JP2006234607A - Cartridge for separating liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for separating a target dissolved substance easily and rapidly from solution containing a dissolved substance, such as protein, without mixing of foreign materials, and further, to provide a demarcating device for preparing a sample for performing an MS analysis easily and rapidly, especially a cartridge for allowing one to visually confirm the progress of situation during separation treatment by the device and the occurrence of troubles. <P>SOLUTION: A unit for placing the sample, a unit for performing separation, concentration, and refinement, and a unit for recovering a separated constituent are set to be a circuit comprising a closed loop, are stored in a cartridge 14 that is detachable from a pump rotor, and further the outer shell of the cartridge 14 is made transparent, thus preventing the mixture of foreign materials and allowing one to visually confirm the progress situation during separation treatment and the occurrence of troubles. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus for separating biological components, particularly solutes typified by biomolecules such as proteins, from stock solutions typified by human plasma, urine and the like.

  In recent years, proteomic analysis research (proteomics) has begun to attract attention as post-genomic research. Since protein, which is a gene product, is thought to be directly linked to disease pathology rather than gene, research results of proteome analysis that comprehensively examines proteins are expected to be widely applicable to diagnosis and treatment. Moreover, it is highly possible that many pathogenic proteins and disease-related factors that could not be found by genome analysis can be found.

  The rapid progress of proteome analysis is technically due to the fact that high-speed structural analysis using mass spectrometers (MS) has become possible. MALDI-ToF-MS (matrix assisted laser desorption ionization) Practical use such as time-of-flight mass spectrometry) enables high-throughput ultra-trace analysis of polypeptides, enables identification of trace proteins that could not be detected in the past, and is a powerful tool for searching for disease-related factors. It has become to.

  The primary purpose of clinical application of proteome analysis is the discovery of biomarker proteins that are induced or lost by disease. Biomarkers behave in relation to pathological conditions, and thus can be diagnostic markers, and are highly likely to be drug discovery targets. In other words, the results of proteome analysis are more likely to be diagnostic markers and drug discovery targets than specific genes, and thus become a post-genome diagnostic and therapeutic trump card (evidence) technology. It can be said that it plays a major role in the promotion of so-called tailor-made medicine (custom-made medicine) because it leads directly to benefits that patients can enjoy, such as responsiveness evaluation and side effect expression prediction.

  When proteomic analysis (clinical proteomics) is introduced into clinical research, it is required to analyze a large amount of specimens quickly and reliably, and because clinical specimens are very small and valuable, they have high resolution, high sensitivity, and high sensitivity. Functional measurements need to be made quickly. Mass spectrometry has been a major driving force, and contributes greatly to the ultra-sensitive and high-throughput characteristics of mass spectrometers. However, although the methods and instruments have been improved rapidly, there is still no situation where proteome analysis can be performed easily and quickly in clinical practice.

  One of the causes is pretreatment of clinical specimens. It is necessary to fractionate and purify proteins from clinical specimens as a treatment prior to mass spectrometry, and this treatment still takes several days, and the pretreatment operation is complicated and requires experience. This is a major obstacle to clinical application. Although it is extremely useful if a systemic disease can be diagnosed or pathologically managed from a small amount of blood or body fluid, many problems arise due to the diversity of proteins contained in plasma.

  It is estimated that there are more than 100,000 human proteins, but it is said that the number of proteins contained in serum alone amounts to about 10,000, and the total serum concentration is about 60-80 mg / mL. High protein content in serum is albumin (molecular weight 66 kDa), immunoglobulin (150-190 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several 100 kDa), etc. / mL). On the other hand, many bioactive proteins such as peptide hormones, interleukins, and cytokines, which are considered to be pathological biomarkers and pathogenesis-related factors, exist only in trace amounts (<ng / mL). The content ratio is actually nano to pico level compared to the high content component of the polymer. In terms of the size of the protein, 70% or less of all types of proteins have a molecular weight of 60 kDa or less, and the above-mentioned trace amounts of biomarker proteins are almost always included in this region (for example, Non-Patent Document 1). . Since these proteins pass through the kidney and are partially excreted in the urine, it is possible to measure not only blood but also urine as a specimen.

