JP2006256995A - Purified solution of biocomponent, and method and apparatus for separating biocomponent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a method for removing excess protein which disturbs the detection of a trace component when carrying out clinic proteome analysis. <P>SOLUTION: The method and apparatus for separating a biocomponent comprises reducing the concentration of a specific protein to a concentration of ≤10% compared to the case not using an affinity ligand and/or antibody against the specific protein from a blood-derived sample by using a separation system using a separation membrane having ≥0.001 and ≤0.5 sieving coefficient of a dextran having 60,000 molecular weight and ≥3 permeation ratio of the dextran having 30,000 molecular weight to the dextran having 60,000 molecular weight and further using the affinity ligand and/or antibody against the specific protein having ≥30,000 molecular weight. The solution obtained by the separation method and apparatus is used for protein analysis targeting the proteome analysis such as mass analysis, electrophoresis and liquid chromatography, and enables the high-sensitivity analysis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and a separation apparatus for separating a specific protein from a blood-derived sample by combining a separation membrane and an antibody.

  In recent years, proteomic analysis (proteomics) research 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 by mass spectrometer (MS) has become possible, and 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.

  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 is introduced into clinical research (clinical proteomics), it is required to analyze a large amount of specimens quickly and reliably, and because clinical specimens are precious 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 techniques and instruments have been improved rapidly, there is still no situation where proteome analysis can be easily and rapidly performed in clinical practice.

  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 content of protein in serum is albumin (molecular weight 66 kDa), immunoglobulin (150-1000 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several hundred kDa), etc. / mL). On the other hand, most 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 protein size, 70% or more of all types of proteins have a molecular weight of 60 kDa or less, and most of the above-mentioned trace amounts of biomarker proteins are mostly 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. In addition, it is essential to recover as much as possible of components having a molecular weight of less than 60,000, but it is essential to exclude specific components having a molecular weight of 30,000 or more.

  Currently, high-performance liquid chromatography (LC) and two-dimensional electrophoresis (2 dimensional-polyacrylamide gel electrophoresis: 2D-PAGE) are used as means for separating high molecular weight proteins. Since only the sample can be processed, the target sample amount is small, and it may not be detected even if an analysis of a specific protein is attempted using a technique such as MS analysis or two-dimensional electrophoresis analysis.

  When this point is resolved, it can be expected that the speed of diagnosis of clinical tests by clinical proteomics will be dramatically improved. Specifically, a device that can efficiently separate and fractionate a target protein group is sufficient.

  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), a centrifuge tube type device for separating high molecular weight components by centrifugal filtration (for example, Nihon Millipore: Amicon Ultra (registered trademark)), Special Table 2002 Fractionation based on the electrophoresis principle disclosed in Japanese Patent No. 542163 (Patent Document 1) (for example, Gradipore: Gradiflow (registered trademark) system), traditional precipitation methods such as ethanol precipitation by Cohn, and chromatography There is a method of fractionation (for example, 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.

  Separation membranes used in artificial kidneys, artificial lungs, plasma separators, etc. have been developed in various sizes depending on the application, and improvements have been made to improve compatibility with biological components. However, there is nothing that suggests a solution to the problems of clinical proteomics (Patent Document 2).

The development of solutions that solve these problems has led to the widespread use of proteome analysis in medical research and clinical settings, enabling faster and more accurate testing and diagnosis, and intractable diseases that have no useful treatment It can be expected to be a powerful tool for investigating the cause of the disease and developing early diagnostic methods.
Japanese translation of PCT publication No. 2002-542163 Patent 3297707 By Anderson NL and Anderson NG, "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

  As described above, when proteome analysis is performed, it is necessary to remove an excessively high molecular weight protein which becomes an obstacle.

  Most recently, a method using an Affi-Gel Blue gel (N. Ahmed et al., Proteomics, On-line version, 2003/06/23) has been published as an effective and improved albumin removal method. Blue gel does not specifically remove albumin alone, but the possibility of simultaneously removing the target protein for proteome analysis cannot be denied, and it is a biological component that can provide an analytical solution for obtaining more information There is no separation solution, biological component separation method and biological component separation apparatus for obtaining it. This is a problem to be solved by the present invention.

The biological component purification solution according to the present invention, the biological component separation method and the biological component separation apparatus for obtaining the same have the following configurations (1) to (20).
(1) Separation in which dextran having a molecular weight of 60,000 obtained from a blood-derived sample has a sieving coefficient of 0.001 or more and 0.5 or less, and a transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 or more. Using a separation system using a membrane, and using an affinity ligand and / or antibody for a specific protein with a molecular weight of 30,000 or more, a specific protein can be selected as compared with the case where an affinity ligand and / or antibody for a specific protein is not used. A biological component separation solution characterized by being reduced to a concentration of 10% or less.
(2) Specific proteins are albumin, immunoglobulin G, transferrin, fibrinogen, immunoglobulin A, α2 macroglobulin, immunoglobulin M, α1 antitrypsin, complement factor C3, haptoglobin, apolipoprotein A1, apolipoprotein B, α1 acidity Glycoprotein, lipid protein a, factor H, ceruloplasmin, complement C4 factor, complement factor B, prealbumin, complement C9 factor, complement C1q factor, complement C8 factor, collagen, myosin, actin, cytokeratin, The biological component purification solution according to (1), which is either keratin or fibronectin (3), wherein a plurality of specific proteins are reduced to a concentration of 10% or less (1) or The biological component purification solution (4) according to (2) Separation membrane wherein the is a hollow fiber membrane (1) biological component separation solution according to any one of the - (3).
(5) The biological component separation solution according to any one of (1) to (3), wherein the separation membrane contains a hydrophilic component.
(6) The biological component separation solution according to (5), which is a separation membrane having a hydrophilic component content of 1% by weight or more.
(7) A method for separating a biological component from a blood-derived sample, wherein the dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and permeates dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000. When a separation system using a separation membrane with a ratio of 3 or more is used, and an affinity ligand and / or antibody for a specific protein having a molecular weight of 30,000 or more is used, and an affinity ligand and / or antibody for a specific protein is not used. A biological component separation method characterized by reducing a specific protein to a concentration of 10% or less as compared to (8) a solution in which an affinity ligand and / or antibody for a specific protein is treated with at least a separation membrane or a separation membrane It is built in a part of the flow path that passes through The biological component separation method according to (7) (9) A unit in which separation membrane modules are combined in multiple stages, and an affinity ligand and / or antibody for a specific protein is incorporated downstream of the separation membrane in the first stage The biological component separation method according to (7) or (8), wherein
(10) The solution treated with the affinity ligand and / or the antibody is further subjected to removal of the solvent using a membrane having a 50% cut-off value of a molecular weight of 1000 or less, according to any one of (7) to (9) The biological component separation method.
(11) The biological component separation method according to any one of (7) to (10), wherein the separation membrane is a hollow fiber membrane.
(12) The biological component separation method according to any one of (7) to (11), wherein the separation membrane contains a hydrophilic component.
(13) The biological component separation method according to (12), wherein the separation component has a hydrophilic component content of 1% by weight or more.
(14) An apparatus for separating a biological component from a blood-derived sample, wherein the dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and permeates dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000. A specific protein having a separation membrane with a ratio of 3 or more and reducing a specific protein having a molecular weight of 30,000 or more to a concentration of 10% or less compared to the case where an affinity ligand and / or antibody for the specific protein is not used Biological component separation device (15) characterized by containing affinity ligand and / or antibody against a flow path through which a separation membrane or a solution treated with the separation membrane passes an affinity ligand and / or antibody for a specific protein Embedded in a part of the living body as described in (14) Separation device (16) A unit in which separation membrane modules are combined in multiple stages, and is characterized in that an affinity ligand and / or antibody for a specific protein is incorporated downstream of the separation membrane in the first stage ( 14) or (15)
The biological component separation apparatus according to 1.
(17) The solution treated with the affinity ligand and / or antibody is characterized in that the solvent is further removed using a membrane having a 50% cutoff value of 1000 or less, preferably 100 or less (14) to (16) The biological component separation device according to any one of the above.
(18) The biological component separation device according to any one of (14) to (17), wherein the separation membrane is a hollow fiber membrane.
(19) The biological component separation device according to any one of (14) to (17), wherein the separation membrane contains a hydrophilic component.
(20) The biological component separation apparatus according to (19), wherein the separation component has a hydrophilic component content of 1% or more.

