WO2020209267A1 - タンパク質含有溶液精製用ポリアミド媒体及びその製造方法 - Google Patents

タンパク質含有溶液精製用ポリアミド媒体及びその製造方法 Download PDF

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WO2020209267A1
WO2020209267A1 PCT/JP2020/015741 JP2020015741W WO2020209267A1 WO 2020209267 A1 WO2020209267 A1 WO 2020209267A1 JP 2020015741 W JP2020015741 W JP 2020015741W WO 2020209267 A1 WO2020209267 A1 WO 2020209267A1
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antibody
polyamide
medium
solution
protein
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French (fr)
Japanese (ja)
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弘樹 谷口
純臣 日高
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Asahi Kasei Medical Co Ltd
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Asahi Kasei Medical Co Ltd
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Priority to EP20788609.4A priority Critical patent/EP3954725B1/en
Priority to US17/601,834 priority patent/US12383884B2/en
Priority to JP2021513652A priority patent/JP7295943B2/ja
Priority to CN202080026156.0A priority patent/CN113692420B/zh
Publication of WO2020209267A1 publication Critical patent/WO2020209267A1/ja
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D65/00—Accessories or auxiliary operations, in general, for separation processes or apparatus using semi-permeable membranes
    • B01D65/02—Membrane cleaning or sterilisation ; Membrane regeneration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/56—Polyamides, e.g. polyester-amides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
    • B01J20/28038—Membranes or mats made from fibers or filaments
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28042—Shaped bodies; Monolithic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/46—Post-polymerisation treatment
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/12—Chemical modification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/18—Details relating to membrane separation process operations and control pH control
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2321/00—Details relating to membrane cleaning, regeneration, sterilization or to the prevention of fouling
    • B01D2321/16—Use of chemical agents
    • B01D2321/164—Use of bases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers

Definitions

  • the present invention relates to a method for producing a polyamide medium for purifying a protein-containing solution, a method for purifying a protein-containing solution, and a polyamide medium for purifying a protein-containing solution.
  • an antibody is a physiologically active substance that controls an immune response, and is obtained from the blood of an immunized animal, a cell culture medium of cells possessing an antibody-producing ability, or an ascites culture solution of an animal.
  • the blood, cell culture medium, and ascites culture medium containing these antibodies are complex contaminants derived from proteins other than antibodies or raw material solutions used for cell culture (hereinafter, these are referred to as impurities). ) Is included, and impurities are removed through a multi-step process.
  • Non-Patent Document 1 As a method for removing impurities from the blood, cell culture solution and ascites culture solution, conventionally, a method using an ion exchange column using an adsorption mechanism or a method using a hydrophobic gel (for example, Non-Patent Document 1 below). (See), and further, a method using a polyamide-containing molded product (see, for example, Patent Document 1 below) and the like have been proposed.
  • a method using a size exclusion column see, for example, Patent Document 2 below
  • a method of performing filtration using a nanofilter, or the like a method of filtering by a size exclusion mechanism is used.
  • Methods for removing things have been proposed.
  • the target protein is recovered by adsorption of the target protein. It has the problem that the rate drops.
  • the present inventors have obtained a polyamide medium for purifying a protein-containing solution that can solve the above-mentioned problems of the prior art by performing a predetermined treatment on the polyamide medium. We have found that it can be obtained and have completed the present invention. That is, the present invention is as follows.
  • a method for producing a polyamide medium for purifying a protein-containing solution A step of treating a polyamide medium before treatment with an acidic or alkaline aqueous solution with an acidic or alkaline aqueous solution.
  • a method for producing a polyamide medium for purifying a protein-containing solution [2] The method for producing a polyamide medium for purifying a protein-containing solution according to the above [1], wherein the polyamide medium is a porous body. [3] The method for producing a polyamide medium for purifying a protein-containing solution according to the above [2], wherein the porous body is a film-like porous body.
  • [4] The method for producing a polyamide medium for purifying a protein-containing solution according to any one of [1] to [3] above, wherein the acidic or alkaline aqueous solution is an acidic aqueous solution having a pH of 5 or less.
  • [5] The method for producing a polyamide medium for purifying a protein-containing solution according to any one of [1] to [3] above, wherein the acidic or alkaline aqueous solution is an alkaline aqueous solution having a pH of 10 or higher.
  • [6] The method for producing a polyamide medium for purifying a protein-containing solution according to any one of [1] to [5] above, wherein the protein-containing solution is an antibody-containing solution.
  • the method for purifying the protein-containing solution according to the above [7] or [7-1], wherein the step of bringing the protein-containing solution into contact with the polyamide medium is a step of filtering the protein-containing solution with the polyamide medium.
  • the sum of the amino groups and the carboxyl groups on the surface of the polyamide medium is 1.01 times or more larger than the sum of the amino groups and the carboxyl groups inside the polyamide medium.
  • the number average molecular weight Mn (S) of the polymer on the surface of the polyamide medium and The number average molecular weight Mn (I) of the polymer inside the polyamide medium is Expressed by the following formula, Polyamide medium for purification of protein-containing solutions.
  • the number average molecular weight Mn (S) of the polymer on the surface of the polyamide medium and The number average molecular weight Mn (I) of the polymer inside the polyamide medium is Expressed by the following formula, Polyamide medium for purification of protein-containing solutions.
  • a method for producing a polyamide medium for removing antibody aggregates [15] The method for producing a polyamide medium for removing antibody aggregates according to the above [14], wherein the polyamide medium is a porous body. [16] The method for producing a polyamide medium for removing antibody aggregates according to the above [15], wherein the porous body is a film-like porous body.
  • a method for removing antibody aggregates from an antibody solution which comprises a step of contacting an antibody solution containing the antibody aggregates with a polyamide medium treated with an acidic or alkaline aqueous solution.
  • a method of removing antibody aggregates [21] The method for recovering an antibody solution having an improved purity of the antibody monomer according to [19] or [19-1] above, wherein the antibody is a monoclonal antibody. [22] The method for removing antibody aggregates from the antibody solution according to [20] or [20-1] above, wherein the antibody is a monoclonal antibody. [23] A polyamide medium for removing antibody aggregates, A polyamide medium for removing antibody aggregates, wherein the sum of amino groups and carboxyl groups on the surface of the polyamide medium is 1.01 times or more larger than the sum of amino groups and carboxyl groups inside the polyamide medium.
  • the polyamide medium for removing antibody aggregates obtained by the method for producing a polyamide medium for removing antibody aggregates according to any one of [14] to [18] above.
  • a polyamide medium for removing antibody aggregates wherein the sum of amino groups and carboxyl groups on the surface of the polyamide medium is 1.01 times or more larger than the sum of amino groups and carboxyl groups inside the polyamide medium.
  • a polyamide medium for removing antibody aggregates The number average molecular weight Mn (S) of the polymer on the surface of the polyamide medium and The number average molecular weight Mn (I) of the polymer inside the polyamide medium is Expressed by the following formula, Polyamide medium for removing antibody aggregates.
  • the polyamide medium for removing antibody aggregates obtained by the method for producing a polyamide medium for removing antibody aggregates according to any one of [14] to [18] above.
