JP2006343220A - Pretreatment method of biocomponent-containing solution and analysis solution purification method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for acquiring a solution from which a material interfering with detection of a trace component when performing clinical proteome analysis is removed. <P>SOLUTION: Undiluted solution is not excessively concentrated or diluted in a fractionation solution circulation circuit, and concentration polarization is kept in a proper range, to thereby remove efficiently an interfering material having a high molecular weight. The solution acquired by a separation film is used for protein analysis such as mass spectrometry, electrophoresis or liquid chromatography, and high-sensitivity analysis becomes possible. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biological component-containing solution, particularly a biological component-containing solution pretreatment method and an analytical solution purification method that can be suitably used to obtain a solution for analysis by separating a specific biological component from human blood, urine, or the like. .

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

  The rapid progress of proteome analysis is largely due to the fact that high-speed structural analysis using mass spectrometers (MS) has become possible. For example, the practical application of MALDI-TOF-MS (matrix assisted laser desorption ionization time-of-flight mass spectrometry) enables high-throughput ultra-trace analysis of polypeptides, and even trace proteins that could not be detected in the past. Has become a powerful tool for the search for disease-related factors.

  By the way, the primary purpose of clinical application of proteome analysis is the discovery of biomarker proteins that are induced or lost by disease. The biomarker protein behaves in relation to a disease state, and thus can be a diagnostic marker and is highly likely to be a drug discovery target. In other words, the results of proteome analysis are more likely to become diagnostic markers and drug discovery targets than specific genes, and thus become a trump card technology for diagnosis and treatment in the post-genome era. It can be said to play a major role in the promotion of so-called tailor-made medical care because it directly leads to benefits that patients can enjoy such as evaluation and prediction of side effects.

  However, when proteomic analysis is introduced into clinical research, it is required to analyze a large amount of specimens quickly and reliably, and individual clinical specimens (solutions containing biological components) are precious and valuable. Sensitive measurements need to be made quickly. Therefore, although mass spectrometers having ultra-high sensitivity and high-throughput characteristics have been rapidly improved, there is still no situation in which proteome analysis can be easily and quickly performed in clinical settings.

In other words, it is necessary to fractionate and purify proteins and peptides in clinical specimens (biological component-containing solutions) before mass spectrometry, and this process takes several days. It is complicated and requires experience.
Furthermore, since proteins and peptides contained in biological component-containing solutions such as blood and body fluids are diverse, there is a problem that fractional purification is not easy. For example, 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 protein concentration in serum is about 60-80 mg / mL. . And the high content protein in human serum is albumin (molecular weight 67 kDa), immunoglobulin (150-190 kDa), transferrin (80 kDa), haptoglobin (> 85 kDa), lipoprotein (several 100 kDa), etc. It exists in a large amount to exceed / mL. On the other hand, many physiologically active proteins such as peptide hormones, interleukins, and cytokines, which are considered to be pathological biomarkers and pathogenesis-related factors, exist only in trace amounts of less than 1 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 less of all types of proteins have a molecular weight of 6760,000 (6.760 kDa) or less (all the above-mentioned trace amounts of biomarker proteins may be included in this region). Therefore, it is not easy to separate and purify them with a separation membrane.

  High-performance liquid chromatography (LC) and two-dimensional electrophoresis (2D-PAGE) are methods for separating and removing high molecular weight proteins from biological component-containing solutions such as blood and body fluids. However, these LC and 2D-PAGE work eventually takes 1-2 days. Therefore, it must be said that it is not practical for diagnosis and treatment in the medical field where an analysis result is desired in the shortest possible time.

  In addition, in order to mainly remove albumin from biological component-containing solutions such as blood and body fluids, a carrier on which an affinity ligand such as a blue dye is immobilized (Non-patent Document 2), and a centrifugal separator that fractionates high molecular weight components by centrifugal filtration. Tube-type equipment (commercialized), fractionation method based on electrophoresis principle (Non-patent Document 3), traditional precipitation methods such as ethanol precipitation by Cohn and chromatographic fractionation methods (eg, non-fractionation) Patent document 4) and the like are also disclosed. However, all of these have problems such as insufficient separation performance, inadequate for a small amount of sample, or mixing with an agent that hinders mass spectrometry or the like. In particular, with the method of separating albumin only by adsorption using albumin as a target, it is difficult to separate other 60,000 or more polymer components such as immunoglobulins even though albumin can be separated.

