaturation that may occur with excessive bubbling. These apparatuses may also prevent leakage and/or contamination of the device, which may allow the use of multiple cartridges either concurrently or sequentially with the same device (e.g., device for in vitro synthesis) without requiring cleaning and/or sterilizing of the device) that may otherwise be necessary. In particular, the methods and apparatuses described herein may be configured so that the sparging may be applied directly from the pneumatic manifold of the system (when in communication with the pneumatic ports of a cartridge) without an expensive and cumbersome interface or sparging apparatus. The device and methods described herein may be configured to monitor the application of bubbles within the cartridge and to dynamically adjust (control) both the application of bubbles, e.g., by controlling the pressure and/or airflow applied from device to the cartridge, as well as the temperature of the material within a chamber of the cartridge, such as the reaction chamber and/or the purification chamber. Sparging (bubbling) and/or temperature may be measured from the cartridge chamber(s) in a non-contact manner, from the device. [0039] For example, described herein are methods of cell-free, in vitro synthesis of biomolecules that include: adding a nucleic acid substrate to a disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within a chamber of the disposable cartridge; controlling, using one or more processors of a device for in vitro synthesis of biomolecules, the application of bubbles through the reaction mixture within the chamber for a combined total of 2 or more hours, while controlling the temperature of the reaction mixture within the chamber; combining, in the cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, washing and eluting the synthesized biomolecule material within the cartridge, wherein the one or more controllers pneumatically controls the transfer of fluid within the cartridge for performing binding, washing or eluting. [0040] The methods (and devices for implementing them) may be configured to detect bubble (sparging) rate within the chamber by sensing pressure applied to the cartridge, e.g., to the sparging emitter within the cartridge, such as the porous matrix (e.g., a hydrophobic matrix). Pressure may be sensed at any point in front of the sparging emitter, such as within the line coupling the pressure source (e.g., air pump) to the cartridge, at the pressure source (pump), within the manifold, etc.

[0041] For example, a method of cell-free, in vitro synthesis of biomolecules may include: attaching a disposable cartridge to a device for in vitro synthesis of biomolecules; adding a nucleic acid substrate into the disposable cartridge with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within a chamber of the disposable cartridge; controlling, using one or more processors of the device for in vitro synthesis of biomolecules, the application of bubbles through the reaction mixture from a porous matrix on a bottom of a chamber for a combined total of 2 or more hours, while controlling the temperature of the reaction mixture within a target range; combining the reaction mixture with a substrate for capturing the synthesized biomolecule in the cartridge; applying negative pressure through the porous matrix to remove flow-through from the chamber; adding wash buffer to the chamber and applying negative pressure through the porous matrix to remove the wash buffer; and adding elution buffer through the chamber and applying negative pressure through the porous matrix to collect the eluted product. In some examples the collection chamber may be swapped out during one or more of these steps to enable separation and analysis of resulting liquids.

[0042] The apparatuses described herein may be configured to apply sparging for a predetermined time period, or for period of time that is dependent upon detecting a signal from within the cartridge (e.g., an indicator of the level of biomolecule generated). For example, the controller of a device as described herein may be configured to apply sparging for between 0-16 hours, 2-24 hours, between 2-18 hours, between 4-24 hours, between 4-18 hours, etc. In some examples the apparatus may be configured to receive a user input for the duration of sparging. The apparatus may be configured to apply sparging continuously until manually stopped. Any of these apparatuses may be configured to adjust or set the level of sparging (e.g., the rate of bubbling), either automatically or manually. For example, the apparatus may be configured to receive a user input on the pressure applied to sparge, the rate of bubbling (bubble/second), or some other metric for sparging. The apparatus may include preset values or ranges of values, which may be used by default and/or may act as limits on user-entered values.

[0043] In any of these apparatuses and methods, sparging may be used for mixing fluid within the cartridge.

[0044] As mentioned, any of these methods and apparatuses may include non-contact sensing and/or application of energy (e.g., thermal energy) from the cartridge and the device. For example, a method of cell-free, in vitro synthesis of a biomolecule may include: adding a nucleic acid substrate to a chamber of a disposable cartridge held within a device for in vitro synthesis of biomolecules comprising one or more processors for pneumatically controlling fluid transfer and biomolecule synthesis within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; sparging, under control of the one or more processors, a gas through the reaction mixture within the chamber; detecting a temperature of the rection mixture; controlling, by the one or more processors, the temperature of the reaction mixture and applying thermal energy through a wall of the cartridge without contacting the wall of the cartridge; and combining, in the cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, flow-through, washing and eluting the synthesized biomolecule material within the cartridge.

[0045] The temperature may be detected by a non-contact technique, such as one or more infrared (IR) sensors on the device that are directed to measuring temperature within the chamber of the cartridge. The temperature may be adjusted (based on sensed temperature) by application of thermal energy using a non-contact technique, such as by the application of radiant heat, including the application of IR energy. This is particularly advantageous in that it may be rapidly applied and regulated, e.g., allowing quick heating and cooling, as cooling may occur quickly when turning off the radiant heating, particularly when sparging. The cartridge may be formed at least partially of a material that is transparent to the applied IR energy, particularly in/around the chamber. Other portions of the cartridge may be protected from the application of heating by IR light by using a material that does not transmit the IR energy as efficiently (or at all), or by including coatings that reflect or absorb the IR energy to limit it from passing into other regions of the cartridge.

[0046] The methods described above may be performed by a device, referred to generally as a device for in vitro synthesis of a biomolecule, that is adapted to hold a cartridge and apply pneumatic force to move, mix, and sparge with the cartridge without transmitting fluid to the cartridge. These devices may be adapted for use with a cartridge including a single chamber for mixing and purifying (and in some cases a single waste port or chamber that may be part of the cartridge or may be part of a separate chamber). In some cases the device may be configured to control operation of a cartridge having multiple interconnected chambers, including separate reaction chambers and purification chambers (and in some examples a plurality of buffer chamber or reservoirs). Thus, the devices described herein may include an interface for coupling (pneumatically coupling) with the cartridge to hold the cartridge in position securely with the sealing pneumatic connection to the pneumatic ports of the cartridge. The device may include a clamp or lock mechanism, as mentioned above. These devices may include a seating or coupling region for coupling to the cartridge (or in some cases, multiple cartridges) in a sealing configuration. The device may also generally include circuitry, such as one or more processors forming part of a controller that may regulate sensing (e.g., temperature sensing, pressure sensing, etc.) both within the device and/or within the cartridge; the controller may also regulate the pump or pumps that may apply pneumatic pressure (both positive and optionally negative pressure/vacuum). The controller (processors) may also be coupled with a pressure manifold that may distribute pressure to one or more of the pneumatic ports of the cartridge in a controlled manner, during operation of the device. The pressure manifold may be controlled by internal valves or switches within the device that may direct pressure from the one or more pumps to a particular output (pneumatic port) using the manifold. The device may also include one or more filters for filtering air into the pump and/or manifold. The filer may be replaceable.

[0047] The device controller may include a memory and/or communications circuitry for communicating with a remote server. In some cases this communications circuitry may be wireless or may be configured for wireless communications. The device controller may also include connections for input and/or output to one or more input devices (e.g., keyboards, dials, switches, touchscreen, or other controls) and one or more output devices (screens, displays, LEDs, etc.). Optionally, output may be sent to a remote device (phone, computer, tablet, etc.) and/or input may be received from a remote device. Any of the devices described herein may include a housing that at least partially covers all or some of the device components (e.g., the controller, manifold, etc.). The seat or coupling for the cartridge(s) may be an opening into or extension of the housing. [0048] For example, a device for cell-free, in vitro synthesis of biomolecules may include: a pneumatic manifold; an air pump or other pressure source coupled to the pneumatic manifold; a cartridge holder configured to hold a cartridge so that one or more pneumatic input ports on the cartridge sealingly mate with the pneumatic manifold; a temperature sensor configured to detect temperature within a chamber of the cartridge; and a controller comprising one or more processors and a memory coupled to the one or more processors, the memory configured to store instructions, that, when executed by the one or more processors, perform a method comprising: applying bubbles through a reaction mixture within a chamber of the cartridge, while controlling the temperature of the reaction mixture at a preset temperature; binding, flow-through, washing and eluting a synthesized biomolecule material within the cartridge, wherein the one or more controllers controls the transfer of liquid within the cartridge without liquid from the cartridge coming in contact with a pump or valve within the device.

[0049] Another example of a device for cell-free, in vitro synthesis of biomolecules may include: a housing; a pneumatic manifold within the housing; an air pump coupled to the pneumatic manifold; a cartridge holder coupled to the housing, the cartridge holder configured to hold a cartridge; a clamping seal configured to secure the cartridge within the cartridge holder so that one or more pneumatic input ports on the cartridge sealingly mate with the pneumatic manifold; a temperature sensor configured to detect temperature within a chamber of the cartridge; and a controller within the housing, the controller comprising one or more processors and a memory coupled to the one or more processors, the memory configured to store instructions, that, when executed by the one or more processors, perform a method comprising: applying of bubbles through a reaction mixture within the chamber while controlling the temperature of the reaction mixture within the chamber; pneumatically combining the reaction mixture with a substrate for capturing the synthesized biomolecule to form a purification substrate; pneumatically removing unbound components; pneumatically controlling washing of the purification substrate; and pneumatically controlling eluting a synthesized biomolecule material from the purification substrate.

[0050] In general, unless the context indicates otherwise, the term “cartridge” is intended to be used broadly to include a case or container including one or more chambers. The cartridge may include a rigid or partially rigid cartridge housing. The cartridge housing may enclose the chamber(s) and may be adapted for interfacing with the devices (e.g., coupling or seat of the device).

[0051] In general, these cartridges may be single-use or limited use (e.g., disposable).

[0052] The cartridges may be configured so that a single chamber is used for the reaction and purification. For example, a disposable cartridge for cell-free, in vitro synthesis of biomolecules may include: a first chamber configured to be connected to a device for cell-free, in vitro synthesis of biomolecules in an upright position, wherein the first chamber comprises an open or openable top and a bottom; a hydrophobic porous matrix at the bottom of the cartridge; a channel extending from the hydrophobic porous matrix and configured to extend into a second chamber; wherein the second chamber comprises a chamber having a threaded connector at a top region that is configured to removably couple to the first chamber directly or indirectly so that the channel extends into the second chamber.

