WO2015041218A1 - 新規抗体精製方法及びそれから得られる抗体(Novel Antibody Purification Method and Antibody obtained therefrom)、並びに陽イオン交換基を用いた新規抗体精製法及びそれから得られる抗体(Novel Antibody Purification method using Cation Exchanger and Antibody obtained therefrom) - Google Patents
新規抗体精製方法及びそれから得られる抗体(Novel Antibody Purification Method and Antibody obtained therefrom)、並びに陽イオン交換基を用いた新規抗体精製法及びそれから得られる抗体(Novel Antibody Purification method using Cation Exchanger and Antibody obtained therefrom) Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- 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
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- 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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/626—Diabody or triabody
Definitions
- the present invention includes a carrier having an affinity ligand for specifically purifying a target molecule (for example, an antibody or an antibody-derived substance, which may be collectively referred to as an antibody or the like hereinafter), and a positive molecule.
- the present invention is a novel method for purifying an antibody or the like (antibody or antibody-derived substance) using a cation exchange group (hereinafter also referred to as a carrier having a cation exchange group or a cation exchange carrier).
- a purification method such as an antibody at pH 4.0 or lower, which is not usually selected as a pH for use of a cation exchange carrier having a carboxyl group as a ligand, and an antibody obtained therefrom.
- Monoclonal antibodies which are the main components of antibody drugs with antibodies as the main drug, are expressed in the culture medium as recombinant proteins mainly using cultured mammalian cells, etc., and purified to high purity by several stages of chromatography and membrane processes. And then formulated.
- Antibody drugs are immunoglobulin G and its related substances, and are formed by fusing an Fc region, which is a constant region of an immunoglobulin molecule, with other functional proteins or peptides in addition to a molecule generally called an antibody. Fc fusion proteins (Fc-containing molecules) are included.
- Fc fusion proteins Fc-containing molecules
- low molecular weight antibodies such as Fab, scFv, and diabody are included.
- these antibody pharmaceuticals include those that are secreted and expressed in the culture supernatant of microorganisms as hosts, and those that are accumulated and expressed in cells or between the outer wall of the cells and the cell membrane, purified and formulated. included.
- the monomer is a 4 quantity consisting of two heavy chain (H chain) molecules consisting of a constant region Fc region and a variable region, and two light chain (L chain) molecules consisting of a variable region.
- H chain heavy chain
- L chain light chain
- An antibody having a body structure is defined as one molecular unit. Multimers of this unit molecule are aggregates, which are the main cause of side effects of antibody drugs.
- the patterning (platforming) of the purification technique has progressed by combining specific unit operations, and in the initial purification process (recovery process), protein A is immobilized as a ligand on a water-insoluble carrier.
- Antibody affinity separation matrices protein A carriers
- a technique is generally used in which antibody is adsorbed on a protein A carrier under neutral conditions and antibody is eluted under acidic conditions. Impurities such as aggregates are removed by a combination of ion exchange chromatography, hydrophobic interaction chromatography and the like after the A chromatography step (Non-patent Document 1, Non-patent Document 2, Non-patent Document 3, Patent Reference 5).
- An affinity ligand has a function of specifically binding to a specific molecule, and an affinity separation matrix (also referred to as an affinity chromatography carrier or affinity carrier) obtained by immobilizing the ligand on a water-insoluble carrier is a biological component or It is used for efficient separation and purification of useful substances from microorganisms including recombinants and cultured mammalian cells.
- an affinity separation matrix also referred to as an affinity chromatography carrier or affinity carrier
- Peptide properties consisting of functional variants (analogous substances) derived from microorganisms such as protein A, protein G, and protein L, or functionally obtained by recombinant expression thereof as antibody affinity ligands that are actually used in industry
- proteinaceous ligands, recombinant proteinaceous ligands such as camel single-chain antibodies and antibody Fc receptors, and chemically synthesized ligands such as thiazole derivatives, are used for purification of antibody drugs and the like.
- Antibody drugs are increasing in demand as ideal drugs because they are less toxic to chemicals and show higher specificity.
- Affinity chromatography typified by protein A chromatography is eluted at an acidic pH, but ion exchange chromatography and hydrophobic interaction chromatography in the latter stage are usually treated at a pH of 5 or higher. It was necessary to adjust pH and ionic strength.
- the cation exchange carrier is usually used at a pH higher than the pKa of its ligand. Further, adsorption / desorption at a pH lower than the isoelectric point (pI) of the protein is performed on the target protein purified by the cation exchange carrier.
- a cation exchange carrier having a sulfo group having a pKa of about 2 or a carboxyl group having a pKa of about 3 to 5 as a ligand is adsorbed and desorbed using a pH 5 to 6 buffer. And purified.
- the pH of the buffer is set between the pKa of the ligand and the pI of the target protein, but the lower the pH used, the greater the positive charge of the protein and the higher the elution ionic strength, The recovery tends to be low.
- the ionic strength of the eluate is high, there is a limitation that the ionic strength must be reduced in the subsequent process construction.
- the pH used is close to or lower than the pKa of the cation exchange ligand, the negative charge of the ligand is protonated and the binding capacity is lowered, and it has not been usually selected. Therefore, for the purification of antibodies using a cation exchange carrier, a buffer solution having a pH of 5 to 6 has been used for a cation exchange carrier having a pKa of 2 to 5.
- Patent Document 8 When an anion exchange chromatography step or a hydrophobic interaction chromatography step is used after the affinity chromatography step (Patent Document 8), when an anion exchange chromatography step is performed after the cation exchange chromatography step (Patent Document 7) Alternatively, when a plurality of target substances are recovered in the cation exchange chromatography step (Patent Document 6), it is necessary to adjust pH and ionic strength.
- the antibody affinity separation matrix shows high specificity to the antibody and can be highly purified, but even if the usage method is strictly set, the separation ability of the monomer (monomer) and aggregates is low, There was a limit to the process for removing aggregates and the like.
- the object of the present invention (first aspect) is an antibody that is the main purpose of affinity purification in the first chromatography step of the purification step of antibody-derived substances such as antibodies or Fc-containing molecules or low-molecular-weight antibodies such as Fab and scFv It is an object of the present invention to provide a novel antibody purification method capable of improving the selective separation characteristics of monomers together with increasing the purity of itself and reducing or omitting the burden on the subsequent impurity removal step with respect to the removal of aggregates and the like.
- the purpose of the present invention (second aspect) is to adjust the pH of an acidic pH eluate, which has been required in the purification of antibodies and the like, with affinity chromatography purification as a recovery chromatography step and highly purified in a subsequent process. It is another object of the present invention to provide an efficient antibody purification method that does not require ionic strength adjustment.
- the present inventor has packed both a carrier having an affinity ligand for an antibody or an antibody-derived substance and a carrier having a cation exchange group in the same column or a linked column.
- a novel separation method having both specific adsorption ability and excellent ability to remove aggregates, etc. was found, and the present invention (first aspect) was completed.
- the present inventor adsorbs and desorbs antibodies and the like on a cation exchange carrier at an acidic pH at which the antibody affinity carrier is eluted, thereby reducing impurities such as antibody aggregates.
- the inventors have found a novel method for separating a cation exchange carrier for obtaining the fraction obtained, and have completed the present invention (second embodiment).
- the gist of the present invention (first aspect) is as follows: [1] A structure in which a carrier 1 having an affinity ligand for an antibody or an antibody-derived substance and a carrier 2 having a cation exchange group are used as a linked or mixed column, and the antibody or antibody-derived substance is adsorbed and eluted as a whole. To purify antibodies or antibody-derived substances.
- a method for purifying an antibody or antibody-derived substance using the carrier 1 having an affinity ligand for the antibody or antibody-derived substance and the carrier 2 having a cation exchange group An antibody or an antibody-derived substance is contained in an integrated column in which a column packed with the carrier 2 is directly connected to the downstream side of the column packed with the carrier 1 or a mixed column packed with a mixture of both the carrier 1 and the carrier 2 Flow the solution and load the antibody or antibody-derived substance onto the column, Next, the antibody or antibody-derived substance purification method, wherein the loaded antibody or antibody-derived substance is eluted by passing the eluate through.
- the purification method according to any one of [2] to [4], wherein a washing solution having a lower ionic strength and a higher pH than the eluate is passed.
- the integrated column or mixed column is equilibrated with an equilibration solution, the antibody or antibody-derived substance-containing solution is loaded, and after the loading, the equilibration solution and the antibody or antibody-derived substance-containing solution are A wash solution having the same or higher ionic strength and a pH higher than that of the eluate is passed, and then a wash solution having a lower ionic strength than the equilibration solution and the antibody or antibody-derived substance-containing solution and a pH higher than that of the eluate.
- the carrier 1 having an affinity ligand is a carrier having protein A or a similar substance as a ligand.
- the antibody or antibody-derived substance is an immunoglobulin G, an immunoglobulin G derivative, or an Fc-containing molecule.
- the carrier 2 having a cation exchange group is a carrier having a carboxyl group as a ligand.
- An integrated column is produced by connecting a column packed with carrier 1 having an affinity ligand before a column packed with carrier 2 having a cation exchange group, and an antibody or an antibody-derived substance under neutral pH conditions
- a mixed column having the carrier 2 having a cation exchange group and a carrier 1 having an affinity ligand in a mixed state is prepared, and an antibody or an antibody-derived substance-containing solution is loaded under a neutral pH condition to have a pH of 4.0.
- the purification method according to any one of [2] to [18], wherein the antibody or antibody-derived substance is eluted using the following acidic buffer.
- the ratio of the carrier 1 and the carrier 2 constituting the mixed column is 1/20 or more and 20/1 or less on a volume basis, according to any one of [2] to [18], [21] Purification method.
- 10% DBC at a retention time of 6 minutes with respect to IgG of carrier 2 is 1/10 times or more and 10 times or less with respect to 10% DBC at a retention time of 6 minutes with respect to IgG of carrier 1 under adsorption conditions
- the gist of the present invention (second embodiment) is as follows: [1] An antibody or antibody-derived substance-containing solution is loaded on the carrier 2 having a cation exchange group having a carboxyl group-containing ligand having a pKa of 4.0 or more, and then an antibody is used using an acidic buffer having a pH of 4.0 or less Alternatively, a method of using a carrier having a cation exchange group for eluting antibody-derived substances. [2] The method according to [1], wherein the carboxyl group-containing ligand is derived from an acidic amino acid.
