WO2016093251A1 - Procédé de purification de substance physiologiquement active - Google Patents

Procédé de purification de substance physiologiquement active Download PDF

Info

Publication number
WO2016093251A1
WO2016093251A1 PCT/JP2015/084446 JP2015084446W WO2016093251A1 WO 2016093251 A1 WO2016093251 A1 WO 2016093251A1 JP 2015084446 W JP2015084446 W JP 2015084446W WO 2016093251 A1 WO2016093251 A1 WO 2016093251A1
Authority
WO
WIPO (PCT)
Prior art keywords
cation exchange
chromatography step
purification method
antibody
exchange chromatography
Prior art date
Application number
PCT/JP2015/084446
Other languages
English (en)
Japanese (ja)
Inventor
弘樹 谷口
一郎 小熊
Original Assignee
旭化成メディカル株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 旭化成メディカル株式会社 filed Critical 旭化成メディカル株式会社
Priority to JP2016563696A priority Critical patent/JPWO2016093251A1/ja
Publication of WO2016093251A1 publication Critical patent/WO2016093251A1/fr

Links

Images

Classifications

    • 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
    • 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
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • 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
    • B01J41/00—Anion exchange; Use of material as anion exchangers; Treatment of material for improving the anion exchange properties
    • B01J41/20—Anion exchangers for chromatographic processes
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • the present invention relates to a method for purifying a physiologically active substance.
  • Immunoglobulin is a physiologically active substance that controls the immune reaction.
  • the antibody is obtained from blood of the immunized animal or a cell culture solution of cells possessing antibody-producing ability or an ascites culture solution of the animal.
  • blood and culture fluids containing these antibodies include proteins other than antibodies, or complex contaminants derived from the raw material used for cell culture, and in order to separate and purify antibodies from those impurity components A complicated and time-consuming operation is usually necessary.
  • Liquid chromatography is important for antibody separation and purification.
  • Chromatographic techniques for separating antibodies include gel filtration chromatography, affinity chromatography, ion exchange chromatography, reverse phase chromatography, and the like, and antibodies are separated and purified by combining these techniques.
  • antibodies having high purity and concentration are produced through the following steps (A) to (C).
  • a step of adding a sample mixed with an antibody and impurities to the column (loading step)
  • counter ions present in the mobile phase are adsorbed using the ion exchange group on the surface of the adsorbent as a stationary phase.
  • the adsorbent beads, membranes such as flat membranes and hollow fibers are employed, and those obtained by binding a cation exchange group or an anion exchange group to these substrates are commercially available as adsorbents.
  • purification can be achieved by eluting the adsorbed physiologically active substance by adsorbing the antibody by bringing a low salt concentration antibody solution into contact with the adsorbent and increasing the salt concentration of the mobile phase. It is a common practice to often separate antibody monomers and antibody dimers and other aggregates. Furthermore, as a better method, purification of the target substance by the flow-through mode described below has been proposed.
  • the flow-through mode is a purification method in which impurities are selectively adsorbed on the adsorbent over the target substance. Therefore, compared with the conventional method using adsorption and elution, the buffer solution (buffer) can be saved and the process can be simplified.
  • a flow-through mode is generally used, and it is performed for the purpose of adsorption removal of a substance having a low isoelectric point.
  • Patent Document 1 discloses an example in which an antibody solution containing eluted protein A is subjected to flow-through purification using multimodal chromatography after the affinity chromatography step. However, the affinity chromatography step and the anion exchange chromatography are disclosed. The continuity with the process is not disclosed.
  • the antibody solution containing the eluted protein A is adjusted in the hydrogen ion index (pH) and then directly processed in the cation exchange chromatography step in the flow-through mode, and further, the cation exchange chromatography.
  • pH hydrogen ion index
  • An example is disclosed in which the antibody solution containing the eluted protein A processed in the step is adjusted in pH and then processed in an anion exchange chromatography step in flow-through mode, and buffer exchange and dilution are not performed.
  • Patent Document 1 does not disclose the continuity between the affinity chromatography step and the anion exchange chromatography step in the purification method.
  • a method for purifying a physiologically active substance comprising a cation exchange chromatography step and an anion exchange chromatography step after an affinity chromatography step, and the bioactivity after elution of the physiologically active substance in the affinity chromatography step.
  • the cation exchange chromatography step and the anion exchange chromatography step are performed in a flow-through manner to purify the physiologically active substance.
  • the cation exchange chromatography step from 30 mmol / L
  • dilution may not be performed, or the volume increase due to dilution may be 2.0 times or less.
  • the cation exchange chromatography carrier may contain a membrane-like substrate and a polymer having a cation exchange group that covers the substrate.
  • the polymer having a cation exchange group may contain a (meth) acrylate unit as a monomer unit.
  • the polymer having a cation exchange group may contain a neutral monomer unit having no charge.
  • the polymer having a cation exchange group may be covalently bonded to the substrate.
  • the polymer having a cation exchange group includes a monomer unit having a cation exchange group, and the weight ratio of the monomer unit having the cation exchange group is lower than the weight ratio of a neutral monomer unit having no charge.
  • the polymer having a cation exchange group may be a copolymer.
  • the cation exchange chromatography carrier may have one or more kinds of cation exchange groups including at least a weak cation exchange group.
  • the cation exchange chromatography carrier may have one or more kinds of cation exchange groups including at least a strong cation exchange group.
  • the main component of the elution buffer used for elution in the affinity chromatography step may be a monovalent acid.
  • the elution buffer used for elution in the affinity chromatography step may be an acetate buffer.
  • the electric conductivity of the elution buffer used for elution in the affinity chromatography step may be 5.0 mS / cm or less.
  • the concentration of the buffer used for elution in the affinity chromatography step may be 100 mmol / L or less.
  • the hydrogen ion index of the eluate containing the physiologically active substance may be adjusted between the cation exchange chromatography step and the anion exchange chromatography step.
  • the electrical conductivity of the eluate containing a physiologically active substance applied to the cation exchange chromatography step and the anion exchange chromatography step may be 5.0 mS / cm or less.
  • virus inactivation may be performed by acid treatment.
