Bulk separation system

Abstract

The invention is an automated separation system comprising a liquid handling device and one or more pipette tip columns, wherein the liquid handling device is equipped with two or more nozzles arranged to receive a pipette tip column comprising separation media, respectively. Furthermore, the system comprises means for applying positive or negative pressure to the intracolumnal lumen above the column bed to effect aspiration of each pipette tip column and dispensing of each pipette tip column. In order to effect sealing of the pressure used to move liquid through the column bed during the separation process without inadvertently ejecting the column from the nozzle, each nozzle is provided with at least one annular protrusion arranged to engage the inside of the pipette tip column which is generally uniformly tapered and does not have any corresponding grooves. Each nozzle can be provided with a sliding ejector to effect removal of the pipette tip column.

Description

Technical Field

[0001] This invention relates to a separation system capable of automatically handling pipette tip columns. More specifically, the chambers and chromatography beds in the packed pipette tip columns used according to the invention have relatively large volumes, high back pressures, or high fluid flow resistance, necessitating requirements for system hermeticity and repeatability. The invention also relates to a method for separating biomolecules using the system according to the invention, which is advantageous for handling larger volumes of liquid compared to conventional pipetting methods. Background Technology

[0002] Since Eppendorf was founded in Germany in the 1960s, manual liquid handling equipment, including pipette tips, has been widely used in laboratories to transport measured volumes of liquids. Since then, the technology has continued to evolve to open up new applications.

[0003] US6,197,259 (Rainin Instruments Co, Inc.) relates to pipette tips described as easy to push. More specifically, the '259 patent describes the problems associated with pushing conventional 'ring-stretch' pipette tips during manual pipetting, which are mounted on a shaft by stretching the pipette tip material to engage with the shaft. To reduce the force required to push the pipette tips, thus making them more suitable for pushing by human thumb pressure, the pipette tips proposed in the '259 patent are not uniformly tapered, but instead include a specially designed annular sealing region that stretches radially outward when mounted on a shaft, but the pipette tip is not tapered in the longitudinal direction.

[0004] US 6,596,240 (Porex Corporation) relates to the field of pipette tips, including sealing strips. In '240, it is described why removing such tips can be difficult and, with repeated use throughout the day, can lead to fatigue and even damage over time. Therefore, a need arises for a method of manufacturing pipette tips that allow for insertion and push-off with minimal force, maintain a good seal, and provide a good fit on a variety of pipettes. Furthermore, a need arises for a method of forming a relatively large, resilient ring on the internal sealing surface of the pipette tip. Typically, the size of this internal ring is limited because it is "undercut" in the mold that forms the tip. To remove the part from the mold, this part must expand, thereby releasing the ring from a groove in the mold core from which the ring is formed. This limits its size and can "smudge" the ring during removal.

[0005] As a solution to the problem, '240 patent proposes a solution in which the tip member includes an elongated tube bracket having a central axis, a protrusion on the inner surface of the bracket, and a groove on the inner surface adjacent to the protrusion. The tip member may have a generally conical shape, and the inner surface is tapered from a rear opening at the proximal end to a tip opening at the distal end.

[0006] US2012 / 0180579 (Perkin Elmer Health Sciences, Inc.) relates to large-volume pipette tips for filling in automated liquid processors. More specifically, the '579 patent application describes problems arising as the size of micropipettes increases, particularly the difficulty of fitting them side-by-side in parallel processing in tray format. As a solution, the '579 patent application proposes a pipette tip whose central portion includes an enlarged cavity (such as a rectangular cavity) designed to accommodate a larger volume than conventional pipette tips, while fitting within the same liquid processor as micropipettes. In this context, the 'large' volume referred to in the '579 patent application means a volume of approximately 5 mL or more, although it states that for even larger pipette tips, the necessary modifications would be immediately apparent to a person skilled in the art.

[0007] US5,200,151 (Dabe Behring Marburg) relates to a fluid dispensing system, and more particularly to a pipette assembly including components suitable for use with disposable pipette tips. To ensure accurate positioning of the disposable pipette tip on the distal end of the rod of the pipette assembly that holds the tip, the proximal lumen of the pipette tip encloses the distal end of the rod and includes a boss surrounding an annular region of the rod, thereby forming a base for the rod and creating an accurate distance between the distal end of the rod and the pipette tip orifice. In a preferred embodiment, the fluid dispensing system is incorporated into an automated analytical instrument.

[0008] US2014 / 0219887 (Agilent) generally relates to pipetting, and more specifically, to automated pipetting, wherein different pipette tips are compatible with the same pipetting device. Thus, pipette tips of different sizes can be coupled to this pipette without modification. Therefore, the same pipette can be used to change different pipette tips, which can be done automatically. The pipette tip can be coupled to an adapter including a proximal end that abuts the pipette and a distal end that abuts the pipette tip. The proximal ends can all have the same geometry and mate with the same pipette. The distal ends can have different geometries and mate with different pipette tips. The pipette can be part of a liquid handling device and is automatically movable to different platform positions. The pipette may include: a locking mechanism for locking the adapter to the pipette or locking the pipette tip directly to the pipette; and a pushing mechanism for pushing the pipette tip from the corresponding adapter.

