JP2006194724A - Separation membrane for protein and/or peptide analysis pretreatment - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a separation film capable of removing a material interfering the detection of a trace component, when analyzing clinical proteome. <P>SOLUTION: Assuming that the molecular weight of a protein or a peptide included in the largest quantity in an undiluted solution to be treated is 4A, an interfering material having a high molecular weight is removed efficiently by using this separation film whose penetration ratio between dextran having a molecular weight of A and dextran having a molecular weight of 4A is 10 or higher. A solution acquired by the separation film is used for protein analysis such as mass spectrometry, electrophoresis or liquid chromatography, to thereby enable high sensitivity analysis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a separation membrane for separating a biological component from a biological component stock solution, particularly human blood, urine and the like to obtain an analysis solution.

  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 trump technology for diagnosis and treatment in the post-genomic era. The identified biomarkers are used to evaluate drug responsiveness of patients. It can be said that it plays a major role in the promotion of so-called tailor-made medicine because it directly leads to the benefits that patients can enjoy, such as predicting the occurrence of side effects.

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

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

  It is estimated that there are more than 100,000 human proteins, but it is said that the number of proteins contained in serum alone amounts to about 10,000, and the total serum concentration is about 60-80 mg / mL. Proteins with high content in human serum are albumin (molecular weight 67 kDa), immunoglobulin (150-190 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several 100 kDa), etc., all of which are 1 mg / mL It exists in large quantities to exceed. On the other hand, many bioactive proteins such as peptide hormones, interleukins, and cytokines that are considered to be pathological biomarkers and pathogenesis-related factors are present in trace amounts of less than 1 ng / mL, and the content ratio is high. Compared to the high-content component of the molecule, it is indeed nano to pico level. In terms of protein size, 70% or less of all types of proteins have a molecular weight of 60,000 (60 kDa) or less, and most of the above-mentioned trace amounts of biomarker proteins are mostly contained in this region (for example, non-proteins) 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, exclude high molecular weight components with a molecular weight of 60,000 or more that interfere with the detection of trace components related to pathogenesis, and recover proteins with a molecular weight of less than 15,000 as much as possible. Is essential first.

Currently, high-performance liquid chromatography (LC) and two-dimensional electrophoresis are used as means for separating and removing high molecular weight proteins.
(2 dimensional-polyacrylamide gel electrophoresis: 2D-PAGE) is used, but LC and 2D-PAGE work alone takes 1-2 days. This required time is much longer than the analysis time of several minutes such as MALDI-TOF-MS and ESI-MS (electrospray ionization mass spectrometry), and the great advantage of high throughput of MS as an analytical tool is clinical proteome analysis So I can't fully demonstrate it. For this reason, for the purpose of obtaining analysis results in the shortest possible time for diagnosis and treatment in the medical field, it must be said that it is not practical at present, and it is difficult to use MS for daily clinical tests. It is a big cause.

  Therefore, if the speed of the means for removing a part or all of the high molecular weight protein from the sample is achieved, it can be expected that the rapidity of the diagnosis of the clinical test by the clinical proteome analysis is dramatically improved. Specifically, there should be a method or apparatus for obtaining a biological component-containing solution in which the composition of the biological component is changed by leaving a target protein group at a high speed with a small amount of sample as an alternative to LC or 2D-PAGE. .

  As a means for removing albumin as a main target substance, a product that has already been put into practical use or a disclosed technique includes a carrier on which an affinity ligand such as a blue dye is immobilized (commercialized), a high molecular weight. Centrifuge tube type fractionation of components by centrifugal filtration (commercialized), fractionation method by electrophoresis principle, fractionation by traditional precipitation methods such as ethanol precipitation by Cohn and chromatography There are methods (for example, Non-Patent Document 2).

  However, all of these have problems such as insufficient separation performance, inappropriate for a small amount of sample, or mixing of a drug that hinders mass spectrometry or the like. In particular, the method of excluding only albumin by targeting albumin is difficult to remove other 60,000 or more polymer components such as immunoglobulin even if albumin can be removed.

  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 no suggestion of solving the problem of the clinical proteome that removes high molecular weight proteins such as albumin with high efficiency (Patent Document 2).

  If a method or device that can solve these problems can be developed, proteome analysis will be widely performed in medical research and clinical settings, enabling more rapid and highly accurate examination and diagnosis, and intractableness without useful treatment It can be expected to be a powerful tool for investigating the causes of various diseases and developing early diagnostic methods.

