• 专利附录
  • 专利说明
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    • 专利摘要:
    MULTIFUNCTIONAL DEVICE AND METHOD FOR SAMPLE PREPARATION It is a device for sample preparation that allows a complete process of binding, washing, elution, exchange and concentration of buffer to be performed without a transfer of samples between the multiple devices. The device includes a reservoir, a column that serves to retain the chromatography medium, a retaining region that serves to maintain a filtration device, and an outlet. The filtration device connects to the retaining region of the centrifugal device, and the assembly can be placed in an optional retainer. The assembly, with or without an optional retainer, can be placed in a conventional centrifuge tube for centrifugation. All steps of binding, washing, elution and buffer concentration can be carried out with the device without any pipetting transfers (and the associated sample losses). The sample preparation device can also be used for connection and washing steps, in the case where the filtration device is not needed, and for buffer exchange and concentration steps, in the case where the medium is not needed.
    公开号:BR102012017457B1
    申请号:R102012017457-0
    申请日:2012-07-13
    公开日:2020-10-27
    发明作者:Christopher A. Scott；Kurt Greenizen；Sara D. Gutierrez；David Briggs；Ralph T. Scaduto；Timothy K. Nadler；Louis Bonhomme；Masaharu Mabuchi；Phillip Clark
    申请人:Emd Millipore Corporation；
    IPC主号:
    专利说明:

    [001] This application claims priority for US Provisional Order with Serial Number 61 / 507,240 filed on July 13, 2011 and for US Provisional Order with Serial Number 61 / 648,631 filed on May 18, 2012, with the descriptions of these incorporated by reference. Background
    [002] Centrifugal filters can be used to separate biological substances such as an antibody enzyme, nucleic acid and protein for the purpose of concentration, desalination, purification, and fractionation. These devices are most commonly used in centrifugal separator instruments, which can consist of a fixed angle rotor configuration or an oscillating or variable angle rotor configuration. The speed of the filtration process and the recovery of the retentate sample are highly evaluated by consumers. Sample recovery values greater than 85% are generally obtained by removing the membrane capsule (sample retainer) and turning it upside down in a receiving tube.
    [003] These devices are typically used to concentrate urine, serum, plasma and cerebrospinal fluid. For example, the measurement of specific proteins in the urine can be important for the diagnosis and management of various disease states, yet the content of these proteins in the urine is generally too small to be detected without first concentrating the proteins. In general, conventional devices include a compartment having a sample reservoir, a filter sealed in the compartment such that the sample must pass through the filter when subjected to an actuating force (such as centrifugation), and a collection chamber that serves to collect the concentrated sample.
    [004] There is a class of protein purification protocols that use antigen-protein affinity to separate the proteins of interest from a mixed sample, such as a cell lysate or serum. Generally, these protocols use small microspheres that are conjugated to antibodies in such a way that they bind to specific proteins from the sample. Once the proteins are effectively linked to the microspheres, there is a need to extract and collect the proteins (elution) from the microspheres for downstream analysis, assay development, etc. Exemplary downstream analysis techniques include two-dimensional gel electrophoresis and mass spectrometry.
    [005] There are a number of processing steps that are necessary in the workflow. These may include balancing the microspheres with a neutral buffer before ligation, washing the microspheres after ligation to remove unbound contaminants, eluting the proteins of interest, changing the buffer of the eluted proteins, concentrating the final diluted sample, and finally recovering the purified protein sample. For affinity purification and immunoprecipitation protocols, the proteins attached to the microspheres are the proteins of interest. For depletion protocols, the unbound fraction (proteins not bound to the microspheres) is the sample of interest.
    [006] The microspheres used in these purification methods are magnetic or non-magnetic. One of the most common non-magnetic microspheres is agarose. Magnetic microspheres such as PureProteome A & G protein, PureProteome albumin and PureProteome albumin and IgG for albumen and serum IgG depletion, Magna ChIP protein microspheres for chromatic immunoprecipitation, and PureProteome nickel magnetic microspheres for recombinant purification with His tag, are commercially available from EMD Millipore.
    [007] When working with magnetic microspheres, current manual methods depend on the use of pipettes to move liquids in the sample tube (plugs, etc.) and move the sample from one device to another. Magnets are used to keep the microspheres next to the sample tube so that the user can pipet the plugs without disturbing the microspheres. There are about 8 pipette steps per sample in a typical binding / washing / eluting workflow.
    [008] For optimal protein binding to microspheres, incubation with these methods is required. The device containing the microspheres and the sample are usually rotated in an end-to-end mixer (from English, “end-over-end”), or placed on a shaker (for example, vortexing) for 10 to 30 minutes. When new buffers are added, such as buffers and washing and eluting, the user will vortex the device for approximately one minute to mix and wash.
    [009] The washing and elution steps need to be repeated several times in order to be effective. For example, the standard protocol is to add wash buffer to the sample bottle, vortex (mix) for approximately one minute, remove the buffer and repeat two or more times. With the magnetic microspheres, the ligation / washing / elution procedure takes about 45 minutes.
    [010] An alternative to magnetic microspheres is agarose microspheres. A commercially available device using agarose microspheres includes a tube with an open bottom and a porous frit positioned on the open bottom. Rather than using pipettes to remove fluids from the sample tube, a bench top centrifuge is used to drive fluids through the frit and into a collection tube - typically a 4 ml or 15 ml tube. The frit pore size is chosen for retaining the microspheres while allowing buffers and proteins to pass through.
    [011] Depending on the size of the centrifuge column used, the workflow can be inconvenient and time-consuming compared to methods using magnetic microspheres. A bench top centrifuge is typically a shared piece of equipment located in a common location; unlike microcentrifuges that each user may have configured on their desktop.
    [012] This process requires 16 pipetting steps per sample and takes about 1 hour to complete.
    [013] For magnetic and agarose workflows, the downstream steps may include changing the carrier buffer and concentrating a diluted sample. In cases where a sample buffer change is desired, perhaps to remove the imidazole-type eluent, the sample is typically transferred to a dialysis membrane tube with clamps or the like, which is then placed inside a buffer exchange for up to 24 hours as the buffer is gradually changed via diffusion.
