WO2007099937A1 - Procede de filtration d'une solution de proteines, etc. et appareil correspondant - Google Patents

Procede de filtration d'une solution de proteines, etc. et appareil correspondant Download PDF

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Publication number
WO2007099937A1
WO2007099937A1 PCT/JP2007/053603 JP2007053603W WO2007099937A1 WO 2007099937 A1 WO2007099937 A1 WO 2007099937A1 JP 2007053603 W JP2007053603 W JP 2007053603W WO 2007099937 A1 WO2007099937 A1 WO 2007099937A1
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WO
WIPO (PCT)
Prior art keywords
nozzle
solution
filter
protein
container
Prior art date
Application number
PCT/JP2007/053603
Other languages
English (en)
Japanese (ja)
Inventor
Hideji Tajima
Masataka Fumoto
Syuuichi Kobayashi
Toshihiro Ishizuka
Original Assignee
Universal Bio Research Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universal Bio Research Co., Ltd. filed Critical Universal Bio Research Co., Ltd.
Publication of WO2007099937A1 publication Critical patent/WO2007099937A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0275—Interchangeable or disposable dispensing tips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter

Definitions

  • the present invention relates to a solution filtration treatment method for proteins and the like and an apparatus thereof (a device for automating pretreatment of plasma or serum samples for mass spectrometry and the method thereof), and particularly, plasma or serum samples derived from living bodies such as humans
  • a solution filtration treatment method for proteins and the like that automates a pretreatment for removing or separating a predetermined protein that may interfere with mass spectrometry and its It relates to equipment.
  • Plasma human plasma
  • Plasma contains various “plasma proteins”. Its concentration shows a wide distribution depending on the type of plasma protein. Serum albumin contained in the blood in the amount of 30-50 mg in 1 mL of blood is the most abundant.
  • column methods such as antibody columns have problems of contamination due to carry-over and difficulty in increasing the amount of analysis and the number of analyzes (throughput). Furthermore, in the clinical field dealing with human plasma, the problem of carry-over and the handling of binoculars have become serious problems, and the column method is not necessarily an appropriate method.
  • the present invention has been made to solve the above problems, and a first object of the present invention is to provide a simple device, inexpensively and reliably, such as albumin, immunoglobulin, etc. High It is to provide a versatile or versatile solution filtration method for proteins and the like and a device capable of performing various treatments including separation or removal of molecular weight proteins and the like.
  • the second purpose is that there is no labor and effort to consistently automate the processing of separation or removal of high molecular weight proteins such as albumin and immunoglobulin, and high reliability and reproducibility.
  • the present invention provides a solution filtration method for protein and the like and a device therefor.
  • a third object is to provide a solution filtration method for proteins, etc., and an apparatus for the same, which can accurately and efficiently carry out treatments such as separation or removal of proteins, etc. of polymer materials such as albumin and immune globulin. That is.
  • Patent Document 1 Japanese Patent No. 3630493
  • Patent Document 2 WO96Z29602
  • Patent Document 3 Japanese Patent Application 2005-144728
  • Patent Document 4 # 112005-3251
  • Patent Document 5 US Pat. No. 6,660,149
  • Non-Patent Document 1 Beckman Coulter, Inc. catalog (PROTEOME LAB IGY, PROTEOME PARTITIONING SOLUTIONS, (BR-9976A)), US, published 20 05
  • Non-Patent Document 2 Latest Mass Spectrometry for Life Sciences Kodansha Kenichi Harada, Published 2002
  • Non-Patent Document 3 Post-genome 'mass-spectrometry chemistry coterie Published by Toshimitsu Niwa 2002
  • the first invention is directed to one or a plurality of nozzles through which a gas sucked or discharged by a suction discharge unit capable of sucking or discharging a specified gas from two or more kinds of gases can pass. Partitioning the inside of the container so that liquid can be stored in a state of being attached to the nozzle on the side of the opening for mounting, An introduction step of introducing a solution containing the predetermined substance into a filter enclosure having a filter capable of separating the predetermined substance by passing the liquid through the mounting opening, and the filter into which the solution is introduced Enclosed volume A mounting step of mounting a container directly or indirectly on the nozzle, and a pressure filtration step of separating the predetermined substance by discharging the gas from the nozzle to the mounted filter container. It is a solution filtration treatment method for proteins and the like.
  • the "two or more kinds of gases” include, for example, the atmosphere, nitrogen, carbon dioxide, oxygen, argon, or a gas obtained by mixing two or more gases selected at their medium strength in various proportions. is there . Moreover, even if the components of these gases are the same, they belong to different types of gases if their pressure, temperature, etc. are different. For example, the case of compressed gas and rare gas for the same gas.
  • a "filter” allows a liquid to pass through a number of penetrating holes or voids of a predetermined size (pore diameter or average void diameter or length) to separate a given substance in the liquid.
  • a penetrating porous solid for separation and capture or removal.
  • the shape is, for example, a block shape, a thin film shape, a thin plate shape, a film shape, or a plate shape.
  • Filter materials include rubber, silicone, cellulose (including regenerated cellulose), nylon, polyethersulfone and other fibrous materials, resin, metal, ceramics, etc., gels, porous bodies, penetrating pores There are quality and water content.
  • the thin film carrier include an ultrafiltration membrane that performs ultrafiltration of proteins.
  • the “block shape” includes a cylindrical shape, a prismatic shape, a spherical shape, and the like.
  • the “predetermined substance” means a molecular weight having a size that can be separated by a predetermined size of the pores or voids of the filter (for example, about twice the size of the pores or voids of the filter)
  • genetic materials such as nucleic acids, biological materials containing biopolymers or low molecules such as proteins, sugars, sugar chains, peptides, pigments, or cells as biological materials , Viruses, plasmids and the like.
  • the pore or void size of the filter is, for example, several nm to several tens nm.
  • the predetermined substance may be a predetermined particle capable of adsorbing the substance.
