Systems, methods, and devices for automated nucleic acid and protein isolation.

Abstract

Purifying target biomolecules, such as nucleic acids or proteins, from biological sources is a time-consuming process typically performed by skilled technicians or scientists due to the highly technical nature of the work. The systems, devices, and methods disclosed herein enable automated bioprocessing and purification of target biomolecules from biological sources. For example, instruments and disposable cartridges are provided for automatically isolating and purifying nucleic acids (such as plasmid DNA from bacterial cultures) or isolating proteins from any biological sample. Such exemplary instruments and cartridges can work in concert to release, mix, and transfer target biomolecules and various reagents and buffers in a timely manner throughout the target biomolecule purification process, resulting in purified target biomolecules with less manual oversight than traditional approaches.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 949,917, filed on December 18, 2019, which is hereby incorporated by reference in its entirety.

[0002] The present disclosure generally relates to systems, methods, and devices for processing biological samples. More specifically, the present disclosure relates to systems, methods, and devices for the automated isolation of nucleic acids and / or proteins from biological and / or environmental sources.

Background Art

[0003] [[ID=1,7]] Certain experimental procedures remain performed using overwhelmingly inefficient manual methods that require individual attention by the scientist or technician performing the procedure. Many of these procedures would benefit from automation. For example, nucleic acid purification or isolation protocols, such as large - scale plasmid preparation from bacterial cultures, are currently time - consuming and inefficient operations that are not fully automated. Prior attempts to automate similar protocols, such as the commercialized embodiments of the systems disclosed in U.S. Patents Nos. 8,404,198 and 9,808,799 and similar products, are plagued by a number of drawbacks, including, for example, not being fully automated and / or not being operable for large numbers of samples. Incremental improvements, such as the introduction of precipitation filters, have reduced the required bench - time. However, even state - of - the - art nucleic acid purification kits still require a significant time investment, for example, taking several hours to purify endotoxin - free plasmids on a maxi, mega, or giga scale and requiring individual attention.

[0004] This is at least in part due to the highly technical nature of nucleic acid purification and the various heterogeneous tasks that need to be performed during such a process. For example, many nucleic acid purification protocols involve moving and routing fluids with different viscosities and densities, and doing so at different times during the purification protocol. Furthermore, during the process of purifying nucleic acids from biological samples, it is crucial that buffers and reagents are homogeneously mixed with the biological sample and / or filtrate, as this can improve the purity and final concentration of the target nucleic acid. To date, incorporating these various fluids into automated processes that can provide timed release, particularly in a way that allows for the mixing of fluids to create homogeneous solutions, has proven challenging.

[0005] Additionally, supplying each of the various buffers and reagents in an automated system can prove problematic and costly. Ideally, the materials containing each buffer / reagent should be made of materials that are chemically compatible (e.g., non-reactive or inert) with the stored solutions, so that the solutions maintain their effectiveness and activity throughout periods of non-use or storage, and until the time these fluids are implemented for their intended purpose.

[0006] The various filtration steps used in many nucleic acid purification protocols provide an additional layer of complexity and difficulty for implementation in automated processes. For example, different steps within nucleic acid purification protocols require selective filtration of the solution based on mechanical and / or ionic means, and subsequent washing or purification of components bound to the filter / membrane. This can generate many times the amount of waste compared to the initial amount of the biological sample, and the isolation or disposal of this waste is a complex factor for automation.

[0007] When performed using conventional field methods, technicians or scientists use a variety of different machines and instruments to carry out nucleic acid purification protocols. This may include, for example, using centrifuges and pipettes to concentrate biological samples, and adding measured volumes of buffer while intermittently stirring or vortexing to homogenize each buffer / reagent in the solution. Numerous different filters / membranes, columns, or magnetic beads may be used under centrifugation or vacuum to further carry out nucleic acid purification protocols, and many of these steps generate waste that is disposed of during the centrifugation / vacuuming step.

[0008] The aforementioned problems worsen as the volume of biological samples increases. Larger volumes of buffers and reagents, as well as more robust filters, membranes, and columns, are generally required when processing large volumes of biological samples. This can lead to more stringent demands on the structural integrity and filtration capacity of various filters / membranes, creating problems when incorporating and monitoring such filters / membranes into automated processes. Furthermore, the large volumes of buffers and reagents used when processing large volumes of biological samples generate more waste. Having the capacity and capability to account for this waste presents an additional and unique technical hurdle for automated processes.

[0009] Furthermore, current nucleic acid purification protocols rely on hands-on human interaction, inherently carrying a risk of contamination, resulting in reduced consistency between sample preparation and processing runs, and requiring continuously skilled technicians to perform these processes. Importantly, large-scale nucleic acid purification is an extremely time-consuming procedure, diverting scientists' attention from central projects or work. These factors make it costly and inefficient, among other things, whether in an academic research institution, a clinical laboratory, or a for-profit company.

[0010] Therefore, there are many shortcomings and problems that can be addressed in the automation of nucleic acid purification, and there is a significant need for systems, methods, and devices that can automate the nucleic acid purification process, in particular those that can incorporate all stages of the purification process into a single consumable element that limits or eliminates user intervention during the nucleic acid purification process.

[0011] Similarly, automated protein purification also requires addressing many similar technical challenges. Protein purification involves not only removing other cellular debris and substances from the target protein, but also ensuring the isolation / purification of the protein while preserving its biological activity. This often necessitates the use of conditions and techniques that do not disrupt the protein's tertiary structure, preserve its native post-translational modifications (e.g., phosphorylation, glycosylation, cysteine ​​disulfide bonds), and avoid introducing unnatural protein modifications (e.g., oxidation, deamidation). While this is difficult to achieve during a practical process, the technical challenges of automating protein purification are substantially increased.

[0012] There is a significant need for systems, methods, and devices that can automate the protein purification process, particularly those that can integrate all stages of the purification process into a single consumable element that limits or eliminates user intervention during the protein purification process. [Overview of the project]

[0013] Implementations of this disclosure solve one or more of the aforementioned or other problems in the art in the automated isolation of target biomolecules, such as target nucleic acids and / or target proteins, from biological and / or environmental sources.

[0014] In particular, one or more implementations may include a device for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from a biological sample. The device may, for example, include: (i) an input reservoir for receiving a biological sample; (ii) a first bioprocessing assembly fluidly communicating with the input reservoir and a lysis buffer reservoir; (iii) a second bioprocessing assembly fluidly communicating with the first bioprocessing assembly and a first elution buffer reservoir; and (iv) a receptacle fluidly communicating with the second bioprocessing assembly. The first bioprocessing assembly may be configured to produce a lysate containing the target biomolecules. The second bioprocessing assembly may include a target biomolecule binding filter configured to hold the target biomolecules from the first bioprocessing assembly, and the receptacle may be configured to receive an output vessel for receiving the target biomolecules in a purified form from the second bioprocessing assembly. Non-limiting embodiments may comprise at least two or more bioprocessing assemblies.

[0015] Apparatus for the automated purification of target biomolecules such as target nucleic acids and / or target proteins may be associated with any number of reservoirs containing reagents and / or buffers suitable for use in the automated isolation of target biomolecules from biological samples. For example, the apparatus may have one or more reservoirs containing resuspension buffer, RNase A (or other enzymes such as proteinase K, tobacco ecchi disease virus (TEV) protease, or universal nuclease), lysis buffer, neutralization buffer, endotoxin removal buffer, chaotropic salt buffer, wash buffer, elution buffer, refolding buffer, isopropanol, 70% ethanol, and / or TE buffer. The contents of these reservoirs may be selected based on the type of target biomolecule to be purified (e.g., whether to automatically purify target nucleic acids or target proteins).

[0016] In some embodiments, the apparatus of this disclosure is used to purify target biomolecules from biological samples. In some embodiments, the biological sample includes bacterial cultures, cell cultures, prokaryotic cell cultures, eukaryotic cell cultures, environmental samples, food or beverage samples, and / or clinical samples (e.g., urine, blood, plasma, saliva, nasal fluid, fecal aqueous solution, cerebrospinal fluid, or other body fluids or exudates). The target biomolecule may be target nucleic acids such as genomic DNA, plasmid DNA, or RNA, or target proteins such as antibodies, cytokines, viral proteins, or other recombinant pharmaceutical proteins, streptavidin, protein A, C-reactive protein (CRP), or natural or recombinant proteins used in functional, structural, or protein interaction assays. In non-limiting embodiments, the target biomolecule may be plasmid DNA isolated from bacterial cultures, cell cultures, biological samples, etc. (i.e., biological samples). In alternative non-limiting embodiments, the target biomolecule may be recombinant antibodies or monoclonal antibodies isolated from eukaryotic cultures (i.e., biological samples). In some non-limiting embodiments, the biological sample includes large quantities of bacterial cultures, large quantities of cell cultures, large quantities of prokaryotic cell cultures, large quantities of eukaryotic cell cultures, large quantities of environmental samples, large quantities of food or beverage samples, or large quantities of clinical samples. In some further non-limiting embodiments, the biological sample may be a small, medium, or large quantity sample.

[0017] In some embodiments where the target biomolecule is a target nucleic acid, an embodiment of the apparatus for automated purification of a target nucleic acid from a biological sample comprises: an input reservoir for receiving a biological sample; a first bioprocessing assembly fluidly communicating with the input reservoir and a lysis buffer reservoir, configured to produce a lysate containing the target nucleic acid; a second bioprocessing assembly fluidly communicating with the first bioprocessing assembly and a first elution buffer reservoir, comprising a nucleic acid binding filter configured to hold the target nucleic acid; and a receptacle fluidly communicating with the second bioprocessing assembly, configured to receive an output container for receiving the purified form of the target nucleic acid.

[0018] In some embodiments where the target biomolecule is a target nucleic acid, embodiments of the apparatus for automated purification of the target nucleic acid from a biological sample may additionally or alternatively include: (i) a first bioprocessing assembly configured to receive a biological sample, comprising a waste separation filter and a plurality of reservoirs fluidly coupled to the waste separation filter; (ii) a second bioprocessing assembly comprising an anion exchange membrane, a wash solution reservoir fluidly coupled to the anion exchange membrane, and a first elution buffer reservoir fluidly coupled to the anion exchange membrane; and (iii) a third bioprocessing assembly comprising a precipitation filter and a second elution buffer reservoir fluidly coupled to the precipitation filter.

[0019] The apparatus or device of the present disclosure may also include a consumable cartridge for use in automated biomolecular purification systems, such as automated targeted nucleic acid purification systems or automated targeted protein purification systems. An exemplary embodiment of the consumable cartridge may include an input reservoir for receiving a bacterial culture, cell culture, or eukaryotic cell culture; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and an elution buffer reservoir; and an output vessel in fluid communication with the second bioprocessing assembly. The first bioprocessing assembly may be configured to produce a lysate from a bacterial or eukaryotic cell culture, the lysate containing, for example, a target nucleic acid. In such embodiments, the second bioprocessing assembly may include a silica filter configured to hold the target nucleic acid, and the output vessel may be configured to receive the target nucleic acid in a purified form from the second bioprocessing assembly. The consumable cartridges may be configured to be associated with an automated nucleic acid purification system for the automatic purification of target nucleic acids without human interaction.

[0020] The methods of the present disclosure may include methods for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from a biological sample. Such exemplary methods may include the steps of: receiving a biological sample in an input reservoir without further human interaction; generating a lysate from the biological sample containing target biomolecules, such as target nucleic acids and / or target proteins, in a first bioprocessing assembly; receiving a target biomolecule-containing portion of the lysate, such as a target nucleic acid-containing portion and / or a target protein-containing portion of the lysate, in a second bioprocessing assembly; holding the target biomolecules on a biomolecule-binding filter (e.g., a nucleic acid-binding filter and / or a target protein-binding filter) in the second bioprocessing assembly; and eluting the purified form of the target biomolecules from the biomolecule-binding filter into an output vessel.

[0021] In some embodiments, the method may further include capturing the cellular contents of a biological sample with a first membrane of a first bioprocessing assembly, and resuspending at least a portion of the cellular contents in one or more of a resuspension buffer, an RNase solution, or a lysis buffer. Resuspending at least a portion of the cellular contents may include, for example, backwashing the first membrane by transferring a resuspension solution, which may include one or more of a resuspension buffer, an RNase solution, or a lysis buffer, through the first membrane via a fluid channel located on the second side of the first membrane.

[0022] The method may additionally include target biomolecule-specific processing steps. For example, in embodiments in which the target biomolecule comprises a target nucleic acid, the method may additionally include mixing the lysate with a neutralizing buffer to form a neutralized lysate and separating the target nucleic acid-containing portion from the waste portion of the neutralized lysate. The method may also, optionally, include mixing an endotoxin removal buffer with the target nucleic acid-containing portion of the lysate. In some examples, retaining the target nucleic acid on a nucleic acid-binding filter in a second bioprocessing assembly includes passing the target nucleic acid-containing portion of the lysate through an anion exchange membrane, removing the target nucleic acid-containing portion of the lysate from the anion exchange membrane, and precipitating the target nucleic acid to desalt and / or concentrate the target nucleic acid. The precipitated target nucleic acid may be further captured in a precipitation membrane according to some disclosed methods.

[0023] Alternatively, retaining the target nucleic acid on a nucleic acid binding filter in a second bioprocessing assembly may include passing the target nucleic acid-containing portion of the lysate through a silica-based or advanced silica-based filter. In such an exemplary method, the target nucleic acid-containing portion of the lysate may be mixed with a chaotropic salt buffer before passing it through the silica-based filter.

[0024] Some embodiments relate to nucleic acid purification equipment that can be used in automated nucleic acid purification processes. Among its features, the equipment can interface with an inserted nucleic acid purification cartridge to control the movement and routing of fluids within the cartridge, control the operation of seals and valves, and control the timing of purification process steps.

[0025] In one embodiment, the purification device includes a casing having an internal compartment configured in a size and shape to receive a purification cartridge, a selectively closable access door providing access to the internal compartment, and a pump assembly disposed within the internal chamber and configured to provide a pumping action through a peristaltic motion. In some embodiments, the device can further include a clamping mechanism disposed within the internal compartment and configured to move between an open position where the internal compartment is accessible and a closed position where the purification cartridge with the clamping mechanism inserted is compressed. The clamping mechanism can thereby assist in maintaining the integrity of the fluid seal of the cartridge during the relatively high pressures that the cartridge can be subjected to during the purification process.

[0026] The device can include one or more sensors for determining the position of components, the operating state of the device, the process status, and / or other displays. For example, one or more position sensors can be utilized to ensure proper insertion of the cartridge, proper cartridge status, and / or secure enclosure of the cartridge prior to starting of automated moving parts. One or more sensors can be communicatively coupled to a controller, which can be configured to automatically control the device operation based at least in part on the information received from the one or more sensors. For example, the controller can be configured to prevent the start of the purification process and / or provide a notification / alarm to the user if it determines that the cartridge is inappropriate, improperly inserted, unable to effectively collect the purified product, loaded with an inappropriate sample, and / or not properly and securely enclosed within the device.

[0027] The machine may also include one or more actuators for interacting with an inserted cartridge to provide, for example, fluid pump transport, seal opening and fluid release, air vent opening, valve control, and / or fluid mixing. In some embodiments, a pump assembly for interfacing with one or more fluid channels of an inserted cartridge includes a camshaft and a plurality of cam members extending laterally from the camshaft. The cam element tips engage the associated fluid channels. The pump assembly is configured such that rotation of the camshaft causes a linear peristaltic movement of the cam element tips, thereby peristaltically compressing the fluid channels and driving fluid movement through the channels.

[0028] In one embodiment, a method for the automated purification of a target nucleic acid from a biological sample includes providing a nucleic acid purification device (i.e., a machine), loading a purification cartridge into an internal compartment of the device through an access door, and initiating a purification procedure using the machine. Initiating the purification procedure causes the machine to automatically purify the target nucleic acid without further human interaction.

[0029] In some embodiments, the method further includes closing the access door of the machine and operating a clamping mechanism of the machine to move to a closed position to compress the inserted cartridge and thereby assist in fluidly sealing the loaded purification cartridge. The method may also include determining that the purification cartridge is fully loaded and / or that the access door is fully closed prior to initiating the purification procedure, such as through the use of one or more position sensors for detecting the position of the cartridge. The method may also include determining that an output container is properly positioned relative to the cartridge, issuing an alarm / notification if it is determined that no output container is present, and / or preventing initiation of the purification procedure.

[0030] In some embodiments, the method may include the step of determining the optical density of a biological sample in a purification cartridge. The instrument may operate as a “smart” instrument capable of changing one or more process parameters in response to received input and / or sensor data. For example, optical density measurement may be used to adjust one or more parameters of the purification procedure, such as the amount of one or more reagents used in the purification procedure, the duration of pump transport through the pump assembly, or the rate of pump transport through the pump assembly.

[0031] In some embodiments, the initial optical density reading is performed before initiating the purification procedure to determine whether the purification cartridge has been used previously. For example, if the optical density reading is substantially equal to the air blank reading, it can be considered that the culture input reservoir of the biological sample cartridge remains undamaged and therefore the cartridge has not been used.

[0032] The systems and apparatus disclosed herein may additionally include, or be associated with, a fluid release system for retaining and selectively releasing fluid. Such exemplary systems may include a flexible gasket; a reservoir located on a first side of the flexible gasket, configured to retain fluid; a fragile seal located between the flexible gasket and the fluid reservoir; and an actuator located on a second side of the flexible gasket, operable to deflect the flexible gasket, thereby causing the fragile seal to break, and thereby selectively releasing fluid from the reservoir.

[0033] Additionally, in some embodiments, the fluid release system includes a flexible air vent, a fragile air seal located on a first side of the flexible air vent, and a vent actuator located on a second side of the flexible air vent. The vent actuator is operable to selectively deflect the flexible air vent toward the fragile air seal, thereby rupturing the fragile air seal to release air into a reservoir.

[0034] The fluid release system may be associated with any number or type of mixing chambers or reservoirs disclosed herein and may allow for the selective release of fluids from these chambers / reservoirs at appropriate times to result in different processes and fluid transfers within the nucleic acid purification system and associated cartridges. Accordingly, embodiments of the present disclosure include an automated system for selectively releasing fluids, further comprising an automated nucleic acid purification system comprising at least one component of the fluid release system disclosed, and a biological sample cartridge for use with the automated nucleic acid purification system, comprising at least one other component of the fluid release system disclosed.

[0035] A method for selectively releasing fluid from a reservoir in an automated process may include bringing a flexible gasket into contact with an actuator, moving the actuator to deflect the flexible gasket toward a fragile seal associated with the reservoir, and causing the flexible gasket to rupture the fragile seal, thereby releasing the fluid from the reservoir.

[0036] A method for selectively releasing fluid from a reservoir may further include bringing a flexible air vent into contact with a vent actuator, moving the vent actuator to deflect the flexible air vent toward a fragile air seal, and causing the flexible air vent to rupture the fragile air seal.

[0037] The systems, methods, and apparatus of this disclosure may also include apparatus for controlled movement of fluids. The apparatus may include a first outer layer having a first side containing a series of channels and a second side. The apparatus may further include a second outer layer located opposite the first side of the first outer layer, and an elastomer layer located between the first and second outer layers. The elastomer layer may include an array of sealing ribs corresponding to the series of channels and may be configured to fluidly separate the channels when the elastomer layer is compressed between the first and second outer layers.

[0038] In some embodiments, the apparatus may additionally include a nominal gap between the elastomer layer and one or both of the first or second outer layers, such that when the elastomer layer is compressed between the first and second outer layers, the compressed portion of the sealing rib is displaced within the nominal gap. Additionally or alternatively, the apparatus may include valves associated with a series of channels, which are selectively movable between a closed position and an open position to restrict or allow the flow of fluid across the valves, respectively.

[0039] In some embodiments, the aperture of the device can provide access to a deflectable portion of the elastomer layer, which comprises a valve sealing rib extending from the elastomer layer toward a first outer layer. The valve sealing rib may contact the first outer layer when the valve is in the closed position and may separate from the first outer layer when the valve is in the open position. Additionally or alternatively, the device may include a plunger that contacts the deflectable portion of the valve. In such embodiments, the plunger may be configured to be passable through the aperture in order to deflect the deflectable portion and move the valve toward the closed position.

[0040] In one embodiment, the device is configured to withstand at least 500 lbf, preferably up to 15,000 lbf, applied along the entire length of the array of sealing ribs. Additionally, the array of sealing ribs can be compressed to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leaking, and / or the sealing ribs are compressed by at least 20%, preferably at least 30%, when the elastomer layer is compressed between the first and second outer layers.

[0041] Embodiments of the present disclosure additionally include methods for controlling fluid movement. Exemplary methods may include providing a device for controlled fluid movement disclosed herein to a system for automated purification of a target nucleic acid or target protein, causing one or more plungers to open valves in the device so that the opening valves allow fluid communication between the upstream and downstream sections of a series of channels, and causing a pump to move the fluid from the upstream section to the downstream section. Disclosed methods for controlling fluid movement may additionally include the step of providing a biological sample containing the target nucleic acid or target protein to the device, where, as provided throughout this application, the biological sample may in some embodiments be a bacterial culture, the target nucleic acid may be plasmid DNA (or a cell culture), and the target protein may be any cellular protein.

[0042] Accordingly, systems, methods, and devices for the automated purification of target biomolecules such as nucleic acids or proteins from biological samples are disclosed.

[0043] Implementations of this disclosure solve one or more of the aforementioned or other problems in the art in the automated isolation of target biomolecules, such as target nucleic acids or proteins, from biological, clinical, and / or environmental sources.

[0044] In particular, one or more implementations may include a device for the automated purification of a target protein from a biological sample, the device comprising: an input reservoir for receiving a biological sample; a first bioprocessing assembly fluidly communicating with the input reservoir and a lysis buffer reservoir, configured to produce a lysate containing the target protein; a second bioprocessing assembly fluidly communicating with the first bioprocessing assembly and a first elution buffer reservoir, comprising a protein-binding support configured to hold the target protein; and a receptacle fluidly communicating with the second bioprocessing assembly, configured to receive an output container for receiving the purified form of the target protein.

[0045] In one embodiment, the device comprises a consumable cartridge for use in an automated protein purification system. In some examples, the consumable cartridge for use in an automated protein purification system comprises: an input reservoir for receiving a biological sample containing a target protein or protein of interest; a first bioprocessing assembly fluidly communicating with the input reservoir and a lysis buffer reservoir, configured to produce a lysate from the biological sample, the lysate containing the target protein; a second bioprocessing assembly fluidly communicating with the first bioprocessing assembly and a elution buffer reservoir, comprising a support or filter configured to hold the target protein; and an output container fluidly communicating with the second bioprocessing assembly, configured to receive the purified form of the target protein. The consumable cartridge is associated with an automated protein purification system and is configured to automatically purify the target protein without human interaction. This cartridge may additionally include one or more components for column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, high-performance protein liquid chromatography, or any combination thereof, in a second bioprocess chamber or an additional bioprocessing chamber. In some examples, these components may be positioned upstream of the target protein binding support. In some examples, one or more of these components may include the target protein binding support.

[0046] In some embodiments, the automated protein purification system may additionally include one or more controllers, including computerized systems that control various fluid transfers, sample transfers, reagent distributions, and other processes.

[0047] The automated protein purification apparatus, system, and consumable cartridges of this disclosure are compatible with a wide range of samples, including but not limited to biological samples, tissues, biopsies, cell lines, cell cultures, cells, cell suspensions, aqueous solutions of urine, saliva, cerebrospinal fluid, blood, serum, plasma, feces, other bodily fluids or exudates, eukaryotic cells, prokaryotic cells or cell suspensions containing prokaryotic cells, selected from the group consisting of rodents, insects, primates, and human cells, bacterial cells, yeast cells, and the like.

[0048] In some embodiments, the first bioprocessing assembly includes a purification filter. In some examples, the purification filter is in fluid communication with an input reservoir and a lysis buffer reservoir, and is configured to separate the target protein-containing portion of the biological sample from the first waste portion of the biological sample.

[0049] In examples where the biological sample is a cell line or tissue containing multiple cells, the target protein-containing portion contains the protein in the cell line or tissue, and the first waste portion contains lysed cells and, optionally, culture medium.

[0050] In some embodiments, the first bioprocessing assembly includes a cell capture or concentration filter. In some embodiments, the cell capture or concentration filter is located upstream of the purification filter. In examples where the cell capture or concentration filter is in fluid communication with an input reservoir and a lysis buffer reservoir, the cell capture or concentration filter is configured to separate the target protein-containing portion of the biological sample from the first waste portion of the biological sample.

[0051] In some embodiments, the apparatus further comprises one or more additional bioprocessing chambers containing reagents for protein purification. In some examples, one or more filters within the apparatus include hollow fiber filters.

[0052] For example, exemplary automated protein purification apparatuses, systems, and consumable cartridges of this disclosure may include a lysis buffer having at least one solubil and a DNase. In some embodiments, the automated protein purification systems disclosed herein may additionally include a cleavage buffer and a protease (e.g., a TEV protease) for removing affinity tags from target proteins. For example, a DNase-treated lysate may be passed through a column or filter that binds to a given affinity tag, and after washing the column, the cleavage buffer and protease can be used to release the target protein from the column / filter. The digested target protein can then be further purified or collected.

[0053] The protein purification systems and apparatus disclosed herein may additionally include, or be associated with, a fluid release system for retaining and selectively releasing fluid. Such exemplary systems may include a flexible gasket; a reservoir located on a first side of the flexible gasket, configured to retain fluid; a fragile seal located between the flexible gasket and the fluid reservoir; and an actuator located on a second side of the flexible gasket, operable to deflect the flexible gasket and break the fragile seal, thereby selectively releasing fluid from the reservoir.

[0054] Additionally, in some embodiments, the fluid release system includes a flexible air vent, a fragile air seal located on a first side of the flexible air vent, and a vent actuator located on a second side of the flexible air vent. The vent actuator is operable to selectively deflect the flexible air vent toward the fragile air seal, thereby rupturing the fragile air seal to release air into a reservoir.

[0055] The fluid release system may be associated with any number or type of mixing chambers or reservoirs disclosed herein and may allow for the selective release of fluids from these chambers / reservoirs at appropriate times to result in different processes and fluid transfers within the protein purification system and associated cartridges. Accordingly, embodiments of the present disclosure additionally include an automated system for selectively releasing fluids, at least one of which is operable by the automated protein purification system, and at least one other component of the disclosed fluid release system is contained within a biological sample cartridge, configured for use with the automated protein purification system.

[0056] A method for selectively releasing fluid from a reservoir in an automated process may include bringing a flexible gasket into contact with an actuator, moving the actuator to deflect the flexible gasket toward a fragile seal associated with the reservoir, and causing the flexible gasket to rupture the fragile seal, thereby releasing the fluid from the reservoir.

[0057] A method for selectively releasing fluid from a reservoir may further include bringing a flexible air vent into contact with a vent actuator, moving the vent actuator to deflect the flexible air vent toward a fragile air seal, and causing the flexible air vent to rupture the fragile air seal.

[0058] The systems, methods, and apparatus of this disclosure may also include apparatus for controlled movement of fluids. The apparatus may include a first outer layer having a first side containing a series of channels and a second side. The apparatus may further include a second outer layer located opposite the first side of the first outer layer, and an elastomer layer located between the first and second outer layers. The elastomer layer may include an array of sealing ribs corresponding to the series of channels and may be configured to fluidly separate the channels when the elastomer layer is compressed between the first and second outer layers.