- An integrated column is prepared by linking a column packed with carrier 1 having an affinity ligand before a column packed with carrier 2 having a cation exchange group, and antibody or antibody-derived substance under neutral pH conditions The method according to any one of [1] to [5], wherein after loading the solution on the integrated column, the antibody or the antibody-derived substance is eluted using an acidic buffer having a pH of 4.0 or lower.
- a mixed column having the carrier 2 having a cation exchange group and a carrier 1 having an affinity ligand in a mixed state is prepared, and an antibody or an antibody-derived substance-containing solution is loaded under a neutral pH condition to have a pH of 4.0.
- the integrated column or mixed column is equilibrated with an equilibration solution, and the antibody or antibody-derived substance-containing solution is loaded, and after this loading, the equilibration solution and the antibody or antibody-derived substance-containing solution are A wash solution having the same or higher ionic strength and a pH higher than that of the eluate is passed, and then a wash solution having a lower ionic strength than the equilibration solution and the antibody or antibody-derived substance-containing solution and a pH higher than that of the eluate.
- the antibody or antibody-derived substance is an immunoglobulin G, an immunoglobulin G derivative, an Fc-containing molecule, or a Fab, scFv, diabody or antigen-binding site-containing molecule according to any one of [1] to [9] how to use.
- An antibody or antibody-derived substance purified by the method according to any one of [1] to [12].
- the affinity chromatography step which is the first step in the purification step of an antibody or an antibody-derived substance such as an Fc-containing molecule or a low-molecular-weight antibody such as Fab or scFv
- main affinity purification is performed.
- the selective separation characteristics of the monomer can be improved, and the load on the subsequent impurity removal step can be reduced.
- the eluate can be directly processed by cation exchange chromatography after the affinity chromatography step, which is the first step of the purification step for antibodies and the like. Since adsorption and desorption of antibodies, etc., to the cation exchange carrier can be performed at the same pH as the elution pH, affinity chromatography purification and cation exchange chromatography purification can be integrated and efficient process construction is possible. .
- a purified monomer antibody can be obtained in a high content (purity).
- FIG. 1 is a diagram showing a conventional flow in which a protein A affinity chromatography step, a virus inactivation step, and a cation exchange chromatography step are performed in this order.
- FIG. 2 is a flowchart showing an example of the present invention, and shows a flow of performing a virus inactivation step after simultaneously performing a protein A affinity chromatography step and a cation exchange chromatography step.
- FIG. 3 is a diagram showing 10% leakage DBC of a carrier having each cation exchange group at pH 3.7, pH 4.2, and pH 4.7.
- FIG. 4 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (hatched line) when a column packed with a carrier having a cation exchange group (cation exchange carrier A) is used (horizontal axis). Is the elution volume (mL), the left vertical axis is the peak area ratio (%), and the right vertical axis is the ionic strength (mM)).
- FIG. 5 is a diagram showing elution of a monomer antibody (outlined) and an aggregate antibody (hatched line) when a column packed with a carrier having a cation exchange group (cation exchange carrier B) is used (horizontal axis).
- FIG. 6 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (hatched line) when a column packed with a carrier having a cation exchange group (cation exchange carrier C) is used (horizontal axis). Is the elution volume (mL), the left vertical axis is the peak area ratio (%), and the right vertical axis is the ionic strength (mM)).
- FIG. 6 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (hatched line) when a column packed with a carrier having a cation exchange group (cation exchange carrier C) is used (horizontal axis).
- cation exchange carrier C carrier having a cation exchange group
- FIG. 7 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (hatched line) when a column packed with a carrier having a cation exchange group (cation exchange carrier D) is used (horizontal axis). Is the elution volume (mL), the left vertical axis is the peak area ratio (%), and the right vertical axis is the ionic strength (mM)).
- FIG. 8 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (shaded line) when a column packed with a carrier having protein A affinity ligand (protein A carrier) is used and the antibody loading is 10 mg.
- FIG. 9 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (shaded line) when a column packed with a carrier having protein A affinity ligand (protein A carrier) is used and the antibody loading is 30 mg.
- the horizontal axis represents the elution volume (mL), the left vertical axis represents the peak area value, and the right vertical axis represents the ionic strength (mM)).
- FIG. 10 is a diagram showing elution of a monomer antibody (white) and an aggregate antibody (shaded line) when a column packed with a carrier having protein A affinity ligand (protein A carrier) is used and the antibody loading is 40 mg.
- the horizontal axis represents the elution volume (mL), the left vertical axis represents the peak area value, and the right vertical axis represents the ionic strength (mM)).
- FIG. 11 shows a column packed with a carrier having a cation exchange group (SP-Sepharose Fast Flow, cation exchange carrier A) just below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- SP-Sepharose Fast Flow, cation exchange carrier A just below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- FIG. 12 shows a monomer antibody when a column packed with a carrier having a cation exchange group (cation exchange carrier A) is connected directly below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- cation exchange carrier A carrier having a cation exchange group
- FIG. 13 shows a monomer antibody when a column packed with a carrier having a cation exchange group (cation exchange carrier B) is connected directly below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- cation exchange carrier B carrier having protein A affinity ligand
- FIG. 14 shows a monomer antibody when a column packed with a carrier having a cation exchange group (cation exchange carrier C) is connected directly below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- cation exchange carrier C carrier having protein A affinity ligand
- FIG. 15 shows a monomer antibody when a column packed with a carrier having a cation exchange group (cation exchange carrier D) is connected directly below a column packed with a carrier having protein A affinity ligand (protein A carrier).
- cation exchange carrier D carrier having protein A affinity ligand
- FIG. 16 shows a monomer antibody (outlined) and an aggregate when a carrier having a protein A affinity ligand (protein A carrier) and a carrier having a cation exchange group (cation exchange carrier D) are mixed in the same column.
- the novel purification method of an antibody or the like (antibody or antibody-derived substance) of the present invention is a method in which a carrier having an affinity ligand for an antibody or the like and a carrier having a cation exchange group are connected or mixed to form an integrated antibody or the like.
- the connection column and the mixing column may be referred to as an integral column and a mixing column, respectively, and “adsorption” means “load”) ”In some cases.
- the purification method of the present invention is a method for purifying an antibody or the like using the carrier 1 having an affinity ligand for the antibody or the like and the carrier 2 having a cation exchange group, and the carrier 1 is filled.
- an antibody or the like is passed through a liquid containing an antibody or the like through an integrated column in which the column filled with the carrier 2 is directly connected to the downstream side of the column or a mixed column in which a mixture of both the carrier 1 and the carrier 2 is filled.
- the loaded antibody and the like are eluted by loading the column and then passing the eluate.
- the carrier 1 having an affinity ligand preferably does not contain a cation exchange group
- the carrier 2 having a cation exchange group preferably does not contain an affinity ligand.
- the separation of the antibody from the cation exchange carrier at an acidic elution pH for elution of the target molecule (antibody etc.) from the affinity carrier, or the affinity carrier and the cation exchange carrier It is characterized in that it is a connected or mixed column and integrally adsorbs and elutes an antibody or the like (in the present invention, the connected column and the mixed column may be referred to as an integrated column and a mixed column, respectively). "May mean" load ").
- the method of using the carrier having a cation exchange group of the present invention is the method of using an antibody or antibody-derived substance-containing solution on a carrier having a cation exchange group having a carboxyl group-containing ligand of pKa 4.0 or higher. After loading, the antibody or antibody-derived substance is eluted using an acidic buffer having a pH of 4.0 or less.
- the carrier 1 having an affinity ligand preferably does not contain a cation exchange group
- the carrier 2 having a cation exchange group preferably does not contain an affinity ligand.
- the present invention (first embodiment, second embodiment) can be carried out by operating a column in which antibody affinity chromatography purification and cation exchange chromatography purification are integrated. That is, since two chromatography steps (see FIG. 1, conventional flow) can be performed in one chromatography step (see FIG. 2, purification flow of the present invention (first embodiment, second embodiment)), the number of steps can be omitted. Production efficiency can be increased without using the solution required in each step. In addition, a high content (high purity) of the antibody monomer can be realized.
- Carrier having affinity ligand refers to the binding of an antigen and an antibody.
- carrier 1, affinity carrier or affinity carrier 1 having affinity ligand refers to the binding of an antigen and an antibody.
- the affinity ligand that can be used in the present invention is not particularly limited as long as it has a feature capable of specifically binding to an antibody or the like as a target molecule.
- a ligand or a chemically synthesized ligand (synthetic compound) is preferred.
- Peptide or protein ligands are more preferable from the viewpoint of specificity to the target molecule.
- affinity ligands for antibodies and the like are protein A, protein G, protein L, protein H, protein D, protein Arp, protein Fc ⁇ R, and antibody binding. It is particularly preferred to be a synthetic peptide ligand and related substances.
- the affinity ligand is more preferably protein A, protein G, protein L, or an analog thereof, and most preferably protein A or an analog thereof.
- the affinity ligand is not particularly limited as long as it has a target molecule binding domain (monomer peptide or protein, single domain), but a large amount in which two or more domains are linked.
- Peptide or protein (multi-domain) is preferable, 2 to 10 is more preferable, and 2 to 8, more preferably 2 to 6 domains are connected to each other, and a multimeric protein is preferable.
- These multimeric proteins may be homopolymers such as homodimers and homotrimers that are linked bodies of a single target molecule-binding domain. If the target molecules are the same, a plurality of types of target molecule-binding domains may be used. It may be a heteropolymer such as a heterodimer or heterotrimer which is a linked body.
- a method for linking the target molecule-binding domains of the affinity ligand of the present invention (first aspect, second aspect)
- a method that does not destabilize the three-dimensional structure of the multimeric protein is preferable. Examples include, but are not limited to, a linkage method via amino acids, a linkage method without involving amino acid residues of a domain sequence, or a linkage method using amino acid residues other than one or more domain sequences. .
- fusion protein obtained by fusing a multimeric protein with another protein having different functions can be preferably used.
- fusion proteins include proteins fused with albumin and GST (glutathione S-transferase), nucleic acids such as DNA aptamers, drugs such as antibiotics, proteins fused with polymers such as PEG (polyethylene glycol), etc.
