Systems, methods, and apparatus for automated separation of nucleic acids from proteins

Abstract

Purification of target biomolecules such as nucleic acids or proteins from biological sources is a time-consuming process that is typically performed by skilled technicians or scientists due to the high degree of technicality of the work. The systems, devices, and methods disclosed herein enable automated bioprocessing and purification of target biomolecules from biological sources. For example, the present disclosure provides an instrument and disposable purification column for automated isolation and purification of nucleic acids (e.g., plasmid DNA from bacterial cultures) or isolation of proteins from any biological sample. Such exemplary instrument and purification column can work in concert to release, mix, and move target biomolecules and various reagents and buffers in a timely manner during the purification of the target biomolecules, resulting in the purified target biomolecules with less human oversight than traditional methods.

Description

Background Technology Technical Field

[0001] This disclosure generally relates to systems, methods, and apparatus for processing biological samples. More specifically, this disclosure relates to systems, methods, and apparatus for the automated separation of nucleic acids and / or proteins from biological and / or environmental sources.

[0002] Related technologies

[0003] Certain laboratory procedures still dominate the process, relying on inefficient manual methods and requiring scientists or lab technicians to concentrate intently while performing them. Many of these procedures would benefit from automation. For example, nucleic acid purification or isolation procedures, such as large-scale plasmid extraction from bacterial cultures, remain time-consuming, inefficient, and not fully automated tasks. Previous attempts at automating these procedures, such as commercial embodiments of the systems disclosed in U.S. Patent Nos. 8,404,198 and 9,808,799, and similar products, have suffered from numerous drawbacks, including incomplete automation and / or inability to operate with large sample volumes. Progressive improvements, such as the introduction of precipitation filters, have reduced the required manual time. However, even state-of-the-art nucleic acid purification kits still require significant time investment; for example, endotoxin-free plasmid purification at the maxi, mega, or giga scale requires hours and individual attention.

[0004] This is at least partly due to the highly technical nature of nucleic acid purification and the diversity of tasks that need to be performed during the process. For example, many nucleic acid purification protocols involve flowing and routing liquids of varying viscosities and densities at different times during the purification process. Furthermore, it is crucial that buffer solutions and reagents be homogeneously mixed with the biological sample and / or filtrate during the purification process from biological samples, as this helps improve the purity and final concentration of the target nucleic acid. Therefore, it has proven difficult to incorporate these diverse liquids into automated processes that can provide delayed release, especially using a method capable of mixing liquids to create a homogeneous solution.

[0005] Furthermore, it has been proven that providing various buffer solutions and reagents separately in automated systems is both problematic and costly. Ideally, the materials containing various buffer solutions and reagents should be chemically compatible with the solutions being stored (e.g., non-reactive or inert materials) so that the solutions remain effective and active during unused or storage periods until they are applied for their intended purpose.

[0006] The various filtration steps employed in many nucleic acid purification protocols further complicate and challenge the application of automated processes. For example, different steps in nucleic acid purification protocols require selective filtration of the solution using mechanical and / or ionic methods, followed by cleaning or purification of the components associated with the filter / membrane. The amount of waste generated in this process is several times the original amount of the biological sample, and the preservation or disposal of these waste products is a complex factor for automation.

[0007] In traditional practice, a technician or scientist uses a variety of machines and instruments to execute nucleic acid purification protocols. This includes, for example, concentrating biological samples using centrifuges and pipettes, adding specific amounts of buffer solutions, and intermittently stirring or vortexing to homogenize each buffer / reagent within the solution. Numerous different filters / membranes, columns, or magnetic beads are used to further the nucleic acid purification protocol under centrifugation or vacuum conditions. Between centrifugation / vacuum steps, many steps generate waste products that require disposal.

[0008] As the volume of biological samples increases, the aforementioned problems are further exacerbated. Processing large-volume biological samples typically requires more buffers and reagents, as well as more robust filters, membranes, and columns. This places more stringent demands on the structural integrity and filtration capacity of various filters / membranes, posing challenges to the incorporation and monitoring of such filters / membranes in any automated process. Furthermore, the use of more buffers and reagents in processing large-volume biological samples generates more waste. The ability to be responsible for such waste presents an additional unique technical hurdle for any automated process.

[0009] Furthermore, current nucleic acid purification protocols rely on hands-on operation and human interaction, inherently carrying a risk of contamination. The lack of consistency between sample preparation and processing procedures necessitates the continuous deployment of highly skilled personnel. Importantly, large-scale nucleic acid purification is an extremely time-consuming process, distracting scientists from central projects or missions. These factors contribute to high costs and inefficiencies—whether in academic or clinical settings or within commercial enterprises.

[0010] Therefore, there is a significant need for automated nucleic acid purification systems, methods, and devices that can solve many unfavorable conditions and problems, especially those that can integrate all stages of the purification process into a single consumable element, thereby limiting or eliminating user intervention in the purification process.

[0011] Similarly, automated protein purification also faces many similar technical challenges. In protein purification, besides removing other cellular debris and materials from the target protein, the isolated / purified protein must also maintain its biological activity. This typically requires conditions and techniques that do not disrupt the protein's tertiary structure, preserve any post-translational modifications (e.g., phosphorylation, glycosylation, cysteine ​​disulfide bonds), and avoid introducing unnatural protein modifications (e.g., oxidation, deamidation). This is difficult to achieve manually, making the technical challenges of automating protein purification significantly greater.

[0012] There is a significant need for systems, methods, and apparatuses capable of protein purification, particularly those that integrate all stages of the purification process into a single consumable element, thereby limiting or eliminating user intervention in the purification process. Summary of the Invention

[0013] The embodiments of this disclosure solve one or more problems or other problems in the art concerning 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 can be incorporated into a single device for the automated purification of target biomolecules such as target nucleic acids and / or target proteins from biological samples. The device includes, for example, (i) an input reservoir for containing the biological sample; (ii) a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir; (iii) a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and a first elution buffer reservoir; and (iv) a container in fluid communication with the second bioprocessing assembly. The first bioprocessing assembly can be used to generate lysates containing the target biomolecules. The second bioprocessing assembly may include a target biomolecule binding filter for retaining the target biomolecules from the first bioprocessing assembly, and the container may be used to contain an output container for containing the purified target biomolecules from the second bioprocessing assembly. Non-limiting examples may include two or more bioprocessing assemblies.

[0015] Devices for the automated purification of target biomolecules (such as target nucleic acids and / or target proteins) can be used in combination with any number of reservoirs containing reagents and / or buffers, appropriately used to automatically separate target biomolecules from biological samples. For example, the device may have one or more reservoirs containing resuspension buffer, RNase A (or other enzymes, such as proteinase K, tobacco etch virus (TEV) enzyme, or totipotent nuclease), lysis buffer, neutralization buffer, endotoxin removal buffer, high-clumping salt buffer, washing buffer, elution buffer, refolding buffer, isopropanol, 70% ethanol, and / or TE buffer. The reservoir contents (e.g., whether to automatically purify target nucleic acids or target proteins) can be selected based on the type of target biomolecule to be purified.

[0016] In some embodiments, the apparatus described herein is used to purify target biomolecules from biological samples. In some embodiments, biological samples include 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 secretions, aqueous solutions of feces, cerebrospinal fluid, or other bodily fluids or exudates). Target biomolecules can be target nucleic acids such as genomic DNA, plasmid DNA, or RNA, or target proteins such as antibodies, cytokines, viral proteins, or other recombinant drug proteins, streptavidin, protein A, C-reactive protein (CRP), or any naturally occurring or recombinant protein used in functional, structural, or protein interaction analyses. As a non-limiting example, the target biomolecule can be plasmid DNA isolated from bacterial cultures, cell cultures, biological samples, etc. (i.e., biological samples). As an alternative, non-limiting example, the target biomolecule can be a recombinant or monoclonal antibody isolated from a eukaryotic culture (i.e., a biological sample). In some non-limiting embodiments, the biological sample includes large-volume bacterial cultures, large-volume cell cultures, large-volume prokaryotic cell cultures, large-volume eukaryotic cell cultures, large-volume environmental samples, large-volume food or beverage samples, or large-volume clinical samples. In some further non-limiting embodiments, the biological sample may be a small-volume, medium-volume, or large-volume sample.

[0017] In some embodiments where the target biomolecule is a target nucleic acid, an embodiment of an apparatus for automatically purifying a target nucleic acid from a biological sample includes: an input reservoir for containing the biological sample; a first bioprocessing component and a lysis buffer reservoir in fluid communication with the input reservoir, the first bioprocessing component being used to generate lysates containing the target nucleic acid; a second bioprocessing component in fluid communication with the first bioprocessing component and the first elution buffer reservoir, the second bioprocessing component including a nucleic acid binding filter for retaining the target nucleic acid; and a container in fluid communication with the second bioprocessing component for holding an output container containing the purified target nucleic acid.

[0018] In some embodiments where the target biomolecule is a target nucleic acid, an embodiment of an apparatus for automatically purifying a target nucleic acid from a biological sample includes: (i) a first bioprocessing assembly for containing the biological sample, the first bioprocessing assembly comprising a waste separation filter and a plurality of reservoirs fluidly connected to the waste separation filter; (ii) a second bioprocessing assembly comprising an anion exchange membrane, a washing solution reservoir fluidly connected to the anion exchange membrane, and a first elution buffer reservoir fluidly connected to the anion exchange membrane; and (iii) a third bioprocessing assembly comprising a precipitation filter and a second elution buffer reservoir fluidly connected to the precipitation filter.

[0019] The apparatus or device described in this disclosure may also include a consumable purification column for use in an automated biomolecule purification system, such as an automated target nucleic acid purification system or an automated target protein purification system. An exemplary embodiment of the consumable purification column may include an input reservoir for containing bacterial cultures, cell cultures, or eukaryotic cell cultures; a first bioprocessing component in fluid communication with the input reservoir and a lysis buffer reservoir; a second bioprocessing component in fluid communication with the first bioprocessing component and a first elution buffer reservoir; and an output container in fluid communication with the second bioprocessing component. The first bioprocessing component may be used to generate lysates from bacterial or eukaryotic cell cultures, wherein the lysates include, for example, target nucleic acids. In this embodiment, the second bioprocessing component may include a silicon-based filter for retaining target nucleic acids, and the output container may be used to contain purified target nucleic acids from the second bioprocessing component. The consumable purification column may be connected to an automated nucleic acid purification system to automatically purify target nucleic acids without human interaction.

[0020] The methods disclosed herein include an automated purification method for target biomolecules, such as target nucleic acids and / or target proteins, from biological samples. This exemplary method may include the following steps: receiving a biological sample at an input reservoir without further human-machine interaction; generating lysates from the biological sample containing target biomolecules (e.g., target nucleic acids and / or target proteins) in a first bioprocessing component; receiving target biomolecules (e.g., target nucleic acids and / or target proteins containing partial lysates) containing the partial lysates in a second bioprocessing component; retaining 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 component; and eluting the purified form of the target biomolecules from the biomolecule binding filter into an output container.

[0021] In some embodiments, the method may further include capturing cellular contents of a biological sample at a first membrane of a first bioprocessing assembly and resuspending at least a portion of the cellular contents in one or more resuspension buffers, deoxyribonuclease solutions, or lysis buffers. The resuspension of at least a portion of the cellular contents may include, for example, backwashing the first membrane by transferring the resuspension, and the resuspension may include one or more of resuspension buffers, ribonuclease solutions, or lysis buffers, from a fluid channel disposed on a second side of the first membrane and passing through the first membrane.

[0022] The method may additionally include target biomolecule-specific processing steps. For example, in some embodiments, where the target biomolecule is composed of a target nucleic acid, the method may additionally include mixing the lysate with a neutralization buffer to form a neutralized lysate, and separating the portion containing the target nucleic acid 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 cases, retaining the target nucleic acid on a nucleic acid-binding filter at a second biological processing component includes passing the target nucleic acid-containing portion of the lysate through an anion exchange membrane and removing the target nucleic acid-containing portion of the lysate from the anion exchange membrane, precipitating the target nucleic acid to remove salts and / or concentrate the target nucleic acid. According to some disclosed methods, the precipitated target nucleic acid may be further captured by a precipitation membrane.

[0023] Alternatively, the target nucleic acid may be retained on a nucleic acid binding filter at a second biological processing component, including passing the target nucleic acid-containing portion of the lysate through a silicon-based or advanced silicon-based filter. In these exemplary methods, the target nucleic acid-containing portion of the lysate may be mixed with a high-cleansing-salt buffer before passing through the silicon-based filter.

[0024] Some embodiments pertain to nucleic acid purification instruments that can be used in automated nucleic acid purification processes. The instrument interfaces with a built-in nucleic acid purification column to control fluid movement and path within the column, control the actuation of seals and valves, and control the timing of purification process steps, among other functions.

[0025] In one embodiment, the purification instrument includes: a sleeve with an internal compartment configured in size and shape to accommodate a purification column; a selectively closable access door leading to the internal compartment; and a pump assembly located within the internal compartment for providing suction via peristaltic movement. In some embodiments, the instrument may further include a clamping mechanism disposed within the internal compartment and for moving between an open position allowing access to the internal compartment and a closed position where the clamping mechanism compresses and inserts the purification column. The clamping mechanism thus helps maintain the integrity of the fluid seal of the column during periods of relatively high pressure that it may be subjected to during purification.

[0026] The instrument may include one or more sensors for determining component position, instrument operating status, process status, and / or other indications. For example, one or more position sensors may be used to ensure proper column insertion, proper column status, and / or secure column closure before initiating automated moving parts. One or more sensors may be communicatively coupled to a controller that may be used to automatically control instrument operation based at least in part on information received from one or more sensors. For example, the controller may be used to prevent the purification process from starting and / or provide notification / alarms to the user when it determines that the column is unsuitable, improperly inserted, cannot effectively collect purified products, contains an unsuitable sample, and / or is not properly and securely closed within the instrument.

[0027] The instrument may also include one or more actuators for interacting with the insert column to, for example, pump fluid, open seals and release fluid, open vents, control valves, and / or mix fluids. In some embodiments, a pump assembly for engaging one or more fluid passages of the insert column includes a camshaft and a plurality of cam elements extending laterally from the camshaft. The tips of the cam elements engage with the associated fluid passages. The pump assembly is used to cause rotation of the camshaft to induce a linear peristaltic motion of the cam element tips, thereby peristally compressing the fluid passages and driving fluid movement through the passages.

[0028] In one embodiment, a method for automatically purifying target nucleic acids from biological samples includes the steps of: providing a nucleic acid purification device (i.e., an instrument), loading a purification column into the internal compartment of the device through an access door, and initiating a purification program using the instrument. The initiation of the purification program enables the instrument to automatically purify the target nucleic acid without further human interaction.

[0029] In some embodiments, the method further includes closing the instrument's access door and moving the instrument's drive clamping mechanism to the closed position to compress the inserted purification column, thereby helping to fluidly seal the loaded purification column. The method may also include the steps of: determining that the purification column is fully loaded and / or determining that the access door is completely closed before initiating the purification program, for example, by using one or more position sensors to detect the column's position. The method may also include the steps of: determining that the output container is correctly placed on the purification column, and issuing an alarm / notification and / or preventing the purification program from starting if the output container is determined to be absent.

[0030] In some embodiments, the method may include the step of determining the optical density of a biological sample in a purification column. 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 determination may be used to adjust one or more parameters of the purification procedure, such as the volume of one or more reagents used in the purification procedure, the duration of pumping via the pump assembly, or the rate of pumping via the pump assembly.

[0031] In some embodiments, an initial optical density reading is taken before starting the purification procedure to determine whether the purification column has been used previously. For example, if the optical density reading is substantially equal to the air blank reading, it can be assumed that the reading indicates the culture inlet reservoir of the biological sample purification column is still intact, and therefore, the purification column has not been used.

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

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

[0034] Fluid release systems can be associated with any number or type of mixing chambers or reservoirs disclosed herein, and can selectively release fluid from these chambers / reservoirs at appropriate times, thereby influencing different processes and fluid movements within the nucleic acid purification system and associated columns. Therefore, embodiments of this disclosure further include automated systems for selective fluid release, comprising an automated nucleic acid purification system consisting of at least one component of the disclosed fluid release system and a biosample purification column used with the automated nucleic acid purification system, and at least one other component including the disclosed fluid release system.

[0035] Methods for selectively releasing fluid from a reservoir during automation may include contacting a flexible gasket 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 break the fragile seal, thereby releasing fluid from the reservoir.

[0036] Methods for selectively releasing fluid from a reservoir may further include contacting a flexible vent with a venting actuator, moving the venting actuator to deflect the flexible vent toward a fragile gas seal, and causing the flexible vent to break the fragile gas seal.

[0037] The systems, methods, and apparatuses described in this disclosure may further include means for controlled movement of fluid. The means may include a first outer layer having a first side and a second side including a series of channels. The means may additionally include a second outer layer arranged opposite the first side of the first outer layer, and an elastomeric layer disposed between the first and second outer layers. The elastomeric layer may include sealing rib structures corresponding to the series of channels and may be used to fluidly separate the channels when the elastomeric layer is compressed between the first and second outer layers.

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

[0039] In some embodiments, the orifice of the device provides access to a deflectable portion of an elastomeric layer, the deflectable portion including a valve sealing rib extending from the elastomeric layer toward a first outer layer. The valve sealing rib is contactable with the first outer layer when the valve is in the closed position and is decoupled from the first outer layer when the valve is in the open position. Alternatively, the device may include a plunger that contacts the deflectable portion of the valve. In these embodiments, the plunger is sized and shaped to allow it to permeate the orifice to deflect the deflectable portion and move the valve toward the closed position.

[0040] In one embodiment, the device is used to withstand a force of at least 500 lbf, preferably up to 15,000 lbf, applied over the entire length of the sealing rib structure. Additionally, the sealing rib structure can be compressed to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leakage, and / or the sealing rib is 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 this disclosure further include methods for controlling fluid movement. An exemplary method may include providing the apparatus disclosed herein for controlled fluid movement to a system for automated purification of target nucleic acids or target proteins, causing one or more plungers to open valves in the apparatus such that the open valves allow fluid communication between upstream and downstream portions of a series of channels, and causing a pump to move fluid from the upstream portion to the downstream portion. The disclosed method for controlling fluid movement may further include the step of providing the apparatus with a biological sample containing the target nucleic acid or target protein, and as described throughout the application, in some embodiments, the biological sample may 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] Therefore, systems, methods, and apparatuses for the automated purification of target biomolecules, such as nucleic acids or proteins, from biological samples are disclosed.

[0043] Embodiments of this disclosure address one or more of the above-mentioned or other problems in the art by automatically isolating target biomolecules, such as target nucleic acids or proteins, from biological, clinical, and / or environmental sources.

[0044] In particular, one or more embodiments may include an apparatus for automatically purifying a target protein from a biological sample, the apparatus comprising: an input reservoir for receiving the biological sample; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir for generating a lysate containing the target protein; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and a first elution buffer reservoir, the second bioprocessing assembly including a protein binding support for retaining the target protein; and a container in fluid communication with the second bioprocessing assembly for receiving an output container for receiving the purified form of the target protein.

[0045] In one embodiment, the device includes a consumable purification column for an automated protein purification system. In some cases, the consumable purification column for an automated protein purification system includes: an input reservoir for receiving a biological sample containing a target protein or related protein; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir for generating lysates containing the target protein from the biological sample; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and an elution buffer reservoir, the second bioprocessing assembly including a support or filter for retaining the target protein; and an output container in fluid communication with the second bioprocessing assembly for receiving the target protein in a purified form, wherein the consumable purification column is associated with and automatically purifies the target protein using an automated protein purification system without human interaction. The consumable purification column may additionally include one or more components in a second or additional bioprocessing chamber for column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, rapid protein liquid chromatography, or any combination thereof. In some cases, these components may be located upstream of a target protein binding scaffold. In some cases, one or more of these components may include a target protein binding support.

[0046] In some embodiments, the automated protein purification system may additionally include one or more controllers, which include computerized systems for controlling various fluid movements, sample movements, reagent dispensing, and other processes.

[0047] The automated protein purification apparatus, system, and consumable purification column disclosed herein are compatible with a variety of samples, including, but not limited to, biological samples, tissues, biopsies, cell lines, cell cultures, cells, cell suspensions, urine, saliva, cerebrospinal fluid, blood, serum, plasma, fecal aqueous solutions, other body fluids or exudates, eukaryotic cells selected from the group consisting of rodent, insect, primate, and human cells, or prokaryotic cells or cell suspensions containing prokaryotic cells, bacterial cells, yeast cells, etc.

[0048] In some embodiments, the first biological processing component includes a clarification filter. In some cases, the clarification filter is in fluid communication with an input reservoir and a lysis buffer reservoir, and the clarification filter is used to separate the protein-containing target protein portion from the first waste portion of the biological sample.

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

[0050] In some embodiments, the first biological processing component includes a cell capture or concentration filter. In some embodiments, the cell capture or concentration filter is disposed upstream of the clarification filter. With the cell capture or concentration filter in fluid communication with the input reservoir and the lysis buffer reservoir, the clarification filter is used to separate the target protein portion containing protein from the first waste portion of the biological sample.

[0051] In some embodiments, the device further includes one or more additional biological processing chambers containing protein purification reagents. In some cases, one or more filters within the device comprise hollow fiber filters.

[0052] For example, exemplary automated protein purification apparatus, systems, and consumable purification columns of this disclosure may include a lysis buffer containing at least one lysis agent and a deoxyribonuclease. In some embodiments, the automated protein purification system described herein may additionally include a cleavage buffer and a proteolytic enzyme (e.g., TEV protease) to remove affinity tags from the target protein. For example, DNase-treated lysates may be passed through a column or filter bound to a given affinity tag, and after washing the column, the target protein may be released from the column / filter using a lysis buffer and a proteolytic enzyme. The digested target protein may 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 to retain and selectively release fluid. Such an exemplary system may include a flexible gasket, a reservoir for containing fluid disposed on a first side of the flexible gasket, a fragile seal disposed between the flexible gasket and the fluid reservoir, and an actuator disposed on a second side of the flexible gasket operable to deflect the flexible gasket and rupture the fragile seal, thereby selectively releasing fluid from the reservoir.

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

[0055] Fluid release systems can be associated with any number or type of mixing chambers or reservoirs disclosed herein and can selectively release fluid from these chambers / reservoirs at appropriate times, thereby influencing different processes and fluid movements within the protein purification system and the associated purification column. Therefore, embodiments of this disclosure also include automated systems for selective fluid release, wherein at least one component is operable by the automated protein purification system, and at least one other component of the disclosed fluid release system is included within a biosample purification column for use with the automated protein purification system.

[0056] Methods for selectively releasing fluid from a reservoir during automation may include contacting a flexible gasket 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 break the fragile seal, thereby releasing the liquid from the reservoir.

[0057] The method for selectively releasing fluid from a reservoir may further include contacting a flexible vent with an exhaust actuator, moving the exhaust actuator to deflect the flexible vent toward a fragile gas seal, and causing the flexible vent to break the fragile gas seal.

[0058] The systems, methods, and apparatuses described in this disclosure may further include means for controlled movement of fluid. The means may include a first outer layer having a first side and a second side including a series of channels. The means may additionally include a second outer layer arranged opposite the first side of the first outer layer, and an elastomeric layer disposed between the first and second outer layers. The elastomeric layer may include sealing rib structures corresponding to the series of channels and may be used to fluidly separate the channels when the elastomeric layer is compressed between the first and second outer layers.

[0059] In some embodiments, the device may additionally include a nominal gap between the elastomeric layer and one or both of the first and second outer layers, such that when the elastomeric layer is compressed between the first and second outer layers, the compressed portion is compressed. The sealing rib moves within the nominal gap. Alternatively, the device may include valves associated with a series of channels that are selectively movable between a closed position and an open position to restrict or allow fluid flow through the valve, respectively.

[0060] In some embodiments, the orifice of the device may provide access to a deflectable portion of the elastomeric layer, the deflectable portion including a valve sealing rib extending from the elastomeric layer toward the first outer layer. The valve sealing rib may contact the first outer layer when the valve is in the closed position and may disengage from the first outer layer when the valve is in the open position. Alternatively, the device may include a plunger that contacts the deflectable portion of the valve. In these embodiments, the plunger is sized and shaped to allow it to permeate the orifice to deflect the deflectable portion and move the valve toward the closed position.

[0061] In one embodiment, the device is used to withstand a force of at least 500 lbf, preferably up to 15,000 lbf, applied over the entire length of the sealing rib structure. Additionally, the sealing rib structure can be compressed to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leakage, and / or the sealing rib is 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 this disclosure also include methods for controlling fluid movement. An exemplary method may include providing the apparatus disclosed herein for controlled fluid movement to a system for automated purification of target biomolecules (e.g., target nucleic acids and / or target proteins), causing one or more plungers to open valves within the apparatus such that the open valves allow fluid communication between upstream and downstream portions of a series of channels, causing a pump to deliver fluid from the upstream portion to the downstream portion. The method for controlling fluid movement described in this disclosure may additionally include the step of providing a biological sample containing the target biomolecules, and as provided throughout the application, in some embodiments, the biological sample may be cells in a cell line, cell culture, tissue, biopsy, blood, serum, plasma, in eukaryotes, prokaryotes, or any other biological material, and the target biomolecules may be any target nucleic acids and / or target proteins derived from these cells.

[0063] Some embodiments describe methods for automatically isolating or purifying target proteins from cell lysates using the devices or systems described herein. Such exemplary methods may include receiving a biological sample at an input reservoir without further human interaction, generating a lysate from the biological sample containing the target protein in a first bioprocessing component, receiving a portion of the target protein containing the lysate at a second bioprocessing component, retaining the target protein on a protein-binding filter or protein-binding carrier at the second bioprocessing component, and eluting the purified form of the target protein into an output container.

[0064] In some embodiments, the method may further include capturing the cellular contents of the biological sample at a first membrane of the first bioprocessing assembly and resuspending at least a portion of the cellular contents in a resuspension buffer, a deoxyribonuclease 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, a ribonuclease solution, a deoxyribonuclease solution, or a lysis buffer, from a fluid channel disposed on a second side of the first membrane and passing through the first membrane.

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

[0066] The method may further include one or more additional steps that contact the lysate containing the target protein with additional protein purification / separation reagents and capture the target protein on a support or filter membrane that can bind to the target protein. Other steps in the method for separating the target protein may include one or more of column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, rapid protein liquid chromatography, or any combination thereof. The target protein can then be eluted from the support or filter for further downstream processing or use.