[0059] In some embodiments, the apparatus may additionally include a nominal gap between the elastomer layer and one or both of the first or second outer layers, such that when the elastomer layer is compressed between the first and second outer layers, the compressed portion of the sealing rib is displaced within the nominal gap. Additionally or alternatively, the apparatus may further include valves associated with a series of channels, the valves being selectively movable between a closed position and an open position to restrict or allow the flow of fluid across the valves, respectively.

[0060] In some embodiments, the aperture of the device can provide access to a deflectable portion of the elastomer layer, the deflectable portion comprising a valve sealing rib extending from the elastomer layer toward a first outer layer. The valve sealing rib may contact the first outer layer when the valve is in the closed position and may separate from the first outer layer when the valve is in the open position. Additionally or alternatively, the device may include a plunger that contacts the deflectable portion of the valve. In such embodiments, the plunger may be configured to be passable through the aperture in order to deflect the deflectable portion and move the valve toward the closed position.

[0061] In one embodiment, the device is configured to withstand at least 500 lbf, preferably up to 15,000 lbf, applied along the entire length of the array of sealing ribs. Additionally, the array of sealing ribs can be compressed to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leaking, and / or the sealing ribs are compressed by at least 20%, preferably at least 30%, when the elastomer layer is compressed between the first and second outer layers.

[0062] Embodiments of the present disclosure additionally include methods for controlling fluid movement. Exemplary methods may include providing a device for controlled fluid movement as disclosed herein in a system for the automated purification of target biomolecules such as target nucleic acids and / or target proteins, causing one or more plungers to open valves in the device so that the opening valves allow fluid communication between the upstream and downstream sections of a series of channels, and causing a pump to move the fluid from the upstream section to the downstream section. Disclosed methods for controlling fluid movement may additionally include the step of providing a biological sample containing the target biomolecules, and as provided throughout this application, the biological sample may, in some embodiments, be a cell line, cell culture, tissue, biopsy sample, blood, serum, plasma, eukaryotic, prokaryotic cells, or any other biological material, and the target biomolecules may be any target nucleic acids and / or target proteins from these cells.

[0063] Some embodiments describe methods for automatically isolating or purifying a target protein from a cell lysate using the apparatus or system described herein. Such exemplary methods may include the steps of: receiving a biological sample in an input reservoir without further human interaction; generating a lysate from the biological sample containing the target protein in a first bioprocessing assembly; receiving the target protein-containing portion of the lysate in a second bioprocessing assembly; holding the target protein on a protein-binding filter or protein-binding support in the second bioprocessing assembly; and eluting the purified form of the target protein from the support or filter into an output vessel.

[0064] In some embodiments, the method may further include capturing the cellular contents of a biological sample with a first membrane of a first bioprocessing assembly, and resuspending at least a portion of the cellular contents in one or more of the following: a resuspension buffer, a DNase solution, an RNase solution, or a lysis buffer. Resuspending at least a portion of the cellular contents may include, for example, backwashing the first membrane by transferring a resuspension solution, which may contain one or more of the resuspension buffer, an RNase solution, a DNase solution, or a lysis buffer, through the first membrane via a fluid channel located on the second side of the first membrane.

[0065] This method may additionally include mixing the lysate with a neutralizing buffer to form a neutralized lysate, and separating the target protein-containing portion from the waste portion of the neutralized lysate. In some embodiments, this method includes mixing an endotoxin removal buffer with the target protein-containing portion of the lysate.

[0066] This method may further include one or more additional steps: contacting a lysate containing the target protein with additional protein purification / isolation reagents; and capturing the target protein on a support or filter capable of binding to the target protein. Additional steps for isolating the target protein may include one or more of the following: column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, high-performance protein liquid chromatography, or any combination thereof. The target protein can then be eluted from the support or filter and further processed or used downstream.

[0067] Accordingly, systems, methods, and devices for the automated purification of target proteins from biological samples are disclosed.

[0068] This summary is provided to introduce a set of concepts in a simplified form, which will be further explained in the detailed description below. This summary is not intended to identify the main or essential features of the claimed subject matter, nor is it intended to be used to indicate the scope of the claimed subject matter.

[0069] Additional purposes and benefits of this disclosure are partially described below, some of which will become apparent from that description or can be acquired through the practice of this disclosure. The features and benefits of this disclosure can be realized and acquired by the equipment and combinations specifically indicated in the attached claims. These and other features of this disclosure will become more fully apparent from the following description and the attached claims or can be acquired through the practice of the disclosure as described below.

[0070] To illustrate how the above and other advantages and features of this disclosure can be obtained, a more specific description of the disclosure, as briefly described above, will be made by reference to the specific embodiments shown in the accompanying drawings. It will be understood that these drawings only illustrate typical embodiments of the disclosure and should therefore not be considered as limiting its scope.

[0071] In drawings, the drawing number may include additional characters. For example, Figure 2 may include Figure 2A and Figure 2B. In this case, the drawing number may generally be used without additional characters (e.g., Figure 2) to refer to all instances of the drawing, while the drawing identifier will include additional characters (e.g., Figure 2A) to refer to a specific instance of the drawing. This disclosure is described and illustrated with further specificities and details using the following attached drawings. [Brief explanation of the drawing]

[0072] [Figure 1] A general system for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from biological samples is illustrated. [Figure 2] The diagram illustrates various components of an exemplary cartridge configured for use in a system for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from biological samples. [Figure 3A] The diagram illustrates various components of an exemplary cartridge configured for use in a system for the automated purification of target nucleic acids from biological samples. [Figure 3B] An exemplary cartridge is illustrated, based on the cartridge of Figure 3A, which consists of a single filter in each of the first and second bioprocessing assemblies. [Figure 3C] Another exemplary cartridge is illustrated, based on the cartridge of Figure 3A, which consists of two filters in the first bioprocessing assembly and a single filter in the second bioprocessing assembly. [Figure 3D] Further illustrative cartridges are shown based on the cartridge of Figure 3A, which consists of a single filter in the first bioprocessing assembly and two filters in the second bioprocessing assembly. [Figure 3E] Further illustrative cartridges are shown, based on the cartridge of Figure 3A, each consisting of two filters in the first and second bioprocessing assemblies. [Figure 4] An exemplary layout of a purification cartridge for automated purification of target nucleic acids from biological samples is illustrated, including an input reservoir, various bioprocessing assemblies and associated membranes, filters, mixing chambers, buffer / reagent reservoirs, pumps, valves, conduits, and an output container for receiving the target nucleic acids of the input biological sample in a purified form. [Figure 5A] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5B]The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5C] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5D] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5E] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5F] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5G] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5H] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5I] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5J] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 5K] The movement of samples and various fluids through an exemplary purification cartridge as they are acted upon by the associated purification equipment is illustrated sequentially. [Figure 6] An exemplary embodiment of a purification cartridge receptacle is illustrated, shown as housing an output container positioned to receive a purified form of target nucleic acid. [Figure 7A]Another embodiment of the exemplary cartridge, configured for use with a system for automated purification of target nucleic acids from biological samples, is illustrated. [Figure 7B] Another embodiment of the exemplary cartridge, configured for use with a system for automated purification of target nucleic acids from biological samples, is illustrated. [Figure 7C] Another embodiment of the exemplary cartridge, configured for use with a system for automated purification of target nucleic acids from biological samples, is illustrated. [Figure 8A] An exemplary embodiment of a fluid release system for retaining and selectively releasing fluid from a reservoir or mixing chamber within a purification cartridge is illustrated. [Figure 8B] An exemplary embodiment of a fluid release system for retaining and selectively releasing fluid from a reservoir or mixing chamber within a purification cartridge is illustrated. [Figure 8C] An exemplary embodiment of a fluid release system for retaining and selectively releasing fluid from a reservoir or mixing chamber within a purification cartridge is illustrated. [Figure 9A] An exemplary embodiment of a flexible air vent for selectively discharging air into the associated reservoir or mixing chamber of a purification cartridge is illustrated. [Figure 9B] An exemplary embodiment of a flexible air vent for selectively discharging air into the associated reservoir or mixing chamber of a purification cartridge is illustrated. [Figure 10] An exemplary embodiment of a purification device capable of automated nucleic acid purification is illustrated. [Figure 11A] A clamping mechanism, which may be included as part of a purification apparatus and may be used to apply compressive force to an inserted purification cartridge to help maintain the integrity of the cartridge's fluid seal during the purification process, is illustrated. [Figure 11B]A clamping mechanism, which may be included as part of a purification apparatus and may be used to apply compressive force to an inserted purification cartridge to help maintain the integrity of the cartridge's fluid seal during the purification process, is illustrated. [Figure 12] A rear view of the purification equipment showing the access point for the manual clamp release mechanism is illustrated. [Figure 13] The access door lock mechanism is illustrated from the perspective of someone inside the equipment's internal compartment. [Figure 14] An embodiment of the access door sensor assembly is shown. [Figure 15] The purification cartridge position sensor assembly is shown in the diagram. [Figure 16] The output container presence sensor assembly is shown in the diagram. [Figure 17] An embodiment of an optical density sensor that may be included in the instrument for measuring the optical density of a biological sample inserted into a purification cartridge is illustrated. [Figure 18A] The illustration shows a pump transport assembly that may be included as part of a purification device and may be used to move and route fluid along the fluid path of an inserted purification cartridge. [Figure 18B] The illustration shows a pump transport assembly that may be included as part of a purification device and may be used to move and route fluid along the fluid path of an inserted purification cartridge. [Figure 19A] The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 19B] The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 19C] The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 19D]The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 19E] The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 19F] The movement of fluid through the fluid channels in the purification cartridge is sequentially illustrated as a result of the operation of the pump assembly engaged with the fluid channels. [Figure 20A] The diagram illustrates a control system that may be included in a device and used to control various components of the device, receive user input, display data, and communicate with other computer devices and / or networks. [Figure 20B] An exemplary method that can be implemented by the control system in Figure 20A to select a purification protocol based at least partially on the received sensor data and to execute the selected purification protocol is illustrated. [Figure 21] A plan view of an exemplary nucleic acid purification cartridge is shown, which has an array of sealing ribs for defining and sealing various fluid channels in the cartridge. [Figure 22] This is a cross-sectional view of the fluid channel of the cartridge, showing an elastomer layer with sealing ribs positioned between two outer layers. [Figure 23A] The diagram illustrates the clamping of the cartridge to compress the sealing rib array and provide sufficient sealing for the purification process. [Figure 23B] The diagram illustrates the clamping of the cartridge to compress the sealing rib array and provide sufficient sealing for the purification process. [Figure 24] A valve mechanism that utilizes an elastomer layer to provide a valve mechanism is illustrated. [Figure 25] The agarose gel analysis of plasmid products isolated using the exemplary systems and nucleic acid purification cartridges disclosed herein is illustrated. [Modes for carrying out the invention]

[0073] Before describing embodiments of this disclosure in detail, it should be understood that this disclosure is not limited to the parameters of the devices, systems, methods, apparatus, products, processes, and / or kits, which are naturally subject to change and are particularly exemplified. Therefore, while specific embodiments of this disclosure are described in detail with reference to specific configurations, parameters, components, elements, etc., the descriptions are illustrative and should not be construed as limiting the scope of the claimed invention. Furthermore, the terms used herein are intended to describe embodiments and are not necessarily intended to limit the scope of the claimed invention.

[0074] Furthermore, unless otherwise implicitly or explicitly understood or stated, it is understood that, with respect to any given component or embodiment described herein, any of the possible candidates or substitutes listed for that component may generally be used individually or in combination with each other. Furthermore, unless otherwise implicitly or explicitly understood or stated, it will be understood that any enumeration of such candidates or substitutes is merely illustrative and not limiting.

[0075] In addition, unless otherwise indicated, numbers representing quantities, components, distances, or other measured values ​​used in the specification and claims should be understood as being modified by the term "about." Accordingly, unless otherwise indicated, numerical parameters described in the specification and appended claims are approximations that may vary depending on the desired characteristics to be obtained by the subject matter presented herein. At a minimum, and without intending to limit the application of the principle of equivalents to the claims, each numerical parameter should be interpreted by applying ordinary rounding techniques, at least in light of the reported significant number of decimal places. Although the numerical ranges and parameters describing the broad range of subject matter presented herein are approximations, the numerical values ​​described in specific examples are reported as accurately as possible. However, any numerical value inherently contains a certain degree of error that inevitably arises from the standard deviation observed in each experimental measurement.

[0076] The headings and subheadings used herein are for structural purposes only and are not intended to be used to limit the scope of the description or claims.

[0077] Overview and advantages of exemplary targeted biomolecule purification systems As described above, many disadvantages and problems can be addressed by automating the purification of target biomolecules, such as target nucleic acids and / or target proteins, especially when processing large volumes of samples. There is a significant need for systems, methods, and devices that can automate the processes of target biomolecule purification, including the respective processes for purifying target nucleic acids and / or target proteins. In particular, there is a significant need for systems, methods, and devices that can incorporate all stages of the purification process into a single consumable element that limits or eliminates user intervention during the purification process.

[0078] Embodiments of this disclosure solve one or more of the aforementioned problems in the art of automated targeted biomolecule purification. For example, as shown in Figure 1, a system 10 for automated purification of targeted biomolecule (such as targeted nucleic acids and / or targeted proteins) can employ a combination of a purification instrument 12 and a purification cartridge 14 to isolate and purify the targeted biomolecule from a biological sample with limited user interaction. In a preferred embodiment, the user adds a biological sample 16 containing the targeted biomolecule to the purification cartridge 14, loads the purification cartridge 14 into the purification instrument 12, where the targeted biomolecule is then automatically isolated, purified, and deposited into an output container 18 associated with the system 10, all without further user interaction with the system. In this way, the targeted biomolecule purification systems of this disclosure (such as nucleic acid purification systems and protein purification systems disclosed herein) can include self-contained systems. In some embodiments, the targeted biomolecule purification apparatus and systems of the present disclosure enable fully automated large-scale targeted biomolecule purification (e.g., endotoxin-free maxiscale plasmid DNA purification from 100–200 mL bacterial culture input or recombinant pharmaceutical protein purification from 500 mL eukaryotic cell culture input) in reduced time compared to manual methods. In some examples, the disclosed systems can automatically isolate and purify targeted biomolecules in less than one hour (e.g., purifying targeted nucleic acids with a total run time of approximately 45 minutes and a setup time of only about 2 minutes).

[0079] Additional advantages can be realized through the implementation forms of the disclosed systems, methods, and devices. For example, the systems disclosed herein may include walkaway benchtop instruments that are robust and capable of processing biological samples of varying input densities (e.g., bacterial cultures, eukaryotic cell cultures, clinical samples, food / beverage samples, or environmental samples) and achieving high-purity target biomolecules such as high-purity nucleic acids ("low-endotoxin" or "endotoxin-free" in the case of plasmid DNA purified from bacterial cultures). In some embodiments, the purification instrument may be cloud-enabled and "smart," equipped with a built-in optical density sensor, as well as communication hardware and software, configured to notify the user of the density of the input culture and / or dynamically trigger different purification protocols depending on the optical density read (e.g., A600) and / or the type of input sample received.

[0080] Additional advantages of the disclosed systems, methods, and devices include, for example, a one-piece purification cartridge design that integrates all reagents, filters, pumps, fluid channels, input reservoirs, waste reservoirs, and final output vessels into a single cartridge. This limits user interaction to a single component of the system, thereby reducing errors, increasing consistency, and significantly reducing the practical time during the targeted biomolecule purification process. The disclosed systems are also much easier to use and can therefore reduce the technical expertise or know-how required to purify targeted biomolecules from biological samples.

[0081] This is in stark contrast to manual kits, which require a technically skilled user to obtain and operate several reagents, purification columns, centrifuges, pipettes, pipette tips, intermediate containers, and final output containers. Similarly, manual and semi-automatic systems require a number of consumable elements that the user operates to achieve the purification of biomolecules such as nucleic acids and / or proteins. Unlike manual processes that require the user to interact with open reagent containers, the disclosed purification cartridges allow the reagents to remain sealed during the setup and purification steps. Some embodiments of this disclosure also allow for the automated release of reagents at various appropriate times in the purification process, which is an improvement over previous manual and semi-automatic processes that require the user to break reagent seals or access reagents directly before and / or during the execution of the protocol.

[0082] In some embodiments, the purification cartridge is a disposable element of the consumables within the system, and in some examples, this can result in lower-cost consumables compared to conventional kits and systems. For example, in some embodiments, the cartridge is made of or incorporates thermoformed plastic, and the use of thermoformed plastic instead of expensive injection molding processes enables a low-cost, high-volume solution for producing consumable purification cartridges. Thermoformed plastic has traditionally been used as a packaging material because it reduces the costs associated with its manufacture. In embodiments of this disclosure in which the purification cartridge is made of thermoformed plastic, or which include thermoformed plastic, such design criterion represents a significant deviation from typical applications, as the product is made from thermoformed plastic rather than the packaging of the standard product. Unexpectedly, the use of thermoformed plastic has resulted in a suitable non-reactive purification cartridge system at a reduced cost without significant loss of practicality.

[0083] As an additional embodiment, some embodiments use a combination of purification equipment and purification cartridge to form a liquid-tight seal within the purification cartridge. This type of sealing can enable a cheaper and simpler manufacturing method, for example, because the purification cartridge does not incorporate traditionally long liquid-tight seals that are often formed by expensive and complex ultrasonic welding. This, apart from, or in combination with, the significantly lower cost of thermoforming, can greatly reduce the cost and manufacturing time of the purification cartridge.

[0084] Furthermore, embodiments of the purification cartridge may include an elastomer layer sandwiched between two thermoformed outer layers. In such embodiments utilizing an elastomer layer between two thermoformed layers, the elastomer layer can be used for valve adjustment purposes, and since only one component is used for each valve instead of individual components for all valves, it allows for simpler assembly of consumables in manufacturing. This design also enables a very simple, reliable, single-axis actuator. For example, the natural elasticity and tendency of the elastomer to bounce back to its original shape also allows the actuator to bring about a change (e.g., valve adjustment or release of fluid in the cartridge) by either pushing the elastomer or releasing pressure from the elastomer, without applying an opposing force and / or without an opposing actuator, thereby simplifying valve adjustment, fluid flow control, and operation in the system.

[0085] Purification cartridge The following disclosure relates to exemplary embodiments of a purification cartridge that may be used to provide automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from biological samples. The purification cartridge may be used in conjunction with other components and / or instruments described herein. For example, the cartridge may be configured to accept and interface with a purification instrument, such as instrument 12 shown and illustrated in Figure 1 or any other purification instrument described herein.

[0086] Cartridge Overview As described above, conventional targeted biomolecule purification procedures, such as nucleic acid purification and / or protein purification, are manual testing processes that require a relatively high level of expertise and time from the laboratory technician. While a well-trained laboratory technician may be able to handle the various operational parameters involved in this process, there is a significant need for systems, methods, and devices that can automate the process of targeted biomolecule purification, such as nucleic acid purification and / or protein purification, particularly those that can incorporate all stages of the targeted biomolecule purification process into a single consumable element that limits or eliminates user intervention during the purification process.

[0087] Designing disposable cartridges that can effectively automate the majority of a process is challenging for several reasons. For example, a cartridge configured to perform all stages of a nucleic acid and / or protein purification process must be able to store and dispense (or otherwise retrieve and dispense on demand) various reagents and buffers associated with each purification protocol. Additionally, the cartridge must be able to move and route fluids of varying viscosities and densities, often by dispensing the appropriate reagents and / or buffers, mixing dissimilar fluids together, and controlling the passage of fluids over and / or through appropriate filters or membranes. Such a cartridge must also be safe for user handling and able to maintain proper fluid separation / sealing throughout the entire biomolecular purification process.

[0088] As described in more detail below, the purification cartridges described herein can satisfy one or more of the challenges mentioned above. For example, a purification cartridge may be configured to be associated with a purification instrument so as to provide a cartridge configured to perform all steps of a biomolecule purification process, by which the user loads a biological sample containing a target biomolecule, such as a target nucleic acid and / or target protein, into the cartridge and inserts the cartridge into the instrument, thereby isolating and purifying the target biomolecule without further user interaction from the user.

[0089] As an additional embodiment, the purification cartridge disclosed herein may be a self-contained solution incorporating each of the many diverse buffers and reagents required for biomolecular purification, such as nucleic acid and / or protein purification, into separate reservoirs, each in such a manner that it can be selectively released at appropriate times during the biomolecular purification process. Additionally, the purification cartridge disclosed herein may consist of multiple interconnected coordinated conduits, valves, and pumps to move and route fluids of varying viscosities and densities throughout the cartridge during the biomolecular purification process. The cartridge may additionally include a mixing chamber equipped with a magnetic stirring rod, which communicates with the cartridge conduits and reservoirs to provide a homogeneous mixture throughout the purification process. Additionally, as described below, the cartridge of this disclosure can be securely sealed as a self-contained consumable, which increases user safety, reduces the risk of reagent / buffer contamination, and provides increased ease of use for automated biomolecular purification processes.

[0090] Figure 2 illustrates a schematic diagram of the general features and components of an exemplary cartridge 20 configured for use in a system for automated purification of target biomolecules (e.g., target nucleic acids and / or target proteins) from biological samples. As shown, the cartridge 20 includes an input reservoir 22 configured to receive a biological sample containing the target biomolecules. In some embodiments, the purification cartridges disclosed herein, including the cartridge 20 illustrated in Figure 2, are preferably configured to process large volumes of sample. Thus, the input reservoir 22 may be sized and shaped to receive about 5 mL to 5 L of biological sample. In some embodiments, the input reservoir may be configured to receive at least 10 mL of biological sample, at least 50 mL of biological sample, at least 100 mL of biological sample, at least 150 mL of biological sample, at least 200 mL of biological sample, at least 250 mL of biological sample, or at least 500 mL of biological sample. In the same or alternative embodiments, the input reservoir may be configured to accept biological samples of less than 50 mL, less than 100 mL, less than 150 mL, less than 200 mL, less than 250 mL, less than 500 mL, less than 1 L, less than 2 L, or less than 5 L. It should be understood that the input reservoir may be sized and shaped to accept amounts of biological sample within a range of amounts having lower and upper limits selected from the aforementioned minimum and maximum amounts. It should also be understood that the systems, apparatus, and cartridges of this disclosure may also be reduced in size and sized and shaped to handle smaller amounts of sample than those disclosed above.

[0091] In a preferred embodiment, the input reservoir is configured to be sized and shaped to receive 50 mL to 250 mL of bacterial culture containing plasmid DNA—target nucleic acid. The input reservoir 22 may be configured to receive other biological samples, such as other cell cultures, eukaryotic cell cultures, clinical samples, or environmental samples, either additionally or alternatively. In one embodiment, the input reservoir 22 is configured to receive up to 1 L, up to 1.5 L, up to 2 L, or any range above, of a clinical sample, such as an aqueous solution of urine, feces, or other bodily fluids or exudates. The input reservoir 22 may be configured to receive a large volume of environmental samples, such as a water sample, either additionally or alternatively.

[0092] In addition to the input reservoir 22, the cartridge 20 may additionally include at least two bioprocessing assemblies 24, 30. A first bioprocessing assembly 24 may be fluid-communicated with the input reservoir 22 and configured to produce a lysate from a biological sample containing a target biomolecule. For example, the first bioprocessing assembly 24 may be fluid-communicated with a lysis buffer reservoir so that the biological sample in the input reservoir 22 can be combined with the lysis buffer in the first bioprocessing assembly 24 to produce a lysate containing a target biomolecule. A second bioprocessing assembly 30 may be fluid-communicated with the first bioprocessing assembly 24 and may include a biomolecule binding filter (e.g., a target nucleic acid binding filter and / or a target protein binding filter) for holding the target biomolecule.

[0093] Cartridge 20 may additionally include a number of reagents and buffers 28 fluidically coupled to a series of valves and pumps 26, as well as first and / or second bioprocessing assemblies 24, 30, to coordinate the movement of various fluids throughout the cartridge 20 during the automated purification process. When used, it should be understood that the pumps of this disclosure are operable to coordinate the movement of various fluids throughout the cartridge by pushing and / or pulling fluids (e.g., operable to apply positive and / or negative pressure) through channels connecting components of the bioprocessing assemblies and internal components within the cartridge. This can be achieved by strategically positioning the pumps within and / or between bioprocessing assemblies so that the pumps are positioned upstream and / or downstream of the fluids being moved. For example, a pump positioned upstream of the fluid can push the fluid through the cartridge, and a pump positioned downstream of the fluid can pull the fluid through the cartridge. As should be understood, a single pump may be operable to pull the fluid in one step of the purification process and push the fluid in another step. For example, a valve may be positioned within a cartridge to pull fluid through a filter / membrane in the first step of the purification process, and may act to push the fluid through the same or a different filter / membrane in later steps of the purification process.

[0094] Continuing to refer to Figure 2, the cartridge 20 may also include a receptacle that is in fluid communication with a second bioprocessing assembly, which is configured to receive an output container 34 for collecting the target biomolecule in a purified form.

[0095] The cartridge 20 in Figure 2 is optionally shown to include a waste reservoir 32. In some embodiments, waste generated during the purification process can be captured and stored in the cartridge itself. This can beneficially allow the cartridge 20 to be a single, self-contained, disposable cartridge, beneficially reducing user exposure to these wastes by collecting all waste produced in the cartridge during the purification process and isolating various chemicals and solutions from the transportable containers. Additionally, by collecting waste in the cartridge, the purification equipment is not exposed to or in contact with the waste, which reduces the possibility of cross-contamination between samples using the same equipment.

[0096] Referring here to Figure 3A, various components of another exemplary cartridge 36 configured for use in a system for automated purification of target nucleic acids are illustrated. Similar to cartridge 20 in Figure 2, cartridge 36 in Figure 3A includes an input reservoir 38, a first bioprocessing assembly 40, a second bioprocessing assembly 56, various reagents and buffers (e.g., lysis buffer, neutralization buffer, RNase A, resuspension buffer, IPA, ethanol, TE buffer, ER wash buffer, wash buffer, elution buffer, chaotropic salt buffer), and an output container 82. Although not shown, it should be understood that cartridge 36 in Figure 3A may additionally include a series of valves and pumps for regulating the movement of various fluids throughout cartridge 36 during the automated purification process. With respect to Figure 2, as described above, valves and pumps may be used to push and / or pull fluid through the cartridge, and some embodiments may include a series of valves spatially configured upstream and downstream of the pump and filter / membrane so that the operation of the pump pushes the fluid through the filter / membrane at one step of the purification process and pulls it through the membrane at different steps of the purification process.

[0097] Continuing to refer to Figure 3A, the illustrated cartridge 36 represents a collection of various exemplary cartridge configurations envisioned within the scope of this disclosure. It should be understood that the spatial configuration and flow between components illustrated within cartridge 36 are not intended to negate the only way of structuring and organizing the cartridges disclosed herein. Rather, the disclosed configurations are essentially exemplary and are intended to serve as schematic diagrams to aid in describing possible representative configurations and components of cartridges envisioned within the scope of this disclosure. As shown in Figure 3A, dashed boxes and arrows indicate potential additions or selections of components that may be used in the exemplary cartridges disclosed herein. Several exemplary selections and configurations are provided in Figures 3B–3E to illustrate more specifically those disclosed in Figure 3A.