    [014] When buffer exchange and concentration are desired, a diafiltration / protein concentration device, such as a centrifugal device with a porous UF membrane sized to retain the proteins, however, allow the buffer to pass through . By controlling the spin time and selecting an appropriate device design, the final concentration can be controlled. For the buffer exchange to be effective, the buffer exchange step needs to be repeated two or three times (as was done with the washing and elution steps). These devices take 30 to 45 minutes and require multiple rotations in a centrifuge. In the Amicon Ultra device commercially available from EMD Millipore, there are 5 pipetting steps for buffer exchange and concentration.
    [015] As protein sample volumes become smaller, potential unwanted sample losses due to the retention volume within a device have become more important than ever. Current data suggest that a loss of 10 pL in a concentrated sample of 50 pL represents 80% protein recovery. If protein loss is reduced by an order of magnitude from 10 pL to 1 pm, protein sample recoveries can be increased from 80% to 98%. An 18% improvement in protein sample retrieval can be quite valuable.
    [016] It is desired to provide a device and method that perform an efficient and effective binding and washing, a buffer exchange and concentration, and / or a complete binding, washing, elution, exchange and buffer concentration process in a single device without the need for pipetting the precious sample between devices, particularly for sample sizes up to about 11 ml. summary
    [017] The problems of the prior art have been overcome by the modalities described here, which, in certain modalities, include a sample preparation device that allows a connection and washing, a buffer exchange and concentration, and / or a complete connection process , washing, elution, exchange and concentration of buffer to be carried out without transferring samples between multiple devices. According to certain modalities, a centrifugal device is provided that includes a reservoir having an inlet, a column to hold the medium, such as a bed of packaged microspheres, a retaining region that serves to receive in a sealing relationship a device filtration, and an outlet. According to certain embodiments, the filtration device includes a compartment having a sample reservoir, one or more, preferably two, substantially vertically oriented membranes (spaced when more than one is present) disposed in the compartment, a drainage channel associated with each membrane in such a way that the fluid that passes through each membrane flows through a respective lower drainage channel in a filtrate collection chamber. The filtration device joins the retaining region of the centrifugal device, and the assembly can be placed in an optional retainer. The assembly, with or without the optional retainer, can be placed in a conventional centrifuge tube for centrifugation. All steps of binding, washing, elution, exchange and buffer concentration can be performed with the device without any pipetting transfers (and the associated sample losses), resulting in superior recovery of the sample of interest. The sample preparation device can also be used for connection and washing steps, in the case where the filtration device is not needed, and for buffer exchange and concentration steps, in the case where the medium is not needed. Multiple buffer changes can be performed on the same device.
    [018] According to certain embodiments, the device may include a retractable feed tube, such as to help reduce the loss of sample solutions that accumulate in the inner wet hole and on the outer surface of the feed tube.
    [019] According to certain modalities, a sample is incubated with the medium in position on the device in such a way that the selected target binds to the medium medium. Then, the remaining free sample can be washed. The sample is purified by eluting the target sample of interest from the medium by adding a buffer that causes the medium to release the captured target back into the solution. Once a sample is purified, it can be concentrated and a useful concentration for analysis and storage (most proteins are more stable when stored at a concentration close to 1 mg / ml).
    [020] According to certain embodiments, the sample preparation device may include a guiding member or diaphragm that can be actuated to evacuate small portions (eg, holding volumes) of sample from the device.
    [021] The sample preparation device results in time savings for binding, washing, buffer exchange, and / or binding, washing, elution and concentration protocols. It is not necessary to pipet the sample, resulting in greater sample recovery. The buffer exchange can be carried out substantially in less time than previously possible, with a single centrifugation step for each of the buffer exchange and washing steps, instead of multiple centrifugation steps previously required. No binding incubation period is necessary.
    [022] According to certain modalities, the mounting interface between the filtration device and the exchange chamber can allow relative movement or separation, such as by mechanical means such as a physical stop or a self-activating geometry subjected to a centrifugal pressure gradient to remove the tip hitch with captured target in order to optimize sample recovery.
    [023] The advantages obtained by the described devices and methods include, but are not limited to, shortened incubation times for affinity separation processes; improved sample concentration on a device platform; improved sample recovery using reverse centrifuge devices; and single centrifuge exchange dilutions. Brief description of the drawings
    [024] Figure 1 is a perspective view, in cross section, of a reservoir / exchange member according to certain modalities; Figure 1B is a perspective view, in cross-section, of a reservoir / exchange member containing a column of pre-packaged microspheres according to certain modalities; Figure 2 is an exploded view of a reservoir / exchange member and the filtration device according to certain modalities; Figure 3 is a vertically oriented cross-sectional side view of a filtration device according to certain modalities; Figure 4 is a perspective view, in cross section, of a reservoir / exchange member and of a filtration device positioned therein according to certain modalities; Figure 5 is an exploded view of a reservoir / exchange member, a filtration device, an optional mounting retainer, a tube and a centrifuge cover according to certain modalities; Figure 6 is a cross-sectional view of an assembly including a reservoir / exchange member, a filtration device, a mounting retainer, a tube and a centrifuge cover according to certain modalities; Figure 7 is a cross-sectional view of an assembly including a reservoir / exchange member, a filtration device, a mounting retainer, a tube and a centrifuge cap, showing the tip of the reservoir / exchange member positioned in the region fixed stop volume of the filtration device, according to certain modalities; Figure 8 is a cross-sectional view of an assembly including a reservoir / exchange member, a filtration device, a mounting retainer, a tube and a centrifuge cap, showing the tip of the reservoir / exchange member raised from the region of fixed stop volume of the filtration device, according to certain modalities; Figure 9 is an exploded perspective view of a connection, washing, elution and concentration device according to certain modalities; Figure 10 is a cross-sectional view showing the volume occupied by the exchange member in the filtration device according to certain modalities; Figure 11 is a cross-sectional view of a reservoir / exchange member having a portion with convolutions to allow axial movement during centrifugation, according to certain modalities; Figure 12 is a cross-sectional view of a reservoir / exchange member having a thin wall portion to allow axial movement during centrifugation, according to certain modalities; Figure 13 is a cross-sectional view of a reservoir / exchange member having a thin wall portion over-molded to allow axial