  • an eluate containing 0.3 g or more of a physiologically active substance per 1 mL volume of the cation exchange carrier may be loaded.
  • the anion exchange chromatography carrier may contain a membrane substrate and a polymer having an anion exchange group that covers the substrate.
  • the polymer having an exchange group having an anion exchange group may contain a (meth) acrylate unit as a monomer unit.
  • the polymer having an anion exchange group may be covalently bonded to the substrate on the membrane.
  • the ligand of the anion exchange chromatography carrier may have two or more alkyl groups having 2 or more carbon atoms.
  • the ligand of the anion exchange chromatography support may be a tertiary amine.
  • treatment with a depth filter may be performed before the cation exchange chromatography step.
  • the step of bringing the physiologically active substance and activated carbon into contact may not be included.
  • an efficient method for purifying a physiologically active substance is provided.
  • Example 2 is a graph of absorbance when the antibody solution according to Example 1 was subjected to size exclusion chromatography. It is an enlarged view of the graph of FIG. It is a table
  • 10 is a table showing the results of Example 7.
  • 10 is a table showing the results of Example 8.
  • 10 is a table showing the results of Example 9.
  • the method for purifying a physiologically active substance is a method for purifying a physiologically active substance including a cation exchange chromatography step and an anion exchange chromatography step after the affinity chromatography step, and the elution of the physiologically active substance in the affinity chromatography step Then, without exchanging the buffer of the eluate containing the physiologically active substance, the cation exchange chromatography step and the anion exchange chromatography step are performed in a flow-through manner to purify the physiologically active substance, and in the cation exchange chromatography step, A cation exchange carrier having a cation exchange group at a density higher than 30 mmol / L is used.
  • the physiologically active substance according to the embodiment is, for example, a monomer component of an antibody protein.
  • An antibody protein which is an example of a physiologically active substance, is a glycoprotein molecule (also referred to as gamma globulin or immunoglobulin) produced by B lymphocytes as a vertebrate infection control mechanism, as is generally defined in biochemistry.
  • the antibody protein purified in the embodiment is used as a human pharmaceutical and has substantially the same structure as the antibody protein in the human body to be administered.
  • the antibody protein may be a human antibody protein, or may be an antibody protein derived from mammals such as non-human bovines and mice.
  • the antibody protein may be a chimeric antibody protein with human IgG and a humanized antibody protein.
  • a chimeric antibody protein with human IgG is an antibody protein in which the variable region is derived from a non-human organism such as a mouse, but the other constant region is substituted with a human-derived immunoglobulin.
  • the humanized antibody protein is a variable region of which complementarity-determining region (CDR) is derived from a non-human organism, but the other framework region (framework region: FR) is derived from a human. It is an antibody protein. Humanized antibody proteins are further reduced in immunogenicity than chimeric antibody proteins.
  • the class (isotype) and subclass of the antibody protein which is an example of the purification target according to the embodiment is not particularly limited.
  • antibody proteins are classified into five classes, IgG, IgA, IgM, IgD, and IgE, depending on the structure of the constant region.
  • the antibody protein targeted by the purification method according to the embodiment may be any of the five classes.
  • IgG has four subclasses, IgG1 to IgG4, and IgA has two subclasses, IgA1 and IgA2.
  • the subclass of the antibody protein targeted by the purification method according to the embodiment may be any.
  • antibody-related proteins such as Fc fusion proteins in which a protein is bound to an Fc region can also be included in antibody proteins targeted by the purification methods according to the embodiments.
  • antibody proteins can also be classified by origin.
  • the antibody protein targeted by the purification method according to the embodiment is any of a natural human antibody protein, a recombinant human antibody protein produced by gene recombination technology, a monoclonal antibody protein, and a polyclonal antibody protein. Also good.
  • human IgG is preferable as an antibody protein targeted by the purification method according to the embodiment from the viewpoint of demand and importance as an antibody drug, but is not limited thereto.
  • impurities removed in the embodiment include antibody aggregates, host cell-derived protein (HCP), DNA, and antibodies containing protein A eluted in the affinity chromatography step.
  • HCP host cell-derived protein
  • Affinity chromatography is often called affinity chromatography, protein A chromatography, or protein A affinity chromatography, and is a chromatography method using protein A.
  • Protein A is derived from Staphylococcus aureus. Protein A has a specifically high affinity for the Fc region of an antibody under neutral conditions. Therefore, when purifying an antibody using natural protein A, first, a solution containing the antibody is brought into contact with a stationary phase having natural protein A as a ligand under neutral conditions, and the natural protein on the carrier is then contacted. An antibody is specifically adsorbed on A. Then, the components that have not been adsorbed on the carrier are washed and removed with a neutral buffer, and then the antibody is released from the natural protein A on the carrier using an acidic solution having a pH of around 3.0.
  • the chromatography material used in the affinity chromatography step is not particularly limited as long as it has protein A, but protein A is generally immobilized on a solid phase.
  • the affinity chromatography step according to the embodiment is performed by a general method without any problem. It is possible to obtain an antibody eluate with higher purity by eluting after washing with a buffer solution having a high electrical conductivity before eluting the antibody from the protein A column. In this embodiment, it is desirable to wash with a buffer solution having a low electrical conductivity after washing with a buffer solution with a high electrical conductivity. By this operation, the electric conductivity of the eluted antibody solution can be kept low, and the purification efficiency in the subsequent ion exchange chromatography step can be ensured.
  • the electric conductivity of such a low electric conductivity washing buffer is preferably 5.0 mS / cm or less, more preferably 4.0 mS / cm or less, and still more preferably 3.0 mS / cm or less.
  • the buffer used for elution from the protein A column is not particularly limited as long as the antibody can be eluted and the impurity removal property of the subsequent ion exchange chromatography step can be ensured.
  • the physiologically active substance is purified by an ion exchange chromatography step without performing buffer exchange of the eluate. Therefore, the adsorption performance tends to be reduced when a polyvalent buffer is used in anion exchange chromatography. Therefore, elution of a physiologically active substance from a protein A column using an elution buffer mainly composed of a monovalent acid is performed. It is desirable to do.
