JP2005214919A - Separation membrane for pretreatment for protein and/or peptide analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separation membrane for obtaining a solution for analysis (specimen to be analyzed) eliminated of the interfering substance at trace analysis, when conducting clinical proteome analysis. <P>SOLUTION: The separation membrane of asymmetric constitution effectively eliminates the interfering substances of high molecular weight. The solution, obtained by this separation membrane, is used for protein analysis, such as mass spectrometry, electrophoresis, liquid chromatography, etc., and enables high-sensitivity analysis. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a separation membrane that separates a biological component from a stock solution containing the biological component, particularly human blood, urine, and the like to obtain an analysis solution (analyte sample).

  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 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 era diagnosis and treatment trump (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 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 protein content in serum is albumin (molecular weight 66 kDa), immunoglobulin (150-1000 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several 100 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 the component having a molecular weight of less than 60,000.

  Currently, high-performance liquid chromatography (LC) and two-dimensional electrophoresis (2 dimensional-polyacrylamide gel electrophoresis: 2D-PAGE) are used as separation methods for these high molecular weight proteins. Since only the sample can be processed, the target sample amount is small and may not be detected even if protein analysis such as MS analysis or two-dimensional electrophoresis analysis is performed.

  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, a device that can efficiently fractionate and separate a target protein group may be used.

  Products that have already been put into practical use with albumin as the main target substance, or disclosed technologies include carriers with immobilized affinity ligands such as blue dyes (for example, “Milpore 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) ") A method of fractionation by the electrophoresis principle disclosed in JP-T-2002-542163 (Patent Document 1) (for example, Gladipore: "Gradiflow (registered trademark)" system), Cohn's ethanol precipitation, etc. For example, a simple precipitation method or a fractionation method by chromatography (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. (Patent Document 2), however, there is no suggestion of solving the problems of the clinical proteome.

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.
JP-T-2002-542163 Japanese Patent No. 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 for the New Chemistry (Vol. 1) Protein (1) Separation, Purification, and Properties”, Tokyo Chemical Doujin, 1990 Cell Engineering Supplement, “Bio-Experiment Illustrated 5”, Shujunsha, 2001

  As described above, when performing clinical proteome analysis, it is necessary to remove excess high molecular weight proteins that would interfere.

  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) is an effective and improved albumin removal method, and a separation method that can provide an analytical solution for obtaining more information There is no. This is a problem to be solved by the present invention.

  Accordingly, an object of the present invention is to provide a separation membrane that can easily obtain an advantageous analytical solution from which excess high molecular weight proteins have been removed.

The separation membrane according to the present invention has the following configurations (1) to (7).
(1) A separation membrane for protein and / or peptide analysis pretreatment, wherein the membrane structure is an asymmetric structure.
(2) The separation membrane according to (1) above, wherein a permeation ratio of a protein having a molecular weight of less than A and a protein having a molecular weight of 4A or more is 20 or more.
(3) The separation membrane according to (1) above, wherein a permeation ratio of a protein having a molecular weight of less than 15,000 and a protein having a molecular weight of 60,000 or more is 20 or more.
(4) The separation membrane according to any one of (1) to (3) above, wherein the membrane material contains a hydrophilic polymer.
(5) The separation membrane according to (4), wherein the hydrophilic polymer is polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylamide, or polyethyleneimine.
(6) The separation membrane according to (4) or (5) above, wherein the content of the hydrophilic polymer is 1 to 10% of the membrane material.
(7) The protein and / or peptide to be analyzed is composed of blood, plasma, serum and other blood-derived substances, urine, ascites, saliva, tears, cerebrospinal fluid, pleural effusion or cells (1) The separation membrane according to any one of (6) to (6).

  The biological component separation solution (analytical solution) obtained by the separation system using the separation membrane in the present invention detects a lot of minute amounts of proteins that have not been detected in the past from biological component-containing stock solutions including blood, serum, and plasma. It becomes possible to do. The present inventors made it possible to obtain a biological component separation solution (analysis solution) having a low concentration of a protein having a molecular weight of 60,000 or more in the total protein using a specific membrane separation unit, and to obtain an excellent analysis result. Succeeded in getting.