[0053] Any of these disposable cartridges for cell-free, in vitro synthesis of polypeptides may include: a first chamber configured to be connected to a system for cell-free, in vitro synthesis of polypeptides (e.g., in an upright position), wherein the first chamber comprises a body (in some examples an elongate cylindrical body) having an open or openable top and a bottom; a porous matrix (in some examples a hydrophobic porous matrix, optionally configured as both a passive valve and sparging emitter, e.g., a hydrophobic frit) at the bottom of the chamber (towards the bottom of the cartridge); a channel extending from the hydrophobic frit and configured to extend into a second chamber; wherein the second chamber comprises a chamber (e.g., tubular chamber) having a threaded connector at a top region. This threaded connector may be configured to removably couple to the first chamber directly or indirectly so that the channel extends into the second chamber. Optionally the cartridge may include a cap or cover configured to couple to the threaded connector at the top region of the second chamber when the second chamber is uncoupled from the first chamber.

[0054] In some examples the second chamber may be configured to removably couple directly to an outer surface of the bottom of the first chamber. The cartridge may include a luer connector extending laterally from the disposable cartridge and configured to couple with the system (e.g., to the coupler of the system). In some examples an outer surface of a bottom region of the first chamber is threaded.

[0055] The first chamber may be configured to hold any appropriate volume, such as between 1 mL and 50 mL of fluid (e.g., 1 mL and 100 mL, between 1 mL and 150 mL, etc.). [0056] Also described herein are cartridges configured to include multiple, connected or connectable, chambers. For example, a disposable cartridge for cell-free, in vitro synthesis of biomolecules may include: a cartridge housing configured to be held within a device for cell- free, in vitro synthesis of biomolecules (e.g., in an upright position); a plurality of chambers within the cartridge housing, including a reaction chamber, a purification chamber, and a plurality of buffer chambers (or buffer reservoirs); a plurality of pneumatically actuated valves, wherein each chamber of the plurality of chamber is connected to a pneumatically actuated valve of the plurality of pneumatically actuated valves near a bottom region of each chamber; a first plurality of pneumatic input ports on the cartridge housing, wherein each of the pneumatic input ports of the first plurality of pneumatic input ports is coupled to a pneumatically actuated valve of the plurality of pneumatically actuated valves; a second plurality of pneumatic input ports on the cartridge housing, wherein each chamber of the plurality of chambers is coupled to a pneumatic input port of the second plurality of pneumatic input ports through an opening at a top region of each chamber of the plurality of chambers; a third pneumatic input port coupled to one or more of the chambers of the plurality of chambers through one or more of the pneumatically actuated valves; a nucleic acid substrate input port at a top of the cartridge in communication with the reaction chamber, wherein the nucleic acid substrate input port further comprises a cover; and at least one porous matrix between the purification chamber and a pneumatically actuated valve of the plurality of pneumatically actuated valves or another chamber.

[0057] For example, a disposable cartridge for cell-free, in vitro synthesis of biomolecules, the cartridge comprising: a cartridge housing configured to be held within a device for cell-free, in vitro synthesis of biomolecules in an upright position; a plurality of chambers within the cartridge housing, including a reaction chamber, a purification chamber, and a plurality of buffer chambers, wherein each chamber is coupled to a shared channel at a bottom region of the cartridge housing; a plurality of pneumatically actuated valves, wherein each chamber is connected to the shared channel through a pneumatically actuated valve; a first plurality of pneumatic input ports on the cartridge housing, wherein each of the pneumatic input ports of the first plurality of pneumatic input ports is coupled to a pneumatically actuated valve of the plurality of pneumatically actuated valves; a second plurality of pneumatic input ports on the cartridge housing, wherein each chamber of the plurality of chambers is coupled to a pneumatic input ports of the second plurality of pneumatic input ports through an opening at a top region of each chamber of the plurality of chambers; a nucleic acid substrate input port at a top of the cartridge in communication with the reaction chamber, wherein the nucleic acid substrate input port further comprises a cover; and at least one porous matrix between the shared channel and the purification chamber.

[0058] Each chamber of the plurality of chambers may be coupled to a shared channel at a bottom region of the cartridge housing that is coupled to the third pneumatic input port.

[0059] Any of these cartridges described herein may be configured to prevent flow of fluid back into the device (from the cartridge) by including one or more (e.g., arranged in series) liquid traps formed in the cartridge housing. For example, each of the chambers of the plurality of chambers includes a liquid trap of the plurality of liquid traps between the opening at the top region of each chamber of the plurality of chambers and the second plurality of pneumatic input ports. The liquid traps may have an input (fluidically coupled to the chamber) on one side that is separated by a trap region from an output. The trap region may be a chamber opening into a lower region relative to the output. The mouth of the output out of the trap may be offset from the mouth of the input into the trap; in some examples the mouth of the input may positioned above or adjacent to the mouth of the output. Generally, fluid must flow into the trap, and be trapped within the trap. The trap region may have a volume of greater than 0.1 mL (e.g., between 0.1 mL and 5 mL, etc.). The trap region may include a capture material (e.g., a porous and/or hydrophilic material.

[0060] The housing of all or a portion of the cartridge, including but not limited to a region over or adjacent to one or more of the chambers (e.g., the reaction chamber, the purification chamber, etc.), may be at least partially optically transparent.

[0061] The cartridge may be pre-loaded with all or some of the material used during the method of cell-free, in vitro synthesis of a biomolecule. For example, the chambers of the cartridge may include freeze-dried (e.g., lyophilized) material forming the buffer, enzyme(s), amino acids, antifoaming agent, salt, etc. or any of the other components. Some of the chambers, e.g., the buffer chambers, such as the wash buffer chamber, binding buffer chamber and/or elution buffer chamber, may include a pre-mixed buffer. Alternatively, the chambers may include dried (e.g., freeze-dried) components that may be controllably combined with water or other resuspension liquid prior to performing the method. In some examples the cartridge may include a chamber or reservoir containing water that may be added by the device (and/or manually) ahead of performing the method using the cartridge. Any of these cartridges may include the substrate for chromatography or ion-exchange purification within the purification chamber (e.g., preloaded). Any of these cartridges may include a chamber for the reaction components that may be combined in the cartridge (under control of the device) to form the reaction mixture. For example, the cartridge may include a chamber holding the one or more enzymes for synthesizing the biomolecule (e.g., in some examples enzymes for translation and/or transcription, such as but not limited to a cell lysate material) and the other synthetic reaction components such as the salts, buffers, nucleotides, amino acids, chemical energy source(s), etc. The enzyme component may be stored separately from the other reaction components.

[0062] Any of the cartridges described herein may include one or more waste chambers. [0063] All or some of the pneumatic input ports may be arranged on the portion of the cartridge to be coupled to (in some examples, seated in) the device. For example, the first and second plurality of pneumatic input ports may be arranged on an outer surface of a bottom region of the cartridge housing. The cartridge may include one or more sealing surfaces that may seal (including lockably sealing) against the interface in the device. For example, the first plurality of pneumatic input ports and the second plurality of pneumatic input ports may comprise a sealing surface. The sealing surface may provide a rigid or compressible (e.g., elastically deformable) surface. In some examples a ring (e.g., O-ring) may be included around each port.

[0064] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0066] FIG. 1 is one example of a cartridge as described herein.

[0067] FIGS. 2A-2B illustrates examples of schematics for transfer of fluids and materials inside a cartridge.

[0068] FIGS. 3A-3B illustrates one example of design schematics for inner workings of a cartridge containing on-cartridge valves.

[0069] FIGS. 3C-3D illustrate an example of design schematics for inner workings of a cartridge containing both on-cartridge valves and pumps.

[0070] FIG. 4 illustrates one example of a device for in vitro synthesis of biomolecules.

[0071] FIG. 5 shows one example of a device for in vitro synthesis of biomolecules can be used for obtaining optical read outs from a cartridge.

[0072] FIG. 6 shows an example of yield comparisons among various cell-free protein expression methods.

[0073] FIG. 7 illustrates an example of protein expression yields for deGFP at various temperatures.

[0074] FIGS. 8 A and 8B shows an example of an example of a device for in vitro synthesis of biomolecules. FIG. 8B shows the device with the upper portion of the outer housing removed. [0075] FIG. 9 shows an example of a mechanical (sealing) clamp of a device for in vitro synthesis of biomolecules that is actuated by a stepper motor that connects and seals a cartridge to a device as described herein.

[0076] FIG. 10A illustrate an example of a cartridge as described herein.

[0077] FIG. 10B shows another example of a cartridge.

[0078] FIG. 11 illustrates one example of a liquid trap that may be included as part of any of the cartridges described herein.

[0079] FIG. 12 schematically illustrates an example of a cartridge.

[0080] FIG. 13 schematically illustrates one example of a pneumatically activated valve as described herein. [0081] FIG. 14 illustrates one example of a method of cell-free, in vitro synthesis of a biomolecule using a device and cartridge as described herein.

[0082] FIG. 15A shows an example of a device coupled to a cartridge using a single chamber for reaction and purification.

[0083] FIG. 15B shows an example of an upper portion of a device for in vitro synthesis of biomolecules similar to that shown in FIG. 15 A.

[0084] FIG. 15C shows a bottom portion (internal) of a device for in vitro synthesis of biomolecules similar to that shown in FIG. 15 A.

[0085] FIG. 15D shows a schematic view of an internal region of portion of the bottom (including the manifold body) a device for in vitro synthesis of biomolecules.

[0086] FIG. 16 is a schematic illustration of an example of an electronics board for a device for in vitro synthesis of biomolecules.

[0087] FIGS. 17A-17C illustrate operation of a device for in vitro synthesis of biomolecules as described herein.

[0088] FIGS. 18A-18B illustrate one example of a cartridge including a single chamber for reaction and purification.

[0089] FIGS. 19A-19B illustrate another example of a cartridge including a single chamber for reaction and purification.

[0090] FIGS. 20A-20L illustrate one example of a method of cell-free, in vitro synthesis of a biomolecule as described herein.

DETAILED DESCRIPTION

[0091] In general, described herein are methods and apparatuses (e.g., devices and systems, including cartridges) for cell-free, in vitro synthesis of a biomolecule. In some examples, the methods and apparatus may relate to portable devices and accompanying cartridges for automated production, purification and formulation of biological materials (biomolecules). The biomolecule may be a protein, RNA, DNA or any combination thereof. Examples of biomolecules that can be produced in the system include protein-based therapeutics, vaccines, antivenoms and lab reagents.

[0092] These apparatuses may be configured as a portable device and may include hardware, software and firmware, for engaging with one or more cartridges in which the cell-free, in vitro synthesis of a biomolecule may be performed. In general, these apparatus and methods may perform cell-free, in vitro synthesis of a biomolecule by controlling operations within the cartridge pneumatically and in an otherwise contact-free manner, in which no liquid or other material from the cartridge is passed into the device holding and operating on the cartridge. [0093] FIG. 1 illustrate an example of a cartridge 1, comprising a cartridge housing forming a first reaction chamber 2 (“A chamber”), and second reaction chamber 3 (“B chamber”). The first and second reaction chambers may be referred to collectively as a reaction module. In some examples, the cartridge may include a single reaction chamber or alternatively a third reaction chamber receiving input from the A chamber and B chamber. In FIG. 1, the cartridge also includes a binding buffer chamber 4, a wash buffer chamber 5, an elution buffer chamber 6 and a purification chamber (configured as a purification column 7) forming part of the purification module. The cartridge in this example also contains a waste chamber 8, a twist-cap 9, an input port and a tube 10 for collecting the final product (synthesized biomolecule).