  • Such monovalent acid is not particularly limited, and examples thereof include acetic acid. Therefore, it is preferable to use an acetate buffer as the elution buffer.
  • the elution buffer used for elution preferably has a low concentration and further a low electrical conductivity from the viewpoint of continuity with the subsequent ion exchange chromatography step.
  • the concentration of the elution buffer is preferably 100 mmol / L or less, more preferably 50 mmol / L or less, and further preferably 30 mmol / L or less from the viewpoint of suppressing the electric conductivity after pH adjustment.
  • the buffer concentration means the concentration of the active ingredient in the buffer.
  • the concentration is usually adjusted from acetic acid and sodium acetate, and is the total concentration of acetic acid and sodium acetate.
  • the Tris buffer it means the concentration of trishydroxymethylaminomethane.
  • the buffer expressed as acetic acid-Tris buffer is the former concentration, and acetic acid-Tris is acetic acid, and Tris-acetic acid is the concentration of Tris.
  • the electric conductivity of the elution buffer is preferably 5.0 mS / cm or less, more preferably 4.0 mS / cm or less, still more preferably 3.0 mS / cm or less / cm, particularly preferably 2.0 mS / cm or less. is there.
  • the eluate from protein A may be used as it is in the next ion exchange chromatography step after adjusting the pH, or may be inactivated by low pH (acidic) treatment.
  • Virus inactivation may be a general method and is not particularly limited, but the pH is preferably 3.5 or less, more preferably 3.4 or less, still more preferably 3.3 or less, and particularly preferably 3. 2 or less.
  • the ion exchange chromatography step is performed after the affinity chromatography step or after virus inactivation.
  • the cation exchange chromatography step or the anion exchange chromatography step can be arbitrarily selected first, in the embodiment, pH adjustment independent of buffer exchange may be performed before the ion exchange chromatography step. I can do it. Moreover, pH adjustment may or may not be performed between the cation exchange chromatography step and the anion exchange chromatography step.
  • impurities can be efficiently removed by adjusting the pH to the basic side before the anion exchange chromatography step.
  • the cation exchange chromatography step is performed after the anion exchange chromatography step, the impurities can be efficiently removed by adjusting the pH to the acidic side before the cation exchange chromatography step.
  • dilute when diluting, dilute by adding an aqueous solution with lower electrical conductivity than demineralized water or an antibody solution to reduce the electrical conductivity. 2.0 times or less, more preferably 1.7 times or less, still more preferably 1.5 times or less, still more preferably 1.3 times or less, still more preferably 1.2 times or less, particularly preferably Is 1.1 times or less.
  • the pH in the cation exchange chromatography step is preferably 4.0 or more, more preferably 5.0 or more, still more preferably 6.0 or more, and particularly preferably 7.0 or more.
  • the electrical conductivity is preferably 5.0 mS / cm or less, more preferably 4.0 mS / cm or less, still more preferably 3.0 mS / cm or less, and particularly preferably 2.0 mS / cm or less.
  • the pH in the anion exchange chromatography step is preferably 5.0 or more, more preferably 6.0 or more, still more preferably 7.0 or more, and particularly preferably 7.5 or more.
  • the electrical conductivity is preferably 5.0 mS / cm or less, more preferably 4.0 mS / cm or less, still more preferably 3.0 mS / cm or less, and particularly preferably 2.0 mS / cm or less.
  • the base used for adjusting the pH to the basic side is not particularly limited.
  • a Tris solution can be used.
  • the concentration of the Tris solution is 1 mol / L, A concentration as high as 2 mol / L is desirable.
  • an acid used when adjusting pH to the acidic side An acetic acid, hydrochloric acid solution, etc. are mentioned.
  • the density per volume of the carrier is higher than 30 mmol / L, preferably higher than 40 mmol / L, more preferably higher than 45 mmol / L, Has a cation exchange group.
  • the density of the cation exchange group is lower than 30 mmol / L, the amount of antibody that can be treated tends to decrease.
  • the cation exchange group is not particularly limited as long as impurities can be removed, but it has one or more kinds of cation exchange groups including a weak cation exchange group. Is desirable.
  • the cation exchange chromatography carrier according to the embodiment may have a strong cation exchange group or a strong cation exchange group as long as it has at least one kind of weak cation exchange group. It is only necessary to have weak cation exchange groups, and the total density of the cation exchange groups is sufficient if the density per volume of the carrier is higher than 30 mmol / L. If the total density of cation exchange groups is lower than 30 mmol / L, the amount of cation exchange groups that can be charged tends to be low, and in addition to the reduction in adsorption capacity, the applicable antibody species tend to be narrow.
  • the density per unit volume of the carrier in the total of cation exchange groups may be higher than 30 mmol / L.
  • the density of weak cation exchange groups is 5 mol / L or more, preferably 10 mol / L or more. If it is preferably 15 mmol / L or more, the charge density can be adjusted.
  • the weak cation exchange group include a carboxylic acid group, a phosphonic acid group, and a phosphoric acid group.
  • the weak cation exchange group can change the amount of charge depending on the pH of the mobile phase. Therefore, the charge density of the cation exchange chromatography carrier can be adjusted by changing the pH of the mobile phase. Therefore, any impurity can be removed by adjusting the pH according to the characteristics of the impurity to be removed.
  • the presence of a strong cation exchange group in the cation exchange chromatography carrier can suppress the performance of the cation exchange chromatography carrier from being greatly influenced by a slight change in pH.
  • strong cation exchange groups include sulfonic acid groups.
  • examples of the cation exchange carrier according to the embodiment include a membrane-like carrier and a bead-like carrier, and a membrane-like substrate is preferable.
  • a membrane-like carrier and a bead-like carrier By being in the form of a membrane, processing at a high flow rate is possible, and more efficient purification is possible.
  • the material of the film substrate is not particularly limited, but is preferably composed of a polyolefin polymer in order to maintain mechanical properties.
  • polyolefin polymers include olefin homopolymers such as ethylene, propylene, butylene, and vinylidene fluoride, two or more types of copolymers of these olefins, or one or more types of olefins, and perhalogenated compounds. Examples thereof include copolymers with olefins.
  • the perhalogenated olefin include tetrafluoroethylene and / or chlorotrifluoroethylene.
  • polyethylene or polyvinylidene fluoride is preferable because it is excellent in mechanical strength and can provide a high adsorption capacity for contaminants such as proteins.
  • the film substrate has, for example, a plurality of pores.