In addition, separation membranes with various pore sizes are used for artificial kidneys, artificial lungs, plasma separators, etc. depending on their applications, and compatibility with biological components (for example, when blood comes into contact with foreign substances) Although it has been improved to improve the state in which coagulation activity and immune activity do not occur (Patent Document 1), the problem that the clinical proteome has to remove high molecular weight proteins such as albumin with high efficiency It does not solve.
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. Cell Engineering Supplement, “Bio-Experiment Illustrated 5”, Shujunsha, 2001 N. Ahmed et al., Proteomics, On-line version, June 23, 2003 Edited by the Japanese Biochemical Society, “Seminar in Experimental Chemistry of Volunteers (Vol. 1) Protein (1) Separation, Purification, and Properties”, Tokyo Chemical Doujin, 1990 Japanese Patent No. 3297707

  The problem to be solved by the present invention is, for example, to remove excess high molecular weight proteins from biological component-containing solutions such as blood and urine during clinical proteome analysis, and to quickly and more reliably remove low molecular weight proteins and peptides. It is to provide a pretreatment method and a solution purification method for a biological component-containing solution that can be separated and purified.

The present invention for overcoming the above problems has the following configurations (1) to (5).
(1) When analyzing a protein and / or peptide in a biological component-containing solution, the biological component-containing solution is circulated and supplied to a separation membrane having a molecular weight of 67,000 and having a sieve coefficient of 0.1 or less. The biological component-containing solution is pretreated and is supplied to the separation membrane after adjusting so that the ratio of total albumin in the liquid / supply-side flow path volume is in the range of 6 to 60 mg / ml. A pretreatment method for a biological component-containing solution characterized by the above.
(2) The biological component-containing solution pretreatment method according to (1), wherein the biological component-containing solution is circulated in a closed circuit.
(3) The pretreatment separation and purification method for a biological component-containing solution according to (1) or (2), wherein a hollow fiber membrane is used as the separation membrane.
(4) The biological component-containing solution is at least one selected from the group consisting of blood-derived substances, urine, ascites, saliva, tears, cerebrospinal fluid, pleural effusion, and a solution obtained by extracting proteins from cells. The pretreatment method for a biological component-containing solution according to any one of (1) to (3) above.
(5) An analytical solution purification method for purifying an analytical solution of protein and / or peptide using the pretreatment method described in any one of (1) to (4) above.

  According to the present invention, an analysis solution capable of more reliably detecting trace amounts of proteins and peptides, which have been difficult to detect in the past, from biological component-containing solutions such as blood, serum, and plasma can be purified in a short period of time. It becomes possible.

  The analytical solution purification method of the present invention is a method in which a solution derived from a living body such as blood, that is, a solution containing a biological component is used as a stock solution to change the composition of protein or peptide. It is what is said. The arrow indicates the liquid flow.

  In FIG. 1, a biological component-containing solution such as blood is sent from an infusion pump 100 to a stock solution tank 101. The biological component-containing solution may be previously contained in the stock solution tank. Then, the biological component-containing solution is fed by the circulation pump 103 and injected into the separation membrane module 105 containing a separation membrane having a sieve coefficient described later via the solution circulation circuit 102 formed of a tube. Thereafter, the permeate that has permeated the separation membrane is fed by the pump 107 and taken out from the treatment liquid recovery port 104 that is the outlet of the permeate, and is used for proteome analysis and the like. On the other hand, the concentrated liquid (biological component-containing solution) that has not permeated the separation membrane is sent again to the stock solution tank 101 and circulated and supplied to the separation membrane module 105.