  Recently, the method using Affi-Gel Blue gel (Non-patent document 3) The method using "Gradiflow" system (Non-patent document 4) and the 6 types of proteins contained in serum at high concentrations are removed with antibodies. Although "Agilent Multiple Affinity Removal System" has been announced as an effective improved albumin removal method, it has not yet been used as an analytical solution for obtaining more information.

With the removal of albumin from plasma as an index, the required method conditions are that the plasma components can flow at high speed, that there is no protein denaturing action, that fine processing has been performed for high functionality, and that it is extremely expensive. It is not. There are still no devices or devices that can solve these problems.
By Anderson NL, 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 Cell Engineering Supplement, “Bio-Experiment Illustrated 5”, Shujunsha, 2001 N. Ahmed et al., Proteomics, On-line version, June 23, 2003 (DL Rothemund et al. (2003), Proteomics, Vol. 3, 279-287) Cell Engineering Supplement, “Bio-Experiment Illustrated 5”, Shujunsha, 2001 Japan, JP 2002-542163 A Japan, Japanese Patent No. 3297707

  In view of the above circumstances, the problem to be solved by the present invention is that it is suitable for proteome analysis by advantageously separating and removing excess high molecular weight proteins that interfere with clinical proteome analysis from biological component-containing solutions. Another object of the present invention is to provide a separation membrane that can easily prepare a biological component-containing solution in which the composition of the biological component is changed.

The separation membrane according to the present invention has the following configurations (1) to (8).
(1) Permeation ratio of dextran having a molecular weight of A and dextran having a molecular weight of 4A in a separation membrane for pretreatment of protein and / or peptide, where the molecular weight of the protein or peptide contained in the largest amount in the processing stock solution is 4A Is a separation membrane characterized by 10 or more.
(2) The separation membrane according to (1) above, wherein a permeation ratio of dextran having a molecular weight of 17,000 and dextran having a molecular weight of 67,000 is 10 or more.
(3) The screening coefficient of dextran having a molecular weight of 17,000 is 0.50 or more, and the screening coefficient of dextran having a molecular weight of 67,000 is 0.05 or less. Separation membrane.
(4) The separation membrane according to (1) to (3) above, wherein the membrane structure is an asymmetric structure.
(5) The separation membrane according to any one of (1) to (4) above, wherein the membrane material contains a hydrophilic polymer.
(6) The separation membrane according to (5), wherein the hydrophilic polymer is polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylamide, or polyethyleneimine.
(7) The separation membrane according to (5) to (6) above, wherein the content of the hydrophilic polymer is 1 wt% or more of the membrane material.
(8) The above-mentioned (1), wherein 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. ) To (7).

  The separation membrane disclosed in the present invention makes it possible to prepare a solution capable of detecting a large amount of a minute amount of protein that has been difficult to detect from biological components such as blood, serum, and plasma.

  The “separation membrane” of the present invention has a permeation ratio of dextran having a molecular weight of 4A and dextran having a molecular weight of 4A when the molecular weight of the protein or peptide contained in the “treatment stock solution” in the largest amount is 4A. The protein or peptide contained in a large amount in the “treatment stock solution” can be removed, and the smaller protein and / or peptide can be recovered to the maximum extent.

  The “treatment stock solution” as used in the present invention is a solution immediately before being treated with a separation membrane (in contact with the separation membrane). For example, when animal blood such as human or serum is used as a processing stock solution, the most abundant component is albumin (molecular weight 67 kDa). In addition, as the “treatment stock solution”, a solution composed of a part of blood components such as animal plasma of humans, urine, saliva, tears, cerebrospinal fluid, ascites, pleural effusion or a protein extracted from cells, etc. A solution containing a protein as a biological substance is exemplified.

  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 membrane materials include cellulose, cellulose acetate, polycarbonate, polysulfone resin, polymethacrylate such as polymethyl methacrylate, polyacrylate, polyamide, polyvinylidene fluoride, polyacrylonitrile, polyester, polyethylene and polypropylene, and derivatives thereof. The material selected from one or more types from the group consisting of can be exemplified. Among these, polysulfone-based resins that are frequently used in dialyzers in recent years are preferable materials because of their good fractionation characteristics.