  The biological component purification solution obtained by the present invention and the biological component separation method and biological component separation apparatus for obtaining the same can detect a large amount of protein that has not been detected in the past from blood and other blood-derived samples. It becomes.

  As a method for obtaining a biological component purification solution, a biological component separation method and a biological component separation device for obtaining the biological component purification solution, the present inventors have a screening coefficient of 0.001 or more for dextran having a molecular weight of 60,000 from a blood-derived sample solution. 5 or less, and a membrane separation system having a permeation ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is used, and specific proteins such as albumin, immunoglobulin G, transferrin, fibrinogen, immunoglobulin A, α2 Macroglobulin, immunoglobulin M, α1 antitrypsin, complement C3 factor, haptoglobin, apolipoprotein A1, α1 acidic glycoprotein, lipid protein a, factor H, ceruloplasmin, complement C4 factor, complement factor B, prealbumin, Complement C9 factor, complement C1q factor, complement C Affinity ligands and / or antibodies for proteins such as factor, collagen, myosin, actin, cytokeratin, keratin, fibronectin, etc. are used to specifically remove specific proteins with a molecular weight of 30,000 or more, typically albumin, and affinity ligands It has also been found that it is preferable to reduce the concentration to 10% or less when no antibody is used.

  The “biological component purification solution” in the present invention is a solution used for protein analysis mainly for proteome analysis, and the “biological component separation method” is a method for obtaining a solution used for protein analysis. A “biological component separation device” is a device / system having a method for obtaining a solution used for protein analysis. The protein analysis method is not particularly limited, but LC (liquid chromatography), 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), nuclear magnetic resonance (NMR), MALDI-TOF-MS and ESI-MS (electro spray ionization) -MS) and the like. These are analysis methods in which the analysis sensitivity is lowered by containing a large amount of a specific protein having a molecular weight of 30,000 or more, such as albumin. The biological component separation solution of the present invention, the biological component separation method, or the biological component By using the solution obtained by the separation device, high sensitivity analysis becomes possible.

  In addition to albumin, high-sensitivity analysis can be achieved by removing specific proteins with a molecular weight of 30,000 or more, such as complements and fibrinogen, which are generally abundant in immunoglobulins and relatively abundant. . Further, by simultaneously removing a plurality of specific proteins having a molecular weight of 30,000 or more, it becomes possible to perform a more sensitive analysis.

  “Albumin” refers to albumin derived from humans, cows, other mammals and birds. The present invention has been found that a solution obtained by a solution separation method and apparatus using albumin as an indicator exhibits excellent characteristics as a protein analysis solution. Specifically, the specific removal of albumin from a solution that has been treated to increase the abundance ratio of substances having a molecular weight of 60,000 or less using a membrane separation system means that a protein having a molecular weight lower than that of albumin is higher. It is possible to obtain information on a large number of proteins that have been concentrated to a concentration and that have been difficult to analyze by the presence of albumin.

  “Immunoglobulins” refers to various immunoglobulins derived from humans, cows, other mammals, fish, amphibians, reptiles and birds and their constituent components. Since immunoglobulins generally have a higher molecular weight than albumin, there are few molecular immunoglobulins in a solution obtained using a membrane separation system that can remove albumin, but the structure has a low molecular weight. Some of the ingredients may be included. In such a case, it is possible to obtain more protein information by removing not only albumin but also immunoglobulins and their fragments. This applies not only to immunoglobulins but also to other proteins such as complement components and fibrinogen.

  “Blood-derived sample” refers to blood from animals such as humans. In addition to blood itself, it includes serum, plasma, and solutions made up of some components of blood such as solutions obtained by diluting and fractionating them. It is.

  In the present invention, “separation” refers to discriminating a protein for recovery from a protein for disposal. In some cases, “fraction” is used to mean “separation”. Fractionation means separating the sample using a method other than the separation method using the membrane pore size. The main protein separation / fractionation methods include aggregation / precipitation based on concentration differences, molecular sieving effect, ionic interaction, hydrophobic interaction, hydrogen bonding, chromatography using specific binding by affinity, etc. Examples include electrophoresis. In order to increase the resolution, it is generally difficult to use a single method, and it is usually achieved by using a plurality of separation modes. For example, a combination of cation chromatography and reverse phase liquid chromatography, a combination of gel filtration and affinity chromatography, or a combination of reverse phase liquid chromatography and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

The method for obtaining the biological component purification solution of the present invention and / or the configuration of the apparatus used for obtaining the biological component purification solution are as follows.
(1) Membrane Separation Step “Membrane separation step” means a step of separating a protein having a molecular weight of 60,000 or more from a sample and a protein having a molecular weight of 60,000 or less, which is a recovery target, from a sample using a separation membrane.

  The “separation membrane” referred to in the present invention is a porous separation membrane, and any of flat membrane type separation membranes such as flat filters and cartridge type filters, and hollow separation membranes such as hollow fiber membranes can be used. it can. In particular, the hollow fiber membrane can be used efficiently because it has a large membrane surface area per amount of processing liquid and can reduce pressure loss.

  In order to increase the membrane surface area per treatment liquid volume, the hollow fiber membrane preferably has a smaller inner diameter, preferably 1000 μm or less, and more preferably 500 μm or less. Further, the flat filter has an advantage that it can be easily formed at a low cost. Examples of the separation membrane material include cellulose, cellulose acetate, polycarbonate, polysulfone, polymethacrylate such as polymethyl methacrylate, polyacrylate, polyamide, polyvinylidene fluoride, polyacrylonitrile, polyester, polyethylene and polypropylene, and derivatives thereof. A material selected from one or more types can be exemplified. Of these, polysulfone, which is often used in dialysis machines in recent years, is a preferred material because of its good fractionation characteristics.

  It is preferable that protein is not adsorbed to the separation membrane as much as possible, and a hydrophilic separation membrane is preferable. In order to be a hydrophilic separation membrane, it is preferable that a hydrophilic component is contained. As a method of containing a hydrophilic component, a hydrophilic monomer and a hydrophobic monomer are copolymerized, a film is formed by blending a hydrophilic polymer and a hydrophobic polymer, or hydrophobic. Examples thereof include a method in which a hydrophilic polymer is bonded and attached to the surface of a film made of the above polymer, and the surface of the film made of a hydrophobic polymer is chemically treated, plasma treated, or radiation treated.

  The hydrophilic component is not particularly limited, but a hydrophilic polymer such as polyalkylene oxide such as polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and polyhydroxyethyl methacrylate is preferable. The hydrophilic component is preferably contained in the film in an amount of 1% by weight or more. These hydrophilic membranes have the effect of suppressing adsorption of necessary proteins and recovering them without waste.

  The “separation membrane module” referred to in the present invention is one in which the separation membrane is housed in a housing. This housing is provided with an inlet through which the solution to be separated flows in and an outlet through which the solution is separated, and a permeate outlet through which the solution separated through the separation membrane flows. The material of the housing is not particularly limited, and examples thereof include those made of plastic such as polycarbonate, polypropylene, polystyrene, polysulfone, and polyethersulfone.