movement during centrifugation, according to certain modalities; Figure 14 is a diagrammatic view of a reservoir / exchange member showing the location of roughness molded on the outer surface of the column, and the exploded detail shows a cross section of the wet surface and the gas boundary layer; Figure 15 is a bottom perspective view of a diaphragm cap according to certain embodiments; Figure 16 is a top perspective view of the diaphragm cap of Figure 15 according to certain embodiments; Figure 17 is a cross-sectional view of a reservoir / exchange member including the diaphragm cap of Figure 15 according to certain embodiments; Figure 18 is a top perspective view of a reservoir / exchange member having a flange modified to accommodate the diaphragm cap of Figure 15 according to certain embodiments; Figure 19 is a cross-sectional view of an assembly including a diaphragm cap in accordance with certain embodiments; Figure 20 is a top perspective view of a reservoir / exchange device showing a diaphragm cap affixed in an open position; Figure 21 is a side view of a reservoir / exchange device showing a diaphragm cap affixed in an open position; Figure 22 is an exploded view of a reservoir / exchange device including a diaphragm and a 15 ml filter according to certain embodiments; Figure 23 is a perspective view of a reservoir-exchange device according to a first alternative embodiment; Figure 24 is a perspective view of a reservoir-exchange device according to a second alternative embodiment; Figure 25 is a perspective view of a reservoir-exchange device according to a third alternative embodiment; Figure 26 is a graph showing device retention volumes with and without diaphragms according to certain modalities; and Figure 27 is a graph that compares a 3-spin procedure to a ligation-wash-elution procedure according to certain modalities. Detailed description of the invention
    [025] First, going back to Figure 1, a reservoir / exchange member 12 is presented according to certain modalities. The member 12 is preferably made of a transparent, low bond material capable of withstanding the forces typically encountered during centrifugation. Suitable materials include clarified polypropylene or polycarbonate. In certain embodiments, member 12 includes a generally cylindrical sample reservoir 14 having an open top (inlet), although other formats are suitable and are within the scope of the embodiments described herein. A top annular flange 16 having an outer diameter larger than the outer diameter of the reservoir 14 that can seat a centrifuge tube cap (not shown in Figure 1) can be provided. The volume or capacity of the reservoir 14 is not particularly limited, and can be chosen based on the size of the sample and / or the size of the filtration device to which the reservoir is to be connected. Exemplary volumes include 3 ml and 11 ml. In certain embodiments, the bottom of reservoir 14 has a frustro-conical shape, tapering downward (in the direction of fluid flow from the open top) and radially inward, converging to a central opening that leads to a column 18 smaller in diameter than that of reservoir 14. The upper portion of column 18 has a diameter and length chosen to maintain a sufficient amount of medium 30 to carry out a connection step, and therefore is positioned downstream in the direction of flow sample during a connection operation, from the sample reservoir 14. In modalities where connections are not desired (for example, a buffer exchange and concentration protocol), the medium can be omitted from column 18. Preferably, the diameter and the column lengths are sufficient to maintain at least 200 microliters of microspheres, creating a packed bed. An exemplary diameter is about 0.635 cm (% inch), with a length of 1.27 cm (14 inch) or more. When a medium is used, a medium retention structure, such as a porous frit 31, can be placed below the medium 20 to hold the medium in position, in the case of a pre-packed column, there would be a need for a medium structure. additional retainer 31a positioned above the medium to contain the medium within the diameter of the column (Figure 1B). The column 18 may include an annular flange 19, which provides a shoulder or stop against which the filtration device 50 is positioned during its use (for example, with an interposed seal 40 (Figure 2), providing a sealing interface liquids between the filtration device 50 and the member 12). Below the annular flange 19, the column has a retaining region 20 of intermediate diameter, which is received in the upper portion of the sample reservoir of the filtration device when positioned on the column during use. The region 20 has an outer diameter smaller than the outer diameter of the annular flange 19. An additional downward diameter portion 21, having an outer diameter smaller than the outer diameter of region 20, sits on a lower region of the reservoir portion samples from the filtration device, above the membranes, when positioned on the column during use.
    [026] The region 22 of column 18 has a finned geometry, tapering radially from a relatively thick upper portion 22a to a relatively thin lower portion 22b, and defines a binding / elution chamber. In the thinner portion 22b, the column tapers radially inward at 22c, converging on a stem 23, preferably centrally located, which has an open bottom end 24, the stem extending axially from the shaped column of fins. The finned feature is shaped to fit within the corresponding filter device 50 with the hollow inner part to maintain a uniform wall thickness. According to certain modalities, the finned feature allows the region 22 of the column 18 to occupy substantially the entire volume between the membranes 12A and 12B of the filtration device 50, thus maintaining the desired sample volume close to the open bottom end 24. Preferably, the rod is cylindrical and tapers radially inward towards the open bottom end 24. The open bottom end 24 allows fluid communication between the reservoir 14 (through any means and fries present, and through the region 22 (binding / elution chamber)) and a downstream device, such as a tube or filtration device.
    [027] According to certain modalities, the medium can be a chromatography medium, such as the medium used to capture selected analytes in a sample and release them when the buffer conditions are appropriately changed. Suitable medium includes microspheres that bind metal chelate, protein A, glutathione, albumin, etc. The medium can be magnetic, non-magnetic, agarose, etc., and can be modified with certain chemicals, such as IMAC, protein A, glutathione, streptavidin, etc. Consequently, the medium can include appropriate chemicals to perform the desired bond.
    [028] According to certain modalities, the lower portion with fins and the column can form a single separable resource that can be fixed to the reservoir 14, such as through a pressure fitting, luer fitting, thread, etc. The separable feature reduces the amount of plastic waste and the costs of available, as the reservoir portion can be washable and reusable. An example device where the column 18 is removable is shown in Figure 23. Although any suitable connection mechanism can be used to connect the column 18 to the reservoir 14, Figure 23 shows an embodiment where the column 18 includes a threaded portion 118 which is threadedly received at the lower portion 144 of the reservoir 14 containing the internal grooves 145 compatible with the threads in the threaded portion 118. The threaded portion 114 is preferably cylindrical and circumscribes the nozzle 146 which is in fluid communication with the reservoir 14. The removal capacity of column 18 allows flexibility in the use of different columns 18 with the same reservoir 14. Figures 24 and 25 show similar modalities where multiple columns can be attached to the reservoir. For example, reservoir 14 'in the embodiment of Figure 24 has two outlets, and two removable columns 18A and 18B can be attached, each to a respective outlet. Similarly, reservoir 14 ”in the embodiment of Figure 25 has four outlets, and four removable columns 18A, 18B, 18C, and 18D can be attached, each to a respective outlet.