  • the number average molecular weight Mn (S) of the polymer on the surface of the polyamide medium and The number average molecular weight Mn (I) of the polymer inside the polyamide medium is Expressed by the following formula, Polyamide medium for removing antibody aggregates.
  • the step comprises contacting the polyamide medium for removing antibody aggregates according to the above [23], [23-1], [24], or [24-1] with an antibody solution containing antibody aggregates.
  • the step comprises contacting the polyamide medium for removing antibody aggregates according to [23], [23-1], [24], or [24-1] with an antibody solution containing antibody aggregates.
  • a method for producing a polyamide medium for purifying a protein-containing solution having a high recovery rate of a target protein in one embodiment. Further, in one embodiment, it is possible to provide a method for purifying a protein-containing solution using a polyamide medium obtained by the production method. Further, in one embodiment, a polyamide medium for purifying a protein-containing solution can be provided.
  • the chromatographic chart of the antibody-containing solution containing an antibody monomer and an antibody aggregate of Example 1 is shown.
  • the enlarged view of the peak part of the chromatographic chart of FIG. 1 is shown.
  • the spectrum of FT-IR of Example 10 and Comparative Example 10 is shown. 1639 cm -1, 1544Cm peak -1 a larger (better described as "water") represents the measurement results of Comparative Example 10, who has been described as toward the peak is small ( "sodium hydroxide” ) Indicates the measurement result of Example 10.
  • the present embodiment a mode for carrying out the present invention (hereinafter, also referred to as “the present embodiment”) will be described in detail. It should be noted that the following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to the following contents. The present invention can be implemented with various modifications within the scope of the gist thereof.
  • the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment includes a step of treating a polyamide raw material with an acidic or alkaline aqueous solution.
  • a polyamide medium having a high recovery rate of the target protein can be obtained.
  • the polyamide raw material that is, the polyamide medium before treatment with an acidic or alkaline aqueous solution refers to an untreated polyamide medium before carrying out the step of treating with an acidic or alkaline aqueous solution.
  • the polyamide raw material may be a medium containing only polyamide or a medium containing a compound other than polyamide.
  • the amount of the target protein adsorbed on the polyamide medium can be reduced, and the target protein can be highly recovered. It can be collected at a rate.
  • the amide bond on the polyamide surface is hydrolyzed by treating the polyamide medium before treatment with an acidic or alkaline aqueous solution, and a hydrophilic functional group is used. It is considered that the appearance of the carboxy group or amino group is reduced in protein adsorption to the polyamide medium, but the present invention is not bound by the mechanism. Further, it is preferable that the polyamide medium for purifying the protein-containing solution obtained by the production method of the present embodiment maintains the strength in the state before being treated with an acidic or alkaline aqueous solution.
  • the polyamide medium for purifying the protein-containing solution obtained by the production method of the present embodiment has a reduced amount of the target protein adsorbed on the polyamide medium and has a strength as compared with the state before treatment with an acidic or alkaline aqueous solution. It is a preferable embodiment that the polyamide medium is maintained.
  • the polyamide medium for purifying the protein-containing solution obtained by the production method of the present embodiment may be a polyamide medium for removing antibody aggregates. That is, the polyamide medium for purifying the protein-containing solution of the present embodiment may be a polyamide medium for purifying the antibody solution. Specifically, when the antibody-containing solution to be purified contains antibody aggregates in addition to the antibody monomer, the aggregates may be removed by a polyamide medium.
  • the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment is a method for producing a polyamide medium for removing antibody aggregates
  • the polyamide medium before being immersed in an alkaline aqueous solution is pH 5 It shall have a step of immersing in the following acidic aqueous solution or alkaline aqueous solution having a pH of 10 or more for 5 minutes or more. As a result, a polyamide medium capable of effectively removing antibody aggregates can be obtained.
  • the protein contained in the protein-containing solution to be purified is not particularly limited as long as it is a protein that can recover the target protein with a high recovery rate by the polyamide medium obtained by the production method of the present embodiment.
  • examples include, but are not limited to, albumin, globulin, fibrinogen and the like.
  • an antibody is a preferable example. That is, a preferable example of the protein-containing solution to be purified is an antibody-containing solution.
  • the protein aggregates are highly selectively removed and the protein monomer is removed.
  • a protein solution with improved purity can be recovered with a high recovery rate. That is, by contacting the antibody solution containing the antibody aggregate with the polyamide medium obtained by the production method of the present embodiment, the antibody aggregate is removed from the antibody solution, and the antibody solution having improved purity of the antibody monomer is obtained. It can be recovered with a high recovery rate.
  • Antibodies include glycoprotein molecules (also referred to as gamma globulin or immunoglobulin) produced by B lymphocytes as a mechanism for preventing infection in vertebrates, as generally defined in biochemistry.
  • the antibody in the antibody solution purified in this embodiment can be used as a human drug, and in such a case, has substantially the same structure as the antibody in the human body to be administered.
  • the antibody may be a human antibody or an antibody protein derived from a mammal such as a cow or mouse other than human. Further, the antibody may be a chimeric antibody protein with human IgG or a humanized antibody.
  • the chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism such as mouse, but the other constant region is replaced with human-derived immunoglobulin. Of the variable regions, the complementarity determining regions (CDRs) are derived from organisms other than humans, but the other framework regions (framework regions: FR) are derived from humans. It is an antibody. Humanized antibodies are even less immunogenic than chimeric antibodies.
  • the antibody class (isotype) and subclass are not particularly limited.
  • antibodies are classified into five classes, IgG, IgA, IgM, IgD, and IgE, depending on the structure of the constant region.
  • the antibody contained in the antibody solution to be purified may be in any of the five classes.
  • IgG has four subclasses of IgG1 to IgG4, and IgA has two subclasses of IgA1 and IgA2.
  • the antibody subclass may be any.
  • An antibody-related protein such as an Fc fusion protein in which a protein is bound to the Fc region can also be included in the antibody to be purified in the present embodiment.
  • antibodies can also be classified by origin.
  • the antibody to be purified in this embodiment may be a natural human antibody, a recombinant human antibody produced by a gene recombination technique, a monoclonal antibody, or a polyclonal antibody.
  • the monoclonal antibody is preferable as the antibody to be purified in the present embodiment from the viewpoint of demand and importance as an antibody drug, but the present embodiment is not limited to this.
  • the antibody examples include a monoclonal antibody or a polyclonal antibody containing any of IgM, IgD, IgG, IgA, or IgE.
  • the antibody may be derived from plasma products or cell culture medium.
  • animal cells or microorganisms can be used as cells.
  • the type of animal cell is not particularly limited, but CHO cell, Sp2 / 0 cell, NS0 cell, Vero cell, PER. Examples include C6 cells.
  • the type of microorganism is not particularly limited, and examples thereof include Escherichia coli and yeast.
  • the antibody aggregates to be removed are the target.
  • examples include a dimer in which two antibody monomers are associated, a trimer in which three target antibody monomers are associated, a multimer in which four or more target antibody monomers are associated, or a mixture thereof. Be done.
  • the multimer may include both a dimer and a trimer.
  • the antibody aggregate may contain a protein other than the target antibody.
  • protein aggregates may be irreversible or reversible aggregates.