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

[0068] This abstract aims to introduce a series of concepts in a simplified form, which will be described in further detail below. This abstract is not intended to identify key or essential features of the subject matter of the claims, nor is it intended to indicate the scope of the subject matter of the claims.

[0069] Additional features and advantages of this disclosure will be set forth in the description which follows, and will become apparent from it, or may be revealed by practice. The features and advantages of this disclosure can be realized by means of the instruments and combinations particularly pointed out in the appended claims. The features and other features of this disclosure will become more apparent from the following description and the appended claims, or may be learned by practice of this disclosure as described below. Attached Figure Description

[0070] To obtain the described methods and other advantages and features of this disclosure, please refer to the specific embodiments shown in the accompanying drawings for a more detailed description of the above disclosure. It should be understood that these drawings depict only typical embodiments of this disclosure and should not be considered as limiting its scope.

[0071] In the accompanying drawings, the numbering may include additional individual letters, such as... Figure 3A This disclosure will describe and explain additional features and details with reference to the accompanying drawings, in which:

[0072] Figure 1 A general system for the automated purification of target biomolecules (e.g., target nucleic acids and / or target proteins) from biological samples is shown;

[0073] Figure 2 Various components of an exemplary purification column are shown for use in a system for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from biological samples;

[0074] Figure 3A Various components of an exemplary purification column used within a system for the automated purification of target nucleic acids from biological samples are shown;

[0075] Figure 3B It shows the basis Figure 3A An exemplary purification column shown is provided with a filter in each of the first and second biological processing components;

[0076] Figure 3C It shows the basis Figure 3A Another exemplary purification column shown has two filters in the first biological processing component and one filter in the second biological processing component;

[0077] Figure 3D It shows the basis Figure 3A Another exemplary purification column shown has one filter in the first biological processing assembly and two filters in the second biological processing assembly.

[0078] Figure 3E It shows the basis Figure 3A Another exemplary purification column shown is equipped with two filters in each of the first and second biological processing components;

[0079] Figure 4 An exemplary layout of a purification column for automated purification of target nucleic acids from a biological sample is shown. The purification column includes an input reservoir, various biological processing components and associated membranes, filters, mixing chambers, buffer / reagent reservoirs, pumps, valves, conduits, and an output container for containing purified target nucleic acids from an input biological sample.

[0080] Figures 5A to 5K The movement of samples and various fluids through an exemplary purification column is shown in sequence under the action of relevant purification instruments;

[0081] Figure 6 An exemplary embodiment of a purification column container is shown, which is used to house an output container for receiving purified target nucleic acids;

[0082] Figure 7A Another embodiment of an exemplary purification column for an automated purification system of target nucleic acids from biological samples is shown;

[0083] Figure 7B Another embodiment of an exemplary purification column for an automated purification system of target nucleic acids from biological samples is shown;

[0084] Figure 7C Another embodiment of an exemplary purification column for an automated purification system of target nucleic acids from biological samples is shown;

[0085] Figures 8A to 8C An exemplary embodiment of a fluid release system is shown for retaining and selectively releasing fluid from a reservoir or mixing chamber within a purification column;

[0086] Figure 9A and 9B An exemplary embodiment of a flexible vent hole for selectively discharging air into an associated reservoir or mixing chamber of a purification column is shown;

[0087] Figure 10 An exemplary embodiment of a purification instrument capable of automated nucleic acid purification is shown;

[0088] Figure 11A and Figure 11B A clamping mechanism is shown, which can be used as part of a purification instrument to apply compressive force to an inserted purification column, thereby maintaining the integrity of the fluid seal of the purification column during purification.

[0089] Figure 12 A rear view of the purification instrument is shown, illustrating the entry point for the manual clamp release mechanism.

[0090] Figure 13 The maintenance door locking mechanism is shown from the perspective of the internal compartment of the instrument.

[0091] Figure 14 An embodiment of the access door sensor assembly is shown.

[0092] Figure 15 The purification column position sensor assembly is shown.

[0093] Figure 16 The output container shows the presence of sensor components.

[0094] Figure 17 An embodiment of an optical density sensor is shown, which may be included in an instrument for measuring the optical density of a biological sample inserted into a purification column;

[0095] Figure 18A and Figure 18B A pumping assembly is shown, which can be used as part of a purification instrument to move and guide fluid along the fluid path of an inserted purification column.

[0096] Figures 19A to 19F The movement of fluid through the fluid channel of the purification column is shown in sequence after the pump assembly meshing with the fluid channel is driven;

[0097] Figure 20A A control system is shown, which may be included in the instrument and is used to control various components of the instrument, receive user input, display data, and communicate with other computer devices and / or networks;

[0098] Figure 20B It shows that it can be made by Figure 20A An exemplary method implemented in the control system for selecting a purification scheme and executing the selected purification scheme based at least in part on received sensor data;

[0099] Figure 21 A plan view of an exemplary nucleic acid purification column is shown, the column having a sealing bone structure for defining and sealing various fluid channels of the column;

[0100] Figure 22 A cross-sectional view of the fluid channel in the column is shown, revealing an elastomer layer and sealing ribs arranged between two outer layers;

[0101] Figure 23A and 23B The purification column is shown to be clamped to compress and seal the bone structure, providing sufficient sealing for the purification process;

[0102] Figure 24 A valve mechanism is shown, which utilizes an elastomer layer to provide a gas distribution mechanism.

[0103] Figure 25 Agarose gel analysis of plasmid products isolated using the exemplary system and nucleic acid purification column of this disclosure is shown. Detailed Implementation

[0104] Before describing the various embodiments of this disclosure in detail, it should be understood that this disclosure is not limited to the parameters of the specific systems, methods, apparatuses, products, processes, and / or components exemplified herein, although such parameters may vary. Therefore, while certain embodiments of this disclosure will be described in detail with reference to specific structures, parameters, components, elements, etc., these descriptions are illustrative and should not be construed as limiting the scope of the claimed invention. Furthermore, the terminology used herein is for the purpose of describing embodiments and is not necessarily intended to limit the scope of the claims.

[0105] Furthermore, for any given component or embodiment described herein, unless expressly or implicitly understood or otherwise stated, it should be understood that any possible candidates or alternatives listed for that component can generally be used alone or in combination with each other. Moreover, unless expressly understood or otherwise stated, any such list of candidates or alternatives is illustrative only and not restrictive.

[0106] Furthermore, unless otherwise stated, figures used in the patent specification and claims to represent quantities, components, distances, or other measurements should be understood as modified by the word "approximately" as defined herein. Therefore, unless otherwise stated, the numerical parameters listed in the patent specification and appended claims are approximations, which may vary depending on the intended characteristics sought to be obtained from the subject matter presented herein. At the very least, no attempt should be made to limit the application of the equivalence doctrine to the scope of the claims; each numerical parameter should be understood at least based on the number of significant figures reported and the application of ordinary rounding. While the numerical ranges and parameters presented herein illustrate that a broad range of the subject matter is approximate, the values ​​described in specific examples are reported as precisely as possible. However, any numerical value necessarily contains a degree of error arising from the standard deviation of the respective test measurements.

[0107] Any headings and subheadings used herein are for organizational purposes only and are not intended to limit the scope of the description or claims.

[0108] Overview and advantages of exemplary target biomolecule purification systems

[0109] As discussed above, some drawbacks and problems can be addressed, such as the automated purification of target biomolecules, including target nucleic acids and / or target proteins, particularly for handling large sample volumes. There is a significant need for systems, methods, and apparatuses capable of automating the purification process of target biomolecules, such as the individual processes for purifying target nucleic acids and / or target proteins. In particular, there is a strong need for systems, methods, and apparatuses that integrate all stages of the purification process into a single consumable element, limiting or eliminating user intervention in the purification process.

[0110] The embodiments of this disclosure solve one or more of the aforementioned problems in automated target biomolecule purification techniques. For example, as Figure 1 As shown, system 10 for automated purification of target biomolecules (such as target nucleic acids and / or target proteins) can use purification instrument 12 and purification column 14 in combination to separate and purify target biomolecules from biological samples with limited user interaction. In a preferred embodiment, the user adds a biological sample 16 containing the target biomolecule to purification column 14 and loads purification column 14 into purification instrument 12, whereby the target biomolecule is subsequently automatically separated, purified, and deposited into output container 18 associated with system 10—all without further user interaction with the system. In this way, the target biomolecule purification system of this disclosure (such as the nucleic acid purification system and protein purification system disclosed herein) can include a self-contained system. In some embodiments, the target biomolecule purification apparatus and system of this disclosure can achieve fully automated large-scale target biomolecule purification (e.g., purification of endotoxin-free maximum-scale plasmid DNA from 100-200 mL bacterial culture input or recombinant drug protein purification from 500 mL eukaryotic cell culture input) in a shorter time compared to manual methods. In some cases, the disclosed systems can automate the separation and purification of target biomolecules in less than an hour (e.g., the total run time for purifying target nucleic acids is about 45 minutes, while the setup time is only about 2 minutes).

[0111] Additional benefits can be achieved by implementing the disclosed systems, methods, and apparatus. For example, the systems disclosed herein may include a portable benchtop instrument that is robust and capable of handling biological samples of varying input densities (e.g., bacterial cultures, eukaryotic cell cultures, clinical samples, food / beverage samples, or environmental samples) and of achieving high purity of target biomolecules, such as high-purity nucleic acids (in the case of plasmid DNA purified from bacterial cultures, "low endotoxin" or "endotoxin-free"). In some embodiments, the purification instrument may be cloud-enabled and "intelligent," with built-in optical density sensors and communication hardware and software for notifying the user of input culture density and / or dynamically triggering different purification protocols based on optical density (e.g., A600) readout and / or received input sample type.

[0112] Other advantages of the disclosed systems, methods, and apparatus include, for example, an integrated purification column design that combines all reagents, filters, pumps, fluid channels, inlet reservoirs, waste reservoirs, and final outlet containers into a single purification column. This limits user interaction to a single component of the system, thereby reducing errors, improving consistency, and significantly reducing hands-on time during the purification of target biomolecules. Furthermore, the disclosed systems are easier to use, thus reducing the technical expertise or trade secrets required to purify target biomolecules from biological samples.

[0113] This contrasts sharply with manual reagent kits, which require a technically skilled user to acquire and operate several reagents, purification columns, centrifuges, pipettes, pipette tips, intermediate containers, and final output containers. Similarly, manual and semi-automatic systems also require numerous user-operated consumable components to purify biomolecules such as nucleic acids and / or proteins. Unlike manual processes (which require user interaction with open reagent containers), the disclosed purification columns allow reagents to remain sealed throughout the setup and purification steps. Some embodiments of this disclosure also allow for the automatic release of reagents at various appropriate times during the purification process, an improvement over previous manual and semi-automatic processes that required the user to break the reagent seal or directly access the reagents before and / or during protocol execution.

[0114] In some embodiments, the purification column is a consumable and disposable component within the system, which in some cases can result in a lower-cost consumable compared to prior art kits and systems. For example, in some embodiments, the purification column is made of, or incorporates, thermoformed plastic. Using thermoformed plastic instead of the more expensive injection molding process provides a lower-cost, larger-volume solution for producing consumable purification columns. Thermoformed plastic has long been used as a traditional packaging material due to its reduced manufacturing costs. In embodiments of this disclosure, where the purification column is made of, or incorporates, thermoformed plastic, this design standard represents a significant difference from typical applications, where the product itself is made of thermoformed plastic, rather than the product packaging being made of thermoformed plastic as is conventional. Surprisingly, the use of thermoformed plastic has resulted in a suitable, non-reactive purification column system that reduces costs without substantially sacrificing practicality.

[0115] As another example, some embodiments combine purification instruments and purification columns, forming a fluid seal within the purification column. This form of seal can make the manufacturing process cheaper and simpler because, for example, the purification column does not contain a traditional, lengthy fluid seal, which is typically formed by expensive and complex ultrasonic welding. This, separated from or combined with the significantly less costly thermoforming process, can greatly reduce the cost and manufacturing time of the purification column.

[0116] Furthermore, embodiments of the purification column may also include an elastomeric layer sandwiched between two thermoformed outer layers. In this embodiment utilizing an elastomeric layer between two thermoformed layers, the elastomeric layer can serve as a valve body, allowing for simpler assembly of consumables during manufacturing, as only one component is used for the valve body, rather than requiring a single component for each valve body. This design also enables very simple and reliable single-axis actuators. For example, the inherent elasticity of the elastomeric element and its tendency to spring back to its original shape also allow the actuator to achieve changes (e.g., valve control or release of fluid within the purification column) by pressing against or releasing pressure from the elastomeric element without applying a counterforce and / or without a counteracting actuator, thus simplifying valve, fluid flow control, and actuation within the system.

[0117] Purification column

[0118] The following disclosure relates to an exemplary embodiment of a purification column for automated purification of target biomolecules, such as target nucleic acids and / or target proteins, from biological samples. The purification column can be used with other components and / or instruments described herein. For example, the purification column can be configured to be received and docked with a purification instrument, such as… Figure 1 Instrument 12 as described and illustrated herein, or any other purification instrument as described herein.

[0119] Purification column overview

[0120] As mentioned above, traditional target biomolecule purification procedures, such as nucleic acid purification and / or protein purification procedures, are manual laboratory processes that require relatively high levels of expertise and time from laboratory technicians. While well-trained laboratory technicians may be able to handle the various operational parameters involved in the process, there is a significant need for systems, methods, and devices that can automate target biomolecule purification processes (such as nucleic acid purification and / or protein purification), particularly those that integrate all stages of the target biomolecule purification process into a single consumable element, thereby limiting or eliminating user intervention in the purification process.

[0121] Designing a single-use purification column capable of effectively automating most of the process is challenging for several reasons. For example, a purification column used to perform all stages of a nucleic acid and / or protein purification process should be able to store and promptly dispense (or otherwise acquire and dispense as needed) the various reagents and buffers associated with their respective purification protocols. The purification column should also be able to move and transport fluids of varying viscosities and densities, typically by dispensing appropriate reagents and / or buffers, mixing different fluids together, and controlling the flow of fluids through and / or through appropriate filters or membranes. Such a purification column should also be safe for the user to operate and able to maintain proper liquid separation / sealing throughout the biomolecule purification process.

[0122] As described in more detail below, the purification columns described herein can address one or more of the challenges mentioned above. For example, the purification column can be configured to be associated with a purification instrument, allowing the user to load a biological sample containing target biomolecules (e.g., target nucleic acids and / or target proteins) into the purification column and insert the column into the instrument, resulting in the separation and purification of the target biomolecules without further user interaction, thus providing a purification column capable of performing all stages of the biomolecule purification process.

[0123] As another example, the purification column disclosed herein can be a self-contained solution, integrating various buffers and reagents required for biomolecule purification (e.g., nucleic acid purification and / or protein purification) in a separate reservoir, and selectively released at appropriate times during the biomolecule purification process. Furthermore, the purification column disclosed herein can be configured with multiple interconnected and cooperating conduits, valves, and pumps to move and deliver fluids of different viscosities and densities throughout the purification column during the biomolecule purification process. The purification column may also include a mixing chamber equipped with a magnetic stir bar, which is in fluid communication with the purification column conduits and reservoir inlet to provide a homogeneous mixture throughout the purification process. Moreover, as provided below, the purification column of this disclosure can be safely sealed as a self-contained consumable, increasing user safety, reducing the risk of reagent / buffer contamination, and increasing ease of use in automated biomolecule purification processes.

[0124] Figure 2 General features and components of an exemplary purification column 20 for automated purification of target biomolecules (e.g., target nucleic acids and / or target proteins) from biological samples within a system are illustrated. As shown, the purification column 20 includes an input reservoir 22 for receiving a biological sample containing the target biomolecules. In some embodiments, the purification column disclosed herein includes... Figure 2The purification column 20 shown is preferably used for processing large-volume samples. Accordingly, the size and shape of the input reservoir 22 can receive approximately 5 mL to 5 L of biological sample. In some embodiments, the input reservoir can be used to hold at least 10 mL, at least 50 mL, at least 100 mL, at least 150 mL, at least 200 mL, at least 250 mL, or at least 500 mL of biological sample. In the same or alternative embodiments, the input reservoir can be used to hold 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 of biological sample. It should be appreciated that the size and shape of the input reservoir can be configured to receive a volume of biological sample within a volume range with lower and upper limits selected from the minimum and maximum volumes described above. It should also be recognized that the systems, apparatus and purification columns disclosed herein can be scaled down and their size and shape can be changed to handle sample volumes smaller than those disclosed above.

[0125] In a preferred embodiment, the input reservoir is sized and shaped to receive 50 mL–250 mL of bacterial cultures containing plasmid DNA—the target nucleic acid. Input reservoir 22 can also be used to receive other biological samples, such as other cell cultures, eukaryotic cell cultures, clinical samples, or environmental samples. In one embodiment, input reservoir 22 is used to contain up to 1 L, up to 1.5 L, or up to 2 L of clinical samples, or any volume range listed above, such as urine, aqueous solutions of excrement, or other bodily fluids or exudates. Input reservoir 22 can also be additionally or alternatively used to contain large volumes of environmental samples, such as water samples or similar samples.

[0126] In addition to the input reservoir 22, column 20 may further include at least two bioprocessing components 24 and 30. A first bioprocessing component 24 may be in fluid communication with the input reservoir 22 and may be used to generate lysates from a biological sample containing target biomolecules. For example, the first bioprocessing component 24 may be in fluid communication with a lysis buffer reservoir so that the biological sample in the input reservoir 22 can be combined with the lysis buffer reservoir in the first bioprocessing component 24 to generate lysates containing target biomolecules. A second bioprocessing component 30 may be in fluid communication with the first bioprocessing component 24, and this component may include biomolecule binding filters (e.g., target nucleic acid binding filters and / or target protein binding filters) for retaining target biomolecules.

[0127] Column 20 may additionally include a plurality of reagents and buffers 28 fluidly connected to the first and / or second bioprocessing components 24, 30, and a series of valves and pumps 26 for coordinating the movement of various fluids within column 20 during automated purification. It should be appreciated that, when using the pumps of the present invention, the pumps of this disclosure can coordinate the movement of various fluids within the column by, for example, pushing and / or pulling fluids through channels connecting to the bioprocessing components within the column (e.g., applying positive and / or negative pressure). This can be achieved by strategically placing the pumps within and / or between the bioprocessing components, such that the pumps are upstream and / or downstream of the fluid to be moved. For example, a pump placed upstream of the fluid can push the fluid through the column, and a pump placed downstream of the fluid can pull the fluid through the column. It should be appreciated that, during purification, a single pump can be used to pull the liquid in one step and push the liquid in another step. For example, in the first step of the purification process, a valve can be installed within the column to allow fluid to pass through a filter / membrane, and in a later step of the purification process, the valve functions to push the fluid through the same or different filters / membranes.

[0128] Continue to refer to Figure 2 The column 20 may further include a container in fluid communication with the second bioprocessing component, the second bioprocessing component being used to contain an output container 34 for collecting purified target biomolecules.

[0129] Figure 2 The purification column 20 shown optionally includes a waste reservoir 32. In some embodiments, waste generated during purification can be captured and stored within the purification column. This advantageously makes column 20 a standalone, single-use biological sample purification column, and by collecting all waste generated within the purification column during purification, it advantageously reduces user exposure to this waste and isolates various chemicals, solutions, and transfer containers. Furthermore, by collecting waste within the purification column, the purification instrument is neither exposed to nor in contact with the waste, which reduces the possibility of cross-contamination between samples using the same instrument.

[0130] Now for reference Figure 3A The figure illustrates various components of another exemplary purification column 36 within a system for the automated purification of target nucleic acids. Figure 2 The purification column 20 shown is similar. Figure 3A The purification column 36 shown includes an input reservoir 38, a first bioprocessing component 40, a second bioprocessing component 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, high-cleavage salt buffer), and an output container 82. Although not shown, Figure 3AThe purification column 36 shown may also include a series of valves and pumps for coordinating the movement of various fluids throughout the purification column 36 during automated purification. (See above regarding...) Figure 2 The valves and pumps can be used to push and / or pull fluid through the purification column. Some embodiments may include a series of valves spatially configured upstream and downstream of the pump and the filter / membrane, such that operation of the pump can cause fluid to pass through the filter / membrane in one step of the purification process and through the membrane in different steps of the purification process.

[0131] Continue to refer to Figure 3A The purification column 36 shown represents a collection of various exemplary purification columns contemplated within the scope of this disclosure. It should be understood that the spatial arrangement and flow between the components shown within purification column 36 are not the only methods for determining the construction and organization of purification columns disclosed herein. Rather, the disclosed structures are exemplary in nature and are intended as schematic diagrams to aid in illustrating possible representative structures and components of purification columns contemplated within the scope of this disclosure. Figure 3A As shown, dashed boxes and arrows indicate the addition or selection of components that may be used in the exemplary purification columns disclosed herein. Figure 3B-3E The document provides some exemplary choices and structures to illustrate this more specifically. Figure 3A The content disclosed in the document.

[0132] Generally speaking, Figure 3A Widely described in and Figure 3B-3E The various possible purification column structures detailed in the text follow similar processing protocols. For example... Figure 3A As shown, the first bioprocessing assembly 40 is fluidly connected to the input reservoir 38. By activating a series of valves and pumps, biological samples received in the input reservoir 38 can be transferred to and processed within the first bioprocessing assembly 40. It should be appreciated that in some embodiments, the input reservoir 38 is a component of the first bioprocessing assembly 40, and one or more purification steps performed within the first bioprocessing assembly 40 can be performed within or using the input reservoir 38. For example, the initial purification step may include homogenization of the biological sample. The biological sample may contain solid or particulate matter, and reducing the particle size of the solid or particulate matter or mixing it more uniformly in the provided medium and / or applied aqueous buffer / solvent is beneficial to the purification process.

[0133] Therefore, 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 method known in the art, including, for example, the use of a magnetic stir bar, high-power beading, vibration, eddy current, or similar methods. For example, a fecal or soil sample can be added to the input reservoir (simultaneously or subsequently by adding a medium and / or an aqueous buffer / solvent), where the biological sample is homogenized and transferred to the first bioprocessing assembly. Additional or alternative homogenization steps can be performed within the first bioprocessing assembly 40 and can be combined with the dissolution of the cellular contents of the biological sample. The biological sample can be combined with a lysis buffer encapsulated within a lysis buffer chamber 42 to achieve lysis of the cellular contents, and in some embodiments, a homogenization component is activated to homogenize the lysed sample.

[0134] In the first biological processing assembly 40, target nucleic acids in a biological sample are partially purified from cell contents and waste media by using one or more filters / membranes. Generally, this involves separating the target nucleic acids after lysing and neutralizing the cell material. For example, a lysis buffer stored in a lysis buffer chamber 42 can be linked to the biological sample in a mixing chamber 44 to form lysates. Neutralization buffer from a neutralization buffer chamber 46, for example, can be combined with the lysates in the mixing chamber 44 and passed through a clarifying filter 52, thereby separating the target nucleic acids containing the biological sample portion 54 from the waste portion of the biological sample.

[0135] As used herein, the terms "filter" or "membrane" include any size-selected and / or electrically charged semi-permeable barrier that can be used to select or separate one or more components from a solution. This includes membranes or columns made of filter paper or material of suitable size. It also includes other filtration methods such as affinity columns, beads (e.g., steel, glass, zirconia, or any other suitable material of suitable size, preferably between 0.1 mm and 2 mm in diameter), or other methods known by the process.

[0136] The partially purified target nucleic acid can then be transferred to a fluidly connected second bioprocessing unit, where it is further purified and concentrated, ultimately collected in an output container 82 as the purified target nucleic acid. The purification steps within the first and second bioprocessing units can be performed efficiently with minimal user interaction.

[0137] The general purification process described above will be detailed in the descriptions of various typical purification columns, such as... Figure 3B-3E As shown.

[0138] like Figure 3BAn embodiment of a purification column for an automated purification system of target nucleic acids from biological samples is shown. The purification column 36a is equipped with a single filter in each of the first and second bioprocessing components 40a and 56a. Specifically, the first bioprocessing component 40a of the illustrated cylinder 36a includes a single filter 52a for filtering target nucleic acid fractions contained in the biological sample from cellular components and media. For example, the filter may be a filter of a suitable size and material appropriate for separating specific target nucleic acids. The above non-limiting example includes a 5 μm nylon filter.

[0139] exist Figure 3B In the embodiment of the purification column 36a, the biological sample can be fed into the reservoir 38 and pass through the filter 52a of the first-step biological processing assembly 40a to remove the medium from the sample. After this process, the cellular portion of the biological sample is retained in the filter 52a. The cells associated with the filter 52a can be lysed using the lysis buffer in the lysis buffer chamber 42 of the purification column 40a to form lysates. Notably, the lysis buffer can be applied to the cell-capturing side of the filter 52a to elute the cells in the filter 52a. Alternatively, the lysis buffer can be pushed in from the back of the filter 52a (e.g., elute the cells through the opposite side of the filter 52a) and then bind with the biological sample in the mixing chamber 44 to form lysates. Neutralization buffer from the neutralization buffer chamber 46 can combine with the lysates in the mixing chamber 44 and enter the filter 52a, thereby separating the target nucleic acid containing the biological sample portion 54 from the waste portion of the biological sample.

[0140] like Figure 3B Further explanation indicates that the target nucleic acid containing the biological sample fraction 54 can be transferred (e.g., using pumps and valves associated with the infrastructure of the purification column 36a) to a second biological processing assembly 56a, wherein the second biological processing assembly 56a is coupled to a single filter or membrane system 76. For example, the single filter or membrane system 76 includes a silicon-based filter 78. In this embodiment, the fraction 54 containing the target nucleic acid in the lysate can be pretreated using an orienting salt buffer automatically obtained from the orienting salt buffer chamber 80 to facilitate the binding of the target nucleic acid to the silicon-based filter 78. Once bound to the silicon-based filter 78, the target nucleic acid can be automatically released and transferred using the rinsing buffer of the washing buffer chamber 66 and subjected to a series of washes through the silicon-based filter 78. Waste generated from the above washing steps can be transferred to a waste reservoir 72. The washed target nucleic acid can be eluted in the silicon-based filter 78 using any suitable low-salt elution buffer known in the art and enters the output container 82 as purified target nucleic acid 84.

[0141] In some embodiments, the first biological treatment component described above may have multiple filters. For example, Figure 3C Another typical purification column 36b shown is equipped with filters 48 and 52 in the first biological treatment assembly 40b, which is connected to... Figure 3B The same second biological treatment component 56a (with a single filter) shown is coupled to it.