[0009] US2017 / 0211129 (Phynexus, Inc.) relates to apparatus and methods for plasmid purification, and describes a pipette tip column and automated method for such purification. More specifically, the '129 patent application addresses the need for large-scale automated and parallel plasmid preparation because, in practice, micrograms to milligrams of plasmid DNA are typically required when using transfection in recombinant DNA technology to obtain protein expression. To obtain these large quantities of plasmid DNA, most researchers perform manual plasmid purification using spinning columns or columns operated via vacuum or gravity. As a solution, patent application '129 proposes an automated method for purifying nucleic acids in a pipette tip column format, which may include the following steps: (a) providing a cell lysate comprising cell debris, liquid, and plasmid DNA; (b) providing a column capable of capturing plasmid DNA; (c) providing a filter device including a filter; (d) passing the cell lysate through the filter device to produce a filtrate; (e) passing the filtrate through the column, wherein a portion of the plasmid DNA in the filtrate is captured by the column; (f) passing a washing solution through the column; and (g) eluting the plasmid DNA by passing an desorption solution through the column, wherein the amount of plasmid DNA eluted from the column is at least 750 μg, wherein steps (e) to (g) are performed at a predetermined time. In this embodiment, step (d) is illustrated using a pipette tip column processed by the ME semi-automated purification system (Phynexus, Inc., San Jose, Calif.), wherein the column is equilibrated with 200 μL of 7M guanidine hydrochloride, and one cycle of back-and-forth flow is performed at 500 μL / min, with a 20-second pause at the end of the aspiration and dispensing steps. The ME semi-automated purification system is designed for PhyTips, which is available from Biotage AB / Phynexus Inc., and has a volume of up to 20,000 μL (20 mL). Alternatively, this method is described in patent application '129 as being designed to operate on a Tecan EVO, Biomek FX, or other robotic liquid processor.

[0010] WO2014 / 140640 (Diagnostics For The Real World Ltd) relates to an apparatus and method for processing biological samples, suitable for downstream applications such as polymerase chain reaction (PCR) and sequencing. More specifically, WO2014 / 140640 describes an automated biological sample processing system including a pipette, a solid-phase material column bound to nucleic acids, a transport device, an air piston device, and an adapter for connecting the pipette to the transport device and the air piston device.

[0011] The adapter is detachably coupled to the transport device and the air piston device for movement with the transport device during sample processing, and the adapter can be coupled to the pipette so that the transport device can controllably position the pipette, thereby enabling the air piston device to controllably draw liquid into and expel liquid from the pipette, and the adapter can be coupled to the column.

[0012] In addition, the adapter includes a filter to prevent the transfer of liquids or aerosols between the pipette or column and the air piston device.

[0013] In summary, there remains a need in the art for improved automated liquid handling systems capable of mounting and pushing filled pipette tips (referred to as pipette tip columns), which can be filled with, for example, chromatography or extraction media (such as SPE or SLE media), particularly large-volume pipette tip columns filled with large volumes of media. There is also a need in the art for simple solutions where the pipette tips are easily mounted onto nozzles, advantageously avoiding the need for complex mechanical structures (such as adapters). Summary of the Invention

[0014] This invention aims to satisfy one or more of the requirements stated above.

[0015] Therefore, a first aspect of the present invention is an automated separation system comprising: a liquid handling apparatus and at least one pipette tip column including a separation medium, wherein the liquid handling apparatus is equipped with: one or more nozzles, such as two or more nozzles, each nozzle being arranged to receive the pipette tip column; and means for applying positive or negative pressure to achieve aspiration of each pipette tip column and dispensing of each pipette tip column, wherein each nozzle is provided with at least one annular protrusion in this system.

[0016] A second aspect of the invention is a method for separating biomolecules from a liquid sample using the system of the invention, wherein the sample is aspirated into each such pipette tip column comprising a separation medium; the biomolecules of the aspirated liquid sample are allowed to bind with the separation medium for a period of time; and the liquid sample comprising any unreacted biomolecules is dispensed from the pipette tip column.

[0017] A third aspect of the invention is a kit for separating biomolecules from a liquid sample using a separation system according to the invention, the kit comprising a 20-40 mL pipette tip column having a bed volume of 4 to 20 mL and comprising a chromatography medium (such as an affinity medium); or an extraction medium, such as a solid-phase extraction (SPE) medium or a supported liquid extraction (SLE) medium. The kit also includes, in a separate compartment, written instructions for performing such separation.

[0018] A column bed volume of 1 to 8 mL, or 1, 2, 3, 4, 5, 6, 7, or 8 mL, can be set (advantageously filled) in a tip volume of 20 to 40 mL. In other embodiments, the tip volume containing the packed bed can be 20 to 50 mL, 20 to 60 mL, 30 to 70 mL, 30 to 80 mL, 40 to 90 mL, 40 to 100 mL, or 50 to 200 mL. The volume of the packed bed can be 1 to 50 mL, 2 to 40 mL, 3 to 30 mL, or 4 to 20 mL.

[0019] Further details, advantages, and embodiments of the invention will become apparent from the dependent claims and from the appended description (including embodiments thereof).

[0020] definition

[0021] Pipette tip: A plastic tube of various shapes used to draw and dispense liquids when connected to a pipette. This definition also includes syringe tips.

[0022] Pipette tip volume: The maximum volume that a pipette tip can hold.

[0023] Filled pipette tip column: A pipette tip with a separation medium positioned inside the tip, typically at the distal end of the tip. Pipette tip columns can be configured from pipette tips, syringes, or similar materials.

[0024] Pipette tip column bed: A bed of separation medium placed inside the pipette tip column. It is usually described by the volume of the bed.

[0025] A filled pipette tip column bed: media, wherein the filter tip is typically located on the distal end of the tip, above the media bed. The filter tip above the media bed may be directly above the bed, or it may be positioned with a gap above the top of the bed.

[0026] Pipette tip column bed volume: The volume of the separation medium bed, usually described in μL or mL.

[0027] Pipette tip column cavity: The volume above the column bed in the pipette tip column.

[0028] Cavity volume: The volume above the top of the bed in the column at the tip of the pipette.

[0029] Pipette tip pump: A pump configured to pump one or more fluids through a pipette tip column by vacuum or pressure.