  • Polyamide medium The polyamide constituting the polyamide medium before treatment with an acidic or alkaline aqueous solution used in the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment or the method for producing a polyamide medium for removing antibody aggregates is formed from an amide bond.
  • the polymer is composed of repeating units, and the monomer unit may be either an aliphatic polyamide or an aromatic polyamide, a mixture thereof, or a plurality of types of aliphatic monomers and aromatic monomers.
  • the polyamide is not limited to the following, but for example, nylon 6, nylon 11, nylon 12 or a combination of hexamethylenediamine and adipic acid obtained by a polycondensation reaction of ⁇ -caprolactam, undecancaprolactam, and lauryllactam.
  • Nylon 66 obtained by polycondensation reaction
  • nylon 610 obtained by polycondensation reaction of hexamethylenediamine and sebacic acid
  • nylon 6T obtained by polycondensation reaction of hexamethylenediamine and terephthalic acid
  • hexamethylenediamine and isophthalic acid hexamethylenediamine and isophthalic acid.
  • Nylon 6I obtained by the polycondensation reaction of ⁇ caprolactam and lauryl lactam
  • nylon 9T obtained by the polycondensation reaction of nonanediamine and terephthalic acid
  • nylon M5T obtained by the polycondensation reaction of methylpentadiamine and terephthalic acid.
  • nylon 612 obtained by a polycondensation reaction
  • polyamide obtained by a polycondensation reaction of paraphenylenediamine and terephthalic acid
  • a polyamide obtained by a polycondensation reaction of metaphenylenediamine and isophthalic acid obtained by the polycondensation reaction of metaphenylenediamine and isophthalic acid.
  • the polyamide medium before treatment with the acidic or alkaline aqueous solution is not particularly limited, and examples thereof include polyamide media (excluding polyamide media having a graft chain). Further, as another embodiment, a polyamide medium having no graft chain is exemplified.
  • the weight average molecular weight of the polyamide used in the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment or the method for producing a polyamide medium for removing antibody aggregates is the weight average molecular weight of the polyamide constituting the polyamide medium before treatment with an acidic or alkaline aqueous solution.
  • a larger one is preferable from the viewpoint of the strength of the polyamide medium.
  • 2000 or more is preferable, more preferably 5000 or more, 10000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, and further preferably 90,000 or more, 95,000 or more. It is over 100,000.
  • the polyamide medium is a fiber
  • it is preferably 20000 or less, more preferably 1,000,000 or less, and further preferably 500,000 or less, 300,000 or less, 200,000 or less.
  • the weight average molecular weight of the polyamide can be measured by a known method such as GPC (Gel Permeation Chromatography).
  • the number average molecular weight of the polyamides constituting the polyamide medium before treatment with an acidic or alkaline aqueous solution used in the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment or the method for producing a polyamide medium for removing antibody aggregates is
  • a larger one is preferable.
  • it is preferably 1000 or more, more preferably 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, and further preferably 20000 or more, 25000 or more, 30,000 or more.
  • the polyamide medium is a fiber
  • it is preferably 1,000,000 or less, more preferably 500,000 or less, and further preferably 100,000 or less, 50,000 or less, 40,000 or less, 30,000 or less.
  • the number average molecular weight of the polyamide can be measured by a known method such as GPC (Gel Permeation Chromatography).
  • the method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment includes a step of treating a predetermined untreated polyamide medium as a raw material with an acidic or alkaline aqueous solution.
  • changes that occur in the polyamide as a raw material are not particularly limited, and examples thereof include hydrolysis and introduction of functional groups.
  • the functional group to be introduced include a hydroxy group, a carboxy group, an amino group, a sulfo group, an aldehyde group, a carbonyl group and a nitro group.
  • the pH of the acidic aqueous solution is preferably 5 or less, more preferably 4 or less, 3 or less, 2 or less, 1.5 or less, etc. from the viewpoint of the progress rate of hydrolysis as the upper limit. .. Further, as the lower limit, 1.0 or more is exemplified from the viewpoint of the strength of the polyamide medium.
  • the acidic aqueous solution examples include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids such as trifluoroacetic acid.
  • the acidic aqueous solution may contain a compound other than the inorganic acid or the organic acid.
  • the immersion time when immersing in an acidic aqueous solution as a treatment method is preferably 5 minutes or more, more preferably 10 minutes, as a lower limit from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before the predetermined treatment, which is the raw material.
  • the above is 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, and more preferably 1 hour or more, 2 for 1 hour or more, depending on the type and form of the polyamide medium and the type of antibody to be applied.
  • Hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 Minutes or less, 20 minutes or less, 10 minutes or less can be appropriately selected.
  • the immersion temperature when immersed in an acidic aqueous solution as a treatment method is preferably 4 ° C. or higher, more preferably 10 ° C., as a lower limit from the viewpoint of the decomposition progress rate of amide bonds on the surface of the polyamide medium before treatment, which is a raw material. As mentioned above, it is 15 ° C. or higher, and more preferably 20 ° C. or higher. Further, as the upper limit, from the viewpoint of the strength of the polyamide medium, it is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, and further preferably 30 ° C. or lower.
  • the lower limit of the liquid passing time when the acidic aqueous solution is passed as the treatment method is preferably 5 minutes or more, more preferably 10 minutes, from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before the predetermined treatment, which is the raw material. Minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, more preferably 1 hour or more in sequence depending on the type and form of the polyamide medium and the type of antibody to be applied. 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • the upper limit is 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, depending on the type and form of the polyamide medium and the type of antibody to be applied. 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the lower limit of the liquid passing temperature when the acidic aqueous solution is passed as the treatment method is preferably 4 ° C. or higher, more preferably, from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before the predetermined treatment as the raw material. Is 10 ° C. or higher, 15 ° C. or higher, and more preferably 20 ° C. or higher.
  • the upper limit is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, still more preferably 30 ° C. or lower, from the viewpoint of the strength of the polyamide medium.
  • the pH of the alkaline aqueous solution is preferably pH 10.0 or higher, more preferably 11.0 or higher, still more preferably 11.0 or higher, as the lower limit from the viewpoint of the decomposition efficiency of the amide bond on the surface of the polyamide medium. It is 12.0 or more, more preferably 12.5 or more, even more preferably 13.0 or more, and particularly preferably 13.5 or more. Further, as the upper limit, from the viewpoint of the strength of the polyamide medium, it is preferably 14.0 or less, more preferably 13.5 or less.
  • alkaline aqueous solution examples include sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, lithium hydroxide aqueous solution and the like, and from the viewpoint of acquisition cost, sodium hydroxide aqueous solution or potassium hydroxide aqueous solution is preferable.
  • the alkaline aqueous solution for treating the polyamide medium may contain a compound other than sodium hydroxide, potassium hydroxide, and lithium hydroxide.