[0142] like Figure 3C As shown, the first bioprocessing component 40b may further include a cell capture filter 48. The biological sample received at the first bioprocessing component 40b can have its non-target nucleic acid fraction removed by the cell capture filter 48. For example, in an embodiment using a bacterial culture as the biological sample, the cell capture filter 48 can retain bacterial cells while allowing the culture supernatant to be delivered to the waste reservoir 50. Lysis buffer or resuspension buffer from the lysis buffer chamber 54 can enter the cell capture filter 48, through which cells are removed and transferred to a cell-containing solution in the mixing chamber 44 to form a lysate. A neutralization buffer chamber 46 may be in fluid communication with the mixing chamber 44, and the neutralization buffer can then be transferred to the mixing chamber 44 to neutralize the lysate. The neutralized lysate can then be passed through a clarification filter 52 to separate the target nucleic acid fraction containing the lysate fraction 54 from the waste fraction of the lysate. In an example of bacterial culture operation, the waste fraction of the lysate includes cellular components of lysed bacterial cells and bacterial genomic DNA.

[0143] It should be noted that although bacterial cultures are used as an example of biological samples, this example is used to illustrate the structural characteristics of the disclosed purification column. Biological samples can also be other samples, such as dirt samples, clinical or forensic samples (e.g., feces, blood, saliva, urine, etc.), water samples, or other samples containing target nucleic acids.

[0144] The first bioprocessing component 40b of the purification column 36b is fluidly connected to the second bioprocessing component 56a, and as described above, the target nucleic acid content portion 54 containing the biological sample can be transferred from the first bioprocessing component 40b to the second bioprocessing component 56a, where it is combined, rinsed and eluted as purified target nucleic acid 84 with a single filter / filtration membrane system 76.

[0145] Figure 3D Another exemplary purification column 36c is shown. The purification column 36c shown is equipped with a single filter 52a (similar to...) within the first biological processing assembly 40a. Figure 3BThe filter shown and discussed is one of two filters in the second bioprocessing component 58b. The lysate target nucleic acid contents 54 are bound to the target nucleic acid capture filter 58b within the second bioprocessing component 56b. Prior to binding to the filter 58b, the lysate target nucleic acid contents 54 may optionally be treated with endotoxin removal (ER) buffer (e.g., from ER rinse buffer chamber 64) as needed, depending on a “low endotoxin” or “endotoxin-free” purification protocol. Once bound to the target nucleic acid capture filter 58b, the target nucleic acid is rinsed and eluted into the output container 82 as purified target nucleic acid 84. Rinsing the target nucleic acid bound to the capture filter 58b may include, for example, filtering various rinse buffers or aqueous solutions containing isopropanol or ethanol with the capture filter 58b and loading them into the waste reservoir 72. These rinse buffers are automatically released from one or more rinse buffer chambers 66 in fluid communication with the capture filter 58b. In a similar manner, the rinsed target nucleic acid can be eluted from the capture filter 58b by, for example, automatic release of distilled water or from an elution buffer obtained from a dH2O / elution buffer chamber 74 fluidly connected to the capture filter 58b.

[0146] like Figure 3D As shown, the capture filter 58b may include a series of filters / membranes 60. The target nucleic acid content portion 54 of the lysate may be bound and rinsed along the series of filters / membranes 60 before elution to the output container 82. In some embodiments, the target nucleic acid content portion 54 of the lysate may be pretreated with a rinsing buffer before binding to the series of filters / membranes 60. The rinsing buffer may be automatically released from the rinsing buffer chamber 66 into the mixing chamber 62 to mix with the target nucleic acid content portion 54 of the lysate. The pretreated target nucleic acid may then be bound to an anion exchange membrane 68, and an additional rinsing step may be performed by automatically flowing rinsing buffer (e.g., rinsing buffer released from one or more different rinsing buffer chambers 66) through 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 rinsing and purification steps.

[0147] In some embodiments, the target nucleic acid is eluted from 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. In this filter, it is desalted by a large volume of ethanol-based wash (e.g., 70% ethanol) before elution to the output container 82.

[0148] Figure 3EAnother exemplary purification column 36d is shown. The purification column 36d is equipped with two filters in each of the first and second biological processing components 40b and 36b. For example, the first biological processing component 40b may include... Figure 3C The same first bioprocessing component 40b as the purification column 36d shown, and the second bioprocessing component 56b may be included in... Figure 3D The same second biological processing unit 56b as the purification column 36c shown.

[0149] Therefore, a purification column for automatically purifying nucleic acids from biological samples may include two or more biological processing components, each connected to at least one filter / membrane. In some embodiments, a first biological processing component may include a clarifying filter, or a first biological processing component may include a cell capture filter and a clarifying filter. Similarly, a second biological processing component may include a silica-based filter, or a second biological processing component may include a precipitation membrane located downstream of an anion exchange membrane. Alternatively, a second biological processing component may include an anion exchange membrane, and the purification column may additionally include a third biological processing component, which includes a precipitation filter for receiving eluted target nucleic acids from the anion exchange membrane.

[0150] Samples were processed using an exemplary purification column.

[0151] Figure 2 The purification column shown and described in Figure 3 can specifically serve as... Figure 4 An exemplary purification column 100. Specifically, Figure 4 Various biological processing components, filters / membranes, buffer / reagent reservoirs, fluid conduits, and valves (e.g., valve 154, etc.) associated with the illustrated purification column 100 are shown. Figure 5A - Also shown in 5K as components marked "V", seals (e.g., seals 156 and seals 158, etc.); in Figure 5A-5K The image also shows a partial cross-sectional view of the component marked "L", the waste storage tank, and the output container. Figure 4 Each of the various components of the purification column 100 shown will be... Figure 5A-5K The exemplary automated nucleic acid purification process is discussed step-by-step. It should be understood that the mechanisms for sealing and discharging liquids throughout the automated nucleic acid purification process are described below with reference to... Figure 5A –5K will be discussed in more detail.

[0152] In one exemplary embodiment, Figure 5A-5KThe purification column 100 can be used with the purification instruments described herein to automate biological processing of biological samples without human intervention, while providing performance at least as good as, if not better than, similar manual processing methods. As mentioned above, human error and lack of reproducibility can occur when scientists or laboratory technicians perform artificial nucleic acid purification protocols for biological samples. When used as part of an automated nucleic acid purification system, the purification column 100 (and other purification columns disclosed herein) can eliminate or reduce human error due to insufficient reproducibility in artificial nucleic acid purification processes. Furthermore, the purification columns provided herein increase ease of use, save labor time, reduce contamination, and improve the efficiency and flexibility of performing nucleic acid separation and purification protocols.

[0153] For example, refer to Figure 5A Technicians, scientists, or other users can add biological samples (such as bacterial cultures or other cell cultures containing plasmid DNA) to culture tank 102. The cartridge 100 can be inserted into a purification instrument that has already initiated a nucleic acid purification protocol. The following is about... Figure 5A The steps discussed in the –5K discussion can be performed automatically without further human interaction. For ease of explanation and description, Figure 5A-5K The components and processing steps described herein will refer to a bacterial culture as the biological sample, while plasmid DNA will be used as the target nucleic acid. It should be noted that other biological samples and target nucleic acids can be processed using the automated nucleic acid purification systems, methods, and apparatus disclosed herein, and are included within the scope of this invention. Secondly, it is important that... Figure 4 and 5A In the disclosed embodiments of –5K, each buffer and reagent discussed and used in the exemplary nucleic acid purification protocol is contained in filter cartridge 100. Similarly, each filter / membrane discussed and used in the upcoming exemplary nucleic acid purification protocol is contained in purification column 100. The discharge, sealing, and movement of fluids in the purification column are typically controlled and / or started by the purification instrument, as detailed below.

[0154] Still referencing Figure 5AA bacterial culture containing plasmid DNA is present in a culture reservoir 102. In some embodiments, air can be pumped through the culture reservoir 102 to disperse any precipitated cells and / or homogenize the culture before it is discharged from the culture reservoir 102. This is particularly advantageous in some embodiments, where the optical density (e.g., A600) of the culture is measured at an optical density window 105 after discharge from the culture reservoir 102, as a more accurate reading can be obtained from a homogenized culture. For example, the optical density of the culture can be reported to the user and / or used by the instrument to show a low yield expected due to insufficient input material (low OD) or to indicate a low yield due to system overload (high OD).

[0155] Regardless of whether optical density measurements are performed, culture reservoir 102 is opened (e.g., by puncturing the fragile seal between the culture reservoir and the fluid channel), and the contents of culture reservoir 102 are pumped (e.g., using pump 104) through the fluid channel within purification column 100 to cell capture filter 106. In cell capture filter 106, the culture medium within the biological sample passes through filter 106 and enters waste reservoir 108. Plasmid DNA containing bacterial cells within the biological sample is retained in cell capture filter 106. In some embodiments, cell capture filter 106 may be a selective filter with a pore gradient between 0.65 μm and 1.2 μm (e.g., a 7.5 mil thick nylon cell capture filter). Additionally, in some embodiments, the cell capture filter is preferably capable of withstanding pressures greater than 20 psi, greater than 30 psi, greater than 40 psi, or greater than 60 psi without leakage, as battery buildup on one side of the filter may lead to low flow conditions and increased filter pressure. In some embodiments, a compliant support gasket (e.g., an elastomeric layer positioned on the cell capture filter) may be added around the filter perimeter opposite the resident elastomeric layer between the outer layers of the purification column to provide a better sealing interface between the purification column and the instrument.

[0156] like Figure 5A A simplified cross-sectional view of the cell capture filter 106 (with support gaskets as shown in element 107) is shown, with the filter 106 sandwiched between the outer layers 115a and 115b of the filter cartridge 100. Biological samples can be extracted from the culture reservoir 102, and between the first outer layer 115a and the first side of the filter 106. The suction or pumping action of the pump 104 causes the culture medium within the biological sample to pass through the filter 106 and into the space between the second side of the filter 106 and the second outer layer 115b. The culture medium is then guided along a fluid conduit and through an open valve into the waste reservoir 108.

[0157] like Figure 5BAs shown, RNase A can be passively mixed with resuspension buffer and dissolution buffer by penetrating a fragile seal that separates resuspension buffer reservoir 110 from RNase A reservoir 112 and lysis buffer reservoir 114. In some embodiments, lysis buffer reservoir 114 may have an elevated, large volume to accommodate the volume of RNase A and resuspension buffer. Gravity flow from resuspension buffer reservoir 110 through RNase A reservoir 112 into lysis buffer reservoir 114 allows these buffers and reagents to be passively mixed in the lysis buffer. Reservoir 114 is used to create resuspension / lysis buffer.

[0158] like Figure 5B A schematic cross-sectional view of the embedded cell capture filter 106 is shown. The combined resuspension / lysis buffer solution can be backwashed on the filter 106 to recapture cells from the filter 106, entraining them in the resuspension / lysis buffer solution, and transferring the cell-containing solution to the first active mixing chamber 116. Figure 5B As shown, the reversible pump 104 allows backwashing on the filter 106 and delivery to the active mixing chamber 116.

[0159] like Figure 5C As shown, cells in a resuspension / lysis buffer solution can be lysed under active mixing conditions in mixing chamber 116. Like the other active mixing chambers discussed below, the first active mixing chamber 116 may include a magnetic stir bar (or other means of stirring and / or mixing solutions within chamber 116) to thoroughly mix the contents in mixing chamber 116 via purification instrument operation. In some embodiments, the magnetic stir bar may enable the mixing speed to reach or exceed 1000 rpm.

[0160] After cell lysates are formed within mixing chamber 116, neutralization buffer can be automatically discharged from neutralization buffer solution tank 118 into mixing chamber 116 (e.g., by puncturing the fragile seal associated with neutralization buffer solution tank 118 and allowing passive transfer from neutralization buffer solution tank 118 to mixing chamber 116). Aggressive mixing of the neutralization buffer with the lysates restores the pH of the lysates to normal, thereby precipitating proteins from the lysates and regenerating genetic material. Due to its circular nature, plasmid DNA can be properly regenerated and remain soluble, while genomic DNA breaks down due to random strand binding. The precipitated cell debris and bound genomic DNA are insoluble in solution and can therefore be separated from the solution.

[0161] Now for reference Figure 5DA smaller volume of ER buffer can be pre-discharged from the ER buffer reservoir 124 into the second active mixing chamber 122 to improve subsequent reaction with subsequent fluids. Simultaneously, or shortly before or after discharging the ER buffer, the neutralized lysate can pass through a clarification filter 118. The clarified lysate (i.e., the target nucleic acid fraction containing the lysate) is extracted through the clarification filter 118 and pumped into the second active mixing chamber 122 by pump 120. The waste fraction of the neutralized lysate is retained in the clarification filter 118.

[0162] In some embodiments, the clarifying filter is prone to collapse during flow and close its porous structure, thereby preventing the filter from performing its desired function. To prevent this, some embodiments may include a thin, semi-rigid, porous backing structure instead of a peripheral support pad to provide support within the clarifying filter and prevent it from collapsing during flow, thus allowing the filter to perform its required function. The clarifying filter can be any clarifying filter known or used in the art and is suitable for separating the nucleic acid-containing portion of the neutralized lysate from the waste-containing portion of the neutralized lysate. For example, the clarifying filter may include a glass fiber-based filter with a pore size greater than about 1 μm and / or less than about 5 μm and a thickness greater than 30 mil. For example, the clarifying filter used within the purification column of this disclosure may include a glass fiber α filter having a pore size of 4.3 μm and a thickness of 45 mil or a glass fiber filter having a pore size of 1 μm and a thickness between 43 mil and 53 mil. Glass fiber-based clarifiers can be used in combination with any suitable semi-rigid clarifier support, such as 1 / 16” thick polyethylene clarifier supports with a pore size greater than 15 μm (e.g., between 15 and 45 μm).

[0163] After the clarified lysate is extracted through the clarifying filter 118 and pumped into the second active mixing chamber 122, it is actively mixed therewith with the ER buffer. The clarified lysate / ER buffer solution is then actively mixed in the second active mixing chamber 122 using a magnetic stir bar provided above with respect to the first active mixing chamber 116 or the like, to remove contaminants from the clarified lysate and improve sample purity.

[0164] Now for reference Figure 5E The clarified lysate is pumped from the second active mixing chamber 122 and passed through an anion exchange membrane structure 126 by pump 128. The plasmid DNA in the clarified lysate binds to the anion exchange membrane structure 126 due to good ion interaction with the charged column, and the waste-containing portion of the clarified 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.

[0165] like Figure 5E As shown in the schematic cross-sectional illustration, the anion exchange membrane 126 is structured to generate an axial flow through a stack of anion exchange membranes. As illustrated in the exemplary cross-sectional illustration, the intermediate layer 117 and the first outer layer 115a on the inlet side form a near-vertical wall approximately 9 mm high. A pore formed by the intermediate layer enters the housing, which can accommodate the stack of anion exchange membranes 126, through the inlet formed by the outer and intermediate layers of the housing as fluid channels. When fluid is extracted through the membrane stack 126, the membrane can ion-capture plasmid DNA while allowing waste portions to pass through the filter assembly 126 and into the waste reservoir 130. It should be noted that the axial flow through the anion exchange membranes 126 can be implemented in different ways or by using stacked membranes of different heights.

[0166] In some embodiments, the anion exchange membrane is a silicone-treated glass fiber membrane. Glass fiber-based anion exchange membranes can be used with any suitable semi-rigid support, such as a 1 / 16” thick polyethylene support filter with a pore size greater than 15 μm (e.g., between 15 and 45 μm).

[0167] After the neutralized lysate passes through the anion exchange membrane 126, the washing buffer in the washing buffer reservoir 132 is automatically discharged and extracted by pump 128 through the anion exchange membrane structure 126. Figure 5F As shown. Any wash solution passing through the anion exchange membrane 126 is collected in the waste storage tank 130. In a preferred embodiment, the wash solution is selected to avoid interfering with membrane-bound plasmid DNA and to remove excess material associated with the plasmid DNA and / or filter 126.

[0168] Now for reference Figure 5G Isopropanol is automatically discharged from the isopropanol reservoir 138 into the third active mixing chamber 136, thereby improving the reaction with subsequent fluids. Simultaneously, or shortly before or after the isopropanol discharge, the elution buffer can be automatically discharged from the elution buffer reservoir 134 via 128 and passed through the anion exchange membrane structure 126. Compared to the anion exchange membrane, the elution buffer provides a better chemical exchange with the plasmid DNA than the anion exchange membrane, thereby separating the plasmid DNA from the membrane and eluting it into the passing elution buffer. This elution solution passes through the anion exchange membrane structure 126 and is pumped into the third active mixing chamber 136. In the third active mixing chamber 136, the elution solution, consisting of the elution buffer and plasmid DNA, is mixed with isopropanol, precipitating the plasmid DNA and several salts from the elution buffer solution.

[0169] The precipitated mixture formed in the third active mixing chamber 136 then passes through the precipitation membrane 138, as... Figure 5HAs shown. Plasmid DNA in the precipitate mixture binds within the precipitator membrane (e.g., based on the pore size of the precipitator membrane), while smaller soluble components and elution buffer pass through the precipitator membrane and are collected in waste reservoir 142. In some embodiments, a precipitator membrane with a pore size greater than or equal to 1 μm and made of or comprising glass fibers is used. For example, the precipitator membrane may comprise a glass fiber filter with a pore size of 1 μm or a 16 mil thick glass fiber D filter.

[0170] In some embodiments, the precipitation filter may not be subjected to the same or similar high pressure as the cell capture filter, but to ensure little or no fluid inflow into the inactive areas of the precipitation filter, a perimeter seal may be associated with the dust collector filter. For example, O-rings (or other functionally similar gaskets) may be located between the outer layers of the purification column and on the opposite side of the precipitation filter, serving as the middle layer of the purification column.

[0171] The precipitator membrane 138 can be washed with an ethanol-based solution to dissolve and remove salts from the plasmid DNA (i.e., desalting). For example... Figure 5I As shown, a 70% ethanol solution can be automatically discharged from ethanol storage tank 144 and extracted through precipitation membrane 138 using 140. The ethanol washing liquid from the storage tank can pass through precipitation membrane 138 along with any soluble salts and enter waste storage tank 142.

[0172] Now for reference Figure 5J After the ethanol wash solution passes through the precipitator filter 138, the pump 140 can continue to operate. This causes air to flow through the precipitator filter 138, drying the plasmid DNA and causing excess liquid (especially ethanol) to evaporate.

[0173] The washed and dried plasmid DNA can be eluted from the precipitation membrane 138 and transferred to the output container 148, as shown. Figure 5K As shown. In this final process, TE buffer can be automatically discharged from TE buffer reservoir 146 and, through the continuous action of pump 140, reach precipitator membrane 138, where plasmid DNA is eluted from precipitator membrane 138 and enters the passing TE buffer. By closing and opening different valves within purification column 100, the eluted plasmid DNA can enter output container 148 from precipitator membrane 138, instead of entering waste reservoir 142 as the liquid previously flowing through precipitator membrane 138.

[0174] The output container 148 can be any container suitable for receiving purified nucleic acids. For example, the output container 148 contains a standard microcentrifuge tube. To ensure that the purified nucleic acids are transferred to the output container rather than remaining in contact with the purification column, the purification column 100 can be designed with a unique shape to overcome the surface tension between the purification column material and the purified nucleic acids. In particular, this unique shape (such as...) Figure 6 (As indicated by arrow 152) This can prevent or reduce the adhesion of purified nucleic acids to the outer walls of the purification column 115a and 115b without them falling into the output container.

[0175] The surface energies of the outer walls 115a and 115b are typically greater than those of the elastic intermediate layer 117 (e.g., the outer walls may be made of or contain PVC, and the surface energy of PVC is much higher than that of most elastic intermediate materials, such as silicone), which causes nucleic acids to preferentially attach to the outer layer rather than the elastic intermediate layer. Accordingly, the output container 148 is placed within the purification column 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 thus aligned outer layer may be attached with a small tag to promote droplet formation and deposition into the output container.

[0176] Once the output container 148 receives the purified nucleic acid, the automated nucleic acid purification protocol is complete, and the user can remove the output container 148 from the purification column 100 via an easily accessible container area.

[0177] It should be noted that the above Figure 4 as well as Figure 5A-5K The specific construction, size, and shape of the various components of the purification column 100 shown are exemplary in nature, while other structures, constructions, shapes, and dimensions are included within the scope of this disclosure. For example, as Figure 7A As shown, another embodiment of the exemplary purification column 160a is also provided for use with a system for automated purification of target nucleic acids in biological samples. As illustrated, the purification column 160a comprises a plurality of components... Figure 4 and Figure 5A-5K The purification column 100 shown uses the same or similar reagents, buffers, filters, and filter membranes. However, purification column 160a includes a separate waste reservoir 164, instead of... Figure 4 and Figure 5A-5K The purification column 100 shown has three independent waste storage tanks 108, 130, and 142. In the case of separate disposal of waste and the purification column, a single waste storage tank (e.g.) Figure 7A The waste storage tank 164 in the middle can advantageously allow users to empty a single waste storage tank instead of entering multiple separate waste storage tanks.

[0178] To further explain Figure 7A The purification column 160a shown is... Figure 4 and Figure 5A-5KThe difference between the purification columns 100 shown is that purification column 160a includes an additional pump 162 located upstream of the lysis buffer reservoir 114 and downstream of the cell capture filter. By placing pumps on both sides of the cell capture filter, for example, suction can be increased when biological samples pass through the cell capture filter from the culture reservoir and when backwashing resuspension / lysis buffer onto the cell capture filter.

[0179] Another embodiment of the purification column 160b is also provided for use with a system for automated purification of target nucleic acids in biological samples, such as... Figure 7B As shown in the figure, purification column 160a contains many [materials related to...]. Figure 4 and Figure 5A-5K The purification columns 100 and 160a shown use the same or similar reagents, buffers, filters, and membranes. Figure 7A The purification column shown is similar to the 160a. Figure 7B The purification column 160b shown includes a separate waste reservoir, rather than... Figure 4 and Figure 5A-5K The purification column 100 shown has three independent waste storage tanks.

[0180] To further explain Figure 7B The purification column 160b shown is... Figure 7A The purification column 160b, as shown in the illustration, is also equipped with a pressure sensor 161 located between the cell capture filter and the first mixing chamber. For example, pressure sensor 161 can monitor the pressure within the purification column during cell capture and / or elution of cells / cell components from the cell capture filter. As mentioned above, the pressure within the purification column increases as the cell capture filter receives more and more cells. Pressure sensor 161 prevents the pressure within the purification column from reaching a breakpoint that would cause the filter or other components within the purification column to fail. Furthermore, pressure sensor 161 can also serve as a means of regulating the system's pumping action. For example, the pump can be powered by a threshold on / off hysteresis system, where the pump fully opens when the system reaches a preset upper threshold (e.g., 10 psi), remains fully closed when it reaches a preset lower threshold (e.g., 2 psi), and reopens when the pressure reaches the upper threshold. It should be noted that users can adjust the system-level thresholds during manufacturing through configurable settings (e.g., accessing a system-related user interface).

[0181] Additionally, the pressure sensor can adjust the pump according to the PID (proportional / integral / derivative) control loop, actively controlling the pump's operating cycle (such as speed) to maintain the set pressure value. In this case, instead of the pump completely shutting down when the system reaches the upper threshold, the pump speed drops sharply as it approaches the upper threshold, thus maintaining the pressure at or near the upper threshold, allowing the pump to continue operating.

[0182] like Figure 7B As shown, the TE buffer reservoir can be separated by two independent outlets. This allows users to perform multiple TE buffer wash cycles while using a single waste reservoir. For example, the first volume of TE buffer can be used to wash away residual ethanol in the mixing chamber through the third mixing chamber and flow into the waste liquid. The remaining TE buffer can be used to rinse the precipitate filter to dissolve the retained target nucleic acid for collection in the finished product tube. This intermediate washing step can improve the efficiency and / or concentration of plasmids obtained by the precipitate filter.

[0183] Figure 7C Another embodiment of the purification column 160c is also provided for use with a system for automated purification of target nucleic acids in biological samples. As shown in the figure, the purification column 160c comprises many components... Figure 4 , Figure 5A - 5K and Figure 7A , 7B The purification columns 100, 160a, and 160b shown use the same or similar reagents, buffers, filters, and membranes. Figure 7C As shown, purification column 160c removes ribonuclease a from a triple stack of lysis buffer and resuspension buffer after cells are resuspended from the cell capture filter before being added to the first mixing chamber. For example, in the example operation, cells may be captured onto the cell capture filter as described above. However, instead of washing the cells removed from the cell capture filter with a mixture of lysis buffer, ribonuclease a, and resuspension buffer, the filter resuspends the cells, and then the cells are introduced into the first mixing chamber via a backwashing step (similar to the steps described above related to Figure 5). The lysis buffer is then pushed into the first mixing chamber through the cell capture filter, thereby lysing the cells. Ribonuclease a may be pre-loaded into the first mixing chamber and introduced into the first mixing chamber with the cells (the resuspension buffer carries the cells from the filter to the mixing chamber) or with the lysis buffer. It should be recognized that in some embodiments, the lysis buffer and resuspension buffer may be added before the cells are resuspended from the cell capture filter into the first mixing chamber.

[0184] Figure 7CThe purification column 160c shown can also be equipped with a recirculation fluid channel connected to a third mixing chamber. For example, before the wash solution flows into the waste reservoir, the recirculation channel can be used to allow the first TE buffer to flow back through the third mixing chamber so that the TE buffer (or other elution solution) can enter the precipitation filter. This is beneficial for improving the efficiency, purity, and / or concentration of target nucleic acids eluted from the precipitation membrane.

[0185] Furthermore, the purification column disclosed herein may include one or more additional bypass valves or vents. For example, a vent may be connected to a cell capture filter so that the pump pressure does not exceed a threshold pressure. This effectively prevents the purification column from being affected by destructive forces due to filter clogging. The bypass valve may also allow unfiltered media to flow into waste liquid. In some cases, the bypass valve can be activated to retrieve media loaded into the purification column that is to be recovered or untreated.

[0186] In addition, multiple windows can be placed throughout the purification column to determine the presence of fluid (e.g., measuring the optical density of a channel that coincides with one of the windows). This is beneficial for dynamic sample handling, such as processing samples that require long filtration times (e.g., fecal samples or dense bacterial cultures with water or urine samples).

[0187] It should be further noted that the purification column layout and design disclosed herein can be adapted to the custom preparation of solutions and reagents using a series of component reservoirs. For example, instead of separate reservoirs for lysis buffer and resuspension buffer, the purification column may have independent reservoirs for each component of buffer or reagent and may generate a specified volume of the desired buffer or reagent (e.g., mixed in a mixing chamber before dispensing and / or directly dispensing to the sample).