[0030] Pump nozzle: The movable part of the pump that provides a seal for the tip of the pipette. In the field of separation, the nozzle is sometimes referred to as the "shaft".

[0031] Annular protrusion: A ridge or raised surface on the pump nozzle that provides a seal for the tip of the pipette.

[0032] Pipette tip column pressure or vacuum: The positive or negative pressure that forces liquid through the pipette tip column during aspiration and dispensing, respectively.

[0033] The aspirated fluid flow from the pipette tip column flows into the pipette tip column and enters the cavity above the column bed.

[0034] Dispensing fluid flow from the pipette tip column: The fluid flows out of the pipette tip column from the cavity above the column bed.

[0035] Pipette tip column back pressure: The resistance to liquid flow through the column when a vacuum or pressure is applied to the column cavity.

[0036] Pipette tip column outlet: The distal end of the pipette tip column.

[0037] Distal end: The end of a pipette tip, syringe tip, pipette tip column, or syringe tip column that is furthest from the connection point with the pump. Attached Figure Description

[0038] Figure 1 A set of exemplary high-sealing pipette pumps and pipette tip columns are shown, illustrating how the seal of the nozzle on the column provides internal cavity pressure and vacuum sealing.

[0039] Figure 2 An illustrative pump nozzle bottom with an annular ridge, a tip column top, and a sliding pusher is shown.

[0040] Figure 3 The top of the tip column, positioned at the bottom of the pump nozzle, is shown, along with a protrusion in the form of a ring ridge that provides air and vacuum seal to the inside of the column cavity above the bed.

[0041] Figure 4 The bottom of the pump nozzle with two annular ridges, the top of the tip column, and the sliding pusher are shown.

[0042] Figure 5 The top of the pipette tip column is shown positioned at the bottom of the pump nozzle.

[0043] Figure 6 The invention illustrates how a sliding pusher device can be used to remove a pipette tip column from a pump nozzle. Detailed Implementation

[0044] In a first aspect, the present invention relates to an automated separation system comprising a liquid handling apparatus and at least one pipette tip comprising a separation medium, wherein the liquid handling apparatus is equipped with: one or more nozzles, such as two or more nozzles, each nozzle being arranged to receive the pipette tip; and means for applying positive or negative pressure to achieve suction and dispensing of each pipette tip, wherein each nozzle has at least one annular protrusion in this system.

[0045] In this context, it should be understood that the term 'liquid processing equipment' refers to equipment well-known in the field of biological processing of liquid samples. Therefore, it can include any standard type of features capable of enabling its operation, including appropriate software for controlling its performance.

[0046] The term 'automatic' here means that the system is capable of performing all or at least some of the standard operations discussed below in an automatic manner. Specifically, where the forces allowed, at least the installation of the pipette tip into the nozzle and the ejection of the pipette tip from the nozzle are performed automatically.

[0047] An annular protrusion is provided on the portion of each nozzle that will contact the attached pipette tip column, and this annular protrusion will be described in further detail below in conjunction with the accompanying drawings. In the field of liquid handling, the term 'nozzle' is sometimes replaced by shaft or manifold head. Advantageously, according to the invention, the nozzle has been configured to allow direct contact with the pipette tip column, thus avoiding the need for additional components such as adapters required to achieve mating.

[0048] In short, this protrusion can advantageously be incorporated as an integral part of the nozzle during its manufacture. Alternatively, the protrusion can be attached in a later stage. In any case, the term 'with protrusion' here means that the protrusion is fixed in place, rather than an O-ring being added. Such nozzles can be molded and manufactured according to standard processes from any suitable plastic or other rigid and non-reactive solid material.

[0049] Therefore, each nozzle of this system may have one or two annular protrusions arranged to seal against the mounted pipette tip column, capable of holding, for example, a liquid volume of at least about 20 mL, such as in the range of about 20 mL to about 40 mL. In one embodiment, the pipette tip column is arranged to hold a packed bed of about 2 mL at its distal end, and to hold about 18 mL of liquid in the cavity volume above the packed bed.

[0050] Each nozzle may be equipped with a sliding pusher that removes the tip from the nozzle when appropriate. A skilled technician will understand that the pipette tip should fit snugly into the nozzle to remain in place during operation and be easily removed after use (e.g., by using a sliding pusher as illustrated in the figure).

[0051] Pipette tip columns (such as homogeneous or substantially homogeneous tapered plastic pipette tips capable of holding at least 20 mL of liquid in a cavity volume) are a conventional design. In some embodiments of the invention, at least 40 mL of liquid can be held. In some embodiments of the invention, at least 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL, and 200 mL of liquid can be held. Those skilled in the art will understand that, since the packed bed of separation medium held between the filter tips is relatively small compared to the liquid volume discussed herein, the figures may be understood to include or not include a packed bed in different embodiments. In any case, the present invention can automatically manage substantially larger volumes compared to the separation of biomolecules (such as plasmids) proposed by the prior art.

[0052] Any plastic or polymer material can be envisioned for use in pipette tip columns, provided they are rigid enough to withstand the pressures described herein and do not react with the liquids and reagents commonly used in biomolecular separation.

[0053] Currently, the inventors have unexpectedly discovered that by providing a protrusion to the nozzle of the liquid handling device, rather than by providing engagement features (such as grooves and protrusions) to the pipette tip as often suggested in the prior art, a manageable force can be used to install and push out conventionally designed pipette tip columns without leakage or premature loss of the pipette tip column. This is surprising given the bed volume of the pipette tip column used, which typically holds approximately 20 mL to approximately 40 mL of lumen fluid, and given that the pipette tip column ultimately needs to be pushed out without problems, the installation of the tip column was found to be difficult. Those skilled in the art will understand that, for specific situations, such as when additional safety features are required, or if higher pressures and / or larger volumes are envisioned, one or more conventional O-rings can be included at the engagement between the nozzle and the pipette tip column.