  Further, the present invention may be modified as shown in FIG. FIG. 2 is a conceptual diagram of a separation system in which the stock solution inlet and the stock solution outlet are directly connected without using the stock solution tank 101 in FIG. The liquid flow is indicated by arrows. In this embodiment, the biological component-containing solution is injected from the injection pump 200 through the three-way valve 201 into the separation membrane module 205 containing the separation membrane, and is sent by the circulation pump 203 through the solution circulation circuit 202 formed of a tube. It is liquefied and circulates. The permeate that has permeated through the separation membrane is taken out from the treatment liquid recovery port 104, which is the permeate outlet, and is used for proteomic analysis and the like.

  Here, in the present invention, when analyzing proteins and peptides in a biological component-containing solution, the biological component-containing solution is circulated and supplied to a separation membrane having a molecular weight of 67,000 and having a sieve coefficient of 0.1 or less. And pre-processing. At this time, the biological component-containing solution is supplied to the separation membrane by adjusting the ratio of the total albumin amount in the liquid / the supply-side flow path capacity to be in the range of 6 to 60 mg / ml.

  The “biological component-containing solution” in the present invention is a biological substance such as a blood-derived substance, urine, saliva, tears, cerebrospinal fluid, ascites, pleural effusion or a solution obtained by extracting proteins from cells. The solution containing is illustrated. The “blood-derived product” is a product derived from the blood of an animal such as a human, and includes not only blood but also a solution composed of some components in blood such as serum and plasma.

  The “separation membrane” as used in the present invention is a porous separation membrane, and any of flat membrane separation membranes such as flat filters and cartridge filters, and hollow separation membranes such as hollow fiber membranes can be used. Can do. The flat membrane type separation membrane has the advantage that it can be easily formed at low cost. On the other hand, the hollow fiber membrane can be used efficiently because it has a large filling membrane area in the same volume and can reduce pressure loss. In order to increase the filling membrane area in a module using hollow fiber membranes, it is necessary to increase the number of filled hollow fiber membranes. Therefore, it is better that the outer diameter is small. However, if the diameter is too small, the membrane area of each film becomes small and pressure loss tends to increase. Therefore, it is preferable to use a hollow fiber membrane having an inner diameter in the range of 50 to 1000 μm and an outer diameter in the range of 60 to 1100 μm.

  Examples of the membrane material include cellulose, cellulose acetate, polycarbonate, polysulfone, polymethacrylic acid ester such as polymethyl methacrylate, polyacrylic acid ester, polyamide, polyvinylidene fluoride, polyacrylonitrile, polyester, polyethylene and polypropylene, and derivatives thereof. Selected from the group consisting of 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 proteins are not adsorbed on the membrane surface as much as possible, and therefore a hydrophilic membrane is preferred. Specifically, a copolymer of a hydrophilic monomer and a hydrophobic monomer, a film formed by blending a hydrophilic polymer and a hydrophobic polymer, or a hydrophobic polymer For example, the surface of a film made of a material having a hydrophilic polymer bonded and adhered thereto, or the surface of a film made of a hydrophobic polymer chemically treated, plasma treated, or radiation treated. Although it does not specifically limit as a hydrophilic component, in order to prevent that the required protein adsorb | sucks to a film surface more highly hydrophilic, such as polyalkylene oxides, such as polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, and polyhydroxyethyl methacrylate. Molecules are preferred. The hydrophilic component is preferably contained in the film in an amount of 1 wt% or more.

  In the present invention, a separation membrane having a molecular weight of 67,000 with a dextran sieve coefficient of 0.1 or less is preferably used, but it is preferably 0.01 or less. If it is small, it is difficult to recover a low molecular weight protein having a molecular weight of about 1 to 30,000, so 0.001 or more is better.

  The aforementioned separation membrane is housed in a housing and used as a separation membrane module. The housing is provided with an inlet through which the biological component-containing solution to be separated flows in and an outlet through which the biological component-containing 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.

Separation membrane module is preferably water permeability is ^{100ml / hr / mmHg / m 2} . If the water permeability is not sufficient, the filtration rate cannot be obtained and the separation rate becomes slow. In addition, when the packed membrane area is increased in order to increase the filtration rate, the amount of protein adsorbed on the membrane surface increases, and a sufficient amount of recovered protein cannot be obtained. Accordingly, it is desirable that the water permeability is higher, and it is preferably 100 ml / hr / mmHg / m ² or more, more preferably 300 ml / hr / mmHg / m ² .