[0188] Accordingly, the organization, layout, and specifications of the components within the purification column can be adjusted according to the number and type of filters / membranes and / or buffers / reagents used in the purification protocol, as well as the volume and steps associated with processing different biological samples and / or different purification protocols, but this requires a skilled person to perform.

[0189] Fluid release mechanism

[0190] The following disclosure relates to an exemplary embodiment of an automated nucleic acid purification system in which, during such automated purification, liquid is selectively released from an input reservoir, a buffer / reagent reservoir, a mixing chamber, or other regions of the purification column. The fluid release system may include interacting components from the disclosed purification instrument and purification column. For example, the actuator of the fluid release system may be associated with and controlled by the purification instrument and interact with a flexible region of the purification column to release fluid.

[0191] Overview of Fluid Release Systems

[0192] Nucleic acid purification procedures are manual, require specialized laboratory skills, and can take several hours to complete. Automation is challenging because it involves a large number of different fluids at specific points in the purification process (at least in part). Previous attempts to replicate the manual process using robotics have been made, but these methods require additional investment in expensive robotic equipment on top of the associated costs of laboratory equipment commonly used in manual processes (centrifuges, vacuum valve assemblies, etc.). Furthermore, these methods fail to reduce the quantity and types of consumables by relying primarily on filter columns and buffers provided in commercial purification kits.

[0193] Furthermore, previous methods have failed to improve the mechanism of fluid release during nucleic acid purification. Instead, previous methods typically used tubing (similar to manual operation) to deliver liquids, or dispensed equal portions of liquid from a large reservoir into various tubing or filter columns. For the former, in addition to monitoring and replacing these additional consumables, necessary injection and spraying of the pipette tip are required, increasing cost and processing time. For the latter, the risk of cross-contamination between samples is high, and special attention is needed to clean and / or sterilize tubing or injection ports between samples. In both cases, the proposed solutions fail to address the need for an independent fluid release system that could reduce the number and / or types of consumables associated with fluid release / transfer during nucleic acid purification and reduce or eliminate the risk of cross-contamination between sample formulations.

[0194] Therefore, this presents several challenges in designing a system capable of selectively releasing appropriate liquids (e.g., samples, buffers, reagents, or combinations thereof) during automated nucleic acid purification procedures. As described in more detail below, the fluid release system described herein addresses one or more of these challenges by adding a flexible gasket between a fluid reservoir on one side and an actuator on the other. The operable actuator deflects the flexible gasket and breaks the fragile seal that holds the fluid within the reservoir, thereby selectively releasing the fluid from the reservoir.

[0195] In some embodiments, the flexible gasket, fragile seal, and reservoir are all components of the purification column, while the actuator is a component of the purification instrument. This facilitates controlled release of fluid from the consumable purification column without cross-contamination of the sample. The actuator is connected to one side of the flexible gasket; pressing the flexible gasket from one side deforms the gasket towards the fragile seal, causing the fragile seal on the other side of the gasket to interact and break. In this way, the actuator can be separated from the fluid through the intermediate gasket, avoiding direct contact with the fluid. The actuator can be repeatedly inserted into the fluid container without requiring cleaning or handling between or inside the sample, and causes minimal contamination.

[0196] Another advantage is that the fluid release system of this disclosure simplifies the fluid release process, reduces processing time, and essentially eliminates the need to know the volume of the released fluid. In other words, the fluid release system of this disclosure can be used to release small or large quantities of fluid because the composition and process of the fluid release are substantially independent of the size of the storage tank. Furthermore, the mechanical device for releasing the fluid is also simple; that is, the system does not require complex mechanical devices that have proven unreliable over time. This can increase the reliability, repeatability, and long-term utility of the system, especially in embodiments using a single-axis drive.

[0197] Fluid release mechanism

[0198] Now refer to Figures 8A-8C The figure illustrates an exemplary embodiment of a fluid release system for retaining and selectively releasing fluid. As shown, the system includes an actuator 166 and a reservoir 168. The reservoir 168 is used to retain fluid and is at least partially defined by an outer layer (e.g., outer layer 115a) of the purification column and a brittle seal 170, which, if intact, can hold the fluid within the reservoir 168.

[0199] As used herein, "fragile seal" refers to a permeable material that can tear, break, or catastrophically fail under mechanical forces, thus losing its sealing function. The fragile seal of this disclosure may consist of a chemically inert material on the reagent side facing the relevant channel or reservoir. In one embodiment, the fragile seal comprises a thin layer of chemically inert material that can break upon the application of external mechanical forces while tightly sealing the fluid. In some cases, the inert material is a plastic, such as polypropylene, polyvinyl chloride, polystyrene, or similar materials. Furthermore, the fragile seal may also include a second reinforcing layer, for example, composed of metal foil. The second reinforcing layer may be fused with the inert material and functionally form a single layer, such that a breakage of the plastic or reinforcing layer is functionally equivalent to a breakage of both layers. Other materials suitable for fragile seals may be selected as known in the art, for example, fragile seals on pre-formed plastic packaging for pharmaceutical filling cavities (such as blister packs), or other fragile seals on some consumer foods. For example, a foil layer with approximately zero moisture loss can be fused and / or used in conjunction with a second reinforcing layer (such as Aclar) to further delay and / or prevent moisture loss.

[0200] A flexible gasket 172 is placed between the fragile seals 170 of the actuator 166. As shown in FIG8A, the flexible gasket 172 is a component of the purification column. This flexible gasket may form part of the intermediate layer 117 (between the outer layers 115a and 115b). For example, the flexible gasket 172 may be an extension of an elastomeric layer sandwiched between the inner and outer thermoformed layers of the purification column, which is similar to that described herein. In these embodiments, the reservoir 168 may be at least partially defined by the flexible gasket 172 in one of the two outer layers of the purification column. As described in more detail herein, the outer layers 115a and 115b of the purification column may be made of or contain thermoformed plastic.

[0201] In some embodiments, such as Figure 8A As shown, the flexible gasket 172 can form a channel or conduit 174 through which fluid can be released from the fluid reservoir 168. To selectively release liquid from the reservoir 168, the actuator 166 moves from a first position (indicated by arrow A) to a second position (indicated by arrow B in Figure 8B). During this process, the actuator 166 contacts a first side of the flexible gasket 172, and with continued axial movement toward the flexible gasket 172, the actuator 166 deflects the flexible gasket 172 toward the fragile seal 170.

[0202] like Figure 8B As shown, the actuator 166 can move a distance toward the reservoir 168, causing the deflected flexible gasket 176 to be punctured or the fragile seal 170 to be punctured. In this way, the actuator 166 will not directly contact the fragile seal 170 or the contents of the reservoir 168. Instead, the surface of the flexible gasket 176 will contact the fragile seal and cause it to break. When some liquid in the reservoir 168 may flow between the deflected flexible gasket 176 and the broken seal 178, during the retraction of the actuator 166, as... Figure 8C As shown, the flexible gasket 172 returns to its original position (e.g., due to its flexible properties) and provides a transparent conduit 174 through which fluid can exit the reservoir 168 through the hole 180 formed by the broken seal 178.

[0203] It is worth noting that the fluid reservoir 168 can be any reservoir or chamber within the purification column, including, for example, an input reservoir, a buffer reagent reservoir, a mixing chamber, etc. Therefore, the fluid within reservoir 168 can be the 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, and combinations thereof, or any other fluid, reagent, buffer, enzyme, or mixture used or formed during nucleic acid purification. Additionally, conduit 174 provides a channel for the released fluid to flow between reservoir 168 and the next or final destination 182 within the purification column.

[0204] Figures 8A-8C The system shown includes components from a nucleic acid purification column and associated instruments. The systems shown in Figures 8A-8C may include components from a protein purification column and associated instruments. For example, actuator 166 may be a component of the purification instrument, while reservoir 168, associated fragile seal 170, and flexible gasket 172 are components of the purification column, as described in more detail herein. In this way, the movement of actuator 166 can be controlled by the instrument, which, as described above, includes computer-executable instructions or other programmable elements that allow fluid release according to time or circumstances by predefined or user-defined protocols. This allows for the dynamic implementation of various nucleic acid purification protocols using any combination of buffers, reagents, and processing parameters without altering the physical components of the instrument, such as the actuator. In other words, the disclosed fluid release system enables multiple uses through a single hardware configuration.

[0205] It is worth noting that the position of the actuators within a nucleic acid or protein purification instrument can also be dynamic. In some embodiments, a purification instrument may have a single actuator that is repositioned throughout the purification protocol, thereby aligning with and causing the breakage of various fragile seals at different times. Alternatively, a purification instrument may have multiple actuators that align with or move between corresponding fragile seals on the purification column.

[0206] In some embodiments, the fragile seal 170 is a foil seal. Alternatively, the fragile seal may be made of a suitable material capable of withstanding the pressure exerted on it by fluid in the associated reservoir or chamber, but which will break when a flexible gasket applies a greater force upon it via an actuator. In some embodiments, the thickness of the fragile seal may be adjusted to accommodate different pressures within the reservoir or by the actuator.

[0207] In some embodiments, such as Figures 8A-8CAs shown, the actuator is blade-shaped. This allows the force applied by the actuator to be concentrated in a smaller area of ​​the fragile seal, thereby reducing the degree of failure that could propagate outwards and enlarge the orifice. It should also be noted that actuators of other shapes can be provided to achieve the same or similar effects. For example, typical actuators can have blade diameters of 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 7 mm, or 10 mm, or smaller than any of the above. The blade diameter can also be selected from the above two diameter ranges (e.g., 1 mm to 10 mm, 2 mm to 5 mm, 3 mm to 7 mm, 2 mm to 4 mm).

[0208] Flexible exhaust port

[0209] In some cases, the fluid released through the mechanisms described above may create negative pressure within the reservoir upon escape. This can result in incomplete drainage of the reservoir (e.g., in a closed system) or intermittent flow from the reservoir or air inflow into the reservoir (e.g., in an open system). Each of these situations can negatively impact the efficiency of the automated purification process and / or the purity or concentration of the output target nucleic acid or protein.

[0210] For example, if the fluid is mixed with suspension, lysate, or eluent and not completely drained, it can alter its desired chemical properties and lead to different or suboptimal results. For instance, if the incompletely drained reservoir contains wash buffer or endotoxin removal buffer, the output target nucleic acid may be contaminated or inconsistent with its intended use. As a non-limiting example, the target nucleic acid could be a plasmid intended for mammalian cell culture analysis. If the plasmid was purified from Gram-negative bacteria and treated with the required volume of endotoxin removal buffer, but not completely cleaned, the toxicity or inflammatory concentration of lipopolysaccharide may still be relevant to the purified plasmid DNA, preventing it from achieving its intended use.

[0211] Intermittent flow and / or gas flow into the fluid reservoir can also negatively impact the efficiency of the automated purification process and / or the purity or concentration of the output target nucleic acid or protein. For example, if the fluid contains surfactants, air flowing into the reservoir can cause bubbles to form in the taut fluid at the lower surface, resulting in incomplete drainage. Many lysis buffers contain surfactants, and incomplete binding of the lysis buffer to the input biological sample can reduce the amount of target nucleic acid that can be purified.

[0212] The fluid release system disclosed herein can solve one or more of the problems described above. For example, a typical fluid release system can be used to release air into a fluid reservoir to facilitate complete drainage of fluid from the reservoir. In some embodiments, such as Figure 9A and9B As shown, the fluid release system includes an exhaust system 184 associated with the fluid reservoir 168. The exhaust system 184 can be used to rupture the fragile gas seal 190 associated with the fluid reservoir 168, thereby directing air into the reservoir 168.

[0213] like Figure 9A and 9B As shown, the venting system 184 is part of the purification column. The venting system 184 includes a flexible vent 188, similar to the flexible gasket described above, which can serve as part of an intermediate layer 117 disposed between the two outer layers 115a, 115b of the column. In some embodiments, the flexible vent 188 is an extension of the elastomeric layer and can partially define the fluid reservoir 168.

[0214] As shown in the figure, the flexible vent 188 is separated from the fluid communication of the reservoir 168 by a fragile gas seal 190. To selectively vent air into the reservoir 168, a movable actuator (not shown) can contact the flexible vent 188 and move continuously axially toward the reservoir 168 (e.g., as shown in the figure). Figure 9A As shown (direction from left to right), the actuator can deflect the flexible vent 188 toward the fragile gas seal 190 until the deflected vent contacts and perforates or otherwise ruptures the fragile gas seal 190. Similar to the flexible gasket described above, the flexible vent 188 can return to its non-deflected position after the deflection force applied by the actuator is removed.

[0215] A flexible vent 188 can form a channel or conduit 192 between the reservoir 168 and the outside air (e.g., through the ruptureable gas seal and vent 186 formed by the flexible vent 188). As shown, the outside air can enter the conduit 192 from the vent 186, moving longitudinally along the reservoir 168 to or above the ruptureable gas seal 190. In some embodiments, the ruptureable gas seal 190 can be positioned at the top of the reservoir 168, oriented away from gravity, and the ruptureable seal (not shown) for releasing liquid from the reservoir 168 can be located at the bottom of the reservoir, oriented towards gravity. In this direction, breaking the ruptureable seal and the ruptureable gas seal can cause the liquid to overflow from the bottom of the reservoir by fluid force under gravity, at which point the air pressure entering the top of the reservoir is equal to the gravity. This allows for complete drainage of the liquid in the reservoir and also prevents blockage or poor drainage due to pressure inequality between the reservoir and the outside air.

[0216] In some embodiments, the fragile gas seal breaks before the breakable seal, allowing the pressure within the reservoir to equalize with the outside air before liquid is released. In other words, the fragile gas seal breaks after the breakable seal to maintain pressure balance when the reservoir is emptied. For reservoirs that are full or near capacity, breaking the fragile gas seal after the breakable seal reduces the likelihood of liquid overflowing through the broken gas seal, as the liquid level may be lower and / or the available volume of the top space of the reservoir may be larger for the fragile gas seal. In some embodiments, the fragile gas seal may be broken by the same actuator as the broken seal described above.

[0217] In some embodiments, the fragile gas seal and the fragile seal break simultaneously or nearly simultaneously. The fragile seal may therefore be damaged by different actuators. Additionally, the movement of the actuators relative to the fragile gas seal can be controlled by the purification instrument, which, as mentioned above, may include computer-executable instructions or other programmable elements that allow air to be vented according to predefined or customer-defined times or conditions. This can facilitate liquid control throughout the nucleic acid purification process, ensuring that the actuators do not come into contact with any liquids and do not contaminate the sample.

[0218] Protein purification columns, instruments and systems

[0219] This disclosure relates to components, automated systems, and methods for purifying target biomolecules from biological samples. The foregoing discussion, in particular, relates to... Figure 3A-5K and Figure 7A The foregoing discussion related to -C illustrates various exemplary component designs and processing workflows for the automated purification of target nucleic acids. Similarly, the embodiments disclosed herein may include similar components and processing workflows for the automated purification of different target biomolecules, i.e., target proteins.

[0220] Protein purification is crucial for characterizing protein function, structure, and interactions. The purification process can involve various steps, including cell lysis, separation of soluble protein components from cell debris, and ultimately, separation of the target protein from product- and process-related impurities. However, many existing methods for extracting and purifying proteins from liquid cell cultures are time-consuming and primarily manual. These methods require specialized expertise and equipment to perform each step. For example, a typical approach involves obtaining cells by dissociating the liquid cell culture, then resuspending the resulting cell pellet in a lysis buffer for further lysis. The lysate is then clarified by an additional centrifugation step, and the clarified lysate is collected. The clarified lysate can then be passed through a protein purification column (or other filter, resin, or protein capture device) for further purification of the desired protein, collected in an elution buffer for use in further downstream workflows.

[0221] The automated systems and accompanying components described herein can automate various steps in the protein purification process and can include automating substantially the entire protein purification process after adding a biological sample containing the target protein to a corresponding protein purification column, as well as operable automated systems. For example, the components and systems disclosed herein are suitable for purifying and / or isolating proteins from bacterial and / or eukaryotic cultures. In one embodiment, the target protein is composed of eukaryotic cells or associated cells such as yeast (e.g., Saccharomyces cerevisiae), or insect cells or rodent cells (e.g., mouse, rat, or hamster cells) such as CHO cells or primate / human cells, such as 293 or COS cells. In another embodiment, the target protein is composed of prokaryotic cells (e.g., as a recombinant protein) or associated prokaryotic cells. Regardless of cell type, automated protein purification systems and associated components and methods are capable of purifying and collecting target proteins from them.

[0222] Similar to the target nucleic acid purification systems and components described above, the automated protein purification column of this disclosure is adapted to receive biological samples containing target proteins via associated culture reservoirs and process the biological samples using a variety of methods. Interconnected bioprocessing components are used to produce purified target proteins. For example, after adding a biological sample containing the target protein (e.g., a prokaryotic / eukaryotic cell culture) to the target protein purification component, the purification column can be processed in a first bioprocessing component for separating the cellular fraction from the sample / culture supernatant. It should be noted that in the case where the target protein is a secreted protein, the supernatant can be further processed rather than discarded. Furthermore, in the case where the target protein is cell-associated, the cellular fraction of the biological sample / culture can be obtained on a cell capture membrane, and the supernatant will be discarded. It should be understood that the above-described nucleic acid purification bioprocessing components and reagents can be used for protein purification.

[0223] As a non-limiting example, cells from a suspension can be added to a target protein purification column and passed through a filter, where cells are captured on a first side of the filter. The filtrate passes through the filter and through a second side of the filter before being introduced into a waste container. After this step, each subsequent step can be selectively detected, optionally by an air / bubble sensor or any other method, to detect the presence of a fluid flow rate or threshold fluid velocity disclosed herein and / or known to those skilled in the art. As described above, the target protein purification column can be associated with a complementary automated purification system that utilizes a pumping action to aspirate the biological sample from the filter along a first flow direction, and may then further enter a second operating state to induce liquid flow in the opposite second flow direction.

[0224] In some embodiments, a suitable cell capture filter system for use in a target protein purification column is selected primarily based on flow rate and filter capacity. Other filter characteristics, such as pore size, are also considered when selecting a suitable cell capture filter system (e.g., for capturing cells containing the target protein). As a non-limiting example, strains of *Escherichia coli* can be engineered to produce recombinant target proteins. Cultures of these *E. coli* can be captured on a membrane system within an automated purification column containing a 0.2-micron hollow fiber filter membrane. As a supplementary example, the cell capture filter associated with a target protein purification column comprises a plurality of porous hollow fibers based on cellulose acetate, which are bent into a series of annular structures. These annular structures effectively provide a large surface area for the filter to capture and / or process biological samples and / or partially purified target proteins. The open ends of these annular structures are embedded in polyurethane or a similar polymer to form end plugs (e.g., machined to ensure the open ends of the annular structures remain open). In one embodiment, the hollow fiber filter can be made from... Laboratories, Inc. (Business Procurement) Filters, such as the MEDIAKAP-25 filter. In addition to any other characteristics or properties of the target protein, other filters and / or cell capture systems can be used based on the size, polarity, and / or presence or absence of the affinity tag, as is well known in the field.

[0225] Furthermore, the bioprocessing components within the target protein purification column of this disclosure also include a fluid mixing chamber and / or may contain a solid support for processing one or more samples. The solid support can be any support used for filtration, washing, staining, elution, collection, processing, or chemical or biological treatment. In some embodiments, the solid support may be selected from filter paper cartridges, filter paper, precipitation membranes, precipitation filters, solid-phase extraction columns, solid-phase extraction boxes, solid-phase extraction disks, resins, membranes (e.g., blot membranes, filter membranes, PVDF membranes, nylon membranes, positively charged nylon membranes, and nitrocellulose membranes), reaction beads (e.g., glass beads and magnetic beads), rigid planar solid supports containing biomolecular arrays (e.g., protein arrays, tissue arrays), microscope slides, and combinations thereof.

[0226] In some embodiments, in one or more bioprocessing chambers, the solid support may include filter paper, a filter, or a filter cartridge. The filter paper, filter, or filter cartridge may include any suitable type of filtration device having appropriate chemical properties, pore size, shape, three-dimensional structure (e.g., symmetrical or asymmetrical three-dimensional structures, including "V"-shaped or funnel-shaped pore structures), and / or surface area for the intended use. That is, the solid support can be selected based on the properties of the target protein and / or the required purification protocol. Furthermore, the solid support may be used for overflow channels, crossflow, tangential flow, or any combination thereof. It should be understood that the filter paper, filter, or filter cartridge may be made of any suitable material, or may be a single-layer or multi-layer filtration device comprising any suitable material, including polyethersulfone, polyethylene, ultra-high molecular weight polyethylene, polypropylene, nylon, cellulose, cellulose triacetate, polyacrylonitrile, polyamide, glass fiber, silica, polysulfone, PVDF, etc. In some embodiments, in one or more processing chambers, the solid support is a precipitation membrane or precipitation filter, or it may also be an imprinted membrane. In some embodiments, the solid support may be a solid-phase extraction column, a solid-phase extraction cartridge, or a solid-phase extraction disc. 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 placed in a column or suspension within the processing chamber.

[0227] As a non-limiting example, embodiments of this disclosure may include a purification column for purifying target proteins, the purification column having a protein capture component that tracks a cell capture sub-component. The protein capture component can be used to clarify lysates, or it can be used to receive and bind target proteins from clarified lysates. Therefore, it should be understood that exemplary purification columns for purifying target proteins in biological samples may include clarification and / or protein capture components that are connected in a flow-through manner to a cell capture component and / or a mixing chamber, in addition to being connected to one or more buffers / washing solutions / reagents. For example, reservoirs containing protease inhibitors, pH difference solutions, washing buffers, elution buffers, etc., may be incorporated into corresponding flow-through connected reservoirs that are sealed as described herein. The envisioned purification column (e.g., as described herein) can be configured according to a protein purification protocol, rather than a nucleic acid purification protocol. Figure 3A-5K and Figure 7A The buffer solutions and processing steps within or between the biological processing components (as shown in -C) are subject to appropriate deletion and / or addition.

[0228] In one embodiment, the automated protein purification column includes sub-components having one or more affinity matrices / columns (e.g., instead of...). Figure 3A-5K and Figure 7A-C provides a dedicated membrane / filter for nucleic acid purification in the automated nucleic acid purification column. Affinity chromatography is an efficient protein purification technique that typically uses only one step to purify proteins to a purity sufficient for analytical characterization. Affinity chromatography is a separation technique based on molecular conformation, where molecules "match" each other, selectively binding to each other in a "lock and key" manner (e.g., antibodies can recognize antigens and specifically bind to them). This technique can use specialized chromatographic resins with ligand-specific receptors (e.g., antibody fragments containing antigen-binding domains) attached to the resin surface. Most commonly, these receptors bind to the target protein in a manner similar to antibody-antigen interactions. This highly specific match between the receptor and its target compound makes affinity column chromatography highly specific as well. The antigen binds to the resin-bound antibody (typically with high affinity), while other sample components and impurities do not bind but instead flow through the affinity column to the waste container. Any bound antigens (usually associated proteins) can then be washed away and eluted from the column, which typically alters the pH, thereby disrupting the interaction between the molecular antigen and the antibody, resulting in a single, high-purity elution peak representing the target protein.

[0229] Therefore, embodiments of the protein purification column disclosed herein may include an affinity column that specifically binds to and captures the target protein while simultaneously allowing residual proteins and cell products to pass through the column and be converted into waste. The column can be washed to remove any loosely bound or residual cell debris (e.g., by opening a reservoir containing wash buffer and aspirating the contents through the column for automated washing). A reservoir within the purification column can be loaded with a solution that can be used to alter the pH of the matrix / column and thus elute the target protein (e.g., into a collection channel). Other washing and / or purification steps can be automated using systems and protein purification columns designed for such applications.

[0230] As a non-limiting example, Thermo Fisher Scientific, through its The product portfolio offers a variety of products for affinity chromatography. The product is based on affinity ligands derived from camelid-derived single-domain antibody fragments (VHH). The affinity receptor is a 12kDa single-domain fragment containing three complementarity-determining regions (CDRs) that form the antigen-binding domain. These affinity receptors are efficiently produced in the yeast *Saccharomyces cerevisiae*, and are therefore not animal-derived. The affinity receptors are then covalently bound to chromatographic beads, generating a resin suitable for protein purification via column chromatography. This process leverages the well-defined specificity and high affinity for the target, creating affinity chromatography resins ideal for single-step protein purification.

[0231] In automated protein purification processes using purification columns, such (and similar) products can help achieve efficient screening, improve affinity and stability, and provide a range of exemplary advantages in the purification process, such as single-step purification, ease of use, minimized purification costs, effective removal of impurities, higher quality products, and increased flexibility.

[0232] Other examples of protein purification products included in one or more sub-components of the automated protein purification column disclosed herein include those for isolating immunoglobulin forms from various environments (e.g., plasma and milk from transgenic animals, or supernatant from mammalian cell cultures). Products. Proteomics Products for protein consumption to support biomarker research applications; HPLC analytical columns—rapid, sensitive, and accurate quantification for small-scale protein preparation from complex mixtures; rapid protein liquid chromatography (FPLC); and / or columns with custom antibody ligands for specific proteins or impurities to adapt affinity purification to new applications.

[0233] In some embodiments, the protein purification columns disclosed herein can be used to run protocols and biological processing procedures selected from: immunoprecipitation, recombinant protein isolation, protein labeling, target protein isolation and segregation, and bead-based automated separation (including magnetic bead-based automated separation). The disclosed protein purification columns can be adapted for use with the automated systems disclosed herein, and / or the systems disclosed herein can be modified to be compatible with the protein purification columns disclosed herein. It should be understood that the functionality of the combined purification columns and systems disclosed herein related to nucleic acid purification can be achieved using corresponding purification columns and systems for protein purification. Therefore, sealing and fluid control mechanisms related to the nucleic acid purification columns and systems provided herein can be used separately and / or associated with the protein purification columns.

[0234] In one embodiment of the automated workflow, the entire cell lysis process—including filtering at least 1 L of cell suspension, any (optional) washing steps, all steps related to loading buffer / solution into appropriate reservoirs or mixing chambers for incubation and / or resuspending of captured and lysed cells, and collecting the clarified lysate—can be completed within 2 hours or less, preferably within 1 hour or less. In one embodiment, when collecting purified target protein from the clarified lysate, all steps involved (including purification of the target protein via a purification column), as well as all washing and elution steps, can be completed within approximately 2 hours or less, preferably within 1 hour. Therefore, in one embodiment, in a single automated process, filtering, lysing, clarifying, and purifying target protein from up to 1 L of cell suspension may take approximately 4 hours or less, preferably approximately 2 hours or less.