  The solution in which the composition of the biological component-containing solution obtained in the present invention is changed is preferably used for protein analysis, particularly protein analysis for proteome analysis. Although it does not specifically limit as an analysis method, LC, 2D-PAGE, nuclear magnetic resonance (NMR), MALDI-TOF-MS, ESI-MS, etc. can be illustrated. These are analytical methods in which the analytical sensitivity is lowered when a specific protein having a molecular weight of 30,000 or more such as albumin is contained in a high content in a part of the solution, and the biological component obtained in the present invention By using a separation solution or a solution obtained by a biological component separation method or a biological component separation device, a highly sensitive analysis can be performed. It is also useful for proteome analysis using MS and electrophoresis.

  MS to which the solution preparation apparatus of the present invention can be directly or indirectly connected is not particularly limited, but preferably, an electrospray ionization type, an atmospheric pressure ionization type, a quadrupole (QQQ) type, a magnetic sector type, a time-of-flight type, MS / MS, MSn, FT-MS type, ion trapping type and combinations thereof. It also includes tandem MS such as MS / MS or MSn (eg MS3). In the case of tandem MS, all types of MS are applicable, but in particular for ion capture, quadrupole-time of flight (Q-TOF), FT-MS, and sector equipment with quadrupole and ion capture It is efficient to use a combination. This allows selective detection of peaks that occur in MS / MS and / or MSn measurements.

  The structure of various trace protein components if further developed by using the above-described analyzer using the biological component separation solution obtained by the present invention or the solution obtained by the biological component separation method or the biological component separation device. Information can be collected. They include not only peptide-mass fingerprint (PMF) but also primary structure information (amino acid sequence) of each peptide.

  In the present invention, in order to change the composition of a blood-derived sample and obtain a solution having a low concentration composition ratio of a specific protein having a molecular weight of 30,000 or more, such as albumin, which is easy to perform protein analysis, A method is adopted in which the permeation ratio, which is a value obtained by dividing the dextran sieving coefficient by the dextran sieving coefficient having a molecular weight of 60,000, is supplied to a separation membrane of 3 or more, and the liquid that has permeated the separation membrane is collected.

  If only a lower molecular weight substance having a molecular weight of 10,000 or less can be efficiently separated, a separation membrane may be combined in multiple stages to eliminate a substance having a high molecular weight. However, the present invention is characterized by a molecular weight of 10,000 to 60,000. It exists in the ability to concentrate a protein which is not high in the molecular weight region (content of 1% or less of the total protein content in plasma). For this method, a separation membrane in which the dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and the permeation ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is 3 or more. It is necessary to use and further combine affinity ligands and antibodies. The separation coefficient of dextran in the separation membrane is preferably 0.01 or more, and the transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is preferably 5 or more, and more preferably 10 or more. The upper limit is not particularly set, but in a membrane separation unit having this value that is too high, there is a concern that the amount of protein recovered in the desired molecular weight region may be reduced. Better.

  A hollow fiber membrane is preferably used for the separation membrane. Hollow fiber membranes have been widely used as a material for dialysis modules, which are conventionally called artificial kidneys, for the purpose of permeating proteins. Separation membranes used in artificial kidneys are designed to prevent as much as possible the penetration of proteins such as albumin, and to allow low-molecular components such as creatinine and urea to permeate. Blood is purified by letting out waste and low molecular weight proteins unnecessary for the living body from the outer space.

  In the separation membrane of the present invention, a method of permeating from the hollow fiber lumen side to the outside through the hollow fiber membrane is preferable. In particular, a method is preferred in which a solution is obtained by allowing a high molecular weight component such as albumin to penetrate as little as possible into the hollow fiber lumen, while allowing a protein component having a molecular weight of 30,000 or less to penetrate. Although similar results can be obtained using a flat membrane separation membrane, the use of a hollow fiber membrane makes it easy to increase the surface area per volume of processing liquid, and there is no operational pressure loss. Since there are few, this invention can be implemented efficiently. Further, the inner diameter of the hollow fiber membrane is preferably 1000 μm or less, and more preferably 500 μm or less. Although there is no particular lower limit, it is preferably 50 μm or more so that the pressure loss of the hollow fiber membrane does not become too large. The hollow fiber type separation membrane preferably contains 1% by weight or more of a hydrophilic component.

  In the present invention, after starting the inflow of the biological component-containing solution, the diluent for the biological component-containing solution is allowed to flow into the stock solution side of the separation membrane. This is because when the diluent is added, excessive concentration on the surface of the membrane is prevented, and deterioration of the permeation ratio of the separation membrane is prevented.

  In addition, it is preferable to use physiological saline or “buffer solution” as the diluent. As the “buffer solution”, MES, BIS-TRIS, ADA, ACES, PIPES, MOPSO, BIS-TRIS PROPANE, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, TRIZMA, HEPPSO, POPSO, TEA, EPPS, TRICINE, GLY-GLY, BICINE, PBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS and the like can be mentioned. Although the above buffer solutions are indicated by abbreviations, the detailed contents include catalogs of reagent manufacturers such as Wako Pure Chemical Industries, Ltd., Sigma Aldrich Japan Co., Ltd. (for example, 2004 edition) and MSDS (safety data sheet). It can be understood by referring.

  The separation membrane unit of the present invention is a module having a separation membrane built-in and having a stock solution inlet and a stock solution outlet connected to a flow path on the stock solution side of the separation membrane, and a permeate exit of the separation membrane, a stock solution inlet and a stock solution outlet A solution circulation circuit having a pump and a separation target liquid inlet in the middle, and a dilution liquid inlet provided at a position upstream of the separation target liquid inlet or at a position in the middle of the solution circulation circuit. The liquid flow circuit for preparing the solution in which the composition of the biological component changed is characterized. It is preferable to combine a plurality of separation membrane modules in multiple stages.

  The unit in which the separation membrane modules are combined in multiple stages includes at least two separation membrane modules, and the separation target liquid outlet of the liquid flow circuit and the separation target liquid inlet of another liquid flow circuit are directly or directly in the liquid flow path. It is an apparatus for preparing a solution in which the composition of a biological component is changed, which is indirectly connected. The first-stage unit is separated under the condition that the flow rate Q1 of the biological component-containing solution flowing into the stock solution side of the separation membrane and the flow rate Q2 of the permeated liquid satisfy 0.005 ≦ Q2 / Q1 ≦ 0.5. The sum Q3 of the flow rate Q2 of the permeated liquid and the flow rate of the biological component-containing solution and the diluted solution flowing into the stock solution side of the separation membrane is substantially equal.

  Further, two modules are prepared, and in the first module, a solution in which the composition of the biological component is changed is obtained while adding the diluent as described above, and then the solution is introduced into the second module. If a solution in which the composition of the biological component is further changed is adjusted while further diluting the diluent, the solution from which the high molecular weight protein more suitable for proteome analysis is removed can be prepared.

  Furthermore, it is possible to prepare additional modules such as third and fourth and perform the same process. The purpose of the separation membrane is to obtain a protein with a molecular weight of 10,000 or more and 60,000 or less. After separation by the membrane, a unit with the function of concentrating the protein by removing the solvent such as excess water is needed as needed. May be combined. With respect to the molecular fractionation performance of the separation membrane used for concentration, the molecular fractionation ability that does not allow the peptide to pass through in physiological saline, that is, the 50% cutoff value is preferably a molecular weight of 1000 or less, more preferably 100 or less. A separation membrane is used. This unit for concentrating proteins is called a “concentration unit”. Concentration will be described in detail in the concentration step (3).