  • predetermined particles are micro-sized, for example, 1 ⁇ m force and several hundreds of meters, and can be retained by adsorbing a nanosized material, for example, lnm force with a size of 10 nm.
  • a filter having a pore diameter of about 0.1 ⁇ m to several hundred ⁇ m can be used. It is possible to separate the predetermined particles and filter the suspension by passing the suspension as a solution in which the predetermined particles are suspended.
  • Directly or indirectly attachable to the nozzle means that the mounting opening and the nozzle are directly connected by fitting or screwing or the like, or the nozzle has air permeability.
  • the nozzle is indirectly attached to the attachment opening through a nozzle attachment member to be attached, such as a chip or an adapter.
  • the “tip” has a large-diameter pipe and a thin-diameter pipe formed in communication with the large-diameter pipe to be thinner than the large-diameter pipe. It has an opening, and the small diameter tube has a mouth that allows liquid to flow in and out by suction and discharge of gas, and includes, for example, a dispensing tip.
  • the nozzle is mounted by, for example, inserting the nozzle into the mounting opening and fitting the nozzle into the mounting opening.
  • the “mountable container” refers to a container that has at least a mounting opening that is mounted on or can be mounted on the member used for suction and discharge, and that can store a liquid therein.
  • Mountable containers include tip-shaped containers.
  • the “chip-shaped container” is a mountable container, and has a mouth portion through which a liquid can be put in and out by suction or discharge of the gas in addition to the mounting opening portion.
  • the “filter enclosure” is a container in which a filter is enclosed in the mountable container, and the filter can store liquid in a state in which the filter is mounted on the nozzle on the mounting opening side. It is provided so as to partition the inside of the container.
  • the filter-enclosed container is called a filter-enclosed chip.
  • the filter is cut between the opening for mounting and the mouth so that the liquid can be stored in the state where the nozzle is mounted on the side of the mounting opening, and by passing the liquid.
  • the predetermined substance can be separated.
  • the tip-shaped container is not limited to having a typical tip shape such as a large diameter tube and a small diameter tube.
  • the filter is accommodated in, for example, a portion corresponding to the thick tube or a portion corresponding to a transition portion between the large tube and the thin tube. It is.
  • the volume of the chip-like container is preferably capable of handling a liquid of, for example, about several / z 1 to several hundred 1 or more.
  • the wearable container is integrally formed, You may make it form so that it can divide into two or three.
  • the mouth portion is not necessarily limited to 1, and there may be a plurality of mouth portions.
  • the outer diameter of the thin tube may be the same as that of the thick tube so that it can be fitted to the container, and only the inner diameter may be formed thinner than the thick tube. In this case, a plurality of thin tubes may be perforated.
  • the material of the mountable container is preferably a translucent material in order to enable optical observation.
  • Examples of the material of the mountable container include polyethylene, polypropylene, polystyrene, acrylic resin, glass, metal, metal compound, and the like.
  • the size is, for example, a size capable of accommodating several microliters of force and several milliliters of liquid in a thin tube. In the pressurizing step, it is preferable that the mouth portion is located above a container provided outside or inserted into the container.
  • a dispensing tip is attached to a nozzle capable of suction and discharge, the liquid is sucked from the container, the liquid is transferred to the storage position of the filter-sealed container, and the mounting is performed. This is performed by discharging the liquid into the opening.
  • the volume of the space in which the liquid can be stored in the mountable container in which the filter is sealed is, for example, several microliters or several milliliters.
  • the liquid storage portion provided outside the filter enclosure can suck the liquid of several microliters or several milliliters into the narrow tube through the mouth of the thin tube. Must be able to be accommodated.
  • the solution introduced in the introduction step includes, for example, plasma, and the predetermined particles suspended in the solution include, for example, albumin, immunoglobulin, a2-macroglobulin, Major proteins contained in plasma 'serum such as 1-lipoprotein or a 1 acidic glycoprotein can be adsorbed and retained.
  • plasma refers to a liquid component obtained by removing solid components, ie, blood cells that are cells, from blood.
  • This plasma contains a wide variety of proteins from high to low molecular weight.
  • Proteins contained in protein plasma include immunoglobulins, albumin (molecular weight 66,000 Da), ⁇ 1-MG (molecular weight 33,000 Da), j8 2 -MG (molecular weight ll, 800 Da).
  • the immunoglobulins include IgM (molecular weight 900,000 Da), IgG (molecular weight 160,000 Da), IgA (molecular weight 150,000 Da), and the like.
  • Da is the atomic mass unit da. Represents Noreton is about 1.660 X 10- 27 kg.
  • the second invention includes a desorption step of detaching the filter enclosure from the nozzle cover after the pressure filtration step, and a different type of filter from the filter enclosed in the filter enclosure.
  • a solution filtration treatment method for proteins and the like which has a mounting step of mounting directly or indirectly on the filter, and a pressure filtration step of discharging the gas from the nozzle to the mounted filter enclosure.
  • the predetermined substance in the solution is capable of adsorbing or adsorbing the protein, or the predetermined substance in the solution.
  • the protein is a solution filtration method for proteins or the like having predetermined particles, wherein the filter has pores or voids having a pore diameter smaller than the size of the predetermined substance.
  • the filter in the reintroduction step is, for example, an ultrafiltration membrane capable of separating proteins or a solution filtration treatment method for proteins such as a microfiltration membrane capable of separating the predetermined particles.
  • ultrafiltration membrane refers to a porous membrane having a pore diameter in the range of lnm to lOOnm.
  • a “microfiltration membrane” is a membrane used to filter solutes or particles of about 0.01 ⁇ m power / zm.
  • the “predetermined particles” are as described in the first invention.
  • a fourth invention is a solution filtration method for proteins, etc., wherein the introduction step, the reintroduction step or the pressure filtration step has a detection step of detecting the amount of liquid introduced into the filter enclosure.