[0094] The cartridges described herein may generally be compatible with a device for controlling cell-free, in vitro synthesis of the biomolecule as will be illustrated below. The device may include a component for seating or receiving (coupling to) one or more cartridge. In some examples the device may hold the cartridge upright (relative to gravity).

[0095] The cartridge may include at least one reactor module for the production of a target biomolecule and one purification module (e.g., in FIG. 1, including binding buffer chamber 4, a wash buffer chamber 5, an elution buffer chamber 6 and a purification chamber/column 7) for separating out the target biomolecule from the reaction components. The flow of fluids within the cartridge is controlled through pneumatic connections 21 (pneumatic ports) established between the cartridge and the device. Pressure (positive or negative) may be applied by drawing a vacuum (negative pressure) or by driving air (or other gas) to delivery positive pressure. In some examples just positive pressure is used. The device may include one or more pumps, and/or a pump manifold to direct and control pressure to the pneumatic ports 21.

[0096] Sealing of these connections (ports) in fluid connection with the source of pressure in the device (e.g., the device manifold in some examples) can be enhanced by using a soft gasket material such as rubber, silicone or elastic polymer. The connection can be further enhanced by mechanical means such as a push-release latch, a motorized mechanism (e.g., a clamp and/or lock, as will be shown in FIG. 9, below), or by using vacuum or magnets. Using at least some of the pneumatic signals from the device in the form of negative pressure can also help maintain the seal.

[0097] Fluid flow may be tightly regulated by the controller of the device, using pressure differential, elastomeric membranes, MEMS devices, hydrophobic frits, rotary pumps or valves or a combination thereof. The cartridge design shown in FIG. 1 is configured so that fluid flow can be solely handled using pressure differential. FIGS. 2A-2B illustrate two designs for handling of fluid flow using on-cartridge pumps and valves. These can be made from elastomeric membranes, MEMS devices, rotary pumps or valves or a combination thereof. [0098] In FIGS. 2 and 2B examples of schematics for transfer of fluids and materials inside a cartridge (such as the cartridge 1 shown in FIG. 1) containing on-cartridge valves, labeled 11-17, or for a cartridge containing both on-cartridge valves 11-18 and pumps 19 and 20 have been illustrated. In FIGS. 2A and 2B the examples show a layout including a first and second reaction chambers 2, 3 of the reaction module, binding buffer chamber 4, wash buffer chamber 5, elution buffer chamber 6 and purification column 7 of a purification module, a waste chamber 8, and a collection chamber 10 for receiving the final product.

[0099] The apparatuses described herein may transfer at least one liquid within the cartridge without the liquid coming in contact with a pump or valve within the device. This is particularly advantageous, as it allows for minimal cross -contamination across runs and enables multiple runs without the need for device clean up between them. In operation, the device controls the cartridge through pneumatic ports 21 to allow for transfer and moving of the reagents within the cartridge. This control may be carried out by generating a pressure differential among chambers 2-6, 8 and 10, e.g., using on-cartridge pumps and valves 11-20 or any combination thereof. In order to help prevent fluids flowing into undesired chambers these apparatuses (e.g., cartridges) may include onboard pumps and/or valves as well as hydrophobic passive valves (e.g., hydrophobic porous matrix, sometimes configured as a frit) to further control flow and prevent incorrect passing of fluid.

[0100] For example, FIGS. 3A-3B and 3C-3D illustrate examples of schematics for different variations of cartridges. For example, FIG. 3A shows a cartridge containing on-cartridge valves 11-17 and FIGS. 3C-3D show another example of a cartridge containing both on-cartridge valves 11-18 and pumps 19 and 20. In some embodiments the device and cartridge are used for production of a protein through cell-free protein synthesis and the purification of the synthesized protein. One such embodiment stores enzymes of transcription and translation (e.g., crude cell lysate) in one of the reaction chambers (e.g., the B chamber) and the reaction buffer including energy sources and amino acids in the second reaction chamber (e.g., the A chamber), for example, 0.2 mM ATP, 0.85 mM GTP, 0.85 mM UTP, 0.85 mM CTP, 31 pg/mL Folinic Acid, 171 pg/mL tRNA, 0.4 mM Nicotinamide Adenine Dinucleotide (NAD), 0.27 mM Coenzyme A (CoA), 4 mM Oxalic Acid, 1.5 mM Spermidine, and 57 mM HEPES buffer, 10 mM Mg(Glu)2, 130 mM K(Glu), 3 mM of each of the 20 amino acids, 2% (w/v) of PEG8000 and 30 mM 3PGA) of the reactor module. Once a nucleic acid substrate (e.g., DNA, RNA, mRNA, etc.) is added to the reaction chamber (e.g., reaction chamber 2) through the twist cap 9 input port, components of chambers 2 and 3 may be mixed and incubated in chamber 3 for 1-16 hours at 16-37°C to carry out protein expression. The process of synthesis may be controlled and regulated by the device. For example, as will be described in greater detail below, in some examples the device may sparge the reaction mixture to increase the surface to volume area (e.g., by bubbling air or other gas through the reaction chamber). The controller of the device may also monitor and control temperature of the reaction mixture. In any of these devices, the controller may be configured to thermally cycle. Thus any of these apparatuses may be configured to cool and heat rapidly. Noncontact heating and/or cooling may be done by cooling the air that is bubbled through the apparatus, as described herein. Any of these methods or apparatuses may include a cooler to cool gas (e.g., air) that is applied.

[0101] Before adding the reaction mixture to the purification module (purification chamber), or during synthesis, the chromatography beads within column 7 (e.g., Ni-NTA beads) are washed and equilibrated by passing the binding buffer (e.g., 5 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol) in chamber 4 through the column 7. Once synthesis step has been completed, the reaction mixture (e.g., contents of chamber 3) may be purified, e.g., moved through column 7 to allow binding of the product to the beads within the chromatography column in this example. The bound product may then be washed using a wash buffer (e.g., 20 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol), stored in chamber 5 in this example. All components until this step may be driven to the waste chamber 8. The product may then elute off the column 7 by passing the elution buffer (e.g., 250 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol), an example of which is shown in chamber 6, through the column 7 and collecting the product in tube 10.

[0102] The reactor module (and therefore the cartridge and device) can be configured to facilitate production of the biomolecule via one of the following processes: continuous exchange cell-free synthesis; fed-batch cell-free synthesis; or batch cell-free synthesis. For continuous exchange synthesis, one or more chambers of the reactor module may be separated by a size exclusion element allowing for feeding of input materials to enzymatic components. For fed- batch synthesis, one or more reagents may be fed to a chamber of the reactor module during the reaction to achieve higher production yields. In other embodiments, batch cell-free synthesis may be used which simply involves transferring reaction components into the reactor module and incubating the mixture. In some embodiments, the device and cartridge enable mixing of the reaction components at the outset or during the synthesis or purification steps.

[0103] In some examples, the biomolecule is, e.g., a polynucleotide. The substrate for capturing the synthesized biomolecule may be, e.g., a silica gel or bead (e.g., coated bead). For example, the device and cartridge may be used for in vitro transcription and purification of RNA from DNA templates. In one such embodiment, polymerase (e.g., T7 RNA polymerase, 25 U/pL final concentration) is stored within chamber 3 of the reactor module while chamber 2 contains transcription buffer, NTPs and inorganic pyrophosphatase (0.005 U/pL final concentration). In this example, the purification column 7 contains silica beads or a silica membrane to capture the RNA post transcription. Binding buffers, wash buffers and elution buffers may also be stored on cartridge and applied during the purification step.

[0104] In yet other embodiments, the device and cartridge are used for in vitro DNA synthesis through a process such as polymerase chain reaction (PCR). In one such embodiment, DNA polymerase is stored within chamber 3 of the reactor module while chamber 2 of the reactor module contains a PCR buffer, a magnesium salt and dNTPs. The purification column 7 contains silica beads or a silica membrane to capture the amplified DNA. Binding buffers, wash buffers and elution buffers may also be stored on cartridge and applied during the purification step.

[0105] The examples shown above in the context of FIGS. 1-3D are not intended to be limited to this variation of the cartridge and device; other device and cartridge designs (as shown and illustrated below) may be used with these same reagents and components.

[0106] Any of these devices may include optical tracking that monitors the production of the raw biomolecular product via the production of a measurable molecular reporter (e.g., Green Fluorescent Protein, Red Fluorescent Protein or GFP11 tag and accompanying components) to track the reaction progress. Optical tracking can directly take place through the body of the cartridge or part thereof without having to move the sample outside the cartridge for measurement purposes. FIG. 5 illustrates an example of a device in which optical tracking may be used. Light from LED 24 is used to excite the fluorescent protein and the emitted light is then filtered using optical filter 25 and measured using sensor 23. The reading can take place in real time as the measurement occurs through the body of the cartridge. In FIG. 5 the configuration can be used for obtaining optical read outs from a cartridge 1. The example includes a sensor 23, an LED 24 and emission filter 25.

[0107] In some examples, an optical system is used to measure product yield. The optical measurement may directly take place through the body of a removable collection tube, cartridge or part thereof without having to move the sample outside the cartridge for measurement purposes.

[0108] Increasing contact between the components of a cell-free protein expression reaction and hydrophobic surfaces (e.g., cartridge body or air) can enhance synthesis yields. Thus, any of the apparatuses (device and cartridges) described herein may be configured to include bubbling of gases through the reaction to achieve higher productivity. To illustrate this, deGFP fluorescent protein was expressed in a 384-well plate, in a shaker incubator at 300 rpm and using the automated device and cartridge system described here. The reaction yields within the cartridge were 83% and 31% higher than those in the 384-well plate and shaker incubator respectively, as illustrated in FIG. 6. FIG. 6 illustrates an example of yield comparisons among various cell-free protein expression methods. deGFP fluorescent protein was expressed in a 384-well plate, in a shaker incubator at 300 rpm and within one embodiment of the device and cartridge system. The data suggests that expression in the device and cartridge system can increase the yields by 83% and 31% compared to 384-well plate and shaker incubator respectively.