- PEG polyethylene glycol
- 10% DBC at a residence time of 6 minutes for IgG of affinity carrier 1 is usually preferably 1 mg / mL or more and 100 mg / mL or less, more preferably 10 mg / mL or more, and further preferably 15 mg / mL. mL or more, even more preferably 20 mg / mL or more, particularly preferably 30 mg / mL or more.
- Such 10% DBC can be obtained by the following equation, for example.
- DBC 10% (V 10% ⁇ V d ) C 0 / V c (where V 10% is the volume of solution when IgG leaks 10%, V d is the volume in the pipe (for example, from the injection to the column inlet) And the internal volume of the pipe from the column outlet to the detector), C 0 is the antibody concentration (mg / mL) of the load solution, and V c is the column volume).
- a predetermined flow rate that is, a predetermined residence time (for example, 1 to 10 minutes, preferably 3 to 6 minutes).
- the volume average particle size of the affinity carrier is, for example, 1 ⁇ m or more and 1000 ⁇ m or less, preferably 5 ⁇ m or more and 500 ⁇ m or less, more preferably 10 ⁇ m or more and 200 ⁇ m or less, and more preferably 150 ⁇ m or less. Is more preferable, 120 ⁇ m or less is even more preferable, and 100 ⁇ m or less is particularly preferable.
- Carrier having cation exchange group in the present invention (first aspect, second aspect) (hereinafter sometimes referred to as carrier 2, cation exchange carrier or cation exchange carrier 2 having cation exchange group) Functions as a cation exchange group under conditions where the target molecule antibody or the like is eluted (desorbed) from the affinity ligand and captures the target molecule, and counter ions such as sodium ion and potassium ion Any cation exchange group that can be eluted (desorbed) depending on the ionic strength in the order of the monomer and the aggregate is immobilized on the water-insoluble carrier.
- the target molecule is recovered with a recovery rate of 80% or more, so that the cation exchange group uses a weakly acidic carboxyl group as a ligand as a cation exchange group.
- a group carboxyl group-containing ligand
- the carboxyl group-containing ligand can be derived from an acidic amino acid, and more preferably is derived from glutamic acid. Since an acidic pH of 4.0 or lower is usually used for elution from the affinity carrier, the cation exchange ligand of the present invention (first embodiment, second embodiment) can be used to obtain a high recovery rate at the pH.
- the pKa of a certain carboxyl group-containing ligand is 4.0 or more.
- the pKa of the carboxyl group-containing ligand is preferably 4.05 or more and 6.5 or less, more preferably 4.10 or more and 6.45 or less. If the pKa is low, the antibody yield may decrease.
- the cation exchange group is a carboxyl group, a sulfone group or the like.
- a carboxyl group is preferable, and a carboxyl group having a pKa of 4.0 or more is more preferable.
- the carboxyl group can be derived from an acidic amino acid, and more preferably is derived from glutamic acid.
- a weak acidic group is preferable.
- a carrier having protein A as an affinity ligand it is preferable to use a cation exchange group having a carboxyl group as a ligand as a cation exchange group.
- the pKa of the cation exchange group is preferably 3.5 or more and 6.5 or less, more preferably 4.0 or more and 6.0 or less, and still more preferably 4.1 or more and 5 or less. .5 or less. If the pKa is low, the antibody yield may decrease.
- the pKa and elution pH of the cation exchange group are determined by cation exchange. It is preferable to satisfy the relationship of the group pKa ⁇ elution pH, more preferably the relationship of the cation exchange group pKa> elution pH.
- a carrier having a cation exchange group is used alone, it has been carried out under the condition of cation exchange group pKa ⁇ elution pH, which is suitable for using a carrier having a cation exchange group and an affinity carrier in combination.
- the charge of the target protein is greatly charged on the positive side during elution, whereas the conversion of the cation exchange group ligand from the negative to the positive side is relatively suppressed. Aggregated antibodies and the like having a further positive charge can be recovered, and the antibody monomer is considered to be easily purified.
- the cation exchange carrier has a volume average particle diameter of, for example, 1 ⁇ m or more and 1000 ⁇ m or less, preferably 5 ⁇ m or more and 500 ⁇ m or less, more preferably 10 ⁇ m or more and 200 ⁇ m or less, More preferably, it is 150 micrometers or less, More preferably, it is 120 micrometers or less, Most preferably, it is 100 micrometers or less.
- the ion exchange capacity of the cation exchange carrier is preferably 0.001 mmol / mL or more and 0.5 mmol / mL or less.
- 10% DBC at a residence time of 6 minutes with respect to IgG of the carrier having a cation exchange group having the carboxyl group-containing ligand is 1 mg / mL or more and 200 mg. / ML or less, more preferably 10 mg / mL or more, further preferably 15 mg / mL or more, even more preferably 20 mg / mL or more, and particularly preferably 30 mg / mL or more.
- such 10% DBC can be calculated
- DBC 10% (V 10% ⁇ V d ) C 0 / V c (where V 10% is the volume of solution when IgG leaks 10%, V d is the volume in the pipe (for example, from the injection to the column inlet) And the internal volume of the pipe from the column outlet to the detector), C 0 is the antibody concentration (mg / mL) of the load solution, and V c is the column volume).
- a predetermined flow rate that is, a predetermined residence time (for example, 1 to 10 minutes, preferably 3 to 6 minutes) and a predetermined pH (for example, 3 to 5, particularly 4). Is preferred.
- Water-insoluble carrier (carrier)
- the “water-insoluble carrier” that can be used in the present invention is a substrate that is insoluble in water and is not particularly limited as long as the antibody affinity ligand and the cation exchange group can be immobilized.
- it consists of inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, and polysaccharides such as crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran.
- Organic carriers, and organic-organic, organic-inorganic, etc. composite carriers obtained by a combination thereof may be mentioned.
- GCL2000 a porous cellulose gel
- Sephacryl S-1000 obtained by covalently crosslinking allyldextran and methylenebisacrylamide
- TOYOPEARL an acrylate-based carrier
- Sepharose CL4B Rapid Run
- an agarose-based crosslinked carrier examples thereof include Agarose Beads and Cellufine, which is a cellulosic crosslinking carrier.
- the water-insoluble carrier used in the present invention is desirably a large surface area from the viewpoint of the processing capacity per unit time, and is a porous substance having a large number of pores of appropriate sizes.
- the form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected.
- the antibody affinity ligand and cation exchange group located on the water-insoluble carrier function in concert, so that their physical distances are close and a certain residence time is obtained, which effectively functions the separation matrix. Therefore, porous beads are preferable.
- a carrier made of a polysaccharide or modified with a monosaccharide or a polysaccharide is preferable in terms of ease of introduction of an antibody affinity ligand. .
- agarose and cellulose carrier are preferable, but not particularly limited thereto.
- a general method can be used as a method for immobilizing an affinity ligand on a water-insoluble carrier or separation matrix.
- the amino group of the antibody affinity ligand may be bound to the carrier via a formyl group introduced on the carrier, and the amino group of the antibody affinity ligand is bound to the carrier via an activated carboxyl group on the carrier. May be.
- these water-insoluble carriers are activated so that the ligand can be covalently bound to the carrier before introducing the antibody affinity ligand, but a commercially available activated carrier may be used or may be activated by itself. good.
- the functional group introduced into the water-insoluble carrier by activation is not particularly limited as long as it is a functional group capable of forming a covalent bond with the affinity ligand.
- an epoxy group epichlorohydrin
- an odor Hydroxy groups aldehyde groups or activated carboxylic acid groups activated with cyanide, N, N-disuccinimidyl carbonate (DSC), etc.
- DSC N-disuccinimidyl carbonate
- NHS N-hydroxysuccinimide
- CDI carbonyldiimidazole
- the protein ligand can be immobilized on the carrier by a method in which a part of the functional group of the protein and a part of the functional group of the carrier are reacted.
- the main functional groups (active groups) on the protein side that can be used for the N-terminal amino acid and lysine (Lys) side chain amino group, the cysteine (Cys) side chain thiol group, or the C-terminal amino acid and glutamic acid ( Glu) side chain and aspartic acid (Asp) side chain carboxyl groups, and the like, but are not limited thereto.
- a method using protein A having a cysteine at the C-terminus has been proposed as a method for controlling the orientation of the ligand and immobilizing the protein antibody affinity ligand on a water-insoluble carrier (US Patent). 6,399,750, Ljungquist C. et al., “Eur. J. Biochem.”, 1989, 186, 557-561).
- a functional group for example, charged amine is added to the linker or spacer. And a method of forming them.
- a method for immobilizing or introducing a cation exchange group into a water-insoluble carrier a method usually used for producing a cation exchange group can be used.
- a method for introducing a carboxymethyl group into a sugar skeleton there are a method for reacting monochloroacetic acid under alkaline conditions, and a method for introducing a sulfuric acid group includes reacting sulfuric acid under alkaline conditions. Is not to be done.
- a carboxyl group can also be introduced by immobilizing the amino acid via the amino group of the amino acid.
- sodium periodate is reacted with diol groups present or introduced on a water-insoluble carrier to activate the carrier, introduce aldehyde groups, and add molecules having amino groups and cation exchange groups in the same molecule
- the cation exchange group that can introduce a cation exchange group by covalently bonding an aldehyde group and an amino group on the support by a reductive amination method by carrying out a reduction treatment after imine formation is directly a water-insoluble carrier. It may be immobilized on the substrate, or may be immobilized via a spacer, a linker or the like.
- the cation exchange group, the spacer or the linker may contain a functional group having other functions
- Their molecular shape is not particularly limited.
- the method of introducing a carboxyl group of an amino acid is preferable as a material for a separation matrix for antibody purification from the viewpoint of toxicity when a ligand is eliminated.
- separation of antibodies and the like from a cation exchange carrier is carried out at an acidic elution pH at which a target molecule is eluted from an affinity carrier, or an affinity carrier and a cation exchange carrier are separated. It is characterized in that it is used as a concatenation or mixing column to adsorb and elute antibodies and the like as a whole.
- a representative example of the first chromatography and the second chromatography used in the purification platform process of the antibody drug for example, a combination of protein A chromatography and cation chromatography is used.
