JP2010051927A - Method of separating biogenic substance using ultra-filtration membrane, module and device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of separating a target biogenic substance with high purity at a high recovery rate and a separation and concentration method. <P>SOLUTION: A solution which is a biogenic substance solution containing the target biogenic substance having a molecular weight of 10-1,000 kDa and a high molecular weight impurity biogenic substance including a dimer of the target biogenic substance and has 1-150 g/L biogenic substance concentration is filtered by cross flow using the ultra-filtration membrane wherein the molecular weight cut off is ≥1.5 and <4.5 times of that of the target biogenic substance and the target biogenic substance is permeated at ≥80% permeability. In such a case, the ratio of the permeability at which the dimer of the target biogenic substance is passed through the ultra-filtration membrane to the permeability at which the target biogenic substance is passed through the ultra-filtration membrane is ≤0.20. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is a cross-flow filtration of a biocomponent solution containing a target biocomponent and a high molecular weight impurity biocomponent containing at least a dimer of the target biocomponent and having a biocomponent concentration of 1 to 150 g / L. Thus, the present invention relates to a method of separating a high molecular weight impurity biological component containing a target biological component and at least a dimer of the target biological component.

  Immunoglobulins (antibodies) are mainly present in blood and body fluids, and recognize and bind to microorganisms such as bacteria and viruses that have entered the body and cells infected with microorganisms as antigens. When an antibody binds to an antigen, the phagocytic cells such as leukocytes and macrophages recognize and phagocytose the antigen-antibody complex and work to remove it from the body, or immune cells such as lymphocytes bind to cause an immune reaction. Or Thus, immunoglobulins play an important role in infection defense mechanisms.

As a method for producing an immunoglobulin, there are a method of separating and purifying from biological components, mainly blood, and a method of separating and purifying from cells such as hybridomas. However, there are 100,000 or more kinds of microorganisms such as proteins, DNA, RNA, viruses and the like as biological components, and it is necessary to separate them from immunoglobulins.
In addition, there are aggregates (mainly dimers) in which a plurality of immunoglobulins are bound together by non-covalent bonds. Immunoglobulin aggregates are considered to be one of the causes of side effects that appear when an immunoglobulin preparation is intravenously injected into the human body, such as shock-like reactions such as cyanosis and decreased blood pressure, respiratory tract symptoms such as dyspnea, and rashes. Yes. These aggregates tend to aggregate with each other to form larger multimers, which can cause cloudiness and precipitation in the solution. In some cases, other proteins, bacteria, viruses, and the like are used as nuclei and bound around them to form huge protein aggregates called antigen-antibody complexes. Once formed, aggregates generally cannot be easily dissociated. Therefore, many methods for removing these aggregates from the solution have been proposed.

Several purification methods have been reported to separate immunoglobulin monomers from the solution in which they are mixed. Examples include ion exchange chromatography, hydrophobic chromatography, gel chromatography, chemical treatment, adsorption, and ultrafiltration membrane.
Ion exchange chromatography and hydrophobic chromatography methods are effective when separating substances with greatly different charges and hydrophobicity, but substances with similar degrees of charge and hydrophobicity, such as immunoglobulin monomers and dimers. However, there are disadvantages such that a sufficient amount of eluent and salt are necessary in addition to sufficient separation.
On the other hand, the gel chromatography method is effective in separating immunoglobulin dimers and dimers having different sizes because of size separation, but cannot process a large amount of immunoglobulins and requires work. There is a disadvantage that the time and cost are long.

Chemical treatment methods that add chemical substances can be processed in large quantities, but it is necessary to remove the chemicals used in the treatment from the solution. This will reduce the transmittance.
Also, the adsorption method does not have high removal efficiency of the immunoglobulin dimer, and further, the work of removing the added adsorbent is required in the same manner as the chemical treatment.

Recently, an ultrafiltration membrane method that has high robustness, is simple, and can process a large amount of immunoglobulins has attracted attention, and there are many reports.
For example, as an ultrafiltration membrane method, a globulin dimer removal method (Patent Document 1) by filtering with an ultrafiltration membrane formed from a polysulfone polymer has been proposed. However, the permeability of the globulin monomer after this method is as low as about 40%, and the filtration rate and the filtration volume are both low, so it cannot be said that the industrial effectiveness is high.

  A method for removing immunoglobulin aggregates by filtering with a regenerated cellulose hollow fiber membrane (Patent Document 2) has also been proposed, but immunoglobulin dimers are hardly removed. It was not a sufficient method for separation.

  In addition, a method of removing immunoglobulin dimers by crossflow filtration using an ultrafiltration membrane (Patent Document 3) has been proposed, but the fractionation performance of immunoglobulin dimers and dimers is proposed. There is no data on the.

  In addition, a method of separating biological components having a molecular weight difference of less than 10 times by cross-flow filtration while maintaining a level in the range of 5 to 100% of the flux at the transition point (Patent Document 4) has been reported. However, only a protein separation of 100,000 or less has been proposed, and no data on the separation of immunoglobulin monomer and dimer is described.

  Also, a method for reducing anti-complement activity by removing aggregates from an aqueous solution of immunoglobulin G fractionated from human plasma and then filtering with a polyolefin porous membrane in the presence of a surfactant (Patent Document 5) However, there is no specific data on the separation of immunoglobulin monomer and dimer, and since a membrane having a large pore of 15 nm or more is used, the anti-complement activity of the membrane is Reduction was not enough.

On the other hand, as biopharmaceuticals other than immunoglobulin, conventionally, interferon, vaccine, insulin, blood coagulation factor, growth hormone, glucagon, erythropoietin, granular colony stimulating factor, osteogenic factor, protein C, soluble receptor, antiviral agent, Vaccines and the like are known, but various side effects due to aggregates are regarded as a problem as with antibodies.
Japanese Patent Publication No.62-3815 JP-A-6-279296 Japanese Patent No. 3746223 Japanese Patent No. 3828143 Japanese Patent Publication No. 7-78025

  An object of the present invention is a biological component solution containing a target biological component and a high molecular weight impurity biological component containing at least a dimer of the target biological component, wherein the biological component concentration is 1 to 150 g / L. By filtering, the target biological component permeates with a transmittance of 80% or more, and the target biological component transmits the ultrafiltration membrane and the target biological component dimer transmits the ultrafiltration membrane. It is an object of the present invention to provide a separation method in which the ratio of the above can be 0.20 or less.

  As a result of intensive research in order to solve the above-mentioned problems, the present inventors have obtained a biological component solution containing a target biological component and a high molecular weight impurity biological component containing at least a dimer of the target biological component, Cross-flow filtration of a solution having a concentration of 1 to 150 g / L allows the target biological component to permeate at a transmittance of 80% or more, and allows the target biological component to pass through the ultrafiltration membrane and the target biological component. The inventors have found a separation method in which the ratio of the permeation rate of the component dimers through the ultrafiltration membrane can be 0.20 or less, and have completed the present invention.

That is, the present invention
[1] Using an ultrafiltration membrane having a molecular weight of the target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component, A biological component solution containing a high molecular weight impurity biological component containing at least a dimer of a target biological component, wherein the biological component concentration of the target biological component and at least the target biological component dimer is 1 to 150 g / L A method of separating a high molecular weight impurity biological component containing a target biological component and at least a dimer of the target biological component by subjecting a solution to cross flow filtration.
[2] Using an ultrafiltration membrane having a molecular weight of the target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component, A biological component solution containing a high molecular weight impurity biological component containing at least a dimer of a target biological component, wherein the biological component concentration of the target biological component and at least the target biological component dimer is 1 to 150 g / L Separating a high molecular weight impurity biological component containing a target biological component and at least a dimer of the target biological component by cross-flow filtering a solution;
A biological component solution containing at least a target biological component and a low molecular weight impurity biological component using an ultrafiltration membrane for concentration / separation having a fractional molecular weight of 0.3 to 1.5 times the target biological component. A separation method comprising a step of concentrating / separating a target biological component by permeating a low molecular weight impurity biological component by cross-flow filtration of a solution having a biological component concentration of 0.1 to 150 g / L .
[3] The separation method according to [1] or [2] above, wherein the concentration of the biological component is 5 to 100 g / L.
[4] The separation method according to any one of [1] to [3], wherein the biological component concentration is subjected to crossflow filtration while being maintained substantially constant.
[5] The separation method according to any one of [1] to [4], wherein the biological component contains at least one of polypeptide, sugar, RNA, and DNA.
[6] The polypeptide contains at least one of antibody, low molecular weight antibody, albumin, blood coagulation factor, interleukin, interferon, cell adsorption factor, cell growth factor, enzyme, riboprotein, and vaccine. The separation method according to [5] above, wherein
[7] The separation method of [6], wherein the antibody is an immunoglobulin.
[8] The method according to [7] above, wherein the immunoglobulin is a monoclonal antibody.
[9] The high molecular weight impurity biological component including at least the dimer of the target biological component includes a complex aggregate composed of the target biological component such as a complex aggregate composed of immunoglobulin and protein A and the eluted affinity ligand. The separation method according to any one of [1] to [8], wherein the separation method is characterized.
[10] At least one of endotoxin, DNA, and CHOP (Chinese hamster ovary cell protein), which is an impurity derived from the starting material of the target biological component, is a high molecular weight impurity biological component containing at least a dimer of the target biological component The separation method according to any one of [1] to [9] above, comprising the above.
[11] The low-molecular-weight impurity biological component contains at least one or more of endotoxin, DNA, and CHOP (Chinese hamster ovary cell protein), which are impurities derived from the starting material of the target biological component. The separation method according to any one of [2] to [10].
[12] The separation method according to any one of [1] to [11], wherein the ultrafiltration membrane is a vinyl alcohol polymer membrane or a sulfone polymer membrane.
[13] A separation method, wherein the separation method according to any one of [1] to [12] is used after an affinity chromatography purification step of the solution.
[14] The separation method according to any one of [1] to [13], wherein the ultrafiltration membrane is a hollow fiber membrane.
[15] The filtration flow rate is maintained at a level exceeding the flow rate at the transition point, and the filtration transmembrane pressure is maintained at a level exceeding the transmembrane pressure at the flow rate transition point. -The separation method in any one of [14].
[16] The separation method according to any one of the above [1] to [15], which is performed using an apparatus including at least one of the following means (a) to (d).
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means [17] An apparatus comprising one or more means of means comprising the module used in the separation method according to any one of [1] to [15] above and the following means (a) to (d).
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means [18] A module using an ultrafiltration membrane and one or more means comprising the following (a) to (d), a module using an ultrafiltration membrane for concentration / separation, and the following (e) to (h) The separation method according to any one of the above [2] to [15], which comprises one or more means consisting of:
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means (e) Means capable of monitoring the concentration of the biological component permeate before concentration (f) Means capable of monitoring the concentration of the biological component permeate after concentration (G) Means capable of controlling the feeding speed of the biological component permeate ) Means capable of controlling the filtration pressure of the ultrafiltration membrane for concentration / separation [19] A module using an ultrafiltration membrane for performing the separation method according to any one of [2] to [15] above and the following A device comprising one or more means comprising (a) to (d), a module using an ultrafiltration membrane for concentration and separation, and one or more means comprising the following (e) to (h): .
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means (e) Means capable of monitoring the concentration of the biological component permeate before concentration (f) Means capable of monitoring the concentration of the biological component permeate after concentration (G) Means capable of controlling the feeding speed of the biological component permeate ) Means for controlling the filtration pressure of the ultrafiltration membrane for concentration and separation

The present invention uses an ultrafiltration membrane having a molecular weight of a target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component. And a biological component solution containing a high molecular weight impurity biological component containing at least a dimer of the target biological component, wherein the biological component concentration is 1-150 g / L, and the target biological component is The ratio of the permeability at which the target biological component permeates the ultrafiltration membrane and the permeability at which the dimer of the target biological component permeates the ultrafiltration membrane can be reduced to 0.20 or less. .
Since the present invention only requires cross-flow filtration with an ultrafiltration membrane, it is an extremely simple operation compared to a method such as chromatography, and can process a large amount of biological component solution. Further, unlike the chemical treatment method, the target biological component is not deactivated or denatured. Furthermore, unlike known membranes, the target biological component can be separated with high recovery and high purity.

  Hereinafter, the biological component separation method and apparatus using the ultrafiltration membrane according to the present invention will be described in detail.

  Examples of the biological component according to the present invention include polypeptides, sugars, RNA, and DNA, and the biological component solution contains at least one of these. Polypeptides according to the present invention refer to peptides and proteins having more than about 10 amino acids. Preferably the polypeptide is a mammalian protein. Examples include antibodies (immunoglobulins), low molecular weight antibodies, albumin, blood coagulation factors, interleukins, interferons, cell adsorption factors, cell growth factors, enzymes, riboproteins, hormones, vaccines and the like.

  More specifically, renin; growth hormone (including human growth hormone and bovine growth hormone); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α-1-antitrypsin; insulin A chain; B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factor (eg, factor VIIIC, factor IX, tissue factor and von Willebrand factor); anticoagulation factor (eg, protein C) Atrial natriuretic factor; pulmonary surfactant; plasminogen activator (eg urokinase or human urine or tissue type plasminogen activator (t-PA)); bombesin; thrombin; hematopoietic growth factor; tumor Necrosis factor-α and tumor necrosis factor-β; Enkephalina RANTES (modulates activation of normally expressed and secreted T cells); human macrophage inflammatory protein (MIP-1-α); serum albumin (eg, human serum albumin); Muellerian inhibitor; relaxin A chain; Relaxin B chain; prorelaxin; mouse gonadotropin related peptide; microbial protein (eg β-lactamase); DNase; IgE; cytotoxic T lymphocyte related antigen (CTLA) (eg CTLA-4); inhibin; activin; blood vessel Endothelial growth factor (VEGF); receptor for hormone or growth factor; protein A or protein D; rheumatoid factor; neurotrophic factor (eg, bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, Newlot Fin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor such as NGF-β); platelet derived growth factor (PDGF); fibroblasts Growth factors (eg, aFGF and bFGF); epidermal growth factor (EGF); transforming growth factor (TGF) (eg, TGF-α and TGF-β (TGF-β1, TGF-β2, TGF-β3, TGF-β4) Or TGF-β5)); insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin Like growth factor binding protein (IGFBP); CD proteins (eg, CD3, CD4, CD8, CD19, CD20, CD34 and CD40) Erythropoietin; osteoinductor; immunotoxin; bone morphogenic protein (BMP); interferon (eg, interferon-α, interferon-β, and interferon-γ); colony stimulating factor (CSF) (eg, M-CSF) , GM-CSF and G-CSF); interleukin (IL) (eg IL-1 to IL-10); superoxide dismutase; T cell receptor; surface membrane protein; decay-promoting factor; viral antigen (eg AIDS envelope) Transport protein; homing receptor; addressin; regulatory protein: integrin (eg, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM); tumor associated antigen (eg, HE) 2 receptor, HER3 receptor or HER4 receptor); and the like or a fragment and / or variant of the above-described polypeptide.