[0109] Any of the devices described herein may control temperature, which may also enhance the yield of the biomolecule significantly. Historically, cell-free protein expression reactions have been carried out at temperatures between 29-37 °C. Experiments carried out at different temperatures indicate that the optimal reaction temperature for cell-free protein expression reactions may be within specific ranges. For example, for some polypeptides the device may optimally be maintained between 22-28 °C and that the yields peak at about 26.5 °C. Accordingly, in some embodiments the cell-free protein expression reactions within the device are optimally carried out at temperatures within a narrow range (e.g., of +/- 0.5 degrees C, +/- 1 degrees C, +/- 1.5 degrees C, +/- 2 degrees C, etc.), such as around a temperature selected between the 22-28 °C range (FIG. 7). FIG. 7 illustrates an example of protein expression yields for deGFP at various temperatures. The optimal range appears to be between 22-28 °C and that the yields peak at about 26.5 °C. This optimal range may be determined for a particular biomolecule and the range or temperature may be set (manually or automatically) in the device so that the device may maintain the reaction chamber within the optimal temperature range (e.g., maintaining the temperature while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C)).

[0110] In other embodiments, the connection between the device and the cartridge is further enhanced using a magnetic connector, mechanical clamp, luer lock or a lid. FIG. 8 illustrates an example of a mechanical clamp actuated by a stepper motor that connects the device to the cartridge and enhances the seal between the two.

[0111] FIG. 4 is another example of a portable device 22 which may hold and operate on a cartridge 1 that is loaded onto it. In FIG. 4, the device is shown with a cover that opens into a seat (shown holding cartridge 1 in an upright position). The device also includes inputs (e.g., one or more buttons) and a display screen, which may be touchscreen. The example cartridge shown includes a twist cap 9 over the input port into which the nucleic acid substrate may be added. [0112] FIGS. 8 A and 8B show another example of a device 800 including a housing with an opening into a seating region 807. The device also includes a touchscreen 805 providing input/output into the device. FIG. 8B shows the device of FIG. 8A with the upper cover removed. In FIG. 8B the seating region 807 is shown and includes a mechanical clamp 815 that may seal the pneumatic ports of the cartridge to an interface 809 for coupling each port to manifold 817. The device also includes a power supply and controller 821, which may include one or more processors and control circuitry for controlling the device, including controlling the pressure manifold (valves, etc.). One or more pumps 819 (e.g., air pumps) may be included to provide positive and/or negative pressure. The device may also include one or more sensors and/or heaters that may also be coupled with and controlled by the controller.

[0113] FIG. 9 shows an example of a portion of a device similar to that shown in FIG. 8A- 8B, and in particular, the mechanical clamp 915; in this example the clamp is actuated by a stepper motor that connects the device to the cartridge 1 and enhances the seal between the two. In FIG. 9, the cartridge 1 is shown loaded into the device. This cartridge example is similar to that shown in FIGS. 10A and 10B. In this example, the cartridge includes a single reaction chamber 931, a binding buffer chamber 933, a wash buffer chamber 935, an elution buffer chamber 937, a purification chamber 939, a collection chamber 941 and a waste chamber 943. Each of these chambers is coupled at its top to an outlet that is fluidically coupled to a liquid trap that also connects to a pneumatic line that may be used to apply pressure from the top of the chambers (each is independently addressable from the top of the chamber in this example).

[0114] For example, FIG. 10A illustrates a first example of a disposable cartridge 1 with an input chamber 26 (e.g., reaction chamber), one receiving chamber 27 (e.g., purification chamber) and one hydrophobic frit 28. The hydrophobic frit is an example of the general use of a porous matrix that may be positioned at a bottom of a chamber. The porous matrix allows entry of gases into the chamber (to provide bubbling) and inhibits liquid from exiting the chamber at low chamber pressures, while allowing liquid to escape the chamber at higher chamber pressures. [0115] For example, FIG. 10B shows a cartridge 1 similar to that shown in FIG. 9, and also includes a single reaction chamber 931, a binding buffer chamber 933, a wash buffer chamber 935, an elution buffer chamber 937, a purification chamber 939, a collection chamber 941 and a waste chamber 943. In this example, the cartridge may include a substrate for capturing the synthesized biomolecule, such as beads for chromatography or ion-exchange purification, within the purification chamber 939. The top of each chamber may include a closeable/lockable cap (shown here as a threaded region) that may be sealed closed. The opening into the reaction chamber 931 may be open or allowed to open/close (and be sealed closed) so that the nucleic acid substrate or other reagents may be added. In FIG. 10B, each chamber is coupled at the top end to an individual pneumatic line, after passing through a liquid trap 1010. The liquid trap may prevent fluid from passing from the chamber into the pneumatic line 1011 that connects to the pneumatic ports 1018 on the bottom end region of the cartridge, where they may mate with a corresponding region in the device to pneumatically connect to the device (e.g., to a pneumatic manifold in the device). In FIG. 10B the bottom ends of each chamber may include a connection to a common or shared channel 1014. Fluid may be passed through the shared channel between the different chambers by the control of a pressure applied from another pneumatic port.

[0116] In FIG. 10B, the cartridge also includes a cartridge housing 1031 configured to be held within the device in an upright position. The housing may be configured (e.g., keyed) so that it can only be inserted in a particular orientation (upright and with the front/back configuration maintained) to prevent confusion.

[0117] The cartridge shown in FIG. 10B may also include a plurality of pneumatically actuated valves. In FIG. 10B the valve seats 1012 are shown. The valve may be a pneumatically actuated valve (an example of which is shown in FIG. 13). In this example each chamber is coupled to the shared channel 1014 through a valve on the cartridge. Thus, each chamber is connected to the shared channel through a pneumatically actuated valve. The shared channel may also be separately valved and may connect to one of the pneumatic input ports 1018 on the cartridge housing.

[0118] Some of the pneumatic input ports may therefore connect to each chamber (in this example at the top of each chamber) and the shared channel. A second subset of the pneumatic input ports may also connect to each valve (not shown in FIG. 10B). This allows the controller to pneumatically actuate the valves of the pneumatically actuated valves in the cartridge in addition to applying pressure to directly move the fluid between the chambers along the shared channel. In FIG. 10B the reaction chamber 931 also includes a nucleic acid substrate input port 1034 at a top of the cartridge in communication with the reaction chamber. The nucleic acid substrate input port may include a cover or lid (not shown).

[0119] Any of these cartridges may also be configured to include a porous matrix that is configured to act as both a sparging outlet (to form bubbles within a chamber) as well at passive valve that limits or prevents the flow of fluid out of the chamber into, e.g., the shared channel. For example in FIG. 10B a porous matrix 1016 may be positioned between the shared channel 1014 and the purification chamber 931; a second porous matrix 1020 may be positioned between the purification chamber 939 and the shared channel 1014.

[0120] FIG. 11 illustrates one example of a trap (liquid trap) 1010 as mentioned above. The liquid trap may fluidly connect an outlet at the top of the chamber to the pneumatic line 1011 and may be configured so that any fluid that passed from the outlet (e.g., including foam due to sparging) may be trapped in the stomach-like chamber 1104 so that gas may instead communicate between the outlet from the chamber 1102 and the pneumatic line inlet 1106. Other fluid traps may be used. In general, these traps provide an offset capture region (stomach region in FIG. 11) that is “lower” than the inlet from the chamber 1102 and the outlet 1106 to the pneumatic line.

[0121] FIG. 12 shows a schematic illustration of a cartridge similar to that shown in FIGS. 10A-10B, including examples of possible chamber volumes beneath each chamber; these volumes are intended as examples only; other, larger or smaller, values may be used. In this example, three solution chambers (Solution A 1201, Solution B 1203, Solution C 1205) are shown, and are all connected, along with the other chambers (Bind 1207, Wash 1209, Elute 1211) and column (Column 1213) to a common shared channel 1231. Each chamber and the column are connected through a valve 1221, which may be a pneumatic valve that is opened/closed by pneumatic force. In FIG. 12, the valves also include a set of redundant valves (circled) 1219 along the shared channel. The schematic also includes a waste chamber 1215 and a product chamber 1217 to receive the synthesized biomolecule. Each chamber is also connected to a pair of valves 1223 (though in some examples a single valve may be used) that connects the channels to atmosphere 1227 and a source of pressure 1225 (e.g., pneumatic line). This allows the controller of the device to pressurize each chamber to control movement of fluid between the chambers.

[0122] FIG. 13 shows one example of a portion of a pneumatic valve that may be used. In this example, the pneumatic valve includes a pneumatic input that may drive movement of a membrane or diaphragm (not shown) seating within the circular (though other shapes may be used) valve seating chamber 1307. The membrane or diaphragm may be driven down by applying positive pressure from the pneumatic input (e.g., from the top), which may close the valve, by blocking the fluid connection between the first input 1303 and the second input 1305. [0123] In operation, a device may control the operation of the cartridge pneumatically by applying air to: open/closed the valve, to pressurize the chambers and/or to sparge within a chamber. For example, FIG. 14 schematically illustrates the operation of a device such as the ones described above. In this example, the cartridge may contain all the reaction components (except for nucleic acid substrate). Components may be in dry form (except for resuspension buffer) and/or may be frozen in the cartridge. The device may be turned on 1403, and a cartridge 1401 placed into the device as shown 1405. The device may push against the cartridge to create a seal around the pneumatic ports, between the ports and the pneumatic connection to the manifold within the device. The template material may then be added into the cartridge 1407, e.g., using pipettor or syringe. As mentioned, the components of the reaction may be preloaded into the cartridge. Alternatively, the cartridge may be loaded, e.g., by opening the input caps on the top of the channels and pipetting in the reaction mixture, purification buffers and substrate (e.g., beads) into the relevant chambers. The caps may be closed to maintain seal. The process may then be started 1409. In some embodiments, the device may insert water, a resuspension mixture or other inputs into the cartridge from a reservoir attached to it.

[0124] During operation of the device 1411, the device may automatically or semi- automatically proceed to each step of the process; semi-automatically may include prompting a user (e.g., on an output device, such as a screen) to complete a particular step or portion of a step. The device may be configured to ensure that the relevant valves are readjusted to default after each step.

[0125] For example, the device may begin the reaction by sparging (e.g., bubbling gas, such as air) through the reaction mixture in the reaction chamber. This may be done, for example, for about 4-16 hours. Sparging may be automatically performed (and regulated) by the device controller. With reference to FIG. 10B, for example, the controller may pneumatically open the valve connecting the shared line 1014 to the pneumatic port 1018, so that positive pressure (air) may be applied to pressurize the shared channel. All of the other valves into the chambers may initially be closed. The controller may then open the reaction chamber 931 port to atmosphere 1034 (at the top of the chamber) and may open the valve to the reaction chamber. Pressurized air may then pass through the porous matrix at a bottom of the chamber to sparge the gas through the reaction mixture. The controller may monitor the temperature and adjust the temperature within the reaction chamber by applying thermal energy to keep the temperature within a target range. The controller may monitor the bubbling (sparging) directly, e.g., optically, or indirectly by monitoring pressure applied, e.g., to the shared channel.