[0054] In some embodiments of the invention, a downward force of 2 to 3 pounds (0.9 to 1.6 N) is required to remove the pipette tip column from the pump nozzle. In some embodiments, a force of 2 to 10 pounds (0.9 to 4.7 N), 3 to 9 pounds (1.6 to 4.2 N), or 4 to 8 pounds (0.8 to 1.4 N) is required to remove the pipette tip column from the pump nozzle. This nozzle is described as the first-generation nozzle in Example 2.

[0055] In other embodiments of this system, each pipette tip column can be mounted to the nozzle using a force ranging from about 50 N to about 160 N.

[0056] In a further embodiment, each pipette tip column of the system is releasable by a push-off using a similar force (i.e., a force ranging from about 50 N to about 160 N). The push-off mechanism must be able to completely eject the column from the nozzle. The column must be completely removed from the nozzle to prevent the automated robot from detecting that the column has not been partially ejected and causing the collapse of the robotic liquid processor. In some specific embodiments of the invention, a force of 3.5 pounds (1.6 N) is required to remove the pipette tip column from the pump nozzle.

[0057] Those skilled in the art will understand that the force required for installing and pushing the pipette tip will be influenced by the specific design of the protrusions on each nozzle. As can be seen from this description, the system according to the invention will include one or more protrusions (e.g., one protrusion on each nozzle), while (contrary to the prior art) the pipette tip column is defined by a uniform slope (i.e., it does not have any grooves corresponding to each nozzle protrusion). Without departing from the spirit of the invention, embodiments with only one protrusion on each nozzle are possible, for example. In such embodiments, the single protrusion may be slightly larger than in cases where two or more protrusions are provided on the nozzle. Therefore, those skilled in the art will understand that the force required to install and / or release each pipette tip column will vary depending on, for example, the number and / or shape and size of the nozzles. Following the principles described herein, those skilled in the art will be able to run simple tests to determine the optimal force for effectively sealing the pipette tip column to the nozzle; and once the separation process has been completed, the pipette tip column can be removed by a simple push.

[0058] Therefore, any invention deviating from the above-defined scope may also be included by the invention as defined in the claims of this invention.

[0059] The pipette tips can be pushed one at a time; or simultaneously; and optionally, their outer surfaces can be provided with means to facilitate pushing them. Such means may be, for example, external ridges or other protrusions that can be used to automatically loosen the pipette tips.

[0060] The advantage of this invention is that the pump enables the mounting of large-filled pipette tip columns and their corresponding nozzles, as discussed above, and the pushing out from their corresponding nozzles, including effective sealing during processing and simple automatic pushing without human intervention. In this context, "large" is understood to be 20 mL or more, as discussed throughout this application.

[0061] This invention can also be used with pipette tip columns of significantly larger volumes (e.g., sample volumes of 2 liters or more, such as 4 liters or more). In this context, it should be understood that the term "pipette" refers to a column that functions similarly to a pipette tip in terms of the ability to aspirate and dispense liquid, but this should not be construed as any limitation on size.

[0062] The preferred pump for this automated separation system is a piston pump, which is arranged to tightly seal and release pressure using a flexible flange as needed for each stage. Piston pumps suitable for this purpose are well known in the art and include elements such as a pump cylinder, pump piston, cylinder space, pressure sensor, and pressure channel. Starting with such a conventional pump, the inventors have provided a sealing device to achieve the handling of the liquid volume discussed herein. More specifically, this sealing device can be made of a suitable material (such as flexible plastic or rubber). Furthermore, to allow the reactivity required for maintaining and releasing pressure separately, this sealing device can be a flexible flange, which is advantageously provided with a suitable lubricant. The sealing flange can be arranged at one or more vertical positions between the pump piston and the cylinder space, such as an annular flange surrounding the piston at a first vertical distance and a second vertical distance from the piston end.

[0063] Therefore, a specific aspect of the invention is a method of using the force discussed above to attach a large pipette tip (such as a 20 to 40 mL pipette tip) to a nozzle on a liquid handling device arranged in this system and / or to push out the nozzle.

[0064] The system according to the invention may include at least two independently arranged pipette tip columns (e.g., two to four columns movable relative to each other). In this context, the term "independently arranged" means that the pipette tip columns are not arranged in a tray or plate-like format, but rather like the larger tubes of a conventional liquid handling system. The method of operation will determine the extent to which these independently arranged pipette tip columns are operated in parallel or one at a time.

[0065] As indicated above and described in detail in the accompanying drawings, each pipette tip is a generally uniformly tapered column and is arranged to provide a tight seal between the inner surface of the pipette tip column and the nozzle. Advantageously, to improve the seal, the pipette tip column may have an inner diameter smaller than the outer diameter of the nozzle. This fit has been described in the field of micropipettes and is well known to those skilled in the art.

[0066] Advantageously, in the event of blockage at the distal end of the pipette tip column, the seal between each nozzle and its corresponding pipette tip column maintains a pressure seal and a vacuum seal for 5 minutes or longer with a pressure loss or vacuum loss of less than 5%, which can be measured using techniques known in the art.

[0067] In the system according to the invention, the means for applying pressure to the tip of the pipette can be provided by an electric motor. Such a drive device is known in the art and is commercially available for use in common liquid handling equipment.

[0068] The separation of biomolecules can advantageously be achieved by filling a pipette tip column with a separation medium capable of interacting with and retaining the target molecule. Therefore, in the system according to the invention, each column may include a filled separation medium, such as a chromatography medium or extraction medium, such as solid-phase extraction (SPE) media, supported liquid extraction (SLE) media, affinity media, ion exchange media, reversed-phase media, normal-phase media, liquid-free media, hydrophobic interaction media, hydrophilic interaction media, and other chromatography media. These chromatography media are well known to those skilled in the art and are readily available from commercial suppliers.