  • the pore diameter is not particularly limited, but is, for example, 5 to 1000 nm, preferably 10 nm or more, more preferably 50 nm or more, still more preferably 100 nm or more, particularly preferably 150 nm or more, or 400 nm or more. From the viewpoint of the surface area of the substrate film, it is preferably 900 nm or less, more preferably 800 nm or less, still more preferably 700 nm or less, and particularly preferably 650 nm or less. If the pore diameter is 5 nm or less, the molecular weight of the separable antibody protein tends to be low. Further, when the pore diameter is 1000 nm or more, the surface area of the film-like substrate is reduced, and the binding capacity of impurities tends to be reduced.
  • the cation exchange carrier according to the embodiment may have a polymer having a cation exchange group for coating the substrate surface on the substrate surface.
  • the polymer may be a copolymer.
  • graft polymerization As a method for fixing the copolymer to the film substrate.
  • examples of the graft polymerization method include a radiation graft polymerization method and a surface living radical polymerization method.
  • any means can be employed to generate radicals on the film substrate, but when the film substrate is irradiated with ionizing radiation This is preferable because uniform radicals are generated on the entire film-like substrate.
  • ionizing radiation ⁇ rays, electron beams, ⁇ rays, neutron rays and the like can be used.
  • electron beams or ⁇ rays are 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 is preferably 1 kGy or more and 1000 kGy or less, more preferably 2 kGy or more and 500 kGy or less, and further preferably 5 kGy or more and 200 kGy or less. If the irradiation dose is less than 1 kGy, radicals tend not to be generated uniformly. Moreover, when the irradiation dose exceeds 1000 kGy, the physical strength of the film-shaped substrate tends to be reduced.
  • radicals are generally generated on a film-like substrate, and then a pre-irradiation method in which the radical is brought into contact with the reactive compound, and the film-like substrate is brought into contact with the reactive compound. And a simultaneous irradiation method in which radicals are generated on a film-like substrate in a state.
  • any method can be applied, but a pre-irradiation method with less oligomer formation is preferable.
  • the graft chain bonding rate (graft rate) by graft polymerization may vary depending on the density of the base film.
  • the substrate film is polyethylene, it is preferably 20% or more from the viewpoint of adsorption capacity, more preferably 25% or more, and still more preferably 30% or more. Further, from the viewpoint of ensuring a mechanically stable strength, it is preferably 200% or less, more preferably 150% or less, and still more preferably 100% or less.
  • w0 is the weight of the porous hollow fiber before the reaction
  • w1 is the weight of the porous hollow fiber into which the graft chain is introduced.
  • the density of the polyvinylidene fluoride is higher than that of polyethylene, so the appropriate graft ratio is different from that of polyethylene.
  • the content is preferably 5% or more, more preferably 10% or more, still more preferably 15% or more from the viewpoint of adsorption capacity. Further, from the viewpoint of ensuring a mechanically stable strength, it is preferably 100% or less, more preferably 80% or less, and still more preferably 70% or less.
  • the copolymer having a cation exchange group since the copolymer having a cation exchange group is fixed to the membrane substrate, it can be adsorbed three-dimensionally compared to the case where the cation exchange group is distributed on the membrane substrate surface. It becomes. The effect is particularly noticeable when it has a graft structure. Therefore, antibody aggregates are more strongly adsorbed than antibody monomers, and antibody monomers can be obtained with high purity.
  • the copolymer preferably contains a neutral monomer unit having no charge in addition to a monomer unit having a cation exchange group in its composition.
  • neutral monomer units include acrylamides, methacrylamides, acrylates, and methacrylate compounds, and more specifically, 2-hydroxyethyl methacrylate, butyl methacrylate, and the like.
  • the mass ratio of the monomer unit having a cation exchange group in the copolymer is preferably lower than the mass ratio of the neutral monomer unit, More preferably, it is 2 times or more, and further preferably 3 times or more.
  • the mass of the monomer having a cation exchange group and the mass of the neutral monomer are determined as follows. I can do it.
  • (Cation exchange group monomer mass) (Cation exchange group density x carrier volume x cation exchange group monomer molecular weight)
  • (Mass of neutral monomer) (Cation exchange carrier mass-substrate carrier mass-cation exchange group monomer mass) From these mass ratios, the mass proportion of monomer units having a cation exchange group and the mass proportion of neutral monomer units can be determined.
  • the copolymer may contain one or more kinds of hydrophilic and / or hydrophobic compounds as monomer units in its composition.
  • hydrophilic and / or hydrophobic monomers By adsorbing these hydrophilic and / or hydrophobic monomers with a monomer having a cation exchange group, the adsorption power of impurities can be improved.
  • the hydrophobic interaction may cause the antibody to be adsorbed on the membrane substrate, resulting in a decrease in the recovery rate of the target antibody. is there. For this phenomenon, it is possible to prevent the antibody from adsorbing to the membrane substrate by introducing a hydrophilic monomer during the polymerization of the copolymer.
  • hydrophilic monomers examples include acrylamide, methacrylamide, and dimethylacrylamide, dimethylmethacrylamide, diethylacrylamide, diethylmethacrylamide, N-methylacrylamide, N-methylmethacrylamide, N-ethylacrylamide, N-ethylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N- (hydroxymethyl) acrylamide, N- (hydroxymethyl) methacrylamide, N- (2-hydroxyethyl) acrylamide, N- (2-hydroxyethyl) methacrylamide, etc.
  • the (meth) acrylamide compound of this is mentioned.
  • hydrophilic monomer as described above, acrylate, methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, 3-hydroxypropyl acrylate, 3-hydroxypropyl methacrylate, 4-hydroxybutyl acrylate, 4-hydroxy And (meth) acrylate compounds such as butyl methacrylate, 2- (dimethylamino) ethyl acrylate, and 2- (dimethylamino) ethyl methacrylate.
  • the adsorption selectivity between the antibody monomer and the antibody aggregate can be increased by introducing a hydrophobic monomer during polymerization of the copolymer and making use of the hydrophobic interaction.
  • a hydrophobic monomer exhibit a stronger hydrophobic interaction than antibody monomers.
  • the difference in the hydrophobic interaction between the antibody monomer and the antibody aggregate can be marked and high selectivity can be realized.
  • hydrophobic monomers include styrenes, alkyl acrylamides, alkyl methacrylamides, alkyl acrylates, and alkyl methacrylates. From the viewpoint of mechanical strength, alkyl acrylamides, alkyl methacrylamides, alkyls, etc. Acrylates and alkyl methacrylates are desirable.
  • the alkyl group a linear or branched alkyl group having 4 or more carbon atoms can substantially exhibit a hydrophobic interaction with the antibody.
  • the copolymer may contain a monomer unit containing two or more polymerizable functional groups in the monomer unit, and a crosslinked structure is constructed by copolymerizing such a monomer.
  • the advantage of the crosslinked structure is that it suppresses the rise of the graft chain and suppresses the fluid flow pressure.