“Membrane separation system” refers to a system having a membrane separation step for separating a protein having a molecular weight of 10,000 or more and 30,000 or less from a blood-derived sample such as serum using a membrane, either alone or in a multistage membrane connected thereto. That is.
(2) Adsorption process "Adsorption process" means a process of adsorbing a specific protein having a molecular weight of 30,000 or more such as albumin in an aqueous solution. As used herein, “adsorption” means that albumin and other specific proteins solubilized in an aqueous solution are captured by interaction with an affinity ligand and / or antibody. An affinity ligand and / or antibody for a specific protein having a molecular weight of 30,000 or more reduces the specific protein to a concentration of 10% or less as compared with the case where the affinity ligand and / or antibody for the specific protein is not used.

  In the present invention, the location of the affinity ligand and / or antibody is not particularly specified as long as it is a site that comes into contact with the liquid treated in the first-stage membrane separation step, but the separation membrane or the solution treated with the separation membrane passes. It is preferably incorporated in a part of the flow path. In the case of a unit in which separation membrane modules are combined in multiple stages, it is preferable that an affinity ligand and / or antibody for a specific protein is incorporated on the downstream side of the first separation membrane. First, it is effective to exclude a polymer component having a molecular weight of 60,000 or more from the first stage separation membrane.

  As the existence form, the affinity ligand and / or antibody immobilized on the beads is used by filling a part or the whole of the circuit, or the affinity ligand and / or antibody is immobilized on the flat filter or the membrane of the hollow fiber module. It is preferable.

  The specific protein of interest to be supplemented is selected from at least one protein present in the blood sample, and usually selected from those having a large abundance, but is analyzed when this biological component separation solution is used for analysis or the like. Those that affect the sensitivity are appropriately selected. Preferably, specifically albumin, immunoglobulin G, transferrin, fibrinogen, immunoglobulin A, α2 macroglobulin, immunoglobulin M, α1 antitrypsin, complement factor C3, haptoglobin, apolipoprotein A1, α1 acidic glycoprotein, lipid Protein a, Factor H, Ceruloplasmin, Complement C4 Factor, Complement Factor B, Prealbumin, Complement C9 Factor, Complement C1q Factor, Complement C8 Factor, Collagen, Myosin, Actin, Cytokeratin, Keratin, Fibronectin It can be illustrated.

  In addition, myosin, actin, cytokeratin, keratin, etc. are components that are not usually contained in blood, but this biological component separation solution can be used for analysis, etc. by mixing from tissues or mixing from the skin during blood collection. When used, it may affect the analytical sensitivity and is selected as a specific protein when it is necessary to remove it.

  Among them, albumin is a preferable substance because it is contained in a large amount in a blood-derived sample. In addition to albumin, it is a particularly preferable selection method to select a protein present in blood at a particularly high concentration, such as immunoglobulins represented by immunoglobulin G and immunoglobulin A, transferrin, haptoglobin, antitrypsin and the like. Moreover, it is preferable to remove a plurality of specific proteins simultaneously. Here, as long as it can be removed by other methods, it may not be selected as a protein to be sailed.

  When the specific protein to be supplemented is an immunoglobulin, it is preferable to use an affinity ligand such as protein A, protein G or protein L instead of using an antibody against the immunoglobulin.

The method for immobilizing the antibody is not particularly limited, but is a method for immobilizing the —NH ₂ terminal of the antibody, a method for immobilizing the sugar moiety of the antibody by oxidation treatment, and immobilization to a ligand such as protein A or protein G. The method can be used as a method for efficiently immobilizing an antibody.

  The antibody to be used may be a polyclonal antibody or a monoclonal antibody without limitation. The protein constituting the antibody is not particularly limited as long as it is an immunoglobulin, but immunoglobulin G is preferred.

  The carrier material used in the present invention is not particularly limited, but the group consisting of cellulose, cellulose acetate, polycarbonate, polysulfone, polymethacrylate, polyacrylate, polyamide, polyvinylidene fluoride, polyacrylonitrile, polyester, polyurethane, polystyrene, polyethylene and polypropylene. It is preferable to use a material containing a polymer selected from one or more types. As for the membrane structure, any of those having a sponge structure close to a uniform structure and those having a two-layer structure of a dense layer and a support layer having a high porosity and maintaining membrane strength can be used.

  Examples of the material form include spherical beads, fibers, etc., fibers knitted fabric, non-woven fabric, flat form using staples, hollow fiber form, etc. It is preferable for the effect of increasing the adsorption surface area. Further, a separation membrane such as a flat membrane or a hollow fiber membrane is particularly preferable because separation and adsorption can be achieved simultaneously.

  The characteristics of the membrane substrate itself include those that have been made hydrophilic to suppress nonspecific protein adsorption and those that have been made hydrophobic to selectively adsorb high molecular weight proteins such as albumin. Depending on each step of adsorption, it is appropriately selected and used.

  For hydrophilic membranes, a copolymer of a hydrophilic monomer and a hydrophobic monomer, a membrane formed by blending a hydrophilic polymer and a hydrophobic polymer, or a hydrophobic polymer For example, the surface of a membrane composed of a hydrophilic polymer is bonded and adhered, or the surface of a membrane composed of a hydrophobic polymer is chemically treated, plasma treated, or radiation treated. The method is not particularly limited. The hydrophilic component is not particularly limited, but a hydrophilic polymer such as polyalkylene oxide such as polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and polyhydroxyethyl methacrylate is preferable. As the hydrophobic membrane, a membrane in which a hydrophobic component is mixed or a hydrophobic ligand is introduced on the membrane surface is used. Examples of hydrophobic components include methacrylate polymers, acrylate esters, olefins such as ethylene and propylene, polymers composed of addition polymerizable compounds having a carbon-carbon double bond such as acrylonitrile and methacrylonitrile, polysulfone, and cellulose. Although a polymer can be illustrated, it will not specifically limit if it can be used as a film | membrane material.

  Furthermore, it is also possible to use a material in which at least one of polyethyleneimine, aminomethylpyridine, polyphenol, blue dye, divalent metal ion, hydrophobic aromatic compound and the like is immobilized.

  The molecular fractionation performance of the membrane for fixing the carrier is not particularly limited, but the sieving coefficient of dextran having a molecular weight of 60,000 is 0.001 or more and 0.5 or less, and the transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000. Is preferably 3 or more.

  The non-adsorbed fraction obtained in this step is subjected to the next re-membrane separation step as necessary. The re-membrane separation step may be omitted.

In the adsorption step and the re-membrane separation step, adsorption or separation performance can be improved by adding various chemicals to the developing buffer. Specifically, a surfactant, an emulsifier, an organic solvent, an alcohol, ethylene glycol, polypropylene glycol, polyethyleneimine, aminomethylpyridine, protamine sulfate, ammonium sulfate, polyphenol, blue dye, chaotropic salt and an aqueous solution used in the process For example, in the adsorption step, ammonium sulfate, polyethylene glycol, polyethyleneimine, chaotropic salt, or the like that promotes the aggregation of albumin is added as appropriate, including at least one substance selected from the group consisting of hydrophobic compounds. It is possible to promote macromolecularization due to protein aggregation of the polymer component, to promote adsorption and to prevent leakage from the membrane, and to efficiently cut off the polymer component. On the other hand, in the re-membrane separation step, the interaction between proteins can be suppressed and molecular weight fractionation can be efficiently performed by appropriately adding a surfactant (such as an amphoteric surfactant or an anionic surfactant). it can.