  For proteomic analysis in general serological tests, it is first essential to exclude high molecular weight components with a molecular weight of 60,000 or more that interfere with the detection of pathogenic trace components.

  Currently, high-performance liquid chromatography (LC) and two-dimensional electrophoresis (2D-PAGE) are used as separation methods for these high molecular weight proteins, but LC and 2D-PAGE are also used. The work alone takes 1-2 days. This required time is much longer than the analysis time of several minutes such as MALDI-TOF-MS and ESI-MS (electrospray ionization mass spectrometry), and the great advantage of high throughput of MS can be fully demonstrated in clinical proteome analysis. I'm not there. For this reason, for the purpose of obtaining analysis results in the shortest possible time for diagnosis and treatment in the medical field, it must be said that it is extremely impractical at the moment, and it is difficult to use MS for daily clinical tests. It has become a big cause.

  If this point is solved, it can be expected that the rapidity of diagnosis of clinical tests by clinical proteome analysis will be dramatically improved. Specifically, any device / apparatus capable of fractionating and separating a target protein group at a high speed with a small amount of sample, which is an alternative to LC or 2D-PAGE, may be used.

  Products that have already been put to practical use with albumin as the main target substance or disclosed technologies include carriers with immobilized affinity ligands such as blue dyes (for example, Japan Millipore: Montage Albumin Deplete Kit (registered trademark)) , Nippon Bio-Rad Co., Ltd .: AffiGel Blue gel (registered trademark), centrifuge tube type apparatus for fractionating high molecular weight components by centrifugal filtration (for example, Nihon Millipore: Amicon Ultra (registered trademark)), electrophoresis principle (E.g., Gradiflow (registered trademark) system), traditional precipitation methods such as ethanol precipitation by Cohn, and fractionation by chromatography (e.g., Non-Patent Document 2). However, these are not sufficient for separation and fractionation, are unsuitable for trace samples, diluted with samples, or mixed with drugs that interfere with mass spectrometry, etc. There is a problem.

  There is a need for a device that has high resolution in a short period of time rather than complicated and time-consuming techniques, although it has high resolution such as 2D-PAGE and liquid chromatography. Most recently, the method using Affi-Gel Blue gel (N. Ahmed et al., Proteomics, On-line version, 2003/06/23) and the method using Gradiflow system (DL Rothemund et al. (2003) , Proteomics, vol. 3, pp279-287) have been published as an effective and improved albumin removal method, and no new simple method with high resolution has been reported.

Moreover, in order to detect a very small amount of protein with high accuracy, it is necessary to prevent contamination by foreign substances.
The foreign substances here include not only proteins but also cells and microorganisms other than the intended ones. Furthermore, for example, in the analysis of a protein in the serum of one patient, a protein in the serum of another patient becomes a foreign substance. There is no device that takes measures against contamination.

  For example, Patent Documents 1 and 2 disclose a method for separating and recovering a protein from a protein solution using a separation membrane. In these examples, the former is only a description of the method and is essential for protein separation. It has not yet been conceived of a specific device having a structure, and the latter does not mention a single separation device comprising all the essential components described as “device”, and Although a sterilization apparatus using ultraviolet irradiation is described, such an apparatus cannot deal with foreign substances other than cells and microorganisms.

  It is also necessary to prevent such a risk because pathogens may leak during processing investigations when the body fluid contains pathogens. There is no device that takes such measures.