  • one of the two or more types of gases is air
  • the introducing step is performed using a dispensing tip attached to the nozzle for suction and discharge of air.
  • the liquid is introduced into the filter enclosure
  • the attaching step attaches the filter enclosure after the dispensing tip is detached from the nozzle
  • the pressure filtration step is a gas other than the atmosphere.
  • the "dispensing tip” is a tip-shaped container that does not contain a filter capable of separating the predetermined substance, and can suck and discharge liquid into the dispensing tip. It is used for dispensing and transferring liquids.
  • a sixth invention is a solution filtration method for protein or the like, wherein the gas other than the atmosphere is compressed nitrogen gas.
  • the pressure of the nitrogen gas in the nitrogen gas compression storage is, for example, 10 kgf / cm 2
  • the pressure to be applied to the filter through the nozzle after being reduced in pressure is, for example, 0.1 kgf / cm 2 it is to several kgf / cm 2.
  • “kgf” represents weight kilogram.
  • the pressure value be variable by providing a pressure regulator. Moreover, it is preferable to provide a pressure reducing valve. In this way, the pressure value can be changed based on the purpose of processing, such as when separating predetermined particles as a predetermined substance, or when directly separating proteins, or the structure of the filter sealing container. Optimal processing can be performed.
  • a solution filtration of protein or the like having a substitution step of replacing the gas remaining in the nozzle with another gas over the pressure filtration step, the introduction step, or the reintroduction step. It is a processing method.
  • the replacement is performed, for example, by supplying a new gas to the nozzle having the residual gas or repeating the suction and discharge.
  • a suction / discharge portion capable of sucking or discharging two or more kinds of gases having a medium force and a gas sucked or discharged by the suction / discharge portion.
  • a filter enclosure having a filter that partitions the inside of the container so that liquid can be stored in the state of being attached to the nozzle, and capable of separating a predetermined substance by passage of the liquid, and a member that can be attached to the nozzle;
  • a solution filtration treatment apparatus for proteins, etc. having a container group having a plurality of containers capable of storing a solution, a specimen, or a reagent, and a moving unit that can move the nozzle head relative to the container group. is there.
  • the "suction or discharge of two or more kinds of gas with a specified force” is performed by switching the supply of the gas to the nozzle by a switching unit using a valve.
  • the “member that can be attached to the nozzle” include an attachable container having the attachment opening, a filter enclosing container (including a filter enclosing tip), a dispensing tip, an adapter, and the like.
  • the ninth invention relates to the structure or processing of the nozzle or a member that can be attached to the nozzle, and the type, amount, pressure, frequency, time, or position of the gas for suction or discharge of the nozzle force.
  • the type of substance includes the nature and size of the substance.
  • the amount of the liquid is the amount of liquid in the filter-sealed container or the dispensing tip, or the amount of liquid in the container, and is detected by, for example, a liquid amount detecting means described later. For example, if the control unit detects the amount of the pre-filter solution, mixture solution or suspension stored in the filter enclosure, the solution, mixture solution or suspension solution is detected according to the amount. The desired concentration can be achieved.
  • the suction / discharge section includes an air suction / discharge section capable of sucking and discharging air through the nozzle, a gas supply section for supplying a gas of a type other than the atmosphere, A protein solution filtration apparatus having a gas supply unit and a switching unit that switches between the air suction and discharge unit and connects to the nozzle.
  • the atmospheric suction / discharge unit is preferably driven by a one-way mechanism.
  • the “one-way mechanism” is a mechanism that can easily perform the forward operation with a small force, and the reverse operation requires a large force and is difficult to execute.
  • the atmospheric suction / discharge unit includes, for example, a cylinder and a plunger built in the cylinder so as to be slidable along the axial direction of the cylinder.
  • the ball screw or the sliding screw is coupled to a nut portion that is screwed into a ball screw or a sliding screw having an axial direction parallel to the cylinder, and the ball screw or the sliding screw is rotated in both forward and reverse directions so that the inside of the cylinder is axially driven. Reciprocate along the direction.
  • the plunger Even if the pressure by the compressed nitrogen gas is strong, the plunger connected to the nut portion hardly moves along the axial direction due to the pressure, so that the influence of the introduction of the compressed nitrogen gas is prevented. Can do.
  • the nozzle has an inlet / outlet through which gas flows in and out at the lower end, communicates with the air suction / discharge unit at the upper end, and communicates with the gas supply unit at the side.
  • the gas inlet is formed.
  • An eleventh aspect of the invention is a solution filtration apparatus for proteins, etc., wherein the gas other than the atmosphere is compressed nitrogen gas, and the gas supply unit is a nitrogen gas compression reservoir.
  • the predetermined substance is the predetermined substance in the solution is a protein or predetermined particles capable of adsorbing or adsorbing the protein, and the filter includes the predetermined substance.
  • This is a solution filtration apparatus for proteins and the like having pores or voids having pore sizes smaller than the size.
  • the “protein” is, for example, plasma albumin, immunoglobulin, a 1-MG or 13 2 -MG as described above.
  • a thirteenth aspect of the present invention is a solution filtration apparatus for proteins, etc., provided with a desorption part for removing a member attached to the nozzle of the nozzle head from the nozzle.
  • the member attached to the nozzle refers to "a member that can be attached to the nozzle" attached to the nozzle.
  • the attachable container attached to the nozzle a filter-enclosed container, or a dispenser. Chip, adapter, etc.
  • a fourteenth aspect of the invention is a solution filtration apparatus for proteins, etc., provided with a liquid amount detection means for detecting the amount of liquid in the filter-enclosed container or the container.
  • control of the type, amount or pressure, number of times, time or position of the suction or discharge of the nozzle force by the control unit is performed by separating or removing a predetermined protein, or It is a solution filtration processing apparatus for proteins and the like, which is performed based on the processing content of either concentration of a protein solution or buffer replacement.