The filtered fraction obtained in this step is subjected to the next concentration step. In the case where the solution can be sufficiently separated and fractionated in the adsorption step and the re-membrane separation step, the concentration step is omitted.
(3) Concentration step “Concentration step” means a step of concentrating proteins in an aqueous solution. As used herein, “concentration” means that excess water and low molecular components having a molecular weight of 1000 or less are removed from an aqueous solution, and the polypeptide portion in the residual liquid is concentrated. In this step, a porous membrane having a molecular sieving effect is used for the flat filter or the membrane of the hollow fiber module, and concentration by molecular sieving is performed. In the case of a small amount of sample, it is effective to use a concentration device in which a flat filter is attached to a centrifugal tube. In the case of a large amount of sample, it is effective to use a hollow fiber.

  The material of the membrane used in the present invention is not particularly limited, but cellulose, cellulose acetate, polycarbonate, polysulfone, polymethacrylate such as polymethyl methacrylate, polyacrylate, polyamide, polyvinylidene fluoride, polyacrylonitrile, polyester, polyurethane, polystyrene, polyethylene In addition, a material containing one or more polymers selected from the group consisting of polypropylene is used. As for the membrane structure, any of those having a sponge structure close to a uniform structure and those having a two-layer structure of a dense layer and a support layer having a high porosity and maintaining membrane strength can be used.

Regarding the molecular fractionation performance of the membrane used in the concentration step, the membrane fraction having a molecular fractionation ability (50% cutoff value is 1000 or less, preferably 100 or less) that does not allow peptides to pass through in physiological saline. An outer filtration membrane is preferably used.
(4) Overall configuration and operating conditions Each process is directly connected by an aqueous solution flow path and can be operated continuously, thereby obtaining an effect that it can be easily and automatically continuously operated. It may be operated. A pump is attached to the tube, and liquid is fed by the pump. However, in a small scale, liquid feeding by a syringe may be performed, and concentration by a centrifugal tube type device may be performed in a concentration process. In addition, “each combined process is directly connected by an aqueous solution flow path and can be operated continuously” as referred to in the present invention is performed by a plurality of apparatuses in which a plurality of processes are connected by an aqueous solution flow path. And a mode in which a plurality of apparatuses that perform a plurality of steps independently are directly connected by an aqueous solution flow path. That is, the hollow fiber module of the first step for efficiently obtaining a protein having a molecular weight of 60,000 or less, and the second hollow fiber for simultaneously performing the step of adsorbing and removing a specific protein such as albumin and the step of concentrating the protein solution A mode in which the modules are directly connected by the aqueous solution flow path is also included.

  Although it is essential to combine the steps (1) and (2) in this permutation, a more excellent effect can be obtained by combining (3). Further, the membrane separation step is further combined as the pretreatment of (1), the concentration step is sandwiched between the steps (1) and (2), and the membrane separation step is combined again after (2). This is determined by the degree of composition of the protein contained in the specimen. Further, the repetition of the separation membrane unit having the functions (1) and (2) and the combination with the process (3) are also appropriately determined depending on the specimen.

  The present invention is suitable for the separation of biomolecules from blood-derived samples, particularly human plasma, serum and the like. The size of each filter and the hollow fiber module and the flow rate of the reflux liquid are appropriately determined depending on the quality and quantity of the sample, but when the so-called desktop size is used, the plasma is 0.1 to 400 mL, preferably 1 to 20 mL. The flow rate is 0.2 to 20 mL / min, preferably 1 to 10 mL / min.

  In addition, the membrane separation system is capable of high-speed processing, and the time required for one treatment is 0.5 to 6 hours. From the viewpoint of preventing sample contamination and biohazard, a series of devices is required once. It is possible to produce a device to be used. In analysis using electrophoresis systems and liquid chromatography, the equipment is reused, so the risk of contamination by the sample and the effect on reproducibility of the regenerated analytical column may be problematic, and the operation is complicated. In addition, it is not necessarily suitable for frequent processing of a large number of specimens. The protein fractionation device according to the present invention can be made disposable, which is a great advantage from the viewpoint of avoiding contamination from the specimen and ensuring the reproducibility of the analysis.

  Hereinafter, one embodiment of the use of the separation membrane of the present invention will be described with reference to the drawings. FIG. 1 is a schematic view showing an example of an apparatus for preparing a solution. The liquid flow is indicated by arrows. A specimen, which is a biological component such as serum, or a biological component-containing solution containing the biological component is passed from the infusion pump 100 through the three-way valve 101 to the first separation membrane module 105 containing a separation membrane having a specific permeation ratio in the first step. The solution is injected and sent through the solution circulation circuit 102 formed of a tube by the circulation pump 103 to circulate. The permeate that has permeated the separation membrane in the first step is obtained from the first-stage membrane unit treatment liquid recovery port 104 that is the outlet of the permeate. This aspect is a basic unit (unit) of one process. When it is desired to further remove a high molecular weight protein, a plurality of modules can be connected. FIG. 2 shows an example in which two modules are connected to perform two-stage processing.

  The permeated liquid is injected into the next process unit through a tube directly connected to the treatment liquid recovery port of the membrane separation unit which is the permeate outlet. The target solution is obtained from a recovery port (104 in the apparatus shown in FIG. 1 and 204 in the apparatus shown in FIG. 2) existing in the most downstream unit in the process.

  FIG. 3 shows an example in which a concentration separation membrane unit is connected to the third stage after the two-stage process, and the three-stage process is performed. The target solution is recovered from the processing liquid recovery port 308 of the concentration unit.

  In the present invention, the removal of a specific protein with an affinity ligand or antibody is limited to the step after substance exclusion in the large molecular weight region by the first-stage separation. That is, in FIG. 1 and FIG. 2, it is between the outside of the 107 film and the recovery port, and in FIG. 3, it is limited to the inside of the 307 film from the 107 film. When the unit for concentration is connected as in the apparatus of FIG. 3, the antibody may be present from the outside of the 107 membrane to the inside of the 207 membrane to prevent the antibody from being mixed into the recovered solution. preferable. FIG. 4 shows an example in which an antibody for removing a specific protein is incorporated into a process after one-stage processing as an antibody module.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples. However, the scope of the present invention is not limited only to these examples (transmission ratio measurement method).
100 hollow fiber membranes with an inner diameter of 200μm are filled in a glass housing with a diameter of about 5mm and a length of 12cm, and the inlet where the solution to be separated flows in and the outlet where it flows out are bonded with epoxy resin chemical reaction type manufactured by Konishi Co., Ltd. A separation membrane module is prepared by potting with an agent Quick Mender (registered trademark). Next, the hollow fiber of the module and the inside of the module are washed with distilled water for 1 hour.

  The method for measuring the transmittance of dextran was as follows. Dextran manufactured by FULKA average molecular weight ~ 1500 (No. 31394), average molecular weight ~ 6000 (No. 31388), average molecular weight 15000 ~ 20000 (No. 31387), average molecular weight ~ 40000 (No. 31389), average molecular weight ~ 60000 ( No.31397) and an average molecular weight of ˜200,000 (No.31398) are dissolved in distilled water so as to be 0.5 mg / mL (3.0 mg / mL in the whole solute), respectively, to prepare a dextran aqueous solution (stock solution). Prepare a pump that circulates the liquid on the stock side and a pump that filters the module, and use ultrafiltered water so that the stock solution circulation flow rate is 5 ml / min and the filtration flow rate is 0.2 mL / min. Adjust. Next, after the filled ultrafiltrated water is replaced with the stock solution, filtration is started at room temperature (25 ° C.). At this time, the stock solution and filtrate at the module outlet are discarded without being returned. The liquid after 60 to 75 minutes is collected, the differential refractive index of dextran in the module stock solution inlet and outlet, and the filtrate is measured, and the sieve coefficient of dextran is calculated from these measured values.