The antibody according to the present invention is used in the broadest sense, and specifically includes a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, a multispecific antibody (for example, a bispecific antibody), or a ligand. As long as the antibody fragment ligand-specific binding region is retained as long as it is modified to include a specific binding region. The antibody herein is directed against the “antigen” of interest. The antigen is preferably a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disease or injury can provide a therapeutic benefit in that mammal. Also included are antibodies to non-polypeptide antigens (eg, tumor-associated glycolipid antigens; see US Pat. No. 5,091,178). Where the antigen is a polypeptide, the antigen can be a transmembrane molecule (eg, a receptor) or a ligand (eg, a growth factor).
The antibody according to the present invention is not limited in any way as long as it can provide a therapeutic benefit,
For example, human antibodies, humanized antibodies, chimeric antibodies, non-human antibodies, antibody fragments can be mentioned.

A humanized antibody according to the present invention is a human immunoglobulin (recipient antibody), wherein the hypervariable region residues of the recipient are non-human species having the desired specificity, affinity and ability (eg, , Mouse, rat, rabbit or non-human primate) hypervariable region residues (
The donor antibody). In some examples, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the donor antibody. These modifications can be made to further refine antibody performance. In general, a humanized antibody comprises substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops are hypervariable of non-human immunoglobulin. Corresponding to the loop and all or substantially all of the FR region is the FR region of the human immunoglobulin sequence. A humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that portion of a human immunoglobulin.

In the chimeric antibody according to the present invention, a part of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in the antibody derived from a specific species or belonging to a specific antibody class or subclass. On the other hand, the remaining part of the chain corresponds in antibodies from other species or belonging to other antibody classes or subclasses as well as fragments of such antibodies (as long as they exhibit the desired biological activity). Identical or homologous to the sequence.

The antibody fragment according to the present invention includes a part of a full-length antibody (generally, an antigen-binding region or a variable region thereof). Examples of antibody fragments are Fab, Fab ′, F (
ab ') 2, Fv fragments; single chain antibody molecules, diabodies; linear antibodies, and multispecific antibodies formed from antibody fragments.

Preferred molecular targets for antibodies encompassed by the present invention include CD polypeptides (eg, CD3, CD4, CD8, CD19, CD20, CD34, and CD40); H
ER receptor family (eg, EGF receptor, HER2, HER3 or H
ER4 receptor); cell adhesion molecule (for example, LFA-1, Mac1, p150, 95,
Av / b containing VLA-4, ICAM-1, VCAM and a or b subunits
Members of 3 integrins (eg, anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors (eg, VEGF); IgE, blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; -4; Polypeptide C
Etc. Soluble antigens or fragments can be conjugated to other molecules as needed and used as an immunogen for antibody production. For transmembrane molecules (eg receptors), these fragments (eg the extracellular region of the receptor) can be used as immunogens. Alternatively, cells that express transmembrane molecules can be used as immunogens.

Specific examples of the antibody according to the present invention include anti-HER2, anti-CD20, anti-IL-8, and anti-VEG.
F, anti-PSCA, anti-CD11a, anti-IgE, anti-Apo-2 receptor, anti-TNF-α, anti-tissue factor (TF), anti-CD3, anti-CD25, anti-CD34
, Anti-CD40, anti-tac, anti-CD4, anti-CD52, anti-Fc receptor, anti-carcinoembryonic antigen (
CEA) antibodies, antibodies specific for breast epithelial cells, antibodies that bind to colon cancer cells, anti-CD33
, Anti-CD22, anti-EpCAM, anti-GpIIb / IIIa, anti-RSV, anti-CMV, anti-HIV
Anti-hepatitis, anti-αvβ3, anti-human renal cell carcinoma, anti-human 17-1A, anti-human colorectal tumor, anti-human melanoma, anti-human squamous cell carcinoma, anti-human leukemia antigen (HLA) antibody, anti-HER2 receptor antibody, Examples include anti-VEGF antibody, anti-IgE antibody, anti-CD20 antibody, anti-CD11a antibody, and anti-CD40 antibody. More specific examples include Muramomab (product name: Orthoclone (OKT3), Rituximab (product name: Ritaxan).
), Basiliximab (product name: Simulect), Daclisumab (product name: Zenapax), Palivizumab (product name: Synagis), Inf
liximab (product name: Remicade), Gemtuzumab zogic
n (Product name: Mylotarg), Alemtuzumab (Product name: Mabcampa
th), Adalimumab (product name: Humira), Omalizumab (product name: Xolair), Vevacizumab (product name: Avastin), Cetux
imab (product name: Erbitux) and the like.

  The molecular weight of the target biological component according to the present invention is 10 to 1,000 kDa. When the biological component is smaller than 10 kDa, the difference in size between the target biological component and the impurity biological component is small, so that size separation by the membrane becomes difficult. On the other hand, ultrafiltration membranes are not suitable for the separation of biological components larger than 1,000 kDa.

The fractionation molecular weight of the ultrafiltration membrane used for separating the target biological component according to the present invention is 1.5 times or more and less than 4.5 times, preferably 2.0 to 3.5 times that of the target biological component. Less, more preferably 2.0 times or more and less than 2.7 times. If it is less than 1.5 times, there is a problem that the amount of permeation decreases, and if it exceeds 4.5 times, there is a problem that the separation performance of the target biological component and the impurity biological component decreases.
The molecular weight cut-off is determined by dead-end filtration using albumin (66,000), γ-globulin (160,000), catalase (232,000), ferritin (440,000), thyroglobulin (669,000), etc. The molecular weight is calculated as the molecular weight at which the blocking rate is 90% from the relationship between the molecular weight and the blocking rate.

The high molecular weight impurity biological component containing at least the dimer of the target biological component according to the present invention refers to the high molecular weight impurity biological component containing the dimer when the biological component described above is the target biological component. Moreover, the high molecular weight impurity biological component containing at least the dimer of the target biological component may contain an aggregate of a trimer or more. Furthermore, the high molecular weight impurity biological component including at least the dimer of the target biological component may include the heterogeneous aggregate of the biological component described above. Examples of the heterogeneous aggregates include aggregates of the above-described biological components and other proteins such as protein A, fatty acids, lipids, and the like. More specifically, a composite aggregate composed of a target biological component such as a composite aggregate composed of immunoglobulin and protein A and an eluted affinity ligand can be mentioned.
Further, the high molecular weight impurity biological component and the low molecular weight impurity biological component containing at least a dimer of the target biological component according to the present invention are endotoxin, DNA, CHOP (Chinese hamster ovary cells) which are impurities derived from the starting material of the target biological component. (Protein) may be included.

  The low molecular weight impurity biological component according to the present invention refers to a biological component having a molecular weight lower than that of the target biological component in the biological component described above. When the target biological component is immunoglobulin (150,000), low molecular weight impurity biological components include insulin (76,000), transferrin (67,000), albumin (66,000), protein A (42,000), Examples include endotoxin, DNA, CHOP (Chinese hamster ovary cell protein), which are aggregates derived from the starting material of the target biological component.

  The ultrafiltration membrane used for removing the low molecular weight impurity biological component and concentrating / separating the target biological component according to the present invention has a fractional molecular weight of 0.3 to 1.5 times that of the target biological component. . If it is 1.5 times or more, the target biological component penetrates, which is not preferable. On the other hand, if it is less than 0.3 times, the removal rate of low molecular weight impurities is not preferable.

  As the ultrafiltration membrane according to the present invention, a membrane having a desired fractional molecular weight can be produced, and it is not limited at all unless a biological component is adsorbed. For example, a vinyl alcohol polymer membrane, a sulfone polymer membrane, Aromatic ether polymer films, fluorine polymer films, olefin polymer films, cellulose films, (meth) acrylic polymer films, (meth) acrylonitrile polymer films, and the like can be given. A vinyl alcohol polymer membrane and a sulfone polymer membrane are preferable.

The sulfone polymer membrane according to the present invention is not particularly limited, and any polymer having a sulfone group in the molecule can be used. Examples of the sulfone polymer include, for example, polysulfone represented by the following formula (1), polyethersulfone represented by the following formula (2), polyarylsulfone represented by the following formula (3), and the like. . In the formula, l, m, and n represent repeating units.
[Chemical 1]
[Chemical 2]
[Chemical 3]

It is also possible to carry out by combining two or more of these polymers. The sulfone polymer can be subjected to various modifications such as esterification, etherification, and epoxidation in the polymer terminal and / or main chain as necessary.
The weight average molecular weight of the sulfone polymer according to the present invention may be 5,000 to 1,000,000. Preferably, it is 10,000 to 500,000, More preferably, it is the range of 2 to 300,000. Within this range, sufficient strength and film formability can be obtained.

  In order to impart hydrophilicity to the sulfone polymer according to the present invention, it is preferable to use a hydrophilic polymer to make it hydrophilic. The type of hydrophilic polymer that imparts hydrophilicity is not particularly limited. For example, polyvinylpyrrolidone, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyethylene glycol-polypropylene glycol block copolymer, polyvinyl alcohol, polyacrylamide, Examples include poly-N, N-dimethylacrylamide, poly-N-isopropylacrylamide, polyhydroxy acrylate, polyhydroxy methacrylate, carboxymethyl cellulose, starch, corn starch, polychitosan, and polychitin. Among these, polyvinyl pyrrolidone has good compatibility with the sulfone polymer, and is particularly preferable for enhancing the hydrophilicity of the entire membrane.

The weight average molecular weight of the hydrophilic polymer that imparts hydrophilicity to the sulfone polymer membrane according to the present invention may be 1,000 to 2,000,000. Preferably it is 5,000 to 1,500,000 or less, More preferably, it is the range of 5,000 to 1,200,000. For example, various grades of polyvinyl pyrrolidone are commercially available from BASF, and have a weight average molecular weight of 9,000 (K17), and similarly 45,000 (K30), 450,000 (K60), 900,000. (K80) and 1,200,000 (K90) are preferably used, and in order to obtain the intended use, characteristics, and structure, each may be used alone or in combination of two or more. . In the present invention, it is most preferable to use K90 alone.
The content of the hydrophilic polymer in the sulfone polymer membrane according to the present invention is not particularly limited as long as the target biological component does not adsorb to the membrane, but it is preferably contained in an amount of 0.1 to 10% by weight. Is 0.3-8 wt%, more preferably 0.5-5 wt%.

The aromatic ether polymer film according to the present invention is not particularly limited, and examples of the aromatic ether polymer film include those represented by the following formula (4).
[Chemical 4]
(R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen, an organic functional group containing 1 to 6 carbon atoms, or an aprotic having 6 or less carbon atoms containing oxygen, nitrogen or silicon. The organic functional groups may be the same or different, and q in the structural formula represents the number of repeating units, and may be a copolymer containing two or more different repeating units.)
The phenolic hydroxyl group at the terminal of the aromatic ether polymer according to the present invention can be subjected to various modifications such as esterification, etherification, and epoxidation as necessary.

The aromatic ether polymer film according to the present invention is mainly composed of an aromatic ether polymer, but contains other high molecular weight substances and additives as long as the characteristics of the aromatic ether polymer are not impaired. You may do it. It is also possible to carry out by combining two or more of these polymers. For example, polystyrene or a derivative thereof may be contained.
The weight average molecular weight of the aromatic ether polymer according to the present invention may be 5,000 to 1,000,000. Preferably, it is 10,000-500,000, More preferably, it is the range of 20,000-300,000. Within this range, sufficient strength and film formability can be obtained.

  In the present invention, it is preferred to use a hydrophilizing agent together with the aromatic ether polymer in order to control the pore size of the membrane and to impart hydrophilicity. The contact between the liquid mixture subjected to separation treatment by hydrophilization and the ultrafiltration membrane made of the aromatic ether polymer of the present invention is improved.

The hydrophilizing agent according to the present invention is not limited as long as it can impart hydrophilicity. It may be a low molecular compound or a high molecular compound.
Examples of the hydrophilizing agent include diethylene glycol, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyethylene glycol-polypropylene glycol block copolymer, polyvinyl alcohol, polyacrylamide, poly-N,
Examples thereof include hydrophilic compounds such as N-dimethylacrylamide, poly-N-isopropylacrylamide, polyvinylpyrrolidone, polyhydroxyacrylate, polyhydroxymethacrylate, carboxymethylcellulose, starch, corn starch, polychitosan, and polychitin. In addition, surfactants, block copolymers, and graft copolymers containing these substances as a hydrophilic segment and a hydrophobic segment can be sufficiently utilized as a hydrophilic agent. For example, a polystyrene-polyethylene glycol block copolymer is preferable.

Since the polystyrene-polyethylene glycol block copolymer according to the present invention has polyethylene glycol having high hydrophilicity in the hydrophilic segment, it can be effectively used as a hydrophilizing agent. Moreover, these can also be used in combination of 2 or more types. Among these, polyethylene glycol and block copolymers and graft copolymers containing polyethylene glycol as hydrophilic segments can be preferably used. Among them, polystyrene-polyethylene glycol block copolymers are particularly aromatic ethers. It can be suitably used as a hydrophilizing agent that improves the hydrophilicity of the polymer film.

The molecular weight of the hydrophilizing agent according to the present invention is appropriately selected depending on the production method and its conditions.
For example, when a non-halogen water-soluble organic solvent is used as a solvent in a wet film formation method,
The solubility of aromatic ether polymers having high solvent resistance is extremely low. Therefore, when blending the hydrophilic agent with the membrane stock solution, it is necessary to appropriately select the molecular weight and the addition amount of the hydrophilic agent in order to obtain a uniformly dissolved membrane stock solution. In order to use a sufficient amount of the hydrophilizing agent, the molecular weight of the hydrophilizing agent is preferably, for example, a number average molecular weight of 300 or more and 100,000 or less. If it is this area | region, it can melt | dissolve enough in the good solvent used for film-forming. A more preferable lower limit is 400 or more, and a particularly preferable lower limit is 500 or more. More preferably, the upper limit is 70,000 or less, and particularly preferably 50,000 or less.

When the hydrophilizing agent according to the present invention is a compound comprising a hydrophobic segment and a hydrophilic segment, the number average molecular weight of the hydrophilic segment of the hydrophilizing agent is preferably 300 or more and 100,000 or less. If it is this area | region, it can melt | dissolve enough in the good solvent used for film-forming. A more preferable lower limit is 400 or more, and a particularly preferable lower limit is 500 or more. More preferably, the upper limit is 70,000 or less, and particularly preferably 50,000 or less.

The polystyrene-polyethylene glycol block copolymer according to the present invention is a block copolymer comprising a segment derived from a polystyrene polymer and a segment derived from a polyethylene glycol polymer.
As the polystyrene polymer forming the segment derived from the polystyrene polymer of the polystyrene-polyethylene glycol block copolymer used in the present invention, a polystyrene polymer comprising a repeating unit represented by the following formula (5) is preferable.
[Chemical 5]
(R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ are hydrogen, a halogen atom excluding fluorine, an organic functional group containing 1 to 6 carbon atoms, or oxygen, nitrogen Or a silicon-containing functional group having 6 or less carbon atoms, which may be the same or different, and s in the structural formula is the number of repeating units. It may be a copolymer including the above.)
The number average molecular weight of the segment derived from the polystyrene-based polymer of the polystyrene-polyethylene glycol block copolymer according to the present invention needs to be 300 or more and 1,000,000 or less. If it is this area | region, it can melt | dissolve sufficiently in the good solvent used for film-forming, and can reduce the elution property with respect to aqueous solution or water-soluble organic solvent. The more preferable lower limit is 500 or more, and the particularly preferable lower limit is 700 or more. The upper limit is more preferably 500,000 or less, and the particularly preferable upper limit is 300,000 or less.