  Regarding the membrane structure, the flow path in the membrane of the separation membrane is preferably an asymmetric structure, and moreover, it is an asymmetric structure consisting of a multilayer structure of a dense layer and a support layer having a high porosity and maintaining membrane strength. preferable. This asymmetric structure is determined when the cross-sectional structure of the film is observed under an observation condition of 1000 times using an electron microscope. Judgment is made based on whether or not there are both a layer in which pores cannot be sufficiently confirmed and a layer in which pores can be confirmed in the thickness direction of the film. When both layers are confirmed, it is recognized as an asymmetric structure.

  Further, in this asymmetric structure, it is preferable that the pores in the vicinity of the surface in contact with the stock solution are the most dense. This is because clogging of the film surface by the solute can be reduced.

  It is preferable that protein is not adsorbed to the membrane as much as possible, and a hydrophilic membrane is preferable. Surface of membrane made by copolymerizing hydrophilic monomer and hydrophobic monomer, blended film of hydrophilic polymer and hydrophobic polymer, or membrane made of hydrophobic polymer Examples include those obtained by binding and adhering a hydrophilic polymer to the surface, and those obtained by subjecting the surface of a film made of a hydrophobic polymer to chemical treatment, plasma treatment, or radiation treatment. 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 wt% or more. These hydrophilic membranes have the effect of suppressing adsorption of necessary proteins and recovering them without waste.

  The sieving coefficient of dextran having a molecular weight A of the separation membrane is preferably larger, and the sieving coefficient of dextran having a molecular weight 4A is preferably smaller. However, when the pore size of the separation membrane is increased and the sieving coefficient of dextran having a molecular weight of A is increased, the sieving coefficient of dextran having a molecular weight of 4A becomes too large to sufficiently remove a protein or peptide contained in a large amount. That is, in order to remove a large amount of protein or peptide contained in the “treatment stock solution” and recover a smaller protein and / or peptide as much as possible, the permeation ratio of dextran having a molecular weight of A and dextran having a molecular weight of 4A is 10 It is preferable to use a separation membrane having the above-described sharp fractionation characteristics with uniform pore diameters. The permeation ratio is more preferably 50 or more, and an upper limit value is not particularly provided. However, in a membrane separation unit having this value that is too high, there is a concern that the sieving coefficient of dextran having a desired molecular weight A will be low. Therefore, it is preferably better than 10,000.

  The transmission ratio can be measured using a module incorporating a separation membrane. A module having at least a raw solution inlet of a biological component-containing solution and an outlet of a permeated liquid permeated through the separation membrane is used on the side of the separation membrane.

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 obtained in the examples were filled in a glass housing having a diameter of about 5 mm and a length of 12 cm, and the inlet into which the solution to be separated flows in and the outlet through which it was discharged were epoxy resin-based chemistry manufactured by Konishi Co. A separation membrane module is produced by potting with a reactive adhesive “Quick Mender”. The module is also provided with a permeate outlet. Next, the hollow fiber of the module and the inside of the module are washed with distilled water for 1 hour.

  Dextran made by FULKA Mr-1500 (No.31394), Mr-6000 (No.31388), Mr15000-20000 (No.31387), Mr-40000 (No.31389), Mr-60000 (No.31397), Mr Dissolve ~ 200000 (No.31398) in distilled water to make 0.5mg / mL each (3.0mg / mL in the whole solute) to make a dextran aqueous 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 replacing the filled ultrafiltrated water with a dextran aqueous solution, filtration is started at room temperature (25 ° C.). After starting, the stock solution and filtrate at the module outlet are discarded without being returned. Collect the module stock solution inlet and outlet liquid and the filtrate 60 to 75 minutes after the start. These solutions are subjected to GPC analysis.

  The concentration measurement by GPC was performed by the following method. 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.

  Calibration of the relationship between the retention time in GPC and the molecular weight of dextran is carried out by measuring monodisperse dextran (Fluxa dextran standards No.31416, No.31417, No.31418) before measuring each sample that has passed through the module. , No.31420, No.31422) is subjected to GPC and the retention time is measured. In actuality, prepare each dextran standard dissolved in No.31416, No.31418, No.31422 and dissolved No.31417, No.31420, and dissolve each dextran standard in 0.5mg / mL. is doing. The relationship between the retention time and the molecular weight is obtained by plotting the retention time of each peak and the molecular weight of a known dextran and obtaining an exponential approximation curve.