  • a suction / discharge part capable of sucking or discharging a designated gas from two or more kinds of gases is provided, and the liquid containing a predetermined substance is sucked.
  • the suction / discharge unit sucks and discharges air to the filter enclosure through the nozzle, attaches the filter enclosure to the nozzle, and again discharges and pressurizes the gas by the suction / discharge unit.
  • the predetermined substance can be separated by filtering using the filter.
  • the filter enclosure used therein is removed, and the filtrate is reintroduced into a new filter enclosure, It is attached and pressure filtration is performed. Therefore, for example, when performing a separation treatment of rare useful proteins in plasma, once a high-molecular-weight protein such as albumin is separated as a predetermined substance, the substance having a purpose different from that of the substance is used. Rare protein can be isolated.
  • a substance containing a target substance once adsorbed on a predetermined particle is separated from the predetermined particle, then the substance is dissociated from the predetermined particle, and then the target substance is further separated. Processing can be performed automatically.
  • the predetermined substance is a predetermined particle and protein capable of adsorbing protein, and a filter capable of separating them is used. Therefore, for example, if the liquid is allowed to pass through after being adsorbed to predetermined micro-sized particles but the predetermined particles are not passed through, and separated using a filter, it is relatively low.
  • the target protein can be separated by applying pressure. This makes it possible to separate proteins easily, inexpensively and reliably. In addition, complex processes can be consistently automated.
  • the amount of liquid introduced into the filter enclosure is measured.
  • the concentration of the liquid can be known, and the reliability of the process Can be increased.
  • the suction and discharge of liquid to the dispensing tip attached to the nozzle is performed by switching between the atmosphere and a gas other than the atmosphere to perform suction and discharge or discharge.
  • a gas other than the atmosphere to perform suction and discharge or discharge.
  • air and compressed nitrogen gas are used as the gas. Therefore, by using properly a gas suitable for introducing the solution into the container and a gas suitable for pressure filtration, the treatment can be performed efficiently and inexpensively with a simple structure. Nitrogen gas is chemically stable and can be easily obtained at low cost.
  • the suction or discharge of the nozzle force regarding the suction or discharge of the nozzle force, the kind, amount, or pressure, the number of times, the time or the position of the gas is changed to a substance such as the structure of the nozzle. Control is performed based on conditions and processing contents. Therefore, pressure filtration can be performed under conditions suitable for the structure of the nozzle and the processing content.
  • appropriate processing of the protein solution can be performed by controlling the suction or discharge of the nozzle according to the content of the separation or removal of the specified protein or the concentration or buffer replacement of the protein solution. Can do.
  • the seventh invention when gases having different specific gravity are sequentially passed or introduced into the nozzle, if there is a gas remaining in the nozzle, it may be difficult to introduce new gas into the nozzle. . Therefore, the treatment can be smoothly advanced by removing the residual gas before the treatment with a new gas is started.
  • FIG. 1 (a) shows an overall perspective view of the protein solution filtration apparatus 10 according to the present embodiment.
  • the apparatus 10 includes a main body 11, a nitrogen gas compression reservoir 12 that compresses and stores nitrogen gas, and a pipe line that connects a nozzle (described later) provided in the main body 11 and the nitrogen gas compression reservoir 12.
  • the main body 11 has a substantially rectangular parallelepiped casing 14, and two side surfaces of the casing 14 are partly cut off, and a lid 14a formed of a transparent plate is fitted into the casing 14 from the outside.
  • the filtration unit 15 can be seen through.
  • a handle 14b is attached to the lid 14a.
  • an operation keyboard 14c is provided on which a user gives instructions, inputs various data, and displays necessary data.
  • the main body 11 is not limited to this form.
  • the main body 11 may be used in a state where it is installed in a refrigeration facility without providing the housing 14 of the main body 11.
  • FIG. 1 (b) shows a state where the lid 14 a is removed from the casing 14 of the main body 11.
  • FIG. 2, FIG. 3, and FIG. 4 are a perspective view, a plan view, and a side view schematically showing the filtration processing unit 15, respectively.
  • the filtration processing unit 15 includes a plunger 17 slidable inside, and performs suction or discharge of the atmosphere, and a plurality of stations (8 stations in this example) arranged in the X-axis direction in the figure.
  • a nozzle head 16 Underneath the cylinder 18 and the cylinder 18 are a nozzle head 16 having eight nozzles 19 each communicating with the cylinder 18, and members that can be attached to the nozzles 19 (including filter enclosing chips described later) ),
  • a container group 22 having a plurality of containers that can store various solutions, specimens, or reagents, and a movement that enables the nozzle head 16 to move in the Y-axis direction and the Z-axis direction in the figure relative to the container group 22 Part (shown in FIGS. 6 and 7).
  • the nozzle head 16 can move the nozzle 19 to each container of the container group 22 so as to cover the entire area of the container group 22.
  • each nozzle 19 communicates with the cylinder 18, and the lower end of the nozzle 19 has an inlet / outlet through which gas can flow in and out, and slightly above the side surface of the nozzle 19,
  • Eight branch pipes 35 communicating with the nitrogen gas compression reservoir 12 through the pipe 13 and the valve 34 have gas inlets connected to each other.
  • the branch pipe 35 or the pipe 13 is flexible.
  • the valve 34 corresponds to the switching unit, and is, for example, an electromagnetic valve, a stop valve, a gate valve, a check valve, or a cock. As a result, when the atmosphere is sucked and discharged, the influence is not given to the pipe line 13.
  • a connection cutoff instruction may be given by a control unit (not shown), and a connection or cutoff instruction may be given based on the detection result of the pressure sensor 21.
  • the eight nozzles 19 provided in the nozzle head 16 are formed so that the chip-like container can be attached thereto.
  • Each nozzle 19 is hydrodynamically connected to the cylinder 18 and the nitrogen gas compression reservoir 12 described above via the conduit 13, the valve 34 and the branch conduit 35.