    [029] Figure 2 is an exploded view of the member 12 and the filtration device 50, with the filtration device 50 shown oriented so that it is received by the column 18 of the member 12. Due to the flattened and tapered configuration of the region 22 of the column 18, and due to the symmetry of the filtration device 50, the filtration device 50 can only fit to the column 18 in one of two ways; the one shown in Figure 2, and the one rotated 180 ° from it. In the embodiment shown, a seal 40 is used to provide a liquid-tight seal between the filtration device 50 and the member 12. Another sealing mechanism can be used, such as O-rings or over-molded seals with adapted shape including seals face, flexible tongue type seals, etc. These overmoulded seals can be integrally molded.
    [030] Turning now to Figure 3, a filtering device 50 suitable for use according to certain modalities is shown. The filtration device 50 is that described in U.S. Publication No. 2009/0078638, the description of which is incorporated by reference. Device 50 includes a sample reservoir 11 for receiving an unfiltered sample, and first and second membranes 12A and 12B arranged on a side wall of device 50 as shown. A holding chamber 14 that defines a fixed stop volume is provided below the membranes 12A and 12B. A collection tip 30 which is generally arc-shaped and protrudes outwardly from the bottom of the device can be provided to locate the fixed stop volume on the center line of the device, and subsequently reduce the variability of the volume of the device. fixed stop as the orientation angle in a centrifuge changes. Preferably, device 50 is made of a solid material that is impermeable to liquids, has low protein binding characteristics, and is strong enough to withstand the gravitational forces (Gs) applied during centrifugation. Suitable materials include acrylic, CYROLITE G20 HiFlo resin, ESTAR HN631 resin and KRATON polymers. In particular, the side panels can be made of a transparent plastic material that allows an operator or user to look through the internal cavity of the device in order to determine the fluid levels before, and after the filtration process. The side panels include a lower drainage channel support that supports the membrane and provides fluid communication to the holding chamber 14. For example, the lower drainage channel support may include a series of spaced longitudinal grooves, channels, or surface textures. that are located below the membrane to capture the filtrate as it passes through the membrane and directs it towards the drainage holes and in a receiving flask. Each membrane is sealed to a respective side panel in such a way that only the fluid that passes through the membrane can escape through the device's drain holes located in the side panels. In certain embodiments, each membrane 12A, 12B is co-extensive with a respective bottom drain channel support and sealed thereto. The geometry of the lower drainage channel is designed to support the membrane and keep it as flat as possible, while allowing sufficient open space below the membrane to allow fluid to flow and pass through the drain holes 18 of the device. It is preferred that the hydraulic fluid resistance is kept as low as possible.
    [031] Suitable membranes include microporous and ultra vaporous membranes, the latter being useful for ultrafiltration. The regenerated cellulose ultrafiltration membranes (for example, the “Ultracel Amicon YM” and “Ultracel PL” membranes available from Millipore Corporation of Bedford, Mass., USA) are well suited for devices aimed at concentrating or desalinating extremely sample liquids diluted or hydrophobic. The use of a hydrophilic membrane having a “hermetic” microstructure promotes good retention with low absorption of protein, DNA, and other macromolecules. Polyethersulfone ultrafiltration membranes (for example, “Amicon PM” and “Biomax PB” also available from Millipore Corporation), or other similar membranes having an “open” microstructure suitable for rapid separation, are best suited for devices intended for concentrate and desalinate more concentrated sample liquids, such as serum, plasma, and conditioned tissue culture.
    [032] Preferably, each membrane 12A, 12B is oriented at a small angle to the longitudinal centerline of the device 10, such that the top of each membrane is spaced from the longitudinal centerline at a distance greater than the membrane bottom. A funnel-shaped configuration is formed. Therefore, the positioning of each membrane takes advantage of the effects of tangential flow during centrifugation. An angle greater than about 0 ° and less than about 5 °, preferably about 3 °, has been found to be suitable.
    [033] The side panels include one or more drainage holes 18 (Figure 2) that are in fluid communication with the holding chamber 14 and allow the filtrate to pass through the device compartment for collection in another compartment. Preferably, each drainage hole 18 is located at the bottom of a respective lower drainage channel groove or channel and is preferably substantially circular in cross-section. The drain holes must be located at a sufficient distance from the side edges of the panels in such a way that the holes are not constricted or otherwise impaired during a heat seal operation that can be used during the manufacture of the device. Preferably, the drain holes 18 are equally spaced from each other and are co-linear.
    [034] Figure 4 shows the member 12 with a filtration device 50 positioned in position on the column 18 of the member 12 according to certain modalities. The seal 40 can be observed by providing a sealing interface between the filtration device 50 and the flange 19 of the column 18. Preferably, the stem 23 of the member 12 is positioned so that it is within the fixed stop volume of the filtration device 50, as discussed in greater detail below.
    [035] As noted in Figure 5, according to certain modalities, an optional mounting retainer 60 can be provided where keeping the member 12 attached to the filtration device 50 is a concern under the centrifugal forces typically applied to the assembly. In certain embodiments, retainer 60 may be made of the same material as member 12, and includes a cylindrical upper retaining sleeve 62 configured to receive reservoir 14 from member 12. An upper annular flange 6 extending radially outwardly provides a seat for the annular flange 16 of the member 12. According to certain modalities, the bottom 63 of the retaining sleeve 62 has a frustro-conical shape to receive the similarly shaped bottom of the reservoir 14. An opening centrally located on the bottom 63 leads to a cylindrical column extending axially 64 having an orifice. The orifice is shaped to receive the filtration device 50, such that a lower portion of the filtration device 50 protrudes axially out of the orifice (Figure 6).
    [036] To assemble components such as for centrifugation, filtration device 50 can be inserted over column 18 of member 12, and then can be inserted into retainer 60. The combination is then placed in a centrifuge tube conventional 70 (for example, 15 or 50 ml), having an outside diameter such that the flange 61 rests on the top surface of the tube 70 (Figure 6). Then, the cap 72 can be threaded onto the tube or otherwise connected to secure the assembly to the tube. When optional retainer 60 is omitted, flange 16 of member 12 rests on the type surface of tube 70.