The development of methods and devices to solve these problems has led to the widespread use of proteome analysis in medical research and clinical settings, enabling faster and more accurate examinations and diagnoses, and intractableness without useful treatments It can be expected to be a powerful tool for investigating the causes of various diseases and developing early diagnostic methods.
By Anderson NL, Anderson NG (Anderson, NG), "The human plasma proteome: history, character and diagnostic prospects (The human plasma proteome: history, character, and diagnostic prospects) ", Molecular & Cellular Proteomics, (USA), The American Society for Biochemistry and Molecular Biology, Inc.), 2002, Volume 1, p845-867. Edited by the Japanese Biochemical Society, “Seminar in Experimental Chemistry of Volunteers (Vol. 1) Protein (1) Separation, Purification, and Properties”, Tokyo Chemical Doujin, 1990 JP 59-116223 Patent No. 3261875

  An object of the present invention is to easily and quickly prepare a device for separating a target solute from a solution containing a solute such as a protein without contamination by a foreign substance, more specifically, a sample for performing MS analysis. It is to provide a fractionation device. In particular, it is an object of the present invention to provide a cartridge capable of visually confirming the progress status and the occurrence of trouble during separation processing by this apparatus.

In order to achieve the above object, the present invention comprises the following arrangement.
(1) A liquid separation cartridge for separating a part or all of a biological component of a body fluid or a liquid containing a biological component,
1) a supply unit for supplying a sample;
2) One or more units for performing one or more operations selected from separation, concentration and purification on the liquid supplied from the supply unit;
3) A cartridge for liquid separation, wherein at least a part of the recovery unit for recovering the component obtained through the unit of 2) is stored in a transparent storage container.
(2) The cartridge for liquid separation according to (1), wherein the cartridge has a squeezing portion on an outer wall for facing a rotor of a rotary tube pump disposed outside the cartridge.
(3) The liquid separation cartridge according to (2), wherein a part of the tube connecting 1) to 3) is disposed outside the pressing portion.
(4) The cartridge for liquid separation according to any one of (1) to (3), wherein the circuits connecting 1) to 3) and the circuit connecting them are closed circuits.
(5) The liquid separation cartridge according to any one of (1) to (4), wherein the unit of 2) includes a separation membrane.
(6) The liquid separation cartridge according to (5), wherein the membrane is a hollow fiber membrane.
(7) A liquid separation apparatus having a rotary tube pump and any one of the liquid separation cartridges (1) to (6) on the apparatus main body.

  By using a device equipped with a transparent cartridge containing a closed circuit according to the present invention, it is possible to easily and quickly separate necessary components without contamination from body fluids while preventing sample contamination and biohazard, It can be concentrated and purified. The apparatus of the present invention can be made into a disposable cartridge, which is a great advantage from the viewpoint of avoiding contamination from the specimen and ensuring the reproducibility of the analysis. In particular, by making the outer shell of the cartridge transparent, it is possible to visually check the progress status and occurrence of troubles during the separation process by this apparatus, and it becomes possible to operate more safely and reliably.

  The term “fraction” as used in the present invention refers to separation of solutes in a solution, and when a plurality of solutes are contained, it refers to separation of all or part of the solutes. In particular, in the case of pretreatment of proteome analysis by MS of body fluid, it means that a protein for recovery is discriminated from a protein for disposal.

  In the present invention, it is necessary that at least a part of the above 1) to 3) is a cartridge that is at least partially incorporated in a transparent container.

  The cartridge of the present invention includes a supply unit for supplying a sample. The method of loading the sample is not particularly limited, but the sample collected in the syringe can be manually or syringe pumped through a three-way stopcock or rubber button, or connected to the bag containing the sample. The method of connecting the pipe to a three-way stopcock and feeding it with a roller pump is preferable in that the sealing property is high and the feeding speed can be controlled.