- the protein A chromatography of the first chromatography usually has a low ability to separate monomers and aggregates and is poor in stability of separation. Elution conditions are selected so that a high recovery rate can be obtained while minimizing denaturation and aggregation of Fc-containing molecules, and removal of aggregates and the like is performed by a subsequent process.
- cation exchange chromatography when cation exchange chromatography is selected as the second chromatography, generally, aggregates and other contaminants are removed in the adsorption / desorption mode. It is necessary to adjust pH and ionic strength of the eluate from the carrier (hereinafter also referred to as protein A carrier) having a pH suitable for adsorption by cation exchange chromatography. Therefore, after setting the conditions for the protein A chromatography step, it is necessary to set the conditions for the cation exchange chromatography step, and while there are various limiting factors, it is said that efficient separation of aggregates and the like is possible. can not cut.
- the affinity carrier and the cation exchange carrier are used to form a linked or mixed column, and adsorption and elution of antibodies and the like are performed as a whole by
- the chromatographic operation of the process can be shortened to one process, and the type and amount of the buffer solution to be used and the working time can be expected to be shortened.
- the novel antibody purification method of the present invention (first aspect, second aspect) is an elution fraction having a high monomer content in a narrow pH range where target molecules are eluted from affinity ligands by setting ionic strength and the like. It is possible to obtain Particularly in the purification of monoclonal antibodies, the elution pH is far away from the isoelectric point of the target molecule, so there is no significant difference in the elution ionic strength range for each antibody, and the conditions for using various target molecules can be set within a narrow range. Can be expected to be possible.
- the elution pH range can be set narrower, and effective washing can be performed by using alkaline CIP (cleaning in place).
- alkaline CIP cleaning in place
- the affinity carrier and cation exchange carrier linked or mixed column can exhibit high specificity by the antibody affinity ligand during adsorption, and the ionic strength can be set within the range of affinity ligand elution conditions. This is superior to other carrier combinations that do not use the affinity carrier 1 in that the use conditions can be easily set.
- the present invention (second embodiment) is used by using a pH higher than the elution pH from the affinity carrier as the elution pH from the cation exchange carrier. ). Further, the present invention (first aspect, second aspect) can be used as an integrated column in which an affinity carrier and a cation exchange carrier are connected or mixed, and can be used as a single column in the process of elution by a single elution operation.
- the present invention (first embodiment, second embodiment) relates to a method for linking an affinity carrier and a cation exchange carrier or using a mixed column.
- a target molecule such as an antibody
- a counter ion of an ion exchange group at a certain concentration or more, and the cation exchange group function does not work under the conditions, and even if it acts, the cation exchange group can be washed with a higher ionic strength.
- Non-specific adsorbate derived from can be removed by washing.
- the ionic strength does not inhibit the adsorption of affinity ligands and can adsorb target substances with high specificity, and by using a washing solution with high ionic strength, it is non-specific to the substrate, linker, spacer, ligand and target molecule.
- the adsorbed molecules can be effectively washed away.
- the culture supernatant in which the recombinant monoclonal antibody is expressed has an ionic strength close to that of body fluids such as humans. Therefore, even if it is directly applied to the linked or mixed column of the present invention (first embodiment, second embodiment), it has high specificity. In addition to being able to maintain the properties, contaminants can be further reduced by a cleaning solution having a higher ionic strength.
- a cleaning solution having a higher ionic strength.
- cation exchange in conjunction with low pH elution from the affinity ligand It is preferred to perform ionic strength-dependent elution of groups.
- the connecting portion is preferably connected by a straight pipe without providing a branch valve in the middle, but a branch valve may be inserted in the middle as long as it can be directly connected as a flow path.
- the function of the coupling or mixing column of the affinity carrier and cation exchange carrier of the present invention can be adjusted by the ratio of the affinity carrier and cation exchange carrier. If the binding capacity of the target substance under neutral conditions of the affinity carrier is greater than the binding capacity of the cation exchange carrier at the acidic elution pH from the affinity carrier, the target substance is eluted from the column even at low ionic strength during acidic elution. When the binding capacity of the affinity carrier is the same or lower than that of the cation exchange carrier, the antibody eluted from the affinity carrier is completely transferred to the cation exchange carrier at low ionic strength. In order to obtain a higher recovery rate, it is necessary to set the elution ionic strength higher. In any case, the recovery rate and the monomer ratio can be controlled by adjusting the ionic strength and / or pH.
- the binding capacity of the cation exchange carrier for IgG with a residence time of 6 minutes is preferably 10 times or less, and more preferably 5 times or less, of the binding capacity of IgG with a residence time of 6 minutes.
- the lower limit is preferably 1/10 or more, and more preferably 1/5 or more.
- the ratio of the binding capacity may be, for example, a ratio obtained from 10% DBC values of the affinity carrier and the cation exchange carrier.
- the ratio of the affinity carrier and the cation exchange carrier (affinity carrier / cation exchange carrier) constituting the integrated column or the mixed column is 1/20 or more and 20 on a volume basis. / 1 or less, more preferably 1/5 or more and 5/1 or less.
- the target molecule to be purified by linking or mixing the affinity carrier and cation exchange carrier of the present invention is an antibody or the like (immunoglobulin G and its related substances).
- Fc fusion protein Fc-containing molecule obtained by fusing the Fc region, which is the constant region of an immunoglobulin molecule, with another functional protein or peptide, and a low molecular weight antibody Used as a raw material for pharmaceuticals.
- the antibody or the like preferably includes immunoglobulin G, an immunoglobulin G derivative, an Fc-containing molecule, and further a low molecular weight antibody such as Fab, scFv, and diabody.
- an integrated column is prepared by connecting a column packed with an affinity carrier before a column packed with the cation exchange carrier.
- a neutral pH condition for example, pH 6 to 9
- the affinity molecule and cation exchange carrier are linked or the target molecule (antibody) is purified using a mixed column.
- it may include a subsequent regeneration process and / or a reuse process such as a CIP process and a re-equilibration process.
- a general affinity column chromatography purification method can be used in the adsorption step. That is, in one example, after adjusting the pH of a protein solution containing an antibody or the like (for example, immunoglobulin G) to be near neutral, the solution is used in the affinity carrier of the present invention (first embodiment, second embodiment). And a cation exchange group (cation exchange carrier) linked or mixed, and the antibody or the like (eg, immunoglobulin G) is specifically adsorbed on the column or affinity carrier packed with the affinity carrier.
- the loading pH is preferably 6 or more, more preferably 6.3 or more and 9 or less, and further preferably 6.5 or more and 8.5 or less.
- an antibody or antibody-derived substance-containing solution having a predetermined concentration is adsorbed on a carrier.
- PBS about 10 mM phosphoric acid, about 150 mM NaCl, etc.
- an equilibration step may be performed before the adsorption step.
- PBS about 10 mM phosphoric acid, about 150 mM NaCl, etc.
- the washing step an appropriate amount of a buffer solution in a condition range in which the affinity ligand functions is passed through to wash the inside of the column.
- the preferable range of pH may be the same range (pH near neutrality) as that of the load, for example, 6 or more is preferable.
- the target molecule such as an antibody (for example, immunoglobulin G) is adsorbed to the affinity carrier.
- impurities may be effectively removed by ionic strength or composition optimization at a pH near neutral.
- Conditions under which the cation exchange carrier does not function during loading and washing are preferred, i.e., use of a washing solution having a pH close to neutral and a high ionic strength above a certain level, and in this process both separation matrices and / or Impurities remaining on the column non-specifically through the immunoglobulin G can be washed.
- the washing step is performed before loading the antibody-containing solution and starting the following elution.
- the number of washing steps is, for example, at least once, preferably two times or more. is there.
- the following examples can mention the suitable example of a washing
- (1) The integrated column or the mixed column is equilibrated with an equilibration solution, and the antibody or antibody-derived substance-containing solution is loaded. After the loading, before the elution start, the equilibration solution and the antibody or antibody-derived substance-containing solution are used. It is preferable to pass a washing solution (second washing solution) having a lower ionic strength and a higher pH than the eluate.
- the washing method used in normal protein A chromatography is also applied to an integral column or a mixed column, and nonspecifically adsorbed on a protein A carrier.
- the impurities adsorbed on the cation exchange carrier can be washed away.
- the above (1) is preferably performed on, for example, an antibody with high purification purity
- the above (2) is preferably performed on, for example, a culture supernatant.
- the second cleaning solution used in the above (1) and (2) is, for example, a solution such as 10 mM Tris / HCl pH 7, and the first cleaning solution used in (2) is, for example, 10 mM Tris 1M NaCl pH 7 It is a solution such as 10 mM Tris pH 7.
- pre-washing may be performed with a solution (for example, PBS) having the same ionic strength and pH as the equilibration solution.
- the column in the ionic strength adjustment step, is replaced with a buffer solution having a low ionic strength in the vicinity of neutrality to prepare for the expression of an ionic strength-dependent elution function by the cation exchange carrier at the time of elution. More preferably, the ionic strength is lower than the ionic strength of the eluate.
- a combination of acidic pH and ionic strength allows the cation exchange separation mode to function at the time of elution from the affinity carrier, and the fraction with a high monomer content is eluted with a low ionic strength. Can be collected in fractions.
- the pH of the eluate the elution pH of an antibody or the like (for example, immunoglobulin G) from an affinity carrier can be applied. Since the pH is determined mainly by the separation conditions determined by the type of affinity carrier and antibody or the like (eg, immunoglobulin G), it does not require special conditions, but it suppresses the formation of aggregates and the like.
- the elution pH of the antibody or the like is 4.0 or less, preferably 3.95 or less, more preferably 3.9 or less, and even more preferably 3.8 or less.
- the elution pH may be, for example, 3.0 or more, preferably 3.2 or more, more preferably 3.5 or more.
- the elution pH of the antibody or the like is preferably less than 5.0, more preferably 4.5 or less, and even more preferably 4.0 or less.
- the elution pH may be, for example, 3.0 or more, preferably 3.2 or more, more preferably 3.5 or more.
- the pH of the eluate is set, for example, between 2 and 4 below 4.0 and below 4.0. However, in order to avoid acid modification of the target molecule, pH 3.0 or more is more preferable, and pH 3.5 or more is particularly preferable.