For example, when the present invention is carried out with a desktop size device, the amount of the biological component-containing solution used is preferably 1 to 400 ml, more preferably 1 to 100 ml, and still more preferably 1 to 10 ml of plasma. Moreover, a preferable filtration flow rate is 0.1-20 ml / min, More preferably, it is 0.2-10 ml / min. If the packed membrane area is too large, the amount of protein adsorbed increases, and if it is too small, the pressure difference between membranes becomes too large when filtration is performed. Therefore, it is necessary to determine the size of the packed membrane area according to the amount of the specimen. When a larger amount of specimen is processed, it is preferable to use a separation membrane having a larger membrane area. For plasma 1 ml, preferably 10~1000cm ^2, more preferably at 10 to 100 cm ^2.

  In the present invention, the biological component-containing solution such as blood is supplied to the separation membrane after being adjusted so that the ratio of the total albumin amount in the liquid / the supply-side flow channel capacity is in the range of 6 to 60 mg / ml. However, when the ratio is adjusted to be in the range of 6 to 60 mg / ml, a diluent is introduced into the separation membrane module together with a biological component-containing solution such as blood. When the stock solution is gradually injected in a closed circuit as shown in FIG. 2, the ratio of the total albumin amount in the solution / the supply-side channel capacity gradually increases. Therefore, although there is a possibility that it falls below this range at the start of injection, in the present invention, the completion of stock solution injection is regarded as the start of processing. Further, the “total albumin amount in the liquid” is the total albumin amount contained in the biological component-containing solution circulated and supplied to the membrane surface, and the “supply side channel capacity” is supplied to the separation membrane. The volume of the flow path through which the solution is circulated.

  It is preferable to use physiological saline or a “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. The buffer solution is abbreviated, but the detailed contents can be understood by referring to catalogs of reagent manufacturers such as Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Japan Co., Ltd. and MSDS (safety data sheet).

  In adjusting the ratio, in addition to adding the diluent, the amount of the undiluted biological component-containing solution itself is prepared, or the volume of the flow path through which the solution supplied to the separation membrane circulates is prepared. The method etc. are mentioned.

  By maintaining the ratio of the total albumin amount in the liquid / supply-side flow path volume within the range of 6 to 60 mg / ml by adding a diluent in this manner, excessive concentration on the separation membrane surface is prevented, This is because deterioration of the permeation ratio of the separation membrane is prevented.

  The flow rate Q1 of the supply liquid supplied to the separation membrane module and the flow rate of the separated permeate from the viewpoint of preventing concentration on the separation membrane surface, preventing deposition of deposits on the membrane surface, and preventing clogging of the membrane holes The ratio Q2 / Q1 of Q2 is desirably 0.5 or less. Further, it is desirable that the flow rate Q2 of the permeated liquid that has passed through the separation membrane and the flow rate Q3 of the diluent supplied to the separation membrane module have a relationship of 0.5 ≦ Q2 / Q3 ≦ 1.5, and Q2 / It is preferable that Q3 is 0.9 to 1.1, that is, approximately 1.

  The solution in which the composition of the biological component-containing solution obtained by the present invention is changed is preferably used for protein analysis and peptide analysis, particularly 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.

  By performing the above analysis using the solution obtained by the method of the present invention, it is possible to collect structural information of protein components and peptide components that were contained only in trace amounts in the original biological component-containing liquid. They include not only peptide-mass fingerprint (PMF) but also primary structure information (amino acid sequence) of each peptide.

  In the present invention, if necessary, a plurality of separation membrane modules may be arranged, and a step of concentrating proteins by removing a solvent such as moisture from the permeate obtained from the above process may be further added. .

(Measurement method of transmission ratio)
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 produced by potting with the agent “Quick Mender”. Next, the hollow fiber of the module and the inside of the module are washed with distilled water for 1 hour.