    [037] For most purification and IP protocols, the connection and washing steps can be performed with a filtration device separate from column 22. The filtration device can then be attached to column 22 and proteins eluted directly to from the device through centrifugation. For depletion in which the unbound fraction is the sample of interest, the filtration device can be left attached to column 22 from the beginning of the process.
    [038] In certain embodiments, the column of medium 30 is concentrated in the medium, preferably microspheres, in a column, as well as a column of chromatography. This creates a packaged bed in which the fluid is conducted through the bed so as to increase the likelihood of interaction between the mobile (throughflow) and stationary (medium) phases. This leads to more efficient binding, washing and elution. In fact, the number of washing and elution steps can be reduced from three to one, with minimal or no binding time (incubation). The recovered protein shows increased activity when only a single concentration step is used. When a bond (incubation) is desirable, frit 31 is preferably a hydrophobic frit, which inhibits sample flow through the frit, thus allowing extended incubation times until it is centrifuged to start the flow. For example, fries that comprise hydrophobic material allow the incubation of agarose in magnetic microsphere solutions without dripping, and when subjected to centrifugal G forces between about 100 and about 700 G, allow the filtrate to pass through the receiving tube. Suitable materials include shirt-synthesized polypropylene produced by Porex Corporation, and a filament extruded polypropylene produced by Filtronna Corporation that has been treated with a surface coating, such as a fluorinated plasma treatment. When no connection is desired, such as for buffer exchange and concentration, the medium can be omitted from the assembly. The frit (medium retention structure) can also be omitted, however, since it does not interfere with the buffer change, it can be left in the device, if desired.
    [039] The finned bottom portion of column 22 allows a new buffer solution to be exchanged more efficiently than conventional methods. Although the present inventors do not adhere to the theory, it is believed to work based on a principle of diafiltration. The compatible geometry between the exchange column and the filtration device optimizes the flow of new buffer through the system. According to certain modalities, the fin geometry preferably fills most of the unused cavity space within the filtration device and keeps the sample volume close to the outlet port 24. Since the new plug inside the column 22 and the sample within the filtration device is in static equilibrium during centrifugation, as the head height of the new buffer decreases, the volume of sample leaving the system at any given time is small while there is a large amount of buffer new flowing through. This leads to high efficiency. As can be seen in Figure 10, the displacement "A" between the membrane surface facing fin 12A (and between the membrane surface facing fin 12B) is greater than the displacement "B" between the surface of the fin facing the surface of the filtration device where no membrane is present (for example, in 81A and 81B), such that the fin does not occlude portions of the membrane that may block the flow through the membrane. In one embodiment, the “A” offset between the fin surface facing each membrane is about 0.0127 cm (0.005 inch) to about 0.0508 cm (0.02 inch), preferably 0.0508 cm (0.02 inch). The displacement "B" between the surface of each fin facing the surface of the filtration device where no membrane is present is between about 0.0508 cm (0.02 inch) and about 0.0127 cm (0.005 inch), preferably, about 0.0127 cm (0.005 inch). Optimally, the amount of sample volume is minimized (to optimize the exchange rate) while optimizing the flow characteristics of the membrane. The fin geometry helps to locate the fluid between the active area of the membranes and the fin, and minimizes the amount of fluid between the inactive membrane regions and the fin.
    [040] Furthermore, by positioning the stem 23 on the fixed stop of the filtration device, the mixture is improved, avoiding the aggregation induced by denaturation, and the drying of the protein is avoided since a new buffer is always available through the buffer exchange column. A more efficient mixture of buffer solutions is obtained because a control volume is formed in the fluid space of the fixed stop volume. Within this control volume, there is a stable flow system. The buffer solution from reservoir 14 enters, and the mixed solution exits through the drainage holes 18. Within the control volume, the flow of buffer solution exiting tip 23 creates and maintains a vortex mix flow. It is this vortex flow that creates the most efficient mix of buffer solutions and sample fluids. As can be seen in Figure 7, an optimal and efficient buffer exchange is achieved when the tip 23 of the exchange device is submerged almost close to the bottom (without coming into contact with the bottom surface that can occlude the exit orifice and obstruct the flow) of the sample volume compartment of the filtration device 50 during centrifugation.
    [041] According to certain modalities, there is an additional benefit in being able to provide a relative movement between the tip 23 and the filtration device 50, such as raising the tip 23 of the exchange device from the sample during the centrifugation once the buffer change has been performed, as shown in Figure 8. This reduces the potential sample loss due to sample adhesion to the external and internal surfaces of the tip due to the surface tension of the materials. The relative movement between the tip and the filtration device can be achieved by mechanical means such as a physical stop or by self-activating geometry subjected to a centrifugal pressure gradient to remove the tip engagement with the captured target to optimize sample recovery. .
    [042] The inclusion of a retractable tip design such as the one shown in Figure 11 helps to reduce the loss of sample solutions that accumulate in the internal wet hole and the outer surface of the tip. Initially, a concentrated protein sample solution is already located at the bottom of the filtration device 50. The buffer exchange solutions are added to the reservoir 14 and allowed to pass through a frit material and into the filtration device. During centrifuge operations, the buffer exchange solution is now found in the filtration device 50 where mixing takes place. This mixture allows the protein sample of saline concentration to be diluted by the buffer exchange solution. When the centrifugation has been completed, the tip end 23 can still extend into the volume of the concentrated sample. The small amount of sample that decreases in the inner hole of the tip's distal end and also lines the surface of the tip's outer wall can be as large as 5 or 6 µl. The most successful mixing behavior occurs because the distal end of the tip is submerged in the concentrated sample volume.
    [043] One option is to perform a secondary spin operation to move this loss from 5 to 6 pL of solution. This may involve stopping the centrifuge and using mechanical maintenance to suspend the entire reservoir out of the sample volume at a distance of 0.254 cm (0.100 inch). However, the use of a secondary spin is undesirable.
    [044] In contrast, a retractable tip design such as that shown in Figures 11 to 13 allows the tips to be attracted to the sample volume due to G forces when the centrifuge increases the centrifugation speed, and elastically removed from the sample volume when the centrifuge rotate to a speed equal to zero, the G forces take the tip 23 to the bottom of the sample volume of the filtration device 50, and promote the most effective mixing behavior that is necessary to obtain the most effective dilution of the buffer solution in a single spin operation. After all the buffer exchange solution has passed through the device, the reduced hydrostatic pressure and the reduced G force during centrifugation cause the tip 23 to be removed from the sample volume. The removed tip allows any residual fluid in the tip's inner hole and on the tip's outer surface to be removed.