[0235] In some embodiments, the processing workflow may include additional processing steps that may increase the total run time. For example, purified proteins may be further purified using complementary chromatographic strategies (e.g., “polishing”) or buffer changes may be performed, including size exclusion, ion exchange, or affinity purification. As a complementary or alternative example, purified proteins may be subjected to proteolysis to remove affinity tags encoded by amino or carboxyl terms in the protein sequence before proceeding to the final purification step. It should be understood that the workflow can be extended, i.e., the size and / or number of various system components can be increased or decreased as needed to accommodate filtration and / or purification requirements for cell suspension volumes greater than or less than 1 L.

[0236] Automated purification instruments

[0237] The following disclosure describes exemplary embodiments of an instrument that can be used for the automated purification of target biomolecules, such as target nucleic acids and / or target proteins. This purification instrument can be used in conjunction with other components described herein. For example, the instrument can be used to house and cooperate with the consumable purification column described above. Although nucleic acid purification is used as an example in describing some functions of this instrument or device, it should be understood that similar instruments (with the modifications described herein) can also be used for protein purification.

[0238] Instrument Overview

[0239] As mentioned above, the conventional procedures for purifying target biomolecules (e.g., target nucleic acids and / or target proteins) are manual laboratory processes that require a relatively high level of laboratory expertise and time. While trained laboratory technicians are capable of handling the various operational parameters involved in the process, designing an instrument that can effectively automate most of the process remains challenging for several reasons. For example, a device for automating the process should be able to efficiently move and deliver fluids of different viscosities and densities, release reagents at specific predetermined times, and, in conjunction with other fluid movement operations, mix different fluids together, process relatively large samples, and perform appropriate fluid separation / sealing throughout the process. Such devices should also be safe to ensure user safety and capable of purifying a given target biomolecule (e.g., a pre-selected target nucleic acid or a pre-selected target protein).

[0240] As detailed below, the purification instruments described herein can address one or more of the challenges described above. For example, the purification instrument may include a pump assembly for providing suction via peristaltic motion so that fluid can be efficiently passed through an inserted consumable purification column; it may also include components for controlling automated mixing of the fluid within the inserted purification column; it may also include components for providing or enhancing the liquid-tight seal on the purification column; and it may include safety mechanisms to protect the user from accidental injury and / or limit the risk of process errors. In some embodiments, the instruments disclosed herein may be used to process large sample volumes (e.g., approximately 5 ml to 5 L, or 10 ml to 500 ml, or approximately 15 ml to 250 ml). It should be appreciated that the size of the system, device, and purification column can also be reduced to purify smaller volumes (e.g., less than 5 ml, such as 0.5 ml to 5 ml).

[0241] Figure 10An isometric view of a purification instrument 300 (also referred to herein as a purification “device”) capable of automating nucleic acid purification is provided. Instrument 300 includes a housing 302 surrounding and defining an internal compartment 304. The internal compartment 304 is sized and shaped to accommodate a sample purification column 301. In a preferred embodiment, the purification column 301 is a consumable purification column, such as those described in more detail elsewhere herein. Instrument 300 includes a selectively closable access door 306 providing access to the internal compartment 304. Within the scope of this disclosure, other means for inserting and / or receiving purification columns are also contemplated, including means for automatically feeding purification columns into the instrument, or other machinery known in the art. Instrument 300 may also include a waste tray (not shown) disposed within the internal compartment 304 for collecting leaks or spills from the purification column and / or the area near the instrument, such as leaks or spills from biological samples or various reagents / buffers used. Waste trays can be selectively inserted or removed for waste removal and cleaning.

[0242] See below for the possible shapes of housing 302 Figure 10 The instrument 300 is shown as a non-limiting example. In this example, the length of the instrument 300 (from access door 306 to rear side 308) is approximately the same as the height of the instrument 300, and the width of the instrument 300 is approximately half its length. These relative dimensions function well in typical laboratory settings. However, it should be understood that the components and features of the instrument 300 do not require a specific shape for the housing 302, provided that the internal compartment 304 can receive the purification column 301. In other embodiments, the housing 302 may be of other shapes, such as having other aspect ratios, length-to-width ratios, and / or width-to-height ratios.

[0243] Furthermore, while the embodiments described above are for receiving the purification column in a substantially vertical direction, other embodiments may allow the purification column to be inserted from different directions. For example, some embodiments may be used to receive the purification column in a substantially horizontal direction. Additionally, although one purification column may be received at a time in the embodiments described above, other embodiments may include multiple compartments, each of which may receive purification columns simultaneously or independently. For example, in these embodiments, multiple samples may be processed in parallel.

[0244] As shown in the figure, the access door 306 may be equipped with a hinge point 310 located near the bottom 312 of the instrument 300, so that the access door 306 can be rotated upward to open and downward to close. This configuration allows the access door 306 to rest against a table surface when open, providing access to the internal compartment 304. In other embodiments, different access doors 306 may be configured, for example, by providing hinge points on the upper, left, or right side of the instrument or by using sliding doors or panel assemblies. While the use of an access door 306 is recommended, it may be omitted in some embodiments.

[0245] In the described embodiment, when the access door 306 is open, the inner surface 314 of the access door 306 can be used as a guide to guide the purification column 301 into the inner compartment 304. For example, a user can place the purification column 301 on the inner surface 314 and then guide the purification column 301 into the inner compartment 304 at a suitable height. The inner surface 314 also includes one or more grooves 316 or other such guide structures that can engage with the purification column 301 to further assist in the correct positioning of the purification column 301 in the inner compartment 304. Other devices and / or structures for guiding the purification column into the compartment are contemplated herein, including, for example, clamping mechanisms for securing the purification column when it is partially inserted into the compartment, and transfer devices for subsequently transporting the purification column further into the compartment for further processing. Other usable devices are known in the prior art.

[0246] In this embodiment, a user interface 318 is also included for accommodating user input and / or displaying instrument information. The user interface 318 is connected to the controller via communication coupling (details are described below – see below). Figure 20A This enables communication with and control of various components of instrument 300. User interface 318 accommodates user input and / or displays information such as the volume of the biological sample placed in the purification column, purification column identification information, selected purification protocol, creation and / or saving of user-defined protocols, sample identification information, instrument identification information, operating instructions for the selectively closable access door 306 or its associated components, the desired concentration of the target nucleic acid to be purified, one or more optical density measurements related to the initial sample and / or samples at different stages of the purification process, and / or the final volume of the eluent containing the target nucleic acid.

[0247] User interface 318 allows interaction with the user via a touchscreen, control panel, mouse, microphone, keyboard, and / or other computer input / output components known in the art. During processing, user interface 318 allows the user to observe the progress of the purification column in performing the purification process and can also issue prompts and warnings indicating that processing is complete or that errors or other problems may occur during processing.

[0248] Instrument clamping mechanism

[0249] See embodiment 320, which can be used as a clamping mechanism for instrument 300 components. Figure 11A and Figure 11B As shown. In some applications, instrument 300 can dock with consumable purification columns with low-cost structures. That is, to reduce manufacturing complexity and associated costs, consumable purification columns can be intentionally designed with minimal inherent sealing. For example, consumable purification columns can be designed to maintain the seal required for fluid separation under ambient pressure, but do not need to withstand the high pressures of the purification process (e.g., as a result of passing biological samples, reagents, and / or other fluids through membranes, filters, resins, and / or microsphere columns in different separation / filtration process steps).

[0250] Consumable purification columns can be designed to provide sufficient sealing force through the clamping mechanism 320 of the instrument 300, enabling them to withstand the high pressures during purification. This design approach allows the instrument to offset the inherent cost of the consumable purification column. It is more economical and efficient to allocate these costs to durable instruments than to disposable, single-use purification columns.

[0251] like Figure 11A and 11B As shown, housing 302 and other components have been removed to better showcase clamping mechanism 320. Clamping mechanism 320 includes components for clamping in the open position (e.g., Figure 11A (as shown) and closing position (as shown) Figure 11B The first plate 322 and the second plate 324 move between (as shown). In the closed position, plates 322 and 324 compress the consumable purification column located therebetween and provide a clamping force sufficient to allow the consumable purification column to withstand the relatively high pressure during purification. Figure 11B As shown, plates 322 and 324 may include a hole 326 that aligns with a consumable purification column assembly (such as a fluid reservoir or consumable purification column channel assembly). Therefore, plates 322 and 324 are used to align and compress the consumable purification column, and to compress the portion of the consumable purification column that needs to be compressed to maintain seal integrity during purification.

[0252] like Figure 11A As shown, the first plate 322 and the second plate 324 are supported within the bracket 328. In this embodiment, the clamping mechanism 320 is used to move the plate 322 laterally relative to the plate 324. That is, the first plate 322 is a movable plate, and the second plate 324 is a fixed plate. The base 330 is mounted in the socket 332, and the base 330 is longitudinally movable relative to the socket 332. The socket 332 is attached to the bracket 328 and / or forms part of the bracket 328, and its function is to hold the base 330 at the edge of the internal compartment and prevent the base 330 from moving laterally.

[0253] like Figure 11B As shown, bracket 328 and socket 332 have been removed to better showcase base 330 and its associated mechanisms. As illustrated, base 330 is connected to first plate 322 via one or more links 334. It is preferable to provide multiple links 334 to better distribute pressure across first plate 322. Links 334 may also include springs to better distribute lateral pressure across the plate. Links 334 may also include various gaskets to adjust the applied pressure.

[0254] A drive screw 336 effectively connects to a motor 338 and passes through a receiver 340. The receiver 340 is attached to a base 330 and is threaded, so that rotation of the drive screw 336 causes the receiver 340 to move longitudinally along the drive screw 336. This causes the base 330 to move longitudinally. Because the socket 332 prevents lateral movement of the base 330, and because the bracket 328 prevents longitudinal movement of the first plate 322, the longitudinal movement of the base 330 causes a corresponding lateral movement of the first plate 332. The end of the connecting rod 334 also moves in the cam path, providing additional mechanical advantage relative to the first plate 322.

[0255] In other embodiments, other mechanisms may be used to provide linear movement of the first plate 322. For example, the base 330 may be effectively connected to a linear actuator, a belt and pulley assembly, a chain and sprocket assembly, a gear and rack assembly, or a combination thereof, instead of connecting a drive screw to the aforementioned location. Some embodiments may include mechanisms that directly push and pull the first plate 322, such as piston-based mechanisms or certain other linear actuators directly applied to the first plate 322. Some embodiments may be used to move plates 322 and 324 relative to each other, rather than keeping one of the plates stationary.

[0256] Since some consumable purification columns may need to withstand pressures of up to 50 to 100 pounds per square inch, the clamping mechanism 320 is used to apply a total sealing force of at least about 500 pounds, or preferably at least about 1,000 pounds, or even better, at least about 2,000 pounds.

[0257] In the event of a power outage during purification, the user can use the manual release mechanism to loosen the clamping mechanism 320 and allow removal of the consumable purification column. Partially processed consumable purification columns may contain biohazardous and / or hazardous chemicals and may need to be removed before servicing, transporting, or further use of instrument 300. In the described embodiment, as... Figure 12 As shown, the manual release mechanism can be accessed through the access point 350 located at the rear side 308 of the instrument 300, but the access point 350 can be located in other positions.

[0258] A manual release mechanism allows commonly used tools (such as screwdrivers or Allen wrenches) inserted through access point 350 to engage the mechanism and allows manual rotation of the drive screw 336 using a rotary tool. The manual release mechanism can also be engaged. For example, once the clamping mechanism is released, the clamping mechanism spring can begin to push the plate away and cause the drive screw 336 to rotate rapidly. Subsequently, a one-way bearing associated with the clamping mechanism begins to rotate to prevent the tool used to activate the manual release clamping mechanism from rotating uncontrollably.

[0259] Other instrument embodiments may omit the clamping mechanism. For example, not all consumable purification columns need to be manufactured to withstand the additional pressure required by the instrument. For instance, if the consumable purification column has a sufficiently inherent seal, if the biological sample contains few cells or is otherwise less viscous (e.g., water or urine samples), and / or if the volume of the biological sample is small relative to the instrument capacity (e.g., about 10 ml), the instrument does not need to include or use a clamping mechanism. It should be understood that while some samples may be “small” relative to the instrument and processing capacity described herein, they may still be large relative to conventional purification instruments and processing capacities.

[0260] In applications where the instrument omits or does not use a clamping mechanism, the associated consumable purification column can be used to withstand the anticipated pressures involved. This consumable purification column may not be able to withstand the aforementioned high pressures (e.g., in cases where approximately 50 to 150 ml of culture / sample volume can be processed through one or more cell capture membranes), but it is still sufficient to withstand the relatively low pressures involved in the aforementioned situations. This consumable purification column can be made of polymeric materials (e.g., thermoforming and / or other suitable polymers), with two separate sides welded, bonded, and / or mechanically locked together along the edges and at any other desired locations.

[0261] Instrument safety mechanism

[0262] Figure 13 The image shown is a view of the door locking mechanism as seen from the angle facing the inner surface 314 of the access door 306. The locking piece 342 (in...) Figure 11A (As can also be seen in the image) It is connected to plate 1 322 (i.e., the moving plate). The corresponding locking slot 344 is set on a part of the access door 306. When the door is closed, the access door 306 is located in the internal compartment. When the access door 306 is closed and the clamping mechanism 320 is driven, the locking piece 342 translates with plate 1 322, thereby entering the locking slot 344. This helps to prevent the access door 306 from being opened after the clamping process has been started.

[0263] The locking mechanism serves as an access barrier, effectively preventing user injury from various moving parts within the internal compartment. The access door 306 is preferably the only exit to the internal compartment. Once closed, the access door 306 and door frame 302 form a barrier separating the user from the internal automated moving parts.

[0264] As an additional or optional precaution, instrument 300 can be used to power the clamping motor 338 only when it is determined that the access door 306 is closed. One or more sensors can be used to detect the open / closed state of the access door 306 to make this decision. Figure 14 In the illustrated embodiment, the door frame 302 includes a pair of redundant Hall effect sensors 346 located within a portion of the door frame 302, defining an access door frame. A pair of corresponding magnets 348 are disposed on the access door 306. When the access door 306 is closed, its position is aligned with the Hall effect sensors 346.

[0265] The positions of Hall effect sensor 346 and magnet 348 can be interchanged, with magnet 348 positioned on the access door frame and Hall effect sensor 346 positioned on access door 306. Although two pairs of sensors / magnets are shown herein, some embodiments may include only one pair or may include two or more pairs of sensors / magnets. Other embodiments may additionally or optionally 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 of all the foregoing.

[0266] Instrument process control mechanism

[0267] Instrument 300 preferably includes one or more features designed to ensure that the various components are properly prepared before the purification process begins. For example, instrument 300 may include features to ensure accurate positioning of the biomolecule purification column before initiating clamping or other steps of the purification process, features to ensure that the inserted purification column is not used, and / or features to ensure that the output container has been accurately positioned for receiving the purified product.

[0268] Figure 15 The illustration shows an embodiment of a column positioning mechanism within an internal compartment of the instrument. A contact switch 352 (e.g., a limit switch) can be positioned within the internal compartment at the point where the inserted column 301 contacts the switch 352. In the illustrated embodiment, the contact switch 352 is positioned at the rear of the internal compartment. In this position, when the column 301 is inserted, its leading edge 305 will advance continuously before reaching the contact switch 352. The contact switch 352 is connected to the instrument controller. The controller may be used to prevent the process from starting before the contact switch 352 has made contact.

[0269] Other embodiments may position the contact switch 352 in other suitable locations within the internal compartment. Other embodiments may additionally or optionally include other types of contacts and / or proximity sensors, such as other types of sensors (e.g., optical sensors, magnetic sensors, etc.) that are associated with other components of the instrument 300 as described herein.

[0270] The post positioning mechanism may also include a locking device 354 for connecting post 301 so that it is secured in place after insertion. The locking device 354 can also provide tactile feedback to the user, alerting them that the post has been accurately positioned. For example, the locking device 354 could be spring-loaded or otherwise biased towards the closed position, and used to "spring" or "switch" the post back to the closed position if displacement occurs during insertion.

[0271] In the illustrated embodiment, during insertion, as the post 301 begins to contact the locking device 354, the engaging part 303 of the post 301 can contact the inclined surface 356 of the locking device 354. Further insertion of the post 301 can cause the locking device 354 to leave the closed position. Once the engaging part 303 has cleared the inclined surface 356, the slot 358 allows the locking device 354 to return to the closed position. Other embodiments may additionally or optionally include other locking device features, such as magnetic couplers, door clamps, ball-loaded tensioning spring pins, door stop balls, and other locking devices known in the art.

[0272] Figure 16 The diagram illustrates other process control mechanisms that Instrument 300 may include. Purification columns typically include an output container for collecting the purified product. Normally, the user can remove the output container before inserting the column into the instrument to label it. However, if the user forgets to reposition the output container inside the column, inserts the column, and starts the purification process, valuable purified product will be spilled onto the instrument plates. This wastes valuable product and may cause mess, requiring cleaning before restarting the instrument.

[0273] exist Figure 16In the illustrated embodiment, contact switch 360 is mounted on a plate (plate 324 in this embodiment) and positioned within column 301 after accurate positioning of output container 307, allowing it to contact the output container 307. Contact switch 360 ensures accurate positioning of output container 307 before the purification protocol begins. For example, contact switch 360 may communicatively couple to a controller, and if contact switch 360 determines that output container 307 is missing, the controller may prevent protocol initiation and / or provide status notification to the user. Other embodiments may additionally or optionally include other types of contact and / or proximity sensors, such as other types of sensors described herein that relate to other components of instrument 300.

[0274] In addition, instrument 300 includes one or more optical density sensors for determining the initial optical density (e.g., OD600 or A600) of a biological sample (e.g., bacterial culture or other cell culture) that has been inserted into a purification column. The optical density can be determined by illuminating the sample using a known wavelength (typically 600 nm, but other wavelengths may be used depending on the sample type, detection target, and / or other special purpose) and measuring the amount of light passing through the sample and reaching the detector opposite the sample. Furthermore, the concentration of nucleic acids in the sample can be measured using optical sensors (e.g., using wavelengths of 230, 260, and / or 280 nm) at the end of the purification process and / or in the middle of the process.

[0275] Instrument 300 may also include one or more other sensors for monitoring protein purity or concentration at absorbance conditions of 210 or 280 nm, or the absorbance ratio at different wavelengths, and for evaluating the decoenzyme / holase status using cytochrome c, heme, and other cofactors or prosthetic groups. In some embodiments, sensors may be additionally or optionally included to monitor pH, conductivity, refractive index, osmotic pressure, redox potential, or protein aggregation.

[0276] Optical density (OD) information obtained by an optical density sensor can be reported to the user, and this information can be used to notify the user whether the biological sample is within a suitable range for processing. For example, the optical density sensor can be communicatively coupled to a controller, and if the measured optical density is not within the priority range, the controller can notify the user and / or prevent the process from proceeding.

[0277] As detailed below, optical density readings can also identify and / or activate specific purification protocols that better optimize a particular type of biological sample. For example, based on a determined sample optical density, the buffer / reagent used to purify the target nucleic acid can be dispersed in different volumes. In another embodiment, the sample mixing time and / or the time spent passing through one or more microporous membranes / filters of the purification column may also vary depending on the determined sample optical density. Optical density sensors measuring samples at different locations on the column and at different stages of the purification process can be located in different places. For example, the presence of fluid at a specific location on the column can be determined by comparing the measured optical density with the expected air impingement flow.

[0278] Optical density information can be used, additionally or selectively, to determine whether the inserted purification column has been used previously. For example, some protocols may require a density reading before placing a biological sample into the purification column inlet reservoir. Before any sample is placed into the column, the initial density reading is approximately equivalent to an air impingement flow reading. A higher reading indicates that the column inlet reservoir has been opened / tapped and infused with a biological sample. For example, a previous sample may have left residue on the sample chamber window. In this case, the controller can notify the user and / or prevent the protocol from proceeding.

[0279] Figure 17 An embodiment of the optical density sensor 362 is illustrated. The illustrated embodiment includes a light source 364 with a wavelength of 595 nm, used as a light-emitting diode (LED) mounted on an aluminum plate. A detector 366, used as a silicon detector mounted on a printed circuit board (PCB), is positioned opposite the light source 364. A sample chamber containing a portion of a biological sample 309 is positioned between the light source 364 and the detector 366. The sample chamber includes a window 368 through which light can be emitted. The optical density sensor 362 may also include one or more other optical components, such as a lens 370, a diffuser 372, a filter, an opening, and / or other optical components.

[0280] The illustrated embodiment is merely one example of a suitable optical density sensor. Furthermore, other optical density sensor configurations known in this art can be utilized. For example, other embodiments may include alternative light source types, alternative detector types, alternative lenses, filters and / or opening structures, alternative optical paths, or alternative sample chamber sizes. As described above, one or more optical density sensors can be used under different wavelength conditions to provide different sample information. For example, suitable wavelengths (e.g., 230, 260, and / or 280 nm) and one or more sensors can be used to measure nucleic acid concentration.

[0281] Instrument pump assembly

[0282] After the purification column is inserted into instrument 300 and the purification process is started, instrument 300 can utilize a series of pump assemblies to move and define the fluid path through the purification column. Pump assemblies can be configured to connect to the purification column (e.g., when the clamping device is closed) and define the fluid path during purification. Pump assemblies can be configured upstream and / or downstream of the various processing sections of the purification column (e.g., individual microporous membranes / filters). That is, pump assemblies can be configured to "push" the sample, reagent, or other fluids downstream of the process, or "pull" the sample, reagent, or other fluids downstream of the process, or both. The pump assemblies described herein are beneficial for the movement and delivery of fluids of varying viscosities and densities.

[0283] Figure 18A An exemplary pump assembly 374 is shown (in a partially exploded view). The pump assembly 374 includes a motor 376, which is effectively connected to a camshaft 378 via a power transmission assembly. As shown, the power transmission assembly may include a gear 379 and / or one or more other power transmission components, such as a belt, pulley, chain, sprocket, etc.

[0284] Connected to the camshaft 378 are multiple cam elements 380 (i.e., "eccentric claws"). As shown, each cam element 380 includes a fixed end 382 for connecting to the camshaft 378 and a pointed tip 384 extending laterally (e.g., vertically) from the camshaft 378. The cam elements 380 are arranged on the camshaft 378 such that rotation of the camshaft 378 causes the cam element tip 384 to produce a linear peristaltic motion. The cam elements 380 and / or other components of the pump assembly 374 may be arranged within a housing 386.

[0285] Figure 18B This is an unfolded view of the cam element tip 384, which contacts the fluid channels 311 / 313 of the purification column. Here, the fluid channel before deflection is shown as 311, and the fluid channel after deflection is shown as 313. The tip 384 can be tilted to achieve effective engagement with the fluid channels 311 / 313, although other cam element shapes can be used depending on specific application requirements and preferences. As shown, movement of the cam element 380 relative to the fluid channels 311 / 313 causes deflection of the fluid channels, thereby displacing the fluid within the fluid channels 311 / 313.

[0286] Figures 19A to 19F The engagement of the pump assembly 374 with the fluid channel 315 of the purification column 301 is explained, and the linear peristaltic motion of the cam element 380 is demonstrated to drive the fluid to move in the fluid channel 315. Figures 19A to 19D The figures shown are cross-sectional plan views, and these plan views are all perpendicular to the side of the pump assembly 374.

[0287] Figure 19A This shows the pump assembly 374 with the camshaft 378 at any 90-degree position. As the camshaft 378 continues to rotate... Figure 19B The 180-degree position shown Figure 19C The position shown is 270 degrees, then rotate to... Figure 19D At the 0-degree (i.e., 360-degree) position shown, the sequential engagement of the cam element 380 with the fluid channel 315 causes the fluid 317 to flow through the fluid channel 315. For example... Figure 19E and Figure 19F As shown, when the camshaft 378 continues to rotate by increasing the number of rotations, the cam element 380 continues to engage with the fluid channel 315 in a corresponding peristaltic manner to further guide the fluid 317 to flow through the fluid channel 315.

[0288] An exemplary embodiment of the pump assembly shows that approximately 0.16 ml of fluid (e.g., approximately 0.05-0.45 ml) can be moved per revolution of the camshaft. When the motor speed is 136 revolutions per minute, the resulting flow rate is approximately 22 ml / min (e.g., approximately 10-35 ml / min). Of course, the flow rate generated by the motor rotation will vary depending on factors such as different motor speeds, different fluid channel dimensions, different numbers or orientations of cam elements, or different fluid viscosities.

[0289] As shown in the pump diagram, pump assemblies are beneficial for moving fluids of varying viscosities and densities. However, other embodiments may additionally or optionally include other fluid delivery methods. For example, the pump assembly may utilize a roller mechanism to compress the fluid channel and then move it linearly along the fluid channel to move the fluid.

[0290] Instrument Control System

[0291] Figure 20A An exemplary control system 388 is illustrated, which can be used to control various components and operations of instrument 300. Controller 390 includes a memory 392a (i.e., a physical storage medium or hardware storage device) and one or more processors 392b (and / or suitable microcontrollers). As described above, controller 390 can be interactively connected to user interface 318 to receive user input information and send display information to user interface 318.

[0292] The controller 390 can also be interactively connected to one or more sensors 399, such as the position sensors described herein. Such position sensors include access door sensors 346 / 348 (e.g., Hall effect sensors), purification column contact switches 352, output container contact switches 360, and optical density sensors 398 (or multiple such sensors). As described above, the controller 390 can be operated based on the measured status of such sensors to send user notifications and / or pause or stop the purification process.

[0293] As described above, taking the optical density sensor 398 as an example, the controller 390 can be used to receive optical density information and send user notifications (to indicate how the optical density information and / or optical density measurement are related to the preferred embodiment range). In some embodiments, the controller 390 can be used to indicate whether an insert purification column has been used previously based on the optical density information received from the optical density sensor 398, and it can also be used to send notifications and / or stop the purification process.

[0294] The controller 390 can also interactively connect to the purification column sensor 396, which is used to connect to an insert-type purification column to obtain identification information from the purification column. For example, the purification column 396 may include a barcode reader and / or other suitable sensor capable of reading purification column identifiers or codes. Identification information may include batch number, expiration date, stock unit number, purification column type, number of uses related to the purification column, and number of times the purification column can be used.