  • the monomer containing two or more polymerizable functional groups in the monomer is not particularly limited, and examples of the polymerizable functional group include olefins.
  • examples of such a monomer include a (meth) acrylamide monomer, a (meth) acrylate monomer, and a monomer mixed with these functional groups.
  • (Meth) acrylamide monomers include N, N'-methylenebisacrylamide, N, N'-ethylenebisacrylamide, N, N'-propylenebisacrylamide, N, N '-(1,2-dihydroxyethylene) bis Acrylamide, N, N'-methylenebismethacrylamide, N, N'-ethylenebismethacrylamide, N, N'-propylenebismethacrylamide, N, N '-(1,2-dihydroxyethylene) bismethacrylamide, etc. Is mentioned.
  • (Meth) acrylate monomers include ethylene glycol diacrylate, 1,3-butanediol diacrylate, 1,4-butanediol diacrylate, 1,5-pentanediol diacrylate, 1,6-hexanediol diacrylate, 1,9-nonanediol diacrylate, 1,10-decanediol diacrylate, neopentyl glycol diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, polyethylene glycol diacrylate, dipropylene glycol diacrylate, tripropylene glycol diacrylate 2-hydroxy-1,3-propanediol diacrylate, 4,4′-thiodibenzenethiol diacrylate, trimethylol proppant Acrylate, pentaerythritol tetraacrylate, ethylene glycol dimethacrylate, 1,3-butanediol dimethacrylate, 1,4-butane
  • a copolymer having a cation exchange group precursor monomer as a monomer unit is produced on the surface of the substrate, and then the cation exchange group precursor is converted into a cation exchange group. Also good.
  • Examples of the functional group of such a cation exchange group precursor include an epoxy group and an ester group, and a cation exchange group precursor monomer having these functional groups is homopolymerized or copolymerized on a substrate,
  • a cation exchange carrier may be obtained by converting a functional group into a cation exchange group.
  • the copolymer provided in the cation exchange chromatography carrier according to the embodiment is fixed to the membrane-like substrate by, for example, covalent bonding.
  • the carrier only needs to have at least a weak cation exchange group and one or more kinds of cation exchange groups so as to finally have a density higher than 30 mmol / L.
  • the amount of antibody load in the cation exchange chromatography step is not particularly limited as long as impurities can be removed, but from the viewpoint of efficient purification, it is preferably 0.3 g or more, more preferably 0.5 g or more, even more preferably from the viewpoint of efficient purification. Is 0.7 g or more, more preferably 0.8 g or more, and particularly preferably 1 g or more.
  • the anion exchange chromatography support according to the embodiment may be in the form of a membrane or a bead, and is not particularly limited. However, if it is in the form of a membrane, a high flow rate is possible, and a more efficient purification process. Construction is possible.
  • the material of the membrane base material of the anion exchange chromatography carrier is not particularly limited, but is preferably composed of a polyolefin polymer in order to maintain mechanical properties.
  • polyolefin polymers include olefin homopolymers such as ethylene, propylene, butylene, and vinylidene fluoride, two or more types of copolymers of these olefins, or one or more types of olefins, and perhalogenated compounds. Examples thereof include copolymers with olefins.
  • the perhalogenated olefin include tetrafluoroethylene and / or chlorotrifluoroethylene.
  • polyethylene or polyvinylidene fluoride is preferable because it is excellent in mechanical strength and can provide a high adsorption capacity for contaminants such as proteins.
  • the film substrate has, for example, a plurality of pores.
  • the pore diameter is not particularly limited, but is, for example, 5 to 1000 nm, preferably 10 nm or more, more preferably 50 nm or more, still more preferably 100 nm or more, particularly preferably 150 nm or more, or 400 nm or more. From the viewpoint of the surface area of the substrate film, it is preferably 900 nm or less, more preferably 800 nm or less, still more preferably 700 nm or less, and particularly preferably 650 nm or less. If the pore diameter is 5 nm or less, the molecular weight of the separable antibody protein tends to be low. Further, when the pore diameter is 1000 nm or more, the surface area of the film-like substrate is reduced, and the binding capacity of impurities tends to be reduced.
  • the anion exchange chromatography carrier may have a structure composed of a base material and a polymer having an anion exchange group that covers the base material.
  • the polymer having an anion exchange group contains acrylate and / or methacrylate as a monomer unit, the hydrophobicity is improved and the impurity removability tends to be improved.
  • the mass ratio of the acrylate monomer and / or methacrylate monomer in the polymer is preferably 20% or more, more preferably 30% or more, still more preferably 40% or more, and still more preferably 50%. Above, particularly preferably 60% or more, and may be 100%.
  • the polymer is more hydrophobic than a polymer consisting only of acrylamide, and improves impurity removal.
  • styrene or the like may be introduced as a method for improving hydrophobicity.
  • styrene or the like has strong hydrophobicity and cannot efficiently remove impurities. There is a tendency, and polymers containing acrylate and methacrylate as monomer units are suitable.
  • the anion exchange chromatographic support When the anion exchange chromatographic support according to the embodiment includes a base material and a polymer having an anion exchange group that covers the base material, the support has a graft structure in which the polymer is covalently bonded to the base material. May be. By fixing the anion exchange group to the graft chain, the anion exchange group is sterically arranged, and the impurities are sterically adsorbed and tend to be removed more efficiently.
  • graft polymerization As a method for forming the graft structure, there is graft polymerization as in the case of the cation exchange carrier.
  • the graft polymerization method include a radiation graft polymerization method and a surface living radical polymerization method.
  • the specific method is also the same as the above-described method for producing a cation exchange carrier.
  • the graft ratio is preferably 10% or more and 250% or less, more preferably 10% or more and 150% or less, and further preferably 20% or more and 100% or less. is there.
  • the graft ratio is 10% or more, the protein adsorption capacity is remarkably increased.
  • the graft ratio is 250% or less, practical physical strength can be obtained.
  • Examples of the graft chain of the anion exchanger according to the embodiment include glycidyl methacrylate. After graft polymerization of glycidyl methacrylate, it is possible to react an epoxy group with an amine to introduce an anion exchange group.
  • the anion exchange ligand according to the embodiment is not particularly limited as long as it exhibits sufficient impurity removability, but the main component is preferably an amino group.
  • the functional group on the amino group is not particularly limited and includes a strong anion exchange group and a weak anion exchange group, but a weak anion exchange group is preferred.
  • Strong anion exchange groups include quaternary ammonium having a trimethylamino group, a triethylamino group, or the like.
  • a tertiary amine is preferable, and from the viewpoint of moderate hydrophobicity, it is preferable to have two or more alkyl groups having 2 or more carbon atoms, such as diethylamino group, dipropylamino group, diisopropyl. There are an amino group, a dibutylamino group and the like, and a diethylamino group is preferable.