The cartridge of the present invention includes a unit that performs at least one operation selected from separation, concentration, and purification on the liquid supplied from the supply unit. These means preferably incorporate a film. The membrane used in the present invention is one kind from the group consisting of cellulose, cellulose acetate, polycarbonate, polysulfone, polymethacrylate, polyacrylate, polyamide nylon, polyvinylidene fluoride, polyacrylonitrile, polyester, polyurethane, polystyrene, polyethylene and polypropylene. By using a filter or hollow fiber containing the material selected above, the target solute component can be separated more efficiently. Any of flat membrane type separation membranes (filters) such as flat filters and cartridge type filters, and hollow separation membranes (hollow fibers) such as hollow fibers can be used. These filters or hollow fibers contain antibodies and their fragments, polyethyleneimine, aminomethylpyridine, polyphenol, blue dyes, divalent metal ions (Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Cu ²⁺ etc.) and hydrophobicity By immobilizing one or more substances (ligands) selected from the group consisting of compounds (methyl group, benzyl group, phenyl group, chloromethyl group, octyl group, lauryl group, etc.), the solute is added to the filter or hollow fiber. Affinity can be imparted. When used for pretreatment of a sample for MS analysis, a function of adsorbing and removing proteins unnecessary for MS analysis such as albumin can be provided. In the present invention, a hollow fiber is particularly preferably used because it has a large surface area per amount of treatment liquid and a small pressure loss in operation. Regarding the molecular fractionation performance of the membrane, the molecular fractionation ability (cutoff value) can be appropriately selected in consideration of the molecular weight of the solute to be collected and the molecular weight of the solute to be removed. It is preferable that a liquid feed pump is installed in this circuit, and the pump can circulate the treatment liquid through the hollow fibers in the module.

  Furthermore, a normal collection container can be used as the collection unit for collecting the components obtained through the unit 2).

  In the cartridge of the present invention, in order to ensure the convenience and stability of mounting, the circuit constituted by the supply unit, the unit for separation, concentration or purification, and the recovery unit is not open to the atmosphere. That is, a closed circuit is preferable. It is preferable that the squeezing part cartridge can be directly attached to and detached from the tube pump, or can be attached to and detached from a portion supporting the tube pump. More preferably, the cartridge is disposable. Furthermore, it is preferable that a part of the outer wall of the cartridge is a squeezing portion that can face the rotor of the rotary tube pump disposed outside the cartridge. Furthermore, it is preferable that a part of the tube connecting each unit is disposed in the vicinity of the outer wall of the pressing portion. As a result, the cartridge of the present invention and the rotary tube pump can be easily connected, and the separation operation can be started easily.

  The direction in which the tube is attached to the squeezed surface of the cartridge matches the direction of the column port. In addition, the same number of grooves as the column are formed on the surface of the cartridge that receives the squeezing, and the tube can be firmly fixed by filling the groove in the groove. When a plurality of tubes arranged in a plurality of grooves are simultaneously squeezed by a single rotor, the squeezing accuracy becomes worse because the rotational shake increases as the distance from the rotor drive unit increases. In order to maintain the accuracy of the squeezing, it is preferable to install the cartridge so that the tube is disposed closer to the rotor drive unit (non-operation side). If the accuracy goes wrong, the tube cannot be pressed down and fixed quantity feeding becomes impossible. In order to easily and accurately attach the cartridge to the main body, it is preferable to provide a guide hole in the pressing portion. If a guide shaft is also provided on the tube pump side, mounting is easily performed by passing the guide shaft through the guide hole. Subsequently, the tube is held at an appropriate distance from the rotating rotor of the peristaltic roller pump by fixing the squeezing unit cartridge, and the solution containing the sample is sent in the circuit in the cartridge by operating the tube pump. .

  The present invention is suitable for the separation of biomolecules from biological components, particularly human plasma, serum, urine, saliva, tears, cerebrospinal fluid, ascites, pleural effusion, amniotic fluid, lymph and the like. The analytical specimen thus obtained is useful for various protein analyzes such as liquid chromatography, electrophoresis, and MS, but is particularly preferably useful for proteome analysis using electrophoresis and MS.