  The biological component separation solution (analysis solution) referred to in the present invention is a solution obtained by subjecting a biological component-containing stock solution such as blood to a specific treatment to change the structure of the contained protein. Here, “blood” refers to the blood of animals such as humans, and includes solutions composed of some components in blood such as serum and plasma. “Biological solution” (biological component-containing undiluted solution) is a solution containing protein with biological substances such as blood, urine, saliva, tears, cerebrospinal fluid, ascites, pleural effusion, or protein extract from cells. That is.

  The “stock solution” is a stock solution for preparing the biological component separation solution (analysis solution) of the present invention. This biological component separation solution (analysis solution) is preferably used for protein analysis because the concentration of protein having a molecular weight of 60,000 or more is low. 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 that lower the analytical sensitivity by containing a large amount of protein that is present in a large amount in some solutions such as albumin. By using this solution, high-sensitivity analysis can be performed. It becomes possible.

  A protein having a molecular weight of less than 15,000 is a protein having a relatively small molecular weight among proteins. If it is blood, its existence ratio is low with respect to the total protein, but there are many types. Here, β2-microglobulin having a molecular weight of 16,000 is used as an index. Proteins having a molecular weight of 60,000 or more correspond to albumin and immunoglobulin transferrin in the case of blood and are present in a high content in the stock solution. Here, albumin having a molecular weight close to 60,000 was used as an index.

The term “separation” as used in the present invention refers to discriminating a protein for recovery from a protein for disposal. The main protein separation / fractionation methods include aggregation / precipitation by concentration difference, molecular sieving effect, ionic interaction, hydrophobic interaction, hydrogen bonding, chromatography using specific binding by affinity, etc. Examples include electrophoresis.
“Membrane” means a porous separation membrane, and any of flat membrane type separation membranes (flat membranes) such as flat filters and cartridge type filters and hollow separation membranes (hollow fiber membranes) such as hollow fibers are used. In general, however, hollow fibers can be used most efficiently because they have 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 inner diameter is preferably smaller, 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 membrane materials include cellulose acetate-based polymers such as cellulose and cellulose triacetate, polycarbonate, polysulfone-based polymers such as polysulfone and polyethersulfone, polymethacrylates such as polymethyl methacrylate, polyacrylates, polyamides, polyvinylidene fluoride, polyacrylonitrile, Examples thereof include one or more materials selected from the group consisting of polyester, polyurethane, polystyrene, polyethylene, polypropylene, and derivatives thereof. Of these, polysulfone, which is often used in dialysis machines in recent years, is a preferred material because of its good fractionation characteristics.

  The “separation membrane module” is a membrane formed by housing a membrane in a housing. The housing is provided with an inlet through which the solution to be separated flows in and an outlet through which the solution to be separated flows out and a separation liquid outlet through which the separated solution flows out. 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 “membrane separation unit” includes a “separation membrane module” and a “tube circuit for circulating the liquid”. Here, “circulation” is a liquid feeding method for returning the liquid discharged from the separation membrane module to the separation membrane module via a tube circuit.

  The “separation system” refers to a system including a membrane separation process in which a stock solution such as blood is fractionated using a membrane.

  The number of membrane separation units in the separation system is preferably 2 or more. The number of membrane separation units is preferably 5 or more, and more preferably 10 or more. There is no particular upper limit, but it is better to be 100 or less because the system becomes complicated.

  In this separation system, it is preferable to use a separation membrane having a permeation ratio of 20 or more between a protein having a molecular weight of less than 15,000 and a protein having a molecular weight of 60,000 or more. The permeation ratio is preferably 50 or more, and more preferably 100 or more. An upper limit value is not particularly provided, but a separation membrane having a too high value collects proteins having a molecular weight of less than 15,000. Since there is concern that the amount will decrease, it is preferably 10000 or less.