    [045] Figure 11 exemplifies how a one-piece reservoir 14 and tip 23 can be configured so that they have a shortened length before centrifugation, and an increase in length during centrifugation, according to certain modalities. Elongation can be achieved by molding or otherwise forming one or more, for example, one to five (three shown), convolutions 90 in a thin wall portion of an elastomeric material that defines column 22, with the purpose of achieving an accordion-like configuration. A suitable material is injection molded silicone, which can stretch up to 50% to 200% without breaking. Other suitable materials may include polyurethanes and other thermoplastic elastomers, and they must have poor adequate nonspecific protein binding performance.
    [046] If greater rigidity is required in the reservoir portion of the device, elastomeric convolutions 90 may be overmoulded at one end of a reservoir precast from polypropylene or an equivalent material.
    [047] Figure 12 shows an example of a simpler design where column 22 includes a portion of straight and thin wall 91. Multiple convolutions have been eliminated from this modality. The thinner walls allow the G forces to extend the axial length of the column 22 during centrifugation. Suitable thin wall thicknesses include between about 0.0381 to about 0.1016 cm (0.015 to about 0.040 inch).
    [048] Figure 13 shows an example of an overmoulded design. The hatched reservoir is precast. Column 22 is overmoulded in the reservoir using a transparent or virtually clear elastomeric material, such as injection-molded liquid silicone (LIM), thermoplastic elastomer, or equivalent material. Suitable materials must have non-specific protein binding performance that does not compromise the recovery of the sample proteins of interest.
    [049] According to certain modalities, the reduction in the sample retention volume can be further improved by reducing the available wet surface area of the outer surface of the feed tube column 22 and / or the tip 23, as well as including a surface rough and more textured on the outer surface of the column and / or the tip. This textured surface can consist of surface roughness (small bumps) that are at least approximately 10 feet in diameter and about 10 feet high. These surface roughnesses can be molded into a device using a low surface energy material, such as polypropylene, polyethylene, PTFE or equivalent. These roughnesses create a surface topography that significantly reduces the wetting of the device's surface. Only the highest points of the asperities are wetted by the fluid flow and come into contact with the sample fluid. The valleys or channels remain non-wet and lined with a thin boundary layer of gas, which in this case would typically be air. This significantly reduces sample losses due to wetting behavior (hydrophobic behavior). This also significantly reduces the opportunity for losses that can occur due to a non-specific protein binding of sample fluids. The combination of a low surface energy material and a rough surface geometry creates what we call the lotus effect, which helps to reduce sample losses associated with superficial fluid retention, and undesirable binding of abundant protein fractions of high interest.
    [050] In cases where the surface roughness of molding on a device can be very difficult or impractical, the same surfaces 22 and 23 can be coated with a silicon solvent emulsion to minimize the surface energy of the device, or can be treated with plasma.
    [051] When further maximization of sample recovery (particularly with high-value sample solutions) is desired, minimizing sample losses due to retention volumes and non-specific protein binding is imperative. As the volumes of protein samples become smaller, undesirable sample losses due to retention within a device have increased in importance. According to certain embodiments, a diaphragm cap having a guiding member or diaphragm can be included in the device to rescue the loss of the accumulating sample solutions, such as in the wet orifice of the feed tube. For example, upon completion of centrifugation, small amounts of sample may decrease in the inner hole at the distal end of the feed tube. This small amount of sample or holding volume can be as large as 5 or 10 pl. Part or all of this retention volume can be evacuated from the internal orifice of the device by activating the guiding member to create pressure in the device and force part or all of this retention volume out of the device.
    [052] Figures 15 and 16 show a diaphragm cap 300 which, in certain embodiments, can be attached, preferably articulated, to the reservoir / exchange member 12. Preferably, diaphragm cap 300 does not interfere with the cap of the device 72. In certain embodiments, the diaphragm cap 300 includes a perimeter 301 and an orientation region 302 located radially inwardly from perimeter 301. Orientation region 302 is descending from perimeter 301 through shoulder 303, and includes an opening 320 to allow air to escape. In certain embodiments, the diaphragm cap 300 is generally circular, with the perimeter 301 being an annular ring, and the orientation region also without circular and having a diameter corresponding to the internal diameter of the inlet (for example, at the location of the flange 16) of the sample reservoir 14 of the member 12. Therefore, the dimensioning of the guidance region 302 allows the outer perimeter edge of the guidance region 302 to engage sealingly with the inner wall of the member 12 as seen in Figure 17.
    [053] In certain embodiments, the top surface of the flange 16 of the reservoir / exchange member 12 includes a lid receiving portion 305, as shown in Figure 18. The lid receiving portion is axially lowered slightly from the remainder of the top surface of the flange 16, and includes a button 306 that extends upward beyond the rest of the flange-type surface 16. The button 306 corresponds in shape to an opening 307 in the perimeter 301 of the lid 300, the opening being 307 is formed in a perimeter portion 308 that is axially lowered slightly (by shoulder height 311, 312) from the remainder of perimeter 301 (Figure 16). A second opening 313 is defined radially inward from the perimeter portion 308 as shown in Figures 16 and 17. In certain embodiments, the top surface 316 of button 306 is wider than the width of opening 307 (best shown) in Figures 19 and 21), thus preventing the lid 300 from unintentionally dislodging from the reservoir / exchange member 12.
    [054] Flange 16 also includes a radially recessed region 307 that is shaped and positioned to cooperate with a tab extending axially 309 over diaphragm cap 300, to allow cap 300 to be fitted to member 12. For -so, when the diaphragm cover 300 is in the closed position as shown in Figure 17, the axially extending flap 309 is positioned in the recessed region 307 of the flange 16. In certain embodiments, the radially recessed region 307 is positioned opposite the portion lid receiving tray 305, and the axially extending flap 309 is similarly positioned opposite perimeter portion 308. Flap 309 and recessed region 307 cooperate to allow simple manual manipulation of the diaphragm cap. For example, the user can move the diaphragm cap from its closed position to its open position while holding the device by simply placing the top of his thumb under the free bottom end of the tap and lifting it up until that is released from the lowered region 307.