[0126] At the end of the reaction time, the device may then prepare the purification chamber to receive the reaction mixture. Thus, the substrate for capturing the synthesized biomolecule may be equilibrated. The device may close the valve into the reaction chamber and may open the valve from the shared channel to the purification chamber 939. As in all of the valves, the controller may open/close them pneumatically, e.g., by applying pressurized air (or in some cases vacuum) to operate the valve. The controller may also open the valve between the binding buffer chamber 933 and the shared channel, and may open the purification chamber port (at the top of the channel) to atmosphere. The controller may pressurize the binding buffer channel port by applying positive pressure from the pneumatic line entering the top of the binding buffer chamber. Optionally, the device may pressurize the shared channel port. Once all of the fluid from the binding buffer chamber has been passed into the purification chamber, the valves may again be all shut. Equilibration of the substrate for capturing the synthesized biomolecule (e.g., beads in some examples) may be completed by transferring the binding buffer from the purification chamber to waste. For example, the device may open the valve(s) connecting the shared channel to the waste chamber and may also open the valve to the purification chamber. The device may pressurize the purification chamber by applying pressure from the pneumatic line entering the top of the chamber. In examples in which the purification chamber includes a porous matrix at the bottom of the purification chamber (e.g., acting as a frit), the controller may ensure the applied pressure exceeds the force threshold for driving fluid though the porous matrix. The waste chamber may be vented to atmosphere or negative pressure applied. The shared channel may optionally be pressurized. After the equilibration fluid is removed, the valves may again be shut.

[0127] The controller may then control the binding by moving the fluid from the reaction chamber 931 to the purification chamber 939. The valves between the purification chamber and the shared channel and the reaction chamber and the shared channel may be opened, and the purification chamber may be open to atmosphere at the top. The reaction chamber may be pressurized (e.g., by applying gas into the top from the pneumatic line specific to the reaction chamber). Optionally, the shared channel may be pressurized. Once the fluid from the reaction mixture is driven up into the purification chamber, air may continue to be driven into the purification chamber to sparge into the purification chamber. This may allow additional mixing and binding. Once binding is complete, the flow-through may be removed. The valves may again be closed, and the valves connecting the shared channel to the waste may be opened (and the waste channel vented to atmosphere), as well as the valve connecting the purification chamber to the shared channel. The purification channel may be pressurized as mentioned above, to drive the flow-through into the waste chamber. Optionally, the shared channel may be pressurized. Once the flow-through is removed, the valves may be shut.

[0128] The substrate for capturing the synthesized biomolecule (now loaded with the synthesized biomolecule) may then be washed. The valves connecting the purification chamber to the shared channel and the valve connecting the wash buffer channel 935 to the shared channel may be opened, the purification chamber vented to atmosphere and the wash buffer chamber may be pressurized. Optionally the shared channel may be pressurized. After transfer of the solution into the purification chamber, air may be continued to be applied to the purification chamber through the bottom (frit) to sparge into the purification chamber for additional mixing. [0129] The wash solution may be removed and sent to the waste container 943, as described above, e.g., by opening just the valves connecting the waste to the shared channel and the valve connecting the purification chamber to the shared channel, pressurizing the purification channel and venting the waste chamber to atmosphere (and optionally pressurizing the shared channel). [0130] The synthesized biomolecule material may then be eluted from the substrate in the purification chamber. The valve between the elution chamber and the common channel and the valve between the purification chamber and the common channel may be opened (all other valves may have already been closed) and the elution chamber may be pressurized while the purification chamber is vented to atmosphere. After transfer of the elution buffer into the purification chamber, air may be sparged into purification chamber for some time as well, optionally, to allow further mixing. Finally, the product may be collected by closing the valves, then opening the valves between the shared channel and the collection chamber 941, opening the valve between the purification chamber and the shared channel, and pressurizing the purification channel while venting the collection chamber to atmosphere. After transfer of the elution buffer to the collection chamber, all of the valves may be closed.

[0131] Once the run is complete, the cartridge may be removed 1412 and the synthesized biomolecule material 1413 may be removed from the cartridge (from the collection chamber) and the waste 1415 (including the spent cartridge) may be disposed.

[0132] FIGS. 15A and 15B illustrate other examples of a devices and cartridge as described herein. In this example, the cartridge includes a single chamber in which the reaction mix and the purification steps may take place. Solutions (binding, washing, elution) may be pipetted manually into the top of the chamber. The device 22 may pneumatically apply positive pressure to bubble (sparge) and negative pressure to remove liquid through the passive valve of a porous matrix at a bottom of the chamber (e.g., hydrophobic frit).

[0133] In FIG. 15A, a porous matrix at a bottom of the chamber is configured as a hydrophobic frit 28, which may act as a passive (pressure-responsive) valve, to separate an input chamber 26 from a receiving chamber 27. The input chamber can act as a reaction or purification chamber, thus forming part or the totality of the reaction module, purification module or both. The receiving module can act as a collection or waste chamber and in some embodiments may be swapped out depending on whether synthesis, waste collection or elution is being carried out. During synthesis or purification, the hydrophobic frit allows for bubbling gases through the reaction for mixing or supply of oxygen, while restricting the flow of the liquid to other chambers. During purification, the frit limits the movement of chromatography components (e.g., NTA beads), enabling separation of waste from eluted products. The pressure differential can be controlled, thus allowing sparging and mixing during synthesis and binding by moving gases into the chamber, while enabling transfer of liquids to receiving chambers during wash and elution steps. While a column may be used for the purification step, the chamber and a frit may be used for batch purification.

[0134] FIGS. 15B-15D illustrate an example of a device for use with the combined reaction/purification cartridge. In FIG. 15B the top portion of the device includes a housing

1501. The device includes a vacuum/pressure outlet line 1503 from a manifold (shown in FIG.

15C), which connects to an air chamber 1511 in the coupling portion of the device. The cartridge 1551 is coupled to the coupler by a luer lock 1517. The cartridge in this example also includes a hydrophobic frit 1515 at the bottom of the chamber. A collection tube 1505 is coupled beneath the air chamber of the coupling portion to receive the outlet of the hydrophobic frit. The collection chamber may be sealed to the device and may include a gasket 1509 and may receive a collection hose 1507 that allows the device to apply negative pressure within the collection chamber. The device in this example also illustrates a temperature sensor, shown as a noncontact (e.g., IR) temperature sensor 1519. Any of the devices described herein may include such a temperature sensor. The device also includes an IR heater (bulb 1513).

[0135] FIG. 15C shows an example of the bottom portion of the device, including valves 1521, 1523, 1525 that couple to a manifold 1529. The manifold receives input (vacuum inlet 1531) from a source of negative pressure (e.g., vacuum side of a pump), and input (pressure inlet 1533) from a source of positive pressure (e.g., pressure side of a pump). The Manifold also includes a pressure sensor port 1535, and a pressure/vacuum outlet 1537. FIG. 15D shows a top view of the device of FIG. 15C (with the valves removed and made partially transparent), also showing the filters that may be included, such as the pressure filter 1541, vacuum filter 1543 and vent filter 1545. The device may also include one or more pumps (for positive and/or negative pressure). The manifold may switch between suction (negative pressure) and positive pressure, and the sensor port may allow connection to a pressure sensor for detecting the applied pressure. The output from the manifold may be provided to the outlet line.

[0136] In general, in the device shown in FIG. 15A-15D the device may couple with a cartridge and may apply positive pressure to bubble (sparge) though a liquid in the chamber of the cartridge using the porous matrix as a sparge outlet and may apply negative pressure to draw fluid through the porous matrix (e.g., hydrophobic frit).

[0137] FIG. 16 schematically illustrates an example of schematics for an electronic board within the device. Any of the devices described herein may include a controller that receives input from sensors and controls. The device also provides output to one or more outputs and controls the operation of a pneumatic manifold. For example, in FIG. 16 the controller includes one or more processor (e.g., CPU 1603) that may include control circuitry 1625, 1615 for controlling operation of the pneumatic controls (e.g., manifold, pumps, etc.). The controller may receive inputs from user inputs (e.g., joystick 1621) as well as sensors (bubbling sensor 1611, pressure sensor 1613, IR sensor 1609), and may provide user output (e.g., screen 1623). The controller may also regulate the power to the device (power supply 1631, power in 1627) or separate power regulation circuitry may be included.

[0138] FIGS. 17A-17C illustrate the operation of the example device shown in FIGS. 15A- 15D, and specially, illustrate the operation of the manifold, as viewed through the partially transparent view shown in FIG. 15D. A single vacuum pump may be used to apply positive or negative pressure by attaching different ends of the vacuum pump to the manifold, which may switch between positive or negative pressure output from the pressure/vacuum outlet that is connected to the outlet line. For example, in a first state, pressure may be applied to the cartridge, e.g., to sparge, as when a reaction mixture is present. In this state, shown in FIG. 17A, the pump is applying positive pressure through manifold from the pressure inlet and out of the pressure/vacuum outlet to the cartridge. The manifold valves may be selected so that the vacuum vent to atmosphere and the pressure output is directed to outlet, as shown. The power applied to the pump may determine the intensity of the pressure applied. In FIG. 17B, the second state, a vacuum state, is shown. In this state the valves are set, e.g., by the controller, so that the vacuum side of the pump is directed to the pressure/vacuum outlet and the pressure side is vented to atmosphere. Finally, FIG. 17C illustrates a third state in which both pressure and vacuum are vented to atmosphere and no pressure (positive or negative) is directed to the pressure/vacuum. [0139] FIGS. 18A and 18B illustrate another example of a cartridge 1800. In FIG. 18A the disposable cartridge includes a chamber 1802 that is configured to be held within a device for cell-free, in vitro synthesis of biomolecules in an upright position. This chamber includes an open or openable top 1804 and a bottom 1805. In this example the cartridge includes a single chamber that is used for both the reaction chamber and for purifying the synthesized biomolecule. The cartridge also includes a hydrophobic porous matrix 1807 (e.g., configured as a hydrophobic frit) at the bottom of the disposable cartridge. In this example a channel 1809 extends from the hydrophobic porous matrix and is configured to extend into a second chamber 1811. The second chamber has a threaded connector 1815 at a top region that is configured to removably couple to the first chamber indirectly by coupling to an air chamber 1813 so that the channel 1809 extends into the second chamber when the second chamber is coupled to the air chamber and therefore to the first chamber, as shown in FIG. 18B. The air connector may also include an inlet on the bottom face that is placed in fluid connection with the second chamber when it is sealed onto the air chamber, so that pressure or vacuum can be applied to positively or negatively pressurize the second chamber and therefore drive sparging or suction. The second chamber may also include a cap 1822 that may fit onto the threads for sealing it when it is removed from the air chamber portion of the cartridge. As shown in FIG. 18B, the assembled cartridge may also include a connector (e.g., a sealing luer-type connector 1823) that couples the connector to the device, as shown.