- the upper limit of the eluate pH is preferably the same as described above.
- the elution pH is generally set between 3.5 and 4.0. It is not limited.
- the elution ionic strength depends on the ratio of the affinity carrier and the cation exchange carrier, but also on the loading amount of the antibody or the like per unit volume (for example, immunoglobulin G). This makes it possible to easily set optimization points.
- the elution conditions of the cation exchange carrier of the present invention (second aspect) or the elution conditions of the present invention (first aspect) can be applied to both salt concentration gradient elution and stepwise elution. Is preferable to set conditions that can achieve antibody recovery and high monomer content by stepwise elution, but gradient elution is easier to set conditions.
- the elution of the antibody or the like may be performed at an acidic pH and an ionic strength gradient, and the elution of the antibody or the like may be performed at an acidic pH and stepwise with an ionic strength.
- the ionic strength adjustment step can be omitted if aggregates and the like remain in the column and do not enter the elution fraction even when the ionic strength and acidic elution pH are combined in the washing step.
- the pH of the eluate is made constant so that the ionic strength of the eluate increases continuously or stepwise.
- the eluate may be any commonly used one such as acetic acid and citric acid.
- the pH of the eluate is preferably 3 or more and 4.0 or less, more preferably 3.1 or more and 3.95 or less, and still more preferably 3.2 or more and 3.90 or less.
- the pH of the eluate is preferably 3 or more and 5 or less, more preferably 3.1 to 4.5, and further preferably 3.2 to 4.0.
- the ionic strength is preferably 0.1 mM or more and 2000 mM or less, more preferably 0.5 mM or more and 1000 mM or less, and further preferably 1 mM or more and 500 mM or less, continuously or stepwise.
- An antibody or the like for example, immunoglobulin G
- purified using a coupled column or mixed column of the affinity carrier and cation exchange carrier of the present invention is antibody affinity separation based on a single separation mode.
- the monomer selectivity is higher than that of the matrix, and the monomer content in the eluate is high.
- CM-Sepharose Fast Flow GE Healthcare; cation exchange carrier A), TOYOPEARL CM-650M (Tosoh; cation exchange carrier B), FRACTOGEL COO (M) (Merck; cation) Exchange carrier C) and Glyoxal-COOH (Preparation Example 1; cation exchange carrier D) were replaced with 1 M KCl (pH 2), and titrated with 0.1 M NaOH to determine pKa and ion exchange capacity. The results are shown in Table 1.
- CM-Sepharose Fast Flow GE Healthcare
- TOYOPEARL CM-650M Tosoh
- Fractogel COO M
- Glyoxal-COOH Preparation Example 1 as a carboxyl group-introduced cation exchange carrier manufactured by Omnifit
- the binding capacity was measured under the following chromatographic conditions using a human polyclonal antibody (Gamma globulin / Nichiyaku: Nippon Pharmaceutical) packed in a column (ID 0.66 cm x Height 7 cm) and prepared to 0.5 mg / mL. did.
- the pH of the load solution was 3.7, 4.2, or 4.7, and the respective binding capacities were measured as 10% leakage dynamic binding capacity (10% DBC).
- the binding capacity (10% DBC) of each carrier is shown in FIG.
- the binding capacity of various cation exchange carriers having a carboxyl group as a ligand was shown in FIG.
- no significant difference was found in the binding capacities of various cation exchange carriers having a carboxyl group as a ligand.
- Protein A chromatography condition column ID 0.66 cm x Height 7 cm, 2.4 mL volume (manufactured by Omnifit) Flow rate: 0.4 mL / min (residence time: 6 minutes), except for CIP, 0.8 mL / min polyclonal antibody (IgG): Gamma globulin Nichiyaku (Nippon Pharmaceutical) Loading solution: 2.5 mg-IgG / mL (PBS, pH 7.4) Equilibration (5 column volumes): PBS, pH 7.4 Load (10mg, 30mg or 40mg) Wash (5 column volumes): PBS, pH 7.4 Wash 2 (4 column volumes): 10 mM Tris / HCl, pH 7 Elution gradient (40 column volumes): A ⁇ B linear gradient A solution: 1 mM citric acid, pH 3.7 B liquid: 250 mM citric acid, pH 3.7 Regeneration (4 column volumes): 50 mM citric acid, 250 mM sodium chloride, pH 3.7 CIP (4 column
- the monomer and aggregate area values of each fraction up to the CIP fraction are shown in FIGS.
- the loaded antibody was recovered in the elution fraction, but the peak top of the aggregate was eluted at the initial stage together with the monomer peak, and separation of the aggregate could not be confirmed at any loading amount.
- the binding capacity (10% DBC) of the protein A carrier was about 50.3 mg / mL.
- Example 1 Separation of antibodies using an integrated column in which a column packed with a protein A carrier and a column packed with a cation exchange carrier B (TOYOPEARL CM-650M) having a carboxyl group as a ligand are connected MabSelect SuRe (GE Healthcare) as a cation exchange carrier B with TOYOPEARL CM-650M (Tosoh) packed in Omnifit columns (ID 0.66 cm x Height 7 cm), and two columns packed with protein A carrier Then, the columns packed with the cation exchange carrier B were connected in this order, and the chromatography operation was carried out as a single column.
- TOYOPEARL CM-650M cation exchange carrier B having a carboxyl group as a ligand
- the monomer recovered by the CIP fraction up to the regenerated fraction of Comparative Example 2 was 99.9%, and the monomer content was 93.5%. Even when the monomer recovery rate was 80%, the monomer content of Comparative Example 2 was 93.4%, whereas the monomer content of Example 1 was 97.1%, and the monomer content was improved. .
- Example 2 Separation of antibodies using an integrated column in which a column packed with a protein A carrier and a column packed with a cation exchange carrier C (Fractogel COO (M)) having a carboxyl group as a ligand are connected.
- a column packed with a protein A carrier and a column packed with a cation exchange carrier C Fractogel COO (M) (Merck) as cation exchange carrier C into Omnifit columns (ID 0.66 cm x Height 7 cm), and two columns are filled with protein A carrier And the column packed with the cation exchange carrier C were connected in this order, and the chromatography operation was carried out as a single column.
- the monomer recovered by the CIP fraction up to the regenerated fraction of Comparative Example 2 was 99.9%, and the monomer content was 93.5%. Even when the monomer recovery rate was 80%, the monomer content of Comparative Example 2 was 93.4%, whereas the monomer content of Example 2 was 99.2%, and the monomer content was improved. .
- Example 3 Separation of antibodies using a column in which a column packed with a protein A carrier and a column packed with a cation exchange carrier D (Glyoxal-COOH) having a carboxyl group as a ligand are connected.
- MabSelect SuRe GE Healthcare
- a protein A carrier Is packed in a column manufactured by Omnifit (ID 0.66 cm x Height 7 cm) as cation exchange carrier D, and two columns packed with protein A carrier, and cation exchange carrier D as cation exchange carrier D.
- the columns were connected in the order of packed columns, and chromatography operation was carried out as one column.
- FIG. 15 shows the area values of monomers and aggregates of each fraction up to the CIP fraction.
- the monomer recovery rate up to the regeneration fraction, the monomer content in the mixture up to the regeneration fraction, the monomer content in the eluate mixture up to 80% monomer recovery rate are comparative examples It is shown in comparison with 2.
- the monomer recovered by the CIP fraction up to the regenerated fraction of Comparative Example 2 was 99.9%, and the monomer content was 93.5%. Even when the monomer recovery rate is 80%, the monomer content of Comparative Example 2 is 93.4%, whereas the monomer content of Example 3 is 99.1%, which improves the monomer content. .
- Example 4 Separation of antibodies using a mixed column in which a protein A carrier and a cation exchange carrier D (Glyoxal-COOH) having a carboxyl group as a ligand are mixed and packed in one column.
- MabSelect SuRe GE Healthcare
- Glyoxal-COOH a cation exchange carrier D having a carboxyl group as a ligand
- MabSelect SuRe GE Healthcare
- Glyoxal-COOH a cation exchange carrier D in a ratio of 4: 1 (volume ratio) and packed into an Omnifit column (ID 0.66 cm ⁇ Height 7 cm) to prepare 2.5 mg / mL. Separation was performed under the chromatographic conditions of Comparative Example 2 using human polyclonal antibody (gamma globulin / Nichiyaku: Nippon Pharmaceutical) as a loading solution.
- the antibody loading was 10 mg.
- the monomer recovery rate up to the regeneration fraction, the monomer content in the mixture up to the regeneration fraction, and the monomer content in the eluate mixture up to 80% monomer recovery rate are comparative examples It is shown in comparison with 2.
- the monomer recovered by the CIP fraction up to the regenerated fraction of Comparative Example 2 was 99.9%, and the monomer content was 96.5%. Even when the monomer recovery rate was 80%, the monomer content of Comparative Example 2 was 97.1%, whereas the monomer content of Example 4 was 99.1%, and the monomer content was improved. .
- the recovery rate is as high as 80% or more. Or, even if the recovery rate is not 80% or more, a new separation mode and method of use that can improve the monomer content can be provided, which can contribute to the improvement in productivity and purification of antibody drug manufacturing processes. .