[0098] In general, the various possible cartridge configurations, broadly characterized in Figure 3A and detailed in Figures 3B–3E, follow similar processing protocols. As shown in Figure 3A, the first bioprocessing assembly 40 is fluidically connected to the input reservoir 38. Through the operation of a series of valves and pumps, the biological sample received in the input reservoir 38 is transferred to the first bioprocessing assembly 40, where it can be processed. It should be understood that in some embodiments, the input reservoir 38 is one component of the first bioprocessing assembly 40, and one or more purification steps performed within the first bioprocessing assembly 40 may be performed in or using the input reservoir 38. For example, the first purification step may include homogenizing the biological sample. The biological sample may contain solid or particulate matter, and the purification process may benefit from a reduction in the size of the solid or particulate matter, or from more uniform mixing in the supplied medium and / or applied aqueous buffer / solvent.

[0099] Accordingly, in some embodiments, the biological sample is homogenized in the input reservoir 38 and / or as a pretreatment step in the first bioprocessing assembly 40. This can be achieved by any means known in the art, including, for example, the use of a magnetic stirring rod, beating of high-power beads, shaking, vortexing, etc. For example, a fecal or soil sample can be added (simultaneously with or subsequently added to the medium and / or aqueous buffer / solvent) to the input reservoir where the biological sample is homogenized and transferred to the first bioprocessing assembly. Additionally or alternatively, the homogenization step can be carried out in the first bioprocessing assembly 40 and can be coupled with the lysis of the cellular contents of the biological sample. The biological sample can be combined with a lysis buffer contained in a lysis buffer chamber 42 to result in the lysis of the cellular contents, and in some embodiments, a homogenizing component is activated to homogenize the lysed sample.

[0100] Within the first bioprocessing assembly 40, the target nucleic acid from the biological sample is partially purified from the cell contents and waste culture medium through the use of one or more filters / membranes. Generally, this involves the lysis of cellular material and the separation of the target nucleic acid after neutralization. For example, the lysis buffer held in the lysis buffer chamber 42 may be combined with the biological sample in the mixing chamber 44 to form a lysate. The neutralization buffer from the neutralization buffer chamber 46 may be combined with the lysate in the mixing chamber 44, for example, and pass through the purification filter 52 to separate the target nucleic acid-containing portion 54 of the biological sample from the waste portion of the biological sample.

[0101] As used herein, the terms “filter” or “membrane” include any size-selective and / or charged semipermeable barrier capable of operating to select or separate one or more components from a solution. This may include, for example, a size-selective filter paper or a film or column of material. It may additionally include other forms or means of filtration, such as affinity columns, beads (e.g., steel, glass, zirconia, or any suitable size, preferably 0.1 to 2 mm in diameter, or other suitable materials), or other means known in the art.

[0102] The partially purified target nucleic acid can then be transferred to a fluidically coupled second bioprocessing assembly, where it is further purified and / or concentrated and finally collected in the output container 82 as purified target nucleic acid. The purification steps in the first and second bioprocessing assemblies can be advantageously achieved with little to no user interaction.

[0103] Next, the generalized purification process described above will be explained in more detail in the context of various exemplary cartridges illustrated in Figures 3B to 3E.

[0104] One embodiment of a cartridge, configured for use with a system for automated purification of target nucleic acids from biological samples, is illustrated in Figure 3B. Cartridge 36a consists of a single filter in each of the first and second bioprocessing assemblies 40a, 56a. In particular, the first bioprocessing assembly 40a of the illustrated cartridge 36a includes a single filter 52a configured to filter the target nucleic acid-containing portion of a biological sample from cellular components and culture medium. The filter may be, for example, a size-selective filter having a selective size and material suitable for the isolation of a particular target nucleic acid. A non-limiting example mentioned above includes a 5 μm nylon filter.

[0105] In an exemplary configuration of cartridge 36a in Figure 3B, a biological sample can pass from the input reservoir 38 to the filter 52a of the first bioprocessing assembly 40a in a first step for removing the medium from the sample. In this way, the cell fraction of the biological sample is retained on the filter 52a. The cells associated with the filter 52a can be lysed using a lysis buffer held in the lysis buffer chamber 42 of cartridge 40a to form a lysate. It should be understood that the lysis buffer can be used to elute cells from the filter 52a by applying the cells to the cell-capturing side of the filter 52a. Alternatively, the lysis buffer can be pushed through the back side of the filter 52a (e.g., the opposite side) to elute cells from the filter 52a and then combine with the biological sample in the mixing chamber 44 to form a lysate. A neutralizing buffer from the neutralizing buffer chamber 46 can be combined with the lysate in the mixing chamber 44, for example, and pass through the filter 52a, thereby separating the target nucleic acid-containing portion 54 of the biological sample from the waste portion of the biological sample.

[0106] As further illustrated in Figure 3B, the target nucleic acid-containing portion 54 of the biological sample can be transferred (for example, using a pump and valve associated with the infrastructure of cartridge 36a) to a second bioprocessing assembly 56a coupled to a single filter / membrane system 76. For example, the single filter / membrane system 76 may include a silica filter 78. In such embodiments, the target nucleic acid-containing portion 54 of the lysate can be pretreated with a chaotropic salt buffer, which is automatically obtained from a chaotropic salt buffer chamber 80, to facilitate the binding of the target nucleic acid to the silica filter 78. Once bound to the silica filter 78, the target nucleic acid can undergo a series of washes via the automatic release and transfer of the wash buffer from the wash buffer chamber 66 through the silica filter 78. Waste from the aforementioned washes can be transferred to a waste reservoir 72. The washed target nucleic acid can be eluted from the silica filter 78 to an output vessel 82 in the form of purified target nucleic acid 84 using any suitable low-salt elution buffer known in the art.

[0107] In some embodiments, the first bioprocessing assembly may have two or more filters. For example, Figure 3C illustrates another exemplary cartridge 36b, which consists of two filters 48, 52 in the first bioprocessing assembly 40b connected to the same second bioprocessing assembly 56a (having a single filter) described above with respect to Figure 3B.

[0108] As shown in Figure 3C, the first bioprocessing assembly 40b may additionally include a cell capture filter 48. A biological sample received in the first bioprocessing assembly 40b may pass through the cell capture filter 48 to remove non-target nucleic acid-containing portions of the biological sample. For example, in embodiments where the biological sample is a bacterial culture, the cell capture filter 48 can retain bacterial cells while allowing the culture supernatant to pass through a waste reservoir 50. Lysis buffer and / or resuspension buffer from the lysis buffer chamber 54 may pass over the cell capture filter 48 to remove cells from the filter 48 and transfer them to the mixing chamber 44, where a lysate is formed from the cell-containing solution. A neutralization buffer chamber 46 may be in fluid communication with the mixing chamber 44, and the neutralization buffer may then be transferred to the mixing chamber 44 where the lysate is neutralized. The neutralized lysate may then pass through a purification filter 52 to separate the target nucleic acid-containing portion 54 of the lysate from the waste portion of the lysate. In the example of bacterial cultures, the aforementioned waste portion of the lysate may include, among other things, cellular components of the lysed bacterial cells, including bacterial genomic DNA.

[0109] While the biological sample is exemplified as a bacterial culture, it should be understood that this example is used to facilitate the illustration of the structural features of the disclosed cartridge. The biological sample may be any other sample, such as a soiled sample, a clinical or forensic sample (e.g., stool, blood, saliva, urine, etc.), a water sample, or any other sample containing the target nucleic acid.

[0110] The first bioprocessing assembly 40b of cartridge 36b is fluidically coupled to the second bioprocessing assembly 56a, and as described above, the target nucleic acid-containing portion 54 of the biological sample can be transferred from the first bioprocessing assembly 40b to the second bioprocessing assembly 56a, where it is bound to a single filter / membrane system 76 as purified target nucleic acid 84, washed, and eluted.

[0111] Figure 3D illustrates yet another exemplary cartridge 36c. The illustrated cartridge 36c consists of a single filter 52a in the first bioprocessing assembly 40a (similar to that illustrated and discussed in Figure 3B) and two filters in the second bioprocessing assembly 58b. The target nucleic acid-containing portion 54 of the lysate is bound to the target nucleic acid capture filter 58b in the second bioprocessing assembly 56b. Prior to binding to the filter 58b, the target nucleic acid-containing portion 54 of the lysate may be optionally pretreated with an endotoxin removal (ER) buffer (e.g., from an ER washing buffer chamber 64) in a “low endotoxin” or “endotoxin-free” purification protocol. Once bound to the target nucleic acid capture filter 58b, the target nucleic acid is eluted into the output vessel 82 in the form of washed and purified target nucleic acid 84. Washing the target nucleic acid bound to the capture filter 58 may include, for example, passing various washing buffers or aqueous solutions containing isopropyl alcohol or ethanol through the capture filter 58b into the waste reservoir 72. These wash buffers are automatically released from one or more wash buffer chambers 66 that are in fluid communication with the capture filter 58b. Similarly, the washed target nucleic acids can be eluted from the capture filter 58b, for example, through the automatic release of distilled water, or in an elution buffer obtained from a dH2O / elution buffer chamber 74 that is fluidly connected to the capture filter 58b.

[0112] As shown in Figure 3D, the capture filter 58b may include a series of filters / membranes 60. The target nucleic acid-containing portion 54 of the lysate may be bound and washed along the series of filters / membranes 60 before being eluted into the output container 82. In some embodiments, the target nucleic acid-containing portion 54 of the lysate may be pretreated with a washing buffer before being bound to the series of filters / membranes 60. The washing buffer may be automatically released from the washing buffer chamber 66 into the mixing chamber 62 for mixing with the target nucleic acid-containing portion 54 of the lysate. The pretreated target nucleic acid may then be bound to an anion exchange membrane 68, where an additional washing step may be performed by automatically passing the washing buffer (e.g., from one or more different washing buffer chambers 66) onto the anion exchange membrane 68. The target nucleic acid may then be eluted from the anion exchange membrane 68 and bound to a precipitation filter 70 for additional washing and purification steps.

[0113] In some embodiments, the target nucleic acid is eluted from the anion exchange membrane 68 and transferred to a mixing chamber, where it is mixed with isopropanol and precipitated from the solution. The precipitated target nucleic acid is then transferred to a precipitation filter 70 for binding, where it is desalted via numerous ethanol-based (e.g., 70% ethanol) washes before being eluted into the output vessel 82.

[0114] Figure 3E illustrates yet another exemplary cartridge 36d. Cartridge 36d consists of two filters in each of the first and second bioprocessing assemblies 40b, 36b. For example, the first bioprocessing assembly 40b may include the same first bioprocessing assembly 40b as the cartridge 36b shown in Figure 3C, and the second bioprocessing assembly 56b may include the same second bioprocessing assembly 56b as the cartridge 36c shown in Figure 3D.

[0115] Therefore, a cartridge for use in the automated purification of nucleic acids from biological samples may include two or more bioprocessing assemblies, each associated with at least one filter / membrane. In some embodiments, the first bioprocessing assembly may include a single purification filter, or alternatively, the first bioprocessing assembly may include a cell capture filter and a purification filter. Similarly, the second bioprocessing assembly may include a single silica-based filter, or alternatively, the second bioprocessing assembly may include a precipitation membrane positioned downstream of the anion exchange membrane. In other words, the second bioprocessing assembly may include an anion exchange membrane, and the cartridge may additionally include a third bioprocessing assembly that includes a precipitation filter for receiving target nucleic acids eluted from the anion exchange membrane.

[0116] Sample processing using an exemplary purification cartridge The cartridges shown and described in Figures 2 and 3 can be specifically embodied as the exemplary cartridge 100 in Figure 4. In particular, Figure 4 illustrates partial cross-sectional views of various bioprocessing assemblies associated with the exemplary cartridge 100, including filters / membranes, buffer / reagent reservoirs, fluid conduits, valves (e.g., valve 154, among others, also shown as the numbered "V" element in Figures 5A–5K), seals (e.g., seals 156 and 158, among others, also shown as the numbered "L" elements in Figures 5A–5K), waste reservoirs, and output containers. Each of the various components of cartridge 100 illustrated in Figure 4 is considered in the context of the exemplary automated nucleic acid purification process stepwise shown in Figures 5A–5K. It should be understood that the mechanisms for sealing and releasing fluids throughout the automated nucleic acid purification process described with reference to Figures 5A–5K will be discussed in more detail below.

[0117] In exemplary embodiments, the cartridge 100 shown in Figures 5A–5K can be used in combination with the purification equipment described herein to provide automated “hand-off” bioprocessing of biological samples while delivering at least equivalent performance, if not superior, to similar manual processing methods. As implied above, scientists or lab technicians tied to a bench while performing manual nucleic acid purification protocols for biological samples are susceptible to human error and lack of reproducibility. When used as part of an automated nucleic acid purification system, cartridge 100 (and other cartridges disclosed herein) can eliminate or reduce human error in the lack of reproducibility that plagues manual nucleic acid purification processes. Additionally, the cartridges provided herein can enhance ease of use, reduce labor time, reduce contamination, and increase efficiency and flexibility in performing nucleic acid isolation and purification protocols.

[0118] For example, referring to Figure 5A, a biological sample such as a bacterial culture or other cell culture containing plasmid DNA can be added to the culture reservoir 102 by a technician, scientist, or other user. Cartridge 100 can be inserted into a purification instrument from which the nucleic acid purification protocol is initiated. The following steps considered with respect to Figures 5A–5K can be performed in an automated process without further human intervention. For ease of illustration and description, the components and processing steps in Figures 5A–5K are made with reference to a bacterial culture as the biological sample and plasmid DNA as the target nucleic acid. Other biological samples and target nucleic acids can be processed using the automated nucleic acid purification systems, methods, and devices disclosed herein and should be understood to be included within the scope of this disclosure. Additionally, it should be understood that each of the buffers and reagents considered and used in the exemplary nucleic acid purification protocols within the embodiments disclosed in Figures 4 and 5A–5K are contained within cartridge 100. Similarly, each of the filters / membranes considered and used in the following exemplary nucleic acid purification protocols is contained within cartridge 100. The release, sealing, and movement of fluid within the cartridge can generally be controlled and / or initiated by the purification equipment, as described in more detail below.

[0119] Continuing to refer to Figure 5A, the bacterial culture containing plasmid DNA is located in the culture reservoir 102. In some embodiments, air can be pumped through the culture reservoir 102 to disperse settled cells and / or homogenize the culture before releasing it from the culture reservoir 102. This can be particularly advantageous in embodiments where the optical density of the culture (e.g., A600) is measured within the optical density window 105, as more accurate readings can be obtained from a homogeneous culture following release from the culture reservoir 102. The optical density of the culture may be reported to the user and / or used by the instrument to indicate, for example, a low yield due to insufficient input material (low OD) or a low yield due to overloading the system (high OD).

[0120] Regardless of whether optical density measurements are performed, the culture reservoir 102 is opened (e.g., by puncturing a fragile seal between the culture reservoir and the fluid channel), and the contents of the culture reservoir 102 are pumped through the fluid channel in the cartridge 100 to the cell capture filter 106 (e.g., via pump 104). At the cell capture filter 106, the culture medium in the biological sample passes through the filter 106 into the waste reservoir 108. Plasmid DNA containing bacterial cells in the biological sample is retained in the cell capture filter 106. In some embodiments, the cell capture filter 106 may be a size-selective filter with a pore gradient of 0.65 μm to 1.2 μm (e.g., a 7.5 mil thick nylon cell capture filter). Additionally, in some embodiments, it is desirable that the cell capture filter withstand pressures above 20 psi, above 30 psi, above 40 psi, or above 60 psi without leakage, as cell accumulation on one side of the filter can lead to low flow conditions and increased pressure on the filter. In some embodiments, a compliant support gasket (e.g., an elastomer layer localized to the cell capture filter) may be added around the filter on the opposite side of the resident elastomer layer between the outer layers of the cartridge to provide a better sealing interface between the cartridge and the instrument.

[0121] As shown in the simplified cross-sectional view of the cell capture filter 106 (support gasket shown as element 107) in Figure 5A, the filter 106 is sandwiched between the outer layers 115a and 115b of the cartridge 100. A biological sample can be drawn from the culture reservoir 102 between the first outer layer 115a and the first side of the filter 106. By suction or pumping action from the pump 104, the culture medium in the biological sample passes through the filter 106 and enters the space provided between the second side of the filter 106 and the second outer layer 115b. The culture medium is then directed along the fluid conduit and through the release valve to the waste reservoir 108.

[0122] As shown in Figure 5B, RNase A can be passively mixed with the resuspension buffer and lysis buffer by piercing the resuspension buffer reservoir 110 with a fragile seal that separates the resuspension buffer reservoir 110 from the RNase A reservoir 112 and the lysis buffer reservoir 114. In some embodiments, the lysis buffer reservoir 114 may have a larger volume overhead to accommodate the volumes of RNase A and resuspension buffer, and a gravity flow from the resuspension buffer reservoir 110 through the RNase A reservoir 112 into the lysis buffer reservoir 114 can allow for passive mixing of these buffers and reagents in the lysis buffer reservoir 114 to create a resuspension / lysis buffer combination.

[0123] As shown in the inserted cross-sectional view of the cell capture filter 106 in Figure 5B, the combined resuspension / lysis buffer can be backwashed over the filter 106 to recapture cells from the filter 106, incorporate them into the resuspension / lysis buffer, and transfer the cell-containing solution to the first active mixing chamber 116. As shown in Figure 5B, the pump 104 can be reversed to allow backwashing over the filter 106 and transfer to the active mixing chamber 116.

[0124] As shown in Figure 5C, cells in the resuspension / lysis buffer can be lysed under active mixing in the mixing chamber 116. Similar to other active mixing chambers discussed below, the first active mixing chamber 116 may include a magnetic stirring rod (or other means for stirring and / or mixing the solution in the chamber 116) that is operable by the purification equipment to actively mix the contents of the mixing chamber 116. In some embodiments, the magnetic stirring rod can stir the mixture at 1000 rpm or more.

[0125] Following the formation of cell lysates in the mixing chamber 116, the neutralizing buffer can be automatically released from the neutralizing buffer reservoir 118 into the mixing chamber 116 (for example, by penetrating the easily breakable seal associated with the neutralizing buffer reservoir 118, allowing for passive transfer from the neutralizing buffer reservoir 118 to the mixing chamber 116). Actively mixing the neutralizing buffer with the lysates allows the pH of the lysates to be restored to normal, leading to protein precipitation from the lysates and the regeneration of genetic material. Due to its circular nature, plasmid DNA can be properly regenerated and maintain its solubility, while genomic DNA is crushed from the solution due to the random association of its strands. The precipitated cell debris and bound genomic DNA become insoluble in the solution and, as a result, can be separated from the solution.

[0126] Referring here to Figure 5D, a smaller amount of ER buffer may be pre-released from the ER buffer reservoir 124 into the second active mixing chamber 122 to improve subsequent contact with the following fluid. Simultaneously with the release of the ER buffer, or within a short period before or after, the neutralized lysate can pass through the purification filter 118. The purified lysate (i.e., the portion of the lysate containing the target nucleic acid) is pulled through the purification filter 118 and pumped by the pump 120 into the second active mixing chamber 122. The waste-containing portion of the neutralized lysate remains trapped within the purification filter 118.

[0127] In some embodiments, the purification filter tends to disintegrate on its own, closing its porous structure during flow, thereby preventing the filter from performing its desired function. To prevent this, some embodiments may include a support over the entire range of the purification filter and a thin, semi-rigid porous backing structure instead of a periphery support gasket to prevent the purification filter from disintegrating during flow, thereby enabling the filter to perform its desired function. The purification filter may be any purification filter known or used in the art that is suitable for separating the waste-containing portion of a neutralized soluble from the nucleic acid-containing portion of the neutralized soluble. For example, the purification filter may include a glass fiber filter having a pore size greater than about 1 μm and / or less than about 5 μm and a thickness greater than 30 mils. For example, the purification filter used in the cartridge of the present disclosure may include a Glass Fiber A filter having a pore size of 4.3 μm and a thickness of 45 mils, or a glass fiber filter having a pore size of 1 μm with a thickness in the range of 43 mils to 53 mils. Glass fiber-based purification filters can be paired with any suitable semi-rigid purification support, such as a 1 / 16-inch thick polyethylene purification support filter having a pore size greater than 15 μm (e.g., pore size of 15-45 μm).

[0128] The purified lysate is drawn through the purification filter 118 and pumped to a second active mixing chamber 122, where it is actively mixed with the ER buffer. The purified lysate / ER buffer may be actively mixed in the second active mixing chamber 122 using a magnetic stirring rod, as provided above with respect to the first active mixing chamber 116, etc., to remove contaminants from the purified lysate for increased sample purity.

[0129] Referring to Figure 5E, the purified lysate is pumped from the second active mixing chamber 122 through the anion exchange membrane structure 126 via the pump 128. Plasmid DNA in the purified lysate binds to the anion exchange membrane structure 126 due to favorable ionic interactions with the charged column, and the waste-containing portion of the purified lysate (i.e., the non-target nucleic acid-containing portion) passes through the anion exchange membrane structure 126 and is deposited in the waste reservoir 130.

[0130] As shown in the inserted cross-sectional view of Figure 5E, the anion exchange membrane 126 is structured to attempt to create axial flow through the stack of anion exchange membranes. As shown in the exemplary cross-sectional schematic, a nearly vertical wall approximately 9 mm high is created by the inlet-side intermediate layer 117 and the first outer layer 115a. The aperture formed by the intermediate layer creates an inlet from the fluid channel formed by the outer and intermediate layers of the cartridge to the housing that holds the stack of anion exchange membranes 126. As the fluid is pulled through the membrane stack 126, the membrane can ionically capture plasmid DNA while allowing waste material to pass through the filter stack 126 into the waste reservoir 130. It should be understood that axial flow through the anion exchange membrane 126 can be created in different ways or by using stacked membranes of different heights.

[0131] In some embodiments, the anion exchange membrane is a silicon-treated glass fiber membrane. The glass fiber anion exchange membrane can be paired with any suitable semi-rigid support, such as a 1 / 16-inch thick polyethylene support filter having a pore size greater than 15 μm (e.g., a pore size between 15 and 45 μm).

[0132] After the neutralized lysate is passed through the anion exchange membrane 126, the wash buffer from the wash buffer reservoir 132 is automatically released, as shown in Figure 5F, and pulled through the anion exchange membrane structure 126 via the pump 128. The wash solution passing through the anion exchange membrane 126 is collected in the waste reservoir 130. In a preferred embodiment, the wash solution is selected so as not to interfere with the membrane-bound plasmid DNA and acts to remove excess material in association with the plasmid DNA and / or filter 126.

[0133] Referring here to Figure 5G, isopropanol may be automatically released from the isopropanol reservoir 138 into the third active mixing chamber 136 to improve contact with the subsequent fluid. Simultaneously with the release of isopropanol, or within a short time interval before or after, the elution buffer may be automatically released from the elution buffer reservoir 134 and through the anion exchange membrane structure 126 via the pump 128. The elution buffer can provide a more favorable chemical interaction with the plasmid DNA than the anion exchange membrane, detaching the plasmid DNA from the membrane and eluting it into the passing elution buffer. This eluent is pumped through the anion exchange membrane structure 126 to the third active mixing chamber 136. In the third active mixing chamber 136, the eluent, consisting of the elution buffer and plasmid DNA, mixes with the isopropanol, and the plasmid DNA precipitates from the elution buffer along with several salts.

[0134] The precipitated mixture formed in the third active mixing chamber 136 then passes over the precipitate membrane 138, as shown in Figure 5H. Plasmid DNA in the precipitated mixture becomes bound to the precipitate membrane (for example, based on the pore size of the precipitate membrane), while smaller soluble components and elution buffer pass through the precipitate membrane and are collected in the waste reservoir 142. In some embodiments, the precipitate membrane, having a pore size of 1 μm or larger, may be made of or contain glass fibers. For example, the precipitate filter may include a glass fiber filter with a pore size of 1 μm or a Glass Fiber D filter with a thickness of 16 mils.

[0135] In some embodiments, the sedimentation filter may not be subjected to the same or similar pressure as the cell capture filter, but a periphery seal can be associated with the sedimentation filter to ensure that little or no fluid escapes into the inactive region of the sedimentation filter. For example, an O-ring (or other functionally similar gasket) can be positioned between the outer layers of the cartridge, as an intermediate layer of the cartridge, on the opposite side of the sedimentation filter.

[0136] The precipitate membrane 138 can be washed with an ethanol-based solution to solubilize plasmid DNA and remove salts (i.e., desalt). As shown in Figure 5I, the 70% ethanol solution is automatically released from the ethanol reservoir 144 and can be pulled through the precipitate membrane 138 via the pump 140. The ethanol washing solution, along with the solubilized salts, can pass through the precipitate membrane 138 and enter the waste reservoir 142.

[0137] Referring here to Figure 5J, the pump 140 can continue operating even after the ethanol washing solution has passed through the sedimentation filter 138. This allows air to move over the sedimentation filter 138, drying the plasmid DNA and causing the evaporation of excess fluid (particularly alcohol).

[0138] The washed and dried plasmid DNA can be eluted from the precipitation membrane 138 and transferred to the output vessel 148, as shown in Figure 5K. During this final process, the TE buffer is automatically released from the TE buffer reservoir 146 and pulled onto the precipitation membrane 138 via the continuous action of the pump 140, allowing the plasmid DNA to be eluted from the precipitation membrane 138 into the passing TE buffer. By closing and opening different valves in the cartridge 100, the eluted plasmid DNA can be routed from the precipitation membrane 138 to the output vessel 148, rather than to the waste reservoir 142, as the fluid before passing through the precipitation membrane 138.

[0139] The output container 148 can be any container suitable for receiving the purified nucleic acid. For example, the output container 148 can include a standard microcentrifuge tube. To ensure that the purified nucleic acid is transferred to the output container rather than remaining attached to the cartridge, the cartridge 100 can incorporate a unique shape that overcomes the surface tension between the cartridge material and the purified nucleic acid. In particular, the unique shape (illustrated by arrow 152 in Figure 6) prevents or reduces the amount of purified nucleic acid adhering to the outer walls 115a, 115b of the cartridge rather than flowing down into the output container.

[0140] The surface energies of the outer walls 115a and 115b are generally greater than those of the elastic intermediate layer 117 (for example, the outer walls may be made of or contain PVC, and the surface energy of PVC is much greater than that of most elastic intermediate materials such as silicon), which allows nucleic acids to preferentially adhere to the outer layer instead of the elastic intermediate layer. Thus, the output container 148 is positioned within the cartridge 100 such that the center of the outer layer 115b is aligned with the center and / or central axis of the output container 148. In some embodiments, the outer layer aligned in this manner may include small tabs that facilitate droplet formation and deposition onto the output container.

[0141] Once the purified nucleic acid is received in the output container 148, the automated nucleic acid purification protocol is completed, and the output container 148 can be removed from the cartridge 100 by the user via an easily accessible receptacle area.