  The separation membrane used in the separation system is preferably a hollow fiber membrane. Even if a flat membrane is used, it is possible to obtain the same result. However, in particular, the hollow fiber membrane can easily increase the membrane surface area per treatment liquid volume, and the present invention can be efficiently carried out. The inner diameter of the hollow fiber membrane is preferably 1 mm or less, and more preferably 0.5 mm 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.

  In the membrane separation unit, it is preferable that the diluent is introduced into the membrane separation unit at least after the biological component stock solution is introduced. When the diluted solution is not sent, the concentration proceeds excessively in the membrane separation unit, and the permeation ratio by the separation membrane deteriorates. In addition, it is preferable to use a solution that is isotonic with the biological component stock solution as the diluent. When the biological component is composed of blood components, it is preferable to use physiological saline or a “buffer solution”. As the above “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.

  The flow rate Q1 of the liquid flowing into the separation membrane module is preferably larger than the flow rate Q2 of the liquid to which the separated liquid is sent. If the flow rate Q1 of the liquid flowing into the separation membrane module is equal to the flow rate Q2 of the liquid to which the separated liquid is sent, the concentration proceeds excessively in the separation membrane module, deposits are deposited on the membrane surface, The ratio in the separation membrane deteriorates due to clogging. In order to suppress the deposition of biological components on the membrane surface, the ratio Q2 / Q1 of the flow rate Q1 of the liquid flowing into the separation membrane module and the flow rate Q2 of the separated liquid sent is 0.5 or less. It is desirable to be. In addition, when the ratio Q2 / Q1 of the flow rate Q1 of the liquid flowing into the separation membrane module and the flow rate Q2 of the separated liquid sent is 0, filtration is not performed, and mass transfer is performed only by diffusion. Since the speed | rate of becomes slow, it is desirable that it is 0.01 or more.

  It is desirable that the flow rate Q2 at which the liquid separated by the separation membrane module is fed and the flow rate Q3 at which the diluting solution flows into the membrane separation unit are equal. When Q2 ≦ 0.5Q3, the volume of the membrane separation unit increases with time, the biological components become too thin, and the separation efficiency deteriorates. In Q2 ≧ 1.5Q3, the volume of the membrane separation unit decreases with time, the concentration of biological components in the separation membrane module proceeds excessively, deposits accumulate on the membrane surface, and the membrane pores become clogged. In addition, the ratio at the separation membrane deteriorates.

The conceptual diagram of the device of the present invention is as shown in FIG. 1 described later, and the actual configuration is as follows.
(1) Fractionation step “Fractionation step” means a step of fractionating a protein having a molecular weight higher than that of albumin in an aqueous solution (biological component-containing stock solution). As used herein, “fractionation” refers to discrimination of proteins by molecular weight. 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 molecular fractionation is performed by a separation sieve. In particular, the use of hollow fibers is effective because the surface area of the fractionation membrane becomes extremely large.

  The material of the membrane used in the present invention is not particularly limited, but cellulose, cellulose acetate polymer such as cellulose triacetate, polycarbonate, polysulfone polymer such as polysulfone and polyethersulfone, polymethacrylate such as polymethyl methacrylate, polyacrylate, polyamide, A material containing at least one polymer selected from the group consisting of polyvinylidene fluoride, polyacrylonitrile, polyester, polyurethane, polystyrene, polyethylene and polypropylene is used. Regarding the membrane structure, it is preferable to use a membrane having an asymmetric structure consisting of a dense layer and a two-layer structure of a support layer having a high porosity and maintaining membrane strength. When an electron microscope is used to observe the cross-sectional structure of the film under 1000 times of observation conditions, an asymmetric structure is defined as having both a layer in which pores cannot be confirmed and a layer in which pores can be confirmed in the film thickness direction. To do. In addition, it is preferable that this asymmetric structure is most dense in the vicinity of the surface in contact with the liquid to be processed. This is because clogging when filtering is performed can be reduced.