  The dextran concentration was measured as follows. The sampled solution is filtered through a filter with a pore size of 0.5 micron, the filtrate is a GPC column (Tosoh TSK-gel-G3000PWXL), the column temperature is 40 ° C, the mobile phase is 1 mL / min of distilled water for liquid chromatography, and the sample injection amount is 100 μl. And measured with a differential refractometer (RI-8020 manufactured by Tosoh Corporation) at a slice time of 0.02 min and a base-line range of 4.5 to 11.0 min. The column is calibrated using monodispersed dextran (Dextran Standard No.31416, No.31417, No.31418, No.31420, No.31422 manufactured by Fluka) immediately before the measurement. In calibration, each dextran standard is divided into No.31416, No.31418, No.31422 and No.31417, No.31420, each dissolved at 0.5 mg / mL, and the retention time and weight average of each peak top The relationship between retention time and weight average molecular weight was obtained by plotting molecular weight and obtaining an exponential approximation curve. For the sieve coefficient, the differential refractive index value (Ci) at the module stock solution inlet, the differential refractive index value at the outlet (Co), and the differential refractive index value (Cf) of the filtrate are measured, and the sieve coefficient (SC) is calculated by the following formula. can do.

SC = 2Cf / (Ci + Co)
The value obtained by dividing the obtained dextran weight average molecular weight 30,000 sieve coefficient by the dextran weight average molecular weight 60,000 sieve coefficient is defined as the transmission ratio.
(Protein analysis by electrophoresis)
The obtained biological component separation solution (analysis solution) was analyzed by electrophoresis. The method is as follows.

  1. Add an equal amount of sample buffer to the biological component separation solution and heat at 100 ° C for 3 minutes to prepare a sample for electrophoresis. As a sample buffer, a mixture of 10 ml of glycerin, 1 g of SDS, 0.05 g of BPB, 25 ml of 0.2 M Tris-HCl pH6.8 (pH adjusted to 6.8 by adding Tris to HCl) and 14 ml of distilled water is used.

  2. Electrophoresis is carried out by the method described in Non-Patent Document 6 using SDS-PAGEmini 4-20% 1.0 mm thickness 10 well (manufactured by TEFCO), which is a commercially available gel for SDS-PAGE.

  3. Remove the gel from the gel cassette and perform fluorescent staining. For fluorescent staining, a commercially available kit Deep Purple Total Protein Stain (manufactured by Amersham Bioscience) is used.

Example 1
Bundling 100 polysulfone hollow fibers taken from Toraysulfone BS-P, an artificial kidney made by Toray, and fixing both ends to a glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber Two were made. The mini-module has an inner diameter of about 7 mm and a length of about 12 cm, and has two dialysate ports like a general hollow fiber membrane dialyzer. The hollow fiber of the mini module and the inside of the module were washed with distilled water. The dextran sieving coefficient of one molecular weight of 60,000 was 0.15, the dextran sieving coefficient of 30,000 was 0.857, and the dextran permeation ratio of 30,000 and 60,000 was 5.7. Thereafter, a PBS solution (manufactured by Seikagaku Corporation, prepared with Dulbecco's PBS (-) powder “Nissui”, hereinafter abbreviated as PBS) was filled to obtain a hollow fiber membrane mini-module (hereinafter abbreviated as mini-module 1). .

  Human serum (SIGMA H1388, Lot 28H8550) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. 1 ml of serum was diluted to 4 ml with PBS, and filtered at 20 ° C for 2 hours at a circulation flow rate of 5 ml / min and a filtration flow rate of 0.2 mL / min. This corresponds to a step of separating a protein having a molecular weight of 60,000 or less for the purpose of recovery). At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. The total amount of albumin in the serum before fractionation was measured by Human Albumin ELISA Quantitation Kit (Cat No. E80-129 manufactured by BETHYL). The result was 22872 μg, and the total amount of albumin in the filtrate obtained in 2 hours The result measured by Albumin ELISA Quantitation Kit was 9456 μg.

  The obtained filtrate is concentrated to 1 mL using a centrifugal separation membrane (Valaspin, 3000 MWCO manufactured by Sartorius), and 4 ml of buffer A (Agilent No. 5185-5987) is added, followed by filtration through a 0.22 μm centrifugal filter. did. 500 ul of this filtrate was applied to a Multiple Affinity Removal Column (No. 5185-5985, manufactured by Agilent), which is a column combining six types of antibodies. The antibodies immobilized on this column are the six types shown in Table 1.

  Next, Buffer A was flowed 10 times the column volume, and the obtained solution was used as a flow-through fraction. Next, the buffer B (Agilent No.5185-5988) was flowed 10 times the column volume of the protein adsorbed on the column, and the resulting solution was used as the adsorption fraction. The flow-through fraction and the adsorbed fraction were concentrated using a centrifugal separation membrane (vivaspin, 3000 MWCO manufactured by Sartorius) and analyzed by SDS-PAGE. The result of the analysis is shown in FIG. There is almost no overlap between the band separated from the pass-through fraction and the band separated from the adsorbed fraction, and the six proteins are almost adsorbed and removed by the antibody, and the concentration of the specific protein is compared to the case where the antibody column is not used. The concentration was 10% or less. It could be preferably used for proteome analysis.

(Comparative Example 1)
40 μL of the same lot of human serum (SIGMA H1388, Lot 28H8550) used in Example 1 was diluted 5 times with the antibody column dedicated buffer used in Example 1 and separated. The flow-through fraction and the adsorbed fraction were each concentrated to 0.2 mL using a centrifugal separation membrane (Sartorius vivaspin, 3000 MWCO), and 5 μL of each was analyzed by SDS-PAGE. The result of the analysis is shown in FIG. Some bands, including albumin, disappeared by the antibody, and the concentration of the specific protein was 10% or less compared to the case where the antibody column was not used, but in a wider molecular weight region than lane 3 in FIG. There were many bands across. It could not be preferably used for proteome analysis.

(Example 2)
Bundling 100 polysulfone hollow fibers taken out from Toraysulfon BS-UL, an artificial kidney made by Toray, and fixing both ends to the glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fibers. Two were made. The mini-module has an inner diameter of about 7 mm and a length of about 12 cm, and has two dialysate ports like a general hollow fiber membrane dialyzer. The hollow fiber of the mini module and the inside of the module were washed with distilled water. Among them, the dextran sieving coefficient of one molecular weight 60,000 was 0.0226, the dextran sieving coefficient of 30,000 was 0.3829, and the dextran permeation ratio of 30,000 and 60,000 was 16.9. Thereafter, a PBS solution (manufactured by Seikagaku Corporation, prepared with Dulbecco's PBS (-) powder “Nissui”, hereinafter abbreviated as PBS) was filled to obtain a hollow fiber membrane mini-module (hereinafter abbreviated as mini-module 1). .

  Human serum (SIGMA H1388, Lot 28H8550) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. 1 ml of serum was diluted to 4 ml with PBS, and filtered at 20 ° C for 2 hours at a circulation flow rate of 5 ml / min and a filtration flow rate of 0.2 mL / min. This corresponds to a step of separating a protein having a molecular weight of 60,000 or less for the purpose of recovery). At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. The total amount of albumin in the serum before fractionation was measured by Human Albumin ELISA Quantitation Kit (Cat No. E80-129 manufactured by BETHYL). The result was 22872 μg, and the total amount of albumin in the filtrate obtained in 2 hours The result measured with Albumin ELISA Quantitation Kit was 791 μg.