[0142] The specific arrangements, sizes, and shapes of the various components of cartridge 100 illustrated and discussed throughout Figure 4 and Figures 5A–5K above are illustrative in nature, and it should be understood that other arrangements, arrangements, shapes, and sizes of the various components are included within the scope of this disclosure. For example, another embodiment of the exemplary cartridge 160a is provided, configured for use with a system for automated purification of target nucleic acids from biological samples, as shown in Figure 7A. As shown, cartridge 160a contains many of the same or similar reagents, buffers, filters, and membranes as cartridge 100 in Figures 4 and 5A–5K. However, cartridge 160a includes a single waste reservoir 164 instead of the three separate waste reservoirs 108, 130, and 142 provided in cartridge 100 in Figures 4 and 5A–5K. In examples where waste and cartridges are located separately, a single waste reservoir, such as waste reservoir 164 in Figure 7A, can be beneficially enabled to empty a single waste reservoir instead of the user having to access multiple separate waste reservoirs.

[0143] As a further example of the differences between cartridge 100 in Figures 4 and 5A-5K and cartridge 160a in Figure 7A, cartridge 160a includes an additional pump 162 positioned upstream of the lysis buffer reservoir 114 and downstream of the cell capture filter. By positioning the pumps on both sides of the cell capture filter, aspiration can be improved, for example, when passing a biological sample from the culture reservoir to the cell capture filter, and additionally when backwashing the resuspended / lysis buffer on the cell capture filter.

[0144] Another embodiment of exemplary cartridge 160b, configured for use with a system for automated purification of target nucleic acids from biological samples, is provided in Figure 7B. As shown, cartridge 160b contains many of the same or similar reagents, buffers, filters, and membranes as cartridges 100 and 160a in Figures 4, 5A–5K, and 7A. Similar to cartridge 160a in Figure 7A, cartridge 160b in Figure 7B contains a single waste reservoir instead of the three separate waste reservoirs provided in cartridge 100 in Figures 4 and 5A–5K.

[0145] As a further embodiment of the difference between cartridge 160b in Figure 7B and that illustrated in Figure 7A, cartridge 160b includes a pressure sensor 161 positioned between the cell capture filter and the first mixing chamber. The pressure sensor 161 can be used, for example, to monitor the pressure in the cartridge during cell capture and / or elution of cells / cellular components from the cell capture filter. As provided above, the pressure in the cartridge may rise as the cell capture filter accepts more and more cells. The pressure sensor 161 can be used to prevent the pressure in the cartridge from rising to a breakpoint where the cartridge's filter or other components fail. Additionally, the pressure sensor 161 can be used as a means to regulate the pumping action of the system. For example, the pump may be powered using a threshold-based on / off hysteresis scheme in which the pump is fully on until it reaches a predetermined upper threshold (e.g., 10 psi), then remains fully off until it reaches a predetermined lower threshold (e.g., 2 psi), at which point the pump is turned on again until the pressure reaches the upper threshold again. It should be understood that thresholds can be adjusted at the system level during manufacturing and / or by the user through configurable settings (e.g., by accessing the user interface associated with the system).

[0146] As a further example, a pressure sensor can be used to regulate a pump according to a PID (proportional / integral / derivative) control loop that attempts to actively control the pump's duty cycle (e.g., speed) to maintain a pressure setpoint. In this case, instead of the pump turning off completely when an upper threshold is reached, the pump speed can be reduced as it approaches the upper threshold so that the pressure can be maintained at or near the upper threshold and the pumping action can continue.

[0147] As further illustrated in Figure 7B, the TE buffer reservoir can be partially divided into two separate outlet ports. This allows for beneficially multiple TE buffer washing cycles while maintaining a single reservoir. For example, the first volume of TE buffer can be washed through a third mixing chamber to wash away residual ethanol from the chamber to the waste. The remaining TE buffer can then be used to wash the entire precipitation filter to solubilize the retained target nucleic acid for collection in the product tube. This intermediate washing step can increase the efficiency and / or concentration of the plasmid obtained from the precipitation filter.

[0148] Another embodiment of exemplary cartridge 160c, configured for use with a system for automated purification of target nucleic acids from biological samples, is provided in Figure 7C. As shown, cartridge 160c contains many of the same or similar reagents, buffers, filters, and membranes as cartridges 100, 160a, and 160b in Figures 4, 5A–5K, 7A, and 7B. As shown in Figure 7C, cartridge 160c can remove RNase A from a triple stack of lysis buffer and resuspension buffer for addition to a first mixing chamber, following the resuspension of cells from the cell capture filter. For example, in the exemplary operation, cells can be captured on the cell capture filter as described above. However, instead of washing the cells from the cell capture filter with a combined solution of lysis buffer, RNase A, and resuspension buffer, the cells can be resuspended from the filter into the first mixing chamber through a backwash step (similar to that described above with respect to Figure 5A). Next, the lysis buffer may be forced into the first mixing chamber through a cell capture filter to induce cell lysis. RNase A may be pre-loaded into the first mixing chamber and discharged into the first mixing chamber simultaneously with the resuspension buffer carrying the cells from the filter into the mixing chamber, or when the lysis buffer is washed into the first mixing chamber. It should be understood that in some embodiments, the lysis buffer and resuspension buffer may be combined before the cells are resuspended from the cell capture filter into the first mixing chamber.

[0149] Cartridge 160c in Figure 7C may additionally have a recirculation fluid channel associated with a third mixing chamber. The recirculation channel can be used, for example, to recirculate the first TE buffer rinse through the third mixing chamber before moving the rinse to a waste reservoir, as a preparation for passing the TE buffer (or other eluent) through the precipitation filter. This can advantageously increase the efficiency, purity, and / or concentration of the target nucleic acid eluted from the precipitation membrane.

[0150] Additionally or alternatively, the cartridges of this disclosure may include one or more additional bypasses or vents. For example, a vent may be associated with a cell capture filter to prevent the pump pressure from exceeding a threshold pressure. This can beneficially prevent destructive forces from being applied to the cartridge due to filter clogging. Bypass valves may be used additionally to move unfiltered media to waste. In some embodiments, such bypass valves may be initiated to recover media loaded into the cartridge that are not to be recollected or otherwise processed.

[0151] Additionally, or alternatively, multiple windows can be arranged throughout the cartridge so that the presence of fluid can be determined (for example, by measuring the optical density of a channel that coincides with one of the windows). This may be beneficial for dynamic sample handling, such as processing samples that may require longer filtration times (e.g., stool samples or high-density bacterial cultures versus water or urine samples).

[0152] It should be further understood that the cartridge layouts and designs disclosed herein can accommodate the custom preparation of solutions and reagents using a series of component reservoirs. For example, instead of having a single reservoir for each of the lysis buffer, resuspension buffer, etc., the cartridge may have separate reservoirs for each component of the buffer or reagent, and can produce specified amounts of each buffer or reagent as needed (e.g., by mixing in a mixing chamber before dispensing, and / or by dispensing directly to the sample).

[0153] Therefore, the organization, layout, and size of the various components within the purification cartridge can be adjusted and / or modified based on the number and type of filters / membranes and / or buffers / reagents used in the purification protocol, as well as the quantities and steps associated with processing different biological samples and / or various different purification protocols, as can be understood by those skilled in the art.

[0154] fluid release mechanism The following disclosure relates to exemplary embodiments of systems for automated nucleic acid purification that may be used during such automated purification processes to selectively release fluid from the input reservoir, buffer / reagent reservoir, mixing chamber, or other areas of a purification cartridge. The fluid release system may include interacting components from the disclosed purification equipment and purification cartridge. For example, an actuator portion of the fluid release system may be associated with the purification equipment and thereby interact with a flexible area of ​​the purification cartridge to release fluid under control.

[0155] Overview of the fluid release system Nucleic acid purification procedures are manual testing processes that require specialized skills and can take many hours to perform. Automation has proven difficult, at least in part, due to the many different fluids required at specific points in the purification process. Previous attempts focused on replicating manual processes using robotics, but these approaches require significant capital investment in expensive robotic equipment, in addition to the additional costs associated with the testing equipment typically used during manual processes (centrifuges, vacuum manifolds, etc.). Furthermore, these approaches failed to reduce the number and types of consumables, as they relied primarily on filtration columns and buffers provided in commercially available purification kits.

[0156] Additionally, previous approaches have failed to improve the mechanism for fluid release during nucleic acid purification processes. Instead, previous approaches continue to move various fluids by pipetting, as in manual processes, or by dispensing aliquots from a large reservoir into various tubes or filtration columns. In the former case, in addition to the need to monitor and replace these additional consumables, there is an increase in additional costs and processing time due to the necessary loading and ejection of pipette tips. As for the latter, there is a high risk of cross-contamination between samples, and special time and care are required to clean and / or sterilize tubes or injection ports between samples. In either case, the proposed solutions fail to address the significant need for a self-contained fluid release system that reduces the number and / or type of consumables associated with fluid release / transfer during nucleic acid purification processes and reduces or eliminates the risk of cross-contamination between sample preparations.

[0157] Therefore, designing a system capable of selectively releasing the appropriate fluid (e.g., sample, buffer, reagent, or combination thereof) during an automated nucleic acid purification procedure presents several challenges. As will be described in more detail below, the fluid release systems described herein can satisfy one or more of the above challenges by including a flexible gasket positioned between one fluid reservoir and the other actuator. The actuator is operable to deflect the flexible gasket, causing it to break a fragile seal holding the fluid in the reservoir, thereby selectively releasing the fluid from the reservoir.

[0158] In some embodiments, a flexible gasket, a fragile seal, and a reservoir are components of the purification cartridge, and an actuator is a component of the associated purification equipment. This beneficially allows for the controlled release of fluid from the consumable cartridge without cross-contamination of the sample. The actuator interfaces with a first side of the flexible gasket and deforms the gasket toward the fragile seal by pushing down on the flexible gasket from the first side, causing the opposite side of the gasket to interact with the fragile seal and break. In this way, the actuator is separated from the fluid by the interposed gasket and does not come into direct contact with the fluid. The actuator can be repeatedly used to puncture the fluid container without requiring cleaning or disposal between or within sample processing, and with little or no risk of contamination.

[0159] As an additional advantage, the disclosed fluid release system simplifies the fluid release process and enables faster processing times while being essentially independent of the amount of fluid being released. That is, the disclosed fluid release system can be used to release small or large amounts of fluid, as the components and processes for fluid release are essentially independent of the reservoir size. Furthermore, the mechanical means for releasing the fluid are simple, meaning that complex mechanisms that may prove unreliable over time are not required. This can enhance the reliability, repeatability, and long-term usefulness of the system, particularly in embodiments utilizing single-axis action.

[0160] fluid release mechanism Referring here to Figures 8A–8C, exemplary embodiments of a fluid release system for retaining and selectively releasing fluid are illustrated. As shown, the system includes an actuator 166 and a fluid reservoir 168. The fluid reservoir 168 is configured to retain fluid and is at least partially bounded by an outer layer of a cartridge (e.g., outer layer 115a) and a fragile seal 170 that acts to retain fluid within the reservoir 168 in a non-destructive state.

[0161] As used herein, the term “easily breakable seal” is intended to include a punctureable material configured to rip, break, or otherwise catastrophically cease to function in response to the application of a mechanical force, thereby ceasing to act as a seal. Easily breakable seals of this disclosure may consist of a chemically inert material facing the reagent side of the associated channel or reservoir. In one embodiment, the easily breakable seal includes a thin layer of chemically inert material configured to rupture in response to an external mechanical force while maintaining a liquid-tight seal. In some examples, the inert material is a plastic such as polypropylene, polyvinyl chloride, or polystyrene. The easily breakable seal may additionally include a second reinforcing layer, for example, made of metal foil. The second reinforcing layer may be fused with the inert material to form a functionally single layer such that a rupture of either the plastic or the reinforcing layer is functionally equivalent to a rupture of both layers. Other suitable materials for easily breakable seals may be selected as known in the art, for example, easily breakable seals formed on pharmaceutically filled cavities of pre-formed plastic packaging (e.g., blister packs), or other easily breakable seals formed on certain consumer foods. For example, a foil layer with nearly zero water vapor loss may be fused and / or used with a second reinforcing layer such as Aclar to further slow and / or prevent water vapor loss.

[0162] A flexible gasket 172 is positioned between the easily breakable seals 170 in the actuator 166. As shown in Figure 8A, the flexible gasket 172 is a component of the refining cartridge. The flexible gasket may be part of an intermediate layer 117 positioned between two outer layers 115a, 115b. For example, the flexible gasket 172 may be an extension of an elastomer layer sandwiched between the inner and outer thermoformed layers of the cartridge, as in the cartridge described herein. In such embodiments, the fluid reservoir 168 may be at least partially defined by the flexible gasket 172 in one of the two outer layers of the cartridge. As described in more detail herein, the outer layers of cartridges 115a, 115b may be made of or include thermoformed plastic.

[0163] In some embodiments, and as shown in Figure 8A, the flexible gasket 172 forms a channel or conduit 174 through which fluid can pass after being released from the fluid reservoir 168. To selectively release fluid from the reservoir 168, the actuator 166 is moved from a first position (indicated by arrow A) to a second position (indicated by arrow B in Figure 8B). In doing so, the actuator 166 contacts the first side of the flexible gasket 172, and as it continues to move axially toward the flexible gasket 172, the actuator 166 deflects the flexible gasket 172 toward the fragile seal 170.

[0164] As shown in Figure 8B, the actuator 166 can move a certain distance toward the reservoir 168, causing the deflected flexible gasket 176 to penetrate or puncture the fragile seal 170. In this way, the actuator 166 does not directly contact the fragile seal 170 or the contents of the fluid reservoir 168. Instead, the surface of the deflected flexible gasket 176 contacts and ruptures the fragile seal. Some fluid from the reservoir 168 may pass between the deflected flexible gasket 176 and the ruptured seal 178, but as shown in Figure 8C, when the actuator 166 is retracted, the flexible gasket 172 returns to its original position (for example, due to its elastic properties), providing a transparent conduit 174 through which the fluid can exit the fluid reservoir 168 via the aperture 180 formed by the ruptured seal 178.

[0165] It should be understood that the fluid reservoir 168 can be any reservoir or chamber in the purification cartridge, including, for example, an input reservoir, a buffer / reagent reservoir, a mixing chamber, etc. As such, the fluid in the fluid reservoir 168 may be any of the following: input sample, resuspension buffer, RNase A, proteinase K, lysis buffer, neutralization buffer, binding buffer, endotoxin removal buffer, washing buffer, elution buffer, isopropanol, 70% ethanol, TE buffer, water, a combination of the above, or any other fluid, reagent, buffer, enzyme, or mixture used or formed within the nucleic acid purification protocol. Additionally, the conduit 174 may provide a path for the released fluid to move between the reservoir 168 and the next or final destination 182 in the purification cartridge.

[0166] The systems illustrated in Figures 8A to 8C may include components from the nucleic acid purification cartridge and associated equipment. The systems illustrated in Figures 8A to 8C may also include components from the protein purification cartridge and associated equipment. For example, actuator 166 may be a component of the purification equipment, while the fluid reservoir 168, associated fragile seal 170, and flexible gasket 172 may be components of the purification cartridge, as described in more detail herein. As such, the movement of actuator 166 may be controlled by equipment that includes computer-executable instructions or other programmable elements that enable time- or situation-dependent fluid release according to a predetermined or user-defined protocol, as described above. This can beneficially enable dynamic implementations of various nucleic acid purification protocols using any of the countless combinations of buffers, reagents, and processing parameters without requiring changes to the physical components of equipment such as actuators. In other words, the disclosed fluid release system allows for multiple uses in a single hardware configuration.

[0167] However, it should be understood that the placement of actuators within nucleic acid or protein purification equipment can also be dynamic. In some embodiments, the purification equipment may have a single actuator that is aligned with various fragile seals and repositioned throughout the purification protocol to cause the rupture of various fragile seals at different times. Additionally or alternatively, the purification equipment may have multiple actuators that are aligned with or movable between corresponding fragile seals on the purification cartridge.

[0168] In some embodiments, the fragile seal 170 is a foil seal. Alternatively, the fragile seal may be made of, or contain, any suitable material that can withstand the pressure applied by the fluid in the associated reservoir or chamber, but will rupture if the force applied by the flexible gasket via the actuator becomes strong. In some embodiments, the thickness of the fragile seal may be adjusted to take into account different reservoir pressures and / or actuator forces.

[0169] In some embodiments, and as illustrated in Figures 8A–8C, the actuator may have a pointed head. This advantageously allows the force applied by the actuator to be concentrated over a smaller area of ​​the fragile seal, thereby causing localized catastrophic defects in the fragile seal that can expand outward to widen the aperture. However, it should be understood that other shapes or forms of actuators may be provided to achieve the same or similar results. For example, exemplary actuators may have tip diameters of 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 7 mm, 10 mm, or any smaller than those mentioned above. The tip diameter can also be selected from any diameter within the range enclosed by two of the aforementioned diameters (e.g., between 1 mm and 10 mm, between 2 mm and 5 mm, between 3 mm and 7 mm, between 2 mm and 4 mm).

[0170] Flexible air vent In some applications, releasing fluid through the mechanisms described above can create negative pressure within the reservoir as the fluid escapes. This can lead to incomplete fluid release from the reservoir (e.g., in a closed system), or alternatively, intermittent flow from the fluid reservoir and / or audible flow of air into the fluid reservoir (e.g., in an open system). Each of the aforementioned can negatively impact the efficiency of the automated purification process and / or the purity or concentration of the output target nucleic acid or output target protein.

[0171] For example, incomplete release can alter the desired chemical properties if the fluid mixes with a suspension, lysate, or eluate, causing different or less optimal effects. For instance, if a reservoir with incomplete release contains wash buffer or endotoxin removal buffer, the outputted target nucleic acid may be contaminated or unsuitable for its intended purpose. As a non-limiting example, the target nucleic acid may be a plasmid intended for use in a mammalian cell culture assay, and if the plasmid is purified from Gram-negative bacteria and incompletely treated with the required amount of endotoxin removal buffer, toxic or inflammatory concentrations of lipopolysaccharide may remain associated with the purified plasmid DNA, making it unsuitable for its intended purpose.

[0172] Intermittent flow into the fluid reservoir and / or gurgling of air can similarly negatively impact the efficiency of the automated purification process and / or the purity or concentration of the output target nucleic acid or target protein. For example, if the fluid contains surfactants, gurgling of air into the fluid reservoir can lead to incomplete fluid release, or cause the fluid under tension to foam or bubble. Many lysis buffers contain surfactants, and incomplete association between the lysis buffer and the input biological sample can reduce the amount of target nucleic acid that can be purified.

[0173] The fluid release systems of this disclosure can solve one or more of the aforementioned problems. For example, an exemplary fluid release system may be configured to discharge air into the fluid reservoir to facilitate the complete or uninterrupted release of fluid from the reservoir. In some embodiments, and as illustrated in Figures 9A and 9B, the fluid release system may include an air vent system 184 associated with the fluid reservoir 168. The air vent system 184 may be operable to rupture a fragile air seal 190 associated with the fluid reservoir 168, thereby routing air into the reservoir 168.

[0174] As shown in Figures 9A and 9B, the air vent system 184 may be part of the purification cartridge. System 184 may include a flexible air vent 188, similar to the flexible gasket described above, and may be part of an intermediate layer 117 located between the two outer layers 115a, 115b of the cartridge. In some embodiments, the flexible air vent 188 is an extension of the elastomer layer and may partially define the fluid reservoir 168.

[0175] As shown, the flexible air vent 188 is separated from fluid communication with the reservoir 168 by the fragile air seal 190. To selectively discharge air into the reservoir 168, an actuator (not shown) can be brought into contact with the flexible air vent 188 and moved in a continuous axial motion toward the reservoir 168 (e.g., from left to right as shown in Figure 9A), and the actuator can deflect the flexible air vent 188 toward the fragile air seal 190 until the deflected air vent contacts and perforates the fragile air seal 190 or otherwise bursts. Similar to the flexible gasket described above, the flexible air vent 188 can return to its non-deflected position following the removal of the deflection force applied by the actuator.

[0176] The flexible air vent 188 can form a channel or conduit 192 between the fluid reservoir 168 and the outside air (for example, through an aperture 186 formed by a ruptured air seal and air vent 188). As shown, access from the outside air to the conduit, conduit 192, can travel longitudinally along the fluid reservoir 168 to begin at the aperture 186 and terminate at or beyond the easily ruptured air seal 190. In some embodiments, the easily ruptured air seal 190 can be positioned at the top of the fluid reservoir 168 in a direction away from gravity, and an easily ruptured seal (not shown) for releasing fluid from the reservoir 168 can be positioned at the bottom of the fluid reservoir in a direction toward gravity. In this orientation, rupture of the easily ruptured seal and easily ruptured air seal may result in air entering at the top of the fluid reservoir, where the pressure is equalized, and the fluid exiting the reservoir through the bottom of the fluid reservoir due to gravity. This can facilitate the complete release of fluid from the reservoir and prevent blockage or gurgling caused by uneven pressure gradients between the fluid reservoir and the outside air.

[0177] In some embodiments, the fragile air seal may be ruptured before the fragile seal so that the pressure in the fluid reservoir can equalize with the outside air before releasing the fluid therefrom. Alternatively, the fragile air seal may be ruptured after the fragile seal to provide equilibrium pressure when the reservoir is empty. For fluid reservoirs that have reached or are near capacity, rupturing the fragile air seal after the fragile seal may reduce the likelihood of fluid escaping through the ruptured air seal when the fluid level may be lower and / or when the amount of available headspace in the fluid reservoir associated with the fragile air seal may be greater. In some embodiments, the fragile air seal may be ruptured by the same actuator as the fragile seal described above.

[0178] In some embodiments, the easily breakable air seal and the easily breakable seal are ruptured simultaneously or nearly simultaneously. Thus, the easily breakable seal can be ruptured by different actuators. Additionally, the movement of the actuator relative to the easily breakable air seal can be controlled by a purification device that includes computer-executable instructions or other programmable elements that enable time- or situation-dependent air discharge according to a predetermined or user-defined protocol, as described above. This can be beneficially enabled to control the fluid flow throughout the nucleic acid purification process without the actuator coming into contact with any fluid or otherwise contaminating the sample.

[0179] Protein purification cartridges, instruments, and systems This disclosure relates to cartridges, automated systems, and methods for purifying target biomolecules from biological samples. The aforementioned discussions, particularly those relating to Figures 3A–5K and 7A–C, illustrate various exemplary cartridge designs and processing workflows for the automated purification of target nucleic acids. Similarly, embodiments disclosed herein may include similar cartridges and processing workflows for the automated purification of different target biomolecules, namely target proteins.

[0180] Protein purification is essential for characterizing the function, structure, and interactions of proteins. Various steps in the purification process may include cell lysis, separation of soluble protein components from cell debris, and finally, separation of the target protein from product and process-related impurities. However, many existing methods for protein extraction and purification from liquid cell cultures are time-consuming, primarily manual processes. These methods require technical expertise and specialized equipment to perform the various steps of the method. For example, one typical procedure involves centrifugation of the liquid cell culture to collect cells, followed by resuspension of the resulting cell pellet in lysis buffer to lyse the cells. The lysate is then purified through an additional centrifugation step, and the purified lysate is decanted for collection. The purified lysate can then be passed through a protein purification column (or other filter, resin, or protein capture means) to further purify the desired protein for collection in elution buffer for further downstream workflows.

[0181] The automated systems and associated cartridges described herein enable the automation of steps in the protein purification process and may include the automation of the entire protein purification process after a biological sample containing the target protein has been added to the corresponding protein purification cartridge and operational automated system. For example, the cartridges and systems disclosed herein are adapted for use in purifying and / or isolating proteins from bacterial and / or eukaryotic cultures. In one embodiment, the target protein is produced by or otherwise associated with eukaryotic cells such as yeast (e.g., Saccharomyces cerevisiae), or insect cells, or rodent cells such as CHO cells (e.g., mouse, rat, or hamster cells), or primate / human cells such as 293 cells or COS cells. In another embodiment, the target protein is produced by or otherwise associated with prokaryotic cells such as E. coli (e.g., as recombinant protein). Regardless of cell type, the automated protein purification systems and associated cartridges and methods can be adapted to purify and collect the target protein therefrom.

[0182] Similar to the targeted nucleic acid purification systems and cartridges described above, the automated protein purification cartridges of this disclosure may be adapted to receive a biological sample containing a target protein via an associated culture reservoir and to process the biological sample through several interconnected bioprocessing assemblies to obtain the purified target protein. For example, after a biological sample containing a target protein (e.g., a culture of prokaryotic / eukaryotic cells) is added to the targeted protein purification cartridge, the cartridge may be processed as described above in a first bioprocessing assembly configured to isolate the cell fraction from the sample / culture supernatant. In instances where the target protein is a secreted protein, it should be understood that the supernatant may be further processed instead of being discarded. Furthermore, in instances where the target protein is cellular, the cell fraction of the biological sample / culture may be captured in a cell-trapping membrane, with the supernatant being discarded. It should be understood that the bioprocessing assemblies and reagents disclosed above for nucleic acid purification may be modified for protein purification.

[0183] As a non-limiting example, cells from a suspension may be added to a target protein purification cartridge and passed through a filter, where the cells are captured by filtration on a first side of the filter. The filtrate may pass through the filter, past its second side, and be directed to a waste receptacle. Completion of this and each subsequent step may be optionally detected by an air / bubble sensor or by any other method for detecting the presence / absence of fluid flow or threshold fluid velocity, as disclosed herein and / or known to those skilled in the art. As described above, the target protein purification cartridge may be associated with a complementary automated purification system that utilizes a pumping action to draw a biological sample through the filter in a first flow direction, and additionally, a second operating state may follow to facilitate the flow of liquid in the opposite second flow direction.

[0184] In some embodiments, a suitable cell capture filter system is selected for use in a target protein purification cartridge, primarily based on its flow rate and filter capacity. Other filter properties, such as pore size, may be one consideration in selecting a suitable cell capture filter system (e.g., for capturing cells containing a target protein). As a non-limiting example, E. coli strains may be engineered to produce recombinant target proteins. Cultures of these E. coli may be captured on a membrane system in an automated purification cartridge equipped with a 0.2 μm hollow fiber filter membrane. In an additional example, a cell capture filter associated with a target protein purification cartridge comprises multiple cellulose acetate porous hollow fibers bent into a series of loops. This collection of loops provides the filter with a large surface area advantageous for capturing and / or processing biological samples and / or partially purified target proteins. The open ends of these loops are potted with polyurethane or a similar polymer to form end plugs (e.g., machined to ensure the open ends of the loops remain open). In one embodiment, the hollow fiber filter may be a MEDIAKAP® filter, such as the MEDIAKAP-25 filter, which is commercially available from SPECTRUM® Laboratories, Inc. Other filters and / or cell capture systems may be used according to the presence of size, polarity, and / or affinity tags, in addition to any other features or properties of the target protein, as are known in the art.

[0185] The bioprocessing assembly within the target protein purification cartridge of this disclosure may additionally include a fluid mixing chamber and / or a solid support for processing one or more samples. The solid support may be any support for filtration, washing, staining, elution, collection, processing, or carrying out chemical reactions or bioprocessing. In some embodiments, the solid support may be selected from one or more of the following: a rigid planar solid support containing a filter cassette, filter paper, precipitation membrane, precipitation filter, solid-phase extraction column, solid-phase extraction cassette, solid-phase extraction disc, a membrane such as a resin, blotting membrane, filter membrane, PVDF membrane, nylon membrane, positively charged nylon membrane and nitrocellulose membrane, reaction beads such as glass beads and magnetic beads, a biomolecular array such as a protein array or tissue array, a microscope slide, and a combination thereof.