[0069] This separation medium is advantageously held in place by arranging filter tips (i.e., filters) on each side of the packed medium, at the top and bottom of the pipette tip column, as is common practice in the field of liquid sample processing of biomolecules and other molecules (such as large organic molecules). In this context, reference is made, for example, to US2017 / 0,211,129 (Phynexus, Inc.), which describes in detail packed pipette tip columns, including the properties of such filter tips, packing levels, etc. Those skilled in the art can easily pack the pipette tip column based on such references or simply using common sense and supplier recommendations. In this system, the medium should advantageously be configured as a packing material, i.e., held in place, unlike a fluidized bed of media.

[0070] The further details provided below, and throughout this specification, concerning the second and third aspects of the invention, also apply to this first aspect.

[0071] In a second aspect, the present invention relates to a method for separating biomolecules or organic molecules from a liquid sample using a system according to the invention. This method can be a sample preparation or 'sample setup' method, commonly known as a step prior to the analysis of target molecules. In this sample preparation method, the medium can remove contaminants from the liquid sample; alternatively, the medium can separate the target molecules from other portions of the liquid sample.

[0072] Therefore, in one mode, the method of the present invention may include: a first step of aspirating a sample into each filled pipette tip column of the system; and a second step of dispensing the purified liquid sample from the pipette tip column. In this mode, the dispensed liquid will include target molecules in an environment much cleaner than before being processed in the pipette tip column.

[0073] In another mode, the method includes the additional steps of: drawing eluent into each filled pipette tip column; allowing the eluent to release the target molecule from the separation medium; and dispensing the eluent comprising the released target molecule from the pipette tip column.

[0074] The isolated biomolecules (i.e., the target of this method) can be selected from the group consisting of: proteins (such as antibodies); protein fragments (such as antibody fragments); polypeptides; and nucleic acids (such as linear DNA, linear RNA, oligonucleotides, genomic DNA, or plasmids). Advantageously, this method is used to isolate plasmids intended for subsequent use in recombinant DNA technology, wherein a large number of plasmids are used as vectors. The target of any method of the present invention is to isolate, in this method, other components of the sample, such as other biomolecules, i.e., proteins, polypeptides, cellular debris components, DNA, and RNA; and the target of any method of the present invention is to isolate from nutrients and other components derived from fermentation liquid culture media, wherein the target biomolecule is produced.

[0075] Depending on the intended use of the isolated target biomolecules, this method can be used as a single separation step; or, as part of a purification protocol, in which case it can be combined with steps such as filtration and centrifugation. For example, according to the invention, a filtration step (e.g., gravity filtration) can be performed prior to treatment in the pipette tip column. Those skilled in the art will understand that, depending on the nature of the initial material, conventional treatment steps (such as cell lysis and, for example, removal of cell debris by centrifugation) can be included in embodiments of the method according to the invention.

[0076] As discussed above regarding the system of the present invention, an advantage of the present invention is that the seal between the nozzle and the pipette tip column unexpectedly exhibits the ability to withstand the high pressures required for forward processing of large volumes of liquid. Therefore, in this method, a positive pressure of 3 to 5 psi (e.g., 4 to 5 psi) can be applied to the pipette tip column without significantly damaging the seal of each pipette tip column nozzle. In some embodiments of the invention, a positive pressure of 2 to 15 psi can be applied to the column cavity without pressure leakage for at least 5 minutes, and the pipette tip is not exposed from the pump nozzle. In some embodiments, a vacuum of 1 to 10 psi can be applied to the column cavity without vacuum leakage for at least 5 minutes. No leakage is defined as a variation of no more than 5%.

[0077] All the details provided in this specification regarding the first aspect also apply to this second aspect of the invention.

[0078] In a third aspect, the present invention relates to a kit for separating biomolecules from liquid samples using a separation system according to the invention. The kit may include: a large-volume pipette tip column, such as 20 to 40 mL in volume, filled with chromatography or solid-phase extraction (SPE) media; and written instructions for performing such separation in a separate compartment.

[0079] In addition, this kit may include one or more of the following in a separate compartment: equilibration buffer, washing solution, and elution solution, all of which are well known to those skilled in the art and are commercially available.

[0080] When understanding the third aspect of the invention, all the details provided in this specification regarding the first and second aspects apply.

[0081] In some embodiments of the invention, even when the internal pressure of the column is as high as preferably 5 psi, the sealing mechanism of the pump nozzle provides a tight fit between the pipette tip and the pipette tip column. In some embodiments of the invention, the column remains sealed and attached to the pump nozzle when the internal pressure of the column is in the range of 4 to 6 psi, 3 to 7 psi, 3 to 8 psi, 3 to 10 psi, or greater than 10 psi, 20 psi, 30 psi, 40 psi, or 50 psi. The seal of the column at the pump nozzle is maintained to -2 psi, 3 psi, 4 psi, 5 psi, 6 psi, 7 psi, 8 psi, 9 psi, or 10 psi. Maintaining the seal is defined as maintaining a pressure leakage of no more than 5% for a period of 2 min, 3 min, 4 min, or 5 min.

[0082] As discussed above, the nozzle seal on the inner head of the pipette tip column features an annular protrusion, such as a ring or a pip. In this context, it should be understood that the term "annular" here means that the protrusion is arranged to generally surround the entire circumference of the nozzle. However, any embodiment in which a minor interruption or deviation is included in such an "annular" shape is intended to be covered by the invention, provided that the advantageous and effective seal discussed herein is achieved. This ring will provide a very tight force on the head of the column, preventing air or vacuum from leaking through the nozzle. Maintaining internal pressure or vacuum is crucial for maintaining reliable and predictable column inflow and outflow. If any leakage occurs, liquid will still flow into or out of the column, but the flow will be unpredictable and incomplete. Predictable flow is particularly necessary for robotic liquid processors. Complete flow is required for the complete capture, washing, and elution of the sample from the column.