[0140] FIGS. 19A-19B illustrate another example of a cartridge similar to that shown in

FIGS. 18A-18B, in which the air connector portion 1913 does not form a part of the cartridge

1900 but instead is part of the coupler of the device. The cartridge thus incudes a first chamber 1902, include a porous matrix 1907 (hydrophobic frit) that couples to the device, as shown in FIG. 19B. The second chamber 1911 (having a removable cap 1922) separately couples to the device, as shown.

[0141] FIGS. 20A-20L illustrate the operation of a device such as the one shown in FIGS. 15A-15D using a cartridge similar to that shown in FIGS. 19A-19B. The device 2000 may be connected to a power supply, such as a wall outlet (e.g., or an adapter for a 12V power source in some examples), as shown in FIG. 20A. The device may be turned on, and a cartridge 2001 (FIG. 20B) may be coupled to the top of the coupler region of the device (e.g., coupling to air chamber, as shown in FIG. 20C). A second chamber 2002 is coupled to the underside of the coupling region (air connector) 2004 to receive the extending channel as shown in FIG. 20D. [0142] FIG. 20E illustrates the addition of the reaction components 2009, forming the reaction mixture, into the cartridge. The porous matrix 2006 (e.g., shown in this example as a hydrophobic frit) prevents the contents from draining into the lower chamber. The control 2011 on the device may then be used to set the bubbling pressure (positive pressure) for sparging, as shown in FIG. 20F. Sparging is occurring, as shown in FIG. 20G, illustrating bubbles 2013 of gas passing through the reaction mixture. The device may maintain the level of bubbles and may prevent over-bubbling, which may result in foam and damage to the reaction components. A pressure sensor and/or an optical sensor are used to help regulate the rate of bubbling. For example, a pressure sensor may monitor pressure within the second chamber (by measuring pressure in the pressure manifold).

[0143] FIG. 20H illustrates the user setting a target temperature for the reaction mixture (e.g., 24C). The device may include temperature sensor, such as a non-contact thermal sensor as shown in FIG. 201, showing an infrared heat sensor 2021. The same device also includes an infrared heat source 2023 (bulb). FIG. 20J illustrates the application of heat by the infrared heat source. Infrared heating and sensing allow cartridges of various sizes and shapes to be used. There is little or no overlap between the IR source emission and the IR sensor’s active range, so they can operate at the same time. Once the synthesis reaction is completed, purification may be performed by adding a substrate 2029 that specifically binds the synthetic biomolecule, as shown in FIG. 20K. Sparging may be applied to mix. This may help the synthetic biomolecule to bind to the substrate (e.g., purification beads). In this example, beads can be added dry, or may be equilibrated first, e.g., with binding buffer. Once binding is complete, the device may be set to vacuum mode, as shown in FIG. 20L, so that the flow through material may be removed from the chamber into the lower waste chamber. In this example, the hydrophobic frit may now keep the substrate (e.g., beads) in the first chamber, while the unbound components (i.e. flow-through) are transferred into the second chamber 2031 for analysis or as waste. The second, bottom, chamber (cartridge) may be removed and/or changed during this procedure.

[0144] An optional step would be to change the bottom cartridge(/collection tube) at this step. Wash solution may be added to the upper chamber and, optionally, sparging may be applied to help mix. The device may again be set to vacuum, drawing the wash solution through the frit and into the waste. The vacuum may be again switched off, and the second, lower, tube changed to a collection tube, as illustrated in FIG. 2 IL. Elution solution may be applied to the upper chamber of the cartridge (similar to 20K) and mixed by applying positive pressure to bubble (sparge). The device may then apply a vacuum into the lower collection tube to draw the eluted product into the collection tube, which may then be removed and capped or sealed.

[0145] Any of the methods (including user interfaces) described herein may be implemented as software, hardware or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions capable of being executed by a processor (e.g., computer, tablet, smartphone, etc.), that when executed by the processor causes the processor to perform any of the steps, including but not limited to: displaying, communicating with the user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.

[0146] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0147] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature. [0148] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/" .

[0149] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the 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 a device in the figures is inverted, elements described as "under" or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0150] Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0151] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0152] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive, and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.

[0153] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0154] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by 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 altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0155] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose 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 above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

What is claimed is:

1. A method of cell-free, in vitro synthesis of a biomolecule, the method comprising: adding a nucleic acid substrate to a chamber of a disposable cartridge coupled to a device for in vitro synthesis of biomolecules comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; sparging a gas through the reaction mixture within the chamber; and combining, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, flow-through, washing and eluting the synthesized biomolecule material within the disposable cartridge, wherein the one or more processors pneumatically controls the transfer of liquid within the disposable cartridge so that liquid from the disposable cartridge does not contact a pump or valve within the device for in vitro synthesis of biomolecules.

2. The method of claim 1, wherein sparging the gas through the reaction mixture comprises applying gas through a porous matrix, wherein the porous matrix allows entry of gases into the chamber and inhibits liquid from exiting the chamber at low chamber pressures, while allowing liquid to escape the chamber at higher chamber pressures, relative to the pressure outside of the chamber.

3. The method of claim 2, wherein the porous matrix comprises a hydrophobic substrate.

4. The method of claim 3, wherein the porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE).

5. The method of claim 1, further comprising performing non-contact temperature sensing of the temperature within the chamber using an infrared heat sensor.

6. The method of claim 5, further comprising controlling temperature within the chamber at a temperature between 16 and 37 degrees Celsius (°C).

7. The method of claim 5, further comprising controlling temperature within the chamber at a temperature between 21 and 28 degrees Celsius (°C). The method of claim 5, wherein controlling temperature comprises using a non-contact heater. The method of claim 5, further comprising maintaining temperature within the chamber while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C). The method of claim 1, further comprising coupling the disposable cartridge to the device for in vitro synthesis of biomolecules. The method of claim 1, wherein sparging is controlled through the reaction mixture by controlling, using one or more processors of the device, pressure from one or more of pressure sources. The method of claim 1, wherein controlling sparging comprises adjusting power to an air pump generating the sparging to maintain a release of air bubbles through the reaction mixture within a preset range. The method of claim 10, further comprising setting the preset range. The method of claim 1, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises pneumatically controlling, by the device for in vitro synthesis of the biomolecule, transfer of the reaction mixture to a second chamber comprising the substrate for capturing the synthesized biomolecule. The method of claim 1, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises controlling, by the device for in vitro synthesis of the biomolecule, the application of bubbles through the substrate for capturing the synthesized biomolecule. The method of claim 1, wherein binding, flow-through, washing and eluting the synthesized biomolecule material within the disposable cartridge comprises applying positive or negative pressure to drive the synthesized biomolecule material through a hydrophobic porous matrix while retaining the chromatography or ion-exchange substrate. The method of claim 1, further comprising applying pneumatic pressure from the device for in vitro synthesis of biomolecules to the disposable cartridge to perform each of: opening and/or closing valves in the disposable cartridge, driving fluid between a plurality of chambers in the disposable cartridge, and applying gas into a porous matrix to cause sparging through the reaction mixture. The method of claim 1, wherein combining the reaction mixture with a substrate for capturing the synthesized biomolecule comprises manually adding the substrate for capturing the synthesized biomolecule to the reaction mixture. The method of claim 1, wherein pneumatically controlling fluid transfer comprises coordinating, by the device for in vitro synthesis, the application of pressure to open and/or close valves in the disposable cartridge and the application of pressure to drive fluid through one or more channels in the disposable cartridge. The method of claim 1, wherein pneumatically controlling binding, washing or elution comprises applying bubbles through the substrate for capturing the synthesized biomolecule. The method of claim 1, wherein the biomolecule comprises a polypeptide. The method of claim 1, wherein the enzymes for synthesizing the biomolecule comprise one or more enzymes for transcription and/or translation. The method of claim 1, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification. A method of cell-free, in vitro synthesis of biomolecules, the method comprising: adding a nucleic acid substrate to a chamber of a disposable cartridge coupled to a device for in vitro synthesis comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; applying a gas, under the control of the one or more processors, through a porous matrix to sparge the gas through the reaction mixture within the chamber; and combining, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by removing unbound components, washing, and eluting or synthesized biomolecule material within the disposable cartridge, wherein the one or more processors applies pressure to drive liquid through the porous matrix for one or more of removing unbound components, washing and eluting.

25. The method of claim 24, wherein the porous matrix allows entry of gases into the chamber and inhibits passing of liquid through the porous matrix at low pressures, while allowing passing of liquid through the porous matrix at higher pressures, relative to the pressure beyond the porous matrix.

26. The method of claim 24, wherein the porous matrix comprises a hydrophobic substrate.

27. The method of claim 24, wherein the porous matrix comprises one or more of: Poly ether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE).

28. The method of claim 24, wherein the porous matrix comprises a hydrophobic frit.

29. The method of claim 24, wherein the nucleic acid substrate is combined with the one or more enzymes for synthesizing the biomolecule to form the reaction mixture prior to adding to the chamber.

30. The method of claim 24, further comprising performing non-contact temperature sensing of the temperature within the chamber using an infrared heat sensor.

31. The method of claim 30, further comprising controlling the temperature within the chamber using a non-contact heater.

32. The method of claim 30, further comprising controlling temperature within the chamber at a temperature between 16 and 37 degrees Celsius (°C).

33. The method of claim 30, further comprising controlling temperature within the chamber at a temperature between 21 and 28 degrees Celsius (°C).

34. The method of claim 30, further comprising maintaining temperature within the chamber while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C).

35. The method of claim 24, further comprising coupling the disposable cartridge to the device for in vitro synthesis of biomolecules.

36. The method of claim 24, wherein applying gas comprises adjusting pressure from one or more pressure sources of the device for in vitro synthesis of biomolecules.

37. The method of claim 24, wherein applying gas comprises adjusting power to an air pump of the device for in vitro synthesis of biomolecules to maintain sparging through the reaction mixture within a preset range.

38. The method of claim 24, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises pneumatically controlling, by the device for in vitro synthesis of the biomolecule, transfer of the reaction mixture to a second chamber comprising the substrate for capturing the synthesized biomolecule.

39. The method of claim 24, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises controlling, by the device for in vitro synthesis of the biomolecule, sparging through the substrate for capturing the synthesized biomolecule.

40. The method of claim 24, further comprising applying pneumatic pressure from the device for in vitro synthesis of biomolecules to the disposable cartridge to perform each of: opening and/or closing valves in the disposable cartridge, driving fluid between a plurality of chambers in the disposable cartridge, and applying gas through the porous matrix to sparge the gas through the reaction mixture.