  Since the retention time at a specific molecular weight is known from the relationship diagram between the retention time and the molecular weight, the differential refractive index of dextran at the retention time corresponding to the specific molecular weight is measured. The concentration is calculated using the refractive index, and the dextran sieving coefficient is calculated from these measured values.

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 molecular weight 15,000 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, 0.2 M Tris-HCl pH6.8 (pH adjusted to 6.8 by adding HCl to Tris) 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 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
18 parts by weight of polysulfone (Solvay Udel (registered trademark) P-3500) and 9 parts by weight of polyvinylpyrrolidone (ISP K30) were added 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 has an inner diameter of 200 μm and a film thickness of 40 μm. When the structure of the hollow fiber is confirmed with an electron microscope (S800 manufactured by Hitachi, Ltd.), the inside surface of the hollow fiber membrane has a dense asymmetric structure. Was.

  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.) or distilled water was filled to obtain a hollow fiber membrane minimodule (hereinafter abbreviated as minimodule (1)). Two mini-modules were created, and one of them (filled with distilled water) was measured for sieve coefficient. The dextran with a molecular weight of 17,000 was found to have a sieve coefficient of 0.71 and a dextran with a molecular weight of 60,000. The coefficient was 0.027 and the transmission ratio was 26. The remaining one (filled with an aqueous PBS solution) was used in 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 outer ports of the mini-module (1) was capped and the other one was connected with a silicone tube and connected to a permeation pump for permeation. On the other hand, the solution inside the hollow fiber membrane was formed by connecting the stock solution inlet and the stock solution outlet of the module with a silicone tube to form a solution circulation circuit so that serum could be circulated using a peristaltic pump. In addition, an inlet for adding an additional PBS aqueous solution was provided in the middle of the solution circulation circuit (FIG. 1).

  Using 4 mL of serum, filtration was carried out 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, after flowing the serum into the solution circulation circuit at 1.0 mL / min, the volume of the filtration was kept constant by adding PBS aqueous solution into the solution circulation circuit at 1.0 mL / min.

  The concentration of albumin in 50 mL of the filtrate obtained in 50 minutes, that is, the composition of the biological component changed, was 32.8 mg / L, the α1-microglobulin concentration was 0.06 mg / L, and the β2 microglobulin concentration was 0.068 mg / L. . The albumin concentration in the human serum used 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 was 119000 μg of albumin, 52.8 μg of α1-microglobulin, and 5.08 μg of β2-microglobulin, whereas the filtrate was 1640 μg of albumin, 3.00 μg of α1-microglobulin, β2-microglobulin. The amount was 3.40 μg, and albumin could be greatly removed while maintaining the amount of β2-microglobulin.
(Example 2)
Using the same hollow fiber (permeation ratio 26) as in Example 1, one minimodule (1) was produced. The shape of the mini module (1) and the number of hollow fibers are the same as in Example 1. Also, 40 hollow fibers (permeation ratio 26) 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)). ) 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 of the ports on the outside of the hollow fiber membrane was capped, and the other was connected with a silicone tube. For the liquid inside the hollow fiber membrane, the inlet and outlet of the stock solution were connected by a silicone tube to form a solution circulation circuit (1), and a peristaltic pump was provided in the middle so that the stock solution could be circulated. A three-way valve was provided in the middle of the solution circulation circuit, and an injection pump was provided on one side via a tube.

  In addition, one mini-module (2) also capped one of the outer ports and the other was connected with a silicone tube. In addition, this mini module was also connected to the solution inside the hollow fiber membrane by connecting the stock solution inlet and outlet with a silicone tube to form a solution circulation circuit (2), and a peristaltic pump was provided in the middle so that the solution could be circulated. A three-way valve was provided in the middle of this solution circulation circuit. In addition, another mini-module (2) also capped one of the outer ports and the other was connected with a silicone tube. In addition, this mini module was also connected to the solution inside the hollow fiber membrane by connecting the stock solution inlet and outlet with a silicone tube to form a solution circulation circuit (3), and a peristaltic pump was provided in the middle so that the solution could be circulated. A three-way valve was provided in the middle of this solution circulation circuit.