  • the valve 34 corresponds to the switching unit, and switches the connection with the nitrogen gas compression store 12.
  • the valve 34, the pipe line 13, and the nitrogen gas compression reservoir 12 are provided separately from the nozzle head 16 and cannot be moved integrally with the nozzle head 16.
  • An elongated plate-like attachment / detachment provided with a plurality of (in this example, eight) through-holes through which the nozzle 19 penetrates below the connection portion of the nozzle 19 with the branch pipe 35.
  • the portion 20 is provided so as to be movable together with the nozzle head 16 so as to be vertically movable with respect to the nozzle head 16.
  • the inner diameter of the through hole is slightly larger than the penetrating nozzle 19 but has an inner diameter smaller than the outer diameter of the upper end opening of the tip-like container to which the nozzle 19 is fitted.
  • the nozzle head 16 On one side surface of the nozzle head 16, there are eight pressure sensors 21 for detecting the pressure inside each nozzle 19, and there is an interval between adjacent nozzles 19 at a position corresponding to each nozzle 19. They are provided at the same interval. Further, as will be described later, the nozzle head 16 is supported by the main body 11 by being coupled to the arms of the moving unit (not shown) on the other side surface of the nozzle head 16.
  • the container group 22 includes an ultrafiltration filter tube (Vivspin 2 manufactured by Sartorius) 23 as eight filter-enclosed chips, and a liquid from a filter unit 23 provided below the filter unit 23.
  • Drain tank 24 for receiving various reagents, such as bicarbonate Dispensing tips 25 arranged in two rows for a common buffer and a spare dispensing, for example, a container 26 containing 50 mM ammonium bicarbonate buffer solution, and a limitation as another type of filter enclosing tip.
  • a container 26 containing 50 mM ammonium bicarbonate buffer solution, and a limitation as another type of filter enclosing tip.
  • a container 26 containing 50 mM ammonium bicarbonate buffer solution, and a limitation as another type of filter enclosing tip.
  • Sample container 30 containing one or more plasma solutions, Dispensing tips 31 for stirring or non-adsorptive transfer arranged in two rows, 2 for plasma dispensing and spare Dispensing tips 32 arranged in rows are arranged on a processing stage 33 shown in FIG. 4 in a matrix of 12 rows x 8 rows, 8 rows each corresponding to each nozzle 19 of the nozzle head 16. Shi It is also of the.
  • the container group 22 can be moved along the X-axis direction of Fig. 2 to detect the liquid level in the ultrafiltration filter unit 23 and the ultrafiltration filter unit 27. It has a liquid level sensing unit 36 equipped with a CCD image sensor as a means.
  • the ultrafiltration filter unit 23 has a tip-like container 38 as a mountable container and a filter 39 enclosed in a chip-like container 38.
  • the chip-like container 38 is formed with a thick tube 42 and one or more thin tubes 41 provided on the lower side of the thick tube 42 and having an inner diameter smaller than the inner diameter of the thick tube 42.
  • the transition portion between the thick tube 42 and the thin tube 41 has a step, and the filter 39 is provided above the step.
  • the upper end of the thick tube 42 has a mounting opening 42a that can be mounted indirectly to the nozzle 19 through a pressurizing adapter 40, and the tip of the thin tube 41 is liquidized by discharging the nitrogen gas. It has a mouth part 41a that can flow out.
  • the filter 39 is partitioned so that liquid can be stored between the mounting opening 42a and the mouth portion 41a while being mounted on the nozzle 19 while being mounted on the mounting opening 42a side.
  • Reference numeral 43 denotes a cylindrical member having the same outer diameter as that of the thick tube 42 in which the thin tube 41 is formed.
  • the ultrafiltration filter unit 27 has a chip-like container 45 and a filter 46 sealed inside the chip-like container 45.
  • the chip-like container 45 includes a thick tube 47, one or more thin tubes 48 provided below the thick tube 47 and formed narrower than the inner diameter of the thick tube 47, and the thick tube 47 and the thin tube 48.
  • Have a step at the transition between The filter 46 is provided above the step.
  • the upper end of the thick tube 47 has a mounting opening 47a that can be mounted indirectly to the nozzle 19 via a pressurizing adapter cap 28, and the tip of the thin tube 48 is liquidized by discharging the nitrogen gas. It has a mouth part 4 8a that can flow out.
  • the filter 46 is partitioned between the mounting opening 47a and the mouth 48a so that liquid can be stored in a state of being mounted on the nozzle 19 on the mounting opening 47a side.
  • Reference numeral 50 denotes a columnar member in which the thin tube 48 is bored, and has a size substantially the same as the outer diameter of the thick tube 47.
  • Reference numeral 49 denotes a container whose upper end can be fitted to the columnar member 50 and receives liquid discharged from the narrow tube 48.
  • reference numerals 51, 52, 53, 54, 55, 56 are containers for accommodating the respective dispensing tips provided on the processing stage 33.
  • FIG. 5 shows a gap between the nozzle 19 in which an ultrafiltration filter unit 27 and an ultrafiltration filter unit 23 are attached to the nozzle 19 and an air supply port 57 of the nitrogen gas compression reservoir 12.
  • the conceptual diagram of the pneumatic path to be connected is shown.
  • the pipe line 13 includes a pressure regulator 58 that adjusts the pressure so that the maximum pressure becomes 2.7 kgf / cm 2 by adjusting the gas amount, a pressure reducing valve 59, and a pressure gauge 60.
  • the pipe 13 is connected to a branch pipe 35 via a valve 34, and the branch pipe 35 is connected to a cylinder 18 and a filter unit 27, which are an air suction / discharge mechanism having a dispensing function.
  • the protein solution filtration apparatus 10 has an information processing unit having a CPU (not shown), various memories, a keyboard, various switches, an input means such as a mouse, a display means, or the like. Provided to be connectable to the information processing unit.