41. The method of claim 24, wherein combining the reaction mixture with a substrate for capturing the synthesized biomolecule comprises pneumatically driving, by the device for in vitro synthesis, the reaction mixture into a second chamber of the disposable cartridge containing the substrate for capturing the synthesized biomolecule.

42. The method of claim 24, wherein pneumatically controlling fluid transfer comprises coordinating, by the device for in vitro synthesis, the application of pressure to open and/or close valves in the disposable cartridge and the application of pressure to drive fluid through one or more channels in the disposable cartridge.

43. The method of claim 24, wherein the biomolecule comprises a polypeptide.

44. The method of claim 24, wherein the enzymes for synthesizing the biomolecule comprise one or more enzymes for transcription and/or translation. The method of claim 24, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification. A method of cell-free, in vitro synthesis of biomolecules, the method comprising: adding a nucleic acid substrate to a chamber of a disposable cartridge that can be connected to a device for in vitro synthesis comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; introducing the reaction mixture into a porous matrix or micro-scale channels within the disposable cartridge to increase surface interactions; controlling the temperature of the reaction mixture within the disposable cartridge; combining, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to enable purification; and pneumatically controlling, by the one or more controllers of device binding, flowthrough, washing or elution to purify a synthesized biomolecule material. The method of claim 46, wherein introducing the reaction mixture into a porous matrix or micro-scale channels within the disposable cartridge comprises introducing the reaction mixture into a microscale channel comprising a porous matrix. The method of claim 47, wherein the porous matrix comprises a hydrophobic substrate. The method of claim 47, wherein the porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE). The method of claim 46, wherein introducing the reaction mixture into a porous matrix or micro-scale channels within the disposable cartridge comprises introducing the reaction mixture into a microscale channel comprising a serpentine channel. The method of claim 46, further comprising applying a gas through the reaction mixture within the disposable cartridge. The method of claim 46, wherein the one or more processors pneumatically controls the transfer of liquid within the disposable cartridge so that liquid from the disposable cartridge does not contact a pump or valve within the device for in vitro synthesis of biomolecules. The method of claim 46, wherein the nucleic acid substrate is combined with the one or more enzymes for synthesizing the biomolecule to form the reaction mixture prior to adding to the chamber. The method of claim 46, wherein controlling the temperature comprises using a noncontact heater. The method of claim 46, further comprising performing non-contact temperature sensing of the temperature within the chamber using an infrared heat sensor. The method of claim 55, further comprising maintaining temperature within the chamber while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C). The method of claim 46, further comprising coupling the disposable cartridge to the device for in vitro synthesis of biomolecules. The method of claim 46, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises pneumatically controlling, by the device for in vitro synthesis of the biomolecule, transfer of the reaction mixture to a second chamber comprising the substrate for capturing the synthesized biomolecule. The method of claim 46, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises controlling, by the device for in vitro synthesis of the biomolecule, sparging through the substrate for capturing the synthesized biomolecule. The method of claim 46, further comprising applying pneumatic pressure from the device for in vitro synthesis of biomolecules to the disposable cartridge to perform each of: opening and/or closing valves in the disposable cartridge, driving fluid between a plurality of chambers in the disposable cartridge, and applying gas to sparge the gas through the reaction mixture. The method of claim 46, wherein combining the reaction mixture with a substrate for capturing the synthesized biomolecule comprises pneumatically driving, by the device for in vitro synthesis, the reaction mixture into a second chamber of the disposable cartridge containing the substrate for capturing the synthesized biomolecule.

62. The method of claim 46, wherein pneumatically controlling fluid transfer comprises coordinating, by the device for in vitro synthesis, the application of pressure to open and/or close valves in the disposable cartridge and the application of pressure to drive fluid through one or more channels in the disposable cartridge.

63. The method of claim 46, wherein the biomolecule comprises a polypeptide.

64. The method of claim 46, wherein the enzymes for synthesizing the biomolecule comprise one or more enzymes for transcription and/or translation.

65. The method of claim 46, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification.

66. A method of cell-free, in vitro synthesis of biomolecules, the method comprising: adding a nucleic acid substrate to a disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within a chamber of the disposable cartridge; controlling, using one or more processors of a device for in vitro synthesis of biomolecules, application of bubbles through the reaction mixture within the chamber for 2 or more hours, while controlling the temperature of the reaction mixture within the chamber; combining, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, washing and eluting the synthesized biomolecule material within the disposable cartridge, wherein the one or more controllers pneumatically controls the transfer of fluid within the disposable cartridge for performing binding, washing or eluting.

67. The method of claim 66, wherein controlling the application of bubbles through the reaction mixture comprises applying gas through a porous matrix, wherein the porous matrix allows entry of gases into the chamber and inhibits liquid from exiting the chamber at low chamber pressures, while allowing liquid to escape the chamber at higher chamber pressures, relative to the pressure outside of the chamber.

68. The method of claim 67, wherein the porous matrix comprises a hydrophobic substrate.

69. The method of claim 68, wherein the porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE).

70. The method of claim 67, wherein the porous matrix comprises a hydrophobic frit.

71. The method of claim 66, wherein controlling the temperature comprises using a noncontact heater.

72. The method of claim 66, further comprising performing non-contact temperature sensing of the temperature within the chamber using an infrared heat sensor.

73. The method of claim 66, further comprising controlling temperature within the chamber at a temperature between 16 and 37 degrees Celsius (°C).

74. The method of claim 66, further comprising controlling temperature within the chamber at a temperature between 21 and 28 degrees Celsius (°C).

75. The method of claim 66, further comprising maintaining temperature within the chamber while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C).

76. The method of claim 66, further comprising coupling the disposable cartridge to the device for in vitro synthesis of biomolecules.

77. The method of claim 66, wherein controlling sparging through the reaction mixture by controlling, using one or more processors of the device, pressure from one or more of pressure sources.

78. The method of claim 66, wherein controlling the application of bubbles through the reaction mixture comprises adjusting power to an air pump generating the bubbles to maintain a release of air bubbles through the reaction mixture within a preset range.

79. The method of claim 66, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises pneumatically controlling, by the device for in vitro synthesis of the biomolecule, transfer of the reaction mixture to a second chamber comprising the substrate for capturing the synthesized biomolecule. The method of claim 66, wherein combining the reaction mixture with the substrate for capturing the synthesized biomolecule comprises controlling, by the device for in vitro synthesis of the biomolecule, the application of bubbles through the substrate for capturing the synthesized biomolecule. The method of claim 66, further comprising applying pneumatic pressure from the device for in vitro synthesis of biomolecules to the disposable cartridge to perform each of: opening and/or closing valves in the disposable cartridge, driving fluid between a plurality of chambers in the disposable cartridge, and applying gas into a porous matrix to cause the application of bubbles through the reaction mixture. The method of claim 66, wherein combining the reaction mixture with a substrate for capturing the synthesized biomolecule comprises pneumatically driving, by the device for in vitro synthesis, the reaction mixture into a second chamber of the disposable cartridge containing the substrate for capturing the synthesized biomolecule. The method of claim 66, wherein pneumatically controlling fluid transfer comprises coordinating, by the device for in vitro synthesis, the application of pressure to open and/or close valves in the disposable cartridge and the application of pressure to drive fluid through one or more channels in the disposable cartridge. The method of claim 66, wherein pneumatically controlling binding, washing or elution comprises applying bubbles through the substrate for capturing the synthesized biomolecule. The method of claim 66, wherein the biomolecule comprises a polypeptide. The method of claim 66, wherein the enzymes for synthesizing the biomolecule comprise one or more enzymes for transcription and/or translation. The method of claim 66, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification. A method of cell-free, in vitro synthesis of biomolecules, the method comprising: connecting a disposable cartridge to a device for in vitro synthesis of biomolecules; adding a nucleic acid substrate into the disposable cartridge with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within a chamber of the disposable cartridge; controlling, using one or more processors of the device for in vitro synthesis of biomolecules, application of bubbles through the reaction mixture from a porous matrix for a combined total of 2 or more hours, while controlling the temperature of the reaction mixture within a target range; combining the reaction mixture with a substrate for capturing the synthesized biomolecule in the disposable cartridge; applying negative pressure through the porous matrix to remove flow-through from the chamber; adding wash buffer to the chamber and applying negative pressure through the porous matrix to remove the wash buffer; and adding elution buffer through the chamber and applying negative pressure through the porous matrix to collect an eluted product. The method of claim 88, wherein controlling the application of bubbles through the reaction mixture comprises applying a gas through the porous matrix, wherein the porous matrix comprises a hydrophobic substrate that allows entry of gas into the chamber through the porous matrix and inhibits passing of liquid through the porous matrix at low pressures, while allowing passing of liquid through the porous matrix at higher pressures, relative to the pressure outside of the chamber. The method of claim 88, wherein the porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE). The method of claim 88, wherein the porous matrix comprises a hydrophobic frit. The method of claim 88, wherein controlling the temperature comprises using a noncontact heater. The method of claim 88, further comprising performing non-contact temperature sensing of the temperature within the chamber using an infrared heat sensor. The method of claim 93, further comprising controlling temperature within the chamber at a temperature between 16 and 37 degrees Celsius (°C). The method of claim 93, further comprising controlling temperature within the chamber at a temperature between 21 and 28 degrees Celsius (°C).

- 50 - The method of claim 93, further comprising maintaining temperature within the chamber while sparging, so that the temperature within the chamber is maintained at a target temperature with +/- 2 degrees Celsius (°C). The method of claim 88, further comprising adjusting a pressure from one or more pressure sources of the device for in vitro synthesis of biomolecules to control the application of bubbles. The method of claim 88, wherein controlling the application of bubbles comprises adjusting power to an air pump of the device for in vitro synthesis of biomolecules to maintain sparging through the reaction mixture within a preset range. The method of claim 88, wherein the biomolecule comprises a polypeptide. . The method of claim 88, wherein the enzymes for synthesizing the biomolecule comprise one or more enzymes for transcription and/or translation. . The method of claim 88, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification. . A device for cell-free, in vitro synthesis of biomolecules, the device comprising: a pneumatic manifold; a pressure source coupled to the pneumatic manifold; a cartridge holder configured to hold a cartridge so that one or more pneumatic input ports on the disposable cartridge sealingly mate with the pneumatic manifold; a temperature sensor configured to detect temperature within a chamber of the disposable cartridge; and a controller comprising one or more processors and a memory coupled to the one or more processors, the memory configured to store instructions, that, when executed by the one or more processors, perform a method comprising: applying sparging through a reaction mixture within a chamber of the disposable cartridge, while controlling the temperature of the reaction mixture at a preset temperature; binding, flow-through, washing and eluting a synthesized biomolecule material within the disposable cartridge, wherein the one or more controllers controls the transfer of liquid within the disposable cartridge

- 51 - without liquid from the disposable cartridge coming in contact with a pump or valve within the device.