  • the immersion time when the polyamide medium is immersed in an alkaline aqueous solution is preferably 5 minutes or more as a lower limit from the viewpoint of sufficiently hydrolyzing the amide bond on the surface of the polyamide medium before a predetermined treatment as a raw material. More preferably, it is 10 minutes or more, 20 minutes or more, 30 minutes or more, and further preferably 40 minutes or more, 50 minutes or more, 55 minutes or more, depending on the type and form of the polyamide medium and the type of protein to be applied. Then, 1 hour or more, 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 Minutes or less, 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the immersion temperature when the polyamide medium is immersed in an alkaline aqueous solution is preferably 4 ° C. or higher as a lower limit from the viewpoint of the decomposition progress rate of amide bonds on the surface of the polyamide medium before a predetermined treatment as a raw material. It is preferably 10 ° C. or higher, 15 ° C. or higher, and more preferably 20 ° C. or higher. Further, as the upper limit, from the viewpoint of the strength of the polyamide medium, 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, still more preferably 30 ° C. or lower is exemplified.
  • the liquid passing time when the alkaline aqueous solution is passed through the polyamide medium is 5 minutes or more as a lower limit from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before a predetermined treatment as a raw material. It is preferably 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, and more preferably depending on the type and form of the polyamide medium and the type of antibody to be applied. , 1 hour or more, 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 Minutes or less, 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the liquid passage temperature when the polyamide medium is passed through an alkaline aqueous solution is set to a lower limit of 4 from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before a predetermined treatment as a raw material.
  • ° C. or higher is preferable, more preferably 10 ° C. or higher, 15 ° C. or higher, and even more preferably 20 ° C. or higher.
  • the upper limit from the viewpoint of the strength of the polyamide medium, it is 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, and further preferably 30 ° C. or lower.
  • the step of treating the polyamide medium before alkali treatment with an alkaline aqueous solution having a pH of 10 or more under the conditions of 4 ° C. or higher and 100 ° C. or lower, 5 minutes or longer and 70 hours or shorter, and the above-mentioned It is preferable to have a step of washing the polyamide medium and a step of bringing the protein-containing solution into contact with the washed polyamide medium. Further, a step of treating the polyamide medium before alkali treatment with an alkaline aqueous solution having a pH of 13 or more under the conditions of 10 ° C. or higher and 30 ° C.
  • the hydrolysis of the amide bond on the surface of the predetermined untreated polyamide medium as a raw material can be caused with high efficiency and a sufficient rate, and the protein-containing solution is purified using the processed polyamide medium.
  • the amount of the target protein adsorbed on the polyamide medium can be reduced, and the target protein can be recovered with a high recovery rate.
  • the method for producing a polyamide medium of the present embodiment is particularly a method for producing a polyamide medium for removing antibody aggregates
  • the polyamide medium before a predetermined treatment as a raw material is immersed in an acidic aqueous solution having a pH of 5 or less for 5 minutes. Immerse in an alkaline aqueous solution having a pH of 10 or more for 10 minutes or more.
  • changes that occur in the polyamide as a raw material are not particularly limited, and examples thereof include hydrolysis and introduction of functional groups.
  • the functional group to be introduced examples include a hydroxy group, a carboxy group, an amino group, a sulfo group, an aldehyde group, a carbonyl group and a nitro group.
  • the upper limit of the pH of the acidic aqueous solution is preferably 5 or less from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before the predetermined treatment, which is a raw material. More preferably, it is 3 or less, 2 or less, and 1.5 or less. Further, the lower limit is preferably 1.0 or more from the viewpoint of the strength of the polyamide medium.
  • the acidic aqueous solution examples include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and organic acids such as trifluoroacetic acid.
  • the acidic aqueous solution may contain a compound other than the inorganic acid or the organic acid.
  • the immersion time when immersing in an acidic aqueous solution as a treatment method is preferably 5 minutes or more, more preferably 10 minutes, as a lower limit from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before the predetermined treatment, which is the raw material.
  • the above is 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, and more preferably 1 hour or more, 2 for 1 hour or more, depending on the type and form of the polyamide medium and the type of antibody to be applied. Time or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 Minutes or less, 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the immersion temperature when immersed in an acidic aqueous solution as a treatment method is preferably 4 ° C. or higher, more preferably 4 ° C. or higher, from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before the predetermined treatment, which is the raw material. It is 10 ° C. or higher, 15 ° C. or higher, and more preferably 20 ° C. or higher. Further, as the upper limit, from the viewpoint of the strength of the polyamide medium, it is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, and further preferably 30 ° C. or lower.
  • the lower limit of the liquid passing time when the acidic aqueous solution is passed as the treatment method is preferably 5 minutes or more, more preferably 10 minutes, from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before the predetermined treatment, which is the raw material. Minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, more preferably 1 hour or more in sequence depending on the type and form of the polyamide medium and the type of antibody to be applied. 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more can be appropriately selected.
  • the upper limit is 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, depending on the type and form of the polyamide medium and the type of antibody to be applied. 2 hours or less, 1 hour or less, 30 minutes or less, 20 minutes or less, 10 minutes or less can be appropriately selected.
  • the lower limit of the liquid passing temperature when the acidic aqueous solution is passed as the treatment method is preferably 4 ° C. or higher, more preferably, from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before the predetermined treatment as the raw material. Is 10 ° C. or higher, 15 ° C. or higher, and more preferably 20 ° C. or higher.
  • the upper limit is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, still more preferably 30 ° C. or lower, from the viewpoint of the strength of the polyamide medium.
  • the pH of the alkaline aqueous solution is preferably pH 10.0 or higher, more preferably 11.0 or higher, still more preferably 12 as the lower limit from the viewpoint of the decomposition efficiency of amide bonds on the surface of the polyamide medium. It is 0.0 or more, even more preferably 12.5 or more, even more preferably 13.0 or more, and particularly preferably 13.5 or more.
  • the upper limit is preferably 14 or less, more preferably 13.5 or less, from the viewpoint of the strength of the polyamide medium.
  • alkaline aqueous solution for example, a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, and a lithium hydroxide aqueous solution are preferable.
  • the alkaline aqueous solution may contain a compound other than sodium hydroxide, potassium hydroxide and lithium hydroxide.
  • the immersion time when the polyamide medium is immersed in an alkaline aqueous solution is preferably 5 minutes or more, more preferably 10 minutes as the lower limit from the viewpoint of sufficiently hydrolyzing the amide bond on the surface of the polyamide medium as a raw material.
  • the above is more preferably 20 minutes or longer, still more preferably 30 minutes or longer, 40 minutes or longer, 50 minutes or longer, 55 minutes or longer, and even more preferably depending on the type and form of the polyamide medium and the type of antibody to be applied.
  • 1 hour or more, 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more can be appropriately selected.
  • the upper limit is 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, from the viewpoint of the strength of the polyamide medium. 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the immersion temperature when the polyamide medium is immersed in an alkaline aqueous solution is preferably 4 ° C. or higher as a lower limit from the viewpoint of the decomposition progress rate of amide bonds on the surface of the polyamide medium before a predetermined treatment as a raw material. It is preferably 10 ° C. or higher, more preferably 15 ° C. or higher, and even more preferably 20 ° C. or higher.
  • the upper limit is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, still more preferably 30 ° C. or lower, from the viewpoint of the strength of the polyamide medium.
  • the liquid passing time when the alkaline aqueous solution is passed through the polyamide medium is 5 minutes or more as a lower limit from the viewpoint of decomposing the amide bond on the surface of the polyamide medium before a predetermined treatment as a raw material. It is preferably 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 55 minutes or more, and more preferably depending on the type and form of the polyamide medium and the type of antibody to be applied. , 1 hour or more, 2 hours or more, 3 hours or more, 5 hours or more, 10 hours or more, 15 hours or more, 20 hours or more, 40 hours or more, 70 hours or more, etc. can be appropriately selected.