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 ultrafiltrated 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. Thereafter, after 60 minutes, the liquid is collected for 15 minutes, the differential refractive index of dextran in the module stock solution inlet, outlet and 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 microns, the filtrate is a GPC column (Tosoh TSK-gel-G3000PW _XL ), the column temperature is 40 ° C, the mobile phase is 1 mL / min of distilled water for liquid chromatography, and the sample injection amount Perform analysis with 100 μl, and measure with a differential refractometer (RI-8020 manufactured by Tosoh Corporation) at slice time of 0.02 min and base-line-range of 4.5 to 11.0 min. Calibration of the column is performed 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 15,000 sieve coefficient by the dextran weight average molecular weight 60,000 sieve coefficient is defined as the transmission ratio.

Example 1
The resin bonded portions at both ends of the dialyzer TS2.1ML manufactured by Toray Industries, Inc. were cut to obtain a hollow fiber membrane. 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 membrane was confirmed with an electron microscope (S800 manufactured by Hitachi, Ltd.), it had an asymmetric structure.

  100 hollow fiber membranes were bundled, and both ends were fixed to a polycarbonate module case with an epoxy-based potting agent so as not to block the hollow portion, thereby producing a mini module. The mini-module has an inner diameter of about 5 mm and a length of about 12 cm. Like a general hollow fiber membrane type 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.

  Two such mini-modules were prepared. One was filled with an aqueous solution of PBS (Dulbecco PBS (-) manufactured by Nissui Pharmaceutical Co., Ltd.), and the remaining one was filled with distilled water. And when the sieve coefficient of one dextran filled with distilled water was measured, the sieve coefficient with a molecular weight of 11000 was 0.542, and the sieve coefficient with a molecular weight of 67,000 was 0.0145. 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 the filtrate and precipitates, and then filtered to 0.45 μm.

  On the other hand, an apparatus as shown in FIG. 1 was prepared using one mini-module described above. First, one of the ports on the outside of the hollow fiber membrane of the mini module was capped, the other was connected with a silicone tube, and a peristaltic pump for performing filtration was provided on the way. Each port inside the hollow fiber membrane was connected with a silicone tube and placed in the stock solution tank, and a peristaltic pump was provided in the middle of the silicone tube on the inlet side so that the stock solution could be circulated. A PBS aqueous solution was placed in the stock solution tank, and the circuit was filled with the PBS aqueous solution with a peristaltic pump.

  Next, empty the stock solution tank containing the PBS aqueous solution and place 1 ml of human serum. Using this human serum as the stock solution, the temperature is 25 ° C, the flow rate of the stock side circulation circuit is 3.0 mL / min, and the filtration flow rate is 0.6 mL. The solution was circulated and supplied to the inside of the hollow fiber membrane while maintaining at / min, and filtration was performed for 4 hours. A filtered volume of PBS aqueous solution was added to the stock solution tank at a rate of 0.6 ml / min and circulated to keep the amount of circulated liquid constant.

  Then, the volume of the filtrate after 4 hours, that is, the volume of the solution in which the composition of the biological component has changed, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 4 hours, albumin Concentration and β2-microglobulin concentration were measured. The albumin concentration was measured with a Human Albumin ELISA Quantitation Kit (Cat No. E80-129, manufactured by BETHYL), and the β2-microglobulin concentration was measured with Grazyme β2-Microglobulin-EIA TEST, manufactured by Sanyo Chemical Industries. The results are shown in Table 1. In addition, the albumin concentration and β2-microglobulin concentration in the circulation circuit at the start of the circulation process cannot be measured because sampling is difficult because the original solution amount is small. Shows the calculated value. In this example, since a membrane having a low albumin permeability is used, it is considered that almost no albumin permeation occurs at the start of circulation. Therefore, calculations were performed assuming that all albumin was present in the circuit. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane (serum + PBS aqueous solution) / supply-side channel capacity is 11.5 mg / ml at the start of the treatment, which is considered to be the largest, and the value is considered to be the smallest. After 4 hours, it was 10.3 mg / ml.

  Moreover, the recovery rate of each protein was calculated | required as follows.