    [055] Figures 20 and 21 show the diaphragm cover 20 in the open position. In the open position, the diaphragm cover 300 remains attached to the flange 16 via button 306. Opening 313 defines a radial geometrical axis around which the cover 300 can articulate between the open position and the closed position, thus defining a built-in hinge. Figure 22 shows an exploded view of an embodiment including a larger filter, such as a 15 ml 50 'filter. In the embodiment shown, a mounting retainer is not used, although one may be present.
    [056] The guiding member or diaphragm 302 consists of a deformed flexible material, and therefore can be easily deflected axially, such as by the user's index finger, when the diaphragm cap is in position in its closed position. Activation of the member 302 in this way creates a force within the device that evacuates the retention fluid in the inner cavity of the feed tube and the distal tube and therefore reduces or eliminates the retention volume.
    [057] The diaphragm cover 300 allows the device assembly to be centrifuged with or without the screw cap 70 in position.
    [058] The diaphragm or guiding member can be elastomeric or thermoformed. Example 1 - Affinity Depletion
    [059] In this protocol, the main contaminants in the sample are selectively bound to the medium while the components of interest remain in solution. Through the thermal of the connection step, the solution is collected for further analysis.
    [060] The microspheres that bind to both albumin and IgG are added to the fully assembled binding, washing, elution and concentration (BWEC) device (for example, Figure 9. A serum sample is added and allowed to interact with the microspheres which selectively remove albumin and IgG from the sample by absorbing them onto the surface of the microspheres through interaction with an immobilized anti-albumin antibody and immobilized protein A. After the incubation step, the microspheres are separated from unbound components in the liquid microspheres are contained by the frit in the BWEC device while the solution containing the analytes and biomarkers of interest passes into the chamber below This chamber can simply be the test tube or it can be a filtration device as a unit Amicon Ultra-O.5 centrifugal filtration system, which provides the benefit that the protein biomarkers in the sample can be concentrated in the same stage of c ntrifugation such as removing microspheres. This is specifically significant for affinity depletion as serum samples typically require a 10-fold dilution prior to microsphere incubation because the albumin and IgG to be removed are in a very high concentration and need to come in contact with a large volume of microspheres to perform complete removal. Once the abundant proteins are removed from the diluted samples, the remaining targets of interest typically need to be concentrated. Therefore, coupling the removal / separation of microspheres and the concentration steps reduces the handling required in the workflow. Example 2 - Affinity Purification
    [061] A typical example of how affinity microspheres are used to purify an analyte of interest is presented in this document. In this case, the microspheres are used to selectively bind the target, the contaminants are washed and then the analyte of interest is eluted from the microspheres by changing the buffer system.
    [062] The immobilized metal affinity chromatography (IMAC) microspheres that are charged with copper are placed in the BWEC device next to a sample containing a fusion protein attached to the 6X His affinity purification tag. It is the 6X His tag that is known to attach to copper-loaded IMAC microspheres (known as his tag microspheres). Once the connection is complete, the device is centrifuged to remove contaminants that remain in the solution while the microspheres are retained by the frit in the device. The microspheres can be washed with additional loading buffer to obtain a clearer purification. However, the initial separation and washes are performed without the filtration device (for example, without an Amicon Ultra-O.5 ml device) and the unbound solution and washes are collected as refuse at the bottom of the centrifuge tube. . Once the washes are complete, the filtration device (for example, an Amicon Ultra-O.5 device) is attached to the sound of the BWEC device and an elution buffer that disassociates the target from the microspheres is added. The purified target is then collected and concentrated in the filtration device in a single turn without the need for additional transfer steps. Example 3 - Changing the Buffer
    [063] Examples 1 and 2 take advantage only of the microsphere handling functionality of the BWEC device. A buffer exchange capability is now described. Ultrafiltration devices have been used for a long time to change buffers. This is done by simply concentrating the sample (for example, 10 times from 500 pl to 50 pl) and then diluting the new buffer back to the original volume. In a single step, this would produce approximately a 10-fold or 90% buffer exchange. Typically, this is insufficient with a maximum in the order of a 99.9% buffer exchange, which requires three separate turns with a typical ultrafiltration device, such as the Amicon Ultra-O.5 device. Additionally, if you simply diluted the sample to the full volume, 1.5 ml in this example, in a single swirl, would not be as effective (96.7%) as three swirls in 0.5 ml each (99.9%) . Although 96.7% may seem close to 99.9%, in fact, there are 33 times more buffer remaining that has been changed to 96.7%. The key to a single successful run is to measure the sample slowly with mixing rather than a single large dilution.
    [064] A protein / DNA sample containing azide or some other undesirable buffer or salt is first added to the fully assembled device (BWEC plus the filtration device, for example, an Amicon Ultra-O.5). Then, it is centrifuged and concentrated to 50 ul. Then 1.5 ml of the new buffer is added to the device and centrifuged again. The device slowly measures the new buffer in the sample and removes the unwanted old buffer, leaving the sample concentrated in the new buffer. Example 4 - Combination of Affinity Purification with Buffer Change
    [065] When an affinity-purified or depleted sample also requires a buffer exchange in addition to the concentration, this can be accomplished simply by combining the purification steps with the buffer exchange.