  • 70 hours or less, 40 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 5 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 Minutes or less, 20 minutes or less, 10 minutes or less, etc. can be appropriately selected.
  • the liquid passing temperature when the polyamide medium is passed through an alkaline aqueous solution is set to a lower limit of 4 from the viewpoint of the decomposition progress rate of the amide bond on the surface of the polyamide medium before a predetermined treatment as a raw material.
  • ° C. or higher is preferable, more preferably 10 ° C. or higher, still more preferably 15 ° C. or higher, and even more preferably 20 ° C. or higher.
  • the upper limit from the viewpoint of the strength of the polyamide medium, it is preferably 100 ° C. or lower, 80 ° C. or lower, 50 ° C. or lower, more preferably 40 ° C. or lower, and further preferably 30 ° C. or lower.
  • the polyamide medium obtained by the production method of the present embodiment is a porous material from the viewpoint of the size of the surface area when the contaminants are removed by adsorption in both the purification of the protein-containing solution and the removal of antibody aggregates. It is preferable to have. Further, when removing impurities by size exclusion, it is preferable that the material is a porous material.
  • the form of the porous body include a film-like substance, a particle-like substance, a monolith, a capillary, a sintered body, and the like, and specific examples thereof include a microporous hollow fiber membrane, a microporous flat membrane, a non-woven fabric, and a woven fabric. Be done.
  • the porous body is in the form of a film, the effect of purifying the protein-containing solution at high speed can be obtained by passing the protein-containing solution, which is preferable.
  • the polyamide medium is a porous membrane
  • the pore size of the porous membrane is small from the viewpoint of removing impurities by adsorption or size exclusion.
  • the average pore size of the polyamide porous membrane is preferably 1000 nm or less, more preferably 500 nm or less, still more preferably 400 nm or less, still more preferably 300 nm or less as the upper limit. Further, as the lower limit, from the viewpoint of the filtration rate, 1 nm or more, 5 nm or more, 10 nm or more, 20 nm or more, and 30 nm or more are preferable.
  • the method for purifying a protein-containing solution of the present embodiment includes a step of bringing the protein-containing solution into contact with a polyamide medium treated with an acidic or alkaline aqueous solution. Specifically, it has a step of bringing the protein-containing solution into contact with the polyamide medium for purifying the protein-containing solution obtained by the method for producing the polyamide medium for purifying the protein-containing solution of the present embodiment described above.
  • the step of bringing the protein-containing solution into contact with the polyamide medium includes, for example, a step of passing the protein-containing solution through the polyamide medium and a step of immersing the polyamide medium in the protein solution, as will be described later.
  • a step of passing the protein-containing solution through the polyamide medium a step of filtering the protein-containing solution with the polyamide medium is exemplified.
  • impurities that can be removed by contacting the polyamide medium with a protein-containing solution include protein aggregates, antibody aggregates, cell host-derived proteins (HCP; Host Cell Proteins) in the biopharmacy manufacturing process, virus particles, and the like. ..
  • the virus particles may have an envelope.
  • the method of recovering the antibody solution and the method of removing the antibody aggregate from the antibody solution of the present embodiment include a step of bringing the antibody solution containing the antibody aggregate into contact with a polyamide medium treated with an acidic or alkaline aqueous solution. .. Specifically, it comprises a step of contacting the polyamide medium for removing antibody aggregates obtained by the method for producing a polyamide medium for removing antibody aggregates of the present embodiment described above with an antibody solution containing antibody aggregates. ..
  • the step of contacting the polyamide medium with the antibody solution containing the antibody aggregate includes, for example, a step of passing the antibody solution through the polyamide medium and a step of immersing the polyamide medium in the antibody solution, as will be described later. Further, as a step of passing the protein-containing solution through the polyamide medium, a step of filtering the protein-containing solution with the polyamide medium is exemplified. As a result, antibody aggregates can be effectively removed from the antibody solution, and an antibody solution with improved purity can be obtained.
  • the protein-containing solution may be an antibody solution, or the antibody solution may contain antibody aggregates.
  • the antibody aggregate can be removed from the antibody solution, and the antibody solution having improved purity of the antibody monomer can be obtained with a high recovery rate.
  • the antibody solution means a solution in which the target antibody is dissolved.
  • the solvent used for the antibody solution may be pure water or a buffer solution.
  • the type of buffer that can be used as a solution is not limited to, for example, tris salt, acetate, Tween, sorbitol, maltose, glycine, arginine, lysine, histidine, sulfonate, phosphate, citric acid, or A buffer solution in which sodium chloride is dissolved can be mentioned.
  • the concentration of the antibody solution used in this embodiment is not particularly limited as long as the antibody is dissolved in the solution.
  • As the lower limit of the concentration of the antibody solution 0.01 mg / mL or more is exemplified, 0.05 mg / mL or more is exemplified as another embodiment, and 0.1 mg / mL or more is exemplified as another embodiment.
  • Yet another embodiment is exemplified by 0.5 mg / mL or more
  • yet another embodiment is exemplified by 1.0 mg / mL or more
  • yet another embodiment is exemplified by 5.0 mg / mL or more. ..
  • the concentration of the antibody solution 100 mg / mL or less is exemplified, 90 mg / mL or less is exemplified as another embodiment, 80 mg / mL or less is exemplified as another embodiment, and still another embodiment is exemplified.
  • 30 mg / mL or less is exemplified as still another embodiment, 25 mg / mL or less is exemplified as yet another embodiment, and 20 mg / mL or less is exemplified as still another embodiment.
  • the concentration of the buffer solution is not particularly limited as long as the above-mentioned predetermined solution is dissolved.
  • As the lower limit of the concentration of the buffer solution 0 mmol / L or more is exemplified depending on the type of the buffer solution, 0.5 mmol / L or more is exemplified as another embodiment, and 1.0 mmol or more as another embodiment.
  • / L or more is exemplified, yet another embodiment is exemplified by 5 mmol / L or more, yet another embodiment is exemplified by 10 mmol / L or more, and yet another embodiment is exemplified by 15 mmol / L or more.
  • 25 mmol / L or more is exemplified.
  • the pH of the buffer solution is not particularly limited, but 4.0 or more is exemplified as the lower limit of pH, 4.5 or more is exemplified as another embodiment, and other embodiments, depending on the type of buffer solution. 5.0 or more is exemplified as an embodiment, 5.5 or more is exemplified as still another aspect, and 6.0 or more is exemplified as still another aspect.
  • As the upper limit of pH 10.0 or less is exemplified, 9.0 or less is exemplified as another aspect, 8.0 or less is exemplified as another aspect, and 8. 5 or less is exemplified, still another embodiment is exemplified by 8.0 or less, yet another embodiment is exemplified by 7.5 or less, and yet another embodiment is exemplified by 7.0 or less. ..
  • the electric conductivity of the buffer solution is not particularly limited, but the lower limit of the electric conductivity is exemplified as 0 mS / cm or more depending on the type of the buffer solution, and 1 mS / cm or more as another embodiment.