[0083] In other embodiments of the invention, the diameter of the pump nozzle is increased such that at least a portion of the nozzle provides a very tight fit with the column. In other embodiments of the invention, an O-ring or similar sealing device is added to the nozzle to provide a tight seal between the pump nozzle and the pipette tip column.

[0084] All these sealing mechanisms that enhance the seal address the issue of easily and predictably pushing the column out of the nozzle. In pipetting technology, pushing is performed by hand force. Electro-actuators can provide additional force; however, the pushing mechanism must be uniform and forceful without damaging the push boss at the top of the pipetting tip column. This is because during use, the pipetting tip column may need to be loaded and pushed more than once (up to 5, 10, or 20 times).

[0085] Detailed description of the attached figures

[0086] Figure 1 The diagram illustrates a high-sealing pipette pump and column for an automated separation system according to the present invention. More specifically, Figure 1 The following are shown: a pump motor 1; a cylindrical piston drive screw 2; a pump piston 3; a pusher device, illustrated here as a pusher rod 4; a pump nozzle sealing device 5, illustrated here as a single protrusion of the nozzle; a pipette tip column 6; a column bed 7; and, lastly, an illustrative container containing a liquid that can be aspirated (such as a sample or buffer fluid 8).

[0087] The nozzle arrangement according to the invention provides effective pressure and vacuum sealing on the column. For example, after sealing for 5 minutes or longer, the pressure loss or vacuum loss is less than 5%.

[0088] Figure 2 A closer view is shown illustrating the arrangement of the pump nozzle bottom 13, the pipette tip column top 14, and the sliding pusher 15. More specifically, the following are shown: the pump nozzle 9; the sliding pusher 10; the pipette tip column top 11; and a protrusion in the form of an annular sealing ridge 12.

[0089] The illustrative annular protrusion provides additional force on the inner wall of the tip of the pipette column, which in turn provides a favorable sealing force. The sealing force is advantageously distributed uniformly across the entire inner surface to prevent air leakage, as even small micro-scratches or defects may allow air to escape and prevent a proper seal.

[0090] Figure 3 The top of the pipette tip column 11, positioned at the bottom of the pump nozzle, is shown. An illustrative sealing device 5 in the form of an annular protrusion provides air and vacuum seals inside the column. Further improvements to the nozzle design, including the protrusion according to the invention, include increasing the nozzle diameter and / or using a tight-fitting O-ring.

[0091] Figure 4 An alternative embodiment of the automatic separation system according to the invention is shown, wherein the pump nozzle bottom 13 is arranged with two annular protrusions in the form of a sealing ridge 12, a tip column top, and a sliding pusher.

[0092] Figure 5 The tip of the pipette column is shown positioned with a strong and advantageous force at the bottom of the pump nozzle. In this exemplary embodiment, two annular ridges 12 provide a vacuum seal on the inner side of the column cavity above the bed.

[0093] Figure 6 This demonstrates how a sliding pusher can remove the top of the pipette tip column from the pump nozzle with a strong and advantageous force.

[0094] The sliding pusher removes the top of the column from the pump nozzle with strong force. Pushing the pipette tip column out of the pump nozzle is difficult. For example... Figure 1 As depicted, a rod can be used to push the column. However, the pusher must not deform the top of the plastic tip of the pipette column. In one purification method, the pipette tip column must be loaded and pushed multiple times.

[0095] Experimental Section

[0096] This embodiment is provided for illustrative purposes only and should not be construed as limiting the invention as defined in the appended claims. All references provided herein, either hereinafter or elsewhere in this specification, are incorporated herein by reference.

[0097] Example 1—Plasmid Purification

[0098] Material

[0099] The automated separation system according to the invention is used for plasmid purification. More specifically, the instrument has two nozzles, each with an annular protrusion as described herein, loaded with a column, sample, vial, tube, and buffer. A packed bed of media is prepared by filling a 4 mL bed of DEAE weakly basic anion exchange resin based on a polyacrylate matrix into the column of a pipette tip. The column hardware containing the 4 mL bed thus prepared is constructed using a 20 mL pipette tip, with the packed bed positioned at the distal end of the column. The sample contains 5 g of *E. coli* cell spheres containing the expression plasmid. The sample is dispersed in TRIS buffer and lysed with 1 M NaOH and SDS surfactant to form a suspension of cell debris and dissolved plasmid. RNase is added to the mixture. The added buffer is color-coded and pre-packaged so that no volume or mass measurement is required throughout the sample preparation process. The sample is then poured into a filter sample reservoir to begin instrument processing. Filtration removes most of the particulate matter by gravity filtration. The removal of particulate matter depends on the process. In plasmid capture, proteins, cell debris, genomic DNA, and other substances are removed, while the plasmid is retained in solution and passed through a filter in the filtrate. The filtration process can take up to an hour, but it begins immediately after filtration starts. The sample is aspirated and transferred via pipette to six 50 mL conical tubes containing equal volumes of filtrate. The pipette tip column is inserted into each conical tube containing the sample and the captured plasmid, and the flow is repeated in series until the entire sample filtrate has been processed. Samples from filtrate directly from the sample reservoir can also be processed. In this case, the pipette tip column is inserted into the reservoir, and the filtrate is aspirated. The column is then moved to the conical tube, and the sample is processed using a reciprocating flow method. Alternatively, the sample is aspirated into the waste liquid, and capture is performed by single aspiration and dispensing through the pipette tip column bed. These processes are repeated until all sample filtrate has been processed. The equipment, including all columns, buffers, vials, and filter reservoirs, was obtained from Biotage AB (Uppsala, Sweden).