It is preferable that protein is not adsorbed to the membrane as much as possible, and a hydrophilic membrane is preferable. These hydrophilic membranes have the effect of suppressing adsorption of necessary proteins and recovering them without waste. 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, polyvinylpyrrolidone, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylamide, and polyethyleneimine is preferable.
(2) Overall configuration and operating conditions Each membrane separation unit is directly connected by a solution flow path and can be operated continuously, so that an effect of simple and automatic continuous operation can be obtained. May be operated. A pump is attached to the tube circuit and liquid is fed by the pump. However, in a small scale, liquid feeding by a syringe or centrifugal operation by a centrifugal tube type device may be performed.

  The present invention is suitable for separation of biological components from biological component-containing stock solutions, particularly human plasma, urine, saliva, tears, cerebrospinal fluid, ascites, pleural effusion and the like. The flow rate of the circulating fluid in each of the above separation units is appropriately determined depending on the quality and quantity of biomaterials such as plasma and urine as raw materials. The reaction is performed at 5 to 100 mL, and the flow rate is 1 to 100 mL / min, preferably 2 to 50 mL / min.

  The membrane separation system is capable of high-speed processing, and the time required for one treatment is 6 hours or less. From the viewpoint of sample contamination and biohazard prevention, a series of devices can be used once. It is possible to produce a device that 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 disposable, which is a great advantage from the viewpoint of avoiding contamination from the specimen and ensuring the reproducibility of the analysis.

  In this way, a liquid having a low composition ratio of a protein having a molecular weight of 60,000 or more in the total protein can be obtained. However, in order to perform a highly sensitive analysis, the composition ratio needs to be less than 0.5. It is preferably less than 0.1, more preferably less than 0.01, and it should be as small as possible.

  The analytical sample (analysis solution) thus obtained is useful for various protein analyzes such as liquid chromatography, electrophoresis, MS, etc., but is particularly preferably useful for proteomic analysis using MS, electrophoresis. It is. The MS to which the apparatus 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.

  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 for obtaining the analytical solution of the present invention will be described with reference to the drawings.

  FIG. 1 is a conceptual diagram of a separation system using a membrane of the present invention (an example of two units of a membrane separation unit). The liquid flow is indicated by arrows. A specimen (stock solution) of a material such as serum is fed from a valve 1 through a solution circulation circuit (tube circuit) 2 by a pump 3, injected into the first separation membrane module 5 and circulated. The liquid processed by the first membrane separation unit is obtained from the processing liquid recovery port 4. This mode is a unit of one unit, and two membrane separation units can be repeated in two stages, and three membrane separation units can be repeated in three stages. The treatment liquid is recovered from the final membrane separation unit. 6 denotes a treatment liquid recovery port of the second stage membrane separation unit, and 7 denotes a second separation membrane module.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited only to these Examples.
(Measurement method of transmission ratio)
Human serum (SIGMA H1388 or equivalent) is centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. A pump for circulating the solution on the stock solution (human serum) side and a pump for filtration are connected to the separation membrane module, and an aqueous PBS solution (Dulbecco PBS (−) manufactured by Nippon Pharmaceutical Co., Ltd.) is filled. Filter the stock solution at a flow rate of 1 ml / min for circulation and 0.2 mL / min for filtration at 20 ° C. At this time, the stock solution at the outlet of the module is discarded without being returned. The liquid after 30 to 60 minutes is collected, the concentrations of albumin and β2 microglobulin in the module stock solution inlet, outlet and filtrate are measured, and the sieve coefficients of albumin and β2 microglobulin are calculated from these measured values. The albumin concentration is measured by outsourcing to SRL Co., Ltd. and using item code 0721 4 (latex agglutination immunoassay).

  In this measurement, an albumin concentration of 0.4 mg / L or less and below the measurement limit is measured by a Human Album ELISA Quantification Kit (Cat No. E80-129) manufactured by BETHYL. The β2 microglobulin concentration is measured by outsourcing to SRL Co., Ltd. and using item code 0210 3 (latex agglutination immunization method). At this time, the average concentration of the concentration at the inlet and outlet of the module is used as the concentration on the stock solution side. The value obtained by dividing the sieve coefficient of β2 microglobulin obtained by the sieve coefficient of albumin 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, 10 ml of glycerin, 1 g of SDS, 0.05 g of BPB, 0.2 M Tris-HCl pH 6.8 (Tris-HCl added to pH 6.8) 25 ml and distilled water 14 ml are used.
2. Electrophoresis is performed by the method described in Non-Patent Document 3 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 CBB staining and silver staining. For silver staining, a commercially available kit, Silver Dye II Kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) is used.