  • 2 mS / cm or more is exemplified as another aspect
  • 3 mS / cm or more is exemplified as still another aspect
  • 4 mS / cm or more is exemplified as yet another aspect
  • yet another aspect is exemplified.
  • 5 mS / cm or more is exemplified as.
  • 100 mS / cm or less is exemplified, 90 mS / cm or less is exemplified as another embodiment, 80 mS / cm or less is exemplified as another embodiment, and further as another embodiment.
  • 70 mS / cm or less is exemplified, yet another embodiment is exemplified by 60 mS / cm or less, yet another embodiment is exemplified by 50 mS / cm or less, and yet another embodiment is exemplified by 40 mS / cm or less. Will be done.
  • the method for purifying a protein-containing solution of the present embodiment when an antibody solution is applied as the protein-containing solution, antibody aggregates are removed from the antibody solution, and an antibody solution having improved purity of the antibody monomer is recovered. Brings the polyamide medium into contact with the antibody solution containing the antibody aggregates as described above.
  • the method of contacting the polyamide medium with the antibody solution is not particularly limited as long as the antibody solution can be brought into contact with the polyamide medium, but as described above, a method of passing the antibody solution through the polyamide medium and immersing the polyamide medium in the antibody solution. The method of doing this can be mentioned. Further, as a method of passing the protein-containing solution through the polyamide medium, a method of filtering the protein-containing solution through the polyamide medium is exemplified.
  • Examples of the method of passing the antibody solution through the polyamide medium include a method of passing the antibody solution through the polyamide medium by a syringe, a pump or the like.
  • a method for passing the antibody solution through the polyamide medium it is sufficient that the antibody solution flowing toward a predetermined portion of the polyamide medium passes through the polyamide medium and the antibody solution can be recovered from the other portion of the polyamide medium.
  • a buffer solution may be passed through the polyamide medium separately from the antibody solution.
  • a method for passing the protein-containing solution through the polyamide medium a method of filtering the protein-containing solution through the polyamide medium is a preferable example.
  • the flow velocity for passing the antibody solution through the polyamide medium is not particularly limited, but the lower limit is 0.1 mL / min or more with respect to 1 mL of the polyamide medium, depending on the type of the antibody solution. 0.5 mL / min or more is exemplified, 1.0 mL / min or more is exemplified as another embodiment, and 5 mL / min or more is exemplified as still another embodiment.
  • polyamide medium for purifying protein-containing solutions, polyamide medium for removing antibody aggregates In the polyamide medium for purifying the protein-containing solution of the present embodiment and the polyamide medium for removing antibody aggregates of the present embodiment, the sum (total number) of the amino groups and carboxyl groups of the polymer on the surface of the polyamide medium is the polyamide medium. It is preferable that the sum (total number) of the amino groups and the carboxyl groups of the polymer in the polymer is 1.01 times or more. As a result, a polyamide medium having high removal selectivity of protein aggregates, for example, antibody aggregates, and excellent strength characteristics can be obtained.
  • the above-mentioned polyamide medium can be obtained by the above-mentioned method for producing a polyamide medium for purifying a protein-containing solution of the present embodiment and the above-mentioned method for producing a polyamide medium for removing antibody aggregates of the present embodiment.
  • the polyamide manufacturing process can be arbitrarily selected, and is not limited to the manufacturing method of the present embodiment described above.
  • the surface also includes the surface of the pores when the polyamide medium is a porous material.
  • the polymer on the surface of the polyamide medium is 10 nm, 5 nm, 3 nm, or 10 nm from the surface, depending on the type, concentration, immersion time, antibody type, type of polyamide medium, form, etc. of the acidic or alkaline aqueous solution used. It means a polymer present in a medium of 2 nm and 1 nm.
  • the polymer inside the polyamide medium means a polymer other than the surface of the polyamide medium as defined above.
  • the polyamide medium for purifying the protein-containing solution and the polyamide medium for removing antibody aggregates of the present embodiment are polyamides, they have functional groups of amino groups and carboxy groups at the polymer terminals.
  • the sum of the functional groups of amino groups and carboxyl groups on the surface of the polyamide medium and the sum of the functional groups inside the polyamide medium tend to be different, and both the selectivity for removing protein aggregates, for example, antibody aggregates and the strength of the polyamide medium are compatible.
  • the sum of the amino groups and the carboxyl groups on the surface is preferably 1.01 times or more, more preferably 1.02 times or more, still more preferably 1. times the sum of the amino groups and the carboxyl groups inside.
  • 03 times or more more preferably 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, 1.1 times or more, 1. 15 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 2.0 times or more, 2.5 times or more, 3.0 times or more 3. 5 times or more, 4.0 times or more, 4.5 times or more, 5.0 times or more, 6.0 times or more, 7.0 times or more, 8.0 times or more, 9.0 times or more, 10.0 times or more These are 15 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, and 100 times or more.
  • the sum of the functional groups of the amino group and the carboxyl group may be calculated by measuring the amino group and the carboxyl group, respectively, and calculating the total number thereof. Further, in light of the mechanism in which an amino group and a carboxyl group are generated one-to-one by hydrolysis of an amide bond in a polyamide medium, the amount of either the amino group or the carboxyl group is measured, and the amount is doubled. Can be sum.
  • the surface of the polyamide medium for purifying the protein-containing solution and the polyamide medium for removing the antibody of the present embodiment can be prepared by appropriately setting the pH of the acidic or alkaline aqueous solution, the immersion time in the alkaline aqueous solution, the immersion temperature, and the like. The sum of the amino group and the carboxyl group can be controlled to be larger than the sum of the internal amino group and the carboxyl group at the magnification shown above.
  • the ratio of a predetermined functional group in the polyamide medium can be determined by the functional group density per mass.
  • the amount of functional groups in the entire polyamide medium can be quantified by, for example, NMR. Further, the amount of functional groups on the surface of the polyamide medium can be analyzed by a known method such as XPS (X-ray photoelectron spectroscopy).
  • the polyamide medium for purifying the protein-containing solution and the polyamide medium for removing antibody aggregates of the present embodiment may differ in the number average molecular weight of the polymer on the surface of the polyamide medium and the number average molecular weight of the polymer inside the polyamide medium. , It is preferable that the inside is larger.
  • the surface also includes the surface of the pores when the polyamide medium is porous.
  • the polymer on the surface of the polyamide medium is 10 nm, 5 nm, 3 nm, or 10 nm from the surface, depending on the type, concentration, immersion time, antibody type, type of polyamide medium, form, etc. of the acidic or alkaline aqueous solution used.
  • the polymer inside the polyamide medium means a polymer other than the surface of the polyamide medium.
  • the ratio of the number average molecular weights at that time is Mn (S) / Mn (I) when the number average molecular weight of the polymer on the surface of the polyamide medium is Mn (S) and the number average molecular weight of the internal polymer is Mn (I). ),
  • Mn (S) / Mn (I) is preferably 0.99 or less from the viewpoint of achieving both selectivity and strength of protein aggregates, for example, antibody aggregates. That is, it is preferable that Mn (S) / Mn (I) ⁇ 0.99.
  • the number average molecular weight of the polyamide medium can be determined by gel permeation chromatography or the like by a known method, and in this case, it is the converted molecular weight of the standard substance.