[0100] method

[0101] All described operations are performed using an automatic separation system according to the invention, including an annular protrusion located on the nozzle.

[0102] Overall procedure:

[0103] 1. Filter the precipitated cell lysate containing the dissolved plasmid to produce a semi-clear lysate. The dissolved plasmid is retained in the filtrate.

[0104] 2. Add proprietary endotoxin removal buffer to the semi-clear lysis buffer and incubate. This binds the endotoxin and prevents it from being captured by the DEAE anion exchange column.

[0105] 3. Equilibrate at room temperature for 30 to 60 minutes.

[0106] 4. Transfer the semi-clear lysis buffer. Transfer the cultured sample to centrifuge tubes. Use 15 mL of sample. Prepare 10 tubes for the next step of capture.

[0107] capture:

[0108] Place the anion exchange column inside the sample conical tube, aspirate the sample into the column, and dispense (absorb and partition) for 4 cycles at a rate of 400 mL / min. Aspirate 12 mL at 400 mL / min with a delay of 45 seconds. Dispense 2 mL at 400 mL / min with a delay of 15 seconds, repeating 6 times. Add a delay at the end of each half-cycle to allow the flow through the column to stop. The plasmid is captured by the DEAE anion exchange column.

[0109] 5. Vacuum washing. Transfer the column and place it in the vacuum washing station. Move the pump downwards to wash the reservoir and aspirate 10 mL of washing buffer.

[0110] Then, 20 mL of air is drawn in at 375 mL / min. 20 mL of air is expelled at 375 mL / min. This process is repeated 5 to 10 times using multiple columns and tips, and pushing the column and tip. Alternatively, in other embodiments, the washing solution is extracted from the tip using only a vacuum.

[0111] 6. Elution. The plasmid is captured in TRIS buffer. Depending on the desired recovery rate, the elution volume can be 5, 10, or 20 mL. Lower elution volumes increase the concentration of recovered plasmid. Higher elution volumes increase the total mass of recovered plasmid.

[0112] Example 2 - Sprayer with enlarged diameter sealing protrusion Mouth.

[0113] Three types of tightly fitting nozzles were used in the embodiments. The first type of nozzle was used in the work described in Embodiments 1 and 3. In this embodiment, the diameter of the protrusion increases unilaterally at the top portion of the pipette tip, reaching a diameter of 20.4 mm. The nozzle diameter at the location without the protrusion and without the pipette string seal is 19.8 mm.

[0114] The second-generation nozzle has a 20.6 mm protrusion that fits at a lower position on the nozzle. The nozzle diameter is 19.8 mm where there is no protrusion and no pipette column seal. This nozzle is also used in the embodiment described in Example 1.

[0115] The third embodiment features a double-ridged protrusion, with the top ridge having a diameter of 20.3 mm and the bottom ridge 20.2 mm. The nozzle diameter at the location without the protrusions and without the pipette column seal is 19.8 mm. Although this diameter is smaller than the second embodiment, the force required for removal is the greatest of the three embodiments. This embodiment is also used in the embodiment described in Example 1.

[0116] Of these three designs, the first generation had the best seal and was the easiest to remove. The third generation had the best seal, but was the most difficult to remove.

[0117] Example 3 - Polypeptide Desalting

[0118] The work described herein is performed by an instrument having a tightly sealed nozzle as described in the first embodiment of Example 2.

[0119] method

[0120] The 20 mL pipette tip column is graduated to 3 mL and fitted with a sieve. 0.5, 1, or 2 g of C18 resin is loaded into the pipette tip. These weights correspond to 1, 2, or 4 mL of resin. The column is washed with ethanol at 190% standard alcohol and the bed is dried under vacuum. A top sieve is attached to create a packed bed column. Resins tested include Isolute C18UC (Biotage, 9220-0500), Isolate C18 EC (Biotage, 9221-0100), MFC18 (Biotage, 240-0005), Sep-Pak (Waters, WAT043350), and C18 reverse silica gel (Sigma, 60757-50G).

[0121] Immerse the column in 20 mL of a wetting solution containing 100% acetonitrile. Perform four 12 mL reciprocating cycles at a flow rate of 400 mL / min, pausing for 60 seconds after each aspiration and dispensing to wet the column. Condition the column in 20 mL of 0.1% TFA solution using the four cycles described above. Add a peptide standard containing trypsin-digested BSA to 10 mL of 0.1% TFA to a final concentration of 1 or 2 mg / mL. Immerse the column in the sample and perform 24 capture cycles of 9 mL at a flow rate of 400 mL / min, pausing for 60 seconds at the end of each aspiration and dispensing to capture the peptide. Wash the column with three consecutive 3 mL samples of 0.1% TFA using four reciprocating cycles. Elute the peptide by applying 3 mL of 40% acetonitrile solution to the top of the column and collect the flow.

[0122] The samples were analyzed for UV / Vis absorption using a NanoDrop spectrophotometer and subjected to polyacrylamide gel electrophoresis.

[0123] result

[0124] Small-scale purification showed very little difference between resins.

[0125] Five different resins, each 500 mg, were packed into columns to screen for the resin exhibiting the highest selectivity and yield for the peptide. For each column, 10 mg of peptide sample was added to 5 mL of capture buffer, and capture was performed through 32 cycles, followed by washing and elution. Purification was repeated. Nanodrop was used to analyze flow-through and elution. Note that the sample and sample buffer contained UV-absorbing impurities, which affected the accuracy of flow-through analysis. However, the analysis revealed trends useful for method development.