The polyethylene glycol polymer forming a segment derived from the polyethylene glycol polymer of the polystyrene-polyethylene glycol block copolymer used in the present invention is a repeating unit represented by the following formula (6) and / or (7). A polyethylene glycol polymer is preferable.
[Chemical 6]
[Chemical 7]
(R ¹⁵ is an organic functional group having 3 or more and less than 30 carbon atoms. Unless hydrophilicity is greatly reduced, R ¹⁶ may contain an ether group, an ester group, a hydroxyl group, or a ketone group. No, u is the number of repeating units.)
The number average molecular weight of the segment derived from the polyethylene glycol polymer of the polystyrene-polyethylene glycol block copolymer used in the present invention needs to be, for example, 300 or more and 100,000 or less. If it is this area | region, sufficient hydrophilic property will be obtained at that time if it can melt | dissolve sufficiently in the good solvent used for film-forming. A more preferred lower limit is 4
00 or more, particularly preferred lower limit is 500 or more, more preferably 70,0 as the upper limit
00 or less, particularly preferably 50,000 or less.

As the composition ratio of the segment derived from the polystyrene polymer of the polystyrene-polyethylene glycol block copolymer according to the present invention and the segment derived from the polyethylene glycol polymer, the segment derived from the polystyrene polymer is all polystyrene-polyethylene. It is necessary that the content is 10% by weight or more and 99% by weight or less of the glycol block copolymer. In this composition ratio, sufficient hydrophilicity can be expressed and elution is suppressed. A more preferred lower limit is 20% by weight or more, and a particularly preferred lower limit is 30% by weight or more,
A more preferred upper limit is 98% by weight or less, and a particularly preferred upper limit is 97% by weight or less.

The block structure of the polystyrene-polyethylene glycol block copolymer according to the present invention is a diblock copolymer composed of two segments, a triblock copolymer composed of three segments, and four or more It may be a multi-block copolymer composed of segments. Moreover, you may be a mixture of these 2 or more types of block copolymers. The number average molecular weights of the respective segments to be constructed may be the same or different.
In the polystyrene-polyethylene glycol block copolymer according to the present invention, the segment derived from the polystyrene polymer and the segment derived from the polyethylene glycol polymer need to be directly chemically bonded at the polymer terminal portion. For production, if necessary, a low molecular compound and / or an organic functional group may be used as a spacer for connecting the segment derived from the polystyrene polymer and the segment derived from the polyethylene glycol polymer. . When the number average molecular weight of the low molecular weight compound and / or the organic functional group is 500 or less, the low molecular weight compound and / or the organic functional group can be expressed without reducing the effect of the segment derived from the polystyrene polymer and the segment derived from the polyethylene glycol polymer. Specific examples include low molecular weight compounds between polystyrene and polyethylene glycol formed when polyethylene glycol is condensed after polymerizing styrene using a radical polymerization initiator having a reactive functional group.

An example of a method for producing the polystyrene-polyethylene glycol block copolymer according to the present invention is a method using a radical polymerization initiator having a reactive functional group. Specifically, an azo radical polymerization initiator having a carboxylic acid group is used, and after chemically converting the carboxylic acid group to an acid chloride group, the terminal has an acid chloride group by radical polymerization of styrene. Polystyrene is obtained. Subsequently, a polystyrene-polyethylene glycol block copolymer can be obtained by condensation with polyethylene glycol (Polymer Papers, 1976, Vol. 33, P131). A polystyrene-polyethylene glycol block copolymer can also be obtained by radical polymerization of styrene using a polyethylene glycol unit-containing polymer azo polymerization initiator. Another example of the synthesis method is a method using living polymerization. Specifically, styrene is polymerized using living radical polymerization with a nitroxide compound to obtain a polymer in which the nitroxide compound is bonded to the polymer terminal. A polymer terminal is converted into a hydroxyl group by hydrolysis, and a polystyrene-polyethylene glycol block copolymer can be obtained by a coupling reaction with polyethylene glycol (Polymer, 1998, Vol. 39, No. 4).
No., P911).

  Examples of the method for hydrophilizing an ultrafiltration membrane made of an aromatic ether polymer using a hydrophilizing agent in the present invention include, for example, a blend method in which a hydrophilizing agent is mixed in advance at the time of film formation, and a solution containing a hydrophilizing agent. Examples thereof include a coating method in which the membrane is immersed and then dried to leave the hydrophilizing agent, and a method in which a hydrophilic acrylic monomer, methacrylic monomer, acrylamide monomer or the like is graft-polymerized on the membrane surface. It is also possible to perform a combination of two or more of these methods. A blending method or a coating method in which the aromatic ether polymer is not chemically modified is preferable, and a blending method capable of performing a hydrophilization treatment in a single step is particularly preferable in terms of production.

The vinyl alcohol polymer film according to the present invention is mainly composed of a vinyl alcohol polymer, but contains other high molecular weight substances and additives as long as the characteristics of the vinyl alcohol polymer are not impaired. Also good. It is also possible to carry out by combining two or more of these polymers.
The vinyl alcohol polymer according to the present invention was copolymerized with polyvinyl alcohol, modified polyvinyl alcohol such as partial acetalization, and ethylene, propylene, vinyl pyrrolidone, vinyl chloride, vinyl fluoride, methyl methacrylate, acrylonitrile, itaconic acid, and the like. Copolymers (including block copolymers and graft copolymers) and derivatives thereof. Among them, a copolymer of ethylene-vinyl alcohol is preferable because it has both stability and chemical resistance, which are characteristics resulting from hydrophobic ethylene, and low protein adsorption, which is a characteristic resulting from hydrophilic vinyl alcohol.

The saponification degree of the vinyl alcohol polymer according to the present invention is 80 to 100 mol%, preferably 85 to 95 mol%.
The polyvinyl alcohol content in the vinyl alcohol polymer chain according to the present invention is preferably at least 30% by weight or more, more preferably 50% by weight or more. If it is less than 30% by weight, it is not preferable because problems such as low hydrophilicity of the film occur.
The weight average molecular weight of the vinyl alcohol polymer according to the present invention is 5,000 to 2,000,000, preferably 10,000 to 900,000, more preferably 50,000 to 800,000. In general, a resin having an average molecular weight exceeding 1,000,000 is difficult to measure by gel permeation chromatography (GPC). Therefore, a viscosity average molecular weight by a viscosity method can be applied as a substitute. An average molecular weight of less than 5,000 is not preferable because the mechanical strength of the film is lowered. An average molecular weight exceeding 2 million is not preferable because uniform melt kneading becomes difficult.

The vinyl alcohol polymer membrane according to the present invention is a membrane made of a vinyl alcohol polymer as a material on the membrane surface with which the separation object contacts, and a substrate made of any material in order to maintain the structure. (Support) may be used. For example, the vinyl alcohol polymer film may be a polymer film composed only of a vinyl alcohol polymer, but a woven or non-woven fabric or porous material may be used as another substrate (support) to increase physical strength. A vinyl alcohol polymer film can also be produced by forming an inorganic material on the base material.
In the present invention, additional treatment may be performed within a range that does not adversely affect the vinyl alcohol polymer film. Examples of the additional treatment include cross-linking treatment and functional group introduction by chemical surface modification.

The fluorine-based polymer film according to the present invention is mainly composed of a fluorine-based polymer, but may contain other high molecular weight substances and additives as long as the characteristics of the fluorine-based polymer are not impaired. It is also possible to carry out by combining two or more of these polymers.
The fluorine-based polymer according to the present invention is a homopolymer of vinylidene fluoride, one kind selected from a monomer group of hexafluoropropylene, pentafluoropropylene, tetrafluoroethylene, chlorotrifluoroethylene, and perfluoromethyl vinyl ether, or It is a copolymer of two types of monomers and vinylidene fluoride. Moreover, the said homopolymer and the said copolymer can also be mixed and used. Among these, vinylidene fluoride is preferable.

  The weight average molecular weight of the fluoropolymer according to the present invention is preferably 50,000 to 5,000,000, more preferably 100,000 to 2,000,000, and further preferably 150,000 to 1,000,000. In general, a resin having an average molecular weight exceeding 1,000,000 is difficult to measure by gel permeation chromatography (GPC). Therefore, a viscosity average molecular weight by a viscosity method can be applied as a substitute. If the average molecular weight is less than 50,000, the melt tension at the time of melt molding becomes small and the moldability is deteriorated, and the mechanical strength of the film is lowered. An average molecular weight exceeding 5 million is not preferable because uniform melt-kneading becomes difficult.

  In order to prevent clogging due to the adsorption of biological components, it is necessary to impart hydrophilicity to the membrane. Examples of the hydrophilization treatment method include, for example, a method in which a fluoropolymer film is immersed in a solution containing a surfactant and then dried to leave the surfactant in the fluoropolymer film. The method of grafting hydrophilic acrylic monomer, methacrylic monomer, etc. on the pore surface of the fluoropolymer film by irradiating the above-mentioned radiation or using a peroxide. A method of mixing, a method of immersing the fluoropolymer film in a solution containing a hydrophilic polymer, and then drying to form a hydrophilic polymer film on the pore surface of the fluoropolymer film. Graft polymerization is most preferable in consideration of the persistence of hydrophilization and the possibility of leakage of hydrophilic additives. In particular, the hydrophilic treatment by the radiation graft polymerization method disclosed in JP-A-62-2179540, JP-A-62-258711, and U.S. Pat. It is preferable in that a uniform hydrophilic layer can be formed on the surface of the pores.

The hydrophilic monomer used in the graft polymerization of the present invention is not particularly limited as long as it is a hydrophilic monomer having a vinyl group. A monomer having one vinyl group is preferable. The hydrophilic monomer according to the present invention is 1% by volume in pure water at 25 ° C. under atmospheric pressure.
It is a monomer that dissolves uniformly when mixed. Examples thereof include hydrophilic vinyl monomers having a hydroxyl group, a sulfonyl group, a sulfone group, an ester group, an amide group, and a urethane group. Among these, a vinyl monomer having one or more hydroxyl groups or a functional group serving as a precursor thereof is preferable. Specifically, (meth) acrylic acid and polyhydric alcohol esters such as hydroxypropyl acrylate and 2-hydroxyethyl methacrylate, alcohols having an unsaturated bond such as allyl alcohol, and vinyl acetate and vinyl propionate. Examples include enol esters.

Furthermore, hydrophilicity is sufficiently imparted by copolymerizing by a graft polymerization method using a hydrophilic vinyl monomer and a crosslinking agent having two or more vinyl groups.
The cross-linking agent used according to the present invention is a cross-linking agent having two or more vinyl groups that can be copolymerized with the hydrophilic monomer, and is introduced by contacting the membrane simultaneously with the hydrophilic monomer. The crosslinking agent preferably has a number average molecular weight of 200 or more and 2,000 or less, more preferably a number average molecular weight of 250 or more and 1,000 or less, and most preferably a number average molecular weight of 300 or more and 600 or less. When the number average molecular weight of the crosslinking agent is 200 or more and 2,000 or less, a high filtration rate of the biological component solution is obtained, which is preferable. In the present invention, any crosslinking agent can be used as long as it has two or more vinyl groups, but a hydrophilic crosslinking agent is preferred.

  Specific examples of the crosslinking agent used in the present invention include divinylbenzene derivatives for aromatic systems, methacrylic acid crosslinking agents such as ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate for aliphatic systems, ethylene glycol diacrylate, Examples include (meth) acrylic acid-based crosslinking agents such as polyethylene glycol diacrylate. A crosslinking agent having three reactive groups such as trimethylolpropane trimethacrylate can also be used. In addition, a mixture of two or more kinds of crosslinking agents can be used. In the present invention, it is most preferable to use polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, or a mixture thereof.

  The graft polymerization method according to the present invention is not limited as long as it is a method for generating radicals. For example, radicals are generated in a fluorine-based polymer film by means such as addition of a radiation initiator, ionizing radiation or chemical reaction. Then, starting from the radical, the monomer is graft-polymerized to the film. In the present invention, any means can be employed for generating radicals in the fluorine-based polymer film, but irradiation with ionizing radiation is preferable in order to generate uniform radicals throughout the film. As types of ionizing radiation, γ rays, electron beams, β rays, neutron rays, and the like can be used, but electron beams or γ rays are most preferable for implementation on an industrial scale. The ionizing radiation is obtained from radioactive isotopes such as cobalt 60, strontium 90, and cesium 137, or by an X-ray imaging apparatus, an electron beam accelerator, an ultraviolet irradiation apparatus, or the like.

  The irradiation dose of ionizing radiation according to the present invention is preferably from 1 kGy to 1,000 kGy. If it is less than 1 kGy, radicals are not generated uniformly, and if it exceeds 1,000 kGy, film strength may be reduced. Graft polymerization is generally divided into two types: a pre-irradiation method in which radicals are generated on a film and then contacted with a reactive compound, and a simultaneous irradiation method in which radicals are generated on the film in a state where the film is in contact with the reactive compound. Is done. In the present invention, any method can be applied, but the pre-irradiation method in which oligomer formation is small is most preferable.

  In the present invention, the contact between the fluorine-based polymer film that has generated radicals, the hydrophilic monomer, and the cross-linking agent can be achieved in the gas phase or in the liquid phase, but in the present invention, the graft reaction can be performed uniformly. A method of contacting in a liquid phase is a preferable method. In order to further promote the graft reaction more uniformly, it is desirable that the hydrophilic monomer and the crosslinking agent are dissolved in a solvent in advance and then contacted with the fluoropolymer film. The solvent for dissolving the hydrophilic monomer and the crosslinking agent is not particularly limited as long as it can be uniformly dissolved. Examples of such solvents include alcohols such as ethanol, isopropanol, and t-butyl alcohol, ethers such as diethyl ether and tetrahydrofuran, ketones such as acetone and 2-butanone, water, and mixtures thereof. .

In the graft polymerization according to the present invention, a reaction solution of 0.3% by volume to 30% by volume in a combined concentration of the hydrophilic monomer and the crosslinking agent is used, and 10 × 10 ^{−5 to} 100 × 100 g per 1 g of the fluoropolymer film. It is desirable to carry out the reaction at a rate of × 10 ⁻⁵ m ³ . If graft polymerization is performed within this range, pores are not filled with the hydrophilic layer, and a film having excellent uniformity can be obtained.
The reaction temperature at the time of graft polymerization according to the present invention is not particularly limited as long as the polymerization reaction occurs, but it is generally carried out at 20 ° C. to 80 ° C.
In the graft polymerization according to the present invention, the hydrophilic monomer may be in any state of gas, liquid or solution when contacting the fluoropolymer film and the hydrophilic monomer, in order to form a uniform hydrophilic layer. Is preferably a liquid or a solution, particularly preferably a solution.