  • the polyamide medium of the present embodiment in which the ratio (Mn (S) / Mn (I)) of the number average molecular weight of the surface polymer to the internal polymer as described above is 0.99 or less is the above-mentioned embodiment of the present embodiment. It is obtained by the method for producing a polyamide medium for purifying a protein-containing solution and the method for producing a polyamide medium for removing antibody aggregates of the present embodiment. Specifically, when the polyamide medium is treated with an acidic or alkaline aqueous solution, a part of the bond chains of the surface polymer is broken, so that the number average molecular weight tends to be larger inside.
  • the polyamide production process can be arbitrarily selected as long as the condition of the number average molecular weight ratio is satisfied, and the method is not limited to the production method of the present embodiment described above.
  • the polyamide medium for purifying the protein-containing solution and the polyamide medium for removing antibody aggregates of the present embodiment appropriately adjust the pH of the acidic or alkaline aqueous solution, the immersion time in the alkaline aqueous solution, the immersion temperature, and the like. By setting, the ratio of the number average molecular weight of the polymer on the surface to the number average molecular weight of the polymer inside can be controlled to the numerical value shown above.
  • Example 1 Immersion of a polyamide film as a polyamide medium in an alkaline aqueous solution A polyamide film as a porous polyamide medium having a circular shape with a diameter of 2.5 cm, a film thickness of 160 ⁇ m, and an average pore size of 0.2 ⁇ m (Whatman (registered trademark): 7402- 002, manufactured by GE Healthcare Co., Ltd., material: polyamide 66) was immersed in a 1.0 mol / L sodium hydroxide solution (pH 14) at room temperature for 24 hours. Next, it was washed with pure water 5 times to completely remove sodium hydroxide.
  • CRL12445 antibody a monoclonal antibody expressed from CHO cell CRL12445 (hereinafter, may be abbreviated as CRL12445 antibody) was prepared.
  • the culture solution containing the monoclonal antibody-producing cells expressed from CRL12445 was filtered using a filtration membrane (manufactured by Asahi Kasei Medical Co., Ltd., trade name BioOptimal (registered trademark) MF-SL), and an antibody-containing solution (culture) containing impurities and antibodies. The supernatant) was acquired.
  • the antibody-containing solution containing the solution was buffer-exchanged with 15 mmol / L tris-HCl buffer (pH 7.0, 5 mS / cm) containing an arbitrary amount of sodium chloride, and the solution was mixed at an arbitrary ratio to mix the antibody aggregate and the antibody.
  • An antibody-containing solution containing a monomer (hereinafter, may be abbreviated as "SM") was prepared.
  • the electric conductivity of the buffer solution was measured using an electric conductivity meter CM-40S (manufactured by DKK-TOA CORPORATION).
  • Comparative Example 1 The polyamide film 2 produced under the same conditions as the polyamide film 1 was used except that the polyamide film was not immersed in the sodium hydroxide aqueous solution. In Comparative Example 1, it was immersed in water. Other conditions were the same as in Example 1. The analysis results by size exclusion chromatography after passing through the polyamide membrane 2 and the antibody monomer recovery rate are shown in [Table 1] below.
  • Example 1 Comparing the results of Example 1 and Comparative Example 1, the treatment of immersing the polyamide membrane in the aqueous sodium hydroxide solution improved the antibody monomer recovery rate while maintaining the amount of antibody aggregates removed. It turned out.
  • Example 2 (1) Treatment of Polyamide Film as Polyamide Medium with Alkaline Aqueous Solution The immersion solution and immersion time in the sodium hydroxide solution are changed, and other conditions are "(1) As a polyamide medium" in [Example 1] above.
  • Polyamide films 3 to 6 and the polyamide film 8 were prepared in the same manner as in "immersion of the polyamide film in an alkaline aqueous solution)".
  • Regarding the polyamide film 7 the same polyamide film as in [Example 1] (Whatman (registered trademark): 7402-002, manufactured by GE Healthcare Japan, material: polyamide 66) was used in the same manner as in [Example 1].
  • the concentration of the antibody-containing solution after passing through the polyamide film 3 to 8 was C3, the amount of the antibody-containing solution was V3, and the antibody monomer ratio was R3.
  • Example 2 Comparing the results of Example 2 and Comparative Example 2, it was found that the antibody monomer recovery rate was improved by immersing the polyamide film in the sodium hydroxide aqueous solution. Since the polyamide films 3 to 6 and the polyamide film 8 have a higher antibody monomer recovery rate than the polyamide film ⁇ , the effect of improving the antibody monomer recovery rate of the polyamide medium of the present invention is alkaline. It became clear that it did not depend on the pH of the solution and the immersion time. Further, since the same effect was obtained in the polyamide film 7, it was shown that the effect of improving the antibody monomer recovery rate of the polyamide medium of the present invention does not depend on the method for treating the alkaline solution.
  • Example 3 The pH, electrical conductivity, and buffer solution of the antibody-containing solution were changed, and under other conditions, the same experiment as in [Example 2] was performed on the polyamide membranes 3 to 5 and the polyamide membrane 8.
  • the antibody-containing solution obtained in ((2) Preparation of antibody-containing solution) and ((3) Purification of antibody-containing solution by affinity column) in [Example 1] was buffered with 15 mmol / L acetate.
  • the buffer was exchanged with a solution (pH 5.5, 15 mS / cm) to obtain an antibody-containing solution.
  • the calculation results of the antibody monomer recovery rate are shown in [Table 4] below.
  • Example 3 Comparing the results of Example 3 and Comparative Example 3, it was found that the antibody monomer recovery rate was improved by immersing the polyamide film in the sodium hydroxide aqueous solution.
  • the amount of antibody monomer recovered by immersing the polyamide membrane in the sodium hydroxide aqueous solution was increased regardless of the liquidity of the antibody-containing solution. It turned out to be effective.
  • Example 4 The antibody type of the antibody-containing solution was changed, and other experiments were carried out in the same manner as in [Example 3] above.
  • Evolocumab manufactured by Astellas Pharma Inc.
  • 15 mmol / L acetate buffer pH 5.5, 15 mS / cm
  • the calculation results of the antibody monomer recovery rate are shown in [Table 5] below.
  • Example 4 Comparing the results of Example 4 and Comparative Example 4, it was found that the antibody monomer recovery rate was improved by immersing the polyamide film in the sodium hydroxide aqueous solution.
  • the effect of improving the amount of antibody monomer recovered by immersing the polyamide film in the sodium hydroxide aqueous solution can be obtained regardless of the antibody type. I found out.
  • Example 5 The method for producing the polyamide film was changed, and other experiments were carried out in the same manner as in [Example 4] above. Specifically, the immersion solution and the immersion time were changed, and the polyamide films 9 to 11 were prepared in the same manner as described above ([Example 1] Treatment of the polyamide film as an alkaline aqueous solution as a polyamide medium). The conditions for producing the polyamide film are shown in [Table 6] below. The calculation results of the antibody monomer recovery rate are shown in [Table 7] below.