  The filtrate obtained by this operation was concentrated by the same method as in Example 1, and separated by the same antibody column as in Example 1. The obtained solution was concentrated and analyzed by SDS-PAGE. As a result, the protein in the high molecular weight region is almost removed from the albumin by membrane separation, and the concentration of the specific protein is 10% or less compared to the case where no antibody column is used, and the protein present in the region having a molecular weight of about 30,000. Was concentrated. It could be preferably used for proteome analysis.

(Example 3)
Bundling 100 polysulfone hollow fibers taken from Toraysulfone TS-ML, an artificial kidney made by Toray, and fixing both ends to the glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber Two were made. The mini-module has an inner diameter of about 7 mm and a length of about 12 cm, and has two dialysate ports like a general hollow fiber membrane dialyzer. The hollow fiber of the mini module and the inside of the module were washed with distilled water. Among them, the dextran sieving coefficient of one molecular weight of 60,000 was 0.007329, the dextran sieving coefficient of 30,000 was 0.244, and the dextran permeation ratio of 30,000 and 60,000 was 33.3. Thereafter, a PBS solution (manufactured by Seikagaku Corporation, prepared with Dulbecco's PBS (-) powder “Nissui”, hereinafter abbreviated as PBS) was filled to obtain a hollow fiber membrane mini-module (hereinafter abbreviated as mini-module 1). .

  Human serum (SIGMA H1388, Lot 28H8550) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. 1 ml of serum was diluted to 4 ml with PBS, and filtered at 20 ° C for 2 hours at a circulation flow rate of 5 ml / min and a filtration flow rate of 0.2 mL / min. This corresponds to a step of separating a protein having a molecular weight of 60,000 or less for the purpose of recovery). At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. The total amount of albumin in the serum before separation was measured by Human Albumin ELISA Quantitation Kit (Cat No. E80-129 manufactured by BETHYL). The result was 22872 μg, and the total albumin amount in the filtrate obtained in 2 hours was determined by Human Albumin. The result measured by ELISA Quantitation Kit was 99 μg.

  The filtrate obtained by this operation was concentrated by the same method as in Example 1, and separated by the same antibody column as in Example 1. The obtained solution was concentrated and analyzed by SDS-PAGE. The results are shown in FIG. Many proteins in the lower molecular weight region than albumin as well as albumin were removed by membrane separation, and the concentration of the specific protein was 10% or less compared to the case where no antibody column was used. The absolute amount of protein that permeated through the membrane was low, and further concentration was necessary to use it for proteomic analysis, but some analytical results could be obtained. It was confirmed that the dextran having a molecular weight of 60,000 has a sieving coefficient of 0.01 or more.

(Comparative Example 2)
Bundled 100 polysulfone hollow fibers produced by trial at Toray, both ends were fixed to the glass tube module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber, and two mini modules were similarly prepared. . The mini-module has an inner diameter of about 7 mm and a length of about 12 cm, and has two dialysate ports like a general hollow fiber membrane dialyzer. The hollow fiber of the mini module and the inside of the module were washed with distilled water. Among them, the dextran sieving coefficient with a molecular weight of 60,000 was 0.000872, the dextran sieving coefficient with a molecular weight of 30,000 was 0.0012, and the dextran permeability ratio with molecular weights of 30,000 and 60,000 was 13.8. Thereafter, a PBS solution (manufactured by Seikagaku Corporation, prepared with Dulbecco's PBS (-) powder “Nissui”, hereinafter abbreviated as PBS) was filled to obtain a hollow fiber membrane mini-module (hereinafter abbreviated as mini-module 1). .

  Human serum (SIGMA H1388, Lot 28H8550) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. 1 ml of serum was diluted to 4 ml with PBS, and filtered at 20 ° C for 2 hours at a circulation flow rate of 5 ml / min and a filtration flow rate of 0.2 mL / min. This corresponds to a step of separating a protein having a molecular weight of 60,000 or less for the purpose of recovery). At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. The total amount of albumin in the serum before separation was measured by Human Albumin ELISA Quantitation Kit (Cat No. E80-129, manufactured by BETHYL). The result was 22872 μg, and the total albumin content in the filtrate obtained in 2 hours The result measured with ELISA Quantitation Kit was 2.3 μg.

  The filtrate obtained by this operation was concentrated by the same method as in Example 1, and separated by the same antibody column as in Example 1. The obtained solution was concentrated and analyzed by SDS-PAGE. Although the concentration of specific proteins was 10% or less compared to the case where no antibody column was used, most of the proteins in the low molecular weight region as well as albumin were removed by membrane separation and detected as specific bands could not. No change in the concentration of a specific protein could be detected. It was confirmed that the dextran having a molecular weight of 60,000 requires a sieve coefficient of at least 0.001 or more.

(Comparative Example 3)
Bundling 100 regenerated cellulose hollow fibers taken from AM-SD, an artificial kidney made by Asahi Medical (currently Asahi Kasei Medical), and fixing both ends to the glass tube module case with an epoxy potting agent so as not to block the hollow part of the hollow fiber Two mini-modules were created in the same way. The mini-module has an inner diameter of about 7 mm and a length of about 12 cm, and has two dialysate ports like a general hollow fiber membrane dialyzer. The hollow fiber of the mini module and the inside of the module were washed with distilled water. Among them, the dextran sieving coefficient of one molecular weight 60,000 was 0.0154, the dextran coefficient of molecular weight 30,000 was 0.0292, and the dextran permeation ratio of 30,000 and 60,000 was 1.9. Thereafter, a PBS solution (manufactured by Seikagaku Corporation, prepared with Dulbecco's PBS (-) powder “Nissui”, hereinafter abbreviated as PBS) was filled to obtain a hollow fiber membrane mini-module (hereinafter abbreviated as mini-module 1). .

  Human serum (SIGMA H1388, Lot 28H8550) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. 1 ml of serum was diluted to 4 ml with PBS, and filtered at 20 ° C for 2 hours at a circulation flow rate of 5 ml / min and a filtration flow rate of 0.2 mL / min. This corresponds to a step of separating a protein having a molecular weight of 60,000 or less for the purpose of recovery). At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. The total amount of albumin in the serum before separation was measured by Human Albumin ELISA Quantitation Kit (Cat No. E80-129, manufactured by BETHYL). The result was 22872 μg, and the total albumin content in the filtrate obtained in 2 hours The result measured with ELISA Quantitation Kit was 269 μg.

  The filtrate obtained by this operation was concentrated by the same method as in Example 1, and separated by the same antibody column as in Example 1. The obtained solution was concentrated and analyzed by SDS-PAGE. As a result, not only albumin but also many proteins in the lower molecular weight region than albumin were removed by membrane separation, and the concentration of the specific protein was 10% or less compared to the case where no antibody column was used. It could not be used preferably. It was confirmed that the transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is required to be 3 or more.

Example 4
An anti-human albumin antibody column was prepared by immobilizing 5 mg of anti-human albumin antibody (Goat Anti-Human Albumin, Policlonal Antibody; Affinity Purified, Academy Bio-Medical Company, Inc.) on HiTrap NHS-activated (manufactured by Amersham Biosciences) did. Excess human albumin solution was applied to this column, and the albumin adsorption capacity of this column was measured and found to be 960 μg. The column was washed with 0.1 M glycine hydrochloride buffer (pH 2.7), washed and equilibrated with PBS. Washing was repeated alternately with glycine hydrochloride buffer and PBS, and it was confirmed that the antibody did not come off the column.

  In the same manner as in Example 2, 100 polysulfone hollow fibers taken out from Tresulfone BS-UL were bundled to produce a minimodule 1, and human serum (SIGMA H1388, Lot 28H8550) was filtered in the same manner as in Example 1. One of the mini-module 1 on the dialysate side was capped, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane was connected at the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. The silicon tube on the dialysate side was connected to the anti-human albumin antibody column so that the solution filtered through the hollow fiber membrane passed through the antibody column.