  The fluoropolymer membrane according to the present invention achieves a high level separation of target biocomponents and impurity biocomponents by introducing a hydrophilic layer having a strong cross-linking structure into a hydrophobic fluoropolymer membrane. be able to. Therefore, it is preferable to perform copolymerization using a crosslinking agent in a proportion of 20 mol% or more and 1,000 mol% or less with respect to the hydrophilic monomer, and preferably, the crosslinking agent is used in an amount of 20 mol% or more and 500 mol% with respect to the hydrophilic monomer. %, And more preferably, the crosslinking agent is used in a proportion of 30 mol% or more and 200 mol% or less with respect to the hydrophilic monomer.

In the present invention, a hydrophilic layer is introduced into a fluorine-based polymer membrane to achieve a high separation performance between a target biological component and an impurity biological component. Therefore, the graft ratio grafted onto the fluoropolymer film is preferably 3% or more and 50% or less, more preferably 4% or more and 30% or less, and most preferably 5% or more and 20% or less. When the graft ratio is less than 3%, the hydrophilicity of the membrane is insufficient, causing a rapid decrease in the filtration rate accompanying protein adsorption. If it exceeds 50%, relatively small pores are filled with the hydrophilic layer, and a sufficient filtration rate cannot be obtained. Here, the graft ratio is a value defined by the following formula (8).
Graft rate (%) =
(Membrane weight after grafting−membrane weight before grafting) / membrane weight before grafting × 100 (8)

The degree of hydrophilicity of the fluoropolymer film according to the present invention can be evaluated by the contact angle. The average value of the advancing contact angle and the receding contact angle at 25 ° C. is preferably 60 ° or less, more preferably 45 ° or less, and further preferably 30 ° or less. Further, as a simple evaluation method, it may be determined that when the fluorine-based polymer membrane is brought into contact with water, water sufficiently permeates into the pores of the membrane so that it has sufficient hydrophilicity.

The olefin polymer film according to the present invention is mainly composed of an olefin polymer, but may contain other high molecular weight substances and additives as long as the characteristics of the olefin polymer are not impaired. It is also possible to carry out by combining two or more of these polymers.
The olefin polymer according to the present invention is a polymer synthesized using olefins or alkenes as monomers, and examples thereof include polyethylene, polypropylene, and poly-4-methyl 1-pentene. Furthermore, the said homopolymer and the said copolymer can also be mixed and used. Among these, polyethylene is preferable.

The weight average molecular weight of the olefin polymer according to the present invention is preferably 50,000 to 5,000,000, more preferably 100,000 to 2,000,000, and further preferably 150,000 to 1,000,000.
In general, a resin having an average molecular weight exceeding 1,000,000 is difficult to measure by gel permeation chromatography (GPC). Therefore, a viscosity average molecular weight by a viscosity method can be applied as a substitute. If the average molecular weight is less than 50,000, the melt tension at the time of melt molding becomes small and the moldability is deteriorated, and the mechanical strength of the film is lowered. An average molecular weight exceeding 5 million is not preferable because uniform melt-kneading becomes difficult.

  In order to prevent clogging due to the adsorption of biological components, it is necessary to impart hydrophilicity to the membrane. Examples of the hydrophilization treatment include a method in which an olefin polymer film is immersed in a solution containing a surfactant and then dried to leave the surfactant in the olefin polymer film. A method of grafting a hydrophilic (meth) acrylic monomer or the like onto the pore surface of an olefin polymer film by irradiating the above-mentioned radiation or using a peroxide. And a method of immersing the olefin polymer film in a solution containing the hydrophilic polymer and then drying to form a hydrophilic polymer film on the pore surface of the olefin polymer film. Graft polymerization is most preferable in view of the persistence of the formation and the possibility of leakage of hydrophilic additives. In particular, the hydrophilic treatment by the radiation graft polymerization method disclosed in JP-A-62-2179540, JP-A-62-258711, and U.S. Pat. It is preferable in that a uniform hydrophilic layer can be formed on the surface of the pores.

The hydrophilic monomer used in the graft polymerization of the present invention is not particularly limited as long as it is a hydrophilic monomer having a vinyl group. A monomer having one vinyl group is preferable. Furthermore, a (meth) acrylic monomer containing a sulfone group, an ester group, an amide group, a hydroxyl group, a sulfonyl group, a urethane group or the like can be suitably used. The hydrophilic monomer according to the present invention is a monomer that dissolves uniformly when mixed with 1% by volume of pure water at 25 ° C. under atmospheric pressure. Examples thereof include hydrophilic vinyl monomers having a hydroxyl group, a sulfonyl group, a sulfone group, an ester group, an amide group, and a urethane group. Among these, a vinyl monomer having one or more hydroxyl groups or a functional group serving as a precursor thereof is preferable. Specifically, (meth) acrylic acid and polyhydric alcohol esters such as hydroxypropyl acrylate and 2-hydroxyethyl methacrylate, alcohols having an unsaturated bond such as allyl alcohol, and vinyl acetate and vinyl propionate. Examples include enol esters.

Furthermore, hydrophilicity is sufficiently imparted by copolymerizing by a graft polymerization method using a hydrophilic vinyl monomer and a crosslinking agent having two or more vinyl groups.
The cross-linking agent used according to the present invention is a cross-linking agent having two or more vinyl groups that can be copolymerized with the hydrophilic monomer, and is introduced by contacting the membrane simultaneously with the hydrophilic monomer. The crosslinking agent preferably has a number average molecular weight of 200 or more and 2,000 or less, more preferably a number average molecular weight of 250 or more and 1,000 or less, and most preferably a number average molecular weight of 300 or more and 600 or less. When the number average molecular weight of the crosslinking agent is 200 or more and 2,000 or less, a high filtration rate of the biological component solution is obtained, which is preferable. In the present invention, any crosslinking agent can be used as long as it has two or more vinyl groups, but a hydrophilic crosslinking agent is preferred.
Here, the hydrophilic crosslinking agent is a crosslinking agent that is uniformly dissolved when mixed with 1% by volume of pure water at 25 ° C. under atmospheric pressure.

Specific examples of the crosslinking agent used in the present invention include divinylbenzene derivatives in aromatic systems,
Examples of the aliphatic type include methacrylic acid-based crosslinking agents such as ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate, and (meth) acrylic acid-based crosslinking agents such as ethylene glycol diacrylate and polyethylene glycol diacrylate. . A cross-linking agent having three reactive groups such as trimethylolpropane trimethacrylate can also be used. In addition, a mixture of two or more kinds of crosslinking agents can be used. In the present invention, it is most preferable to use polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, or a mixture thereof from the viewpoint of the permeability of the target biological component and the removal performance of the impurity biological component.

  The graft polymerization method according to the present invention is not limited as long as it is a method for generating radicals. For example, radicals are generated in an olefin polymer film by means of addition of a radiation initiator, ionizing radiation, chemical reaction, or the like. Then, starting from the radical, the monomer is graft-polymerized to the film. In the present invention, any means can be employed for generating radicals in the olefin polymer film, but irradiation with ionizing radiation is preferable in order to generate uniform radicals throughout the film. As types of ionizing radiation, γ rays, electron beams, β rays, neutron rays, and the like can be used, but electron beams or γ rays are most preferable for implementation on an industrial scale. The ionizing radiation is obtained from radioactive isotopes such as cobalt 60, strontium 90, and cesium 137, or by an X-ray imaging apparatus, an electron beam accelerator, an ultraviolet irradiation apparatus, or the like.

  The irradiation dose of ionizing radiation according to the present invention is preferably from 1 kGy to 1,000 kGy. If it is less than 1 kGy, radicals are not generated uniformly, and if it exceeds 1,000 kGy, film strength may be reduced. Graft polymerization is generally divided into two types: a pre-irradiation method in which radicals are generated on a film and then contacted with a reactive compound, and a simultaneous irradiation method in which radicals are generated on the film in a state where the film is in contact with the reactive compound. Is done. In the present invention, any method can be applied, but the pre-irradiation method in which oligomer formation is small is most preferable.

  In the present invention, the contact between the olefin polymer film that has generated radicals, the hydrophilic monomer, and the cross-linking agent can be achieved in the gas phase or in the liquid phase, but in the present invention, the graft reaction can be performed uniformly. A method of contacting in a liquid phase is a preferable method. In order to further promote the graft reaction more uniformly, it is desirable that the hydrophilic monomer and the crosslinking agent are dissolved in a solvent in advance and then contacted with the olefin polymer film. The solvent for dissolving the hydrophilic monomer and the crosslinking agent is not particularly limited as long as it can be uniformly dissolved. Examples of such solvents include alcohols such as ethanol, isopropanol, and t-butyl alcohol, ethers such as diethyl ether and tetrahydrofuran, ketones such as acetone and 2-butanone, water, and mixtures thereof. .

The graft polymerization according to the present invention uses a reaction solution of 0.3% by volume to 30% by volume with a concentration of a hydrophilic monomer and a cross-linking agent, and 10 × 10 ^{−5 to} 100 with respect to 1 g of the olefin polymer membrane. It is desirable to carry out the reaction at a rate of × 10 ⁻⁵ m ³ . If graft polymerization is performed within this range, pores are not filled with the hydrophilic layer, and a film having excellent uniformity can be obtained.
The reaction temperature at the time of graft polymerization according to the present invention is not particularly limited as long as the polymerization reaction occurs, but it is generally carried out at 20 ° C. to 80 ° C.
In the graft polymerization according to the present invention, when the olefin polymer film and the hydrophilic monomer are brought into contact with each other, the hydrophilic monomer may be in any state of gas, liquid or solution, but forms a uniform hydrophilic layer. For this purpose, a liquid or a solution is preferable, and a solution is particularly preferable.

  The olefin polymer membrane according to the present invention achieves a high level separation of the target biological component and the impurity biological component by introducing a hydrophilic layer having a strong cross-linked structure into the hydrophobic olefin polymer membrane. be able to. Therefore, it is preferable to perform copolymerization using a crosslinking agent in a proportion of 20 mol% or more and 1,000 mol% or less with respect to the hydrophilic monomer, and preferably 20 mol% or more of the crosslinking agent with respect to the hydrophilic monomer, The crosslinking agent may be used in a proportion of 500 mol% or less, more preferably 30 mol% or more and 200 mol% or less with respect to the hydrophilic monomer.

The present invention introduces a hydrophilized layer into an olefin polymer membrane to achieve a high separation performance between the target biological component and the impurity biological component. Therefore, the graft ratio grafted on the olefin polymer film is preferably 3% or more and 50% or less, more preferably 4% or more and 30% or less, and most preferably 5% or more and 20% or less. When the graft ratio is less than 3%, the hydrophilicity of the membrane is insufficient, causing a rapid decrease in the filtration rate accompanying protein adsorption. If it exceeds 50%, relatively small pores are filled with the hydrophilic layer, and a sufficient filtration rate cannot be obtained. Here, the graft ratio is a value defined by the following formula (9).
Graft rate (%) =
(Membrane weight after grafting−membrane weight before grafting) / membrane weight before grafting × 100 (9)

  The degree of hydrophilicity of the olefin polymer film according to the present invention can be evaluated by the contact angle. The average value of the advancing contact angle and the receding contact angle at 25 ° C. is preferably 60 degrees or less, more preferably 45 degrees or less, and still more preferably 30 degrees or less. In addition, as a simple evaluation method, when the olefin polymer membrane is brought into contact with water, it may be determined that the membrane has sufficient hydrophilicity if water spontaneously permeates into the pores of the membrane.

The (meth) acrylic polymer film according to the present invention is not particularly limited, and examples of the (meth) acrylic polymer include those represented by the following formula (10).
[Chemical Formula 10]
^{(Wherein, R 17} and ^{R 18} represents an alkyl group or an aralkyl group having 1 to 14 carbon atoms.
The hydrogen atom of the alkyl group or the hydrogen atom of the aralkyl group may be substituted with an alkoxy group having 1 to 10 carbon atoms. In the formula, v and w represent repeating units. )
Among them, poly (meth) acrylic acid and poly (meth) acrylic acid ester can be used. Preferred are polyacrylic acid, polymethacrylic acid, polymethyl acrylate, polyethyl acrylate, polymethyl methacrylate, polyethyl methacrylate, and a copolymer of two or more thereof. Various modifications such as esterification, etherification, epoxidation, and sulfonylation can be performed on the polymer terminal and / or main chain as necessary.

The (meth) acrylic polymer film according to the present invention is mainly composed of poly (meth) acrylic acid ester, but other high molecular weight substances and additives are added within the range that does not impair the properties of poly (meth) acrylic acid ester. You may contain the thing. It is also possible to carry out by combining two or more of these polymers.
As the weight average molecular weight of the (meth) acrylic polymer according to the present invention, 5,000 to 1,000,000 can be used. Preferably, it is 10,000-500,000, More preferably, it is the range of 20,000-300,000. Within this range, sufficient strength and film formability can be obtained.

  In the present invention, it is preferable to use a hydrophilic polymer together with the (meth) acrylic polymer in order to control the pore size of the membrane and to impart hydrophilicity. Examples of the hydrophilic polymer include polyvinyl pyrrolidone, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyethylene glycol-polypropylene glycol block copolymer, polyvinyl alcohol, poly (meth) acrylamide, poly-N, N-dimethyl (meth) acrylamide, Examples thereof include poly-N-isopropyl (meth) acrylamide, polyhydroxy acrylate, polyhydroxy methacrylate, carboxymethyl cellulose, starch, corn starch, polychitosan, and polychitin. Among them, polyvinyl pyrrolidone is particularly preferable for improving the hydrophilicity of the entire film because of its good compatibility with (meth) acrylic polymers.

  The weight average molecular weight of the hydrophilic polymer that imparts hydrophilicity to the (meth) acrylic polymer film according to the present invention may be 1,000 to 2,000,000. Preferably it is 5,000 to 1,500,000 or less, More preferably, it is the range of 5,000 to 1,200,000. For example, various grades of polyvinyl pyrrolidone are commercially available from BASF and have a weight average molecular weight of 9,000 (K17), and similarly 45,000 (K30), 450,000 (K60), 900,000. (K80) and 1,200,000 (K90) are preferably used, and in order to obtain the intended use, characteristics, and structure, each may be used alone or in combination of two or more. .

The (meth) acrylonitrile film according to the present invention is not particularly limited, and examples of the (meth) acrylonitrile polymer include those represented by the following formula (11).
[Chemical 11]
(In the formula, R ¹⁹ and R ²⁰ represent hydrogen or a methyl group. R ²¹ represents an alkyl group or an aralkyl group having 1 to 14 carbon atoms. The hydrogen atom of the alkyl group or the hydrogen atom of the aralkyl group represents a carbon number. (It may be substituted by an alkoxy group of 1 to 10. In the formula, x and y represent repeating units.)
Among these, poly (meth) acrylonitrile, poly (meth) acrylonitrile acid ester, and the like can be used. Polyacrylonitrile and polymethacrylonitrile are preferable.