[0186] In some embodiments, the solid support in one or more bioprocessing chambers may comprise filter paper, filters, or filter cassettes. The filter paper, filters, or filter cassettes may comprise any suitable type of filter having appropriate chemical properties, pore size, shape, three-dimensional configuration such as a symmetric or asymmetric three-dimensional configuration including a "V" or funnel-shaped pore configuration, and / or surface area for the intended application. That is, the solid support may be selected based on the properties of the target protein and / or the desired purification protocol. Additionally, the solid support may be constructed for through-fraction, cross-flow, tangential flow, or any combination thereof. It should be understood that the filter paper, filters, or cassettes may be monolayer or multilayer filters made of or containing any suitable material such as polyethersulfone, polyethylene, ultra-high molecular weight polyethylene, polypropylene, nylon, cellulose, cellulose triacetate, polyacrylonitrile, polyamide, glass fiber, silica, polysulfone, PVDF, etc. In some embodiments, the solid support in one or more of the processing chambers may be a precipitation membrane or precipitation filter, or alternatively, it may be a blotting membrane. In some embodiments, the solid support may be a solid-phase extraction column, a solid-phase extraction cassette, or a solid-phase extraction disk. In some embodiments, the solid support may include a plurality of beads, such as coated beads, coated glass beads, glass beads, magnetic beads, or coated magnetic beads. The beads may be packed into a column or suspended in a processing chamber.

[0187] As a non-limiting example, embodiments of the present disclosure may include a cartridge for purifying a target protein, having a protein capture assembly following a cell capture subassembly. The protein capture assembly may be configured to purify a lysate, or alternatively, to receive and bind the target protein from the purified lysate. It should be understood that, as such, an exemplary cartridge for purifying a target protein from a biological sample may include, in addition to one or more buffer / wash / reagent chambers, a purification and / or protein capture assembly fluidly connected to the cell capture assembly and / or mixing chamber. For example, reservoirs containing protease inhibitors, pH differentiation solutions, wash buffers, elution buffers, etc., may be contained in each fluidly connected reservoir, sealed as described herein. Omissions and / or additions of buffers and processing steps within and between the bioprocessing assemblies of a hypothetical cartridge (e.g., as shown in Figures 3A–5K and 7A–C) may be adapted to follow a protein purification protocol instead of a nucleic acid purification protocol.

[0188] In one embodiment, the automated protein purification cartridge includes a subassembly having one or more affinity matrices / columns (instead of nucleic acid purification-specific membranes / filters, for example, provided in the automated nucleic acid purification cartridges of Figures 3A-5K and 7A-C). Affinity chromatography is an effective technique for protein purification, often enabling single-step purification of proteins to a level of purification sufficient for analytical characterization. Affinity chromatography is a molecular structure-based separation technique in which molecules that "fit" with each other selectively bind in a "lock and key" manner (e.g., an antibody may recognize and specifically bind to an antigen). This technique can use application-specific chromatography resins having ligand-specific receptors (e.g., fragments of antibodies containing antigen-binding domains) attached to the resin surface. In most cases, these receptors bind to target proteins in a manner similar to antibody-antigen interactions. This highly specific fit between the receptor and its target compound also enables highly specific affinity column chromatography. Antigens bind to the resin-bound antibody (usually with high affinity), while other sample components and impurities do not bind and instead flow through the affinity column to the waste container. Next, the bound antigen (typically the target protein) can be washed and eluted from the column. Often, changing the pH causes the interaction between the molecular antigen and the antibody to be lost, resulting in a single, high-purity elution peak representing the target protein.

[0189] Accordingly, embodiments of the protein purification cartridge disclosed herein may include an affinity column contained therein that can specifically bind to and capture a target protein while allowing residual proteins and cell products to pass through the column into waste. The column may be washed to remove loosely bound or residual cell debris from the column (e.g., automatically by opening a reservoir containing a wash buffer and pumping the contents through the column). The reservoir in the cartridge may contain a solution that can operate to alter the pH of the matrix / column, thereby causing the elution of the target protein (e.g., into a collection tube). Other washing and / or purification steps may be performed automatically using systems and protein purification cartridges that are operable for such use.

[0190] As a non-limiting example, Thermo Fisher Scientific offers a variety of products for affinity chromatography through its CaptureSelect® product portfolio. CaptureSelect® products are affinity ligands based on single-domain antibody fragments (VHHs) derived from camelid animals. The affinity receptors are 12kDa single-domain fragments containing three complementarity determination regions (CDRs) that form an antigen-binding domain. These affinity receptors are efficiently produced in the yeast Saccharomyces cerevisiae and are therefore not of animal origin. The affinity receptors are then covalently bound to chromatography beads to generate resins suitable for protein purification via column chromatography. These can be beneficially developed with defined specificity and high binding affinity to targets, enabling the creation of affinity chromatography resins well suited for single-step protein purification.

[0191] Such (and similar) products can beneficially enable selectivity, affinity, and stability during automated protein purification processes made possible by the cartridges that utilize them, enabling exemplary benefits such as single-step purification, ease of use, minimized purification costs, effective impurity removal, higher quality products, and increased flexibility in the purification process.

[0192] Additional examples of protein purification products that may be contained within one or more subassemblies of the automated protein purification cartridges disclosed herein include Antibody Toolbox® products for separating immunoglobulin formats from a wide range of environments such as plasma, milk from genetically modified animals, or supernatants from mammalian cell cultures; Proteomics Toolbox® products for protein depletion to support biomarker research applications; and HPLC analytical chromatography columns—columns with custom antibody ligands against specific proteins or impurities that enable rapid, highly sensitive, and accurate quantification of proteins from complex mixtures and small-scale sample preparation, high-performance protein liquid chromatography (FPLC), and / or affinity purification tailored to novel applications.

[0193] In some embodiments, the protein purification cartridges disclosed herein may be used to perform protocols and bioprocessing procedures selected from immunoprecipitation, recombinant protein isolation, protein labeling, target protein separation and isolation, and automated bead separation, including automated magnetic bead separation. The disclosed protein purification cartridges may be further adapted for use in the automated systems disclosed herein, and / or the systems disclosed herein may be modified to correspond to the protein purification cartridges disclosed herein. It should be understood that the functions of the combined cartridges and systems disclosed herein with respect to nucleic acid purification may be realized in the corresponding cartridges and systems configured for protein purification. As such, sealing mechanisms and fluid control mechanisms associated with the nucleic acid cartridges and systems provided herein may be further used, and / or associated with the protein purification cartridges.

[0194] In one embodiment of the automated workflow, the entire cell lysis process, including filtration of a cell suspension up to at least 1 L, any (optionally manually) washing steps, all steps related to incubation and / or resuspension of captured cells and loading of buffer / solution into a suitable reservoir or mixing chamber for cell lysis, and collection of the purified lysate, can be completed within 2 hours, preferably within 1 hour. In one embodiment, all steps involved in collecting the purified target protein from the purified lysate, including purification of the target protein through a purification column with all washing and elution steps, can be completed within about 2 hours, preferably within 1 hour. Thus, in one embodiment, filtration, lysis, purification, and purification of target protein from a cell suspension up to at least 1 L can be completed in a single automated process within about 4 hours, preferably within about 2 hours.

[0195] In some embodiments, the process workflow may include additional processing steps that may increase the total amount of execution time. For example, purified proteins may be further purified (e.g., "polished") or buffer-exchanged by complementary chromatographic strategies, which may include size exclusion, ion exchange, or affinity purification. As an additional or alternative example, purified proteins may be proteolytically treated to remove affinity tags encoded in the amino or carboxyl-terminal protein sequence, followed by a final purification step. As should be understood, the workflow may be scalable to accommodate filtration and / or purification requirements for amounts of cell suspension greater than or less than 1 L by increasing or decreasing the size and / or number of various system components as needed.

[0196] automatic purification equipment The following disclosure relates to exemplary embodiments of instruments that may be used to provide automated purification of target biomolecules such as target nucleic acids and / or target proteins. The purification instruments may be used in conjunction with other components described herein. For example, the instruments may be configured to receive and interface with consumable purification cartridges such as those described above. While some features of the instruments or apparatus are described using nucleic acid purification as an example, it will be understood that similar instruments (with modifications described herein) are also useful for protein purification.

[0197] Equipment Overview As described above, conventional protocols for purifying target biomolecules such as target nucleic acids and / or target proteins are manual testing processes that require a relatively high level of expertise and time from the laboratory technician. While a well-trained laboratory technician can handle the various operational parameters associated with the process, designing equipment that can effectively automate the majority of the process is challenging for several reasons. For example, a device configured to perform an automated process must be able to effectively move and route fluids of different viscosities and densities, provide reagent release in combination with other fluid movement operations at specific required times, mix dissimilar fluids together, process relatively large volumes of samples, and maintain proper fluid separation / sealing throughout the process. Such a device must also be safe for the user to operate and function to purify a given target biomolecule, such as a pre-selected target nucleic acid or pre-selected target protein.

[0198] As will be described in more detail below, the purification equipment described herein can satisfy one or more of the above challenges. For example, the purification equipment may include a pump assembly configured to provide a pumping action through peristaltic motion to effectively move fluid through an inserted consumable cartridge, components for controlling the automatic mixing of fluid in the inserted cartridge, components for providing or enhancing a liquid-tight seal on the cartridge, and safety mechanisms for protecting the user from accidental injury and / or limiting the risk of process errors. In some embodiments, the equipment of this disclosure may be configured to process large quantities of sample (e.g., about 5 ml to 5 l, or 10 ml to 500 ml, or about 15 ml to 250 ml). It should be understood that the systems, apparatus, and cartridges can also be reduced in size to purify smaller quantities (e.g., less than 5 ml, such as 0.5 ml to 5 ml).

[0199] Figure 10 provides an isometric view of a purification instrument 300 (which may also be referred to herein as the purification “apparatus”) capable of automated nucleic acid purification. The instrument 300 includes a casing 302 that surrounds and defines an internal compartment 304. The internal compartment 304 is sized and shaped to receive a sample purification cartridge 301. In a preferred embodiment, the purification cartridge 301 is a consumable cartridge, such as the cartridges shown and described in more detail elsewhere herein. The instrument 300 includes a selectively closable access door 306 that provides access to the internal compartment 304. Other forms of inserting and / or receiving cartridges are envisioned within the scope of this disclosure, including, for example, automatically feeding cartridges into the instrument or other mechanisms known in the art. The instrument 300 may also include a waste tray (not shown) located within the internal compartment 304 and configured to collect leaks or spills from the cartridge and / or surrounding areas of the instrument, such as leaks / spills of biological samples or various reagents / buffers used. The waste tray may be selectively insertable and removable to facilitate waste removal and cleaning.

[0200] The apparatus 300 in Figure 10 provides one non-limiting embodiment of the possible shapes of the casing 302. In this embodiment, the length of the apparatus 300 (from the access door 306 to the rear 308) is approximately the same as the height of the apparatus 300, and the width of the apparatus is approximately half the length. These relative dimensions appear to work well in a typical laboratory setup. However, it will be understood that the components and features of the apparatus 300 do not require a particular shape of the casing 302, as long as the internal compartment 304 can accommodate the cartridge 301. In other embodiments, the casing 302 may form other shapes, such as other length-to-height, length-to-width, and / or width-to-height ratios.

[0201] Furthermore, while the illustrated embodiment is configured to receive cartridges in a substantially vertical orientation, other embodiments may be configured to allow cartridges to be inserted in different orientations. For example, some embodiments may be configured to receive cartridges in a substantially horizontal orientation. Additionally, while the illustrated embodiment is configured to receive one purification cartridge at a time, other embodiments may include multiple compartments, each capable of receiving purification cartridges simultaneously or independently. Such embodiments may, for example, be able to process multiple samples in parallel.

[0202] As shown, the access door 306 may consist of a hinge point 310 located near the underside 312 of the equipment 300, such that the access door 306 pivots downward to open and pivots upward to close. This configuration allows the access door 306 to be placed on a benchtop when open, providing easy access to the interior compartment 304. Other embodiments may configure the access door 306 differently, such as by including a hinge point on the upper, right, or left side of the equipment, or by utilizing a sliding door or panel assembly. While the access door 306 is preferred, some embodiments may omit the door.

[0203] In the illustrated embodiment, when the access door 306 is open, the inner surface 314 of the access door 306 can function as a guide for orienting the cartridge 301 into the internal compartment 304. For example, a user can place the cartridge 301 on the inner surface 314 and then orient the cartridge 301 into the internal compartment 304 at the appropriate height. The inner surface 314 may also include one or more grooves 316 or other such guiding structures, which allow engagement with the cartridge 301 to further assist in correctly positioning the cartridge 301 within the internal compartment 304. Other mechanisms and / or structures for guiding the cartridge into the compartment are envisioned herein and include, for example, a gripping mechanism configured to secure the cartridge when the cartridge is partially inserted into the compartment, and a subsequent moving mechanism for further transporting the cartridge into the compartment for processing. Other mechanisms may be used as are known in the art.

[0204] The illustrated embodiment also includes a user interface 318 for receiving user input and / or displaying instrument information. The user interface 318 is communicably connected to a controller (described in more detail below - see Figure 20A) to enable communication with and control over various components of the instrument 300. The user interface 318 is configured to receive user input and / or display information related, for example, the amount of biological sample placed in the cartridge, cartridge identification information, a selected purification protocol, creation and / or storage of a user-defined protocol, sample identification information, instrument identification information, operating instructions for the selectively closable access door 306 or related components, a desired concentration of the target nucleic acid to be purified, one or more optical density measurements of the initial sample and / or related to the sample at various stages in the purification process, and / or the final volume of the eluent containing the target nucleic acid.

[0205] The user interface 318 may be configured to allow interaction with the user via a touchscreen, control pad, mouse, microphone, keyboard, and / or other computer input / output components known in the art. During processing, the user interface 318 may provide the user with the opportunity to observe the progress of the purification process being performed on the cartridge and may provide alarms indicating completion of processing or any errors or other problems that may occur during processing.

[0206] Equipment clamping mechanism Figures 11A and 11B illustrate embodiments of a clamping mechanism 320 that may be included as part of the instrument 300. In some applications, the instrument 300 may interface with consumable cartridges having a relatively low-cost construction. That is, consumable cartridges may be intentionally designed with minimal inherent sealing to reduce manufacturing complexity and associated costs. For example, a consumable cartridge may be designed with sealing sufficient to maintain fluid separation at ambient pressure, but not necessarily the high pressures experienced during the purification process (e.g., as a result of passing biological samples, reagents, and / or other fluids through membranes, filters, resins, and / or bead columns for various separation / filtration process steps).

[0207] The consumable cartridges may instead be designed to rely on the clamping mechanism 320 of the instrument 300 to provide sufficient sealing force to allow the cartridges to withstand the high pressures they experience during the refining process. This design approach beneficially allows the instrument to offset the costs inherent in the cartridges. These costs are more economically efficient when used with durable instrumentation than with single-use consumable cartridges.

[0208] In Figures 11A and 11B, the casing 302 and other components have been removed to better illustrate the clamping mechanism 320. The clamping mechanism 320 includes a first plate 322 and a second plate 324 configured to move between an open position (shown in Figure 11A) and a closed position (shown in Figure 11B). In the closed position, plates 322 and 324 compress the cartridge positioned between them, providing sufficient clamping force to allow the cartridge to withstand the relatively high pressures associated with the purification process. As best shown in Figure 11B, one or both of plates 322 and 324 may include an aperture 326 that aligns with components of the cartridge, such as the cartridge's fluid reservoir or channel components. Thus, plates 322 and 324 are configured to align with and compress portions of the cartridge that require compression to maintain the integrity of the seal during the purification process.

[0209] As shown in Figure 11A, the first and second plates 322 and 324 are supported within the frame 328. In this embodiment, the clamping mechanism 320 is configured to move the first plate 322 laterally relative to the second plate 324. That is, the first plate 322 is a translational plate, and the second plate 324 is a stationary plate. The foot 330 is positioned within the socket 332 and is capable of longitudinal translation relative to the socket 332. The socket 332 is attached to and / or is part of the frame 328 and functions to maintain the foot 330 relative to the perimeter of the internal compartment and to prevent lateral movement of the foot 330.

[0210] In Figure 11B, the frame 328 and socket 332 have been removed to better illustrate the foot 330 and its associated mechanism. As shown, the foot 330 may be connected to the first plate 322 by one or more linkages 334. Preferably, multiple linkages 334 are provided to better distribute compressive forces across the first plate 322. The linkages 334 may also include springs to better distribute lateral compressive forces across the plate. The linkages 334 may also include various shims to adjust the amount of force applied.

[0211] The drive screw 336 is operably connected to the motor 338 and screwed into the receiver 340. The receiver 340 is attached to the foot 330, and the rotation of the drive screw 336 causes the receiver 340 to be threaded so that it translates longitudinally on the drive screw 336. This causes the foot 330 to move longitudinally. The longitudinal movement of the foot 330 causes the corresponding lateral movement of the first plate 322, as the foot 330 is prevented from moving laterally by the socket 332 and the first plate 322 is prevented from moving longitudinally by the frame 328. The end of the linkage 334 also moves within a cam path, which provides an additional mechanical advantage to the first plate 322.

[0212] Other embodiments may utilize other mechanisms to provide linear motion of the first plate 322. For example, instead of a drive screw, the foot 330 may be operably connected to a linear actuator, a belt and pulley assembly, a chain and sprocket assembly, a gear and gear rack assembly, or a combination thereof. Some embodiments may include a mechanism that directly pushes or pulls the first plate 322, such as a piston-based mechanism or some other linear actuator mechanism applied directly to the first plate 322. Some embodiments may be configured to move each plate 322, 324 relative to one another, rather than having one plate stationary.

[0213] Since some cartridges may need to withstand pressures of up to 50-100 psi, the clamping mechanism 320 is configured to apply a total sealing force of at least about 500 lbf, more preferably at least about 1,000 lbf, or even more preferably at least about 2,000 lbf.

[0214] If power is lost during the refining process, the user may utilize a manual release mechanism to allow the clamp mechanism 320 to be released and the cartridge removed. Partially processed cartridges may contain biohazardous and / or hazardous chemicals that may need to be removed before the equipment 300 can be serviced, transported, or used further. In the illustrated embodiment, as shown in Figure 12, the manual release mechanism is accessible via an access point 350 located on the rear 308 of the equipment 300, although the access point 350 may be located in other locations.

[0215] The manual release mechanism is configured so that a common tool (e.g., a screwdriver, hex wrench) inserted through the access point 350 can make contact with the mechanism and rotate the drive screw 336 by rotating the tool. The manual release mechanism may also be clutched. For example, when the clamp is released, the clamp spring force may begin to push the plates apart, causing the drive screw 336 to rotate rapidly. A one-way bearing associated with the release mechanism may then begin to rotate to prevent uncontrolled rotation of the tool used to initiate the manual release of the clamp.

[0216] Other embodiments of the instrument may omit the clamping mechanism. For example, not all cartridges are necessarily manufactured to utilize additional compression from the instrument. For example, if the cartridge has sufficient inherent sealing, if the biological sample contains few cells or is otherwise of low viscosity (e.g., water or urine samples), and / or if the volume of the biological sample is small relative to the instrument's capacity (e.g., about 10 ml), the instrument may not necessarily include or use a clamping mechanism. It will be understood that some samples may be "small" relative to the capacity of the instruments and processes described herein, but such samples may still be considered large relative to conventional purification instruments and processes.

[0217] In applications where the instrument omits or does not utilize a clamping mechanism, the associated cartridge may be constructed to inherently withstand the expected pressure. Such cartridges may not necessarily be able to withstand the high pressures described above (for example, culture / sample volumes of around 50–150 ml may be processed through one or more cell-trapping membranes), but they are still constructed adequately to withstand the relatively low pressures involved in the aforementioned scenarios. Such cartridges may be formed using polymer materials (e.g., thermoformable and / or other suitable polymers) in which two separate sides are fused, bonded, and / or mechanically locked together along the edges and other arbitrary desired positions.

[0218] Safety mechanism of the equipment Figure 13 illustrates the door lock mechanism as viewed from a viewpoint facing the inner surface 314 of the access door 306. The locking tab 342 (also seen in Figure 11A) is attached to the first plate 322 (i.e., the “movable plate”). The corresponding locking slot 344 is located in a portion of the access door 306 that is present within the interior compartment when the door is closed. When the access door 306 is closed and the clamping mechanism 320 is activated, the locking tab 342 translates together with the first plate 322, thereby entering the locking slot 344. This initiates the clamping process and prevents the access door 306 from opening.

[0219] The illustrated locking mechanism functions as an access barrier to beneficially prevent user injury from various moving parts within the internal compartment. Preferably, the access door 306 is the only opening providing access to the internal compartment. Once closed, the access door 306 and casing 302 form a barrier that isolates the user from the internally moving automated parts.

[0220] As an additional or alternative precaution, the device 300 may be configured to allow power to be supplied to the clamp motor 338 only when it is determined that the access door 306 is closed. This determination may be made using one or more sensors configured to detect whether the access door 306 is open or closed. In the embodiment shown in Figure 14, the casing 302 includes a pair of redundant Hall effect sensors 346 in a portion of the casing 302 that defines the access door frame. A pair of corresponding magnets 348 are positioned within the access door 306 in a location that aligns with the Hall effect sensors 346 when the access door 306 is closed.

[0221] The positions of the Hall effect sensor 346 and the magnet 348 may be reversed such that the magnet 348 is inside the access door frame and the Hall effect sensor 346 is inside the access door 306. Although two pairs of sensors / magnets are shown here, some embodiments may include only a single pair or more than two pairs. Other embodiments may additionally or alternatively include other types of contact and / or proximity sensors, such as capacitive or inductive proximity sensors, infrared proximity sensors, optical sensors, eddy current sensors, mechanical switches (e.g., limit switches), or combinations thereof.

[0222] Process control mechanism of equipment The instrument 300 preferably includes one or more features that function to ensure the proper preparation of various components before commencing the purification process. For example, the instrument 300 may include features that ensure the proper positioning of the biomolecular purification cartridge before commencing clamping or other steps of the purification process, features that ensure the inserted purification cartridge is unused, and / or features that ensure the output container for receiving the purified product is properly positioned.

[0223] Figure 15 illustrates an embodiment of a cartridge positioning mechanism positioned within an internal compartment of the equipment. A contact switch 352 (e.g., a limit switch) may be positioned within the internal compartment at a position where the inserted cartridge 301 contacts the switch 352. In the illustrated embodiment, the contact switch 352 is positioned on the rear side of the internal compartment. In this position, as the cartridge 301 is inserted, its leading edge 305 advances until it reaches the contact switch 352. The contact switch 352 is communicatively connected to an equipment controller. The controller may be configured to prevent the process from starting unless the contact switch 352 is in contact.

[0224] Other embodiments may position the contact switch 352 in other suitable locations within the internal compartment. Other embodiments may additionally or alternatively include other contact and / or proximity sensors, such as other types of sensors described herein in relation to other components of the device 300 (e.g., optical sensors, magnetic sensors, etc.).

[0225] The cartridge positioning mechanism may also include a latch 354 configured to engage with the cartridge 301 to help hold the cartridge in place once it has been inserted to the correct position. The latch 354 may also be configured to provide tactile feedback to the user to indicate that the cartridge is in the correct position. For example, the latch 354 may be spring-loaded or otherwise biased toward a closed position and configured to return to the closed position with a "snap" or "click" after being displaced during cartridge insertion.

[0226] In the illustrated embodiment, as the cartridge 301 begins to contact the latch 354 during insertion, the engaging feature 303 of the cartridge 301 can contact the inclined surface 356 of the latch 354. Further insertion of the cartridge 301 moves the latch 354 away from the closed position. Once the engaging feature 303 clears the inclined surface 356, the groove 358 allows the latch 354 to return to the closed position. Other embodiments may additionally or alternatively include other latching functions such as magnetic couplings, roller catches, ball tension catches, bullet catches, and other latching mechanisms known in the art.

[0227] Figure 16 illustrates another process control mechanism that the instrument 300 may include. The purification cartridge typically includes an output container for collecting the purified product. Often, the user removes the output container before inserting the cartridge into the instrument, for example, to label the output container. However, if the user forgets to reposition the output container within the cartridge, and the cartridge is inserted and the purification process is started, the valuable purified product will be dispensed onto the floor of the instrument. This is likely to waste valuable product and create a mess that requires cleanup before the instrument can be used again.

[0228] In the embodiment shown in Figure 16, the contact switch 360 is mounted on one of the plates (in this embodiment, the second plate 324) and positioned to contact the output container 307 when the output container 307 is properly positioned on the cartridge 301. The contact switch 360 ensures that the output container 307 is properly positioned before the purification protocol is initiated. For example, the contact switch 360 may be communicatively coupled to a controller, which may operate to prevent the protocol from being initiated and / or to provide a status notification to the user if the contact switch 360 determines that the output container 307 is not present. Other embodiments may additionally or alternatively include other contact and / or proximity sensors, such as other types of sensors described herein in relation to other components of the device 300.

[0229] The instrument 300 may also include one or more optical density sensors configured to determine the initial optical density (e.g., OD600 or A600) of a biological sample (e.g., a bacterial culture or other cell culture) inserted into a purification cartridge. Optical density can be determined by illuminating the sample with light of a known wavelength (typically about 600 nm, but other wavelengths may be available depending on the sample type, the object being measured, and / or other specific application needs) and measuring the amount of light that passes through the sample and reaches a detector on the opposite side of the sample. Optical sensors may also be used at the end of the purification process and / or in the middle of the process to measure the concentration of nucleic acids in the sample (e.g., using wavelengths of 230, 260, and / or 280 nm).

[0230] The instrument 300 may also include one or more additional sensors to monitor protein purity or concentration by assessing absorbance at 210 or 280 nm, or the ratio of absorbances at different wavelengths, or the state of apoenzymes versus holoenzymes having cytochrome, heme, and other cofactors or prosthetic groups. In some embodiments, additional or alternative sensors may be included to monitor pH, conductivity, refractive index, osmotic pressure, redox potential, or protein aggregation.

[0231] Optical density (OD) information acquired by an optical density sensor may be reported to the user and used to inform the user whether the biological sample is within a suitable range for processing. For example, the optical density sensor may be communicatively coupled to a controller, which may operate to provide notifications to the user and / or prevent protocol intervention if the measured OD is outside a suitable range.

[0232] As will be described in more detail below, OD readings can also be used to determine and / or initiate specific purification protocols that are better optimized for a given biological sample type. For example, the buffers / reagents used to purify a target nucleic acid may be dispersed in different amounts based on the determined OD of the sample. In another embodiment, the timing / duration of mixing the sample and / or passing it through one or more membranes / filters in the cartridge may also vary according to the determined OD of the sample. Optical density sensors may be placed in various locations to take measurements of the sample in different sections of the cartridge and at different stages of the purification process. For example, an optical density sensor may be used to determine whether fluid is present in a particular part of the cartridge by, for example, comparing the measured OD to an expected air blank.