  • treatment with a depth filter may be performed before the cation exchange chromatography step and the anion exchange chromatography step.
  • the treatment capacities of the cation exchange chromatography step and the anion exchange chromatography step tend to be greatly improved.
  • a depth filter is less expensive than a cation exchange carrier or an anion exchange carrier, which leads to cost reduction.
  • depth filters examples include Emphaze (registered trademark), AEX Hybrid Purifier (3M company), and Zeta Plus (registered trademark, 3M company).
  • the activated carbon treatment can further improve the purity of the target antibody, but the target antibody is also adsorbed, so that the antibody recovery rate is lowered, which is not preferable.
  • the antibody of the target substance tends to be recovered with high purity by purifying with an anion exchanger having moderate hydrophobicity as described above, or by adding treatment with a depth filter. It is in. *
  • a hollow fiber cation exchange membrane having a carboxylic acid group was produced by radiation graft polymerization according to the following procedure. A solution obtained by dissolving 3.08 g of 2-hydroxyethyl methacrylate, 1.54 g of butyl methacrylate and 0.57 g of methacrylic acid in 240 mL of a 50 vol% aqueous t-butyl alcohol solution and bubbling with nitrogen for 30 minutes was used as a reaction solution.
  • a polyethylene porous hollow fiber (3.00 g, 15 cm, 15 fibers) having an outer diameter of 3.0 mm, an inner diameter of 2.0 mm, and an average pore diameter of 0.25 ⁇ m was placed in a sealed container, and the air in the container was replaced with nitrogen. Thereafter, while cooling with dry ice from the outside of the container, ⁇ rays 25 kGy were irradiated to generate radicals.
  • the obtained polyethylene porous hollow fiber having radicals was transferred to a glass container and decompressed to 200 Pa or less to remove oxygen in the reaction tube. 140 mL of the reaction solution adjusted to 40 ° C. was introduced into this, and allowed to stand for 16 hours. Thereafter, the hollow fiber was washed with methanol and vacuum dried in a vacuum dryer to obtain 5.31 g of a cation exchange membrane 1 having a graft rate of 77%.
  • the volume of one hollow fiber obtained was measured and found to be 1.05 mL.
  • This hollow fiber was hydrophilized with ethanol and replaced with water. After removing water, 10 mL of 0.1 mol / L sodium hydroxide aqueous solution was added. After standing for 1 hour, the aqueous sodium hydroxide solution was taken out and 10 mL of pure water was added. After further standing for 1 hour, pure water was recovered to recover sodium hydroxide remaining in the membrane. The collected sodium hydroxide solution was integrated and titrated with 0.1 mol / L hydrochloric acid. As a result, 7.93 mL was required for neutralization.
  • the weak cation exchange group of the membrane reacted with sodium hydroxide was 184 ⁇ mol.
  • the weak cation exchange group density could be determined, and the cation exchange group density was 175 mmol / L.
  • This was modularized (membrane volume 0.25 mL) to obtain a cation exchange membrane according to a cation exchange membrane production example.
  • the mass ratios of the cation exchange group monomer unit and the neutral monomer unit having no charge were 0.103 and 0.897, respectively.
  • FIG. 1 is an example of a chromatograph obtained by measuring an antibody solution under the above conditions.
  • FIG. 2 is an enlarged view of the chromatograph. What appears at the peaks of (1) and (2) in FIG. 2 is an antibody aggregate, and the peak at (3) is a monomer.
  • Example 1 In Example 1, a culture supernatant containing 0.163 g / L of AE6F4 antibody (human monoclonal antibody) was used as an antibody protein, and a series of purification steps by affinity chromatography, cation exchange chromatography, and anion exchange chromatography were buffered. We went without exchanging.
  • AE6F4 antibody human monoclonal antibody
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 48 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is passed through.
  • a 1 mol / L Tris buffer was added to the eluate to adjust the pH to 7.0 to obtain an antibody solution.
  • the antibody solution had an electrical conductivity of 1.7 mS / cm.
  • the antibody solution used for the cation exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates. As shown in FIG. 3, the ratio of the aggregate (1) in the antibody solution was 0.72%, the ratio of the aggregate (2) was 1.04%, and the ratio of the monomer was 98.24%. It was. The HCP content was 298 ppm, and the protein A content was 3 ppm.
  • HCP was measured using CYGNUS TECHNOLOGIES CHINESE HAMSTER OVARY Host Cell Proteins-3rd Generation ELISA kit, Protein A was measured using CYGNUS TECHNOLOGIES PROTEIN A ELISA UV.
  • an anion exchange carrier (Capto Q, GE Healthcare Japan) having a strong anion exchange group having a volume of 1 mL was used.
  • Tris buffer When 1 mol / L Tris buffer was added to the antibody solution collected in the cation exchange step to adjust the pH to 7.8, the electric conductivity was 1.8 mS / cm. Thereafter, the antibody solution was brought into contact with Capto Q.
  • the amount of antibody solution added to Capto Q was 82 mL (concentration of 2.96 mg / mL), the flow rate was 1.0 mL / min, and the temperature was 25 ° C.
  • Example 2 In Example 2, a culture supernatant containing 0.163 g / L of AE6F4 antibody (human monoclonal antibody) is used as an antibody protein, and a series of purification steps by affinity chromatography, anion exchange chromatography, and cation exchange chromatography are buffered. We went without exchanging.
  • AE6F4 antibody human monoclonal antibody
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 48 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is passed through.
  • a 1 mol / L Tris buffer was added to the eluate to adjust the pH to 7.8 to obtain an antibody solution.
  • the electric conductivity of the obtained antibody solution was 1.8 mS / cm.
  • the antibody solution used for the anion exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates. As shown in FIG. 3, the ratio of aggregate (1) in the antibody solution was 0.83%, the ratio of aggregate (2) was 1.12%, and the ratio of monomer was 98.05%. It was. The HCP content was 317 ppm, and the protein A content was 3 ppm.
  • Example 3 In Example 3, a culture supernatant containing 0.163 g / L of AE6F4 antibody (human monoclonal antibody) was used as an antibody protein, and a series of purification steps by affinity chromatography, cation exchange chromatography, and anion exchange chromatography were buffered. We went without exchanging.
  • AE6F4 antibody human monoclonal antibody
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 48 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is passed through.
  • a 1 mol / L Tris buffer was added to the eluate to adjust the pH to 7.0 to obtain an antibody solution.
  • the obtained antibody solution had an electrical conductivity of 1.7 mS / cm.
  • the antibody solution used for the cation exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates. As shown in FIG. 3, the percentage of aggregate (1) in the antibody solution was 0.79%, the percentage of aggregate (2) was 1.16%, and the percentage of monomer was 98.05%. It was. The HCP content was 390 ppm, and the protein A content was 3 ppm.
  • the amount of antibody solution added to QyuSpeed D was 82 mL (2.99 mg / mL concentration), the flow rate was 1.5 mL / min, and the temperature was 25 ° C.