The monomer composition constituting the (meth) acrylonitrile polymer according to the present invention has a (meth) acrylonitrile content of at least 50% by weight, preferably 60% to 100% by weight.
The content of one or more vinyl compounds copolymerizable with (meth) acrylonitrile is 50% by weight or less, preferably 0% by weight to 40% by weight. The vinyl compound is not particularly limited as long as it is a known compound having copolymerizability with (meth) acrylonitrile, and preferred copolymer components include methyl (meth) acrylate and (meth) acrylic acid. Examples include ethyl, vinyl acetate, sodium (meth) acrylic sulfonate, p (para) -styrene sulfonic acid soda, hydroxyethyl methacrylate, ethyl triethylammonium methacrylate, ethyl trimethylammonium methacrylate, and vinylpyrrolidone. Can do. Examples thereof include acrylonitrile-methyl acrylate-PVP copolymer.
Various modifications such as esterification, etherification, epoxidation, and sulfonylation can be performed on the polymer terminal and / or main chain as necessary.

The (meth) acrylonitrile-based polymer film according to the present invention is mainly composed of a (meth) acrylonitrile-based polymer, but other high molecular weight substances and additives are added within the range not impairing the properties of the (meth) acrylonitrile-based polymer. You may contain the thing. It is also possible to carry out by combining two or more of these polymers.
The weight average molecular weight of the (meth) acrylonitrile polymer according to the present invention may be 5,000 to 1,000,000. Preferably, it is 10,000-500,000, More preferably, it is the range of 20,000-300,000. Within this range, sufficient strength and film formability can be obtained.

In the present invention, it is preferable to use a hydrophilic polymer together with the (meth) acrylonitrile polymer in order to control the pore size of the membrane and to impart hydrophilicity. Examples of hydrophilic polymers include polyvinyl pyrrolidone, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyethylene glycol-polypropylene glycol block copolymer, polyvinyl alcohol, poly (meth) acrylamide, poly-N, N-dimethyl (meth) acrylamide, Examples thereof include poly-N-isopropyl (meth) acrylamide, polyhydroxy acrylate, polyhydroxy methacrylate, carboxymethyl cellulose, starch, corn starch, polychitosan, and polychitin.
Among them, polyvinylpyrrolidone is particularly preferable in terms of improving compatibility with the (meth) acrylonitrile polymer and enhancing the hydrophilicity of the entire film.

  The weight average molecular weight of the hydrophilic polymer imparting hydrophilicity to the (meth) acrylonitrile-based polymer film according to the present invention can be 1,000 to 2,000,000. Preferably it is 5,000 to 1,500,000 or less, More preferably, it is the range of 5,000 to 1,200,000. For example, various grades of polyvinyl pyrrolidone are commercially available from BASF and have a weight average molecular weight of 9,000 (K17), and similarly 45,000 (K30), 450,000 (K60), 900,000. (K80) and 1,200,000 (K90) are preferably used, and in order to obtain the intended use, characteristics, and structure, each may be used alone or in combination of two or more. .

In the purification of polypeptides, the affinity chromatography purification step is an essential step. In particular, affinity chromatography using protein A or the like as a ligand is used. The separation principle is based on the specific interaction between the immunoglobulin and other biological components for the ligand. Specifically, biological components including immunoglobulins, contaminating proteins, sugar chains, and nucleic acids are passed through affinity chromatography to specifically bind only to the immobilized immunoglobulin. The specifically bound immunoglobulin is removed from the immobilized ligand using a low pH, high pH, high salt, competitive ligand, etc., and recovered to obtain a purified immunoglobulin.
However, the problem with the affinity chromatography purification step is that it requires contact with a low pH or a high salt in order to elute the immunoglobulin from the ligand. Aggregates are by-produced, and the ligand itself (for example, protein A) is eluted to form a complex aggregate with the immunoglobulin.
Then, by performing the separation method according to the present invention, the aggregate and the complex can be removed, and the immunoglobulin can be sufficiently purified.

  Protein A according to the present invention includes protein A recovered from natural sources, synthetically produced protein A (eg, by peptide synthesis or by recombinant techniques), and CH2 / CH3 regions (eg, And variants thereof that retain the ability to bind to proteins having the Fc region). Protein A can be purchased commercially from Repligen, Pharmacia and Fermate. Protein A is generally immobilized as a ligand on a solid support material. Furthermore, protein A column refers to an affinity chromatography resin or column containing a chromatographic solid support matrix to which protein A is covalently bonded. Examples of affinity chromatography ligands other than protein A include protein L and protein G. These protein L and protein G are also those recovered from natural sources, synthetically produced (eg, by peptide synthesis or recombinant techniques), and CH2 / CH3 regions (eg, Fc And variants thereof that retain the ability to bind to a protein having a region. Furthermore, an affinity chromatography purification method using an affinity chromatography resin or column prepared by combining protein A, protein L and protein G is also included.

The thickness of the ultrafiltration membrane of the present invention is preferably 15 to 2,000 μm, more preferably 15 to 1,000 μm, and most preferably 20 to 500 μm. If the film thickness is less than 15 μm, the strength of the ultrafiltration membrane tends to be insufficient, such being undesirable. On the other hand, if it exceeds 2,000 μm, the permeation performance of the target biological component tends to be insufficient, which is not preferable.
The structure of the ultrafiltration membrane according to the present invention is not particularly limited as long as the fractionation performance can be expressed. However, in order to satisfy the high fractionation performance and the high throughput of the target biological component, a hollow fiber or a flat membrane is used. An inclined structure having a dense layer on one surface, both surfaces, or the inner surface is preferable.

The structure of the dense layer of the ultrafiltration membrane according to the present invention is not particularly limited as long as the fractionation performance can be expressed, but in order to satisfy the high fractionation performance and the high throughput of the target biological component, The diameter of the polymer particles forming the layer is preferably 100 nm or less, more preferably 60 nm or less, and particularly preferably 30 nm or less. In the case of an ultrafiltration membrane using phase separation, a large number of polymer particles are formed at the initial stage of phase separation, and the polymer particles grow and connect with the progress of phase separation to form a dense layer. The polymer particles referred to here are polymer pseudo-particles that are grown and connected.
When the ultrafiltration membrane according to the present invention has a dense layer on the hollow fiber, flat membrane surface, or inner surface, the thickness of the dense layer is usually 100 μm in order to improve the permeation of the biological component solution. The thickness is preferably 10 μm or less, more preferably 1 μm or less, particularly preferably 0.5 μm or less, and most preferably 0.2 μm or less.

  The ultrafiltration membrane according to the present invention is highly selected on the membrane surface by treating under special conditions using a biological component solution containing a target biological component and a high molecular weight impurity biological component containing at least a dimer of the target biological component. A highly permeable cake layer is formed. The diameter of the polymer pseudoparticle plays an extremely important role in the performance of the cake layer.

The porosity of the ultrafiltration membrane according to the present invention is preferably 30 to 95%, more preferably 40 to 90%, and most preferably 50 to 85%. If the porosity is less than 30%, the filtration rate becomes insufficient, and if it exceeds 95%, the strength of the ultrafiltration membrane becomes insufficient. The porosity is a numerical value obtained from the apparent volume obtained from the cross-sectional area and length of the membrane, the weight of the membrane, and the true density of the membrane material.

The shape of the ultrafiltration membrane according to the present invention is not particularly limited as long as the fractionation performance can be expressed.For example, various shapes such as a hollow fiber shape, a flat membrane shape, and a tube shape can be used. A hollow fiber shape having a large filtration effective membrane area compared to the volume is effective.
The membrane surface structure of the ultrafiltration membrane according to the present invention is not particularly limited, and examples thereof include single holes such as a circle and an ellipse, continuous holes continuously connected, net-like fine holes, slit-like fine holes, and the like.

  The ultrafiltration membrane in the present invention may be an ultrafiltration membrane as long as the surface of the membrane to be contacted with an object to be separated. In order to maintain the structure, a substrate (support) made of any material can be used. Good. For example, a woven fabric, a nonwoven fabric, a porous inorganic material or the like is used as another base material (support) to increase physical strength, and a membrane obtained by molding an ultrafiltration membrane on these base materials.

In general, cross-flow filtration and dead-end filtration are performed as general-purpose filtration methods as filtration methods. Cross-flow filtration is a process in which a liquid to be treated containing fine particles is filtered while being supplied to a membrane to separate fine particles having different diameters. While the fine particles (gel layer) deposited on the membrane surface are scraped off by the shearing force generated by the parallel flow of the fine particle solution, the stable performance of the gel layer is maintained over a long period of time to maintain the separation performance. On the other hand, dead-end filtration
Since the protein solution is allowed to flow perpendicularly to the membrane surface, deposits accumulate on the membrane surface, the permeation resistance gradually increases with the filtration time, and the permeation protein concentration changes. Also called vertical filtration or normal filtration.

Cross-flow filtration according to the present invention is a method in which a protein solution is allowed to flow parallel to the membrane surface, and a material deposited on the membrane surface is pushed away by shearing force, thereby establishing a dynamic equilibrium and forming a constant protein concentration. This is a filtration system that enables operation. It is also called cross flow filtration, parallel filtration or tangential flow filtration.

The biocomponent concentration of the target biocomponent according to the present invention and at least the dimer of the target biocomponent is preferably 1 to 150 g / L, preferably 5 to 100 g / L, capable of expressing fractionation performance. More preferably, 5-50 g / L is good. The concentration ratio of the target biocomponent dimer to the target biocomponent (dimer concentration of target biocomponent / target biocomponent concentration) is usually 0.2 or less, preferably 0.1 or less.
The lower limit of the concentration of at least the target biocomponent dimer and the target biocomponent according to the present invention is 1 g / L or more, preferably 5 g / L or more. When the concentration is less than 1 g / L, the fractionation performance is rapidly reduced.
On the other hand, when the concentration is higher than 150 g / L, the transmissivity of the target biological component is clearly reduced due to membrane blockage.
Since a cake layer with extremely high permselectivity is formed only when the concentration of the dimer of the target biological component and the target biological component is at least in the special range described above, high fractionation performance and high throughput of the target biological component Is achieved. Therefore, the upper limit of the concentration is 150 g / L or less, preferably 100 g / L or less, and more preferably 50 g / L or less.

  The linear velocity according to the present invention is the velocity of the solution flowing parallel to the film surface. The linear velocity is not particularly limited as long as the fractionation performance can be expressed. For example, the linear velocity is 1 to 200 cm / second, preferably 10 to 100 cm / second. It is preferred to maintain the filtration flow rate at a level that exceeds the flow rate at the transition point. Further, it is particularly preferable to maintain the filtration flow rate at a level exceeding the flow rate at the transition point, and further maintain the filtration transmembrane pressure at a level exceeding the transmembrane pressure at the flow rate transition point.

The filtration pressure according to the present invention is not particularly limited as long as the fractionation performance can be expressed. However, in order to achieve a high throughput, the pressure exceeds the pressure at the transition point (TPM: transmembrane pressure). It is preferable to implement.
The transition point means a predetermined point on the flow rate vs. TMP curve obtained as follows. Experimental values of flow rate (Jf) vs. TMP are collected in short path length modules where the inlet and outlet TMP are ± 10% of each other or in full length modules where the same conditions are obtained using recirculated filtrate. The experimental value is applied to the curve obtained by the following equation (12).
Jf = Jmax × TMP / (k + TMP) (12)
(Where Jmax (asymptotic value) and k are determined by linear regression of 1 / Jf vs. 1 / TMP;
(This gives a slope of k / Jmax and an intercept of 1 / Jmax)
Next, the transition point is determined from the graph according to the following criteria. An intercept of a tangent line passing through the origin with respect to the curve obtained by Jf = Jmax and Expression (12) (tangent line is Jf = Jmax × TMP / k) is obtained. A straight line is then drawn through the intercept and perpendicular to the tangent on the curve. The transition point is defined by the intersection of this latter straight line and curve.
Mathematically, this transition point (Jf ^* ) is expressed by the following formula (13).
(Jmax−Jf ^* ) ⁴ = −Jmax × k ² (Jmax−2Jf ^* ) (13)

Patent Document 1 describes that in order to separate a target biological component and an impurity biological component having a molecular weight ratio of less than 10 times, it is essential to filter at a pressure equal to or lower than TMP. That is, there is a problem that the processing amount becomes small.
However, in the separation method of the present invention, the separation performance can be maintained even when a high molecular weight impurity biological component including a target biological component and at least a dimer of the target biological component is filtered at a pressure higher than TMP. The filtration rate, that is, the throughput is also large.

The concentration change of the biological component during the cross-flow filtration described in the present invention is not limited at all unless the membrane occlusion or the degradation of the fractionation performance occurs, but when the concentration of the target biological component before filtration is 100, It is preferable that it is 50-200, More preferably, it is 70-150, More preferably, it is 80-120. In particular, it is more preferable to perform cross flow filtration while maintaining the biological component concentration substantially constant. In order to achieve high fractionation performance and permeation performance in a short time, it is preferable to filter the biological component solution at a constant concentration (constant concentration filtration). Here, “while maintaining the concentration substantially constant” means “while maintaining the concentration with slight fluctuation”. For example, a slight concentration fluctuation that occurs when the concentration is controlled by an operation or an apparatus is included. However, when the remaining amount of the biological component solution decreases in the latter half of the filtration and the filtration becomes difficult, in order to recover the biological component remaining in the membrane or in the apparatus piping, a dilute solution, buffer solution, water, physiological saline When water is added and filtration is performed, it is not necessary to perform filtration at a constant concentration.

  The apparatus for performing separation of high molecular weight impurities biological components by crossflow filtration according to the present invention is not limited at all as long as it is a device that can control biological component concentration, linear velocity, pressure, etc. The concentration of the biological component solution is monitored with an absorptiometer, and the linear velocity with respect to the device for supplying the diluent and the ultrafiltration membrane to keep the concentration of the biological component solution constant and the pressure across the ultrafiltration membrane A cross flow filtration device in which a device for controlling the flow is integrated.

  Specifically, a cross flow filtration apparatus as shown in FIG. The concentration of the immunoglobulin solution in the biological component solution tank (4) is monitored by the concentration controller (11) incorporating the absorbance meter, and the signal is sent to the liquid feeding pump 1 (2) to control the rotation. The biological component concentration in the biological component solution tank (4) is controlled while adding the diluent in the diluted solution tank (1). Furthermore, pressure and flow rate are monitored by pressure / flow rate controller 1 (12) with pressure gauge 1 (5), pressure gauge 2 (6) and flow meter (10), and tangent to ultrafiltration membrane module (8). Control is performed by sending a signal to the liquid feed pump 2 (3) and the regulating valve (7) so that the linear velocity in the direction and the pressure across the ultrafiltration membrane become set values. A device capable of measuring the concentration of the permeate in the permeate tank (9) and the ratio of the target biocomponent and the high molecular weight impurity biocomponent, such as an absorptiometer or GPC, is connected to the crossflow filtration device. Also good.