  Next, the silicone tube connected to the outer port of the mini-module (1) of the solution circulation circuit (1) and the three-way valve provided in the middle of the solution circulation circuit (2) were joined. Further, a silicone tube connected to the outside port of the mini-module (2) of the solution circulation circuit (2) and a three-way valve provided in the middle of the solution circulation circuit (3) were joined. These tubes and three modules were filled with an aqueous PBS solution to create a system in which mini-modules were connected in three stages in series (FIG. 2).

  After adding 4 mL of serum at 0.2 mL / min from the three-way valve of the solution circulation circuit (1) through the infusion pump, each of the solution circulation circuit (1), the solution circulation circuit (2), and the solution circulation circuit (3) The hollow fiber inner liquid was circulated at a flow rate of 5.0 mL / min, and filtration was carried out at 20 ° C. for 4 hours at a filtration flow rate of 0.2 mL / min. At this time, the volume of the filtered volume was kept constant by adding an aqueous PBS solution at a rate of 0.2 mL / min from the same location as the serum charging valve to circulate.

  After 4 hours, 47 mL of the filtrate obtained from the solution circulation circuit (3), that is, the solution in which the composition of the biological component was changed, was obtained. 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 the sample for electrophoresis heated at 100 ° C. for 3 minutes was analyzed by electrophoresis. The results are shown in lane 3 of FIG. Lanes 2 in FIG. 3 are electrophoretograms of diluted human serum before treatment with the separation system of Comparative Example 1. As can be seen from the three lanes in FIG. 3, it was confirmed that there were few proteins at a molecular weight of 67,000 or more and many proteins at a molecular weight of 17,000 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. 3, 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 67,000 or more, and only a small amount of protein was observed at a molecular weight of 17,000 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 67,000 or more and many proteins at a molecular weight of 17,000 or less. It was confirmed that there is.

  As can be seen from FIG. 3, a large amount of protein was present at a molecular weight of 67,000 or more, and only a small amount of protein was observed at a molecular weight of 17,000 or less.

It is the schematic of the separation system used in Example 1 of this invention. (One membrane separation unit) It is the schematic of the separation system used in Example 2 of this invention. (3 membrane separation units) Lane 2 is the human serum of Comparative Example 1, and Lane 3 is an electrophoretogram of the biological component separation solution obtained in Example 2.

Explanation of symbols

100 Injection 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 Module lower port 202 Second stage solution circulation circuit 203 Second stage circulation pump 204 Second stage Treatment liquid recovery port 205 of the second membrane separation unit 206 Second separation membrane module 206 Lower port 302 of the second stage module Third stage solution circulation circuit 303 Third stage circulation pump 304 Treatment liquid collection port of the third stage membrane separation unit 305 Third separation membrane module 306 Lower port 1 of third stage module 1 lane Rainbow colored protein molecular weight markers
2 2 lanes Human serum 3 3 lanes Example 2 filtrate

Claims (8)

  In the separation membrane for protein and / or peptide analysis pretreatment, when the molecular weight of the protein or peptide contained in the largest amount in the processing stock solution is 4A, the molecular weight A dextran calculated by the GPC method and the molecular weight 4A dextran A separation membrane for protein and / or peptide analysis pretreatment characterized by having a permeation ratio of 10 or more.   The separation membrane for protein and / or peptide analysis pretreatment according to claim 1, wherein a permeation ratio of dextran having a molecular weight of 17,000 and dextran having a molecular weight of 67,000 is 10 or more.   2. The protein according to claim 1, wherein the dextran having a molecular weight of 17,000 has a sieving coefficient of 0.50 or more, and the dextran having a molecular weight of 67,000 has a sieving coefficient of 0.05 or less. Or separation membrane for peptide analysis pretreatment.   The separation membrane for protein and / or peptide analysis pretreatment according to any one of claims 1 to 3, wherein the membrane structure is an asymmetric structure.   The separation membrane for protein and / or peptide analysis pretreatment according to any one of claims 1 to 4, wherein the membrane material contains a hydrophilic polymer.   6. The separation membrane for protein and / or peptide analysis pretreatment according to claim 5, wherein the hydrophilic polymer is polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, polyhydroxyethyl methacrylate, polyacrylamide, or polyethyleneimine. .   The separation membrane for protein and / or peptide analysis pretreatment according to any one of claims 5 and 6, wherein the content of the hydrophilic polymer is 1 wt% or more of the membrane material.   8. 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 for protein and / or peptide analysis pretreatment according to any one of the above.