  • 10 mL of a buffer (pH 7.8) having the same composition as the antibody solution was passed at a flow rate of 1.5 mL / min to wash QyuSpeed D.
  • a total of 92 mL of solution was collected in the flow-through process and the washing process.
  • the collected solution was analyzed by size exclusion chromatography (SEC) and ELISA, and the contents of aggregate components, HCP and protein A were decreased. The results are shown in FIG.
  • Example 4 In Example 4, a culture supernatant containing 0.163 g / L of AE6F4 antibody (human monoclonal antibody) is used as an antibody protein, and a series of purification steps by affinity chromatography, anion exchange chromatography, and cation exchange chromatography are buffered. We went without exchanging.
  • AE6F4 antibody human monoclonal antibody
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 48 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is passed through.
  • a 1 mol / L Tris buffer was added to the eluate to adjust the pH to 7.8 to obtain an antibody solution.
  • the electric conductivity of the obtained antibody solution was 1.8 mS / cm.
  • the antibody solution used for the anion exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates. As shown in FIG. 3, the ratio of the aggregate (1) in the antibody solution was 0.78%, the ratio of the aggregate (2) was 1.21%, and the ratio of the monomer was 98.01%. It was. The HCP content was 365 ppm, and the protein A content was 3 ppm.
  • Example 5 In Example 5, a culture supernatant containing 0.163 g / L of AE6F4 antibody (human monoclonal antibody) was used as an antibody protein, and a series of purification steps of affinity chromatography, cation exchange chromatography, and anion exchange chromatography were buffered. We went without exchanging. In the anion exchange chromatography step, 4 g or more of antibody was loaded per carrier volume.
  • AE6F4 antibody human monoclonal antibody
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 180 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is further passed through.
  • a 1 mol / L Tris buffer was added to the eluate to adjust the pH to 7.0 to obtain an antibody solution.
  • the obtained antibody solution had an electrical conductivity of 1.7 mS / cm. The same operation was performed twice in total, and each eluate was integrated.
  • the antibody solution used for the cation exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates.
  • the ratio of the aggregate (1) in the antibody solution was 0.56%
  • the ratio of the aggregate (2) was 1.79%
  • the ratio of the monomer was 97.65%.
  • the HCP content was 349 ppm and the protein A content was 4 ppm.
  • Example 6 virus inactivation and neutralization were performed by low pH treatment after the affinity chromatography step, and a series of purification steps of cation exchange chromatography and anion exchange chromatography were performed without exchanging the buffer.
  • phosphate buffer (20 mmol / L sodium phosphate + 150 mmol / L NaCl (pH 8.0)) is passed through, and 180 mL of Tris / acetate buffer (100 mmol / L (pH 8.0)) is further passed through. After washing, 360 mL of 25 mmol / L acetate buffer (pH 3.4) was passed as an eluent to elute the antibody from the column.
  • Example 7 In Example 7, a series of purification steps of affinity chromatography, cation exchange chromatography, and anion exchange chromatography are performed without exchanging the buffer, and pH adjustment is not performed between the cation exchange step and the anion exchange step. The test was carried out under three conditions of pH 7.0, 7.5, and 8.0.
  • the obtained antibody solutions had electrical conductivities of 2.1, 2.2, and 2.2, respectively.
  • the antibody solution used for the cation exchange chromatography process mentioned later was prepared by mixing with the antibody solution which is the same solution composition as the obtained antibody solution, and contained many aggregates.
  • Example 8 In Example 8, a series of purification steps of infinity chromatography, cation exchange chromatography, and anion exchange chromatography were performed without exchanging the buffer, and QyuSpeed D, Mustang Q (an anion exchange membrane) were used in the anion exchange step. Pall Corporation) and Natriflo HD-Q (NATRIX Separations) were used.
  • the affinity chromatography step of Example 8 was performed in the same manner as the affinity chromatography step of Example 5.
  • the ratio of aggregate (1) in the antibody solution was 0.76%
  • the ratio of aggregate (2) was 1.91%
  • the ratio of monomer was 97.33%.
  • the HCP content was 572 ppm
  • the protein A content was 6 ppm.
  • the flow rates were 1.5 ml / min, 1.1 mL / min, and 1.2 mL / min, respectively.
  • a buffer having the same composition as the antibody solution was passed through to wash the anion exchange membrane.
  • the buffers used for washing were 10 mL, 7.2 mL, and 8.0 mL, respectively.
  • the collected solution was analyzed by size exclusion chromatography (SEC) and ELISA, and the contents of aggregate components, HCP and protein A were decreased. The results are shown in FIG.
  • Example 9 In Example 9, a series of purification steps of finity chromatography, cation exchange chromatography, and anion exchange chromatography were performed without exchanging the buffer, and Mustang S (Pall Corporation) was used in the cation exchange step.
  • the affinity chromatography step of Example 9 was performed in the same manner as the affinity chromatography step of Example 5.
  • the ratio of aggregate (1) in the antibody solution was 0.68%, the ratio of aggregate (2) was 1.79%, and the ratio of monomer was 97.53%.
  • the HCP content was 511 ppm and the protein A content was 5 ppm.
  • Example 10 Treatment with a depth filter was performed after the affinity chromatography step, and a series of purification steps of cation exchange chromatography and anion exchange chromatography were performed without exchanging the buffer.
  • the affinity chromatography step of Example 10 was performed in the same manner as the affinity chromatography step of Example 5 except that the pH after elution was adjusted to 8.
  • the ratio of aggregate (1) in the antibody solution was 0.81%, the ratio of aggregate (2) was 1.89%, and the ratio of monomer was 97.3%.
  • the HCP content was 541 ppm and the protein A content was 5 ppm.
  • Example 10 (Depth filter, cation exchange chromatography, anion exchange chromatography process)
  • the antibody solution was converted into an emphaze (registered trademark) AEX Hybrid Pierfier (8.5 mL), a device in which four 0.25 mL cation exchange membranes 1 were connected in parallel, 0.25 mL of QyuSpeed D, was passed through a device connected in series.
  • emphaze registered trademark
  • AEX Hybrid Pierfier 8.5 mL
  • the amount of the antibody solution added was 800 mL (concentration of 2.60 mg / mL), the flow rate was 1.5 mL / min, and the temperature was 25 ° C.
  • 40 mL of a buffer (pH 8.0) having the same composition as the antibody solution was passed at a flow rate of 2.5 mL / min and washed.
  • a total of 840 mL of solution was collected in the flow-through process and the washing process.
  • the ratio of the aggregate (1) was 0%
  • the ratio of the aggregate (2) was 0.32%
  • the proportion of the monomer was 99.68%
  • the monomer recovery rate was 90%.
  • the HCP content was 3 ppm
  • the protein A content was reduced to 1 ppm or less.
  • the treatment with the depth filter increased the throughput in the cation exchange chromatography process and the anion exchange chromatography process, and the antibody monomer could be recovered with high purity and high yield.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