    [066] The IMAC microspheres are loaded into the BWEC device next to a sample containing a 6X His-linked fusion protein. Once the connection is complete, the device is centrifuged to remove contaminants that remain in solution while the microspheres are retained by the frit in the device. The microspheres can be washed with additional loading buffer to obtain a clearer purification. Once the washes are complete, the filtration device (for example, an Amicon Ultra-O.5 device) is attached to the outlet of the BWEC device and an elution buffer that disassociates the target from the microspheres and is added. The purified target is then collected and concentrated in the filtration device in a single spin without the need for additional transfer steps. To remove the imidazole, which is typically used in the elution buffer, 1.5 ml of PBS can be added to the device and spun again. PBS will have no impact on microspheres and vice versa. PBS will be slowly measured in the previously eluted sample, removing the imidazole, replacing it with PBS. Example 5 - Retention volumes
    [067] Retention volumes were evaluated with connection-wash-elution-concentration (BWEC) devices and diaphragm caps. The devices were pre-washed with 1.5 ml BSA (1 mg / ml PBS) at 4000 xg for 2 minutes, and then 0.5 ml BSA (1 mg / ml PBS) was added to each of the devices after assembly with a 0.5 pm filter device (AMICON ULTRA 0.5 ml 10K, available from EMD Millipore Corporation), followed by centrifugation for 15 minutes at 4000 xg. Retention volumes were calculated by weight difference of the devices before and after activating the diaphragm. The results are shown in Figure 26, and demonstrate that activation of the diaphragm results in the recovery of more than 1.5 pl of sample compared to no diaphragm. Example 6
    [068] Binding-washing-eluting devices (BWE) were evaluated in exchange for buffer. 50 pl of 10 mM Tris, pH 7.5, 1 M NaCI were distributed to a filter device (AMICON ULTRA 0.5 ml 10K, available from EMD Millipore Corporation) and mounted in exchange tubes and centrifuged at 4000 xg for 15 minutes after adding 1.5 ml of 10 mM Tris, pH 7.5 to the exchange tube. The retentates were collected by reverse centrifugation for 2 minutes at 1000 xg and the final volume was adjusted to 100 pl with 10 mM Tris. Conductivities were measured after adding 4.9 ml Milli-Q of water. For the control of 3 turns, the buffer exchange was performed by three consecutive washes with 0.5 ml. Figure 27 shows that the connection-wash-elution step is performed in an equivalent way to the 3-spin method despite only a single spin.
    权利要求:
    Claims (16)
    [0001]
    1. Device for sample preparation, CHARACTERIZED by the fact that it comprises a reservoir / exchange member having a sample reservoir, a column extending axially from said reservoir, and a spaced outlet in fluid communication with said reservoir of samples, and a filtering device fixedly sealed or capable of being fixedly sealed to said reservoir / exchange member, said filtering device comprising one or more spaced membranes and a holding chamber defining a fixed stop volume, wherein said outlet of said reservoir / exchange member is positioned in said fixed stop volume when said filtration device is sealingly attached to said reservoir / exchange member.
    [0002]
    2. Sample preparation device according to claim 1, CHARACTERIZED by the fact that it also comprises an assembly retainer comprising a retaining sleeve configured to receive said sample reservoir and an opening that leads to a column extending axially having an orifice shaped to said filtering device.
    [0003]
    3. Sample preparation device, according to claim 1, CHARACTERIZED by the fact that said outlet comprises an opening in a rod extending axially from said column.
    [0004]
    4. Device for sample preparation according to claim 3, CHARACTERIZED by the fact that said rod is axially extendable and axially retractable within said fixed stop volume.
    [0005]
    5. Sample preparation device according to claim 4, CHARACTERIZED by the fact that said rod is fixed at one end by said reservoir and comprises an overmolded portion of elastomer which is axially ex-tensile and axially retractable within said fixed stop volume.
    [0006]
    6. Sample preparation device, according to claim 4, CHARACTERIZED by the fact that said rod comprises one or more convolutions that allow it to be axially extendable and axially retractable within said fixed stop volume.
    [0007]
    7. Sample preparation device according to claim 1, CHARACTERIZED by the fact that the filtration device comprises two spaced membranes, and has a volume between said spaced membranes, and said column comprising a shaped chamber for occupy said volume in such a way that said chamber is displaced from each of said membranes.
    [0008]
    8. Device for sample preparation, according to claim 7, CHARACTERIZED by the fact that said chamber of said column tapers radially inwards towards said outlet.
    [0009]
    9. Sample preparation device according to claim 1, CHARACTERIZED by the fact that it also comprises a diaphragm cap comprising a guiding member positioned on said reservoir / exchange member.
    [0010]
    10. Sample preparation device according to claim 1, CHARACTERIZED by the fact that said column comprises a chromatography medium.
    [0011]
    11. Device for sample preparation according to claim 1, CHARACTERIZED by the fact that said column comprises a frit.
    [0012]
    12. Sample preparation device according to claim 11, CHARACTERIZED by the fact that said frit comprises a hydrophobic material.
    [0013]
    13. Device for sample preparation according to claim 1, CHARACTERIZED by the fact that said column is removable from said reservoir / exchange member.
    [0014]
    14. Sample preparation method, CHARACTERIZED by the fact that it comprises: providing a sample preparation device comprising a reservoir / exchange member having a sample reservoir, a column extending axially from said reservoir, said column containing a means, and a spaced outlet and in fluid communication with said sample reservoir, and a filtration device attached to said reservoir / exchange member, said filtration device comprising one or more spaced membranes and a holding chamber defining a fixed stop volume; selectively attaching a target to said medium, washing said sample attached to said medium to remove contaminants, and eluting said target from said medium in said filtration device by applying a centrifugal force in a single step.
    [0015]
    15. Method, according to claim 14, CHARACTERIZED by the fact that it further comprises providing an orientation member in said reservoir / exchange member, and reducing any retention volume of said sample in said device by moving said guiding member axially.
    [0016]
    16. Method for performing a buffer exchange on a sample, CHARACTERIZED by the fact that it comprises: providing a device for sample preparation comprising a reservoir / exchange member having a sample reservoir, a column extending axially from the said reservoir, and a spaced outlet and in fluid communication with said sample reservoir, and a filtration device attached to said reservoir / exchange member, said filtration device comprising one or more spaced membranes and a holding chamber defining a fixed stop volume; introducing a sample containing a first buffer into said sample tank; concentrating said sample by applying centrifugal force to said sample; introducing a second buffer into said sample reservoir; apply centrifugal force to said sample again; and recovering the resulting concentrated sample from said filtration device.
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    法律状态:
    2013-07-30| B03A| Publication of an application: publication of a patent application or of a certificate of addition of invention|
    2018-02-06| B25G| Requested change of headquarter approved|Owner name: EMD MILLIPORE CORPORATION (US) |
    2018-12-11| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|
    2020-04-14| B09A| Decision: intention to grant|
    2020-10-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/07/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161507240P| true| 2011-07-13|2011-07-13|
    US61/507.240|2011-07-13|
    US201261648631P| true| 2012-05-18|2012-05-18|
    US61/648.631|2012-05-18|BR122019027405-3A| BR122019027405B1|2011-07-13|2012-07-13|device and method for sample preparation|
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