Protein recovery rate (%) = (filtrate x protein concentration in the filtrate)
(Stock solution volume x protein concentration in the stock solution)
As a result of the calculation as described above, the recovery rate of albumin was 0.075%, the recovery rate of β2-microglobulin was 100%, and while maintaining the amount of β2-microglobulin, albumin could be significantly removed. .

(Example 2)
One mini-module was prepared in the same manner as in Example 1, and an apparatus as shown in FIG. 2 was prepared. The mini module was capped on one of the outer ports and the other one connected to 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. An inlet for adding an additional PBS aqueous solution was provided in the middle of the solution circulation circuit, and the solution circulation circuit was filled with the PBS aqueous solution.

  In addition, 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.

  Next, filtration was performed at 25 ° C. for 60 minutes at a circulation flow rate of 3 ml / min and a filtration flow rate of 0.6 ml / min using 4 ml of a solution obtained by diluting the serum three times with a PBS aqueous solution. At this time, after 3 times diluted serum was flowed into the solution circulation circuit at 0.6 ml / min, the volume of the filtration was added to the solution circulation circuit at a volume of 0.6 ml / min with PBS solution to keep the circulating fluid volume constant. Kept.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the flow channel on the supply side is 36.2 mg / ml at the start of treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. It was 35.9 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.408%, the recovery rate of β2-microglobulin was 100%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.

(Example 3)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum 4 times with PBS aqueous solution was used.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the flow channel on the supply side is 22.5 mg / ml at the start of treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. It was 22.3 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.229%, the recovery rate of β2-microglobulin was 76.0%, and albumin was significantly removed while maintaining the amount of β2-microglobulin. I was able to.

Example 4
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum 8 times with an aqueous PBS solution was used.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the supply-side channel capacity is 11.8 mg / ml at the start of the treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. It was 11.6 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.245%, the recovery rate of β2-microglobulin was 100%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.

(Example 5)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a 16-fold diluted serum solution in PBS was used.

Then, the filtrate volume, albumin concentration and β2-microglobulin concentration after 60 minutes, and the volume, albumin concentration and β2-microglobulin concentration of the solution remaining in the circulation circuit after 60 minutes passed are obtained. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the flow channel on the supply side is 6.7 mg / ml at the start of the treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. But it was 6.7mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.169% and the recovery rate of β2-microglobulin was 94%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.
(Example 6)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of undiluted serum was used, and the supply liquid flow path volume was changed by increasing the supply liquid flow path diameter.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the flow channel on the supply side is 35.9 mg / ml at the start of treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. It was 35.7 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.032%, the recovery rate of β2-microglobulin was 87%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.

(Example 7)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum 4 times with PBS aqueous solution was used, and the supply liquid flow path volume was changed by changing the supply liquid flow path diameter.

Then, the filtrate volume, albumin concentration and β2-microglobulin concentration after 60 minutes, and the volume, albumin concentration and β2-microglobulin concentration of the solution remaining in the circulation circuit after 60 minutes passed are obtained. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total albumin amount / supply-side channel capacity in the solution supplied to the hollow fiber membrane is 15.0 mg / ml at the start of the treatment, which is considered to be the largest, and after 60 minutes, which is believed to be the smallest. It was 14.9 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.063%, the recovery rate of β2-microglobulin was 90%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.
(Example 8)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum 4 times with PBS aqueous solution was used, and the supply liquid flow path volume was changed by changing the supply liquid flow path diameter.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 1. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the supply-side channel is 7.5 mg / ml at the start of the treatment that is considered to be the largest, and after 60 minutes, the value is considered to be the smallest. But it was 7.5mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.039% and the recovery rate of β2-microglobulin was 84%, and albumin was largely removed while maintaining the amount of β2-microglobulin. I was able to.

(Comparative Example 1)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of undiluted serum was used.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 2. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the flow channel on the supply side is 105.0 mg / ml at the start of treatment, which is considered to be the largest, and after 60 minutes, which is thought to be the smallest. 99.8 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 3.81%, and the recovery rate of β2-microglobulin was 84%. That is, albumin and β2-microglobulin could not be sufficiently separated.

(Comparative Example 2)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum with PBS aqueous solution twice was used.