[0295] The instrument is not limited to the number and / or type of sensors shown. For example, in some embodiments, one or more flow rate sensors, temperature sensors, time delay sensors, volume sensors, weight sensors, and / or other types of sensors for measuring other parameters of the purification process may be additionally or optionally included.

[0296] The controller 390 can also be interactively connected to one or more actuators 397. For example, when connected to the clamping motor 338, it can control the movement of the clamping mechanism; when connected to the pump assembly 374, it can control the fluid movement within the purification column. The series of actuators 397 may also include one or more sealing actuators 391 and / or valve actuators 393. The sealing and valve actuators are used to selectively disrupt fluid seals in the purification column and control the opening and closing of valves in the purification column (this is described in more detail elsewhere herein). The series of actuators 397 may also include one or more mixing actuators 395. In a preferred embodiment, the mixing actuator includes a rotatable magnet positioned aligned with a corresponding magnetic stirring element disposed within the mixing chamber of the purification column.

[0297] The controller 390 can also be interactively connected to the communication hardware 394. The communication hardware 394 may include a router and / or other network hardware. The router and / or other network hardware is used to provide communication services for one or more of the following: a network (e.g., a local area network (LAN), a wide area network (WAN), a cloud network), an external server system, an external computer device, a distributed computer system, and a wired or wireless network. The communication hardware 394 may also include components that enable data upload or download via direct connection, such as an Ethernet port, a PCMCIA slot, or a Universal Serial Bus (USB) port.

[0298] In some embodiments, as part of a “smart” purification system, controller 390 is configured to automatically modify the purification protocol based on input information received from one or more sensors. For example, the measured optical density of the input sample can be used to adjust one or more operating parameters of the purification procedure. Such variable operating parameters may include, for example, the volume of one or more reagents moved by the instrument used in the purification procedure, the pumping time, and / or the pumping rate driven by the pumping assembly. For instance, a cell capture / filtration step is typically performed as the initial part of the purification procedure. When the optical density of the input sample is high, this cell capture / filtration step will be preferentially performed for a longer period. As another example, when the optical density of the input sample is high, a larger volume of one or more reagents may be preferentially used.

[0299] Furthermore, the control system 388 can communicate with a user inventory database to track the usage and availability of consumable products (e.g., buffers, reagents, purification columns) within the user's organization or laboratory. The control system or user inventory database can be associated with one or more rules. These rules can prompt the user when a threshold or trigger condition is met. For example, when available inventory falls below a threshold level, a rule can prompt the user to purchase additional consumables (or encourage a purchase). Importantly, any number or type of trigger conditions associated with the system described herein can be predetermined and / or modified to meet the needs or preferences of individual users.

[0300] As a further example Figure 20B An exemplary method 400 for performing a purification procedure is described. Method 400 can be implemented using a suitable computer control system, such as... Figure 20A The control system 388 is shown. In the method 400 shown, the control system can be used to receive sensor data from one or more sensors to determine one or more process states (step 402). The one or more sensors mentioned above may include... Figure 20AAny or all of the sensors 399 shown and / or other sensors described herein (e.g., temperature sensors, volume sensors, weight sensors, cartridge scanners (such as barcode or QR code scanners), etc.). Process status may include any measurement operations performed by such sensors. Furthermore, process status data may also include user-inputted and / or information obtained from a specific purification column, such as the type of purification column (e.g., anion exchange and precipitation or silica-based capture), sample type (e.g., cell culture, environmental, clinical, food, or forensic samples), etc.

[0301] The control system can then compare the determined process state with a range of different solutions stored in a solution library (step 404). The solution library may be stored in the memory 392a of the controller 390 and / or accessible via network communication between the controller 390 and one or more server systems, a "cloud" database, etc. The solution library includes different purification solutions related to different possible process states. For example, the solution library may include purification solutions associated with "low" initial optical density readings, different purification solutions associated with "medium" initial optical density readings, and different purification solutions associated with "high" initial optical density readings. Of course, the numerical values ​​of the different solutions are not necessarily limited to low / medium / high classifications. For example, the number of different solutions can be differentiated and categorized based on any desired granularity level numerical range.

[0302] Each individual purification protocol can define the process steps and parameters required to perform the purification process. For example, a purification protocol can define whether cell capture and / or cell lysis is used, the duration of cell capture and / or cell lysis, the reagents used, the amount of reagents used, whether and / or when a mixing process is performed in one or more purification steps, the duration and / or rate of one or more mixing steps, the pumping duration and / or rate between one or more purification steps, whether and / or when one or more valves in the purification column are opened and closed, whether and / or when one or more seals in the purification column are punctured (to allow appropriate liquid release or gas venting), the type of nucleic acid capture involved, the duration of nucleic acid capture, the type of protein to be captured, the duration of target protein capture, or a combination thereof.

[0303] Then, the control system can select a purification scheme from the scheme library based on the determined process state (step 406). For example, based on a specific set of determined process states, a purification scheme can be selected as its best match; or based on mutually exclusive corresponding process states, a purification scheme can be selected as its unique match from a series of purification schemes.

[0304] The control system then controls the actuators of one or more instruments to perform operations related to the selected purification protocol, based on the purification protocol selected by the operator (in step 408). The actuators of the one or more instruments mentioned herein may include... Figure 20A This refers to any or all of the actuators 397 shown. For example, if a specific stirring speed and duration are required in a certain process step according to the currently selected purification protocol, the controller 390 will drive the corresponding stirring actuator 395 to complete the part of the task specified in the selected purification protocol. In another example, if a specific amount of reagent needs to be used during the purification process, and this reagent needs to be added multiple times throughout the purification process, the controller 390 will control the closed actuators 391, pump assemblies 374, and / or valve actuators 393 associated with the process to complete the reagent addition operation along a specific pipeline path, according to the requirements of the selected purification protocol. In a third example, if the selected purification protocol requires a specific call capture duration (e.g., an estimate based on an initial optical density value), the controller 390 will correspondingly control the relevant closed actuators 391, pump assemblies 374, and / or valve actuators 393 as required, thereby ensuring that the duration of the call capture process meets the relevant requirements.

[0305] In some implementations, method 400 can be iterative. As indicated by arrow 410, while the selected purification protocol is being executed, controller 390 can continue to receive additional sensor data to process one or more updated process states. The system can then compare the updated process states with the contents of the protocol library and update / modify the executing purification protocol accordingly based on the latest acquired measurement results. This allows the system to re-optimize the purification procedure between each two steps based on the results of the previous process step. For example, if sensor data shows that the system has collected a small amount of lysate in the cell capture step, which is clearly inconsistent with the expected result based on the initial optical density value of the input sample, the system can update / modify the initially selected purification protocol to better adjust the amount and / or timing of reagent use, stirring operations, pumping operations, and other similar parameters in subsequent processes based on the amount of lysate detected.

[0306] The control system 388 of this disclosure may contain and use various computer systems, including dedicated or general-purpose computer hardware, or communicate with such computer systems. The control system 388 within the scope of this patent may also include physical and other forms of computer-readable media for carrying or storing various computer-executable instructions and / or data structures. The aforementioned computer-readable media may be any existing form of storage medium, and such storage medium should be accessible on various general-purpose or dedicated computer systems. The computer-readable medium for storing computer-executable instructions should be a physical storage medium (such as various hardware storage devices). The computer-readable medium for carrying computer-executable instructions should be a transmissible medium. Therefore, embodiments of this patent may include at least two completely different types of computer-readable media, for example, but not limited to, computer-readable hardware storage media and computer-readable media for data transmission.

[0307] The aforementioned computer-readable hardware storage media include random access memory, read-only memory, electrically erasable programmable read-only memory, read-only optical disc drives or other types of optical storage media (such as CDs, DVDs, etc.), disk storage or other magnetic storage devices, or any other medium that can be used to store the required program code, which may be computer-executable instructions or related data structures, and should be accessible and readable using a general-purpose or special-purpose computer system.

[0308] The term "network" in this disclosure refers to one or more data links used for the transmission of electronic data between multiple computer systems and / or modules and / or other electronic devices. When information is transmitted or provided to a computer via a network or other communication connection (which may be a wired connection, a wireless connection, or a hybrid wired-wireless network), the computer should treat that connection as a transmission medium. This transmission medium may include network connections and / or data links for carrying required program code, which may be computer-executable instructions or related data structures, and should be accessible and readable using general-purpose or special-purpose computer systems. Hybrid forms of the aforementioned transmission medium also fall within the scope of computer-readable media.

[0309] Furthermore, program code that exists in the form of computer-executable instructions or data structures and is transmitted to various computer system components can be automatically transferred by the system from a computer-readable transmission medium to a computer-readable hardware storage medium (and vice versa). For example, when a computer system receives computer-executable instructions via a network or data link, it caches them in the random access memory (RAM) of a network interface module (such as a network interface card) and ultimately transmits the instructions to the computer system's RAM and / or to a more secure computer-readable hardware storage medium within the computer system. Therefore, computer-readable hardware storage media belongs to the same category of components as other computer system components that also (or even primarily) use transmission media.

[0310] Computer-executable instructions include various instructions and data, such as instructions and data used to implement specific functions or sets of functions on general-purpose computers (target computing devices, etc.), special-purpose computers (sensors and / or target computing devices, etc.), or special-purpose processing devices. Computer-executable instructions can take various forms, such as binary files, intermediate format instructions such as assembly language, or even source code.

[0311] The functions described herein may be performed additionally or selectively, but at least some of the functions should be implemented on one or more hardware logic components. For example, illustrative hardware logic components that may be used include, but are not limited to, field-programmable gate arrays (FPGAs), application-specific integrated circuits (ASICs), application-specific standard products (ASSPs), microcontroller systems (SoCs), complex programmable logic control devices (CPLDs), etc.

[0312] Furthermore, the wireless communication between computer systems mentioned in this disclosure can be based on all wireless solutions within the art, including Bluetooth, wireless personal area networks, ultra-wideband (UWB), and wireless local area networks. Accordingly, it should be noted that even if a particular wireless solution (or corresponding component) has been specifically mentioned or described in all disclosed embodiments, we can still replace the specific wireless solution mentioned in the embodiments with any other wireless solution within the art, provided that the disclosed functions are achieved and / or similar functions are implemented or substantially similar tasks are performed.

[0313] Fluid seals and valve actuators

[0314] The following disclosure relates to exemplary embodiments of a target biomolecule purification column, such as a target nucleic acid purification column or a target protein purification column, which can be used to automate the purification of selected target biomolecules. Fluid seal and valve actuation embodiments can be used with other components described herein. For example, the seal and valve actuation components can be integrated with the consumable (e.g., disposable) purification columns mentioned above and used in devices such as the target biomolecule purification instruments mentioned above (e.g., target nucleic acid purification instruments or target protein purification instruments), and / or in one or more fluid release mechanisms described above. In preferred embodiments, the fluid seal, valve actuation, fluid lines and connections, fluid reservoirs, and membrane / filters are primarily used to reduce or remove leachates that may contaminate or covalently alter the purified biomolecules, particularly the purified target proteins.

[0315] Overview of Seals and Valve Actuation Devices

[0316] Traditional nucleic acid or protein purification procedures must be performed manually in a laboratory, requiring advanced laboratory skills and considerable time. Previous attempts to automate the purification process have largely focused on improving the sample-carrying purification column, resulting in the invention of rigid polymer purification columns. Because these columns require precise control of the fluid flow along a specific path at specific times, fluid mixing, and the control of fluid flow through the purification column to successfully complete the purification process, they are typically complex and costly, especially when dealing with large numbers of samples.

[0317] It is particularly important to note that different types of fluids must be sealed before mixing operations, meaning the fluids must be completely isolated from each other. Sealing and isolating these fluids becomes more challenging due to the need to move them along relatively complex paths and meet various time parameters. For example, pumping fluids along the desired path can create pressure differentials that may pressure the fluid seals designed to separate the fluid channels of the purification column. While conventional purification columns use seals that can withstand these pressure differentials to prevent leakage, this significantly increases the manufacturing cost of related components and / or the purification column itself. For instance, manufacturing longer fluid seals often requires complex ultrasonic welding between injection-molded parts. These additional costs and expenses make "consumable," "disposable," "single-use," or "disposable" purification columns uneconomical, even if such columns are readily available to users and offer advantages like ease of use and time savings.

[0318] Therefore, ensuring that a well-designed purification column guarantees effective fluid sealing and valve actuation performance presents numerous challenges. As will be described in many specific details below, the purification column mentioned in this paper can effectively address one or more of the challenges mentioned above by employing more efficient sealing and valve actuation devices. For example, as will be described in many specific details below, the purification column contains an elastomeric layer located between two relatively rigid outer layers. This elastomeric layer contains numerous sealing ribs that, when the elastomeric layer is compressed by the two outer layers, act as a fluid seal for the fluid channels formed in the outer layers.

[0319] This structural design utilizes cheaper materials and simpler manufacturing methods. For example, this purification column does not require a large fluid seal length, thus eliminating the need for lengthy and complex ultrasonic welding operations. By transferring pressure to external instruments or components, costs and expenses can be shifted from consumable purification columns (typically used only a limited number of times) to other devices used for longer periods.

[0320] This design offers an additional advantage: the elastomeric layer can also be used to achieve valve actuation. Therefore, all purification column valve bodies consist of a single component, whereas in traditional designs, each valve body requires its own independent component. These advantages, along with the structural components that enable them, will be described in detail below.

[0321] Sealing strip

[0322] Figure 21 The purification column 501 is illustrated schematically, showing a series of fluid channels formed within it due to numerous polymer sealing ribs 522. The sealing ribs 522 divide the purification column into different sections, thus limiting the flow of each fluid channel. As shown in the figure, some of the fluid channels formed by the sealing ribs ultimately constitute relatively narrow fluid paths 504, while others form relatively wide and larger fluid reservoirs 506. As used herein, a "reservoir" generally refers to a portion of a purification column used for storing, mixing, filtering, separating, and / or realizing fluid reactions, and the narrow channels formed between these reservoirs are primarily used for transporting fluid between them.

[0323] Figure 22 The image shown is a cross-sectional view of a purification column 501, which can be seen to be composed of multiple different layers, which further form various fluid channels. Figure 22The document also shows a cross-sectional view of fluid channel 524. The purification column cross-section shown here is vertical, and the purification column in the preferred embodiment is oriented the same way. However, it should be understood that the features described herein do not imply that all application instances should be vertical. In practice, other orientations may be used depending on specific application requirements and / or preferences.

[0324] The embodiment described includes a first outer layer 508. The first outer layer 508 consists of a first side 510 (e.g., an inner side) and a second side 512 (e.g., an outer side). A second outer layer 514 is arranged opposite to the first side 510 of the first outer layer 508. Similarly, the second outer layer 514 also consists of a first side 516 (e.g., an inner side) and a second side 518 (e.g., an outer side).

[0325] As shown in the figure, the first outer layer 510 may have outwardly projecting grooves or channels, thereby forming the channel wall 526 of the fluid channel 524. The cross-sectional shape of the fluid channel may be arc-shaped as shown in the examples, while its inner portion is typically wider than its outer portion (e.g., the bottom or "lowest point" of the fluid channel). In the embodiment shown herein, the channel wall 526 is convex, moving from the lowest point to a turning point 527 opposite to the lowest point. Other embodiments may include fluid channels with different cross-sectional shapes, including polygonal, for example, rectangular cross-sectional shapes.

[0326] Located between the first outer layer 510 and the second outer layer 514 is an elastomer layer 520. The elastomer layer 520 includes sealing rib structures 522. As shown, a pair of sealing ribs 522 extend toward and contact the first side 510 of the first outer layer 508, thereby positioning the sealing ribs 522 on either side of the fluid channel 524. The sealing ribs 522 thus define the width of the fluid channel 524 and restrict the "upward" and "downward" (from) fluid flow within the channel 524. Figure 22 (From the perspective of) movement. As shown in the figure, the sealing ribs 522 can be oriented such that the vertex (i.e., the innermost point) of each rib 522 corresponds to or exceeds the opposite inflection point 527 (relative to the lowest point of the channel).

[0327] In the illustrated embodiment, a nominal gap 521 may exist between the elastomeric layer 520 and the first outer layer 508 and / or the second outer layer 514. The nominal gap 521 provides space for the elastomeric material of the sealing strips 522 to deflect during compression. The sealing strips 522 are also preferably spaced slightly further from the fluid channel wall 526 so that, during compression, these sealing strips do not deflect and over-cover the fluid channel.

[0328] The elastomeric layer 520 can be formed from a variety of suitable elastomeric materials, including thermosetting elastomers and thermoplastic elastomers. Non-limiting examples of suitable elastomers include natural rubber, isoprene rubber, butadiene rubber, chloroprene rubber, butyl rubber, halogenated butyl rubbers such as chloroprene rubber and bromoprene rubber, styrene-butadiene rubber, nitrile rubber, ethylene propylene rubber, ethylene propylene rubber, epichlorohydrin rubber, polyacrylic rubber, silicone rubber, fluorosiloxanes, fluororubber, perfluororubber, polyether block amide (PEBA), chlorosulfonated polyethylene, ethylene-vinyl acetate, closed-cell foams, and combinations thereof. For example, the elastomeric layer can have a Young's modulus of about 5 to about 500 MPa, or about 10 to about 100 MPa.

[0329] Outer layers 508 and 514 can be formed from a variety of suitable materials. Outer layers 508 and 514 preferably have greater rigidity (e.g., a higher Young's modulus) than the elastomeric layer 520. Preferably, outer layers 508 and 514 are formed from polymeric materials that are readily manufactured in thermoforming processes. Non-limiting examples include polyethylene, polypropylene, polycarbonate, polyethylene terephthalate, polystyrene, polyvinyl chloride, other polymers, and combinations thereof. In general, outer layers 508 and 514 can have a Young's modulus of about 500 to about 4000 MPa.

[0330] Figure 22 The illustrated embodiment includes a fluid channel 524 formed in a first outer layer 508 and a sealing rib 522 extending toward the first outer layer 508. Although not shown here, the purification column may also include one or more fluid channels formed in a second outer layer 514 and associated sealing ribs extending from the elastomeric layer 520 toward the second outer layer 514.

[0331] When the elastomer layer 520 is compressed between the first and second outer layers 508 and 514, the sealing rib 522 is used to seal the fluid channel 524, thereby separating it from any other fluid channel. Figure 23A and 23B An example of a purification column 501 clamped between a first clamping plate 532 and a second clamping plate 534 is shown. Figure 23A Clamping plates 532 and 534 are shown before compression of purification column 501. Figure 23B The purification column 501 is shown to be completely compressed between clamping plates 532 and 534.

[0332] The clamping plates 532 and 534 are configured in size and shape such that they are located on both sides of the purification column 501 to allow compression of the sealing bone portion of the purification column 501 while preventing other portions, such as the reservoir 506, from being compressed. For example, the clamping plates 532 and 534 may include a hole 536 that allows most of the reservoir 506 to avoid compression, while the bottom sealing bone is positioned to accept compression.

[0333] The sealing rib structure is designed to withstand compression of at least approximately 500 lbf, or preferably up to approximately 15,000 lbf, applied over the entire length of the sealing rib structure. When compressed under these types of loads, the sealing rib may deflect by approximately 20% to approximately 30%, or more. Such compression has been shown to adequately maintain a sealing force of approximately 4.5 lbf per inch, capable of maintaining a seal under pressures exceeding 50 to 60 psi.

[0334] Compression can be provided by the clamping mechanism of the purification instrument, such as those described elsewhere herein. However, other embodiments may utilize other clamping mechanisms whose operation does not necessarily require the actuation of the purification instrument. For example, some embodiments may include a purification column having a consumable intermediate section and two reusable plates, the column being located in the intermediate section and tightened before use to compress the intermediate section.

[0335] Valve body

[0336] The purification column 501 may include multiple valves at different locations, which can be selectively opened and closed during the purification process to guide the flow of fluid. For example, one or more valves may be located near each reservoir of the purification column and used to control the inflow and / or outflow of fluid from the reservoir during purification.

[0337] Figure 24 An exemplary valve mechanism 540 is shown in cross-section. Regarding the valve component of valve mechanism 540, the valve component is advantageously formed by utilizing the same elastomer layer 520 used elsewhere in purification column 501 to form a sealing rib 522, thereby reducing the number of necessary components and simplifying the manufacture of the purification column.

[0338] As shown, the elastomeric layer 520 includes a deflectable portion 542 extending through a hole 528 formed in the second outer layer 514. The deflectable portion 542 is shown here as a "dome"-shaped structure. However, other embodiments may provide deflectable portions of other shapes. The deflectable portion 542 is biased outwards, such that an opening 530 is provided between the fluid channels 523 and 525 formed in the first outer layer 508.

[0339] In the open state, fluid 503 can freely pass through the channel 546 formed between fluid passages 523 and 525. The valve can be actuated and moved to the closed state when the plunger 505 or other suitable structure engages with the deflectable portion 542 and deflects it toward the channel 546. The side of the deflectable portion 542 facing the channel 546 may include a sealing rib 544 that can engage with the channel 546 under pressure from the plunger 505 to help close the opening 530 and seal the fluid passage 525 from the fluid passage 523. When the plunger 505 retracts, the deflectable portion 542 springs back to its normal geometry, thereby reopening the valve.

[0340] The deflectable portion 542 is preferably capable of remaining closed at pressures exceeding 30 psi, or even exceeding 50 to 60 psi. This translates to a force of approximately 5 lbf required on the deflectable portion 542 to hold it in the closed position. Therefore, the deflectable portion 542 should preferably be designed to withstand forces of approximately 3 to 7 lbf, and / or up to approximately 50 to 60 psi, without failing or “rupturing,” allowing fluid to pass through.

[0341] The plunger 505 may be part of a purification instrument in which the purification column 501 can be processed. For example, multiple plungers may be mounted on one or both clamps of the purification instrument described herein.

[0342] The cross-sectional shapes of the sealing strips 522 and 544, as shown in the foregoing examples, are semi-circular geometries (e.g., generally resembling O-ring geometry). Other embodiments may include other cross-sectional shapes, including other curved or polygonal shapes, such as triangles.

[0343] It should be understood that any processing systems, valves, seals, etc. disclosed above regarding any target biomolecule purification system and purification column can be utilized and adapted, where appropriate, to systems and purification columns specifically configured for the automated purification of target nucleic acids or target proteins.

[0344] Definition of Terminology Abbreviations

[0345] To aid in understanding the scope and content of this written description and the appended claims, some terms are defined directly below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0346] As used herein, the terms “approximately,” “about,” and “roughly” mean a quantity or condition that is close to the specifically stated quantity or condition, which still performs the desired function or achieves the desired result. For example, the terms “approximately,” “about,” and “roughly” may refer to a quantity or condition that differs from the specifically stated quantity or condition by less than 10%, or less than 5%, or less than 1%, or less than 0.1%, or less than 0.01%.

[0347] The term “buffer” in this context refers to any suitable buffer, wash buffer, resuspension buffer, lysis buffer, neutralization buffer, RNase (or other enzyme), binding buffer / solution, elution / collection buffer, or precipitation buffer that may be used with or without any additional suitable reagents, depending on the protocol being performed. Suitable buffers, their composition, and methods of use are disclosed in the following U.S. Patent references: USPat. No. / USPat. Publication Date: January 26, 2012, No. 6,914,137, No. 2006 / 0154247, No. 2007 / 0117972, No. 6,242,220, No. 5,990,301, No. 7,214,508, No. 7,109,322, and No. 6,297,371, all of which are incorporated herein by reference in their entirety.

[0348] The exemplary resuspension buffers of the systems and methods described herein may include any suitable biologically acceptable buffer, such as Tris, TAPS, Bicine, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES, acetate, or the like, with a pH value between about 3.5 and about 9, about 5 and about 8, or about 6.5 and about 7.5. Furthermore, the resuspension buffer may include 1 mM to 100 mM, 5 mM to 50 mM, or 10 mM to 20 mM chelating agents, such as EDTA, EGTA, ALA, BAPTA, defarasirox, deferiprone, deferoxamine, DTPA, dimercaprol, DMPS, DMSA, or the like. The resuspension buffer optionally includes a ribonuclease, such as one or more ribonucleases selected from any one or more ribonucleases: ribonuclease A, ribonuclease H, ribonuclease I, ribonuclease III, ribonuclease L, ribonuclease P, ribonuclease PhyM, ribonuclease T1, ribonuclease T2, ribonuclease U2, ribonuclease VI, ribonuclease V, ribonuclease, ribonuclease PEI, ribonuclease II, ribonuclease R, ribonuclease D, ribonuclease T, exonuclease I, exonuclease II, etc. In some embodiments, the resuspension buffer optionally contains lysozyme or a carbohydrate (e.g., a sugar) between about 1 mM and about 500 mM, about 10 mM and about 200 mM, about 20 mM and about 100 mM, or about 30 mM and about 75 mM. Typical sugars include glucose, fructose, galactose, mannose, maltose, and lactose.

[0349] For example, the resuspension buffer can be an aqueous solution containing 50 mM Tris-HCl (pH 8.0) and 10 mM EDTA. In another embodiment, the resuspension buffer can be an aqueous solution containing 50 mM Tris-HCl (pH 8.0), 2.4 mg / ml RNase A, and 10 mM EDTA. In another non-limiting example, the resuspension buffer can be an aqueous solution containing 50 mM Tris (pH 7.4), 100 pg / ml RNase A1, 10 mM EDTA, and 5 mM glucose.

[0350] For lysis solutions or buffers, suitable lysis solutions or buffers may include a combination of one or more denaturants and one or more lipid disruptors in an aqueous carrier medium. Denaturants may be nucleic acid denaturants, such as basic salts. Suitable basic salts include sodium hydroxide, potassium hydroxide, and calcium hydroxide. Suitable lipid disruptors include ionic surfactants. Typical ionic surfactants include sodium cholate, sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC), n-lauroyl sarcosinate, hexadecyltrimethyl bromide (CTAB), and bis(2-ethylhexyl)sulfonate succinate.

[0351] As a non-limiting example, the lysis buffer used herein may be an aqueous solution containing 1% (v / v) sodium dodecyl sulfate (SDS) and 200 mM sodium hydroxide. As another non-limiting example, a typical lysis solution may be an aqueous solution containing about 10 mM to about 500 mM, about 50 mM to about 250 mM, about 100 mM to about 200 mM NaOH and up to about 10% (v / v) SDS, up to about 5% (v / v) SDS, or up to about 1% (v / v) sodium dodecyl sulfate (SDS). Additional reagents may be present in the lysis buffer, which will be readily identifiable by a skilled person.