L'invention concerne un procédé de purification d'une substance physiologiquement active, ledit procédé comprenant une étape de chromatographie d'affinité suivie d'une étape de chromatographie d'échange de cations et d'une étape de chromatographie d'échange d'anions, dans lequel : après l'élution de la substance physiologiquement active dans l'étape de chromatographique d'affinité, la substance physiologiquement active est purifiée par la mise en œuvre de l'étape de chromatographie d'échange de cations et de l'étape de chromatographie d'échange d'anions au moyen du procédé à écoulement continu, sans échange du tampon de l'éluat qui contient la substance physiologiquement active ; et, dans l'étape de chromatographie d'échange de cations, un support d'échange de cations ayant un groupe échangeur de cations à une densité supérieure à 30 mmol/l est utilisé.
PCT/JP2015/084446 2014-12-08 2015-12-08 Procédé de purification de substance physiologiquement active WO2016093251A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2016563696A JPWO2016093251A1 (ja) 2014-12-08 2015-12-08 生理活性物質の精製方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014248375 2014-12-08
JP2014-248375 2014-12-08

Publications (1)

Publication Number Publication Date
WO2016093251A1 true WO2016093251A1 (fr) 2016-06-16

Family

ID=56107428

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/084446 WO2016093251A1 (fr) 2014-12-08 2015-12-08 Procédé de purification de substance physiologiquement active

Country Status (2)

Country Link
JP (1) JPWO2016093251A1 (fr)
WO (1) WO2016093251A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018045344A1 (fr) * 2016-09-01 2018-03-08 Takeda Vaccines, Inc. Procédés de production de virus pour produire des vaccins
WO2018047906A1 (fr) * 2016-09-09 2018-03-15 旭化成メディカル株式会社 Support pour chromatographie par échange de cations forts et son utilisation
CN111471101A (zh) * 2020-04-13 2020-07-31 国药集团武汉血液制品有限公司 一种去除人免疫球蛋白类制品中残留的IgA的方法
CN113784772A (zh) * 2019-05-03 2021-12-10 波诺和迪塔股份公司 从角豆提取物中分离松醇的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010528076A (ja) * 2007-06-01 2010-08-19 エフ．ホフマン−ラ ロシュ アーゲー 免疫グロブリン精製
JP2013189427A (ja) * 2012-03-12 2013-09-26 Emd Millipore Corp フロースルー式での生物製剤からのタンパク質凝集体の除去
WO2016013609A1 (fr) * 2014-07-25 2016-01-28 旭化成メディカル株式会社 Vecteur de chromatographie d'échange d'ions positifs et son procédé d'utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010528076A (ja) * 2007-06-01 2010-08-19 エフ．ホフマン−ラ ロシュ アーゲー 免疫グロブリン精製
JP2013189427A (ja) * 2012-03-12 2013-09-26 Emd Millipore Corp フロースルー式での生物製剤からのタンパク質凝集体の除去
WO2016013609A1 (fr) * 2014-07-25 2016-01-28 旭化成メディカル株式会社 Vecteur de chromatographie d'échange d'ions positifs et son procédé d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"Mustang(R) Chromatography Capsules and Cartridges", PALL CORPORATION, 2014, Retrieved from the Internet <URL:https://www.pall.com/ps/PDFGenerator?URL=http://www.pall.com/main/Biopharmaceuticals/printpdf.page%3Flid=gri78lef%26country=DE%26industry=Biopharmaceuticals> [retrieved on 20160224] *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018045344A1 (fr) * 2016-09-01 2018-03-08 Takeda Vaccines, Inc. Procédés de production de virus pour produire des vaccins
WO2018047906A1 (fr) * 2016-09-09 2018-03-15 旭化成メディカル株式会社 Support pour chromatographie par échange de cations forts et son utilisation
JPWO2018047906A1 (ja) * 2016-09-09 2019-04-04 旭化成メディカル株式会社 強カチオン交換クロマトグラフィー担体及びその使用方法
CN109661575A (zh) * 2016-09-09 2019-04-19 旭化成医疗株式会社 强阳离子交换层析载体及其使用方法
US11801505B2 (en) 2016-09-09 2023-10-31 Asahi Kasei Medical Co., Ltd. Strong cation exchange chromatographic matrix and method for using same
CN113784772A (zh) * 2019-05-03 2021-12-10 波诺和迪塔股份公司 从角豆提取物中分离松醇的方法
CN113784772B (zh) * 2019-05-03 2023-08-01 波诺和迪塔股份公司 从角豆提取物中分离松醇的方法
CN111471101A (zh) * 2020-04-13 2020-07-31 国药集团武汉血液制品有限公司 一种去除人免疫球蛋白类制品中残留的IgA的方法

Also Published As

Publication number Publication date
JPWO2016093251A1 (ja) 2017-05-25

Similar Documents

Publication Publication Date Title
JP6437553B2 (ja) 陽イオン交換クロマトグラフィー担体及びその使用方法
JP5981133B2 (ja) 強カチオン交換基を有する温度応答性吸着剤、及びその製造方法
JP6253584B2 (ja) 温度応答性クロマトグラフィーによる抗体の精製方法
JP6163541B2 (ja) 抗体タンパク質の精製方法
JP6665184B2 (ja) 混床イオン交換吸着剤
WO2016093251A1 (fr) Procédé de purification de substance physiologiquement active
US11801505B2 (en) Strong cation exchange chromatographic matrix and method for using same
JP7032046B2 (ja) 生理活性物質の連続的な定流速精製方法
EP2986625A2 (fr) Ligands en mode mixte
WO2012121409A1 (fr) Procédé de purification d&#39;une substance physiologiquement active au moyen d&#39;un support de chromatographie sensible à la température
JP2014129319A (ja) 抗体タンパク質の精製方法
WO2017126496A1 (fr) Procédé de purification de protéine
JP6621176B2 (ja) タンパク質の精製方法
JP2016108287A (ja) カチオン交換クロマトグラフィー担体を用いた生理活性物質の精製方法
WO2014003142A1 (fr) Anticorps
JP2015124177A (ja) 吸着材、吸着材の製造方法、及び抗体の精製方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15866686

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2016563696

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15866686

Country of ref document: EP

Kind code of ref document: A1