  • Example 5 Comparing the results of Example 5 and Comparative Example 5, it was found that the antibody monomer recovery rate was improved by immersing the polyamide membrane in the sodium hydroxide aqueous solution. From this, the present invention can obtain the effect of improving the antibody monomer recovery rate regardless of whether the solution used for treating the polyamide medium is an acidic aqueous solution or an alkaline aqueous solution, and the solution concentration and immersion time of immersion are also limited. It turned out not to be done.
  • Example 6 Only the method for producing the polyamide film was changed, and the same experiment as in [Example 4] was performed under other conditions.
  • the polyamide film 12 was prepared by changing only the immersion temperature and using the same method as in ((1) Immersing the polyamide film as a polyamide medium in an alkaline aqueous solution) in [Example 1].
  • the conditions for producing the polyamide film are shown in [Table 8] below.
  • the calculation results of the antibody monomer recovery rate are shown in [Table 9] below.
  • Example 6 Comparing the results of Example 6 and Comparative Example 6, it was found that the antibody monomer recovery rate was improved by immersing the polyamide film in the sodium hydroxide aqueous solution. From this, it was found that the present invention is not limited to the treatment temperature with the aqueous sodium hydroxide solution, and the desired effect can be obtained.
  • Example 7 (1) Immersion of a polyamide film in an alkaline aqueous solution A polyamide film as a porous polyamide medium having a circular shape with a diameter of 9.0 cm, a thickness of 170 ⁇ m and an average pore size of 0.2 ⁇ m (Whatman®: 7402-009, GE Health). Care Co., Ltd., Material: Polyamide 66) was immersed in a 1.0 mol / L sodium hydroxide solution (pH 14) at room temperature for 1 hour. Next, it was washed with pure water 5 times to completely remove sodium hydroxide. The obtained polyamide film was replaced with methanol, and the solvent was completely removed by vacuum drying to obtain a polyamide film 13. Further, the immersion time was changed to prepare the polyamide films 14 and 15 under the same conditions as described above. The conditions for producing the polyamide film are shown in [Table 10] below.
  • Example 7 Comparing the results of Example 7 and Comparative Example 7, it was found that the Young's modulus and the maximum stress of the polyamide film did not change regardless of the presence or absence of immersion in the sodium hydroxide aqueous solution. That is, according to the present invention, it has been found that a practically sufficient mechanical strength can be maintained.
  • protein preparations such as antibody drugs, virus removal filters and their prefilters are required to have properties that they are not easily deformed even when a pressure is applied to the membrane and are not easily torn even when a high pressure is applied to the membrane. From this point of view, it was found that the Young's modulus and the maximum stress did not change even when immersed in an alkaline aqueous solution, and the membrane strength could be maintained as a protein purification filter, which was practically preferable.
  • Example 8 (1) Immersion of Polyamide Membrane in Alkaline Aqueous Solution A polyamide membrane as a porous polyamide medium having a diameter of 2.5 cm, a thickness of 160 ⁇ m, and an average pore diameter of 0.2 ⁇ m (Whatman®: 7402-002, GE Health). (Care) was immersed in a 1.0 mol / L sodium hydroxide solution (pH 14) at room temperature for 1 hour. Next, it was washed with pure water 5 times to completely remove sodium hydroxide. The obtained polyamide film was replaced with methanol, and the solvent was completely removed by vacuum drying to obtain a polyamide film 16. The conditions for producing the polyamide film are shown in [Table 12] below.
  • Measuring device HLC-8320GPC (manufactured by Tosoh) Column: TSKgel GMH HR- H (S) x 3 (4.6 mm I.D. x 15 cm) Column temperature: 40 ° C Eluent: Na trifluoroacetic acid 5 mmol / L Calibration curve: polymethyl methyllate (12 points)
  • Example 8 Comparing the results of Example 8 and Comparative Example 8, it was found that even if the polyamide film was immersed in an aqueous sodium hydroxide solution, the weight average molecular weight and the number average molecular weight were not affected. This means that the hydrolysis by alkaline immersion has not progressed to the inside of the polyamide film. Moreover, from the results of Examples 1 to 6 and Comparative Examples 1 to 6, the adsorption behavior of the antibody to the polyamide film changes depending on the presence or absence of treatment with an alkaline aqueous solution, so that the surface properties of the polyamide film have changed. It is shown. Therefore, from the results of Examples 1 to 6, Example 8, and Comparative Examples 1 to 6 and Comparative Example 8, the treatment with the alkaline aqueous solution changes the surface properties of the polyamide film, but does not affect the internal properties. I found out.
  • Example 9 (1) Immersion of Polyamide Membrane in Alkaline Aqueous Solution
  • the polyamide film 17 is immersed in the same method as ((1) Soaking in an alkaline aqueous solution of a polyamide film) in [Example 8] except that the immersion time of the sodium hydroxide aqueous solution is 40 hours. Made.
  • the preparation conditions for the polyamide film 17 are shown in [Table 14] below.
  • Example 9 Comparing the results of Example 9 and Comparative Example 9, it was found that the rubidium concentration on the surface of the polyamide film was increased by treating the polyamide film with an alkaline aqueous solution. From this, it was found that the amount of carboxyl groups on the surface was increased by treating the polyamide film with an alkaline aqueous solution. That is, it was found that the treatment with an alkaline aqueous solution made the surface of the polyamide film hydrophilic, leading to an increase in the amount of the target protein recovered.
  • Example 10 Immersion of polyamide sheet in alkaline aqueous solution 66 nylon sheet (model number: 107-14301, thickness: 0.3 mm, manufactured by Kokugo) is immersed in 1.0 mol / L sodium hydroxide aqueous solution (pH 14) for 13 days at room temperature. did. Next, it was washed with pure water 5 times to completely remove sodium hydroxide. The obtained sheet was immersed in a 0.001 mol / L hydrochloric acid solution for 1 hour, washed with distilled water, and air-dried to obtain a polyamide sheet (1).
  • the production conditions for the polyamide sheet (1) are shown in [Table 16] below.
  • a polyamide sheet (2) was prepared under the same conditions as the polyamide sheet (1) in [Example 10] except that the polyamide sheet was not immersed in an aqueous sodium hydroxide solution, and was used as a measurement sample. In Comparative Example 10, it was immersed in water. Other conditions were the same as in [Example 10] above.
  • the production conditions for the polyamide sheet (2) are shown in [Table 16] above.
  • the measurement results are shown in FIG. In FIG. 3, "sodium hydroxide” indicates the measurement result of the polyamide sheet (1) immersed in the aqueous sodium hydroxide solution, and "water” in FIG. 3 means the aqueous sodium hydroxide solution. It is shown that it is the measurement result of the polyamide sheet (2) which was not immersed in water but was immersed in water.
  • Example 10 Comparing the results of Example 10 and Comparative Example 10, by treating the polyamide sheet in an alkaline aqueous solution, 1639 cm -1, the peak intensity of 1544cm -1 was reduced. From this, it was found that the treatment of the polyamide sheet with an alkaline aqueous solution reduced the amide bond on the surface. When combined with the results of Example 8 and Comparative Example 8, it was found that hydrolysis proceeded on the surface of the polyamide sheet by the treatment with the alkaline aqueous solution.
  • the method for producing a polyamide medium for purifying a protein-containing solution of the present invention has industrial applicability in the field of antibody-containing solution production technology in which the selectivity for removing antibody aggregates is improved.

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