(Example 1)
Add 18 parts by weight of polysulfone (Solvay Udel (registered trademark) P-3500) and 9 parts by weight of polyvinylpyrrolidone (ISP K30) to a mixed solvent of 72 parts by weight of N, N′-dimethylacetamide and 1 part by weight of water. And dissolved at 90 ° C. for 10 hours to obtain a stock solution. This film forming stock solution was sent to a spinning discharge section having a temperature of 30 ° C., and discharged from an outer tube of an orifice type double cylindrical die having an outer diameter of 0.35 mm and an inner diameter of 0.25 mm. A solution consisting of 60 parts by weight of N, N′-dimethylacetamide and 40 parts by weight of water was discharged from the inner tube as the core liquid. The discharged film forming stock solution passes through a dry zone of 350 mm in an atmosphere with a temperature of 30 ° C., then passes through a 40 ° C. coagulation bath (water bath), passes through a water washing step at 80 ° C. for 30 seconds, and a spinning speed of 40 m / min. And wound up into bundles. The obtained hollow fiber membrane had an inner diameter of 200 μm and a film thickness of 40 μm. When the structure of the hollow fiber was confirmed with an electron microscope (S800 manufactured by Hitachi, Ltd.), it had an asymmetric structure.

  100 hollow fibers were bundled, and both ends were fixed to a glass tube module case with an epoxy-based potting agent so as not to block the hollow portion of the hollow fiber, thereby producing a mini module. The mini-module has an inner diameter of about 5 mm and a length of about 17 cm. Like a general hollow fiber membrane dialyzer, it has two hollow fiber inner ports (blood ports) and an outer port (dialysate port). ). The hollow fiber of the mini module and the inside of the module were washed with distilled water. Thereafter, an aqueous solution of PBS (Dulbecco PBS (-)) manufactured by Nissui Pharmaceutical Co., Ltd. was filled to obtain a hollow fiber membrane minimodule (hereinafter abbreviated as minimodule 1). Two mini-modules were prepared, and the transmission ratio of one of them was measured and found to be 149. The remaining one was used for the following experiment. Human serum (SIGMA H1388, Lot 122K0424) was centrifuged at 3000 rpm for 15 minutes to remove precipitates, and then filtered to 0.45 μm. One of the dialysate side ports of the mini module 1 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 (FIG. 2).

  FIG. 2 is a schematic view of the separation system used in Example 1 (one unit of the membrane separation unit). The liquid flow is indicated by arrows. Serum and diluent (PBS) are sent from the valve 1 through the solution circulation circuit (tube circuit) 2 by the pump 3, injected into the first separation membrane module 5, and circulated. The liquid processed by the first membrane separation unit is obtained from the processing liquid recovery port 4. This processing liquid is recovered from the separation system.

  After adding 4 ml of serum at 10 ml / min, filtration was performed at 20 ° C. for 50 minutes at a circulation flow rate of 10 ml / min and a filtration flow rate of 1.0 mL / min. At this time, a filtered volume of PBS was added to the fractionation system at 1.0 ml / min to keep the amount of circulating liquid constant. The filtrate obtained in 50 minutes had an albumin concentration of about 50 ml, an albumin concentration of 32.8 mg / l, an α1-microglobulin concentration of 0.06 mg / l, and a β2 microglobulin concentration of 0.068 mg / l. The albumin concentration was 29800 mg / L, the α1-microglobulin concentration was 13.2 mg / L, and the β2-microglobulin concentration was 1.27 mg / L. The human serum used as the stock solution had an albumin amount of 119000 μg, an α1-microglobulin amount of 52.8 μg and a β2-microglobulin amount of 5.08 μg, whereas the filtrate had an albumin amount of 1640 μg, an α1-microglobulin amount of 3.00 μg, a β2-microglobulin amount. The amount of microglobulin was 3.40 μg, and albumin could be significantly removed while maintaining the amount of β2-microglobulin.