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Abstract
Description
[1] 抗体または抗体由来物質に対するアフィニティリガンドを有する担体1と陽イオン交換基を有する担体2を用いて連結または混合カラムとし、一体として抗体または抗体由来物質の吸着および溶出を行うことを特徴とする抗体または抗体由来物質の精製方法。
[2] 前記抗体または抗体由来物質に対するアフィニティリガンドを有する担体1と陽イオン交換基を有する担体2を用いた抗体または抗体由来物質の精製方法であり、
前記担体1を充填したカラムの下流側に前記担体2を充填したカラムを直結した一体型カラムまたは前記担体1と担体2の両方の混合物が充填された混合型カラムに、抗体または抗体由来物質含有液を通液して抗体または抗体由来物質をカラムに負荷し、
ついで溶出液を通液することで負荷した抗体または抗体由来物質を溶出させることを特徴とする抗体または抗体由来物質の精製方法。
[3] 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のグラジエントで行う[1]または[2]に記載の精製方法。
[4] 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のステップワイズで行う[1]または[2]に記載の精製方法。
[5] 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する[2]~[4]のいずれかに記載の精製方法。
[6] 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液と同じまたはより高いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液し、次いで平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する[2]~[4]のいずれかに記載の精製方法。
[7] 前記アフィニティリガンドを有する担体1がプロテインA、プロテインG、プロテインLまたはそれらの類縁物質をリガンドとする担体である[1]~[6]のいずれかに記載の精製方法。
[8] 前記アフィニティリガンドを有する担体1がプロテインAまたはそれらの類縁物質をリガンドとする担体である[1]~[7]のいずれかに記載の精製方法。
[9] 前記抗体または抗体由来物質が、免疫グロブリンG、免疫グロブリンG誘導体、または、Fc含有分子である[1]~[8]のいずれかに記載の精製方法。
[10] 前記抗体または抗体由来物質が、Fab、scFv、diabody、または抗原結合部位含有分子である[1]~[9]のいずれかに記載の精製方法。
[11] 前記陽イオン交換基を有する担体2が、カルボキシル基をリガンドとする担体である[1]~[10]のいずれかに記載の精製方法。
[12] 前記カルボキシル基が酸性アミノ酸に由来する[11]に記載の精製方法。
[13] 前記抗体または抗体由来物質の溶出pHが5.0未満である[1]~[12]のいずれかに記載の精製方法。
[14] 前記担体1のIgGに対する滞留時間6分での10%DBCが1mg/mL以上100mg/mL以下である[1]~[13]のいずれかに記載の精製方法。
[15] 前記担体2のイオン交換容量が0.001mmol/mL以上0.5mmol/mL以下である[1]~[14]のいずれかに記載の精製方法。
[16] 前記担体1の体積平均粒径が1μm以上1000μm以下であり、前記担体2の体積平均粒径が1μm以上1000μm以下である[1]~[15]のいずれかに記載の精製方法。
[17] pKaが4.0以上であるカルボキシル基含有リガンドを有する陽イオン交換基を有する担体2に抗体または抗体由来物質含有溶液を負荷した後、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する[1]~[16]のいずれかに記載の精製方法。
[18] 前記カルボキシル基含有リガンドを有する陽イオン交換基を有する担体2のIgGに対する滞留時間6分での10%DBCが1mg/mL以上200mg/mL以下である[1]~[17]のいずれかに記載の精製方法。
[19] 前記陽イオン交換基を有する担体2を充填したカラムの前にアフィニティリガンドを有する担体1を充填したカラムを連結して一体型カラムを作製し、中性pH条件で抗体または抗体由来物質含有溶液を前記一体型カラムに負荷した後に、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する[2]~[18]のいずれかに記載の精製方法。
[20] 前記一体型カラムを構成する担体1と担体2の割合が、体積基準で、1/20以上20/1以下である[2]~[19]のいずれかに記載の精製方法。
[21] 前記陽イオン交換基を有する担体2をアフィニティリガンドを有する担体1と共に混合状態で有する混合型カラムを作製し、中性pH条件で抗体又は抗体由来物質含有溶液を負荷してpH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する[2]~[18]のいずれかに記載の精製方法。
[22] 前記混合型カラムを構成する担体1と担体2の割合が、体積基準で、1/20以上20/1以下である[2]~[18]、[21]のいずれかに記載の精製方法。
[23] 吸着条件下における担体1のIgGに対する滞留時間6分での10%DBCに対する、担体2のIgGに対する滞留時間6分での10%DBCが、1/10倍以上10倍以下である[1]~[22]のいずれかに記載の精製方法。
[24] [1]~[23]のいずれかに記載の精製方法で精製された抗体または抗体由来物質。
[1] pKaが4.0以上であるカルボキシル基含有リガンドを有する陽イオン交換基を有する担体2に抗体または抗体由来物質含有溶液を負荷した後、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する陽イオン交換基を有する担体の使用方法。
[2] 前記カルボキシル基含有リガンドが酸性アミノ酸に由来する[1]に記載の使用方法。
[3] 前記カルボキシル基含有リガンドを有する陽イオン交換基を有する担体2のIgGに対する滞留時間6分での10%DBCが1mg/mL以上200mg/mL以下である[1]または[2]に記載の使用方法。
[4] 前記陽イオン交換基を有する担体2のイオン交換容量が0.001mmol/mL以上0.5mmol/mL以下である[1]~[3]のいずれかに記載の使用方法。
[5] 前記陽イオン交換基を有する担体2の体積平均粒径が1μm以上1000μm以下である[1]~[4]のいずれかに記載の使用方法。
[6] 前記陽イオン交換基を有する担体2を充填したカラムの前にアフィニティリガンドを有する担体1を充填したカラムを連結して一体型カラムを作製し、中性pH条件で抗体または抗体由来物質含有溶液を前記一体型カラムに負荷した後に、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する[1]~[5]のいずれかに記載の使用方法。
[7] 前記陽イオン交換基を有する担体2をアフィニティリガンドを有する担体1と共に混合状態で有する混合型カラムを作製し、中性pH条件で抗体又は抗体由来物質含有溶液を負荷してpH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する[1]~[5]のいずれかに記載の使用方法。
[8] 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液よりも低イオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する[6]または[7]に記載の使用方法。
[9] 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液と同じまたはより高いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液し、次いで平衡化溶液および抗体または抗体由来物質含有溶液よりも低イオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する[6]または[7]に記載の使用方法。
[10] 前記抗体または抗体由来物質が免疫グロブリンG、免疫グロブリンG誘導体、Fc含有分子、またはFab、scFv、diabodyもしくは抗原結合部位含有分子である[1]~[9]のいずれかに記載の使用方法。
[11] 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のグラジエントで行う[1]~[10]のいずれかに記載の使用方法。
[12] 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のステップワイズで行う[1]~[10]のいずれかに記載の使用方法。
[13] [1]~[12]のいずれかに記載の使用方法で精製された抗体または抗体由来物質。
例えば、本発明(第一態様)の精製方法は、前記抗体等に対するアフィニティリガンドを有する担体1と陽イオン交換基を有する担体2を用いた抗体等の精製方法であり、前記担体1を充填したカラムの下流側に前記担体2を充填したカラムを直結した一体型カラムまたは前記担体1と担体2の両方の混合物が充填された混合型カラムに、抗体等含有液を通液して抗体等をカラムに負荷し、ついで溶出液を通液することで負荷した抗体等を溶出させることを特徴とするものである。なお、本発明(第一態様)において、アフィニティリガンドを有する担体1は、好ましくは陽イオン交換基を含まず、陽イオン交換基を有する担体2は、好ましくはアフィニティリガンドを含まない。
本発明(第一態様、第二態様)における、「アフィニティリガンドを有する担体」(以下、アフィニティリガンドを有する担体1、アフィニティ担体またはアフィニティ担体1と称する場合がある)とは、抗原と抗体の結合に代表される、特異的な分子間の親和力に基づいて、ある分子の集合から標的(目的)の分子を選択的に捕集(結合)する物質がリガンドとして水不溶性担体に固定化されたものを示す。
第一態様、第二態様において、前記アフィニティリガンドは、標的分子結合ドメイン(単量体ペプチドまたは蛋白質、単ドメイン)を有していれば特に制限されないが、2個以上のドメインが連結された多量体ペプチドまたは蛋白質(複ドメイン)が好ましく、2~10個がより好ましく、2~8個、更に2~6個のドメインが連結された多量体蛋白質であることが好ましい。これらの多量体蛋白質は、単一の標的分子結合ドメインの連結体であるホモダイマー、ホモトリマー等のホモポリマーであっても良いし、標的分子が同一であれば、複数種類の標的分子結合ドメインの連結体であるヘテロダイマー、ヘテロトリマー等のヘテロポリマーであってもよい。
斯かる10%DBCは、例えば以下の式により求めることができる。
DBC10%=(V10%-Vd)C0/Vc(式中、V10%は、IgGが10%漏出した時の溶液体積、Vdは配管内体積(例えば、インジェクションからカラム入り口までの配管内体積およびカラム出口から検出器までの配管内体積を含む)、C0は負荷液の抗体濃度(mg/mL)、Vcはカラム体積)。DBC10%を測定する場合、所定の流速、すなわち、所定の滞留時間(例えば1分~10分、好適には3~6分)で行うことが好適である。
本発明(第一態様、第二態様)における、「陽イオン交換基を有する担体」(以下、陽イオン交換基を有する担体2、陽イオン交換担体または陽イオン交換担体2と称する場合がある)は、アフィニティリガンドから標的分子である抗体等が溶出(脱離)する条件下で陽イオン交換基として機能し標的分子を捕捉できると共に、ナトリウムイオン、カリウムイオン等のカウンターイオンにより、該標的分子の単量体(モノマー)、凝集体の順序でイオン強度依存的に溶出(脱離)出来る陽イオン交換基が水不溶性担体上に固定化されているものであれば良い。アフィニティリガンドからの標的分子の溶出pHである酸性pH域において、80%以上の回収率で標的分子を回収するため、陽イオン交換基として、弱酸性基であるカルボキシル基をリガンドとする陽イオン交換基(カルボキシル基含有リガンド)の利用が好ましい。また、前記カルボキシル基含有リガンドは、酸性アミノ酸に由来することが可能で、より好ましくはグルタミン酸に由来するものである。通常、アフィニティ担体からの溶出にはpH4.0以下の酸性pHが用いられることから、当該pHで高い回収率を得るには、本発明(第一態様、第二態様)の陽イオン交換リガンドであるカルボキシル基含有リガンドのpKaは4.0以上である。前記カルボキシル基含有リガンドのpKaは、好ましくは4.05以上6.5以下、より好ましくは4.10以上6.45以下である。pKaが低いと、抗体収率が低下する虞がある。