  • the information processing unit includes the discharge amount, pressure, frequency, time, or position of the nozzle in the structure of the nozzle, a member attached to the nozzle or a filter-enclosed chip, a solution to be processed, or the solution.
  • a control unit is provided for controlling based on the material condition such as the type, concentration or amount of the substance, or the coordinate position including the storage position, and the processing content for the liquid.
  • the main body 11 has the nozzle head 16 and the container group 22 provided below.
  • the nozzle head 16 has eight nozzles 19 and eight cylinders 18 communicating with the nozzles 19 (not shown in FIGS. 6 and 7). Is provided with a plunger 17 slidable therein.
  • the eight plungers 17 are attached to the plunger drive plate 69 at their upper ends.
  • the plunger drive plate 69 is connected to a nut portion 64 that is screwed into the ball screw 65, and in conjunction with the nut portion 64 that moves up and down as the ball screw 65 rotates, the eight plungers 17 Are moved up and down all at once.
  • the ball screw 65 is pivotally supported by an inverted L-shaped ball screw support member 68 attached to a head mounting plate 70 to which the nozzle head 16 is attached.
  • a position sensor 96 is provided on the horizontal plate of the ball screw support member 68, and a rod 95 provided on the plunger drive plate 69 so as to protrude upward is provided between the light emitting element and the light receiving element of the position sensor 96. The position of the plunger 17 is detected by blocking.
  • the ball screw 65 is rotationally driven by a timing belt (not shown) spanned between the motor shaft 63 of the motor 62 also attached to the head mounting plate 70 and the upper end portion 66 of the ball screw 65. Is done.
  • the nozzle 19 on the upper side of the detachable portion 20 is provided with a hole 21a, which communicates with the pressure sensor 21 via a rubber tube 21b. 19 pressure can be measured.
  • a connecting portion 35a connected to the branch pipe 35 for introducing the compressed nitrogen gas is provided in a portion of the nozzle 19 opposite to the side where the hole 21a is provided.
  • the head mounting plate 70 to which the nozzle head 16 is mounted is provided with two guide members 71a, 72a in the vicinity of both edges thereof, It is provided so as to be slidable in the vertical direction by engaging with two guide pillars 73 and 74 extending in the vertical direction (Z-axis direction).
  • the head mounting plate 70 is connected to the head vertical drive plate 77 via springs 75 and 76.
  • the head vertical drive plate 77 has both edges Two guide members 71b and 72b are provided in the vicinity of the portion, and are respectively supported so as to be slidable in the vertical direction by engaging with two guide columns 73 and 74 extending in the vertical direction.
  • the head upper / lower drive plate 77 is attached to a nut portion 79 that is screwed with a ball screw 78 extending in the vertical direction at the center thereof.
  • the ball screw 78 is rotationally driven by a motor 80 via a coupler 81.
  • the motor 80 is provided on a rack 82, and the rack 82 is provided on a frame 83.
  • the large vertical plate 86 and the small vertical plate 89 for supporting the track are attached to the casing 14 so as to sandwich the frame 83. It is fixed.
  • the large vertical plate 86 is provided with rails 87 and 88 along the Y-axis direction, and the wall portion of the frame 83 that faces the large vertical plate 86 is provided.
  • Pieces 84 and 85 are provided at corresponding positions outside 83b and slidably engaged with the rails 87 and 88.
  • the small vertical plate 89 is provided with a rail 90 along the Y-axis direction, and a piece 91 is provided at a corresponding position inside the wall portion 83a facing the small vertical plate 89 of the frame 83. It is provided and slidably engages with the rail 90.
  • the large vertical plate 86 is provided with a ball screw 93 extending in the Y-axis direction, and a nut portion 94 is screwed into the ball screw 93, and the frame 83 is formed of the nut portion. It is linked to 94. Further, as shown in FIG. 7A, the ball screw 93 is rotationally driven by a motor 92.
  • step S1 the plasma is stored in advance at ⁇ 80 ° C. (protein concentration: 60-8 Omg / ml) at 500 ⁇ l before being subjected to the treatment, and the sample is thawed at room temperature.
  • step S2 the plasma is filtered with a filter having a pore diameter of 0.22 ⁇ m (for example, a Millipore Millex GV 0.22 ⁇ m syringe filter) using, for example, a dispensing tip attached to the nozzle.
  • a filter-enclosed tip (not shown) enclosed in a tip is attached to the nozzle 19 and subjected to pressure filtration by sucking and discharging using the cylinder 18, etc., and the filtrate 100-400 1 is stored in the sample container 30 of the container group 22.
  • Container 29 contains rubmine and IgG adsorption resin (Amersham Biosciences) and PBS buffer solution, and about 9-10 ml of 50 mM ammonium bicarbonate buffer solution is contained in container 26 of each lane. deep.
  • each nozzle 19 of the nozzle head 16 is moved in the Y-axis direction to the position of the dispensing tip 32 for lml, and then moved in the Z-axis direction to thereby move the dispensing tip. 3 Insert the nozzle 19 into the upper end opening of 2, and attach it by pressing it.
  • the nozzle head 16 equipped with the dispensing tip 32 is moved to the position of the container 30, and the branch pipe 35 and the nozzle 19 are connected using the valve 34.
  • the plasma fluid in the container 30 to 70 1 is aspirated.
  • the nozzle head 16 With the plasma fluid sucked, the nozzle head 16 is moved along the Y-axis direction to the container 29 containing the albumin and IgG adsorption resin and PBS solution, and moved in the Z-axis direction. Then, the plasma is discharged into the container 29 using the cylinder 18 and mixed.
  • the container 29 1.2 ml of the albumin IgG adsorption resin is suspended in advance in 2.8 ml of PBS solution.