(Example 2)
One mini-module 1 was prepared using the same hollow fiber (permeation ratio 149) as in Example 1. The shape of the mini module 1 and the number of hollow fibers are the same as in the first embodiment. Also, 40 hollow fibers (permeation ratio 149) similar to those in Example 1 were fixed to a glass tube module case having an inner diameter of about 5 mm and a length of about 12 cm, and a mini module (hereinafter abbreviated as mini module 2) Two pieces were prepared in the same manner as in Example 1. The minimodule was used for the following experiments.

  Human serum (SIGMA, H1388, Lot 122K0424) was centrifuged at 3000 rpm for 15 minutes to remove the filtrate and precipitates, and then filtered to 0.45 μm. First, one mini module 1 was prepared, one on the dialysate side was capped, and one was connected to a silicone tube. The liquid inside the hollow fiber membrane was connected to the inlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. This was used as the first stage membrane separation unit. Furthermore, one mini-module 2 was capped on one side of the dialysate side, and one was connected to a peristaltic pump by connecting a silicone tube. The liquid inside the hollow fiber membrane of the mini module 2 was also connected to the outlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. This was used as the second stage membrane separation unit. Furthermore, another mini-module 2 was capped on one side of the dialysate side, and one was connected with a silicone tube and connected to a peristaltic pump. The liquid inside the hollow fiber membrane of the mini module 2 was also connected to the outlet and outlet with a silicone tube so that serum could be circulated using a peristaltic pump. This was used as the third stage membrane separation unit. Next, the silicone tube on the dialysate side of the mini-module is connected to the silicone tube of the next stage membrane separation unit via a valve, and the entire separation system is filled with PBS to create a separation system with a three-stage membrane separation unit. (FIG. 3).

  FIG. 3 is a schematic diagram of the separation system used in Example 2 (example of three units of membrane separation unit). The liquid flow is indicated by arrows. Serum and diluent (PBS) are sent from the valve 1 through the solution circulation circuit (tube circuit) 2 by the pump 3, injected into the first separation membrane module 5, and circulated. The liquid processed by the first membrane separation unit is obtained from the processing liquid recovery port 4. Next, this processing liquid is injected into the second separation membrane module 7 and circulated. The liquid processed by the second membrane separation unit is obtained from the processing liquid recovery port 6. Further, this processing liquid is injected into the third separation membrane module 9 and circulates. The liquid processed by the third membrane separation unit is obtained from the processing liquid recovery port 8. This processing liquid is recovered from the separation system.

  After 4 ml of serum is added to the first stage membrane separation unit at 0.2 ml / min, the flow rate is 5.0 mL / min for both the first stage membrane separation unit, the second stage membrane separation unit, and the third stage membrane separation unit. The hollow fiber inner liquid was circulated in and filtered at 20 ° C. for 4 hours at a filtration flow rate of 0.2 mL / min. At this time, PBS of a filtered volume was added to the fractionation system at 0.2 ml / min to keep the amount of circulating liquid constant. The filtrate obtained in 4 hours was about 47 ml. When this filtrate was concentrated to 4 ml using Sartorius vivaspin20 (3000 MWCO type), the albumin concentration was 0.38 mg / l, the β2 microglobulin concentration was 0.583 mg / L, and the albumin concentration in the human serum used as the stock solution Was 31200 mg / L, and the β2-microglobulin concentration was 1.19 mg / L. The human serum used as the stock solution had an albumin amount of 124800 μg and a β2-microglobulin amount of 4.76 μg, whereas the filtrate had an albumin amount of 19 μg and a β2-microglobulin amount of 2.92 μg, and the β2-microglobulin amount The albumin could be greatly removed while maintaining

  The sample was concentrated 10-fold using Sartorius vivaspin500 (3000 MWCO type), an equal amount of sample buffer was added, and 25 μl of electrophoresis sample heated at 100 ° C. for 3 minutes was analyzed by electrophoresis. The results are shown in lane 3 of FIG. Lanes 2 in FIG. 4 are electrophoretograms of diluted human serum before treatment with the separation system of Comparative Example 1. As can be seen from the 3 lanes in FIG. 4, it was confirmed that there were few proteins at a molecular weight of 60,000 or more and many proteins at a molecular weight of 15,000 or less.