  Further, a cross flow filtration device as shown in FIG. 2 may be used. A device capable of monitoring the concentration between the biological component solution tank (4) and the ultrafiltration membrane module (8), for example, a UV flow cell (13) is provided, and the biological component in the biological component solution tank (4) The concentration is monitored by the concentration controller 1 (11), the signal is sent to the liquid feed pump 1 (2) to control the rotation, and the biological component solution is added while adding the diluent in the dilution solution tank (1). The biological component concentration in the tank (4) is controlled. Furthermore, pressure and flow rate are monitored with pressure / flow rate controller 1 (12) by pressure gauge 1 (5), pressure gauge 2 (6) and flow meter (10), and tangent to ultrafiltration membrane module (8). Control is performed by sending a signal to the regulating valve (7) so that the linear velocity in the direction and the pressure across the ultrafiltration membrane become set values. A device capable of measuring the concentration of the permeate in the permeate tank (9) and the ratio of the target biocomponent and the high molecular weight impurity biocomponent, for example, an absorptiometer or GPC is connected to the crossflow filtration device. Also good.

  Usually, when the biological component solution is removed with an ultrafiltration membrane, the concentration of the obtained biological component solution in the permeate often decreases from the filtrate. For example, when a biological component solution of 1 to 150 g / L is filtered. The concentration of the permeated biological component solution is 0.1 to 150 g / L. Therefore, it is preferable to concentrate and separate the permeated biological component solution by crossflow filtration. The cross flow filtration conditions for concentrating / separating the permeated biological component solution are not particularly limited as long as the target biological component solution can be obtained. 01 cm / second or more, preferably 0.1 / second or more is preferable, and the upper limit is 100 cm / second or less, preferably 75 cm / second or less. The lower limit of the pressure is 0.01 MPa or more, preferably 0.02 MPa or more, and the upper limit is 0.5 MPa or less, preferably 0.3 MPa or less.

The present invention uses an ultrafiltration membrane having a molecular weight of a target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component. And a biological component solution containing a high molecular weight impurity biological component containing at least a dimer of the target biological component, wherein the biological component concentration is 1 to 150 g / L, A step of separating a high molecular weight impurity biological component including at least a dimer of a target biological component, and an ultrafiltration membrane for concentration / separation having a fractional molecular weight of 0.3 to 1.5 times that of the target biological component And using a biological component solution containing at least a target biological component and a low molecular weight impurity biological component and having a biological component concentration of 0.1 to 150 g / L by cross-flow filtration, It is preferred that is transmitted through the partial comprising the step of concentrating and separating the target biological component.
The separation step and the concentration / separation step may be performed in any order. Following the step of separating the high molecular weight impurity biological component from the biological component solution, a method of providing a step of permeating the low molecular weight impurity biological component and concentrating / separating the target biological component, or passing the low molecular weight impurity biological component from the biological component solution In addition, following the step of concentrating / separating the target biological component and the high molecular weight impurity biological component, a method of providing a step of permeating the target biological component and separating the high molecular weight impurity biological component may be employed.

  The concentration of the biological component solution after concentration / separation obtained in the concentration / separation step of the biological component solution according to the present invention depends on the type and use of the target biological component and the subsequent purification step (for example, the step of removing viruses). Set the concentration ratio according to the purpose. Usually, the lower limit is preferably 0.5 g / L or more, more preferably 1 g / L or more, and the upper limit is preferably 300 g / L or less, more preferably 150 g / L or less.

The concentration / separation step is a step of concentrating / separating the target biological component and low molecular weight impurity biological component solution obtained in the separation step or the target biological component and high molecular weight impurity biological component solution. A device including a device capable of monitoring the concentration of the biological component solution, and a device capable of controlling the liquid feeding speed and filtration pressure of the solution of the target biological component and the low molecular weight impurity biological component are preferable. Specifically, an apparatus as shown in FIG. 3 is illustrated.
In the concentration / separation step, the biological component permeate is fed to the ultrafiltration membrane module (24) for concentration / separation using the liquid feed pump 3 (15). At that time, the pressure gauge 3 (16), the pressure gauge 4 (17), and the flow meter 2 (22) monitor the filtration pressure and the filtration flow rate, and the pressure / flow rate controller 2 (18) sends a signal to the adjustment valve 2 (19). To adjust the tangential line speed and the pressure across the ultrafiltration membrane to the set values for the concentration / separation ultrafiltration membrane module (24). Further, the concentration of the biological component solution is monitored by the concentration controller 2 (14) and the absorbance meter (20), and the concentration / separation degree is adjusted by the concentration controller 2 (14) and the pressure / flow rate controller 2 (18).
The liquid that permeates the concentration / separation ultrafiltration membrane module (24) can be reused as a diluent, and is sent to the diluent tank (1). The concentration of the concentrated biological component solution is monitored with an absorbance meter (20), and the biological component solution is recirculated to the permeate tank (9) until the target concentration is reached. ) To change the flow path and collect the biological component concentrated / separated liquid in the biological component recovery liquid tank (21).

  The solvent or diluent used in the preparation of the biological component solution according to the present invention is not limited at all unless it causes denaturation or aggregation of the biological component. For example, PBS, physiological saline, N- [Tris ( Hydroxymethyl) methyl] glycine (Tricine), N, N-bis (2-hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1- Dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO) ), 2-amino-2-methyl-1-propanol (AMP), N-cyclohexyl- -Good buffer agents such as aminopropane sulfonic acid (CAPS) and piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), acetate, glycine, citrate, phosphate, veronal, borate And buffers such as succinate, tris (hydroxymethyl) aminomethane, and imidazole.

The concentration of the buffering agent according to the present invention is not limited as long as it does not cause denaturation or aggregation of biological components, but it is, for example, 1 mM to 1 M, preferably 10 mM to 500 mM, more preferably 50 mM to 200 mM. .
The pH of the buffer according to the present invention is not limited as long as it does not cause denaturation or aggregation of biological components. For example, pH 3 to pH 10, preferably pH 4 to pH 9, and more preferably pH 5 to pH 8 is good. .

  The additive to be added to the solvent or diluent used in the preparation of the biological component solution according to the present invention is not limited as long as it does not cause denaturation or aggregation of the biological component. For example, inorganic salts and surfactants Amino acids, stabilizers, preservatives and the like.

Examples of inorganic salts according to the present invention include sodium chloride, potassium chloride, magnesium chloride,
Examples include calcium chloride and magnesium sulfate. As its concentration, for example, 1 mM
˜1M, preferably 10 mM to 500 mM, more preferably 50 mM to 200 mM.

The surfactant according to the present invention is not limited as long as it does not affect the fractionation performance. For example, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene lauryl ether [for example, Emulgen 120: Manufactured by Kao Corporation]
Polyoxyethylene alkyl phenyl ether [eg, polyoxyethylene octyl phenyl ether (eg, Triton X-100: manufactured by Rohm and Haas), polyoxyethylene isooctyl phenyl ether, polyoxyethylene nonyl phenyl ether, etc.], polyethylene glycol Nonionic surfactants such as monolaurate, cationic surfactants such as stearyltrimethylammonium chloride and alkylbenzyldimethyl, anionic surfactants such as cholic acid, deoxycholic acid, sodium polyoxyethylene alkylphenol ether sulfate, stearyl Examples include betaine and betaine type surfactants such as 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine. The concentration is 0.001% by weight to 1% by weight, although it depends on the type of surfactant.

The amino acid according to the present invention is not limited as long as it does not affect the fractionation performance. For example, lysine, arginine, cysteine, alanine, glycine, serine, proline, etc., and salts such as hydrochlorides of these amino acids Is mentioned. The concentration is 0.01 wt% to 20 wt%, although it depends on the type of amino acid.

Examples of the stabilizer according to the present invention include glucose, sorbitol, sucrose, glycerol,
Examples include amino acids such as polyethylene glycol and arginine. The concentration is preferably 1 to 50% by weight.

The preservative according to the present invention is not limited as long as it does not affect the fractionation performance,
For example, sodium azide etc. are mentioned. The concentration is preferably 0.001% by weight to 1% by weight.

Since the separation method according to the present invention is a method of separation by size fractionation, purification of synthetic pharmaceuticals and refinement of various beverages such as sake, beer, wine, happoshu, tea, oolong tea, vegetable juice, fruit juice, It can also be used for applications such as separation of fine particles from treated water, separation for oil-water separation and liquid gas separation, separation for purification of water and sewage.

The separation membrane module in the present invention is, for example, a flat membrane or a hollow fiber membrane accommodated in a casing, and is separated from one or more liquid inlets into which a liquid containing at least a separation target is poured into the casing. Is provided with at least one liquid outlet for deriving. The casing used for the module is assembled from one or more casing parts.
The material of the casing component can be selected as necessary, such as metal, glass, thermoplastic resin, thermosetting resin, and the like. A suitable material is a thermoplastic resin material having transparency in which the inside can be observed. Specifically, polymethyl methacrylate, polystyrene, hard vinyl chloride resin, polyethylene terephthalate, polypropylene, polystyrene butadiene copolymer, polycarbonate. And polymethyl methacrylate. Particularly preferred are transparent amorphous resins, such as polystyrene butadiene copolymer, polycarbonate, and polymethyl methacrylate.

The method of manufacturing the casing parts used when assembling the casing used for the module according to the present invention is not limited as long as the molding process is possible. For example, welding, press molding, injection molding, reaction injection molding, super Examples include sonic pressure bonding, plasma fusion, and adhesion using an adhesive. These may be used alone or in combination of two or more. A particularly preferable method for producing a casing component is a method of sealing with an injection molded product using a thermoplastic resin having transparency as a material and an appropriate adhesive.

Casings and / or casing parts used in the modules according to the invention are surfaces which are in contact with and / or not in contact with the liquid to be separated during molding and / or after molding and / or during assembly and / or after assembly. Surface processing can be performed. There are various methods for surface treatment. For example, when hydrophilizing, hydrophilic polymer coating or surface oxidation by plasma treatment in air, etc., when hydrophobizing, water repellent and / or mold release When the agent is applied and oxygen permeation is reduced, various inorganic coats including a silicon oxide film can be applied by vapor deposition or the like. Adhesive control at the interface of the same and / or different materials is facilitated during module assembly by performing hydrophilic treatment on the casing and / or casing material, and various types of temporary use during assembly by performing hydrophobic treatment The peelability from the protective film or the like can be improved.

The structure of the module according to the present invention differs depending on the shape of the membrane used and the separation object in the liquid. For example, if the separation object is a substance that can be separated in size, the module structure can be separated. The liquid is appropriately accommodated in the casing so that the liquid to be circulated only through one or a plurality of flat membranes, and has a structure having a liquid inflow port upstream of the liquid flow path and a liquid outflow port downstream. Further, a holding material intended to hold a flat membrane such as a metal mesh or a non-woven fabric and a flat membrane can be combined and accommodated in a casing to be modularized. Furthermore, the flat membrane can be folded and accommodated in a pleated shape in order to accommodate a large membrane area.

  The method for producing the ultrafiltration membrane according to the present invention is not limited in any way, and examples thereof include a melt film forming method and a wet film forming method. The melt film-forming method is a method in which a film material and a plasticizer are uniformly mixed by heating and then phase separation is generated by cooling, and a film is obtained by extracting the plasticizer from the obtained film film. It is. In addition, the wet film formation method involves contacting a membrane stock solution in which a membrane material is dissolved in a good solvent with a coagulation liquid composed of another solvent that is miscible with the good solvent in the membrane stock solution but is incompatible with the membrane material. In this method, concentration-induced phase separation is generated from the contact surface to obtain a membrane.

The method for producing the ultrafiltration membrane according to the present invention is not limited as long as an ultrafiltration membrane having the desired fractionation performance can be produced. As an example, a wet film forming method of a sulfone polymer film will be described.
As a method for producing a sulfone polymer film, for example, a wet film forming method can be mentioned. The wet film formation method is to bring a membrane stock solution in which a membrane material is dissolved in a good solvent into contact with a coagulation liquid composed of another solvent that is miscible with the good solvent in the membrane stock solution but is incompatible with the membrane material. In this method, concentration-induced phase separation is generated from the contact surface to obtain a membrane.

  The good solvent used in the wet film-forming method according to the present invention is preferably a solvent capable of dissolving 10 g or more in 100 g pure water at 20 ° C. and dissolving 5% by weight or more of the sulfone polymer of the film material. Is not limited as long as it is miscible with water, and specific examples include N-methyl-2-pyrrolidone, N, N-dimethylacetamide, dimethyl sulfoxide, N, N-dimethylformamide, and γ-butyrolactone. It is done. These can be used in combination of two or more.

  The membrane stock solution used in the wet film-forming method for obtaining the ultrafiltration membrane according to the present invention is not limited at all as long as it can produce a sulfone polymer membrane having the desired structure and performance. %, The lower limit of the concentration range of the sulfone polymer is 1% by weight or more, preferably 2% by weight or more, and particularly preferably 3% by weight or more. Moreover, as an upper limit, the solution which melt | dissolved uniformly in 45 weight% or less, Preferably it is 35 weight% or less, Most preferably, it is 25 weight% or less is used suitably. The hydrophilic polymer has a lower limit of 0.1% by weight or more, preferably 0.5% by weight or more, and an upper limit of 20% by weight or less, preferably 10% by weight or less, and a uniformly dissolved solution is preferably used. . In addition, the lower limit of the temperature of the membrane stock solution is preferably 0 ° C. or higher, preferably 10 ° C. or higher, particularly preferably 25 ° C. or higher, and the upper limit is a good solvent boiling point or lower in the membrane stock solution. If it is this temperature condition, the viscosity suitable for processing into a film | membrane preferable as a film | membrane stock solution can be obtained.

  The coagulation liquid used in the wet film formation method for obtaining the sulfone polymer film according to the present invention is any substance that can cause concentration-induced phase separation when in contact with the membrane stock solution and can form a film from the contact surface. Although it does not limit at all, For example, a pure water, a monoalcohol solvent, a polyol solvent, or these 2 or more types of mixed liquids are used suitably. Examples of the monoalcohol solvent include methanol, ethanol, propanol and the like. Examples of the polyol solvent include ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, glycerin, propylene glycol, 1,2-butanediol, 1,4-butanediol, and the like. Polyvinyl alcohol, polyethylene glycol, polypropylene glycol, polyethylene oxide, polyethylene glycol-polypropylene glycol block copolymer, polyacrylamide, polyvinyl pyrrolidone, polyhydroxy acrylate, polyhydroxy methacrylate, polyacrylic acid, polymethacrylic acid, polyitaconic acid in the coagulation liquid Water-soluble polymers such as polyfumaric acid, polycitraconic acid, poly-p-styrene sulfonic acid, poly-p-styrene sulfonic acid soda, N, N-dimethylacrylamide, carboxymethyl cellulose, starch, corn starch, polychitosan, polychitin It is also possible to add. Although it depends on the molecular weight and the amount of the water-soluble polymer to be added, the filtration performance can be improved by adding these.

The coagulation liquid may contain a good solvent such as N-methyl-2-pyrrolidone, N, N-dimethylacetamide, dimethyl sulfoxide, N, N-dimethylformamide, and γ-butyrolactone. In particular, when using a coagulation liquid containing a good solvent in a non-solvent, the composition differs depending on the composition of the membrane stock solution, the contact temperature between the membrane stock solution and the coagulation solution, etc. In this case, 90% by weight or less is preferable as the weight% of the good solvent. Within this range, concentration-induced phase separation necessary and sufficient for forming a film can be sufficiently achieved.