[0352] For a neutralizing solution, a suitable neutralizing solution refers to one or more agents capable of neutralizing surfactants or alkaline solutions in a suitable aqueous carrier medium. In some embodiments, the neutralizing solution contains about 0.5 M to about 5 M of acetate, with a pH greater than 4. A typical neutralizing solution includes a 3.1 M aqueous solution of potassium acetate with a pH of about 5.5.

[0353] In some embodiments, such as protein purification, non-ionizing buffers and reagents that do not destroy protein structure and function can be used.

[0354] A suitable wash buffer refers to a salt of 0.1 mM to about 100 mM, a suitable biological buffer of about 0.5 mM to about 500 mM, in a suitable aqueous carrier medium, such that the pH of the wash buffer is at least about 6.0 or higher, and at least 5% (v / v), at least 10% (v / v), or at least 15% (v / v) alcohol, such as ethanol or isopropanol. In an exemplary embodiment, the wash buffer may be an aqueous solution with a pH of 5.0 containing 812.5 mM sodium chloride (NaCl) and 100 mM sodium acetate trihydrate. In another non-limiting example, the wash buffer may be a solution with a pH of 5.0 comprising 1.5 mM sodium chloride and 100 mM sodium acetate trihydrate. Optionally, the wash buffer contains about 0.01% (v / v) to up to about 10% (v / v) of a nonionic detergent, such as... X-100, amphoteric surfactants (CHAPS), or NP-40. In some embodiments, the addition of such detergents enhances the removal of endotoxins in the formulation. Therefore, a typical endotoxin removal or washing buffer may contain 10% (v / v) of such detergents. X-100, 750 mM sodium chloride (NaCl), and 50 mM zwitterionic buffer (MOPS) at pH 7.0. As another non-limiting example, an endotoxin removal or washing buffer contains 1 M sodium chloride (NaCl), 50 mM zwitterionic buffer (MOPS), pH > 8.0, 15% (v / v) isopropanol, and 0.5% (v / v) isopropanol. X-100.

[0355] In some embodiments, ultrapure water or distilled water can be used to elute target nucleic acids. Alternatively, TE buffer or elution buffer can be used. TE buffer comprises an aqueous carrier medium containing 10 mM tris(hydroxymethyl)aminomethane (Tris) and 0.1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0. Elution buffer comprises salts in a suitable aqueous carrier medium, up to about 150 mM of biological buffer at pH 5.0–9.0, and up to about 10 mM of chelating agent. Typical elution buffers comprise 100 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl) and 1250 mM sodium chloride (NaCl) at pH 8.5. Another non-limiting example of an elution buffer includes a combination of 10 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl) and 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0.

[0356] In some embodiments, the equilibration buffer may optionally be passed through a membrane or filter used in any of the first or second biological processing components prior to use. In these embodiments, the equilibration buffer comprises 1 M salt, 500 mM biological buffer with a pH between 5.0 and 9.0, about 10% (v / v) nonionic detergent, and about 20% (v / v) ethanol. Typical equilibration buffers include, for example, 750 mM sodium chloride (NaCl), 50 mM zwitterionic buffer (MOPS) at pH 7, 15% (v / v) isopropanol, and about 0.15% (v / v) ethanol. X-100.

[0357] In some embodiments, the precipitation buffer may optionally be passed through a precipitation filter before use. The precipitation buffer comprises up to 5 M potassium acetate and up to 500 M biological buffer with a pH between approximately 5.0 and 9.0. A typical precipitation buffer comprises 3.1 M potassium acetate at pH 5.5.

[0358] As used herein, the term "large volume" includes any volume greater than about 5 mL, and particularly any volume greater than about 10 mL or a volume range between 10 mL and 5 L. The term "large volume" as used herein should be understood to generally refer to biological samples, including up to 250 mL, 10–250 mL, 50–200 mL, 100–150 mL, 100 mL–2 L, or any range or value between the two. For example, plasmid DNA can be purified from large volumes of bacterial cultures using the disclosed systems, methods, and apparatus with a large volume of 150 mL. As an additional, non-limiting example, large volume includes up to 1 L of urine. It should be noted that the systems, methods, and apparatus disclosed herein can be suitably adapted to process biological samples of different volumes, including smaller volumes than those described above.

[0359] The term “target biomolecule” as used in this article is intended to be understood as a nucleic acid or protein derived from a biological or environmental source (as defined in this article).

[0360] 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 can be one or more of a gene, regulatory sequence, genomic DNA, plasmid DNA, cDNA, or RNA. As described herein, the target nucleic acid can take any of the foregoing forms and have any length, although in preferred embodiments, the target nucleic acid constitutes a plasmid obtained from a bacterial culture.

[0361] As used herein, the term "target protein" is intended to be understood as a protein sequence of biological or environmental origin. This sequence can be a polypeptide, oligopeptide, glycoprotein, lipoprotein, phosphoprotein, membrane protein, or any protein form. As outlined herein, target proteins can take any of the aforementioned protein forms and can be of any length, although in preferred embodiments, the target protein remains in its biologically active state and structure, which may include natural post-translational modifications and may be uncontaminated or chemically unmodified.

[0362] Various aspects of this disclosure, including apparatus, systems, and methods, may be illustrated with reference to one or more embodiments or implementations that are exemplary in nature. As used herein, the term "exemplary" means "serving as an example, instance, or illustration" and is not necessarily to be construed as being preferred or more advantageous than other disclosed embodiments. Furthermore, references to "implementation" of this disclosure or the invention include specific references to one or more embodiments thereof, and are intended to provide illustrative examples without limiting the scope of the invention (as determined by the appended claims rather than by the following description).

[0363] As used in this specification, words appearing in the singular form include their plural counterparts, and words appearing in the plural form include their singular counterparts, unless otherwise implied or explicit. Therefore, it should be noted that, as used in this specification and the appended claims, the singular forms “an,” “a,” and “the” include plural references unless the context explicitly specifies otherwise. For example, a reference to a singular reference (e.g., “widget”) includes one, two, or more references, unless otherwise implied or explicit. Similarly, unless the content and / or context explicitly indicate otherwise, a reference to multiple references should be interpreted as including a single reference and / or multiple references. For example, a reference to a plural reference (e.g., “widget”) does not necessarily require multiple such references. Rather, it is understood that, regardless of the inferred number of references, one or more references herein are estimated unless otherwise stated.

[0364] As used herein, directional terms such as “top,” “bottom,” “left,” “right,” “up,” “down,” “upper,” “lower,” “proximal,” and “distal” are used only to indicate relative directions and not to limit the scope of this disclosure and / or the invention claimed herein.

[0365] in conclusion

[0366] The terms and expressions used herein are descriptive rather than restrictive, and their use is not intended to exclude any equivalents of the features or portions shown and described, but various modifications are recognized within the scope of the claimed invention. Therefore, it should be understood that while preferred embodiments, exemplary embodiments, and optional features have been explicitly disclosed in part, modifications and variations of the concepts disclosed herein can be adopted by 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 and various variations and / or modifications of the inventive features shown herein, and the principles shown and followed in this disclosure (which may be applicable to those skilled in the art) can be applied to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the claims, and are considered to be within the scope of this disclosure.

[0367] It should also be recognized that, according to certain embodiments of this disclosure, systems, devices, products, kits, methods, and / or processes may include, contain, or otherwise incorporate the properties or features (e.g., components, members, elements, parts, and / or portions) described in other embodiments disclosed and / or described herein. Therefore, various features of certain embodiments may be compatible with, combined with, cover, and / or incorporated into other embodiments of this disclosure. Consequently, the disclosure of certain features related to specific embodiments of this disclosure should not be construed as limiting the application of or inclusion of said features in particular embodiments. Rather, it should be recognized that other embodiments may also include said features, members, elements, parts, and / or portions without necessarily departing from the scope of this disclosure.

[0368] Furthermore, unless a feature is described as needing to be combined with another feature, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. Moreover, to avoid obscuring aspects of exemplary embodiments, well-known aspects of illustrative systems, methods, devices, etc., are not described in detail herein. However, these aspects are also contemplated herein.

[0369] Overall, all references cited in this application are compiled together, provided that they do not contradict the disclosures herein. It will be apparent to those skilled in the art that methods, apparatuses, apparatus elements, materials, procedures, and techniques not specifically described herein can be applied to the practice of the invention as broadly disclosed herein without the need for excessive experimentation. All known functional equivalents of the methods, apparatuses, apparatus elements, materials, procedures, and techniques specifically described herein are intended to be included in this invention.

[0370] When a group of materials, ingredients, components, or compounds is disclosed herein, it should be understood that all such individual items and all their sub-items are disclosed separately. When the Markush group or other groupings are used herein, all individual items of the group, as well as all possible combinations and sub-combinations, are intended to be individually included in this disclosure. Unless otherwise stated, formulations or combinations of each component described or illustrated herein may be used to practice the invention. Whenever a range is given in the specification, such as a temperature range, time range, or component range, all intermediate ranges and sub-ranges, as well as all individual values ​​contained within a given range, are intended to be included in the invention.

[0371] All modifications within the equivalent meaning and scope of the claims should be included within their scope.

[0372] Example

[0373] The following examples are provided to illustrate implementation of the various embodiments disclosed herein and are intended to be exemplary, and necessarily limit the scope and / or content of the disclosure herein.

[0374] Example 1

[0375] The instrument and accompanying disposable purification column disclosed herein are used directly from bacterial cultures for a fully automated plasmid DNA purification protocol.

[0376] To culture bacteria using a target nucleic acid (e.g., plasmid DNA), a single colony containing the relevant plasmid is selected from a rigorously screened fresh streak plate and inoculated with 1 ml of starter culture in LB medium (containing an appropriate selective antibiotic, ampicillin in this case). The culture is incubated at 37°C with vigorous shaking (300 rpm) for 8 hours. Next, the starter culture is diluted 1:1000 in LB medium containing an appropriate selective antibiotic in a 5 L flask. 1 L of the culture is incubated at 37°C for 14 hours with vigorous shaking (300 rpm) to promote bacterial growth and amplification. The bacterial culture is then examined to ensure it reaches approximately 2–6 x 10⁻⁶ at 600 nm (A600). 9 Cells were selected at a density of 1 cell / mL or an absorbance of 2.0–6.0, and then plasmid DNA was purified.

[0377] The bacterial cells used in this specific case were the top 10 and DH5α Escherichia coli, but other E. coli strains have been used in additional studies that yielded similar results. The plasmid used in this specific case was the high-copy pGL 4.50, but other plasmids with different backbones and different sized inserts, both high-copy and low-copy, have yielded similar results in similar experiments.

[0378] The instrument prototype and the accompanying disposable purification column prototype (e.g., purification column 160b in Figure 7b) are used for fully automated plasmid DNA purification from 150 ml (maximum scale) bacterial cultures. PureLink TM The Expi endotoxin-free Maxi plasmid purification kit was used side-by-side as a control, strictly following the manufacturer's instructions.

[0379] Figure 1The results of three instrument runs and one manual preparation using the PureLink kit for the pGL4.50 plasmid / first 10 cells are shown, with a culture OD600 of 3.0. Three instrument runs and one manual preparation using the PureLink kit for the pGL4.50 plasmid / DH5a cells resulted in a culture OD600 of 4.5. Table 1 below records the plasmid DNA equivalent (µg), plasmid concentration in approximately 1 ml of eluted plasmid in the output tube, and plasmid purity measured using nanodroplets (A260 / 280, A260 / 230). Agarose gel analysis of the plasmid products is shown below. Figure 25 As shown.

[0380] Table 1

[0381]

[0382] For pGL4.50 plasmid / first 10 cells, the automatically purified plasmid equivalent was 755–803 μg, and for pGL4.50 plasmid / DH5a cells, it was 881–982 μg, very similar to manual preparation using a PureLink anion exchange column-based kit. A260 / 280 purity was high for all samples (e.g., greater than 1.9). A260 / 230 purity was high for all samples (e.g., greater than 2.2). Agarose gel analysis showed high purity and good integrity of the purified plasmid products.

[0383] Table 2 below shows the results of a total of 25 plasmid purifications on two instrument prototypes. The average equivalent of high-copy plasmids obtained from 150 ml of infused bacterial culture was 796 μg, comparable to manual maxi-prep. Plasmid purity is as follows: Figure 25 The requirements are met. The total run time of the instrument prototype is approximately 50 minutes, significantly faster than manual preparation, which requires at least 1.5 hours or more for a standard anion exchange membrane column.

[0384] Table 2: Summary of a total of 25 plasmid purifications performed on purification columns 1 and 2 (each purification column was designed as follows). Figure 7B As shown; results were entered based on 150 ml of bacterial culture.

[0385]

[0386] As shown in the figure, the instrument prototype and the accompanying purification column prototype enable fully automated plasmid DNA purification, directly from bacterial cultures (without centrifugation), and provide high yields of clean plasmid DNA, significantly faster than "classic" manual purification tools.

[0387] Additional Implementation Examples

[0388] The following are exemplary, non-limiting embodiments that include one or more of the features described above.

[0389] Example 1: An apparatus for automatically purifying biomolecules such as target nucleic acids or target proteins from biological samples, comprising: an input reservoir for containing biological samples; a first bioprocessing component in fluid communication with the input reservoir and a lysis buffer reservoir, the first bioprocessing component being used to generate lysates containing target biomolecules such as target nucleic acids or target proteins; a second bioprocessing component in fluid communication with the first bioprocessing component and a first elution buffer reservoir, the second bioprocessing component including a target biomolecule binding filter for retaining target biomolecules, such as a target nucleic acid binding filter or a target protein binding filter; and a container in fluid communication with the second bioprocessing component, the container being used to hold an output container receiving target biomolecules such as target nucleic acids or target proteins from the second bioprocessing component.

[0390] Example 2. The apparatus according to Example 1, wherein the apparatus comprises a consumable purification column.

[0391] Example 3. The device according to any one of Examples 1-2, wherein the input reservoir is used to accommodate large-capacity clinical samples, large-volume environmental samples, food samples, or beverage samples.

[0392] Example 4. The device according to any one of Examples 1-3 further includes a light density detector window disposed between the input storage tank and the first biological processing component, through which the light density of the biological sample can be detected.

[0393] Example 5. The device according to any one of Examples 1-4, wherein the first biological treatment component includes a clarification filter.

[0394] Example 6. According to the device described in Example 5, the clarification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the clarification filter is used to separate the target nucleic acid portion of the biological sample from the first waste portion of the biological sample.

[0395] Example 7. According to the device described in Example 5 or 6, the first biological treatment component includes a cell capture filter disposed upstream of the clarification filter.

[0396] Example 8. In the device according to Example 7, the cell capture or concentration filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the cell capture or concentration filter is used to separate the target nucleic acid portion of the biological sample from the first waste portion of the biological sample.

[0397] Example 9. According to the device described in Example 7 or 8, the lysis buffer reservoir is fluidly connected to the cell capture filter, enabling the cell capture filter to be backwashed and allowing the backwash solution to enter the clarifying filter.

[0398] Example 10. The device according to Example 9 further includes a first mixing chamber disposed between the clarifying filter and the cell capture filter, the first mixing chamber being used to contain the backwash solution.

[0399] Example 11. The apparatus according to Example 10 further includes a neutralization buffer reservoir in fluid communication with the first mixing chamber, the first mixing chamber being used to contain the backwash liquid and the neutralization buffer, and to mix the backwash liquid and the neutralization buffer to form a neutralized lysate.

[0400] Example 12. According to the device described in Example 11, the clarification filter is in fluid communication with the first mixing chamber, and the clarification filter is used to separate the second waste portion and the target nucleic acid-containing portion of the biological sample.

[0401] Example 13. The apparatus according to any one of Examples 9-12, wherein the clarifying filter includes the cell capture filter for concentrating the cellular components of the biological sample in a first purification step and clarifying the neutralized lysate in a subsequent second purification step.

[0402] Example 14. The device according to any one of Examples 1-13, wherein the target biomolecule binding filter of the second bioprocessing component comprises a silicon-based filter or a microsphere column having affinity for the target nucleic acid.

[0403] Example 15. According to the device described in Example 14, the second biological processing component further includes a purification reagent reservoir in fluid communication with the target biomolecule binding filter (e.g., the nucleic acid binding filter).

[0404] Example 16. The device according to Example 14 or 15, wherein the target biomolecule binding filter, such as the nucleic acid binding filter, is in fluid communication with the elution buffer reservoir and the output container.

[0405] Example 17. The device according to any one of Examples 1-16, wherein the second bioprocessing component includes a second mixing chamber disposed between the first bioprocessing component and the target biomolecule binding filter.

[0406] Example 18. According to the device described in Example 17, the second mixing chamber is in fluid communication with the endotoxin removal buffer reservoir.

[0407] Example 19. The device according to any one of Examples 1-18, wherein the target biomolecule binding filter is a nucleic acid binding filter comprising an anion exchange membrane.

[0408] Example 20. The device according to Example 19, wherein the second biological treatment component includes a precipitation membrane disposed downstream of the anion exchange membrane.

[0409] Example 21. According to the device described in Example 20, the anion exchange membrane is used to separate the third waste portion of the biological sample from the target nucleic acid-containing portion.

[0410] Example 22. The device according to Example 20 or 21 further includes a second elution buffer reservoir, wherein the first elution buffer reservoir is fluidly connected to the anion exchange membrane, allowing the target nucleic acid to be eluted through the anion exchange membrane, and the second elution buffer reservoir is fluidly connected to the precipitation membrane, allowing the target nucleic acid to be eluted through the precipitation membrane.

[0411] Example 23. The apparatus according to any one of Examples 20-22, wherein the precipitation membrane is used to separate the fourth waste portion of the biological sample from the target nucleic acid-containing portion.

[0412] Example 24: The apparatus according to any one of Examples 20-23, wherein the precipitating membrane is in fluid communication with a precipitant storage tank, the precipitant storage tank optionally containing isopropanol.

[0413] Example 25: The apparatus according to any one of Examples 20-24, wherein the precipitated membrane is in fluid communication with a washing / desalination solution storage tank, the washing / desalination solution storage tank optionally containing about 70% ethanol.

[0414] Example 26: The device according to any one of Examples 20-25, wherein the second biological treatment component includes a third mixing chamber disposed between and in fluid communication with the anion exchange membrane and the precipitation membrane, wherein the third mixing chamber is fluidly connected to the precipitant storage tank and / or the desalination solution storage tank, and is disposed between the precipitation membrane and the precipitant storage tank and / or the desalination solution storage tank.

[0415] Example 27. The device according to any one of Examples 1-26, wherein the output container is selectively detachable from the device.

[0416] Example 28 The device according to any one of Examples 1-27, wherein the size and shape of the input reservoir are suitable for receiving biological samples of at least 5 mL, preferably at least 100 mL or up to 2 L.

[0417] Example 29. An embodiment of an apparatus for automatically purifying nucleic acids from a biological sample includes: an input reservoir for containing the biological sample; a first bioprocessing component and a lysis buffer reservoir in fluid communication with the input reservoir, the first bioprocessing component being used to generate lysates containing target nucleic acids; a second bioprocessing component in fluid communication with the first bioprocessing component and the first elution buffer reservoir, the second bioprocessing component including a nucleic acid binding filter for retaining the target nucleic acids; and a container in fluid communication with the second bioprocessing component for holding an output container containing purified target nucleic acids. Another embodiment of an apparatus for automatically purifying target nucleic acids from a biological sample includes: a first bioprocessing component for containing the biological sample, the first bioprocessing component including a waste separation filter and a plurality of reservoirs fluidly connected to the waste separation filter; a second bioprocessing component including an anion exchange membrane, a washing solution reservoir fluidly connected to the anion exchange membrane, and a first elution buffer reservoir fluidly coupled to the anion exchange membrane; and a third bioprocessing component including a precipitation filter and a second elution buffer reservoir fluidly connected to the precipitation filter.

[0418] Example 30: The device according to Example 29, wherein the first biological processing component further includes a cell capture filter, and the first of the plurality of reservoirs includes a resuspension buffer reservoir.

[0419] Example 31. The device according to Example 30, wherein the input reservoir is fluidly connected to a first side of the cell capture filter, and the resuspension buffer reservoir is fluidly coupled to a second side of the cell capture filter.

[0420] Example 32. An apparatus according to any one of Examples 1-31, wherein the apparatus is a consumable purification column used in an automated nucleic acid purification system, the consumable purification column being associated with the automated nucleic acid purification system to achieve automated purification of the target nucleic acid without human interaction.

[0421] Example 33. A method for automatically purifying a target nucleic acid from a biological sample, comprising: receiving the biological sample in an input reservoir; generating a lysate containing the target nucleic acid from the biological sample in a first biological processing component without further human interaction; receiving the target nucleic acid containing a portion of the lysate in a second biological processing component; retaining the target nucleic acid on a nucleic acid binding filter in the second biological processing component; and eluting the purified form of the target nucleic acid from the nucleic acid binding filter into an output container.

[0422] Example 34. The method of Example 33 further includes capturing the cellular contents of the biological sample at a first membrane of the first biological processing component.

[0423] Example 35. The method of Example 33 or 34 further includes measuring the optical density of the biological sample prior to generating the lysate, optionally wherein the optical density is measured prior to capturing the cell contents at the first membrane.

[0424] Example 36. The method according to Example 35, wherein the biological sample comprises a bacterial culture, and the optical density measurement of the biological sample comprises automatically measuring the absorbance of the bacterial culture at approximately 600 nm.

[0425] Example 37. The method of any of the embodiments of Examples 33-36 further includes resuspending at least a portion of the cell contents in one or more resuspension buffer solutions, ribonuclease solutions, or lysis buffers. Optionally, resuspending the at least a portion of the cell contents includes backwashing the first membrane.

[0426] Example 38. According to the method of Example 37, the process of backwashing the first membrane includes transferring a resuspension solution containing one or more resuspension buffers, ribonuclease solutions, or lysis buffers from a fluid channel disposed on a second side of the first membrane and through the first membrane.

[0427] Example 39. The method described in any of Examples 33-38 further includes mixing the lysate with a neutralization buffer to form a neutralized lysate, and separating the portion containing the target nucleic acid from the waste portion of the neutralized lysate.

[0428] Example 40. The method according to any of Examples 33-39 further includes mixing the endotoxin removal buffer with the target nucleic acid fraction of the lysate.

[0429] Example 41. The method according to any of Examples 33-40, wherein retaining the target nucleic acid on the nucleic acid binding filter at the second biological processing component includes passing the target nucleic acid-containing portion of the lysate through an anion exchange membrane.

[0430] Example 42. The method according to Example 41 further includes removing the target nucleic acid portion of the lysate 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 on the precipitation membrane.

[0431] Example 43. The method according to any of Examples 33-42, wherein retaining the target nucleic acid on the nucleic acid binding filter at the second biological processing component includes passing the target nucleic acid-containing portion of the lysate through a precipitation membrane, thereby capturing the target nucleic acid at the precipitation membrane.

[0432] Example 44. The method according to any of Examples 33-43, wherein retaining the target nucleic acid on the nucleic acid binding filter at the second biological processing component includes passing the target nucleic acid-containing portion of the lysate through a silicon-based filter, mixing the target nucleic acid-containing portion of the lysate with a dissociation salt buffer before passing the target nucleic acid-containing portion of the lysate through the silicon-based filter, and optionally washing the silicon-based filter with an alcohol-based washing solution.

[0433] Example 45. The method according to Example 44, wherein eluting the purified form of the target nucleic acid from the nucleic acid binding filter includes elution with water or TE buffer under low-salt conditions.

[0434] Example 46. The method described in any of Examples 33-45 is implemented using the device described in any of Examples 1-32.

[0435] Example 47. An apparatus for automatically purifying target nucleic acids or target proteins from biological samples, comprising: a housing having an internal compartment sized and shaped to accommodate a purification column; optionally, a selectively closable access door providing access to the internal compartment; and a pump assembly disposed within the internal chamber for providing pumping action via peristaltic movement.

[0436] Example 48. The device according to Example 47 further includes a clamping mechanism disposed in the internal compartment, the clamping mechanism being configured to move between an open position and a closed position. When in the open position, it can enter the internal compartment; when in the closed position, the clamping mechanism clamps onto the inserted purification column, enabling the biological sample to be processed. Optionally, when in the closed position, the clamping mechanism operates to fluidly seal the biological sample purification column.

[0437] Example 49. The device according to Example 48 further includes a controller, wherein the controller supplies power to the clamping mechanism only when it is determined that the access door is closed.

[0438] Example 50. The device according to Example 48 or 49 further includes a locking mechanism for locking the access door in the closed position when the clamping mechanism is moved to the closed position.

[0439] Example 51. The device according to any of Examples 48-50, wherein the clamping mechanism is powered by a motor, optionally wherein the motor is operatively connected to a worm gear or drive screw, the worm gear or drive screw being rotatable to cause movement of the clamping mechanism.

[0440] Example 52. The device according to Example 51 further includes a manual release mechanism operably connected to the clamping mechanism, enabling the clamping mechanism to be manually released independently of the motor.

[0441] Example 53. The device according to any of Examples 48-52, wherein the housing includes an open end to which an access door is connected and a closed end opposite to the open end, the open end and the closed end defining a longitudinal direction, wherein the clamping mechanism is configured to move in a direction transverse to the longitudinal direction.

[0442] Example 54. The device according to any of Examples 47-53 further includes: an optical density sensor for measuring the optical density of a biological sample placed in a biological sample purification column; and an optical density sensor for measuring the concentration of target biomolecules, such as the concentration of target nucleic acids and / or target proteins of the purified product.

[0443] Example 55. The device according to any of Examples 47-54 further includes a user interface for displaying instrument information and receiving user input, the user interface being used to accommodate user input related to one or more biological sample volumes, selected purification protocols, operating instructions for selectively closing maintenance doors, desired concentrations of target biomolecules such as target nucleic acids or target proteins, or the final volume of eluent containing target biomolecules.

[0444] Example 56. The device according to any of Examples 47-55, wherein the inner compartment includes a position sensor for determining that the purification column has been fully inserted into the inner compartment, and the limit switch is positioned such that it is actuated when the purification column is in contact with the leading edge of the purification column when it is fully inserted into the inner compartment.

[0445] Example 57. The device according to any of Examples 47-56 further includes one or more access door sensors for determining whether the access door is in an open or closed position, wherein the one or more access door sensors optionally include one or more Hall effect sensors and one or more corresponding magnets.

[0446] Example 58. The device according to any of Examples 47-57 further includes an output container sensor for detecting the presence of an output container at the insertion of the purification column.

[0447] Example 59. The device according to any of the embodiments 47-58 further includes one or more rotatable magnets configured to direct an electromagnetic field inward to the internal compartment.