[0233] OD information may be used, additionally or alternatively, to determine whether an inserted cartridge has been used previously. For example, certain protocols require an OD reading before placing a biological sample into the cartridge's input reservoir. The initial OD reading of the cartridge before the sample is placed should be substantially the same as the air blank reading. A higher reading may indicate that the cartridge's input reservoir has previously been unsealed / broken and filled with a biological sample. For example, a previous sample is likely to leave residue in the sample chamber window. In such environments, the controller may act to provide notification to the user and / or to prevent the protocol from being overridden.

[0234] Figure 17 schematically illustrates one embodiment of the optical density sensor 362. The illustrated embodiment includes a light source 364, which is mounted on an aluminum plate and configured as a light-emitting diode (LED) having a wavelength of 595 nm. A detector 366, which is configured as a silicon detector mounted on a printed circuit board (PCB), is located on the opposite side of the light source 364. A sample chamber, which is filled with a portion of a biological sample 309, is located between the light source 364 and the detector 366. The sample chamber includes a window 368 through which the synchrotron radiation can pass. The optical density sensor 362 may also include one or more additional optical components, such as a lens 370, a diffuser 372, a filter, an aperture, and / or other optical components.

[0235] The illustrated embodiment is one example of a suitable optical density sensor. Other optical density sensor configurations known in the art may also be used. For example, other embodiments may include alternative light source types, alternative detector types, alternative lenses, filtering, and / or aperture arrays, alternative optical paths, or alternative sample chamber dimensions. As described above, one or more optical density sensors may also be used at different wavelengths to provide different information about a sample. For example, one or more sensors may be used to measure the concentration of nucleic acids using suitable wavelengths (e.g., 230, 260, and / or 280 nm).

[0236] Equipment pump assembly Once the purification cartridge is inserted into the instrument 300 and the purification process is initiated, the instrument 300 operates to move and route fluid through the cartridge using a series of pump assemblies. The pump assemblies are positioned to engage with the purification cartridge (e.g., when the clamp mechanism is closed) to controllly direct the fluid during the purification process. The pump assemblies may be positioned upstream and / or downstream of each processing section of the cartridge (e.g., each membrane or filter). That is, the pump assemblies may be positioned to "push" the sample, reagent, or other fluid of the process further downstream, to "pull" the sample, reagent, or other fluid of the process further downstream, or both. The pump assemblies described herein are usefully capable of moving and routed fluids of varying viscosities and densities.

[0237] Figure 18A illustrates an exemplary pump assembly 374 (in an exploded view). The pump assembly 374 includes a motor 376 operably connected to a camshaft 378 via a power transmission assembly. The power transmission assembly may include a gear 379 as shown, and / or one or more other power transmission components such as a belt, pulley, chain, sprocket, etc.

[0238] Attached to the camshaft 378 are a plurality of cam elements 380 (i.e., “finger parts”). As shown, each cam element 380 includes a mounting end 382 configured to be attached to the camshaft 378 and a tip 384 extending laterally (e.g., vertically) from the camshaft 378. The cam elements 380 are arranged on the camshaft 378 such that the rotation of the camshaft 378 causes a linear peristaltic motion of the cam element tip 384. The cam elements 380 and / or other components of the pump assembly 374 may be located within the housing 386.

[0239] Figure 18B is a magnified view of the tip 384 of a cam element in contact with the fluid channels 311 / 313 of the cartridge. Here, the fluid channel before deflection is shown as 311, and the fluid channel after deflection is shown as 313. The tip 384 may be tilted to provide effective engagement with the fluid channels 311 / 313, but other cam element shapes may be used depending on the needs and preferences of the particular application. As illustrated, the movement of the cam element 380 relative to the fluid channels 311 / 313 deflects the fluid channels, thereby displacing the fluid contained within that portion of the fluid channels 311 / 313.

[0240] Figures 19A to 19F illustrate the engagement between the pump assembly 374 and the fluid channel 315 of the cartridge 301, and sequentially show how the linear peristaltic motion of the cam element 380 can drive the movement of fluid through the fluid channel 315. Figures 19A to 19D show a cross-sectional plan view of the pump assembly 374 from a viewpoint perpendicular to the side.

[0241] Figure 19A shows a pump assembly 374 with a camshaft 378 in any 90-degree position. As the camshaft 378 continues to rotate to the 180-degree position shown in Figure 19B, the 270-degree position shown in Figure 19C, and then to the 0 (i.e., 360)-degree position shown in Figure 19D, the sequential engagement of the cam element 380 with the fluid channel 315 moves the fluid 317 through the fluid channel 315. As the camshaft 378 continues to rotate through additional rotations, the cam element 380 continues to engage with the fluid channel 315 in a corresponding peristaltic motion to further direct the fluid 317 through the fluid channel 315, as shown in Figures 19E and 19F.

[0242] One exemplary embodiment of the pump assembly is shown to move approximately 0.16 ml of fluid (e.g., approximately 0.05 to 0.45 ml) per rotation of the camshaft. At a motor RPM of 136, the resulting flow rate was approximately 22 ml / min (e.g., approximately 10 to 35 ml / min). Of course, different flow rates can be achieved using different motor speeds, different fluid channel dimensions, different numbers or orientations of cam elements, different fluid viscosities, etc.

[0243] The pump illustrated in the pump assembly enables the efficient movement of fluids of varying viscosities and densities. However, other embodiments may include, additionally or alternatively, other means of fluid transport. For example, the pump assembly may utilize a roller mechanism that compresses a fluid channel and then moves the fluid by moving linearly across the fluid channel.

[0244] Equipment control system Figure 20A schematically illustrates an exemplary control system 388 that may be used to control various components and operations of the device 300. The controller 390 includes a memory 392a (i.e., a physical storage medium or hardware storage device) and one or more processors 392b (and / or a suitable microcontroller). As described above, the controller 390 may be communicably coupled to the user interface 318 to receive user input via the user interface 318 and to transmit display information to the user interface 318.

[0245] The controller 390 may also be communicatively coupled to one or more sensors 399, such as access door sensors 346 / 348 (e.g., Hall effect sensors), cartridge contact switch 352, output container contact switch 360, and optical density sensor 398 (or more such sensors), as described herein. As described above, the controller 390 may transmit user notifications and / or act to pause or stop the refining process based on the determined state of such sensors.

[0246] For example, as described above with respect to the optical density sensor 398, the controller 390 may be configured to receive OD information and send a user notification indicating how the OD information and / or associated OD measurements relate to a preferred protocol range. In some embodiments, the controller 390 may be configured to indicate whether an inserted cartridge has been used previously based on the OD information received from the optical density sensor 398, send a notification, and / or stop the purification process accordingly.

[0247] The controller 390 may also be communicatively coupled to a cartridge sensor 396 configured to interface with the inserted cartridge and obtain identification information from the cartridge. For example, the cartridge 396 may include a barcode reader and / or other suitable sensor capable of reading an identifier or code placed on the cartridge. The identification information may include a lot number, expiration date, inventory management unit number, cartridge type, number of uses associated with the cartridge, and number of remaining uses associated with the cartridge.

[0248] The apparatus is not limited to the number and / or types of sensors shown. For example, some embodiments may additionally or alternatively include one or more flow rate sensors, temperature sensors, time-lapse sensors, volume sensors, weight sensors, and / or other sensor types for measuring other parameters of the purification process.

[0249] The controller 390 may also be communicatively coupled to one or more actuators 397, such as a clamp motor 338 to control the operation of the clamp mechanism and a pump assembly 374 to control the movement of fluid through the cartridge via the pump assembly 374. The set of actuators 397 may also include one or more seal actuators 391 and / or valve actuators 393 (and described in more detail elsewhere herein) configured to selectively break fluid seals in the cartridge and to control the opening and closing of valves in the cartridge, respectively. The set of actuators 397 may also include one or more mixing actuators 395. In a preferred embodiment, the mixing actuator includes a rotatable magnet positioned to align with a corresponding magnetic stirring element positioned within the mixing chamber of the cartridge.

[0250] The controller 390 may also be communicatively connected to communication hardware 394. The communication hardware 394 may include routers and / or other networking hardware configured to provide communication with one or more of the following via wired or wireless connections: networks (e.g., local area networks (LANs), wide area networks (WANs), cloud-based networks), external server systems, external computer devices, distributed computer systems, and the internet. The communication hardware 394 may also include components that enable data uploading or downloading via direct connections such as Ethernet ports, Personal Computer Memory Card International Association (PCMCIA) slots, or Universal Serial Bus (USB) ports.

[0251] In some embodiments, the controller 390 is configured to automatically modify the purification protocol based on inputs received from one or more sensors as part of a “smart” purification system. For example, the determined optical density of the input sample may be used to adjust one or more operational parameters of the purification procedure. Such variable operational parameters may include, for example, the amount of one or more reagents moved by the instruments used in the purification procedure, the timing of pump transport, and / or the rate of pump transport via the operation of the pump transport assembly. As an example, the cell capture / filtration step is typically performed as the first part of the purification procedure. This cell capture / filtration step may be performed for a longer period of time, preferably when the optical density of the input sample is higher. As another example, the use of larger amounts of one or more reagents may be preferred when the optical density of the input sample is higher.

[0252] The control system 388 may also be enabled to communicate with a user inventory database to track the use and availability of consumables (e.g., buffers, reagents, cartridges) within the user's organization or laboratory. The control system or user inventory database may be associated with one or more rules that prompt (or simply cause) the user to purchase additional consumables when threshold or trigger conditions are met, such as when available stock falls below a threshold level. It should also be understood that any number or type of trigger conditions associated with the systems described herein may be predetermined and / or modified to meet the requirements or preferences of individual users.

[0253] As a further example, FIG. 20B illustrates an exemplary method 400 for performing a purification procedure. Method 400 may be implemented using a suitable computer control system such as the control system 388 shown in FIG. 20A. In the illustrated method 400, the control system may be configured to receive sensor data from one or more sensors to determine one or more process states (step 402). The one or more sensors may include, for example, any or all of the sensors 399 shown in FIG. 20A and / or other sensors described herein, temperature sensors, volume sensors, weight sensors, cartridge scanners (e.g., barcode or QR code scanners), etc. The process state may include any measurements made by such sensors. The process state data may additionally include information input by the user, such as cartridge type (e.g., anion exchange and precipitation or silica-based capture), sample type (e.g., cell culture, environmental, clinical, food, or forensic sample), and / or information obtained from a particular cartridge.

[0254] Next, the control system may compare the determined process state to a set of different protocols stored within a protocol library (step 404). The protocol library may be stored within the memory 392a of the controller 390 and / or accessed via network communication between the controller 390 and one or more server systems, a "cloud" database, etc. The protocol library includes various purification protocols associated with various possible process states. For example, the protocol library may include a purification protocol associated with a "low" initial OD reading, a different purification protocol associated with a "medium" initial OD reading, and a different purification protocol associated with a "high" initial OD reading. Of course, the number of different protocols need not be limited to a low / medium / high classification, but may be distinguished, for example, based on a numerical range at any desired level of granularity.

[0255] Each individual purification protocol can define the desired process steps and parameters for performing the purification process. For example, whether cell capture and / or cell lysis are utilized, the duration of cell capture and / or cell lysis, the reagents utilized, the amount of reagents utilized, whether mixing is performed and / or when it is performed in one or more purification steps, the duration and / or rate of one or more mixing steps, the duration and / or rate of pump transport between one or more purification steps, whether to open and close one or more valves within the cartridge, and / or when to open and close them, whether to pierce one or more seals within the cartridge to allow release of the corresponding fluid or expulsion of gas and / or when to pierce them, the type of nucleic acid capture involved, the duration of nucleic acid capture, the type of captured protein, the duration of target protein capture, or combinations thereof are defined.

[0256] The control system can then select a purification protocol from the protocol library based on the determined process state (step 406). The purification protocol can be selected, for example, as the best match for a particular set of determined process states or as the only match within a set of purification protocols having mutually exclusive corresponding process states.

[0257] Next, the control system may execute the selected purification protocol by operating one or more instrument actuators according to the selected purification protocol (step 408). One or more instrument actuators may include any or all of the actuators 397 illustrated in Figure 20A. For example, if the selected purification protocol requires a specific mixing rate and / or duration at a particular process step, the controller 390 operates the appropriate mixing actuator 395 accordingly to execute that part of the selected purification protocol. In another example, if the selected purification protocol requires a specific amount of reagent to be used and released at a specific time during the purification procedure, the controller 390 operates the associated seal actuator 391, pump assembly 374, and / or valve actuator 393 accordingly to appropriately route and utilize the reagent according to the selected purification protocol. In another example, if the selected purification protocol requires a specific call capture duration (for example, as estimated based on the first OD reading), the controller 390 operates the associated seal actuator 391, pump assembly 374, and / or valve actuator 393 accordingly to allow for an appropriate duration for the cell capture step.

[0258] In some implementations, method 400 can operate iteratively. As indicated by arrow 410, while the selected purification protocol is being executed, the controller 390 may continue to receive additional sensor data to determine one or more updated process states. The updated process states can then be compared with the protocol library, and the purification protocol may be updated / revised "on the fly" according to the most recently acquired measurements. This is beneficially possible to re-optimize the purification procedure between each process step based on the results of the previous process step. For example, if, following the cell capture step, sensor data indicates that less lysate was collected than expected based on the initial OD reading of the input sample, the initially selected purification protocol may be updated / revised to better match the subsequent use and / or timing of reagents, mixing, pump transport, etc., with the measured amount of lysate.

[0259] The control system 388 of the present disclosure may include, utilize, or communicate with a dedicated or general-purpose computer, including computer hardware. Within the scope of the present invention, the control system 388 may also include physical and other computer-readable media for carrying or storing computer-executable instructions and / or data structures. Such computer-readable media may be any available media accessible by a general-purpose or special-purpose computer system. A computer-readable medium for storing computer-executable instructions is a physical storage medium (e.g., a hardware storage device). A computer-readable medium carrying computer-executable instructions is a transmission medium. Therefore, embodiments of the present invention may comprise at least two distinctly different types of computer-readable media, namely, a computer storage medium and a transmission computer-readable medium.

[0260] Computer-readable hardware storage media include RAM, ROM, EEPROM, CD-ROM or other optical disk storage devices (such as CDs and DVDs), magnetic disk storage devices or other magnetic storage devices, or any other media that can be used to store desired program code means in the form of computer-executable instructions or data structures and can be accessed by a general-purpose or dedicated computer.

[0261] A “network” is defined as one or more data links that enable the transmission of electronic data between computer systems and / or modules and / or other electronic devices. When information is transmitted to or provided to a computer via a network or another communication connection (either wired, wireless, or a combination of wired and wireless), the computer appropriately considers that connection to be a transmission medium. A transmission medium may include networks and / or data links that can be used to carry desired program code in the form of computer executable instructions or data structures and that can be accessed by general-purpose or dedicated computers. The above combination also falls within the scope of computer-readable media.

[0262] Furthermore, upon reaching various computer system components, program code in the form of computer-executable instructions or data structures can be automatically transmitted from a transmission computer-readable medium to a computer-readable hardware storage medium (or vice versa). For example, computer-executable instructions or data structures received via a network or data link may be buffered in RAM within a network interface module (e.g., a "NIC") and then finally transmitted to the computer system RAM and / or the computer system's less volatile computer-readable hardware storage medium. Thus, computer-readable hardware storage mediums may also be included in computer system components that utilize (or primarily utilize) transmission media.

[0263] Computer executable instructions include instructions and data that cause a general-purpose computer (e.g., a destination computing device), a dedicated computer (e.g., a sensor cap and / or destination computing device), or a dedicated processing device to perform a specific function or set of functions. Computer executable instructions can be, for example, binary, intermediate format instructions such as assembly language, or even source code.

[0264] The functions described herein may be performed, additionally or alternatively, at least in part, by one or more hardware logic components. For example, exemplary types of hardware logic components that may be used include, but are not limited to, field-programmable gate arrays (FPGAs), program-specific integrated circuits (ASICs), program-specific standard products (ASSPs), system-on-chip systems (SOCs), complex-programmable logic devices (CPLDs), and others.

[0265] Furthermore, wireless communication between any of the disclosed computer systems may be performed using any wireless protocol known in the art, including, for example, Bluetooth, ZigBee, ultra-wideband (UWB), and Wi-Fi. Therefore, where a particular wireless protocol (or related component) is specifically noted or described in any of the disclosed embodiments, it should be understood that the noted particular wireless protocol may be interchangeable with any other wireless protocol known in the art while maintaining any disclosed functionality and / or performing the same or substantially similar tasks related thereto.

[0266] Fluid seals and valve mechanisms The following disclosure relates to exemplary embodiments of target biomolecule purification cartridges, such as target nucleic acid purification cartridges or target protein purification cartridges, which may be used in automated purification processes for selected target biomolecules. Embodiments of fluid seals and valves may be used in conjunction with other components described herein. For example, seal and valve components may be integrated with consumable (e.g., single-use) purification cartridges as described above, used with target biomolecule purification equipment (such as target nucleic acid purification equipment or target protein purification equipment) as described above, and / or included in one or more fluid release mechanisms as described above. In preferred embodiments, fluid seals, valve mechanisms, flow path tubes and connections, fluid reservoirs, and membranes / filters are designed to minimize or eliminate leachates that may contaminate or covalently modify the purified biomolecules, particularly the purified target proteins.

[0267] Overview of seal and valve mechanisms Traditional nucleic acid or protein purification procedures are manual testing processes that require a relatively high level of expertise and time from the laboratory technician. Several previous attempts to automate steps in the purification process have typically focused on sample transport cartridges, usually formed as rigid polymer cartridges. Such cartridges are often relatively complex and expensive, especially when dealing with large volumes of samples, because they require routing fluids in specific amounts and at specific times, mixing the fluids, and controlling the fluid's passage through the cartridge to successfully execute the purification procedure.

[0268] In particular, different types of fluids need to be fluidically sealed and kept separate from each other until the appropriate mixing step is required. The challenges associated with maintaining these fluid seals are exacerbated by the need to move the fluids in relatively complex patterns and under various time parameters. For example, pumping fluids to create a desired fluid pathing generates associated pressure differences, which can stress the fluid seals necessary to separate the various fluid channels in a cartridge. To prevent leakage, conventional cartridges are formed with seals that can inherently withstand these pressure differences, which can significantly increase the cost of the cartridge components and / or manufacturing. For example, creating long, liquid-tight seals often requires complex, long ultrasonic welding between injection-molded parts. These costs make "consumable," "disposable," "single-use," or "one-time use" cartridges less economically viable, despite the greater convenience and time-saving potential such cartridges could offer the user.

[0269] Therefore, designing a purification cartridge that can provide effective fluid sealing and valves presents several challenges. As will be described in more detail below, the purification cartridges described herein can satisfy one or more of the above challenges by utilizing effective sealing and valve mechanisms. For example, as will be described in more detail below, the purification cartridge may include an elastomer layer positioned between two relatively rigid outer layers. The elastomer layer includes a number of ribs that fluidly seal fluid channels formed in one or both of the outer layers when the elastomer layer is compressed between the two outer layers.

[0270] This structure beneficially allows for the use of less expensive materials and simpler manufacturing methods. For example, such cartridges do not require long liquid-tight seals and therefore do not require long and complex ultrasonic welding. By offloading the compression function to external equipment or components, costs are beneficially shifted from cartridges (which typically have much more limited use) to more permanent devices.

[0271] As an additional advantage, the elastomer layer can be used to provide valve function. Therefore, in contrast to individual components for each valve, only one component is needed to form the foundation of all cartridge valves. These advantages and the structural components capable of providing them are described in more detail below.

[0272] Sealing ribs Figure 21 schematically illustrates a purification cartridge 501 having a series of fluid channels defined by the presence of multiple polymer ribs 522. The ribs 522 function to separate different sections of the cartridge, thereby defining the boundaries of distinct fluid channels. As shown, some of the defined fluid channels form relatively narrow fluid pathways 504, while others form relatively wide, larger fluid reservoirs 506. As used herein, “reservoirs” typically refers to a portion of a cartridge configured to store, mix, filter, separate, and / or react fluids, while the narrower pathways between these reservoir spaces are primarily configured to transport fluids from one reservoir to another.

[0273] Figure 22 illustrates a cross-sectional view of a cartridge 501 to illustrate the multiple layers that may be included to form the cartridge and define various fluid channels. The diagram in Figure 22 shows the fluid channels 524 in cross-section. The cartridge section is shown here as vertically oriented, which is the orientation adopted by the cartridge in a preferred embodiment. However, it will be understood that the features described herein do not require vertical orientation, and other orientations may also be utilized according to the needs and / or preferences of a particular application.

[0274] The illustrated embodiment includes a first outer layer 508. The first outer layer 508 includes a first side 510 (i.e., the inside) and a second side 512 (i.e., the outside). The second outer layer 514 is located on the opposite side of the first side 510 of the first outer layer 508. The second outer layer 514 similarly includes a first side 516 (i.e., the inside) and an outside 518 (i.e., the outside).

[0275] As shown, the first outer layer 510 may include an outwardly extending indentation or groove that defines the wall surface 526 of the fluid channel 524. The cross-sectional shape of the fluid channel may be substantially curved, as in the illustrated embodiment, where the inner section is wider than the outer section (i.e., the bottom or "nadir" of the fluid channel). In the illustrated embodiment, the wall surface 526 rises, exits the nadir, and moves to an inflection point 527 located on either side of the nadir. Alternative embodiments may include fluid channels having different cross-sectional shapes, including polygons such as a rectangular cross-sectional shape.

[0276] An elastomeric layer 520 is disposed between a first outer layer 510 and a second outer layer 514. The elastomeric layer 520 includes an array of sealing ribs 522. As shown, a pair of sealing ribs 522 extend toward a first side 510 of the first outer layer 508 and contact the first side 510 of the first outer layer 508 such that the sealing ribs 522 are disposed on each side of a fluid channel 524. Thereby, the sealing ribs 522 define the width of the fluid channel 524 and restrict “upward” and “downward” (from the perspective of FIG. 22) movement of fluid within the channel 524. As shown, the sealing ribs 522 can be oriented such that the apex (i.e., the most inner point) of each rib 522 is aligned with or beyond an inflection point 527 that faces the bottom of the channel.

[0277] In the illustrated embodiment, a nominal gap 521 can exist between the elastomeric layer 520 and the first outer layer 508 and / or the second outer layer 514. The nominal gap 521 allows space for the elastomeric material of the sealing ribs 522 to deflect when compressed. The ribs 522 also preferably are spaced a small distance from the fluid channel wall 526 such that when compressed, the ribs 522 do not deflect and overly cover the fluid channel.

[0278] The elastomer layer 520 can be formed from a variety of suitable elastomer materials, including thermosetting elastomers and thermoplastic elastomers. Non-limiting examples of suitable elastomers include natural rubber, isoprene rubber, butadiene rubber, chloroprene rubber, halogenated butyl rubbers such as butyl rubber, chlorobutyl rubber, and bromobutyl rubber, styrene-butadiene rubber, nitrile rubber, ethylene propylene rubber, ethylene propylene diene rubber, epichlorohydrin rubber, polyacrylic rubber, silicone rubber, fluorosilicone, fluoroelastomers, perfluoroelastomers, polyether block amide (PEBA), chlorosulfonated polyethylene, ethylene vinyl acetate, closed-cell foam, and combinations thereof. For example, the elastomer layer may have a Young's modulus of about 5 to about 500 MPa, or about 10 to about 100 MPa.

[0279] The outer layers 508 and 514 can be formed from a variety of suitable materials. Preferably, the outer layers 508 and 514 have higher rigidity (e.g., higher Young's modulus) than the elastomer layer 520. Preferably, the outer layers 508 and 514 are formed from polymer materials that are readily manufactured by a thermoforming process. Non-limiting examples include polyethylene, polypropylene, polycarbonate, polyethylene terephthalate, polystyrene, polyvinyl chloride, other polymers, and combinations thereof. For example, the outer layers 508 and 514 may have a Young's modulus of about 500 to about 4,000 MPa.

[0280] The embodiment shown in Figure 22 includes a fluid channel 524 formed in the first outer layer 508 and a sealing rib 522 extending toward the first outer layer 508. Although not shown herein, the cartridge may also include one or more fluid channels formed in the second outer layer 514, with associated sealing ribs extending from the elastomer layer 520 toward the second outer layer 514.

[0281] The rib 522 is configured to seal the fluid channel 524 when the elastomer layer 520 is compressed between the first and second outer layers 508 and 514, thereby fluidly separating it from other fluid channels. Figures 23A and 23B illustrate an embodiment of a cartridge 501 that is clamped between a first clamp plate 532 and a second clamp plate 534. Figure 23A shows the clamp plates 532 and 534 before being compressed against the cartridge 501, and Figure 23B shows the cartridge 501 fully compressed between the clamp plates 532 and 534.

[0282] The clamp plates 532 and 534 are sized and shaped to be positioned on either side of the cartridge 501, allowing compression of the portion of the cartridge 501 containing the sealing ribs while leaving other sections, such as the reservoir 506, uncompressible. For example, plates 532 and 534 may include an aperture 536 that allows a large portion of the reservoir 506 to avoid compression when the underlying ribs are positioned to be compressed.

[0283] The sealing rib array is configured to withstand compressions of at least about 500 lbf, preferably up to about 15,000 lbf, applied along the entire length of the array. When compressed under these types of loads, the sealing ribs may deflect by about 20% to about 30% or more. Such compression has been shown to adequately maintain a sealing force of about 4.5 lbf per inch, capable of maintaining a seal at pressures of 50–60 psi or more.

[0284] Compression may be provided by a clamping mechanism of the purification apparatus, as described elsewhere in this specification with respect to the purification apparatus. However, other embodiments may utilize other clamping mechanisms that do not necessarily have to operate as a result of the operation of the purification apparatus. For example, some embodiments may include a cartridge having an intermediate section of consumables and two reusable plate sections positioned above the intermediate section and which can be tightened to compress the intermediate section before use.

[0285] valve The purification cartridge 501 may include a number of valves at different locations that can be selectively opened and closed to direct the flow of fluid during the purification procedure. For example, one or more valves may be located near each reservoir in the cartridge and may be configured to control the movement of fluid into and out of the reservoirs during the purification process.

[0286] Figure 24 illustrates an exemplary valve mechanism 540 in cross-section. The valve components of the valve mechanism 540 can be beneficially formed using the same elastomer layer 520 used elsewhere in the cartridge 501 to form sealing ribs 522, thereby reducing the number of components required and simplifying the manufacture of the cartridge.

[0287] As shown, the elastomer layer 520 includes a deflectable portion 542 extending through an aperture 528 formed in the second outer layer 514. The deflectable portion 542 is illustrated here as a “dome” shaped structure. However, other embodiments may provide deflectable portions of other shapes. The deflectable portion 542 is biased outward so that an opening 530 is provided between fluid channels 523 and 525 formed in the first outer layer 508.

[0288] In the open state, the fluid 503 can freely pass through the beam 546 formed between the fluid channels 523 and 525. The valve can be actuated and moved to the closed state when a plunger 505 or other preferred structure engages with the deflectable portion 542 and deflects it toward the beam 546. The side of the deflectable portion 542 facing the beam 546 may include a sealing rib 544 that, under compression from the plunger 505, engages with the beam 546 to help close the opening 530 and seal the fluid channel 525 away from the fluid channel 523. When the plunger 505 is retracted, the deflectable portion 542 springs back to its normal shape, thereby reopening the valve.