The MS that can be applied directly or indirectly after this device is not particularly limited, but as an ionization part, an electrospray ionization type, an atmospheric pressure ionization type, a fast atom collision type, a quadrupole type, a cyclotron resonance type, a magnetic sector type, A matrix-assisted laser destructive ionization type or the like is used in appropriate combination with a mass analyzer such as an ion capture type, a time-of-flight type, or a Fourier transform type. In this case, it can also be used as tandem MS or FT-MS such as MS / MS and MS ⁿ . In the case of tandem MS, all types of MS can be applied, but it is particularly efficient to use a combination of ion trapping type, quadrupole-time of flight (Q-TOF) type, FT-MS, etc. .

  By analyzing with the combination with this device, it is possible to collect the structural information of various trace protein components, but not only the peptide-mass fingerprint (PMF) but also the primary structure information of each peptide (amino acid) Array).

  Hereinafter, one embodiment of the method for separating protein components in an aqueous solution and the protein fractionation apparatus of the present invention will be described with reference to the drawings, but the present invention is not limited thereto.

(Example)
1, 2 and 3 are diagrams of the fractionation device of the present invention. In FIG. 1, the separation unit is constituted by three-stage units. The circuit assembly was as follows.

  A three-way joint 2a and a joint 2c were connected to the rubber button 2b. Using a flexible tube, the joint 2c and the lower nozzle 6a of the hollow fiber membrane module 5a were connected so as to crawl on the curved surface of the multi-channel compression unit 8. Furthermore, the back 12 with a tube was connected to the three-way joint 2a. The hollow fiber membrane modules 5a, 5b, 5c constituting the separation part, and the hollow fiber membrane module 5d constituting the concentrating part and the multi-channel type pressing part 8 are integrated, and each hollow fiber membrane module 5a, 5b, 5c, 5d is integrated. A flexible tube is connected to the upper nozzles 4a, 4b, 4c, and 4d provided at the upper end of each, and each tube is installed so as to run over the curved surface of the multi-channel type squeezing portion 8, and the lower nozzles 6a, 6b, 6c, Each connected to 6d. Next, the trunk nozzle 7a of the hollow fiber membrane module 5a and the lower nozzle 6b of the hollow fiber membrane module 5b are connected, and the lower nozzle 7b of the hollow fiber membrane module 5b and the lower nozzle 6c of the hollow fiber membrane module 5c are connected to the hollow fiber. The trunk lower nozzle 7c of the membrane module 5c and the lower nozzle 6d of the hollow fiber membrane module 5d were connected by piping. The fuselage lower nozzle 7d of the hollow fiber membrane module 5d and the three-way joint 2a were connected by piping. Further, the lower nozzle 6d of the hollow fiber membrane module 5d and the recovery container cap 12 were connected, and the upper nozzle 4d of the hollow fiber membrane module 5d and the recovery container cap 12 were connected. A collection container cap 12 was sealed in a collection container 11 that was transparent and became a collection unit. All the hollow fiber membrane modules, nozzles, tubes, joints, backs 13 with tubes, collection containers 11 and collection container caps 12 described above were closed circuits. The closed circuit was filled with an aqueous buffer which is a mobile phase.

  As shown in FIG. 2, the circuit is housed in a housing container 13 having a window 15 made of a partially transparent plastic, thereby forming a cartridge 14.

  And the apparatus shown in FIG. 3 was assembled. The liquid separator M is equipped with a rotor 9 of a multi-channel rotary tube pump. In addition, a rotatable roller is mounted around each rotor. The cartridge was fixed by pushing the cartridge in such a manner that the guide shafts 8a and 8b penetrate the guide holes 8a and 8b of the pressing portion 8 existing in the cartridge. The fixed cartridge 14 is moved in parallel to the tube pump rotor 9 side, and is disposed on the curved surface of the outer wall of the squeezing unit 8 and the squeezing unit 8 in a rotatable manner around the rotor of the tube pump. A liquid delivery system was formed with the tubes.