  1 ml of serum was diluted to 4 ml with PBS, and filtration was performed at 20 ° C. for 2 hours at a flow rate of circulation flow rate of 5 ml / min and filtration flow rate of 0.2 mL / min. At this time, PBS was added to the serum for the filtered volume, and the amount of circulating liquid was kept constant. From the results of Example 2, the total amount of albumin in the serum before separation / fractionation is 22872 μg, and the total amount of albumin in the filtrate before passing through the antibody column obtained in 2 hours is predicted to be 791 μg. The total amount of albumin in the filtrate after passing through the antibody column obtained in 2 hours was measured with a Human Albumin ELISA Quantitation Kit, and the result was 1 μg or less. The concentration of albumin was 10% or less compared to the case where no antibody column was used, and could be preferably used for proteome analysis. As a result, the separation by the membrane and the albumin removal by the antibody can be simultaneously performed in the connected circuit.

It is a membrane separation unit schematic diagram of the separation system of the present invention. 1 is a schematic diagram of a separation system of the present invention. (Two membrane separation units) 1 is a schematic diagram of a separation system of the present invention. (Example of 2 membrane separation units and 1 concentration unit) 1 is a schematic diagram of a separation system of the present invention. (Example of two membrane separation units, one affinity ligand or antibody-containing module, and one concentration unit) 2 is an electrophoresis (SDS-PAGE) photograph of each fraction obtained in Example 1. FIG. Albumin that was present in a large amount in the sample before the antibody column (Multiple Affinity Removal Column) treatment has almost disappeared from the fraction passed through the antibody column. 2 is an electrophoretogram of each fraction obtained in Comparative Example 1. Even when the separation membrane-untreated serum is treated with an antibody column, many proteins are present in the high molecular weight region. 2 is an electrophoresis photograph of each fraction obtained in Example 3. FIG. The resulting solution has a low protein content and the band is light overall.

Explanation of symbols

100 infusion pump
101 Three-way valve 102 Solution circulation circuit 103 Circulation pump 104 Membrane separation unit treatment liquid recovery port 105 Separation membrane module 106 Lower port of module 107 Semi-permeable membrane inside module (image)
202 Second stage solution circulation circuit 203 Second stage circulation pump 204 Treatment liquid recovery port 205 of second stage membrane separation unit Second separation membrane module 206 Lower port 207 of second stage module Half of second stage module Permeable membrane (image)
302 Concentration Unit Solution Circulation Circuit 303 Concentration Unit Circulation Pump 304 Concentration Unit Filtrate Outlet 305 Concentration Membrane Module 306 Concentration Module Lower Port 307 Semipermeable Membrane Inside Concentration Module (Image)
308 Treatment solution recovery port of the concentration unit 400 Affinity ligand or antibody-containing module 1 Molecular weight marker for electrophoresis (RPN5800)
2 Filtrate 3 obtained in Example 1 Antibody column passage fraction 4 of the filtrate obtained in Example 1 Antibody column adsorption fraction 5 of filtrate obtained in Example 1 Human serum 6 of Example 1 Antibody column passage of human serum Fraction (Comparative Example 1)
7 Antibody column adsorption fraction of human serum 8 Filtrate obtained in Example 3 Antibody column flow-through fraction of filtrate obtained in Example 3 Antibody column adsorption fraction of filtrate obtained in Example 3

Claims (20)

  A biological component separation solution obtained from a blood-derived sample, wherein the dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and the transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is 3 Using the separation system using the separation membrane as described above, and further using an affinity ligand and / or antibody for a specific protein having a molecular weight of 30,000 or more, compared with a case where an affinity ligand and / or antibody for a specific protein is not used, A biological component separation solution, wherein a specific protein is reduced to a concentration of 10% or less.   Specific proteins are albumin, immunoglobulin G, transferrin, fibrinogen, immunoglobulin A, α2 macroglobulin, immunoglobulin M, α1 antitrypsin, complement C3 factor, haptoglobin, apolipoprotein A1, apolipoprotein B, α1 acidic glycoprotein, Lipid protein a, factor H, ceruloplasmin, complement factor C4, complement factor B, prealbumin, complement factor C9, complement factor C1q, complement factor C8, collagen, myosin, actin, cytokeratin, keratin, fibronectin The biological component purification solution according to claim 1, wherein   The biological component purification solution according to claim 1 or 2, wherein a plurality of specific proteins are reduced to a concentration of 10% or less.   The biological component separation solution according to any one of claims 1 to 3, wherein the separation membrane is a hollow fiber membrane.   The biological component separation solution according to any one of claims 1 to 3, wherein the separation membrane contains a hydrophilic component.   The biological component separation solution according to claim 5, wherein the separation component has a hydrophilic component content of 1% by weight or more.   A method for separating a biological component from a blood-derived sample, wherein a dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and a transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is 3 Using the separation system using the separation membrane as described above, and further using an affinity ligand and / or antibody for a specific protein having a molecular weight of 30,000 or more, compared with a case where an affinity ligand and / or antibody for a specific protein is not used, Biological component separation method characterized by reducing specific protein to a concentration of 10% or less   The biological component according to claim 7, wherein an affinity ligand and / or an antibody for a specific protein is incorporated in at least a part of a separation membrane or a channel through which a solution treated with the separation membrane passes. Separation method   The unit according to claim 7 or 8, wherein the separation membrane module is a multi-stage unit, and an affinity ligand and / or an antibody against a specific protein is incorporated downstream of the first separation membrane. The biological component separation method described.   The biological component separation method according to any one of claims 7 to 9, wherein the solvent is further removed from the solution treated with the affinity ligand and / or the antibody using a membrane having a 50% cutoff value of a molecular weight of 1000 or less. .   The biological component separation method according to any one of claims 7 to 10, wherein the separation membrane is a hollow fiber membrane.   The biological component separation method according to claim 7, wherein the separation membrane contains a hydrophilic component.   The biological component separation method according to claim 12, wherein the separation component has a hydrophilic component content of 1% by weight or more.   An apparatus for separating a biological component from a blood-derived sample, wherein a dextran having a molecular weight of 60,000 has a sieving coefficient of 0.001 or more and 0.5 or less, and a transmission ratio of dextran having a molecular weight of 30,000 and dextran having a molecular weight of 60,000 is 3 An affinity ligand for a specific protein that has a separation membrane as described above, and reduces the specific protein with a molecular weight of 30,000 or more to a concentration of 10% or less compared to the case where an affinity ligand and / or antibody for the specific protein is not used. And / or a biological component separation device containing an antibody   The biological component separation according to claim 14, wherein an affinity ligand and / or an antibody for a specific protein is incorporated into a separation membrane or a part of a flow path through which a solution treated by the separation membrane passes. apparatus   The unit according to claim 14 or 15, wherein the separation membrane module is a multi-stage unit, and an affinity ligand and / or an antibody against a specific protein is incorporated downstream of the first-stage separation membrane. The biological component separation device described.   17. The solution treated with an affinity ligand and / or an antibody is further subjected to removal of the solvent using a membrane having a 50% cutoff value of a molecular weight of 1000 or less, preferably 100 or less. Biological component separation device.   The biological component separation device according to any one of claims 14 to 17, wherein the separation membrane is a hollow fiber membrane.   The biological component separation device according to any one of claims 14 to 17, wherein the separation membrane contains a hydrophilic component. The biological component separation device according to claim 19, wherein the separation component has a hydrophilic component content of 1% or more.