[0289] The deflectable portion 542 can preferably remain closed under pressures reaching 30 psi or more, or 50–60 psi or more. This corresponds to a force of about 5 lbf required to hold the deflectable portion 542 in the closed position. Therefore, the deflectable portion 542 should preferably be configured to withstand a force of about 3–7 lbf and / or a pressure of up to about 50–60 psi without failing to allow fluid to pass through or "cracking".

[0290] The plunger 505 may be part of a purification apparatus capable of processing the cartridge 501. For example, multiple plungers may be mounted on one or both of the clamp plates of the purification apparatus described herein.

[0291] In the embodiments described above, the cross-sectional shapes of the sealing ribs 522 and 544 are shown as having a semicircular shape (for example, substantially similar to the shape of an O-ring). Other embodiments may include other cross-sectional shapes, including other curved shapes or polygons such as triangles.

[0292] It should be understood that any of the aforementioned processing systems, valves, seals, etc., disclosed above with respect to either a targeted biomolecule purification system or cartridge, may be used and adapted, as needed, for use in systems and cartridges specifically configured for the automated purification of targeted nucleic acids or targeted proteins.

[0293] Abbreviation list of defined terms To facilitate understanding of the scope and content of this document and the attached claims, several selected terms are defined below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates.

[0294] As used herein, the terms “approximately,” “about,” and “substantially” describe a quantity or condition that is close to a specific stated quantity or condition that still performs the desired function or achieves the desired result. For example, the terms “approximately,” “about,” and “substantially” may refer to a quantity or condition that deviates by less than 10%, or less than 5%, or less than 1%, or less than 0.1%, or less than 0.01%, from the specifically stated quantity or condition.

[0295] The term “buffer” in this context refers to any suitable buffer, washing solution, resuspension buffer, lysis buffer, neutralization buffer, RNase (or other enzyme), binding buffer / solution, elution / collection buffer, or precipitation buffer, which may be used with or without additional suitable reagents, depending on the protocol being performed. Suitable buffers, as well as their compositions and methods of use, are disclosed in the following U.S. Patent References, namely U.S. Patents / Publications No. 6,914,137, 2006 / 0154247, 2007 / 0117972, 6,242,220, 5,990,301, 7,214,508, 7,109,322, and 6,297,371, all of which are incorporated herein by reference.

[0296] Exemplary resuspension buffers according to the systems and methods described may include any suitable bioacceptable buffer, such as Tris, TAPS, Bisine, Trisine, HEPES, TES, MOPS, PIPES, Cacodylate, MES, Acetates, etc., having pH values ​​of approximately 3.5–approximately 9, approximately 5–approximately 8, or approximately 6.5–approximately 7.5. Additionally, the resuspension buffer may contain chelating agents such as EDTA, EGTA, ALA, BATA, defalasiloxane, deferiprone, deferoxamine, DTPA, dimercaprol, DMPS, DMSA, etc., in concentrations of 1 mM–100 mM, 5 mM–50 mM, or 10 mM–20 mM. The resuspension buffer may optionally contain ribonucleases such as endoribonucleases and / or exoribonucleases selected from one or more of the following: RNase A, RNase H, RNase I, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, RNase VI, RNase V, PNPase, RNase PEI, RNase II, RNase R, RNase D, RNase T, exoribonuclease I, exoribonuclease II, etc. In some embodiments, the resuspension buffer may optionally contain lysozymes and / or carbohydrates such as sugars in concentrations of about 1 mM to about 500 mM, about 10 mM to about 200 mM, about 20 mM to about 100 mM, and about 30 mM to about 75 mM. Exemplary sugars include glucose, fructose, galactose, mannose, maltose, and lactose.

[0297] For example, the resuspension buffer may be an aqueous solution containing 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA. In an additional example, the resuspension buffer may be an aqueous solution containing 50 mM Tris-HCl (pH 8.0), 2.4 mg / ml RNase A, and 10 mM EDTA. In yet another non-limiting example, the resuspension buffer may be an aqueous solution containing 50 mM Tris (pH 7.4), 100 pg / ml RNase Al, 10 mM EDTA, and 5 mM glucose.

[0298] With respect to dissolving solutions or buffers, a suitable dissolving solution or buffer may contain one or more denaturing agents in combination with one or more lipid-destroying agents in an aqueous carrier medium. The denaturing agents may be nucleic acid denaturing agents such as alkaline salts. Suitable alkaline salts may include sodium hydroxide, potassium hydroxide, calcium hydroxide, etc. Suitable lipid-destroying agents may include ionic surfactants. Exemplary ionic surfactants include sodium cholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC), N-lauroyl sarcosine salt, cetyltrimethylammonium bromide (CTAB), bis(2-ethylhexyl) sulfosuccinate, etc.

[0299] As a non-limiting example, the lysis buffer used herein may be an aqueous formulation containing 1% (v / v) SDS and 200 mM sodium hydroxide. As another non-limiting example, the exemplary lysis solution may be an aqueous solution containing about 10 mM to about 500 mM, about 50 mM to about 250 mM, or about 100 mM to about 200 mM NaOH in combination with up to about 10% (v / v) SDS, up to about 5% (v / v) SDS, or up to about 1% (v / v) SDS. It should be understood that additional agents may be present in the lysis buffer, as will be readily apparent to those skilled in the art.

[0300] With regard to the neutralization solution, a suitable neutralization solution may contain one or more agents capable of neutralizing the surfactant / alkaline solution present in the dissolution solution in a suitable aqueous carrier medium. In some embodiments, the neutralization solution may contain a suitable acetate in a concentration of about 0.5 M to about 5 M with a pH greater than 4. An exemplary neutralization solution may contain an aqueous solution of potassium acetate in a concentration of about 3.1 M with a pH of about 5.5.

[0301] In some embodiments, such as for protein purification, non-chaotropic buffers and reagents that do not disrupt the structure and function of proteins can be used.

[0302] A suitable wash buffer may contain, in a suitable aqueous carrier medium, 0.1 mM to about 100 mM of salt, about 0.5 mM to about 500 mM of a suitable biological buffer, and at least 5% (v / v), at least 10% (v / v), or at least 15% (v / v) of a suitable alcohol such as ethanol or isopropyl alcohol. In an exemplary embodiment, the wash buffer may be an aqueous solution containing 812.5 mM NaCl and 100 mM sodium acetate trihydrate (pH 5.0). In another non-limiting example, the wash buffer may be a formulation containing 1.5 NaCl and 100 mM sodium acetate trihydrate at pH 5.0. Optionally, the wash buffer may contain about 0.01% (v / v) to a maximum of about 10% (v / v) of a suitable nonionic detergent, such as TRITON® X-100, CHAPS, or NP-40. The addition of such detergents may, in some embodiments, enhance the removal of undesirable endotoxins from the preparation. Thus, an exemplary endotoxin removal / washing buffer may comprise 10% (v / v) TRITON® X-100, 750 mM NaCl, and 50 mM MOPS (pH 7.0). In another non-limiting example, an endotoxin removal / washing buffer may comprise 1 mM NaCl, 50 mM MOPS, pH > 8.0, 15% (v / v) isopropyl alcohol, and 0.5% (v / v) TRITON® X-100.

[0303] In some embodiments, ultrapure water or distilled water may be used to elute the target nucleic acid. Alternatively, a TE buffer or elution buffer may be used. A TE buffer may include, for example, an aqueous carrier medium containing 10 mM Tris (pH 8.0) and 0.1 mM EDTA. A preferred elution buffer may include, in a suitable aqueous carrier medium, a suitable biological buffer up to about 150 mM with a pH of 5.0 to 9.0, and / or a suitable chelating agent up to about 10 mM. An exemplary elution buffer may include, for example, 100 mM Tris-HCl (pH 8.5) and 1250 mM NaCl. Another non-limiting example of an elution buffer includes 10 mM Tris-HCl (pH 8.0) combined with 1 mM EDTA.

[0304] In some embodiments, the equilibration buffer may be optionally passed through a membrane or filter used in either the first or second bioprocessing assembly before use. In such embodiments, the equilibration buffer may contain up to 1 M of salt, up to 500 mM of a suitable biological buffer with a pH of 5.0–9.0, up to about 10% (v / v) of a suitable nonionic detergent, and up to about 20% (v / v) of alcohol. An example equilibration buffer may contain, for example, 750 mM NaCl, 50 mM MOPS (pH 7), 15% (v / v) of isopropyl alcohol, and about 0.15% (v / v) of TRITON® X-100.

[0305] In some embodiments, the precipitation buffer may be optionally passed through a precipitation filter before use. The precipitation buffer may include, for example, potassium acetate up to 5 M and a suitable biological buffer up to 500 mM with a pH of about 5.0–9.0. An exemplary precipitation buffer may include, for example, 3.1 M potassium acetate at pH 5.5.

[0306] As used herein, the term “large volume” includes any volume greater than approximately 5 mL, preferably any volume greater than approximately 10 mL, or any value or range of values ​​between 10 mL and 5 L. It should be understood that “large volume” as used herein is generally made with reference to a biological sample and may include volumes up to 250 mL, 10–250 mL, 50–200 mL, 100–150 mL, 100 mL–2 L, or any range or value in between. For example, plasmid DNA may be purified from a large volume of bacterial culture using the disclosed systems, methods, and devices, where a large volume is 150 mL. As an additional non-limiting example, a large volume may include up to 1 L of urine. It should be understood that the systems, methods, and devices disclosed herein may be adapted, as needed, to process different volumes of biological samples, including smaller volumes than those described above.

[0307] As used herein, the term “target biomolecule” is intended to be understood as nucleic acid or protein (as these terms are defined) derived from a biological or environmental source.

[0308] As used herein, the term “target nucleic acid” is intended to be understood as a nucleic acid sequence derived from a biological or environmental source, and may be one or more of a gene, regulatory sequence, genomic DNA, plasmid DNA, cDNA, or RNA. As outlined herein, the target nucleic acid may take any of the aforementioned forms and may be of any length, but in a preferred embodiment, the target nucleic acid constitutes a plasmid obtained from a bacterial culture.

[0309] As used herein, the term “target protein” is intended to be understood as a protein sequence derived from a biological or environmental source, and may be a polypeptide, oligopeptide, glycoprotein, lipoprotein, phosphorylated protein, membrane protein, or any protein. As outlined herein, a target protein may take any of the aforementioned forms and may be of any length, but in preferred embodiments, the target protein is in its biologically active state and conformation, which may include natural post-translational modifications and may be free from contamination or chemical modifications.

[0310] Various aspects of this disclosure, including devices, systems, and methods, may be illustrated by reference to one or more essentially exemplary embodiments or implementations. Where used herein, the term “exemplary” means “serving as an example, case, or illustration” and should not necessarily be construed as being preferable or advantageous to other embodiments disclosed herein. In addition, references to “implementations” of this disclosure or the invention include specific references to one or more embodiments thereof, and are intended to provide exemplary examples without limiting the scope of the invention, as set forth in the appended claims, rather than the following description.

[0311] Where used herein, unless otherwise implicitly or explicitly understood or stated, a singular word encompasses its plural equivalents, and a plural word encompasses its singular equivalents. Therefore, note that the singular forms “a,” “an,” and “the” as used herein and in the appended claims include multiple subjects unless explicitly indicated otherwise. For example, a reference to a single referent (e.g., “widgets”) includes one, two, or more referents unless specifically implicitly or explicitly understood or stated otherwise. Similarly, references to multiple referents should be interpreted as including a single referent and / or multiple referents unless explicitly indicated otherwise by the content and / or context. For example, a reference to a plural referent (e.g., “widgets”) does not necessarily require multiple such referents. Instead, it will be understood that, regardless of the presumed number of referents, one or more referents are contemplated herein unless specifically stated.

[0312] As used herein, terms indicating direction, such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “proximal,” and “distal,” are used herein solely to indicate relative directions and are not intended to otherwise limit the scope of the invention disclosed or claimed.

[0313] knot The terms and expressions used herein are for illustrative purposes only and are not limiting. In using such terms and expressions, there is no intention to exclude the exhibited and described features or their equivalents, but it is recognized that various modifications are possible within the scope of the claimed invention. Therefore, while the invention has been partially and concretely disclosed by preferred embodiments, exemplary embodiments, and optional features, it should be understood that modifications and variations of the concepts disclosed herein are possible for those skilled in the art, and such modifications and variations are considered to be within the scope of the invention, as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the invention, as well as various modifications and / or changes to the features of the invention shown herein, and additional uses of the principles shown herein that arise for those skilled in the relevant art and owners of this disclosure may be made with respect to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and should be considered within the scope of this disclosure.

[0314] It will be understood that a system, device, product, kit, method, and / or process according to a particular embodiment of this disclosure may include, incorporate, or otherwise include characteristics or features (e.g., components, members, elements, parts, and / or parts) described in other embodiments disclosed and / or described herein. Accordingly, various features of a particular embodiment may be compatible with, combined with, included in, and / or incorporated in other embodiments of this disclosure. Therefore, the disclosure of a particular feature relating to a particular embodiment of this disclosure should not be construed as limiting the application or inclusion of such feature to a particular embodiment. Rather, it will be understood that other embodiments may include such features, members, elements, parts, and / or parts without necessarily departing from the scope of this disclosure.

[0315] Furthermore, unless a feature is described as requiring another feature in combination with it, any feature herein can be combined with any other feature of the same or different embodiments disclosed herein. Moreover, various well-known embodiments, such as exemplary systems, methods, and apparatus, are not described in particular detail herein to avoid obscuring the embodiments of the exemplary models. However, such embodiments are contemplated herein as well.

[0316] All references cited herein are incorporated herein by reference in their entirety to the extent that they do not conflict with the disclosures herein. It will be apparent to those skilled in the art that methods, devices, device elements, materials, procedures, and techniques other than those specifically described herein can be applied to the implementation of the invention as broadly disclosed herein without relying on excessive experimentation. All known functional equivalents in the art of the methods, devices, device elements, materials, procedures, and techniques specifically described herein are intended to be incorporated herein.

[0317] Where a group of materials, compositions, components, or compounds is disclosed herein, it is understood that all individual members of those groups and all their subgroups are disclosed separately. Where a Markush group or other group is used herein, all individual members of the group, and all possible combinations and partial combinations of the group, are intended to be included in this disclosure individually. All formulations or combinations of components described or illustrated herein can be used to carry out the invention unless otherwise specified. Wherever a range, for example, a temperature range, a time range, or a composition range is indicated in the specification, all intermediate and subranges, as well as all individual values ​​included within the given range, are intended to be included in this disclosure.

[0318] All modifications that fall within the equivalent meaning and scope of the claims should be included within those scopes. [Examples]

[0319] The following examples are provided to illustrate various implementations of the embodiments disclosed herein, and are illustrative in nature, and are not intended to unnecessarily limit the scope and / or content of the disclosures provided herein.

[0320] Example 1 The instruments and accompanying single-use cartridges disclosed herein were used for a fully automated plasmid DNA purification protocol directly from bacterial cultures.

[0321] To grow a bacterial culture containing a target nucleic acid (e.g., plasmid DNA), a single colony of bacteria containing the plasmid of interest was taken from a newly streaked selective plate and inoculated with a 1 mL starter culture in LB medium (containing an appropriate selective antibiotic, in this case, ampicillin). The culture was incubated at 37°C for 8 hours with vigorous shaking (300 rpm). Next, the starter culture was diluted 1:1000 in LB medium containing an appropriate selective antibiotic in a 5 L flask. The 1 L culture was incubated at 37°C for approximately 14 hours with vigorous shaking (300 rpm) to promote bacterial growth and amplification. The bacterial culture was then incubated until the cell density reached approximately 2–6 x 10⁶ cells. 9 Cells / mL or absorbance at 600nm is 2.0-6.0 (A 600 After confirming that the condition had been successfully reached, the DNA was then processed for plasmid DNA purification.

[0322] The bacterial cells used in this particular example were Top10 and DH5α Escherichia coli, but other E. coli strains have also been used in additional studies yielding similar results. The plasmid used in this particular example was high-copy pGL4.50, but other plasmids with various backbones, and inserts of various sizes, high-copy and low-copy plasmids have also been used in similar experiments yielding similar results.

[0323] The instrument prototype and its accompanying single-use cartridge prototype (e.g., cartridge 160b in Figure 7b) were used for fully automated plasmid DNA purification from 150 ml of bacterial culture (maxiscale). The PureLink® Expi Endotoxin-Free Maxi Plasmid Purification Kit was used alongside as a control, strictly following the manufacturer's protocol.

[0324] Figure 1 shows the OD of the pGL4.50 plasmid / Top10 cells and culture. 600 =3.0 was measured using three instrumental runs and one manual preparation with the PureLink kit, as well as the OD of pGL4.50 plasmid / DH5a cells and cultures. 600 The results for 4.5 are shown for three instrumental runs and one manual preparation using the PureLink kit. Table 1 below captures the plasmid DNA yield (micrograms), plasmid concentration in the output tube at approximately 1 ml elution volume, and plasmid purity measured by Nanodrop (A260 / 280, A260 / 230). Agarose gel analysis of the plasmid product is shown in Figure 25. [Table 1]

[0325] For pGL4.50 plasmid / Top10 cells, the plasmid yield from automated purification was 755–803 micrograms, and for pGL4.50 plasmid / DH5a cells, it was 881–982 micrograms, which was nearly identical to manual preparation with a PureLink anion exchange column-based kit. A260 / 280 purity was high for all samples (e.g., above 1.9). A260 / 230 purity was also high for all samples (e.g., above 2.2). Agarose gel analysis demonstrated the high purity and integrity of the purified plasmid products.

[0326] Table 2 below shows the results of a total of 25 plasmid purifications performed with the two instrument prototypes. The average yield for high-copy plasmids obtained from 150 ml of input bacterial culture was 796 micrograms, which is comparable to manual maxi preparation. Plasmid purity met the requirements, as described above for Figure 25. The total run time for the instrument prototypes was approximately 50 minutes, which is significantly faster than manual preparation, which takes at least 1.5 hours or more for typical anion-exchange membrane-based columns. [Table 2]

[0327] As shown, the instrument prototype and accompanying cartridge prototype enable fully automated plasmid DNA purification directly from bacterial cultures (without centrifugation), resulting in significantly faster and higher yields of clean plasmid DNA than "classic" manual purification kits.

[0328] Description of additional embodiments The following are exemplary, non-limiting embodiments that incorporate one or more of the features described above.

[0329] Embodiment 1. An apparatus for the automated purification of biomolecules such as target nucleic acids or target proteins from a biological sample, comprising: an input reservoir for receiving a biological sample; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir, configured to produce a lysate containing a target biomolecule such as a target nucleic acid or target protein; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and a first elution buffer reservoir, configured to hold a target biomolecule, including a target nucleic acid binding filter or a target protein binding filter; and a receptacle in fluid communication with the second bioprocessing assembly, configured to receive an output container for receiving a target biomolecule such as a target nucleic acid or target protein from the second bioprocessing assembly.

[0330] Embodiment 2. The apparatus according to Embodiment 1, wherein the apparatus includes a consumable cartridge.

[0331] Embodiment 3. The apparatus according to any one of Embodiments 1 to 2, wherein the input reservoir is configured to receive a large volume of clinical samples, a large volume of environmental samples, a food sample, or a beverage sample.

[0332] Embodiment 4. The apparatus according to any one of Embodiments 1 to 3, further comprising an optical density detector window positioned between an input reservoir and a first bioprocessing assembly, wherein the optical density detector window is configured to enable detection of the optical density of a biological sample.

[0333] Embodiment 5. The apparatus according to any one of Embodiments 1 to 4, wherein the first bioprocessing assembly comprises a purification filter.

[0334] Embodiment 6. The apparatus according to Embodiment 5, wherein the purification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the purification filter is configured to separate the target nucleic acid-containing portion of the biological sample from the first waste portion of the biological sample.

[0335] Embodiment 7. The apparatus according to Embodiment 5 or 6, wherein the first bioprocessing assembly comprises a cell capture filter, and the cell capture filter is located upstream of the purification filter.

[0336] Embodiment 8. The apparatus according to Embodiment 7, wherein a cell capture or concentration filter is in fluid communication with an input reservoir and a lysis buffer reservoir, and the cell capture or concentration filter is configured to separate the target nucleic acid-containing portion of the biological sample from the first waste portion of the biological sample.

[0337] Embodiment 9. The apparatus according to Embodiment 7 or 8, wherein the lysis buffer reservoir is fluidically connected to the cell capture filter to allow backwashing of the cell capture filter and passage of backwash to the purification filter.

[0338] Embodiment 10. The apparatus according to Embodiment 9, further comprising a first mixing chamber positioned between a purification filter and a cell capture filter, wherein the first mixing chamber is configured to receive backwashing.

[0339] Embodiment 11. The apparatus according to Embodiment 10, further comprising a neutralizing buffer reservoir in fluid communication with the first mixing chamber, wherein the first mixing chamber is configured to receive backwash and neutralizing buffer and to provide a mixture of backwash and neutralizing buffer to form a neutralized soluble product.

[0340] Embodiment 12. The apparatus according to Embodiment 11, wherein the purification filter is in fluid communication with the first mixing chamber, and the purification filter is configured to separate the second waste portion from the target nucleic acid-containing portion of the biological sample.

[0341] Embodiment 13. The apparatus according to any one of Embodiments 9 to 12, wherein the purification filter comprises a cell capture filter, the cell capture filter configured to concentrate the cellular components of a biological sample in a first purification step and to purify the neutralized lysate in a second, subsequent purification step.

[0342] Embodiment 14. The apparatus according to any one of Embodiments 1 to 13, wherein the target biomolecule binding filter of the second bioprocessing assembly includes a silica-based filter or a column of beads having affinity for the target nucleic acid.

[0343] Embodiment 15. The apparatus according to Embodiment 14, further comprising a purification reagent reservoir in fluid communication with a target biomolecule binding filter, such as a nucleic acid binding filter, for the second bioprocessing assembly.

[0344] Embodiment 16. The apparatus according to Embodiment 14 or 15, wherein a target biomolecule binding filter, such as a nucleic acid binding filter, is in fluid communication with an elution buffer reservoir and an output container.

[0345] Embodiment 17. The apparatus according to any one of Embodiments 1 to 16, wherein the second bioprocessing assembly comprises a second mixing chamber positioned between the first bioprocessing assembly and the target biomolecule binding filter.

[0346] Embodiment 18. The apparatus according to Embodiment 17, wherein the second mixing chamber is in fluid communication with the endotoxin removal buffer reservoir.

[0347] Embodiment 19. The apparatus according to any one of Embodiments 1 to 18, wherein the target biomolecule binding filter is a nucleic acid binding filter comprising an anion exchange membrane.

[0348] Embodiment 20. The apparatus according to Embodiment 19, wherein the second bioprocessing assembly comprises a precipitation membrane located downstream of the anion exchange membrane.

[0349] Embodiment 21. The apparatus according to Embodiment 20, wherein the anion exchange membrane is configured to separate the third waste portion from the target nucleic acid-containing portion of the biological sample.

[0350] Embodiment 22. The apparatus according to Embodiment 20 or 21, further comprising a second elution buffer reservoir, wherein the first elution buffer reservoir is fluidly connected to an anion exchange membrane to enable elution of target nucleic acids from the anion exchange membrane, and the second elution buffer reservoir is fluidly connected to a precipitate membrane to enable elution of target nucleic acids from the precipitate membrane.

[0351] Embodiment 23. The apparatus according to any one of Embodiments 20 to 22, wherein the precipitation membrane is configured to separate the fourth waste portion from the target nucleic acid-containing portion of the biological sample.

[0352] Embodiment 24. The apparatus according to any one of Embodiments 20 to 23, wherein the precipitation membrane is in fluid communication with the precipitation reagent reservoir, and the precipitation reagent reservoir optionally contains isopropanol.

[0353] Embodiment 25. The apparatus according to any one of Embodiments 20 to 24, wherein the precipitated membrane is in fluid communication with a washing / desalting solution reservoir, and the washing / desalting solution reservoir optionally contains about 70% ethanol.

[0354] Embodiment 26. The apparatus according to any one of Embodiments 20 to 25, comprising a third mixing chamber, the second bioprocessing assembly being positioned between an anion exchange membrane and a precipitation membrane and in fluid communication with them, wherein the third mixing chamber is fluidly connected to a precipitation reagent reservoir and / or a desalting solution reservoir and is positioned between the precipitation membrane and the precipitation reagent reservoir and / or the desalting solution reservoir.

[0355] Embodiment 27. The apparatus according to any one of Embodiments 1 to 26, wherein the output container is selectively removable from the apparatus.

[0356] Embodiment 28. The apparatus according to any one of Embodiments 1 to 27, wherein the input reservoir is sized and shaped to accept at least 5 mL, preferably at least 100 mL, or up to 2 L of a biological sample.

[0357] Embodiment 29. An embodiment of an apparatus for automated purification of target nucleic acids from a biological sample comprises: an input reservoir for receiving a biological sample; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir, configured to produce a lysate containing the target nucleic acid; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and a first elution buffer reservoir, comprising a nucleic acid binding filter configured to hold the target nucleic acid; and a receptacle in fluid communication with the second bioprocessing assembly, configured to receive an output container for receiving the purified form of the target nucleic acid. Another embodiment of the apparatus for automated purification of target nucleic acids from biological samples comprises a first bioprocessing assembly configured to receive a biological sample, the first bioprocessing assembly comprising a waste separation filter and a plurality of reservoirs fluidly coupled to the waste separation filter; a second bioprocessing assembly comprising an anion exchange membrane, a wash solution reservoir fluidly coupled to the anion exchange membrane, and a first elution buffer reservoir fluidly coupled to the anion exchange membrane; and a third bioprocessing assembly comprising a precipitation filter and a second elution buffer reservoir fluidly coupled to the precipitation filter.

[0358] Embodiment 30. The apparatus according to Embodiment 29, wherein the first bioprocessing assembly further comprises a cell capture filter, and the first reservoir of a plurality of reservoirs comprises a resuspension buffer reservoir.

[0359] Embodiment 31. The apparatus according to Embodiment 30, wherein an input reservoir is fluidically connected to a first side of the cell capture filter, and a resuspension buffer reservoir is fluidically connected to a second side of the cell capture filter.

[0360] Embodiment 32. The apparatus according to any one of Embodiments 1 to 31, wherein the apparatus is a consumable cartridge for use in an automated nucleic acid purification system, and the consumable cartridge is configured to be associated with the automated nucleic acid purification system to enable automated purification of target nucleic acids without human interaction.

[0361] Embodiment 33. A method for automated purification of a target nucleic acid from a biological sample, comprising: receiving the biological sample in an input reservoir; producing a lysate from the biological sample in a first bioprocessing assembly without further human interaction, wherein the lysate contains the target nucleic acid; receiving the target nucleic acid-containing portion of the lysate in a second bioprocessing assembly; holding the target nucleic acid on a nucleic acid-binding filter in the second bioprocessing assembly; and eluting the purified form of the target nucleic acid from the nucleic acid-binding filter into an output container.

[0362] Embodiment 34. The method according to Embodiment 33, further comprising capturing the cellular contents of a biological sample with a first membrane of a first bioprocessing assembly.

[0363] Embodiment 35. The method according to Embodiment 33 or 34, further comprising measuring the optical density of a biological sample before producing a lysate, wherein the optical density is measured before the cell contents are captured by the first membrane.