(Comparative Example 1)
2 μl of sample of human serum (SIGMA, H1388, Lot 122K0424) diluted 100 times with sample buffer was analyzed by electrophoresis. The results are shown in lane 2 of FIG.

  In FIG. 4, one lane is Rainbow colored protein molecular weight markers (Amersham LIFE SCIENCE, RPN756 Batch58), two lanes are human serum of Comparative Example 1, and three lanes are electrophoresis of the biological component separation solution obtained in Example 2. It is a photograph. As can be seen from the two lanes, human serum had a large amount of protein at a molecular weight of 60,000 or more, and only a small amount of protein was observed at a molecular weight of 15,000 or less. On the other hand, as can be seen from 3 lanes, the concentrated solution of the filtrate of Example 2 treated with the separation system had few proteins at a molecular weight of 60,000 or more and many proteins at a molecular weight of 15,000 or less. I was able to confirm.

  As can be seen from FIG. 4, a large amount of protein was present at a molecular weight of 60,000 or more, and only a small amount of protein was observed at a molecular weight of 15,000 or less.

It is a conceptual diagram of the separation system using the membrane of the present invention (example of two units of membrane separation unit). It is the schematic of the separation system used in Example 1 of this invention (1 unit of a membrane separation unit). It is the schematic of the separation system used in Example 2 of this invention (example of 3 units of a membrane separation unit). It is a figure which shows the electrophoresis photograph of a solution.

Explanation of symbols

1 valve in Fig. 1 2 in Fig. 1 Solution circulation circuit (tube circuit)
1 pump 3 in FIG. 1 4 treatment liquid recovery port of the first stage membrane separation unit 5 in FIG. 1 first separation membrane module 6 in FIG. 1 treatment liquid recovery port of the second stage membrane separation unit 7 in FIG. Second separation membrane module 1 in FIG. 2 2 in FIG. 2 Solution circulation circuit (tube circuit)
2 of FIG. 2 4 of FIG. 2 4 process liquid recovery port of the first stage membrane separation unit 5 of FIG. 2 1st separation membrane module 1 of FIG. 3 2 of FIG. 3 Solution circulation circuit (tube circuit)
3 of FIG. 3 4 of FIG. 3 4 process liquid recovery port of the first stage membrane separation unit 5 of FIG. 3 1st separation membrane module 6 of FIG. 3 7 process liquid recovery port of the second stage membrane separation unit Second Separation Membrane Module Fig. 3 8 Third Stage Membrane Separation Unit Treatment Liquid Recovery Port 9 Fig. 3 9 Third Separation Membrane Module Fig. 4 Lane 11 (Rainbow colored protein molecular weight markers)
Lane 22 in FIG. 4 (human serum of Comparative Example 1)
FIG. 4, 33 lanes (biological component separation solution obtained in Example 2)

Claims (7)

A separation membrane for protein and / or peptide analysis pretreatment, wherein the membrane structure is an asymmetric structure. The separation membrane according to claim 1, wherein a permeation ratio between a protein having a molecular weight of less than A and a protein having a molecular weight of 4A or more is 20 or more. The separation membrane according to claim 1, wherein a permeation ratio between a protein having a molecular weight of less than 15,000 and a protein having a molecular weight of 60,000 or more is 20 or more. The separation membrane according to any one of claims 1 to 3, wherein the membrane material contains a hydrophilic polymer. The separation membrane according to claim 4, wherein the hydrophilic polymer is polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylamide, or polyethyleneimine. The separation membrane according to claim 4 or 5, wherein the content of the hydrophilic polymer is 1 to 10% of the membrane material. The protein and / or peptide to be analyzed is composed of blood, plasma, serum and other blood-derived substances, urine, ascites, saliva, tears, cerebrospinal fluid, pleural effusion or cells. The separation membrane according to any one of the above.

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