[0448] Example 60. The device according to any of Examples 47-59, wherein the pump assembly includes a camshaft coupled to a motor via a power transmission assembly, a plurality of cam elements attached to the camshaft and extending laterally from the camshaft toward the purification column upon insertion, the cam elements optionally including beveled tips for engaging the purification column upon insertion.

[0449] Example 61. The device according to Example 60, wherein the arrangement of the cam elements causes the rotation of the cam shaft to induce a linear peristaltic motion at the tip of the cam elements.

[0450] Example 62. A system comprising the apparatus and purification column according to any one of Examples 47-62, wherein the purification column is the apparatus according to any one of Examples 1-32.

[0451] Example 63. A method for automatically purifying target nucleic acids from biological samples, comprising: providing an apparatus (or system) as described in any of Examples 47-62; loading a purification column into an internal compartment of the apparatus through an access door; and initiating an apparatus purification program, wherein the initiation of the purification program enables the instrument to automatically purify the target nucleic acid without further human interaction.

[0452] Example 64. The method according to Example 63 further includes closing the equipment access door and moving the clamping mechanism to the closed position to clamp the loaded purification column and fluid-tighten it.

[0453] Example 65. The method according to Example 64 further includes determining that the purification column is fully loaded and / or that the access door is completely closed before starting the purification process.

[0454] Example 66. The method according to any of Examples 63-65 further includes determining whether the position of the output container is correct and providing a warning when it is determined that there is no output container.

[0455] Example 67. The method according to any of Examples 63-66 further includes determining the optical density of the biological sample within the purification column, optionally wherein the determined optical density of the input sample is used to adjust one or more parameters of the purification procedure, said one or more parameters including the volume of one or more reagents used in the purification procedure, the duration of pumping through the pump assembly, or the rate of pumping through the pump assembly.

[0456] Example 68. According to the method of Example 67, a higher optical density reading, relative to a lower optical density reading, requires one or more of the following parameters: a larger volume of one or more reagents used, a longer pumping time for one or more fluid motion steps, and a higher fluid flow rate during one or more fluid motion steps.

[0457] Example 69. The method according to any of Examples 63-68, wherein an initial optical density reading is obtained before starting the purification procedure to determine whether the purification column has been used before, wherein the optical density reading is substantially equal to the air blank reading, indicating that the culture input reservoir of the biological sample purification column is intact.

[0458] Example 70. The method according to any of Examples 63-69 further includes loading a large volume of biological sample, approximately 5 ml to 5 L, 10 ml to 500 ml, or 15 ml to 250 ml, into a purification column.

[0459] Example 71. A control system for controlling an automated purification apparatus (or system) according to any of Examples 47-62, comprising: one or more processors; and one or more hardware storage devices having computer-executable instructions thereon, which can be executed by the one or more processors to cause the control system to at least: receive sensor data from one or more sensors to determine one or more process states of the automated purification apparatus; compare the determined process states with a set of different protocols stored in a protocol library; select a purification protocol from the protocol library based on the determined process states; and implement the selected purification protocol by causing one or more instrument actuators of the automated purification apparatus to operate according to the selected purification protocol.

[0460] Example 72. The control system according to Example 71, wherein one or more sensors include an optical density sensor, a door sensor, a purification column proximity or contact sensor, an output container proximity or contact sensor, a purification column scanner, a timer, a temperature sensor, a weight sensor, a volume sensor, or a combination thereof.

[0461] Example 73. The control system according to Example 71 or 72, wherein the determined process state includes at least a determined optical density of the initial input sample, and optionally, wherein the selected purification protocol provides a cell capture duration based on the determined optical density of the initial input sample.

[0462] Example 74. A control system according to any of Examples 71-73, wherein the selected purification scheme determines: whether cell capture and / or cell lysis is used, the duration of cell capture and / or cell lysis, the reagent to be used, the volume of the reagent to be used, whether and 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 pumping between one or more purification steps, whether and when one or more valves in the purification column are opened and closed, whether and when one or more seals in the purification column are punctured to allow the corresponding fluid release or gas venting, the type of target biomolecule capture involved, the duration of target biomolecule capture, or a combination thereof.

[0463] Example 75. A fluid release system for retaining and selectively releasing fluid, comprising: a flexible gasket; a reservoir disposed on a first side of the flexible gasket for retaining 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, wherein the actuator is operable to deflect the flexible gasket, thereby causing the fragile seal to rupture and selectively release fluid from the reservoir.

[0464] Example 76. The system according to Example 75, wherein the flexible gasket is an elastomer.

[0465] Example 77. The system according to Example 75 or 76, wherein the flexible gasket is part of an intermediate layer disposed between two outer layers, and the reservoir is optionally defined at least in part by the flexible gasket and one of the two outer layers.

[0466] Example 78. The system according to any of Examples 75-77, wherein the fragile seal comprises a chemically inert material, and the fragile seal may optionally comprise a reinforcing layer, wherein the chemically inert material is bonded or fused to the surface of the reinforcing layer such that the chemically inert material is positioned toward and / or forms the sidewall defining the reservoir.

[0467] Example 79. The system according to any of Examples 75-78, wherein the fragile seal comprises a puncture-resistant material and is constructed to catastrophically fail under the mechanical force applied by the actuator.

[0468] Example 80. A system according to any of Examples 75-79, wherein the fluids include nucleic acid purification reagents, protein purification reagents, input sample, resuspension buffer, RNase A, DNase, proteinase K, lysis buffer, neutralization buffer, ionizing salt buffer, non-ionizing salt buffer, binding buffer, endotoxin removal buffer, washing buffer, elution buffer, isopropanol, ethanol, water, or TE buffer.

[0469] Example 81. The system according to any of Examples 75-80 further includes a fluid passage in fluid communication with the storage tank, the fluid passage being used to receive fluid when fluid is released from the storage tank.

[0470] Example 82. The system according to any one of Examples 75-81 further includes a flexible vent, a fragile gas seal disposed on a first side of the flexible vent, and a venting actuator disposed on a second side of the flexible vent. Optionally, the venting actuator is operable to selectively deflect the flexible vent toward the fragile gas seal, thereby breaking the fragile gas seal to release air into the storage tank.

[0471] Example 83. An automated system for selectively releasing a fluid, comprising: a fluid release system according to any one of Examples 75-82; a biosample purification column for use in conjunction with an automated target biomolecule purification system, the biosample purification column including the device according to any one of Examples 1-32, the automated system optionally including a control system according to any one of Examples 71-74, and optionally including an automated purification device or system according to any one of Examples 47-62.

[0472] Example 84. A method for selectively releasing fluid from a reservoir during an automation process, comprising: contacting a flexible gasket 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 break the fragile seal, thereby releasing fluid from the reservoir.

[0473] Example 85. The method according to Example 84, wherein the fluid is released from the reservoir without the actuator being in direct contact with the fluid.

[0474] Example 86. The method according to Example 84 or 85 further includes retracting the actuator from the broken fragile seal, and removing the flexible gasket from the broken fragile seal as the actuator retracts.

[0475] Example 87. The method according to any one of Examples 84-86 further includes: contacting the flexible vent with the venting actuator; moving the venting actuator to deflect the flexible vent toward the fragile gas seal; and causing the flexible vent to break the fragile gas seal, wherein breaking the fragile gas seal allows air to be released into the storage tank.

[0476] Example 88. The system described in any of the embodiments of Examples 75-83 is implemented according to the method described in any of the embodiments of Examples 84-87.

[0477] Example 89. A device for controlled fluid motion, comprising: a first outer layer including a first side and a second side, the first side including a series of channels; a second outer layer disposed opposite to the first side of the first outer layer; an elastomeric layer disposed between the first outer layer and the second outer layer, the elastomeric layer including sealing rib structures corresponding to the series of channels, the elastomeric layer being used to separate the channels from the fluid when compressed between the first outer layer and the second outer layer.

[0478] Example 90. The device according to Example 89, wherein a first channel in a first series of channels includes a storage tank, and optionally, one or more of the first outer layer or the second outer layer is a thermoformed polymer.

[0479] Example 91. The device according to Example 89 or 90 further includes a nominal gap between the elastomer layer and the first outer layer and / or the second outer layer, such that when the elastomer layer is compressed between the first outer layer and the second outer layer, the compressed portion of the sealing strip is displaced within the nominal gap.

[0480] Example 92. The device according to any of the embodiments 89-91 further includes valves associated with a series of channels, the valves being selectively movable between a closed position and an open position to restrict or allow fluid to pass through the valves, respectively.

[0481] Example 93. The device according to any of Examples 89-92, wherein the valve has a hole in the second outer layer.

[0482] Example 94. The device according to Example 93, wherein the orifice provides a passage to a deflectable portion of the elastomeric layer, the deflectable portion including a valve sealing rib extending from the elastomeric layer toward a first outer layer.

[0483] Example 95. The device according to Example 94, wherein when the valve is in the closed position, the valve sealing rib contacts the first outer layer; when the valve is in the open position, the valve sealing rib separates from the first outer layer.

[0484] Example 96. The device according to Example 94 or 95 further includes a plunger in contact with a deflectable portion of the valve, the plunger being sized and shaped to pass through the orifice to deflect the deflectable portion and move the valve toward a closed position.

[0485] Example 97. The device according to any of Examples 94-96, wherein the valve further includes a flange formed in the first outer layer and extending toward the orifice, optionally wherein the flange contacts the valve sealing rib when the valve is in the closed position.

[0486] Example 98. The device according to any of Examples 94-98, wherein when the valve is fully moved to the open position, the deflectable portion extends beyond the orifice.

[0487] Example 99. The device according to any one of Examples 89-98, wherein the second outer layer includes a second series of channels disposed on a first side of the second outer layer, the first side of the second outer layer facing the first side of the first outer layer.

[0488] Example 100. The device according to Example 99, wherein the sealing rib structure includes 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.

[0489] Example 101. The device according to any of Examples 89-100, wherein the sealing rib structure is arranged along the contour of a series of channels.

[0490] Example 102. The device according to Example 101, wherein each channel includes a lowest point and forms a surface rising from the lowest point to an inflection point located on the opposite side of the lowest point, optionally wherein the sealing ribs include apexes, the sealing ribs being arranged such that the apexes are located at the inflection point of the channel or extend beyond the inflection point relative to the respective lowest point of the channel.

[0491] Example 103. A method for controlling fluid movement, comprising: providing the apparatus according to any one of Examples 89-102 to a system for automatically purifying target nucleic acids or target proteins; causing one or more plungers to open valves within the apparatus, the opened valves enabling fluid communication between upstream and downstream portions of a series of channels; and causing a pump to move fluid from the upstream portion to the downstream portion.

[0492] Example 104. The method according to Example 103 further includes one or more of the following requirements: compressing the sealing rib structure to withstand a fluid pressure of at least 30 psi, preferably at least 60 psi, before leakage; constructing the device to withstand a force of at least 500 lbf, preferably up to 15,000 lbf, applied over the entire length of the sealing rib structure; or compressing the sealing rib by at least 20%, preferably at least 30%, when the elastomer layer is compressed between the first and second outer layers.

[0493] Example 105. A biological sample purification column, comprising at least two of the following: the apparatus according to any one of Examples 1-32; the apparatus according to any one of Examples 47-62; the control system according to any one of Examples 71-74; the system according to any one of Examples 75-83; and the apparatus according to any one of Examples 89-102.

[0494] Example 106. An apparatus for automatically purifying a target protein from a biological sample, comprising: an input reservoir for containing the biological sample; a first bioprocessing assembly in fluid communication with the input reservoir and a lysis buffer reservoir, the first bioprocessing assembly for generating a lysate containing the target protein; a second bioprocessing assembly in fluid communication with the first bioprocessing assembly and a first elution buffer reservoir, the second bioprocessing assembly including a protein binding support for retaining the target protein; and a container in fluid communication with the second bioprocessing assembly for holding an output container containing the target protein.

[0495] Example 107. The apparatus according to Example 106, wherein the apparatus comprises a consumable purification column.

[0496] Example 108. In the apparatus according to Example 106 or 107, the first biological treatment component includes a clarification filter.

[0497] Example 109. According to the apparatus described in Example 108, the clarification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the clarification filter is configured to separate the target protein-containing component from the first waste portion of the biological sample.

[0498] Example 110. The device according to any one of Examples 106-109 is characterized in that the first biological treatment structure includes a cell capture or concentration filter, wherein optionally, the cell capture or concentration filter is located upstream of the clarification filter.

[0499] Example 111. According to the apparatus described in Example 110, the cell capture and concentration filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and is configured to separate the target protein-containing component from the first waste portion of the biological sample.

[0500] Example 112. The apparatus of one of Examples 106-111 includes one or more biological processing chambers equipped with protein purification reagents.

[0501] Example 113. The device according to any of Examples 106-112 further includes one or more filters, wherein the one or more filters comprise hollow fiber filters.

[0502] Example 114. A method for automatically separating or purifying target proteins from bacterial cell lysis using the apparatus described in any of Examples 106-113.

[0503] Example 115. In the method of Example 114, the target protein is separated using a cell division step, column chromatography, affinity chromatography, gel filtration chromatography, ion exchange chromatography, rapid protein liquid chromatography, or a combination of these methods.

[0504] Example 116. The samples in the methods of Example 114 or 115 include biological samples, tissues, biopsies, cell cultures, cells, cell suspensions, urine, saliva, cerebrospinal fluid, blood, serum, plasma, aqueous solutions of feces, other bodily fluids or secretions, and eukaryotic cells or cell suspensions composed of prokaryotic cells selected from mouse, insect, primate, and human cells or prokaryotic cells.

[0505] Example 117. The method according to any of Examples 114-116, wherein the biological sample refers to a cell line or tissue containing a number of cells, and the component containing the target protein includes the protein in the cell line or tissue and a first waste portion containing lysed cells and culture medium (optionally).

[0506] Example 118. A biological sample purification column comprising at least two of the following: the apparatus according to any one of Examples 106-117; the apparatus according to any one of Examples 47-62; the control system according to any one of Examples 71-74; the system according to any one of Examples 75-83; and the apparatus according to any one of Examples 89-102.

Claims

1. A system comprising an apparatus for automatically purifying a target biomolecule from a biological sample, comprising: Purification column, the purification column comprising: Input storage tanks used to hold biological samples; A first bioprocessing component in fluid communication with the input reservoir and the lysis buffer reservoir, the first bioprocessing component being used to generate lysates containing target biomolecules; A second bioprocessing component in fluid communication with the first bioprocessing component and the first elution buffer reservoir, the second bioprocessing component including a target biomolecule binding filter for retaining target biomolecules; A container in fluid communication with the second biological processing component, the container being used to hold an output container containing target biomolecules from the second biological processing component; An outer shell having internal compartments, the dimensions and shape of which are adapted to house the purification column; A pump assembly, disposed within the internal compartment, is used to provide pumping action via peristaltic movement; and A clamping mechanism is disposed within the internal compartment and configured to move between an open position and a closed position. In the open position, the internal compartment is accessible, and in the closed position, the clamping mechanism clamps onto an inserted purification column, enabling the biological sample to be processed. In the closed position, the clamping mechanism operates to compress the purification column and thus assist in a fluid seal of the purification column.

2. The system according to claim 1, characterized in that, The biological sample may be a cell culture, a clinical sample, an environmental sample, or a food sample.

3. The system according to claim 1 further includes a light density detector window disposed between the input storage tank and the first biological processing component, through which the light density of the biological sample can be detected.

4. The system according to claim 1, characterized in that, The first biological treatment component includes a clarification filter.

5. The system according to claim 4, characterized in that, The clarification filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the clarification filter is used to separate the target nucleic acid portion of the biological sample from the first waste portion of the biological sample.

6. The system according to claim 4, characterized in that, The first biological processing component includes a cell capture filter or a concentration filter, wherein the cell capture filter or concentration filter is disposed upstream of the clarification filter.

7. The system according to claim 6, characterized in that, The cell capture or concentration filter is in fluid communication with the input reservoir and the lysis buffer reservoir, and the cell capture or concentration filter is used to separate the target nucleic acid portion of the biological sample from the first waste portion of the biological sample.

8. The system according to claim 6, characterized in that, The lysis buffer reservoir is fluidly connected to the cell capture filter, enabling the cell capture filter to be backwashed and allowing the backwash solution to enter the clarification filter.

9. The system of claim 8 further includes a first mixing chamber disposed between the clarifying filter and the cell capture filter, the first mixing chamber being configured to contain the backwash solution.

10. The system of claim 9, further comprising a neutralization buffer reservoir in fluid communication with the first mixing chamber, the first mixing chamber being configured to contain the backflushing solution and the neutralization buffer, and to mix the backflushing solution and the neutralization buffer to form a neutralized lysate.

11. The system according to claim 10, characterized in that, The clarification filter is in fluid communication with the first mixing chamber, and the clarification filter is used to separate the second waste portion and the target nucleic acid-containing portion of the biological sample.

12. The system according to claim 10, characterized in that, The clarification filter includes the cell capture filter, which is used to concentrate the cellular components of the biological sample in a first purification step and to clarify the neutralized lysate in a subsequent second purification step.

13. The system according to claim 1, characterized in that, The target biomolecule binding filter of the second biological treatment component includes a silicon-based filter or a microsphere column with affinity for the target biomolecule.

14. The system according to claim 13, characterized in that, The second biological processing component also includes a purification reagent reservoir in fluid communication with the target biomolecule binding filter.

15. The system according to claim 13, characterized in that, The target biomolecule binding filter is in fluid communication with the elution buffer reservoir and the output container.

16. The system according to claim 15, characterized in that, The second bioprocessing component includes a second mixing chamber disposed between the first bioprocessing component and the target biomolecule binding filter.

17. The system according to claim 16, characterized in that, The second mixing chamber is in fluid communication with the endotoxin removal buffer reservoir.

18. The system according to claim 1, characterized in that, The target biomolecule binding filter is a nucleic acid binding filter containing an anion exchange membrane.

19. The system according to claim 18, characterized in that, The second biological treatment component includes a precipitation membrane disposed downstream of the anion exchange membrane.

20. The system according to claim 19, characterized in that, The anion exchange membrane is used to separate the third waste portion of the biological sample from the portion containing the target nucleic acid.

21. The system of claim 19 further includes a second elution buffer reservoir, wherein the first elution buffer reservoir is fluidly connected to the anion exchange membrane to allow the target nucleic acid to be eluted through the anion exchange membrane, and the second elution buffer reservoir is fluidly connected to the precipitation membrane to allow the target nucleic acid to be eluted through the precipitation membrane.

22. The system according to claim 19, characterized in that, The precipitation membrane is used to separate the fourth waste portion of the biological sample from the portion containing the target nucleic acid.

23. The system according to claim 19, characterized in that, The precipitating membrane is in fluid communication with the precipitant storage tank.

24. The system according to claim 19, characterized in that, The precipitated membrane is in fluid communication with the washing / desalination solution storage tank.

25. The system according to claim 23, characterized in that, The second biological treatment component includes a third mixing chamber disposed between and in fluid communication with the anion exchange membrane and the precipitation membrane, wherein the third mixing chamber is fluidly connected to the precipitant storage tank and is disposed between the precipitation membrane and the precipitant storage tank.

26. The system according to claim 24, characterized in that, The second biological treatment component includes a third mixing chamber disposed between and in fluid communication with the anion exchange membrane and the precipitation membrane, wherein the third mixing chamber is fluidly connected to the desalination solution storage tank and is disposed between the precipitation membrane and the desalination solution storage tank.

27. The system according to claim 1, characterized in that, The output container can be selectively removed from the system.

28. The system according to claim 1, characterized in that, The input reservoir is sized and shaped to accommodate at least 5 mL of biological samples.

29. The system according to claim 1, characterized in that, It also includes a selectively closable access door that provides access to internal compartments.

30. The system of claim 29 further includes a controller, wherein the controller supplies power to the clamping mechanism only when it is determined that the access door is closed.

31. The system of claim 29 further includes a locking mechanism for locking the access door in the closed position when the clamping mechanism is moved to the closed position.

32. The system according to claim 1, characterized in that, The clamping mechanism is powered by a motor.

33. The system of claim 32 further includes a manual release mechanism operably connected to the clamping mechanism, enabling the clamping mechanism to be manually released independently of the motor.

34. The system according to claim 29, characterized in that, The housing includes an open end to which the access door is connected and a closed end opposite the open end, the open end and the closed end defining a longitudinal direction through the open end and the closed end, wherein the clamping mechanism is configured to move in a direction transverse to the longitudinal direction.

35. The system according to claim 1, further comprising: A light density sensor is used to measure the light density of a biological sample placed inside the purification column.

36. The system of claim 1 further includes a user interface for displaying instrument information and receiving user input, the user interface being configured to accommodate user input relating to one or more of the following: biological sample volume, selected purification scheme, operating instructions for a selectively closable access gate, or desired concentration of target biomolecules.

37. The system according to claim 1, characterized in that, The inner compartment includes a position sensor for determining that the purification column has been fully inserted into the inner compartment, and the limit switch is positioned such that it is activated when the purification column is fully inserted into the inner compartment and comes into contact with the leading edge of the purification column.

38. The system according to claim 1, characterized in that, It also includes one or more access door sensors for determining whether the access door is in the open or closed position.

39. The system of claim 1 further includes an output container sensor for detecting the presence of an output container at the insertion of the purification column.

40. The system of claim 1, further comprising one or more rotatable magnets positioned to direct an electromagnetic field inward into the inner compartment.

41. The system according to claim 1, characterized in that, The pump assembly includes a camshaft connected to a motor via a power transmission assembly, a plurality of cam elements attached to the camshaft and extending laterally from the camshaft toward the purification column upon insertion.

42. The system according to claim 41, characterized in that, The cam element causes a linear peristalsis at the tip of the cam element due to the rotation of the cam shaft.

43. An apparatus for automatically purifying target nucleic acids from biological samples, comprising: A first biological processing component for containing the biological sample, the first biological processing component including a waste separation filter and a plurality of storage tanks fluidly connected to the waste separation filter; The second biological treatment component includes an anion exchange membrane, a washing liquid storage tank fluidly coupled to the anion exchange membrane, and a first elution buffer storage tank fluidly coupled to the anion exchange membrane. . The third biological treatment component includes a precipitation filter and a second elution buffer reservoir fluidly connected to the precipitation filter. The device is configured to be fluid-sealed by a clamping mechanism of a separate device for automatically purifying target biomolecules from a biological sample when the device is inserted into the separate device.

44. The device according to claim 43, characterized in that, The first biological processing assembly further includes a cell capture filter, wherein the first of the plurality of reservoirs includes a resuspension buffer reservoir.

45. The device according to claim 44, characterized in that, The input reservoir is fluidly connected to the first side of the cell capture filter, and the resuspension buffer reservoir is fluidly connected to the second side of the cell capture filter.

46. ​​A control system for controlling the system according to any one of claims 1-42, comprising: One or more processors; . One or more hardware storage devices storing computer-executable instructions that can be executed by one or more processors to enable the control system to at least: Receive sensor data from one or more sensors to determine the status of one or more processes in the automated purification device; The determined process state is compared with a set of different solutions stored in the solution library; Select a purification protocol from the protocol library based on the determined process status; . The selected purification scheme is executed by causing one or more instrument actuators of the automated purification device to operate according to the selected purification scheme.

47. The control system according to claim 46, characterized in that, The one or more sensors include a door sensor, a box-type proximity or contact sensor, an output container proximity or contact sensor, a box scanner, a timer, a temperature sensor, a weight sensor, a volume sensor, or a combination thereof.

48. The control system according to claim 46, characterized in that... The determined process state includes the determined optical density of at least one initial input sample.

49. The control system according to claim 46, characterized in that, The selected purification protocol defines one or more of the following: whether cell capture is used, whether cell lysis is used, the duration of cell capture, the duration of cell lysis, the reagents to be used, the amount of reagents to be used, whether mixing is performed at one or more purification steps, when one or more purification steps are performed, the duration of one or more mixing steps, the rate of one or more mixing steps, the duration of pumping between one or more purification steps, the rate of pumping between one or more purification steps, whether valves in one or more boxes are opened and closed, when valves in one or more boxes are opened and closed, whether one or more seals in the box are punctured to allow the corresponding fluid release or venting, when one or more seals in the box are punctured to allow the corresponding fluid release or venting, the type of target biomolecule capture involved, or the duration of target biomolecule capture.

50. The system according to claim 1, characterized in that, It also includes a fluid release system for retaining and selectively releasing fluid, including: Flexible washers; A reservoir is provided on the first side of the flexible gasket, which is used to retain fluid; A fragile seal positioned between a flexible gasket and a fluid reservoir. An actuator on the second side of the flexible gasket, wherein the actuator is operable to deflect the flexible gasket, thereby causing the fragile seal to break and selectively releasing fluid from the reservoir.

51. The system according to claim 50, characterized in that, The flexible gasket is an elastomer.

52. The system according to claim 50, characterized in that, The flexible gasket in question is part of an intermediate layer disposed between two outer layers.

53. The system according to claim 50, characterized in that, The fragile seal comprises a chemically inert material.

54. The system according to claim 50, characterized in that, The fragile seal includes a puncture-resistant material designed to fail in response to the application of mechanical force by the actuator.

55. The system according to claim 50, characterized in that, The fluids include nucleic acid purification reagents, protein purification reagents, input samples, resuspension buffer, RNase A, DNase, proteinase K, lysis buffer, neutralization buffer, ionization salt buffer, non-ionization salt buffer, binding buffer, endotoxin removal buffer, washing buffer, elution buffer, isopropanol, ethanol, water, or TE buffer.

56. The system according to claim 50, characterized in that, It also includes a fluid passage in fluid communication with the storage tank, the fluid passage being configured to receive the fluid when fluid is released from the storage tank.

57. The system according to claim 50, characterized in that, It also includes a flexible vent, a fragile air seal disposed on a first side of the flexible vent, and an venting actuator disposed on a second side of the flexible vent.

58. An automated system for selectively releasing fluid, comprising: The system according to any one of claims 50-57; . Biological sample box for use in conjunction with the system according to any one of claims 1-42.

59. A control system for controlling the system according to claim 35, comprising: One or more processors; as well as One or more hardware storage devices thereon store computer-executable instructions, which can be executed by the one or more processors to enable the control system to at least: Receive sensor data from optical sensors to determine one or more process states of the automated purification device; compare the determined process states with a set of different protocols stored in a protocol library; A purification scheme is selected from the scheme library based on the determined process status; as well as The selected purification scheme is implemented by causing one or more instrument actuators of the automated purification device to operate in accordance with the selected purification scheme.

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