103. The device of claim 102, further comprising a non-contact heater configured to heat a material within the chamber of the disposable cartridge without contacting the disposable cartridge.

104. The device of claim 103, wherein the non-contact heater is an infrared heater.

105. The device of claim 102, wherein the temperature sensor is an infrared thermal sensor.

106. The device of claim 102, wherein the disposable cartridge holder is configured to clamp the disposable cartridge in a vertical position so that the one or more pneumatic input ports on the disposable cartridge sealingly mate with the pneumatic manifold.

107. The device of claim 102, wherein the controller is configured to monitor one or both of pressure applied to the chamber and sparging within the chamber, further wherein the controller is configured to adjust power to the air pump to maintain sparging through the reaction mixture within a preset range.

108. The device of claim 107, wherein the controller is configured to receive a user input of the preset range.

109. The device of claim 102, wherein the controller is further configured to receive a user input of a target temperature for the temperature of the reaction mixture.

110. The device of claim 102, wherein the controller is further configured to pneumatically combine, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule.

111. The device of claim 102, wherein the controller is further configured to mix the reaction mixture with a substrate for capturing the synthesized biomolecule by sparging.

112. A device for cell-free, in vitro synthesis of biomolecules, the device comprising: a housing; a pneumatic manifold within the housing; a pressure source coupled to the pneumatic manifold;

- 52 - a cartridge holder coupled to the housing, the cartridge holder configured to hold a cartridge; a clamping seal configured to secure the cartridge within the cartridge holder so that one or more pneumatic input ports on the cartridge sealingly mate with the pneumatic manifold; a temperature sensor configured to detect temperature within a chamber of the cartridge; and a controller within the housing, the controller comprising one or more processors and a memory coupled to the one or more processors, the memory configured to store instructions, that, when executed by the one or more processors, perform a method comprising: sparging a reaction mixture within the chamber while controlling the temperature of the reaction mixture within the chamber; pneumatically combining the reaction mixture with a substrate for capturing the synthesized biomolecule; pneumatically removing unbound components; pneumatically controlling washing of the purification substrate; and pneumatically controlling eluting a synthesized biomolecule material from the purification substrate. . The device of claim 112, further comprising a non-contact heater configured to heat a material within the chamber of the cartridge through a wall of the cartridge. . The device of claim 113, wherein the non-contact heater is an infrared heater. . The device of claim 112, wherein the temperature sensor is an infrared thermal sensor. . The device of claim 112, wherein the cartridge holder is configured to hold the cartridge in a vertical position. . The device of claim 112, wherein the controller is configured to monitor one or both of pressure applied to the chamber and an optical sensor detecting bubbles within the reaction mixture and adjusting power to the air pump to maintain sparging through the reaction mixture within a preset range. . The device of claim 117, wherein the controller is configured to receive a user input of the preset range.

- 53 -

. The device of claim 112, wherein the controller is further configured to receive a user input of a target temperature for the temperature of the reaction mixture. . They device of claim 112, wherein the controller is further configured to mix the reaction mixture with the substrate for capturing the synthesized biomolecule by sparging the purification substrate. . The device of claim 112, wherein the controller is configured to apply pneumatic pressure to open and close valves in the cartridge, to drive fluid between a plurality of chambers in the cartridge and sparge within one or more chambers of the plurality of chamber in the cartridge. . A disposable cartridge for cell-free, in vitro synthesis of biomolecules, the cartridge comprising: a first chamber configured to be held within a device for cell-free, in vitro synthesis of biomolecules in an upright position, wherein the first chamber comprises an open or openable top and a bottom; a hydrophobic porous matrix at the bottom of the disposable cartridge; a channel extending from the hydrophobic porous matrix and configured to extend into a second chamber; wherein the second chamber comprises a chamber having a threaded connector at a top region that is configured to removably couple to the first chamber directly or indirectly so that the channel extends into the second chamber. . The disposable cartridge of claim 122, further comprising a cap configured to couple to the threaded connector at the top region of the second chamber when the second chamber is uncoupled from the first chamber. . The disposable cartridge of claim 122, wherein the second chamber is configured to removably couple directly to an outer surface of the bottom of the first chamber. . The disposable cartridge of claim 122, further comprising a luer connector extending laterally from the disposable cartridge and configured to couple with the device for cell- free, in vitro synthesis of polypeptides. . The disposable cartridge of claim 122, wherein an outer surface of a bottom region of the first chamber is threaded.

- 54 -

. The disposable cartridge of claim 122, wherein the first chamber is configured to hold between 1 mL and 50 mL of fluid. . A disposable cartridge for cell-free, in vitro synthesis of biomolecules, the disposable cartridge comprising: a cartridge housing configured to be held within a device for cell-free, in vitro synthesis of biomolecules in an upright position; a plurality of chambers within the cartridge housing, including a reaction chamber, a purification chamber, and a plurality of buffer chambers; a plurality of pneumatically actuated valves, wherein each chamber of the plurality of chamber is connected to a pneumatically actuated valve of the plurality of pneumatically actuated valves near a bottom region of each chamber; a first plurality of pneumatic input ports on the cartridge housing, wherein each of the pneumatic input ports of the first plurality of pneumatic input ports is coupled to a pneumatically actuated valve of the plurality of pneumatically actuated valves; a second plurality of pneumatic input ports on the cartridge housing, wherein each chamber of the plurality of chambers is coupled to a pneumatic input port of the second plurality of pneumatic input ports through an opening at a top region of each chamber of the plurality of chambers; a third pneumatic input port coupled to one or more of the chambers of the plurality of chambers through one or more of the pneumatically actuated valves; a nucleic acid substrate input port at a top of the cartridge in communication with the reaction chamber, wherein the nucleic acid substrate input port further comprises a cover; and at least one porous matrix between the purification chamber and a pneumatically actuated valve of the plurality of pneumatically actuated valves or other another chamber of the plurality of chambers. . The disposable cartridge of claim 128, wherein the at least one porous matrix comprises a hydrophobic substrate. . The disposable cartridge of claim 128, wherein the at least one porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE).

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131. The disposable cartridge of claim 128, wherein each chamber of the plurality of chambers is coupled to a shared channel at a bottom region of the cartridge housing that is coupled to the third pneumatic input port.

132. The disposable cartridge of claim 128, further comprising a plurality of liquid traps formed in the cartridge housing, wherein each of the chambers of the plurality of chambers includes a liquid trap of the plurality of liquid traps between the opening at the top region of each chamber of the plurality of chambers and the second plurality of pneumatic input ports.

133. The disposable cartridge of claim 128, wherein the cartridge housing is at least partially optically transparent over the reaction chamber.

134. The disposable cartridge of claim 128, wherein the plurality of buffer chambers comprises a wash buffer chamber, a binding buffer chamber and an elution buffer chamber.

135. The disposable cartridge of claim 128, wherein the first plurality of pneumatic input ports and the second plurality of pneumatic input ports are arranged on an outer surface of a bottom region of the cartridge housing.

136. The disposable cartridge of claim 128, wherein the first plurality of pneumatic input ports and the second plurality of pneumatic input ports further comprising a sealing surface.

137. The disposable cartridge of claim 128, wherein the purification chamber comprises a substrate for capturing the synthesized biomolecule.

138. The disposable cartridge of claim 128, further comprising a chamber for one or more enzymes for transcription or/or translation material and a chamber for a reaction buffer including chemical energy sources and amino acids.

139. A disposable cartridge for cell-free, in vitro synthesis of biomolecules, the disposable cartridge comprising: a cartridge housing configured to be held within a device for cell-free, in vitro synthesis of biomolecules in an upright position;

- 56 - a plurality of chambers within the cartridge housing, including a reaction chamber, a purification chamber, and a plurality of buffer chambers, wherein each chamber is coupled to a shared channel at a bottom region of the cartridge housing; a plurality of pneumatically actuated valves, wherein each chamber is connected to the shared channel through a pneumatically actuated valve; a first plurality of pneumatic input ports on the cartridge housing, wherein each of the pneumatic input ports of the first plurality of pneumatic input ports is coupled to a pneumatically actuated valve of the plurality of pneumatically actuated valves; a second plurality of pneumatic input ports on the cartridge housing, wherein each chamber of the plurality of chambers is coupled to a pneumatic input ports of the second plurality of pneumatic input ports through an opening at a top region of each chamber of the plurality of chambers; a nucleic acid substrate input port at a top of the cartridge in communication with the reaction chamber, wherein the nucleic acid substrate input port further comprises a cover; and at least one porous matrix between the shared channel and the purification chamber. ethod of cell-free, in vitro synthesis of a biomolecule, the method comprising: adding a nucleic acid substrate to a chamber of a disposable cartridge held within a device for in vitro synthesis of biomolecules comprising one or more processors for pneumatically controlling fluid transfer and biomolecule synthesis within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; sparging, under control of the one or more processors, a gas through the reaction mixture within the chamber; detecting a temperature of the reaction mixture; controlling, by the one or more processors, the temperature of the reaction mixture by applying thermal energy through a wall of the disposable cartridge without contacting the wall of the disposable cartridge; and combining, in the disposable cartridge, the reaction mixture with a substrate for capturing the synthesized biomolecule to purify synthesized biomolecule material by binding, flow-through, washing and eluting the synthesized biomolecule material within the disposable cartridge.

. The method of claim 140, further comprising adjusting the sparging by adjusting the pressure applied to a sparging emitter within the chamber. . The method of claim 141, wherein the sparging emitter comprises a porous matrix. . The method of claim 142, wherein the porous matrix comprises a hydrophobic substrate. . The method of claim 142, wherein the porous matrix comprises one or more of: Polyether Ether Ketone (PEEK), Polyethylene (PE), Ultra-High Molecular Weight Polyethylene (UHMWPE), Polypropylene (PP) or Polytetrafluoroethylene (PTFE). . The method of claim 142, wherein the substrate for capturing the synthesized biomolecule is a substrate for chromatography or ion-exchange purification. . A method of cell-free, in vitro synthesis of a biomolecule, the method comprising: adding a nucleic acid substrate to a chamber of a disposable cartridge coupled to a device for in vitro synthesis of biomolecules comprising one or more processors for pneumatically controlling fluid transfer within the disposable cartridge, wherein the nucleic acid substrate is combined with one or more enzymes for synthesizing the biomolecule to form a reaction mixture within the chamber; sparging a gas through the reaction mixture within the chamber from a hydrophobic porous matrix by applying gas through the porous matrix at a low pressure; and eluting synthesized biomolecule out of chamber through the hydrophobic porous matrix by applying a low pressure across the hydrophobic porous matrix, wherein the one or more processors pneumatically controls the transfer of liquid within the disposable cartridge so that liquid from the disposable cartridge does not contact a pump or valve within the device for in vitro synthesis of biomolecules.

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