  • the albumin 'IgG adsorption resin contained in the container 29 is added to 1.2 ml of albumin' IgG adsorption resin (manufactured by Amersham, the amount of slurry is 4 ml) in another filter unit 27 in advance. Then, 2.8 ml PBS solution is added and, for example, centrifugal filtration (1000 g) is performed five times, or the filter unit 27 is attached to the nozzle 19 and the nitrogen gas compression reservoir 12 is used. The resin was washed and equilibrated with PBS in advance by pressure filtration.
  • step S4 the nozzle head 16 in which the plasma, albumin 'IgG adsorption resin, and PBS solution stored in the container 29 are stored and the dispensing tip 32 is attached to each nozzle 19 is
  • the tip 32 is moved in the Y-axis direction to the position of the container 51, which is the original storage position of the tip 32.
  • the dispensing tip 32 is detached by moving the detachment portion 20 downward.
  • the nozzle head 16 to which the dispensing tip 32 has been detached moves in the Y-axis direction to the position of the dispensing tip 31 having a capacity of 4 ml, and the nozzle 19 is moved to the upper end of the dispensing tip 31 in the Z-axis direction.
  • step S5 4 ml of the suspension in which the resin in which the albumin and IgG in the plasma are adsorbed and suspended in the container 29 is suspended is aspirated by the dispensing tip 31 using the cylinder 18.
  • the sample is transferred in the Y-axis direction, placed on the ultrafiltration filter unit 27, and discharged into the ultrafiltration filter unit 27.
  • the nozzle head 16 is moved along the Y-axis to the position of the containers 54 and 53 that accommodate the dispensing tip 31, and the dispensing tip 31 is detached by the detaching portion 20 there.
  • the nozzle head 16 is moved to the position of the pressure adapter cap 28 of the ultrafiltration filter unit 27, the nozzle head 16 is lowered in the Z-axis direction, and the adapter cap 28 is moved to the nozzle. Attach to 19.
  • the nozzle head 16 is moved to the position of the ultrafiltration filter unit 27, the nozzle head 16 is lowered along the Z axis, and the nozzle 19 is moved through the adapter cap 28 to Attach at the upper end of the ultrafiltration filter unit 27 containing the suspension.
  • the valve 34 is switched to release the connection between the nozzle 19 and the cylinder 18 which is the gas suction / discharge unit, and the nitrogen gas compression reservoir 12 and the conduit 13 are connected.
  • the suspension in the ultrafiltration filter unit 27 is subjected to pressure filtration (O.lkgf / cm 2 ) for 3 minutes,
  • the albumin and IgG adsorbed resin and PBS are separated by a filter 46. These times or discharge amounts are instructed by the control unit.
  • the valve 34 Before attaching the adapter cap 28 and the ultrafiltration filter unit 27 to the nozzle 19 of the nozzle head 16, the valve 34 is switched and the nitrogen gas compression reservoir 12 and the conduit 13 are connected. It is preferable to supply nitrogen gas into the nozzle 19 so that the atmosphere in the nozzle is replaced with nitrogen gas.
  • step S6! / After the resin separation, the detachable part 2 is removed from the nozzle head 16. After the ultrafiltration filter unit 27 is detached using 0, the nozzle head 16 is moved to the position of a new dispensing tip 31 and moved in the Z-axis direction so that the tip of the nozzle is separated. Install dispensing tip 31 by pushing it into tip 31. Further, the valve 34 is switched to connect the cylinder 18 and the nozzle 19 which are atmospheric suction / discharge sections. The nozzle head 16 is transferred to a container 49 provided on the lower side of the ultrafiltration filter unit 27, and 2.8 ml of the filtrate is sucked and transferred to the position of the ultrafiltration filter unit 23. Housed in the outer filtration filter unit 23. In the ultrafiltration filter unit 23, a filter 39 made of a 3 kDa ultrafiltration membrane is enclosed. Next, the valve 34 is switched again, and the nozzle 19 and the nitrogen gas compression reservoir 12 are connected via the pipe line 13.
  • step S7 the ultrafiltration filter unit 23 is concentrated under pressure of 2.7 kgf / cm 2 under a temperature condition of 4 ° C to about 0.2 m.
  • the liquid volume is detected by the liquid volume detecting means, and the liquid volume is detected.
  • Pressure is applied accordingly to achieve the concentration.
  • 2.6 ml of 50 mM ammonium bicarbonate solution is added from the container 26, and pressure filtration is performed again.
  • a total of 4 pressurizations are performed, including 3 times of concentration buffer exchange work to this bicarbonate solution.
  • the protein measurement result in the human plasma processed by the protein solution filtration processing device according to the embodiment of the present invention and the same processing are performed, such as installation of the filter-enclosed chip on the centrifugal device. Therefore, stability and repeatability can be improved without centrifugal filtration and manual operation by comparing the measurement results with centrifugal filtration, i.e., centrifugal filtration.
  • step S5 in FIG. 8 that is, the process of removing albumin and IgG from human plasma is based on the manual operation using a centrifuge and the pressure filtration process according to the embodiment of the present invention. Compared. The plasma after the treatment was fragmented into peptides with proteolytic enzymes and measured with a high performance liquid chromatography (LC) mass spectrometer (MS).
  • LC liquid chromatography
  • MS mass spectrometer
  • the time axis was aligned using the I-OPAL method (international application PCT / JP2004 / 004621) in order to align the elution time of the LC, which fluctuates with each measurement.
  • the signal peak was detected by the peak detection program of the I-OPAL analysis tool.
  • the standard substance used as a marker when aligning the time axis in the I OPAL alignment program was added before the sample measurement.
  • the correlation coefficient (cosine correlation), which is an index of coincidence when 3 to 6 measurement results obtained by the OPAL method are overlaid, is calculated on the third floor using the apparatus according to the embodiment of the present invention.
  • the measurements were 0.9233, 0.9251, and 0.9445, respectively, while the six measurements that had undergone centrifugal filtration and manual pretreatment were between 0.8816 force and 0.9489, and the average was
  • the number of signal peaks obtained was 25229 from three measurements using the apparatus according to the embodiment of the present invention, and was manually performed. It was 27475 from 6 measurements, though it was pretreated by.