[0126] The flow and elution data show some variation between columns, which is expected for screening experiments. Without accounting for this variation, there is virtually no difference in capture performance or elution between the various resins (Table 1).

[0127] Due to its low variability in replication, MFC18 was chosen for development of methods for subsequent extensions.

[0128] Table 1: Small-scale resin screening

[0129]

[0130] Resin expansion and impaired flow

[0131] MFC18 was extended to a 2 mg column for subsequent method development. However, flow problems were observed. The MFC18 column could not be fully wetted and conditioned, resulting in low performance. The increased resin volume revealed an unexpectedly high back pressure. The back pressures of the five resins tested in small-scale trials were retested when the loaded volume was increased from 500 mg to 2 g. Based on these studies, C18 UC exhibited the lowest back pressure and is the best candidate for extension.

[0132] Efficient recycling is scalable.

[0133] The optimized method was further tested to determine if the separation could be used for larger sample volumes requiring increased mass recovery. 38 mg of the peptide was added to 20 mL of capture buffer. Purification using Isolute (Biotage, 9220-0500) was repeated. Capture completion was assessed by removing 1 μL after 8 and 32 capture cycles and analyzing the elution using NanoDrop.

[0134] Approximately 80% of the peptides were captured after 8 cycles, and this percentage was increased to approximately 90% after 32 cycles. The captured peptides were completely recovered during elution, with approximately 34 mg of the initial 38 mg measured.

[0135] Table 2: Plasmid Recovery

[0136]

Claims

1. An automated separation system comprising a liquid handling device and at least one pipette tip, wherein the pipette tip includes a separation medium, and the liquid handling device is equipped with: one or more nozzles, each nozzle being arranged to receive the pipette tip; and means for applying positive or negative pressure to achieve aspiration and dispensing of each pipette tip, wherein in the automated separation system, each nozzle has at least one annular protrusion, and each pipette tip does not have a groove corresponding to the at least one annular protrusion.

2. The automatic separation system according to claim 1, wherein each nozzle is provided with at least two annular protrusions, said at least two annular protrusions being arranged to seal against the tip column of the installed pipette.

3. The automatic separation system according to claim 1, wherein each nozzle is provided with a sliding pusher.

4. The automated separation system according to any one of claims 1-3, wherein each pipette tip column is capable of holding at least 20 mL of liquid volume.

5. The automated separation system according to any one of claims 1-3, wherein each pipette tip column is uniformly tapered and arranged to provide a tight seal between the inner surface of the pipette tip column and the nozzle.

6. The automatic separation system according to any one of claims 1-3, wherein the seal between each nozzle and the tip column of the pipette is arranged to maintain a pressure seal or vacuum seal for 5 minutes or longer when the pressure loss or vacuum loss is less than 5%.

7. The automatic separation system according to any one of claims 1-3, wherein each pipette tip column can be mounted to the nozzle using a force in the range of 50N to 160N.

8. The automated separation system according to any one of claims 1-3, wherein each pipette tip column can be released by pushing with a force in the range of 50N to 160N.

9. The automated separation system according to any one of claims 1-3, wherein the means for applying pressure to the tip column of the pipette is provided by an electric motor.

10. The automated separation system according to any one of claims 1-3, wherein the automated separation system comprises at least two independently arranged pipette tip columns.

11. The automated separation system according to any one of claims 1-3, wherein each pipette tip column comprises a filled separation medium.

12. The automated separation system according to any one of claims 1-3, wherein the separation medium is selected from the group consisting of: chromatography media, solid phase extraction (SPE) media, and supported liquid extraction (SLE) media.

13. The automated separation system according to any one of claims 1-3, wherein each pipette tip column is defined by a uniform slope.

14. The automated separation system according to any one of claims 1-3, wherein each pipette tip column is uniformly conical.

15. The automatic separation system of claim 1, wherein the liquid handling device is equipped with two or more nozzles.

16. The automated separation system according to any one of claims 1-3, wherein each pipette tip column is capable of holding liquid in the range of 20 mL to 40 mL.

17. The automated separation system according to any one of claims 1-3, wherein the automated separation system comprises two to four independently arranged pipette tip columns.

18. A method for separating biomolecules from a liquid sample using an automated separation system according to any one of claims 1 to 17, wherein: The sample is aspirated into each of these pipette tip columns; the biomolecules of the aspirated liquid sample are allowed to react with the separation medium for a period of time; and a liquid sample comprising any unreacted biomolecules is dispensed from the pipette tip column.

19. The method of claim 18, the method further comprising the steps of: drawing eluent into each filled pipette tip column; allowing the eluent to release biomolecules from the separation medium; and dispensing eluent comprising the released biomolecules from the pipette tip column.

20. The method according to claim 18 or 19, wherein the isolated biomolecule is selected from the group consisting of: proteins; polypeptides; and nucleic acids.

21. The method of claim 18 or 19, wherein a positive pressure of 3 to 5 psi is applied to the pipette tip column without significantly damaging the seal of each nozzle and its corresponding pipette tip column.

22. The method of claim 20, wherein the nucleic acid is linear DNA, linear RNA, or a plasmid.

23. The method of claim 18 or 19, wherein a positive pressure of 4 to 5 psi is applied to the pipette tip column without significantly damaging the seal of each nozzle and its corresponding pipette tip column.

24. A kit for separating biomolecules from a liquid sample using an automated separation system according to any one of claims 1 to 17, the kit comprising: A 20 to 40 mL pipette tip column is filled with chromatography medium, supported liquid extraction (SLE) medium, or solid phase extraction (SPE) medium. And, in a separate compartment, written instructions for carrying out such separation.

25. The kit of claim 24, wherein the kit comprises, in a separate compartment: One or more balanced buffer solutions; Washing liquid; And, the elution buffer.

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