The film forming temperature in the wet film forming method according to the present invention is not limited as long as it is a temperature at which the film stock solution and the coagulating liquid are brought into contact with each other to cause concentration induced phase separation, but the lower limit of the film forming temperature is 0. ℃
Above, preferably 10 ° C. or higher, particularly preferably 25 ° C. or higher. The upper limit is a temperature below each boiling point of the membrane stock solution or coagulation liquid, preferably a temperature lower by 5 ° C. or more from each boiling point, particularly preferably a temperature lower by 10 ° C. or more from the boiling point. In the case of a hollow fiber membrane, it depends on the temperature of the double nozzle. In a flat membrane, it is determined by the coagulation liquid temperature.

  The membrane stock solution used in the wet film formation method for obtaining the sulfone polymer membrane according to the present invention, the coagulation solution, particularly the coagulation solution that passes through the inside of the yarn during the production of the hollow fiber membrane (hereinafter referred to as the internal coagulation solution) is dissolved after uniform dissolution. It is desirable to remove the gas. By removing the dissolved gas, film defects due to foaming of the dissolved gas can be remarkably improved. Further, by removing oxygen from the dissolved gas, the oxidation reaction to the material due to film processing at a high temperature is reduced. This step may be omitted when no gas is dissolved in the membrane stock solution, the coagulation solution, and the internal coagulation solution.

When producing a hollow fiber membrane by the wet film-forming method according to the present invention, in order to further promote the coagulation by the membrane stock solution and the internal coagulation solution discharged from the double spinning nozzle, It can be provided and brought into contact with a coagulation liquid (hereinafter referred to as an external coagulation liquid) filled in the coagulation tank.
When producing a hollow fiber membrane by the wet film-forming method of the present invention, the cross-sectional structure of the hollow fiber membrane is not only a uniform structure but also various non-uniform structures. And the temperature and humidity of the space from the spinneret to the external coagulation liquid surface can be adjusted. Although there is no limitation as long as the temperature and humidity of the space can be adjusted, for example, the lower limit of the free running distance is 0.001 m, preferably 0.005 m, particularly preferably 0.01 m or more,
The upper limit is 2.0 m, preferably 1.5 m, particularly preferably 1.2 m or less. The temperature in the space from the spinneret to the external coagulation liquid surface is 10 ° C. or higher, preferably 20 ° C. or higher, particularly preferably 25 ° C. or higher as the lower limit. The humidity varies depending on the temperature, but the lower limit is 0%, preferably 10%, particularly preferably 30% or more, and the upper limit is 100% or less.

The winding speed in the case of producing a hollow fiber membrane by the wet film-forming method according to the present invention is as follows: various factors as production conditions, the shape of the nozzle, the composition of the spinning stock solution, the composition of the internal and external coagulating solutions, and the stock solution Although the speed may vary depending on the temperature of each coagulating liquid, a speed of approximately 300 m / hour to 9,000 m / hour is selected.
In the wet film-forming method according to the present invention, after the solidification by the coagulating liquid, the solvent removal can be promoted by immersing in a solvent removal tank in order to increase the strength of the film. The solvent removal liquid is a solvent that can remove the remaining solvent after concentration-induced phase separation by the coagulation liquid, and any solvent that does not dissolve the membrane can be used. In general, water, ethanol or the like is often used.

The undried ultrafiltration membrane of the present invention obtained by the wet film formation method is not limited at all as long as the temperature does not cause membrane breakage during drying. Dry within a temperature range below the temperature. Preferable drying temperature is 30 ° C. or higher, 150 ° C. or lower,
More preferably, it is 50 degreeC or more and 140 degrees C or less. The time required for drying is determined by the relationship with the drying temperature, but is generally selected from 0.01 hours to 48 hours.

The permeation rate according to the present invention is the target biological component amount or impurity biological component amount contained in the target biological component and impurity biological component mixed solution before filtration, and the target biological component that has permeated through the ultrafiltration membrane and emerged into the filtrate. It is calculated from the amount or the amount of impurities biological component.
When the target biological component is used as a medicine, health food, or cosmetic, it is better to remove the impurity biological component as much as possible due to problems such as side effects. Therefore, the transmittance according to the present invention is preferably 80% or more, preferably 85% or more, more preferably 90% or more. Further, the transmittance ratio between the target biological component and the impurity biological component is preferably 0.20% or more, preferably 0.15% or more, more preferably 0.1% or more. The lower the transmittance ratio, the lower the transmittance of one biological component, the higher the other transmittance, and the higher the fractionation performance.

Examples of the method for measuring the transmittance according to the present invention include a method of calculating from results such as high performance liquid chromatography, nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, etc. However, the present invention is not limited to these.

The present invention will now be described by way of examples and comparative examples, but is not limited thereto.

[Production example]
<Method for producing hollow fiber membrane (PSf-1)>
1650 g of N, N-dimethylacetamide (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter abbreviated as DMAc), 280 g of polysulfone (P1700, manufactured by UCC, hereinafter abbreviated as PSf) and 110 g of polyvinylpyrrolidone (K-90, BASF (hereinafter abbreviated as PVP) was added and poured into a 5,000 × 10 ⁻⁶ m ³ reactor for a membrane stock solution. The pressure reduction and nitrogen substitution were repeated 5 times while stirring the reactor. Thereafter, the temperature of the solution in the reactor was raised to 60 ° C. to obtain a uniform PSf DMAc solution. After confirming uniform dissolution, stirring was stopped at this stage, and degassing was performed under reduced pressure. Thereafter, the pressure was returned to the same pressure as the atmospheric pressure, and a spinning membrane stock solution maintained at 60 ° C. was obtained.
480 g of pure water was mixed with 520 g of DMAc and added to a 3,000 × 10 ⁻⁶ m ³ reactor for the internal coagulation liquid. Depressurization and nitrogen substitution were repeated 5 times to obtain an internal coagulating liquid.
The internal coagulating liquid and the membrane stock solution were passed through a double spinning nozzle (inner diameter 100 μm, slit width 50 μm, outer diameter 300 μm) maintained at 60 ° C. Each flow rate was appropriately adjusted according to the winding speed during spinning.
The obtained hollow fiber membrane had an idling distance of 0.6 m and an external coagulation liquid (
After being solidified by being guided into the pure water), it was wound up with a winder. The winding speed was 2,400 m / hour to 4,800 m / hour.
Thereafter, the obtained hollow fiber membrane was repeatedly immersed and washed with pure water at 60 ° C., and thereafter 70
It dried for 6 hours with the hot air dryer of degreeC. By this production method, a sulfone polymer membrane having a molecular weight cut-off of 360 kDa, an inner diameter of 179 μm, and a film thickness of 39 μm could be produced.
An electron micrograph of the inner dense layer of the hollow fiber membrane is shown in FIG. The diameter of the polymer particles forming the dense layer is about 30 nm.

<Method for producing hollow fiber membrane (PSf-2, PSf-3)>
Each hollow fiber membrane (PSf-2, PSf-3) is subjected to the same conditions as in the production method of (PSf-1) except that the composition of the internal coagulation solution (pure water / DMAc) and the polysulfone concentration in the membrane stock solution are changed. Produced. The results are shown in Table 2.

[Method for Measuring CHOP (Chinese Hamster Ovary Cell Protein) Concentration]
The concentration of CHOP was measured by enzyme-linked immunosorbent assay (ELISA) using goat anti- (host cell protein) antibody. The entire purified goat anti-CHOP antibody was immobilized in a well of a microtiter plate. A dilution of the biocomponent solution containing CHOP was incubated in the wells, followed by incubation with the whole anti-CHOP conjugated with peroxidase. The horseradish peroxidase was then quantified by reading the absorbance at 492 nm using o-phenylenediamine. Based on the principle of sandwich ELISA, the concentration of peroxidase corresponded to the concentration of CHOP. The measurement range of ELISA was 5 ng / mL to 320 ng / mL. Depending on the concentration of the biological component solution, it was diluted and measured.

[Measurement method of endotoxin concentration]
Since endotoxin is prone to adsorption and inactivation, the endotoxin concentration was measured immediately after sampling. 200 μL of the sample was placed in a limlith reagent for dialysis solution (for 0.2 mL of dialysis rimlith reagent manufactured by Wako Pure Chemical Industries, Ltd.), and vortexed after 3 seconds. The gelation time of the Limlith reagent was measured with a toxinometer (ET-301 manufactured by Wako Pure Chemical Industries, Ltd.). At this time, the set temperature is 37 ° C. and the measurement time is 200 min. In the same manner, an endotoxin standard (derived from Wakken E. coli UKT-B) was measured and used as a calibration curve.

[Method for measuring DNA concentration]
DNA concentration was assessed according to the THRESHOLD Total DNA assay (Molecular Devices, Corp., Sunnyvale, CA).

[Electron microscope measurement method]
Cut the sample to an appropriate size, fix it on the sample stage using conductive tape, apply osmium coating using a hollow cathode CVD osmium coater (type HPC-1S) manufactured by Vacuum Device Co., Ltd. A sample was used. Using a high resolution scanning electron microscope (S-4700) manufactured by Hitachi, Ltd., structural analysis of the cross section of the membrane was performed at an acceleration voltage of 1 kV, a working distance of 5 × 10E-3 m, and a predetermined magnification.

<Measurement of molecular weight cutoff>
Using each ultrafiltration membrane, 1 wt% bovine albumin (Sigma-Aldrich, molecular weight 60,000) and bovine γ-globulin (Invitrogen, molecular weight 150,000), ferritin (Sigma-Aldrich, molecular weight 450,000) ) At a constant pressure dead end of 0.010 MPa. The amount of albumin, γ-globulin, and ferritin in the immunoglobulin permeate that permeated within 5 minutes from the start of filtration was measured, and the capture rate of each protein captured on the membrane was calculated. A calibration curve between the molecular weight and the capture rate of each protein was prepared, the molecular weight at the capture rate of 90% was determined from the calibration curve, and the value was determined as the fractional molecular weight.

<Calculation method of throughput, transmittance, transmittance and transmittance ratio>
The amount of treatment, the amount of permeation, the transmittance, and the transmittance ratio were calculated using the volume and concentration of biological components before and after filtration. For the concentration measurement of target biological components and impure biological components, high-performance liquid chromatographic measurement (two columns G3000SWXL made by Tosoh Corp., SC8020 system made by Tosoh Corp., UV8020 detector made by Tosoh Corp.) ) And obtained from the absorption peak area ratio at a wavelength of 280 nm.
First, the treatment amount of the biological component permeated through the membrane was calculated by the following formulas (14) to (16).
Amount of biological component processed (g)
= Target biological component throughput (W1) + impurity biological component throughput (W2) (14)
W1 (g) = Va × A1a / A1s × Ka (15)
W2 (g) = Va × A2a / A2s × Ka (16)
Va: Volume of biological component solution before filtration (L)
A1s: HPLC peak area of target biological component of 1 g / L (mV · sec)
A2s: HPLC peak area of ionic impurities of 1 g / L (mV · sec)
A1a: HPLC peak area (mV · sec) of the target biological component before filtration
A2a: HPLC peak area (mV · sec) of impurity biocomponent before filtration
Ka: dilution factor of biological component solution before filtration

Next, the permeation amount (P1) of the target biocomponent and the permeation amount (P2) of the impurity biocomponent were calculated by the following equations (17) and (18).
P1 (g) = Vf × A1f / A1s × Kf (17)
P2 (g) = Vf × A2f / A2s × Kf (18)
Vf: Permeate volume (L)
A1s: HPLC peak area of target biological component of 1 g / L (mV · sec)
A2s: HPLC peak area of ionic impurities of 1 g / L (mV · sec)
A1f: HPLC peak area (mV · sec) of the target biological component in the permeate
A2f: HPLC peak area (mV · sec) of impurity biological components in the permeate
Kf: dilution factor of permeate

Further, the transmittance of the target biological component and the transmittance of the impurity biological component were calculated by the following formulas (19) and (20).
Permeability of target biological component (%) = P1 / W1 × 100 (19)
Impurity biological component transmittance (%) = P2 / W2 × 100 (20)
As a result, the transmittance ratio (fractionation performance) was calculated by the following formulas (21) and (22). When the molecular weight of the target biological component is smaller than the molecular weight of the impurity biological component, (21) is used. On the other hand, when the molecular weight of the target biological component is larger than the molecular weight of the impurity biological component, the transmittance ratio is calculated using (22). .
Transmittance ratio = impurity biological component transmittance / target biological component transmittance (21)
Transmittance ratio = transmittance of target biological component / transmittance of impurity biological component (22)
The lower the transmittance ratio, the higher the fractionation performance of the target biological component and the impurity biological component.

<Example of production of anti-SCF antibody>
(1) Preparation of immunogen SCF cDNA isolated from a cDNA library of HeLa cells that highly express SCF was incorporated into an animal cell expression vector pBCMGS-neo, which was then used as mouse fibroblast cell line Balb / 3T3 cells. The resulting transfectant was used as an immunogen.
(2) Production of hybridoma (a) Immunization The above transfectants were intraperitoneally administered to 8-week-old Balb / c mice (female) at intervals of 2 weeks. The effect of immunity was evaluated by the reactivity of peripheral blood serum collected from the tail vein of mice with the immunogen. After confirming the effect, final immunization and cell fusion were performed.
(B) Cell fusion Four days after the final immunization, the spleen cells of the immunized mouse and the mouse myeloma-derived cell line SP-2 were subjected to cell fusion according to a conventional method.
(C) Screening of anti-SCF antibody-producing hybridoma As an anti-SCF antibody-producing hybridoma screening method, an indirect antibody method using a transfectant and its parent cell line (Balb / 3T3) as an antigen was used. A hybridoma producing an antibody that binds to the transfectant and does not bind to the parent cell line (Balb / 3T3) was selected and cloned.
(D) Purification of antibody The culture supernatant of the SCF-expressing clone was subjected to ultrafiltration concentration, and then mixed with an equal amount of a binding buffer (BioRad Protein MAPS buffer). Protein A-Sepharose CL-4B (manufactured by Pharmacia) was equilibrated with a binding buffer, and the mixture was allowed to flow through the column to bind the antibody. Then, the column was washed with the binding buffer. A 0.2M Glycine-HCl buffer (pH 3.0) was passed through the column for elution to obtain a pre-purification anti-SCF antibody. Next, purification is performed in the order of DEAE-Sepharose FF (manufactured by GE Healthcare), Phenyl-Sepharose HP (manufactured by GE Healthcare), and Sephadex-G75 (manufactured by GE Healthcare). Isolated.
The target biological component and impurity biological component contained in the pre-purification anti-SCF antibody and the post-purification anti-SCF antibody were analyzed by the method described above. The results are shown in Table 1.