  • the relative standard deviation (RSD) indicates how much the peak intensity for each measurement before overlaying by alignment is changed, and the protein according to the embodiment of the present invention. 15.8% when using a solid solution filtration device, and 13.7% when using centrifugal filtration and manual work.
  • the number of signal peaks in which the RSD exceeded 20% was 6292 when using the apparatus according to the embodiment of the present invention, and 5548 when using the centrifugal filtration and manual work. Yes, all were less than a quarter.
  • FIG. 9 (a) and FIG. 9 (b) show the case where the treatment is performed using the apparatus according to the embodiment of the present invention and the case where the pretreatment is performed by centrifugal filtration and manual operation, respectively.
  • the frequency distribution of RSD values is shown when the signal intensity fluctuations are expressed in relative standard deviation (RSD) by superimposing the results of measurements 6 to 6 times.
  • the horizontal axis is the RSD value divided in 10% increments, with the leftmost region corresponding to 0 to 10% and the rightmost region corresponding to more than 50%.
  • the vertical axis represents the number of signal peaks whose RSD values are in the corresponding range. It can be seen that approximately the same number of distribution shapes can be obtained as those using the device according to the embodiment of the present invention and centrifugal filtration and manual operation. If the device according to the embodiment of the present invention is used, centrifugal filtration and manual operation are obtained. It has been shown that the processing can be performed without the need for the user, and the user's effort can be eliminated or alleviated with a simple device structure and scale.
  • the filter-enclosed chip is not limited to the illustrated shape, and may have various shapes as described above.
  • each valve for connecting to and shutting off from the compressed nitrogen gas reservoir is provided corresponding to each nozzle 19, but each branch of the branch pipe 35 is provided.
  • a solenoid valve may be provided for each, and connection and disconnection may be controlled based on the detection result of each pressure sensor corresponding to each cylinder. If an abnormality is detected in the pressure in the nozzle, the valve of the corresponding branch is shut off, the supply of compressed gas to the nozzle is stopped, and an accident is prevented or wasteful processing is performed. You can cancel. Further, the nitrogen gas compression reservoir may be prepared in the number of nozzles connected to each nozzle. Industrial applicability [0100] The present invention relates to a protein filtration treatment method and apparatus.
  • the present invention relates to fields that require handling of in vivo proteins such as immune system, plasma and serum, for example, industrial fields, agricultural fields such as food, agricultural products, and marine products processing, pharmaceutical fields, hygiene, health, immunity, diseases , Medical fields such as genetics, and science fields such as chemistry or biology.
  • the present invention is particularly effective when a series of processes using a large number of reagents and substances are successively executed in a predetermined order.
  • FIG. 1 is a perspective view showing an appearance of a protein solution filtration apparatus according to an embodiment of the present invention.
  • FIG. 2 is a perspective view schematically showing a filtration treatment unit of the protein solution filtration apparatus according to the embodiment of the present invention.
  • FIG. 3 is a plan view showing a filtration treatment unit of the protein solution filtration apparatus according to the embodiment of the present invention.
  • FIG. 4 is a side view schematically showing a filtration treatment unit of the protein solution filtration treatment apparatus according to the embodiment of the present invention.
  • FIG. 5 is a conceptual diagram of a pneumatic path of the protein solution filtration apparatus according to the embodiment of the present invention.
  • FIG. 6 is a front view showing the mechanism of the main body of the protein solution filtration apparatus according to the embodiment of the present invention.
  • FIG. 7 is a side view showing the mechanism of the main body of the protein solution filtration apparatus according to the embodiment of the present invention.
  • FIG. 8 is a flowchart of processing according to the embodiment of the present invention.
  • FIG. 9 is a frequency distribution diagram of relative standard deviations showing the results of processing using the protein solution filtration processing apparatus according to the embodiment of the present invention, centrifugal filtration, and manual processing. Explanation of symbols
  • Nitrogen gas compression storage (nitrogen gas supply unit, suction discharge unit)

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé permettant de filtrer une solution de protéines, etc., dans lequel, si l'on considère les protéines de masse moléculaire élevée, la séparation et d'autres traitements peuvent s'effectuer facilement, de manière sûre et à coût réduit ; ainsi qu'un appareil correspondant. Le procédé consiste à disposer d'un récipient renfermant un filtre qui comprend : un récipient pouvant se monter qui est pourvu d'une ouverture de montage capable de se fixer directement ou indirectement sur une ou plusieurs lignes de buses par lesquelles peut passer un gaz qui a été évacué par aspiration à l'aide d'une unité d'évacuation par aspiration capable d'évacuer par aspiration un gaz désigné entre deux types de gaz ou plus ; ainsi qu'un filtre, enfermé dans le récipient pouvant se monter, qui effectue une séparation de manière à maintenir un liquide dans un état lui permettant d'être placé dans des buses du côté de l'ouverture de montage et qui est capable de séparer une substance donnée lors du passage du liquide. Le procédé comprend : une étape qui consiste à introduire dans le récipient, par l'ouverture de montage, une solution renfermant une substance donnée ; une étape de fixation qui consiste à fixer sur la buse correspondante le récipient contenant le filtre dans lequel a été introduite la solution ; et une étape de filtration par pressurisation qui permet de séparer la substance donnée en évacuant le gaz par la buse dans le récipient contenant le filtre monté.
PCT/JP2007/053603 2006-02-28 2007-02-27 Procede de filtration d'une solution de proteines, etc. et appareil correspondant WO2007099937A1 (fr)

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JP5703171B2 (ja) * 2011-08-19 2015-04-15 富山産業株式会社 濾過装置
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CN107255689B (zh) * 2017-06-28 2019-10-25 中国科学院青岛生物能源与过程研究所 一种阵列式动态分散固相萃取装置及萃取方法

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