Then, the filtrate volume, albumin concentration and β2-microglobulin concentration after 60 minutes, and the volume, albumin concentration and β2-microglobulin concentration of the solution remaining in the circulation circuit after 60 minutes passed are obtained. It was measured. The results are shown in Table 2. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the supply-side channel is 64.9 mg / ml at the start of processing considered to be the largest, and after 60 minutes, the value is considered to be the smallest. It was 63.6 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 1.34%, and the recovery rate of β2-microglobulin was 99%. That is, albumin and β2-microglobulin could not be sufficiently separated.
(Comparative Example 3)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a serum diluted 40-fold with an aqueous PBS solution was used.
Then, the filtrate volume, albumin concentration and β2-microglobulin concentration after 60 minutes, and the volume, albumin concentration and β2-microglobulin concentration of the solution remaining in the circulation circuit after 60 minutes passed are obtained. It was measured. The results are shown in Table 2. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the supply-side channel is 2.3 mg / ml at the start of the treatment that is considered to be the largest, and after 60 minutes, the value is considered to be the smallest. It was 2.2 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 1.13%, and the recovery rate of β2-microglobulin was 79%. That is, albumin and β2-microglobulin could not be sufficiently separated.
(Comparative Example 4)
An apparatus was prepared and filtered in the same manner as in Example 2 except that 4 ml of a solution obtained by diluting serum 4 times with PBS aqueous solution was used, and the supply liquid flow path volume was changed by changing the supply liquid flow path diameter.

  Then, the volume of the filtrate after 60 minutes, the albumin concentration and the β2-microglobulin concentration, and the volume of the solution remaining in the circulation circuit after 60 minutes, the albumin concentration and the β2-microglobulin concentration were determined. It was measured. The results are shown in Table 2. Moreover, a calculated value is shown about the albumin density | concentration and (beta) 2-microglobulin density | concentration in the liquid in the circulation circuit at the time of a circulation process start. As a result, the total amount of albumin in the solution supplied to the hollow fiber membrane / the capacity of the supply-side channel is 5.0 mg / ml at the start of the treatment, which is considered to be the largest, and after 60 minutes, the value is considered to be the smallest. It was 5.08 mg / ml. Furthermore, as a result of obtaining the recovery rate of each protein, the recovery rate of albumin was 0.021%, and the recovery rate of β2-microglobulin was 67%. That is, albumin and β2-microglobulin could not be sufficiently separated.

1 is a conceptual diagram of a separation system used in Example 1. FIG. It is a conceptual diagram of the separation system used in Examples 2-8 and Comparative Examples 1-4.

Explanation of symbols

100 Injection pump 101 Stock solution tank 102 Solution circulation circuit 103 Circulation pump 104 Treatment liquid recovery port 105 Separation membrane module 106 Module lower port 107 Filtration pump 200 Injection pump 201 Three-way valve 202 Solution circulation circuit 203 Circulation pump 204 Membrane Separation unit treatment liquid recovery port 205 Separation membrane module 206 Lower port of module

Claims (5)

  When analyzing proteins and / or peptides in a biological component-containing solution, the biological component-containing solution is circulated and supplied to a separation membrane having a molecular weight of 67,000 and a dextran sieving coefficient of 0.1 or less. The biological component-containing solution is supplied to the separation membrane by adjusting the ratio of the total albumin amount in the liquid / the supply-side channel capacity to be in the range of 6 to 60 mg / ml. A pretreatment method for a biological component-containing solution.   2. The biological component-containing solution pretreatment method according to claim 1, wherein the biological component-containing solution is circulated in a closed circuit.   The method for pretreatment separation and purification of a biological component-containing solution according to claim 1 or 2, wherein a hollow fiber membrane is used as the separation membrane.   The biological component-containing solution is at least one selected from the group consisting of blood-derived substances, urine, ascites, saliva, tears, cerebrospinal fluid, pleural effusion, and a solution obtained by extracting proteins from cells. Item 4. A pretreatment method for a biological component-containing solution according to any one of Items 1 to 3. An analytical solution purification method for purifying an analytical solution of protein and / or peptide using the pretreatment method according to claim 1.

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