第一態様において、その好ましい態様では、陽イオン交換基が、カルボキシル基、スルホン基等であることが推奨される。中でも、カルボキシル基が好ましく、pKaが4.0以上のカルボキシル基がより好ましい。また、前記カルボキシル基は、酸性アミノ酸に由来することが可能で、より好ましくはグルタミン酸に由来するものである。さらに、アフィニティリガンドからの標的分子の溶出pH域において、局所的な酸性環境の形成を避けることが好ましく、弱酸性基であることが好ましい。たとえば、プロテインAをアフィニティリガンドとする担体を用いる場合は、陽イオン交換基としてカルボキシル基をリガンドとする陽イオン交換基の利用が好ましい。
従来では、陽イオン交換基を有する担体を単独で使用する際、陽イオン交換基pKa<溶出pHの条件で行われていたところ、陽イオン交換基を有する担体とアフィニティ担体を併用する為の好適な条件を本発明で見出したものである。前記関係を満足する限り、溶出時に標的タンパク質(抗体等)の電荷がプラス側に大きく帯電するのに対して、陽イオン交換基のリガンドがマイナスからプラス側への転化が比較的抑えられる為、プラスの電荷をさらに帯びた凝集抗体等を回収することができ、抗体モノマーが精製されやすくなると考えられる。
DBC10%=(V10%-Vd)C0/Vc(式中、V10%は、IgGが10%漏出した時の溶液体積、Vdは配管内体積(例えば、インジェクションからカラム入り口までの配管内体積およびカラム出口から検出器までの配管内体積を含む)、C0は負荷液の抗体濃度(mg/mL)、Vcはカラム体積)。DBC10%を測定する場合、所定の流速、すなわち、所定の滞留時間(例えば1分~10分、好適には3~6分)、所定pH(例えば3~5、特に4)で行うことが好適である。
本発明(第一態様、第二態様)に用いることのできる「水不溶性担体」は、水に不溶な基材であって、抗体アフィニティリガンドと陽イオン交換基を固定化できれば特に制限されないが、例えば、ガラスビーズ、シリカゲルなどの無機担体、架橋ポリビニルアルコール、架橋ポリアクリレート、架橋ポリアクリルアミド、架橋ポリスチレンなどの合成高分子や、結晶性セルロース、架橋セルロース、架橋アガロース、架橋デキストランなどの多糖類からなる有機担体、さらにはこれらの組み合わせによって得られる有機-有機、有機-無機などの複合担体などが挙げられる。市販品としては、多孔質セルロースゲルであるGCL2000、アリルデキストランとメチレンビスアクリルアミドを共有結合で架橋したSephacryl S-1000、アクリレート系の担体であるTOYOPEARL、アガロース系の架橋担体であるSepharose CL4B、Rapid Run Agarose Beads、および、セルロース系の架橋担体であるCellufineなどを例示することができる。
また、これらの水不溶性担体は、抗体アフィニティリガンド導入の前にリガンドが担体に共有結合できるように活性化されるが、市販の活性化担体を用いても良いし、自ら活性化を行っても良い。
リンカーを利用する固定化技術としては、担体とリガンドの距離を確保し、立体障害を排除して高性能化を図る方法の他、リンカーまたはスペーサーの中に官能基(例えば、帯電アミン)を付与、形成させる方法等が挙げられる。抗体アフィニティリガンドの固定化時にリンカーまたはスペーサー部分にリガンドを効果的に集積し、固定化収率の向上による分離性能の向上が検討されてきている。たとえば、リンカーアームの一部としてNHS活性化されたカルボン酸で誘導体化されたアガロース担体への蛋白質性リガンドの固定化技術が挙げられる(米国特許第5,260,373号、特開2010-133733、特開2010-133734)。
また、リンカーやスペーサーとは別に担体に会合性基を利用し、抗体アフィニティリガンドを担体に集積した後に、会合性基と抗体アフィニティリガンドの間に共有結合を形成させずに、水不溶性担体上に抗体アフィニティリガンドを個別に固定化する方法も提案されている(特開2011-256176)。
抗体医薬品の精製プラットホームプロセスに利用される第一クロマトグラフィーと第二クロマトグラフィーの代表例として、例えばプロテインAクロマトグラフィーと陽イオンクロマトグラフィーの組み合わせが用いられる。
(1)前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液(第2の洗浄液)を通液することが好ましく、(2)前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液と同じまたはより高いイオン強度の、かつ溶出液よりも高いpHの洗浄液(第1の洗浄液)を通液し、次いで平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液(第2の洗浄液)を通液することも好ましい。上記(1)および(2)の方法は、通常のプロテインAクロマトグラフィーで使用される洗浄方法を、一体型カラムまたは混合型カラムにも適用するものであり、プロテインA担体に非特異的に吸着した不純物を洗い流すことができ、陽イオン交換担体に吸着した不純物をも洗い流すことができる。また、上記(1)は、例えば精製純度の高い抗体に対して行われることが好適であり、上記(2)は、例えば培養上清に対して行われることが好適である。上記(1)および(2)で使用される第2の洗浄液は、例えば10mM Tris/HCl pH7等の溶液であり、上記(2)で使用される第1の洗浄液は、例えば10mM Tris 1M NaCl pH7、10mM Tris pH7等の溶液である。なお、上記(1)および(2)の前に、平衡化溶液と同じイオン強度およびpHの溶液(例えば、PBS)で前洗浄してもよい。
第一態様、第二態様において、例えば、抗体等の溶出を酸性pHでかつイオン強度のグラジエントで行ってもよく、抗体等の溶出を酸性pHでかつイオン強度のステップワイズで行ってもよい。なお、洗浄工程のイオン強度と酸性溶出pHの組み合わせでも凝集体等がカラムに残留し溶出画分に混入しない場合は、イオン強度調節工程を省略することが出来る。
カルボキシル基導入担体の調製(調製例1)
4%アガロースビーズとして冷水に置換したLow density Glyoxal 4 Rapid Run(ABT社)を湿潤体積として4mLを反応容器にとり、冷やした1Mグルタミン酸(pH6)で5回洗浄し、回収後にスラリーの液量を7mLとした。クロマトチャンバー内で2時間転倒攪拌した後に、1Mのジメチルアミンボラン水溶液を0.5mL追加投入し、クロマトチャンバー内で1時間30分攪拌した。更に、一晩室温で転倒攪拌した。遠心して担体を沈降させた後に液面が6mLになるように上清を除去した中に、20mgの水素化ホウ素ナトリウムを直接加え、室温で更に2時間転倒攪拌した。水、0.1Mクエン酸、0.1M水酸化ナトリウム、および0.5MのNaClを添加したPBSで十分に洗浄し、グルタミン酸のアミノ基を介し還元的アミノ化法でアルデヒド基にカルボキシル基を導入したアガロース担体を得た。本陽イオン交換担体をGlyoxal-COOHとした。
カルボキシル基導入陽イオン交換担体として、CM-Sepharose Fast Flow(GEヘルスケア;陽イオン交換担体A)、TOYOPEARL CM-650M(東ソー;陽イオン交換担体B)、FRACTOGEL COO(M)(メルク;陽イオン交換担体C)、Glyoxal-COOH(調製例1;陽イオン交換担体D)を1M KCl(pH2)で置換し、0.1M NaOHで滴定してpKaとイオン交換容量を求めた。結果を表1に示した。
カルボキシル基導入陽イオン交換担体として、CM-Sepharose Fast Flow(GEヘルスケア)、TOYOPEARL CM-650M(東ソー)、Fractogel COO(M)(メルク)、Glyoxal-COOH(調製例1)をOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、0.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、以下のクロマトグラフィー条件にて結合容量を測定した。負荷液のpHを3.7、4.2、または、4.7として、それぞれの結合容量を10%漏出の動的結合容量(10%DBC)として測定した。
カラム:ID 0.66cm x Height 7cm、2.4mL容(Omnifit社製)
流速:0.4mL/分(滞留時間:6分)
ポリクローナル抗体(IgG):ガンマグロブリン・ニチヤク(日本製薬)
負荷液:0.5mg-IgG/mL(5mMクエン酸:pH3.7、4.2、または、4.7)
平衡化液:5mMクエン酸(pH3.7、4.2、または、4.7)
溶出液:50mMクエン酸、0.5M塩化ナトリウム(pH3.7)
CIP液:0.1M水酸化ナトリウム、1M塩化ナトリウム
中和・再平衡化液:5mMクエン酸(pH3.7、4.2、または、4.7)
各クロマトグラフィー溶出液をゲルろ過に供し、凝集体とモノマーを分画し、そのエリア値の比較からモノマー含量を求めた。以下にゲルろ過条件を示した。
カラム:Superdex 200 10/300 GL (ID 1cm x Height 30cm)(GEヘルスケア)
流速:0.5mL/分
検出波長:214nm
負荷液:100μL/Injection(吸光度値が1を超えない範囲に希釈)
溶離液:PBS(pH7.4)
陽イオン交換担体を用いたpH5緩衝液中での抗体の分離
陽イオン交換担体として、CM-Sepharose Fast Flow(GEヘルスケア)、TOYOPEARL CM-650M(東ソー)、Fractogel COO(M)(メルク)、Glyoxal-COOH(調製例1)をOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、0.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、以下のクロマトグラフィー条件にて分離を行った。溶出フラクションには、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
カラム:ID 0.66cm x Height 7cm、2.4mL容(Omnifit社製)
流速:0.4mL/分(滞留時間:6分)、ただし、CIP以降は、0.8mL/分
ポリクローナル抗体(IgG):ガンマグロブリン・ニチヤク(日本製薬)
負荷液:0.5mg-IgG/mL(10mMクエン酸、pH5)
平衡化(5カラム体積):10mMクエン酸、pH5
負荷(30mg)
洗浄(5カラム体積):10mMクエン酸、pH5
溶出グラジエント(40カラム体積): A→Bリニアグラジエント
A液:10mMクエン酸、pH5
B液:250mMクエン酸、pH5
再生(4カラム体積):50mMクエン酸、250mM 塩化ナトリウム、pH5
CIP(4カラム体積):0.1M水酸化ナトリウム、1M塩化ナトリウム
中和・再平衡化(4カラム体積):10mMクエン酸、pH5
フラクション:1カラム体積
プロテインA担体を用いた抗体の分離
プロテインA担体として、MabSelect SuRe(GEヘルスケア)をOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、以下のクロマトグラフィー条件にて分離を行った。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
カラム:ID 0.66cm x Height 7cm、2.4mL容(Omnifit社製)
流速:0.4mL/分(滞留時間:6分)、ただし、CIP以降は、0.8mL/分
ポリクローナル抗体(IgG):ガンマグロブリン・ニチヤク(日本製薬)
負荷液:2.5mg-IgG/mL(PBS、pH7.4)
平衡化(5カラム体積):PBS、pH7.4
負荷(10mg、30mgまたは40mg)
洗浄(5カラム体積):PBS、pH7.4
洗浄2(4カラム体積):10mM Tris/HCl、pH7
溶出グラジエント(40カラム体積): A→Bリニアグラジエント
A液:1mMクエン酸、pH3.7
B液:250mMクエン酸、pH3.7
再生(4カラム体積):50mMクエン酸、250mM 塩化ナトリウム、pH3.7
CIP(4カラム体積):0.1M水酸化ナトリウム、1M塩化ナトリウム