  Further, a syringe pump 1 is attached above the rotor 9 of the multichannel tube pump. Each rotor 9 was provided with a motor (not shown).

  Returning to FIG. 1, the actual operation will be described. The liquid flow is indicated by arrows. After inserting the needle of the syringe 1 enclosing a stock solution such as serum into the rubber button 2b of the supply unit, the sample was introduced at a speed controlled by a syringe pump. The administered stock solution was fed to the hollow fiber membrane module 5a of the separation unit while being mixed with the mobile phase and being conveyed by a roller (not shown) of the rotor 9a driven by a motor. The filtrate produced while circulating through the hollow fiber membrane module 5a by the roller (not shown) of the rotor 9b driven by the motor exits from the body nozzle 7a, and is fed to the latter hollow fiber membrane module 5b by the roller of the rotor 9b ( (Not shown). The filtrate of the hollow fiber membrane module 5b was further transported to the subsequent hollow fiber membrane module 5c. Thus, the solute of the undiluted solution was fractionated by the hollow fiber membrane modules 5a, 5b and 5c forming the separation part. The filtrate of the hollow fiber membrane module 7c was transported to the hollow fiber membrane module 7d of the concentration unit. The filtrate produced during circulation through the hollow fiber membrane module 7d exited from the body nozzle 7a and was returned to the supply section via the joint 2a. The filtrate of the hollow fiber membrane module 7c was transported to the hollow fiber membrane 7d in the concentration part. The liquid circulation and liquid feeding in the separation unit and the concentration unit were performed by a roller (not shown) of the rotor 9b. After the designated time has elapsed, the rotary rotors 9a and 9b are stopped, and the rotary rotor 9c driven by the motor is started. As a result, the air in the recovery container 10 pushed out the concentrated liquid in the circuit in the concentrating section, and the concentrated liquid as the obtained component was recovered in the recovery container 11 through the lower nozzle 6d.

  Since the storage container constituting the cartridge was transparent, it could be confirmed that there was no abnormality in operation, and it was confirmed that the desired component was accumulated in the collection unit.

The external view of the system which consists of a supply part, the unit which performs liquid operation, and a collection | recovery unit, and a pressing part. FIG. 2 is an external view of a cartridge in a state where the apparatus of FIG. 1 is stored in a storage container. A liquid separation apparatus equipped with the cartridge of the present invention. (A) is a plan view, (B) is a left side view.

Explanation of symbols

5a, 5b, 5c: Hollow fiber membrane module 8: Squeezed portion 8a, 8b: Guide hole 9: Tube pump rotor 11: Collection container 14: Cartridge 15: Window M: Liquid separator

Claims (7)

A liquid separation cartridge for separating a part or all of a biological component of a body fluid or a liquid containing a biological component,
1) a supply unit for supplying a sample;
2) One or more units for performing one or more operations selected from separation, concentration and purification on the liquid supplied from the supply unit;
3) A cartridge for liquid separation, wherein at least a part of the recovery unit for recovering the component obtained through the unit of 2) is stored in a transparent storage container. The cartridge for liquid separation according to claim 1, wherein the cartridge has a pressing portion on an outer wall for confronting a rotor of a rotary type tube pump disposed outside the cartridge. 3. The liquid separation cartridge according to claim 2, wherein a part of the tube connecting 1) to 3) is disposed outside the pressing portion. The cartridge for liquid separation according to any one of claims 1 to 3, wherein 1) to 3) and a circuit connecting them are closed circuits. The cartridge for liquid separation according to any one of claims 1 to 4, wherein the unit (2) includes a separation membrane. 6. The liquid separation cartridge according to claim 5, wherein the membrane is a hollow fiber membrane. A liquid separation apparatus having a rotary tube pump and the liquid separation cartridge according to claim 1 on an apparatus main body.

Images