[0364] Embodiment 36. The method according to Embodiment 35, wherein the biological sample comprises a bacterial culture, and the optical density of the biological sample is measured by automatically measuring the absorbance of the bacterial culture at approximately 600 nm.

[0365] Embodiment 37. The method according to any one of Embodiments 33 to 36, further comprising resuspending at least a portion of the cell contents in one or more of a resuspension buffer, an RNase solution, or a lysis buffer, and optionally, resuspending at least a portion of the cell contents includes backwashing the first membrane.

[0366] Embodiment 38. The method according to Embodiment 37, wherein backwashing the first membrane includes transferring a resuspension solution, comprising one or more of the following: a resuspension buffer, an RNase solution, or a lysis buffer, through the first membrane via a fluid channel located on the second side of the first membrane.

[0367] Embodiment 39. The method according to any one of Embodiments 33 to 38, further comprising mixing the lysate with a neutralizing buffer to form a neutralized lysate, and separating the target nucleic acid-containing portion from the waste portion of the neutralized lysate.

[0368] Embodiment 40. The method according to any one of Embodiments 33 to 39, further comprising mixing an endotoxin removal buffer with the target nucleic acid-containing portion of the lysate.

[0369] Embodiment 41. The method according to any one of Embodiments 33 to 40, wherein the second bioprocessing assembly holds the target nucleic acid on a nucleic acid binding filter, and the portion of the lysate containing the target nucleic acid is passed through an anion exchange membrane.

[0370] Embodiment 42. The method according to Embodiment 41, further comprising removing the portion of the lysate containing the target nucleic acid from the anion exchange membrane, precipitating the target nucleic acid to desalt and / or concentrate the target nucleic acid, and optionally capturing the precipitated target nucleic acid with a precipitation membrane.

[0371] Embodiment 43. The method according to any one of Embodiments 33 to 42, wherein the second bioprocessing assembly holds the target nucleic acid on a nucleic acid binding filter, and the portion of the lysate containing the target nucleic acid passes through the precipitate membrane to capture the target nucleic acid in the precipitate membrane.

[0372] Embodiment 44. The method according to any one of Embodiments 33 to 43, wherein the second bioprocessing assembly holds the target nucleic acid on a nucleic acid binding filter, passing the portion of the lysate containing the target nucleic acid through a silica-based filter, mixing the portion of the lysate containing the target nucleic acid with a chaotropic salt buffer before passing it through the silica-based filter, and optionally washing the silica-based filter with an alcohol-based washing solution.

[0373] Embodiment 45. The method according to Embodiment 44, wherein eluting the target nucleic acid in a purified form from the nucleic acid binding filter is performed using water or TE buffer under low-salt conditions.

[0374] Embodiment 46. The method according to any one of Embodiments 33 to 45, performed using the apparatus described in any one of Embodiments 1 to 32.

[0375] Embodiment 47. Apparatus for automated purification of target nucleic acids or target proteins from a biological sample, comprising: a casing having an internal compartment configured to receive a purification cartridge; an optionally selectably closable access door providing access to the internal compartment; and a pump assembly disposed within an internal chamber and configured to provide pump transport action through peristaltic motion.

[0376] Embodiment 48. The apparatus according to Embodiment 47, further comprising a clamping mechanism located within an internal compartment, configured to move between an open position in which the internal compartment is accessible and a closed position in which the clamping mechanism is clamped to a purification cartridge inserted to allow processing of a biological sample, wherein optionally, when in the closed position, the clamping mechanism operates to fluidly seal the biological sample cartridge.

[0377] Embodiment 49. The apparatus according to Embodiment 48, further comprising a controller which routes power to the clamping mechanism only when it determines that the access door is closed.

[0378] Embodiment 50. The apparatus according to Embodiment 48 or 49, further comprising a locking mechanism configured to lock the access door in the closed position when the clamping mechanism is moved to the closed position.

[0379] Embodiment 51. The apparatus according to any one of Embodiments 48 to 50, wherein the clamping mechanism is powered by a motor, and optionally the motor is operably connected to a rotatable worm gear or drive screw that causes movement of the clamping mechanism.

[0380] Embodiment 52. The apparatus according to Embodiment 51, further comprising a manual release mechanism operably connected to the clamping mechanism in order to enable manual release of the clamping mechanism independently of the motor.

[0381] Embodiment 53. The apparatus according to any one of embodiments 48 to 52, wherein the casing comprises an open end to which an access door is attached and a closed end opposite the open end, the open end and the closed end defining a longitudinal direction, and the clamping mechanism is configured to move in a direction transverse to the longitudinal direction.

[0382] Embodiment 54. The apparatus according to any one of Embodiments 47 to 53, further comprising an optical density sensor for measuring the optical density of a biological sample received in a biological sample cartridge, an optical density sensor for measuring the concentration of a target biomolecule such as a target nucleic acid concentration or a target protein concentration of a purified product, or both.

[0383] Embodiment 55. The apparatus according to any one of Embodiments 47 to 54, further comprising a user interface for displaying instrument information and for receiving user input, wherein the user interface is configured to receive user input relating to one or more of the following: the volume of a biological sample, a selected purification protocol, instructions for operation of a selectively closable door, a desired concentration of a target biomolecule such as a target nucleic acid or target protein, or the final volume of an eluent containing the target biomolecule.

[0384] Embodiment 56. The apparatus according to any one of Embodiments 47 to 55, wherein the internal compartment includes a position sensor for determining that the purification cartridge is fully inserted into the internal compartment, and a limit switch is positioned to be activated when it comes into contact with the leading edge of the purification cartridge when the purification cartridge is fully inserted into the internal compartment.

[0385] Embodiment 57. The apparatus according to any one of Embodiments 47 to 56, further comprising one or more access door sensors configured to determine whether an access door is in an open or closed position, wherein one or more access door sensors optionally comprises one or more Hall effect sensors and one or more corresponding magnets.

[0386] Embodiment 58. The apparatus according to any one of Embodiments 47 to 57, further comprising an output container sensor configured to detect the presence of an output container in an inserted purification cartridge.

[0387] Embodiment 59. The apparatus according to any one of embodiments 47 to 58, further comprising one or more rotatable magnets arranged to direct an electromagnetic field inward into an internal compartment.

[0388] Embodiment 60. The apparatus according to any one of Embodiments 47 to 59, wherein the pump assembly comprises a camshaft connected to a motor via a power transmission assembly, and a plurality of cam elements mounted on the camshaft, extending laterally toward the purification cartridge when inserted, and the cam elements optionally include angled tips that engage with the purification cartridge when inserted.

[0389] Embodiment 61. The apparatus according to Embodiment 60, wherein the cam elements are arranged such that the rotation of the camshaft causes a linear peristaltic motion of the tips of the cam elements.

[0390] Embodiment 62. A system comprising the apparatus described in any one of Embodiments 47 to 62 and a purification cartridge, wherein the purification cartridge is the apparatus described in any one of Embodiments 1 to 32.

[0391] Embodiment 63. A method for automated purification of a target nucleic acid from a biological sample, comprising: providing an apparatus (or system) according to any one of Embodiments 47 to 62; loading a purification cartridge into an internal compartment of the apparatus through an access door; and initiating a purification procedure in the apparatus, wherein initiating the purification procedure causes the apparatus to automatically purify the target nucleic acid without further human interaction.

[0392] Embodiment 64. The method according to Embodiment 63, further comprising closing the access door of the apparatus and moving the clamping mechanism to a closed position to clamp and fluidly seal the loaded purification cartridge.

[0393] Embodiment 65. The method according to Embodiment 64, further comprising determining that the purification cartridge is fully loaded and / or that the access door is fully closed before initiating the purification procedure.

[0394] Embodiment 66. The method according to any one of Embodiments 63 to 65, further comprising determining whether an output container is properly positioned and providing a warning when it is determined that an output container is not present.

[0395] Embodiment 67. The method according to any one of Embodiments 63 to 66, further comprising determining the optical density of a biological sample in a purification cartridge, wherein the determined optical density of the input sample is used to adjust one or more parameters of the purification procedure, the one or more parameters including the amount of one or more reagents used in the purification procedure, the duration of pump transport through the pump assembly, or the rate of pump transport through the pump assembly.

[0396] Embodiment 68. The method according to Embodiment 67, wherein a higher optical density reading results in one or more of the following compared to a lower optical density reading: a larger amount of one or more reagents used, a longer pumping time in one or more fluid transfer steps, and a higher fluid velocity in one or more fluid transfer steps.

[0397] Embodiment 69. The method according to any one of Embodiments 63-68, wherein the initial optical density reading is taken before initiating the purification procedure to determine whether the purification cartridge has been used previously, and an optical density reading substantially equal to the air blank reading indicates that the culture input reservoir of the biological sample cartridge remains undamaged.

[0398] Embodiment 70. The method according to any one of Embodiments 63 to 69, further comprising loading a large quantity of biological sample having an amount of about 5 ml to 5 L, or 10 ml to 500 ml, or about 15 ml to 250 ml, into a purification cartridge.

[0399] Embodiment 71. A control system for controlling an automatic purification apparatus (or system) described in any one of Embodiments 47 to 62, comprising one or more processors and one or more hardware storage devices on which computer executable instructions that can be executed by the one or more processors are stored, wherein the control system causes at least one or more sensors to receive sensor data in order to determine one or more process states of the automatic purification apparatus, compares the determined process state with a set of different protocols stored in a protocol library, selects a purification protocol from the protocol library based on the determined process state, and executes the selected purification protocol by operating one or more instrument actuators of the automatic purification apparatus according to the selected purification protocol.

[0400] Embodiment 72. The control system according to Embodiment 71, wherein one or more sensors include an optical density sensor, an access door sensor, a cartridge proximity or contact sensor, an output container proximity or contact sensor, a cartridge scanner, a timer, a temperature sensor, a weight sensor, a volume measurement sensor, or a combination thereof.

[0401] Embodiment 73. A control system of Embodiment 71 or 72, wherein the determined process state includes at least the determined optical density of the initial input sample, and optionally, the selected purification protocol provides a cell capture duration based on the determined optical density of the initial input sample.

[0402] Embodiment 74. A control system according to any one of Embodiments 71 to 73, defining whether the selected purification protocol utilizes cell capture and / or cell lysis, the duration of cell capture and / or cell lysis, the reagents used, the amount of reagents used, whether and / or when mixing is performed in one or more purification steps, the duration and / or rate of one or more mixing steps, the duration and / or rate of pump transport between one or more purification steps, whether and / or when one or more valves in the cartridge are opened and closed, whether and / or when one or more seals in the cartridge are punctured to allow the release of the corresponding fluid or the discharge of gas, the type of target biomolecule capture involved, the duration of target biomolecule capture, or a combination thereof.

[0403] Embodiment 75. A fluid release system for holding and selectively releasing a fluid, comprising: a flexible gasket; a reservoir disposed on a first side of the flexible gasket and configured to hold a fluid; a fragile seal disposed between the flexible gasket and the fluid reservoir; and an actuator disposed on a second side of the flexible gasket, the actuator being operable to deflect the flexible gasket to break the fragile seal and selectively release the fluid from the reservoir.

[0404] Embodiment 76. The system according to Embodiment 75, wherein the flexible gasket is an elastomer.

[0405] Embodiment 77. The system according to Embodiment 75 or 76, wherein the flexible gasket is part of an intermediate layer positioned between two outer layers, and the reservoir is optionally defined at least partially by the flexible gasket and one of the two outer layers.

[0406] Embodiment 78. The system according to any one of Embodiments 75 to 77, wherein the fracturing seal comprises a chemically inert material, and the fracturing seal also optionally comprises a reinforcing layer, wherein the chemically inert material is associated with or fused to the surface of the reinforcing layer such that the chemically inert material is positioned toward and / or forms a side wall defining the reservoir.

[0407] Embodiment 79. The system according to any one of embodiments 75 to 78, comprising a punctureable material, wherein the easily breakable seal is configured to catastrophically break in response to the application of a mechanical force applied by an actuator.

[0408] Embodiment 80. The system according to any one of Embodiments 75 to 79, wherein the fluid comprises a nucleic acid purification reagent, a protein purification reagent, an input sample, a resuspension buffer, RNase A, DNase, proteinase K, a lysis buffer, a neutralization buffer, a chaotropic salt buffer, a non-chaotropic salt buffer, a binding buffer, an endotoxin removal buffer, a washing buffer, an elution buffer, isopropanol, ethanol, water, or a TE buffer.

[0409] Embodiment 81. The system according to any one of embodiments 75 to 80, further comprising a fluid channel in fluid communication with a reservoir, wherein the fluid channel is configured to receive fluid when fluid is released from the reservoir.

[0410] Embodiment 82. The system according to any one of Embodiments 75 to 81, further comprising a flexible air vent, a fragile air seal disposed on a first side of the flexible air vent, and a vent actuator disposed on a second side of the flexible air vent, wherein optionally the vent actuator is operable to selectively deflect the flexible air vent toward the fragile air seal, thereby rupturing the fragile air seal to discharge air into a reservoir.

[0411] Embodiment 83. An automated system for selectively releasing a fluid, comprising a fluid release system according to any one of Embodiments 75 to 82, and a biological sample cartridge for use with an automated target biomolecule purification system, comprising an apparatus according to any one of Embodiments 1 to 32, wherein the automated system also optionally comprises a control system according to any one of Embodiments 71 to 74, and optionally comprises an automated purification apparatus or system according to any one of Embodiments 47 to 62.

[0412] Embodiment 84. A method for selectively releasing fluid from a reservoir in an automated process, comprising: bringing a flexible gasket into contact with an actuator; moving the actuator to deflect the flexible gasket toward a fragile seal associated with the reservoir; and causing the flexible gasket to rupture the fragile seal, thereby releasing the fluid from the reservoir.

[0413] Embodiment 85. The method according to Embodiment 84, wherein the fluid is released from the reservoir without the actuator coming into direct contact with the fluid.

[0414] Embodiment 86. The method of Embodiment 84 or 85, further comprising pulling the actuator away from the torn fragile seal, and the flexible gasket moving away from the torn fragile seal in response to the actuator being pulled away.

[0415] Embodiment 87. The method according to any one of Embodiments 84 to 86, further comprising bringing a flexible air vent into contact with a vent actuator, moving the vent actuator to deflect the flexible air vent toward a fragile air seal, and causing the flexible air vent to rupture the fragile air seal, wherein when the fragile air seal is ruptured, air is discharged into a reservoir.

[0416] Embodiment 88. The method according to any one of Embodiments 84 to 87, performed using the system described in any one of Embodiments 75 to 83.

[0417] Embodiment 89. Apparatus for controlled movement of a fluid, comprising: a first outer layer having a first side and a second side, the first side comprising a series of channels; a second outer layer located on the opposite side of the first side of the first outer layer; and an elastomer layer located between the first outer layer and the second outer layer, the elastomer layer comprising an array of sealing ribs corresponding to the series of channels, configured to fluidly separate the channels when the elastomer layer is compressed between the first outer layer and the second outer layer.

[0418] Embodiment 90. The apparatus according to Embodiment 89, wherein the first channel of a series of channels comprises a reservoir, and optionally one or more of the first outer layer or the second outer layer is a thermoformable polymer.

[0419] Embodiment 91. The apparatus according to Embodiment 89 or 90, further comprising a nominal gap between the elastomer layer and one or both of the first outer layer or the second outer layer, wherein when the elastomer layer is compressed between the first and second outer layers, the compressed portion of the sealing rib is displaced within the nominal gap.

[0420] Embodiment 92. The apparatus according to any one of embodiments 89 to 91, further comprising a valve associated with a series of channels, wherein the valve is selectively movable between a closed position and an open position to restrict or allow the flow of fluid across the valve, respectively.

[0421] Embodiment 93. The apparatus according to any one of embodiments 89 to 92, wherein the valve comprises an aperture in the second outer layer.

[0422] Embodiment 94. The apparatus according to Embodiment 93, wherein the aperture provides access to a deflectable portion of the elastomer layer, and the deflectable portion comprises a valve sealing rib extending from the elastomer layer toward a first outer layer.

[0423] Embodiment 95. The apparatus according to Embodiment 94, wherein the valve sealing rib contacts the first outer layer when the valve is in the closed position and separates from the first outer layer when the valve is in the open position.

[0424] Embodiment 96. The apparatus according to Embodiment 94 or 95, further comprising a plunger that contacts a deflectable portion of a valve, wherein the plunger is sized and shaped to pass through an aperture in order to deflect the deflectable portion and move the valve toward a closed position.

[0425] Embodiment 97. The apparatus according to any one of Embodiments 94 to 96, wherein the valve further comprises a balm formed in a first outer layer and extending toward an aperture, wherein optionally, when the valve is in the closed position, the balm contacts a valve sealing rib.

[0426] Embodiment 98. The apparatus according to any one of embodiments 94 to 98, wherein when the valve is moved to the fully open position, the deflectable portion extends outside and beyond the aperture.

[0427] Embodiment 99. The apparatus according to any one of embodiments 89 to 98, wherein the second outer layer comprises a second series of channels arranged on the first side of the second outer layer, and the first side of the second outer layer faces the first side of the first outer layer.

[0428] Embodiment 100. The apparatus according to Embodiment 99, wherein the arrangement of sealing ribs comprises a first set of sealing ribs extending toward a first outer layer and a second set of sealing ribs extending toward a second outer layer.

[0429] Embodiment 101. The apparatus according to any one of embodiments 89 to 100, wherein the arrangement of sealing ribs is arranged to follow the contour of a series of channels.

[0430] Embodiment 102. The apparatus according to Embodiment 101, wherein each channel has a bottom and forms a surface rising from the bottom to an inflection point located on the opposite side of the bottom, and optionally, sealing ribs are arranged such that they include a vertex and the sealing ribs are located at the inflection point of the channel or beyond the inflection point relative to the bottom of each channel.

[0431] Embodiment 103. A method for controlling fluid movement, comprising: providing an apparatus described in any one of Embodiments 89 to 102 to a system for automated purification of a target nucleic acid or target protein; causing one or more plungers to open valves in the apparatus; allowing the opening valves to enable fluid communication between an upstream section and a downstream section of a series of channels; and causing a pump to move the fluid from the upstream section to the downstream section.

[0432] Embodiment 104. The method according to Embodiment 103, further comprising one or more of the following: the array of sealing ribs is compressed to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leaking; the device is configured to withstand at least 500 lbf, preferably up to 15,000 lbf, applied along the entire length of the array of sealing ribs; or the sealing ribs are compressed by at least 20%, preferably at least 30%, when the elastomer layer is compressed between the first and second outer layers.

[0433] Embodiment 105. A biological sample cartridge comprising at least two of the following: the apparatus described in any one of Embodiments 1 to 32, the apparatus described in any one of Embodiments 47 to 62, the control system described in any one of Embodiments 71 to 74, the system described in any one of Embodiments 75 to 83, and the apparatus described in any one of Embodiments 89 to 102.

[0434] Embodiment 106. Apparatus for automated purification of a target protein from a biological sample, comprising: an input reservoir for receiving a biological sample; a first bioprocessing assembly having fluid communication with the input reservoir and a lysis buffer reservoir, configured to produce a lysate containing the target protein; a second bioprocessing assembly having fluid communication with the first bioprocessing assembly and a first elution buffer reservoir, comprising a protein-binding support configured to hold the target protein; and a receptacle having fluid communication with the second bioprocessing assembly, configured to receive an output container for receiving the target protein.

[0435] Embodiment 107. The apparatus according to Embodiment 106, wherein the apparatus comprises a consumable cartridge.

[0436] Embodiment 108. The apparatus according to Embodiment 106 or 107, wherein the first bioprocessing assembly comprises a purification filter.

[0437] Embodiment 109. The apparatus according to Embodiment 108, wherein the purification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the purification filter is configured to separate the target protein-containing portion of the biological sample from the first waste portion of the biological sample.

[0438] Embodiment 110. The apparatus according to any one of Embodiments 106 to 109, wherein the first bioprocessing assembly comprises a cell capture or concentration filter, and optionally the cell capture or concentration filter is located upstream of the purification filter.

[0439] Embodiment 111. The apparatus according to Embodiment 110, wherein a cell capture or concentration filter is in fluid communication with an input reservoir and a lysis buffer reservoir, and the cell capture or concentration filter is configured to separate the target protein-containing portion of the biological sample from a first waste portion of the biological sample.

[0440] Embodiment 112. The apparatus according to any one of Embodiments 106 to 111, further comprising one or more additional bioprocessing chambers having reagents for protein purification.

[0441] Embodiment 113. The apparatus according to any one of Embodiments 106 to 112, further comprising one or more filters, one or more of which are hollow fiber filters.

[0442] Embodiment 114. A method comprising automatically isolating or purifying a target protein from a cell lysate using the apparatus described in any one of Embodiments 106 to 113.

[0443] Embodiment 115. The method according to Embodiment 114, wherein the target protein is isolated by one or more of the following steps: cell lysis, column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, high-performance protein liquid chromatography, or a combination thereof.

[0444] Embodiment 116. The method according to Embodiment 114 or 115, wherein the sample comprises a biological sample, tissue, biopsy, cell culture, cells, cell suspension, aqueous solution of urine, saliva, cerebrospinal fluid, blood, serum, plasma, feces, other bodily fluids or exudates, eukaryotic cells, prokaryotic cells, or a cell suspension containing prokaryotic cells selected from the group consisting of rodents, insects, primates, and human cells.

[0445] Embodiment 117. The method according to any one of Embodiments 114 to 116, wherein the biological sample is a cell line or a tissue containing a plurality of cells, the target protein-containing portion contains a protein in the cell line or tissue, and the first waste portion comprises lysed cells and optionally, a culture medium.

[0446] Embodiment 118. A biological sample cartridge comprising at least two of the following: the apparatus described in any one of Embodiments 106 to 117, the apparatus described in any one of Embodiments 47 to 62, the control system described in any one of Embodiments 71 to 74, the system described in any one of Embodiments 75 to 83, and the apparatus described in any one of Embodiments 89 to 102.

Claims

1. A system having an apparatus for the automated purification of target biomolecules from biological samples, wherein the system has a purification cartridge, The aforementioned purification cartridge is An input reservoir for receiving the aforementioned biological sample, A first bioprocessing assembly, which is in fluid communication with the input reservoir and the lysis buffer reservoir, and is configured to produce a lysate containing the target biomolecule, A second bioprocessing assembly, which is in fluid communication with the first bioprocessing assembly and the first elution buffer reservoir, includes a target biomolecule binding filter and is configured to hold the target biomolecule; A receptacle that is in fluid communication with the second bioprocessing assembly, and is configured to receive an output container for receiving the target biomolecule from the second bioprocessing assembly, The aforementioned system further, A casing having an internal compartment, configured to be of a size and shape for receiving the aforementioned purification cartridge, A pump assembly is located within an internal chamber and configured to provide pumping action through peristaltic motion. The system further comprises a clamping mechanism located within the internal compartment, wherein the clamping mechanism is configured to move between an open position in which the internal compartment is accessible and a closed position in which the clamping mechanism is clamped to a purification cartridge inserted to allow processing of the biological sample, and when in the closed position, the clamping mechanism operates to fluidly seal the biological sample cartridge.

2. The system according to claim 1, wherein the purification cartridge further comprises an optical density detector window positioned between the input reservoir and the first bioprocessing assembly, the optical density detector window being configured to enable detection of the optical density of the biological sample.

3. The system according to claim 1 or 2, wherein the first bioprocessing assembly comprises a purification filter and a cell capture filter, the cell capture filter being located upstream of the purification filter.

4. The system according to claim 3, wherein the lysis buffer reservoir is fluidly connected to the cell capture filter to allow backwashing of the cell capture filter and backwashing to the purification filter, and further comprises a first mixing chamber positioned between the purification filter and the cell capture filter, wherein the first mixing chamber is configured to receive the backwash.

5. The system according to any one of claims 1 to 4, wherein the target biomolecule binding filter of the second bioprocessing assembly includes a silica-based filter or a column of beads having affinity for the target biomolecule.

6. The system according to any one of claims 1 to 5, wherein the second bioprocessing assembly comprises a second mixing chamber positioned between the first bioprocessing assembly and the target biomolecule binding filter, and the second mixing chamber is in fluid communication with an endotoxin removal buffer reservoir.

7. The system according to any one of claims 1 to 6, wherein the target biomolecule binding filter is a nucleic acid binding filter comprising an anion exchange membrane, the second bioprocessing assembly comprises a precipitation membrane disposed downstream of the anion exchange membrane, and the anion exchange membrane is further configured to separate a third waste portion from the target nucleic acid-containing portion of the biological sample.

8. The system according to any one of claims 1 to 7, wherein the output container is selectively removable from the apparatus, and / or the input reservoir is sized and shaped to accept at least 5 mL of biological sample.

9. The system according to any one of claims 1 to 8, wherein the casing comprises an open end to which an access door is attached and a closed end opposite to the open end, the open end and the closed end define a longitudinal direction, and the clamping mechanism is configured to move in a direction transverse to the longitudinal direction.

10. The system according to any one of claims 1 to 9, wherein the pump assembly comprises a camshaft connected to a motor via a power transmission assembly, a plurality of cam elements mounted on the camshaft, extending laterally therefrom toward the refining cartridge when inserted, the cam elements include angled tips that engage with the refining cartridge when inserted, and the cam elements are arranged such that rotation of the camshaft causes linear peristaltic motion of the tips of the cam elements.

11. Furthermore, it has a fluid release system for retaining and selectively releasing fluid, and the fluid release system is Flexible gasket and A reservoir disposed on the first side of the flexible gasket, configured to hold the fluid, A breakable seal is placed between the flexible gasket and the reservoir, The system according to any one of claims 1 to 10, comprising: an actuator disposed on the second side of the flexible gasket, wherein the actuator is operable to deflect the flexible gasket in order to break the easily breakable seal and selectively release the fluid from the reservoir.

12. The system according to claim 11, further comprising: a flexible air vent; a fragile air seal disposed on a first side of the flexible air vent; and a vent actuator disposed on a second side of the flexible air vent, wherein the vent actuator is operable to selectively deflect the flexible air vent toward the fragile air seal, thereby rupturing the fragile air seal to discharge air into the reservoir.

13. The aforementioned purification cartridge further, A first outer layer comprising a first side and a second side, wherein the first side comprises a series of channels, A second outer layer is positioned on the opposite side of the first side of the first outer layer, The system according to any one of claims 1 to 12, comprising: an elastomer layer disposed between the first outer layer and the second outer layer, wherein the elastomer layer comprises an arrangement of sealing ribs corresponding to the series of channels, and is configured to fluidly separate the channels when the elastomer layer is compressed between the first outer layer and the second outer layer.

14. The system according to claim 13, further comprising a valve associated with the series of channels, wherein the valve is selectively movable between a closed position and an open position to restrict or allow the flow of fluid across the valve, respectively, the valve comprising an aperture in the second outer layer, the aperture providing access to a deflectable portion of the elastomer layer, the deflectable portion comprising a valve sealing rib extending from the elastomer layer toward the first outer layer.

15. The system according to claim 14, wherein the valve sealing rib contacts the first outer layer when the valve is in the closed position, separates from the first outer layer when the valve is in the open position, and the deflectable portion extends outside and beyond the aperture when the valve is moved completely to the open position.

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