[Example 1]
The number was taken out so that the total cross-sectional area of the hollow portion of the hollow fiber membrane (PSf-1) was 0.01 m ² to prepare a yarn bundle.
As a biological component mixed solution, 500 mL of 10 g / L blood donated globenin-I-Nichiyak (manufactured by Nippon Pharmaceutical Co., Ltd.) containing 50 g / L of lysine hydrochloride was used. When the concentrations of immunoglobulin monomer (molecular weight: 150 kDa), dimer (molecular weight: 300 kDa) and multimer in the biological component mixed solution were measured by GPC, 9.1 g / L and 0.8 g / L, It contained 0.1 g / L.
The module prepared above and the biological component mixed solution are connected to a crossflow filtration apparatus as shown in FIG. The liquid feed pump is rotated so that the linear velocity in the hollow fiber membrane is 10 cm / second, and the average of the pressure on the hollow fiber membrane side and the pressure on the outlet side is 0.027 MPa (pressure at the transition point is 0.005 MPa). Adjusted with valve 1. Since the concentration of the biological component mixed solution increases as the cross flow filtration proceeds, the aqueous PBS solution is added and controlled so that the absorbance during filtration becomes the absorbance of the biological component mixed solution before filtration. After performing cross flow filtration at 25 ° C. for 5 hours, the concentrations of the immunoglobulin monomer and dimer in the biological component mixed solution and permeate were measured by GPC. The results (Table 2), the amount of transmitted immunoglobulin mer (Protein A) is 167 g / m ^2, the immunoglobulin dimer (protein B) permeation amount was 2.0 g / m ^2. Furthermore, as a result of calculating the transmittance and transmittance ratio (fractionation performance) of the immunoglobulin monomer and dimer, the monomer transmission rate and the dimer transmission rate were 90.2% and 12. The transmittance ratio was 0.14.

[Example 2]
The number was taken out so that the total cross-sectional area of the hollow portion of the hollow fiber membrane (PSf-2) was 0.01 m ² to prepare a yarn bundle.
As a biological component mixed solution, 500 mL of 10 g / L bovine albumin (manufactured by SIGMA) was used. When the concentrations of bovine albumin monomer (molecular weight: 69 kDa), dimer (molecular weight: 128 kDa) and multimer in the biological component mixed solution were measured by GPC, 9.5 g / L and 0.4 g / L, It contained 0.1 g / L.
The module prepared above and the biological component mixed solution are connected to a crossflow filtration apparatus as shown in FIG. Since the concentration of the biological component mixed solution increases as the cross flow filtration proceeds, the aqueous PBS solution is added and controlled so that the absorbance during filtration becomes the absorbance of the biological component mixed solution before filtration. After cross-flow filtration at 25 ° C. for 5 hours, the concentrations of bovine albumin monomer and dimer in the biological component mixed solution and permeate were measured by GPC. As a result (Table 2), the permeation amount of bovine albumin monomer (protein A) was 79 g / m ² and the permeation amount of bovine albumin dimer (protein B) was 0.5 g / m ² . Further, the transmittance and transmittance ratio (fractionation performance) of bovine albumin monomer and dimer were calculated. As a result, the monomer transmittance and the dimer transmittance were 83.9% and 13. The transmittance ratio was 0.16.

[Comparative Example 1]
The biological component mixed solution was separated by the same method as in Example 1 except that the hollow fiber membrane (PSf-1) was changed to the hollow fiber membrane (PSf-3). As a result (Table 2), the permeation amount of immunoglobulin monomer (protein A) was 335 g / m ² and the permeation amount of immunoglobulin dimer (protein B) was 28.6 g / m ² . Furthermore, as a result of calculating the transmittance and transmittance ratio (fractionation performance) of the immunoglobulin monomer and dimer, the monomer transmission rate and the dimer transmission rate were 96.5% and 93.%, respectively. The transmittance ratio was 0.97.
[Example 1], [Example 2], and [Comparative Example 1] are described in Table 2 above.

[Example 3] Two-stage filtration The number of the hollow fiber membranes (PSf-1 and PSf-2) was taken out so that the total cross-sectional area of the hollow portion was 0.01 m ² to prepare a yarn bundle.
As a biological component mixed solution, 0.7 g / L bovine albumin (manufactured by SIGMA) containing lysine hydrochloride 50 g / L, 9.3 g / L donated globenin-I-Nichiyaku (immunoglobulin monomer: 8.6 g) / L, immunoglobulin dimer: 0.7 g / L,) was used in an amount of 500 mL.
The hollow fiber membrane (PSf-1) module produced above and the biological component mixed solution are connected to a crossflow filtration device as shown in FIG. As the cross flow filtration proceeds, the concentration of the biological component mixed solution on the side of the retentate increases, so that an aqueous PBS solution is added and controlled so that the absorbance during filtration becomes the absorbance of the biological component mixed solution before filtration. After cross-flow filtration at 25 ° C. for 5 hours, the permeation amount of immunoglobulin monomer, immunoglobulin dimer and bovine albumin was measured. As a result, the permeability of the immunoglobulin monomer was 90.2%, the permeability of the immunoglobulin dimer was 12.3%, and the permeability of bovine albumin was 99.0%. Moreover, the biological component mixed solution on the filtrate side was diluted and the concentration thereof was 1 g / L. They are listed in Table 3.
Next, the biological component mixed solution and the hollow fiber membrane (PSf-2) module produced above are connected to a cross flow filtration device as shown in FIG. As the cross flow filtration proceeds, the concentration of the biological component mixed solution on the side of the retentate increases, so that an aqueous PBS solution is added and controlled so that the absorbance during filtration becomes the absorbance of the biological component mixed solution before filtration. After cross-flow filtration at 25 ° C. for 5 hours, the permeation amount of immunoglobulin monomer, immunoglobulin dimer and bovine albumin was measured. As a result, the transmission rate of the immunoglobulin monomer was 4.8%, the transmission rate of the immunoglobulin dimer was <1%, and the transmission rate of bovine albumin was 96.0%. Further, the biological component mixed solution on the retentate side was concentrated and the concentration was 10 g / L. They are listed in Table 3.
From the above results, it was found that immunoglobulin dimer and bovine albumin can be removed and immunoglobulin monomer can be purified by performing two-stage filtration.

[Example 4] Two-stage filtration Anti-SCF antibody monomer and dimer aggregates, CHOP hollow fiber membrane (PSf-1) were obtained under the same filtration conditions as in Example 3 except that the pre-purification anti-SCF antibody was used. The first-stage crossflow filtration used was performed. As a result, the transmittance of immunoglobulin monomer was 90.2%, the transmittance of immunoglobulin dimer was 12.3%, and the filtrate concentration of CHOP was 10E2 ng / ml. Moreover, the biological component mixed solution on the filtrate side was diluted and the concentration thereof was 1 g / L. They are listed in Table 3.
Subsequently, second-stage crossflow filtration using a hollow fiber membrane (PSf-2) was performed in the same manner as in Example 3. As a result, the permeability of the immunoglobulin monomer was 4.8%, the permeability of the immunoglobulin dimer was <1%, and the concentration of the retentate in CHOP, DNA, and endotoxin was 10 ng / ml or less, 1 pg / ml respectively. Results of less than ml and less than 1 EU / ml were obtained. Further, the biological component mixed solution on the retentate side was concentrated and the concentration was 10 g / L. They are listed in Table 3.
From the above results, it was found that the immunoglobulin dimer can be purified by removing the immunoglobulin dimer, CHOP DNA, and endotoxin by performing two-stage filtration.

[Example 5]
The number was taken out so that the total cross-sectional area of the hollow portion of the hollow fiber membrane (PSf-1) was 0.005 m ^2, and a yarn bundle for evaluating the performance of human immunoglobulin monomer / dimer fractionation was prepared. The yarn bundle is connected to a cross flow filtration device as shown in FIG.
Next, a 0.01 g / L human immunoglobulin solution containing 50 g / L lysine hydrochloride was set in the apparatus, the wire bundle in the hollow fiber membrane was 10 cm / second, and the hollow fiber membrane entry side pressure and exit side pressure were The liquid feed pump 1 (2) was rotated so that the average of was 1010 MPa, and was adjusted with the adjustment valve (7). During filtration, an aqueous PBS solution containing 50 g / L lysine hydrochloride in the diluted solution tank (1) is added to the biological component solution tank (4) so that the human immunoglobulin concentration is always constant. After forming a cake layer on the surface of the membrane by filtration for 10 minutes, it was replaced with a 0.25% human immunoglobulin solution, and the immunoglobulin dimer permeability / monomer permeability ratio (immune ratio for 1 minute) (Globulin dimer permeability / monomer permeability) was measured.
In the same manner as described above, 0.1 g / L and 1 g / L, 10 g / L human immunoglobulin solutions and a diluent as a blank were used, and after filtration in the same manner, 0.25% human immunity It was replaced with a globulin solution, and the immunoglobulin dimer permeability / monomer permeability ratio (immunoglobulin dimer permeability / monomer permeability) for 5 minutes was measured.
As a result, when the cake layer was formed at 0 g / L, 0.01 g / L, and 0.1 g / L, the immunoglobulin dimer permeability / monomer permeability ratios were 0.63 and 0.00, respectively. 67, 0.59. From this, it was found that at a concentration of 1 g / L or less, a cake layer capable of separating immunoglobulin monomer and dimer in a short time was not formed. On the other hand, when the cake layer was formed at 1 g / L and 10 g / L, the ratio of immunoglobulin dimer permeability / monomer permeability was 0.20 and 0.14, respectively. It turned out that the cake layer which can isolate | separate a monomer and a dimer is formed.
From the above results, it is possible to separate an immunoglobulin monomer and a dimer in a short time, that is, a resolution with a ratio of the transmittance of an immunoglobulin dimer to that of an epithelial globulin monomer is 0.2 or less. It has been found that a concentration of 1 g / L or more is necessary to form a cake layer for expressing the above.

The separation method according to the present invention can be suitably used in the field of separation and purification of biopharmaceuticals such as nucleic acids, sugars, proteins, vaccines, blood fraction preparations and the like.

It is a figure which illustrates the crossflow filtration apparatus of this invention. It is a figure which illustrates the crossflow filtration apparatus of this invention. It is a figure which illustrates the concentration / isolation | separation process of this invention. It is an electron micrograph of the inner surface dense layer of the hollow fiber membrane of the present invention.

Explanation of symbols

1 Dilution solution tank 2 Liquid feed pump 1
3 Liquid feed pump 2
4 Tank for biological component solution 5 Pressure gauge 1
6 Pressure gauge 2
7 Adjustment valve 1
8 Ultrafiltration membrane module 9 Tank for permeate 10 Flow meter 11 Concentration controller 1
12 Pressure and flow controller 1
13 UV flow cell 14 Concentration controller 2
15 Liquid feed pump 3
16 Pressure gauge 3
17 Pressure gauge 4
18 Pressure / Flow Controller 2
19 Adjustment valve 2
20 Absorbance meter 21 Biological component recovery liquid tank 22 Flow meter 2
23 Switching valve 24 Ultrafiltration membrane module for concentration / separation

Claims (19)

  Using an ultrafiltration membrane having a molecular weight of the target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component, the target biological component and at least the target biological component A biological component solution containing a high molecular weight impurity biological component containing a dimer of components, wherein the biological component concentration of the target biological component and at least the target biological component dimer is 1 to 150 g / L A separation method comprising separating a high molecular weight impurity biological component containing a target biological component and at least a dimer of the target biological component by cross-flow filtration. Using an ultrafiltration membrane having a molecular weight of the target biological component of 10 to 1,000 kDa and a fractional molecular weight of 1.5 to 4.5 times that of the target biological component, the target biological component and at least the target biological component A biological component solution containing a high molecular weight impurity biological component containing a dimer of components, wherein the biological component concentration of the target biological component and at least the target biological component dimer is 1 to 150 g / L Separating the high molecular weight impurity biological component including the target biological component and at least a dimer of the target biological component by cross-flow filtration;
A biological component solution containing at least a target biological component and a low molecular weight impurity biological component using an ultrafiltration membrane for concentration / separation having a fractional molecular weight of 0.3 to 1.5 times the target biological component. A separation method comprising a step of concentrating / separating a target biological component by permeating a low molecular weight impurity biological component by cross-flow filtration of a solution having a biological component concentration of 0.1 to 150 g / L .   The separation method according to claim 1 or 2, wherein the biological component concentration is 5 to 100 g / L.   The separation method according to any one of claims 1 to 3, wherein the cross-flow filtration is performed while the biological component concentration is maintained substantially constant.   The separation method according to any one of claims 1 to 4, wherein the biological component contains at least one of polypeptide, sugar, RNA, and DNA.   The polypeptide contains at least one of antibody, low molecular weight antibody, albumin, blood coagulation factor, interleukin, interferon, cell adsorption factor, cell growth factor, enzyme, riboprotein, and vaccine. The separation method according to claim 5.   The separation method according to claim 6, wherein the antibody is an immunoglobulin.   The separation method according to claim 7, wherein the immunoglobulin is a monoclonal antibody.   The high molecular weight impurity biological component containing at least a dimer of a target biological component includes a composite aggregate composed of a target biological component such as a complex aggregate composed of immunoglobulin and protein A and an eluted affinity ligand. The separation method according to claim 1.   The high molecular weight impurity biological component including at least the dimer of the target biological component contains at least one of endotoxin, DNA, and CHOP (Chinese hamster ovary cell protein), which are impurities derived from the starting material of the target biological component. The separation method according to claim 1, wherein:   The low molecular weight impurity biological component contains at least one of endotoxin, DNA, and CHOP (Chinese hamster ovary cell protein), which are impurities derived from the starting material of the target biological component. The separation method according to any one of the above.   The separation method according to claim 1, wherein the ultrafiltration membrane is a vinyl alcohol polymer membrane or a sulfone polymer membrane.   The separation method according to claim 1, wherein the separation method is used after an affinity chromatography purification step of the solution.   The separation method according to claim 1, wherein the ultrafiltration membrane is a hollow fiber membrane. 15. The filtration flow rate is maintained at a level exceeding the flow rate at the transition point, and further, the filtration transmembrane pressure is maintained at a level exceeding the transmembrane pressure at the flow rate transition point. The separation method according to crab. The separation method according to any one of claims 1 to 15, which is carried out using an apparatus comprising one or more means selected from the following (a) to (d).
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane means An apparatus comprising one or more means of the module used in the separation method according to any one of claims 1 to 15 and means comprising the following (a) to (d).
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane means Modules using ultrafiltration membranes and one or more means comprising the following (a) to (d), modules using ultrafiltration membranes for concentration / separation and means comprising the following (e) to (h) The separation method according to claim 2, comprising one or more of the following.
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means (e) Means capable of monitoring the concentration of the biological component permeate before concentration (f) Means capable of monitoring the concentration of the biological component permeate after concentration (g) Means capable of controlling the feeding speed of the biological component solution (h) Means that can control the filtration pressure of the ultrafiltration membrane for concentration and separation A module using an ultrafiltration membrane and one or more of the following means (a) to (d) for performing the separation method according to any one of claims 2 to 15, A device comprising a module using a filtration membrane and one or more means comprising the following (e) to (h).
(B) Means capable of monitoring the concentration of the biological component solution (b) Means capable of controlling the concentration of the biological component solution (c) Means capable of controlling the linear velocity of the biological component solution (d) Control of the filtration pressure of the ultrafiltration membrane Means (e) Means capable of monitoring the concentration of the biological component permeate before concentration (f) Means capable of monitoring the concentration of the biological component permeate after concentration (G) Means capable of controlling the feeding speed of the biological component permeate ) Means for controlling the filtration pressure of the ultrafiltration membrane for concentration and separation