中和・再平衡化(4カラム体積):PBS、pH7.4
プロテインA担体を充填したカラムとスルホプロピル基をリガンドとする陽イオン交換担体(SP-Sepharose Fast Flow)を充填したカラムとを連結した一体型カラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体としてSP-Sepharose Fast Flow(GEヘルスケア)をそれぞれOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2本のカラムをプロテインA担体を充填したカラム、陽イオン交換担体を充填したカラムの順で連結し、1体のカラムとしてクロマトグラフィー操作を実施した。2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、以下のクロマトグラフィー条件にて分離を行った。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
カラム:ID 0.66cm x Height 7cm、2.4mL容(Omnifit社製)の連結体
流速:0.4mL/分(滞留時間:6分)、ただし、CIP以降は、0.8mL/分
ポリクローナル抗体(IgG):ガンマグロブリン・ニチヤク(日本製薬)
負荷液:2.5mg-IgG/mL(PBS、pH7.4;10mM リン酸、150mM NaCl等)
平衡化(5カラム体積):PBS、pH7.4
負荷(40mg)
洗浄(5カラム体積):PBS、pH7.4
洗浄2(4カラム体積):10mM Tris/HCl、pH7
溶出グラジエント(40カラム体積): A→Bリニアグラジエント
A液:1mMクエン酸、pH3.7
B液:250mMクエン酸、pH3.7
再生(4カラム体積):50mMクエン酸、250mM 塩化ナトリウム、pH3.7
CIP(4カラム体積):0.1M水酸化ナトリウム、1M塩化ナトリウム
中和・再平衡化(4カラム体積):PBS、pH7.4
プロテインA担体を充填したカラムとpKaが4.0未満のカルボキシル基をリガンドとする陽イオン交換担体A(CM-Sepharose Fast Flow)を充填したカラムとを連結した一体型カラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体AとしてCM-Sepharose Fast Flow(GEヘルスケア)をそれぞれOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2本のカラムをプロテインA担体を充填したカラム、陽イオン交換担体Aを充填したカラムの順で連結し、1体のカラムとしてクロマトグラフィー操作を実施した。2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、比較例3のクロマトグラフィー条件にて分離を行った。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
カラム:ID 0.66cm x Height 7cm、2.4mL容(Omnifit社製)の連結体
流速:0.4mL/分(滞留時間:6分)、ただし、CIP以降は、0.8mL/分
ポリクローナル抗体(IgG):ガンマグロブリン・ニチヤク(日本製薬)
負荷液:2.5mg-IgG/mL(PBS、pH7.4;10mM リン酸、150mM NaCl等)
平衡化(5カラム体積):PBS、pH7.4
負荷(40mg)
洗浄(5カラム体積):PBS、pH7.4
洗浄2(4カラム体積):10mM Tris/HCl、pH7
溶出グラジエント(40カラム体積): A→Bリニアグラジエント
A液:1mMクエン酸、pH3.7
B液:250mMクエン酸、pH3.7
再生(4カラム体積):50mMクエン酸、250mM 塩化ナトリウム、pH3.7
CIP(4カラム体積):0.1M水酸化ナトリウム、1M塩化ナトリウム
中和・再平衡化(4カラム体積):PBS、pH7.4
プロテインA担体を充填したカラムとカルボキシル基をリガンドとする陽イオン交換担体B(TOYOPEARL CM-650M)を充填したカラムとを連結した一体型カラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体BとしてTOYOPEARL CM-650M(東ソー)をそれぞれOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2本のカラムをプロテインA担体を充填したカラム、陽イオン交換担体Bを充填したカラムの順で連結し、1体のカラムとしてクロマトグラフィー操作を実施した。2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、比較例3のクロマトグラフィー条件にて分離を行った。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
表2の各連結カラムの評価結果について、再生画分までのモノマー回収率、再生画分までの混合液中のモノマー含量、モノマー回収率80%までの溶出液混合液中のモノマー含量を比較例2と比較して示した。比較例2の再生画分までのCIP画分までに回収できたモノマーは99.9%であり、モノマー含量は93.5%であった。モノマー回収率が80%の時点で比較しても、比較例2のモノマー含量が93.4%であるのに対し、実施例1のモノマー含量は97.1%であり、モノマー含量が向上した。
プロテインA担体を充填したカラムとカルボキシル基をリガンドとする陽イオン交換担体C(Fractogel COO(M))を充填したカラムとを連結した一体型カラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体CとしてFractogel COO(M)(メルク)をそれぞれOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2本のカラムをプロテインA担体を充填したカラム、陽イオン交換担体Cを充填したカラムの順で連結し、1体のカラムとしてクロマトグラフィー操作を実施した。2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、比較例3のクロマトグラフィー条件にて分離を行った。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
表2の各連結カラムの評価結果について、再生画分までのモノマー回収率、再生画分までの混合液中のモノマー含量、モノマー回収率80%までの溶出液混合液中のモノマー含量を比較例2と比較して示した。比較例2の再生画分までのCIP画分までに回収できたモノマーは99.9%であり、モノマー含量は93.5%であった。モノマー回収率が80%の時点で比較しても、比較例2のモノマー含量が93.4%であるのに対し、実施例2のモノマー含量は99.2%であり、モノマー含量が向上した。
プロテインA担体を充填したカラムとカルボキシル基をリガンドとする陽イオン交換担体D(Glyoxal-COOH)を充填したカラムとを連結したカラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体DとしてGlyoxal-COOHをそれぞれOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2本のカラムをプロテインA担体を充填したカラム、陽イオン交換担体Dを充填したカラムの順で連結し、1体のカラムとしてクロマトグラフィー操作を実施した。2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、比較例3のクロマトグラフィー条件にて分離を行った。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
表2の各連結カラムの評価結果について、再生画分までのモノマー回収率、再生画分までの混合液中のモノマー含量、モノマー回収率80%までの溶出液混合液中のモノマー含量を比較例2と比較して示した。比較例2の再生画分までのCIP画分までに回収できたモノマーは99.9%であり、モノマー含量は93.5%であった。モノマー回収率が80%の時点で比較しても、比較例2のモノマー含量が93.4%であるのに対し、実施例3のモノマー含量は99.1%であり、モノマー含量が向上した。
プロテインA担体とカルボキシル基をリガンドとする陽イオン交換担体D(Glyoxal-COOH)とを1つのカラム内に混合充填した混合型カラムを用いた抗体の分離
プロテインA担体としてMabSelect SuRe(GEヘルスケア)を、陽イオン交換担体DとしてGlyoxal-COOHをそれぞれ4:1(体積比)に混合してOmnifit社製のカラム(ID 0.66cm x Height 7cm)に充填し、2.5mg/mLに調製したヒトポリクローナル抗体(ガンマグロブリン・ニチヤク:日本製薬)を負荷液として、比較例2のクロマトグラフィー条件にて分離を行った。抗体負荷量は、10mgとした。なお、1カラム体積は、1本分の2.4mLとして操作した。溶出液には、最終濃度として50-100mMのアルギニンを添加し、pH5~6としてゲルろ過クロマトグラフィーにてモノマー含量の測定を行った。また、そのエリア値分析から各分画のモノマーおよび凝集体量を求めた。
表3の各連結カラムの評価結果について、再生画分までのモノマー回収率、再生画分までの混合液中のモノマー含量、モノマー回収率80%までの溶出液混合液中のモノマー含量を比較例2と比較して示した。比較例2の再生画分までのCIP画分までに回収できたモノマーは99.9%であり、モノマー含量は96.5%であった。モノマー回収率が80%の時点で比較しても、比較例2のモノマー含量が97.1%であるのに対し、実施例4のモノマー含量は99.1%であり、モノマー含量が向上した。
すなわち、本発明(第二態様)の使用方法を用いることで、アフィニティ担体から標的分子の溶出を行う酸性溶出pHで陽イオン交換担体からの抗体等の分離を行うこと、または、アフィニティ担体と陽イオン交換担体を連結または混合カラムとし、一体として抗体等の吸着、および溶出を行うことにより、抗体医薬品の製造プロセスの生産性向上と高純度化に寄与できる。
Claims (37)
- 抗体または抗体由来物質に対するアフィニティリガンドを有する担体1と陽イオン交換基を有する担体2を用いて連結または混合カラムとし、一体として抗体または抗体由来物質の吸着および溶出を行うことを特徴とする抗体または抗体由来物質の精製方法。
- 抗体または抗体由来物質に対するアフィニティリガンドを有する担体1と陽イオン交換基を有する担体2を用いた抗体または抗体由来物質の精製方法であり、
前記担体1を充填したカラムの下流側に前記担体2を充填したカラムを直結した一体型カラムまたは前記担体1と担体2の両方の混合物が充填された混合型カラムに、抗体または抗体由来物質含有液を通液して抗体または抗体由来物質をカラムに負荷し、
ついで溶出液を通液することで負荷した抗体または抗体由来物質を溶出させることを特徴とする抗体または抗体由来物質の精製方法。 - 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のグラジエントで行う請求項1または2に記載の精製方法。
- 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のステップワイズで行う請求項1または2に記載の精製方法。
- 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する請求項2~4のいずれかに記載の精製方法。
- 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液と同じまたはより高いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液し、次いで平衡化溶液および抗体または抗体由来物質含有溶液よりも低いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する請求項2~4のいずれかに記載の精製方法。
- 前記アフィニティリガンドを有する担体1がプロテインA、プロテインG、プロテインLまたはそれらの類縁物質をリガンドとする担体である請求項1~6のいずれかに記載の精製方法。
- 前記アフィニティリガンドを有する担体1がプロテインAまたはそれらの類縁物質をリガンドとする担体である請求項1~7のいずれかに記載の精製方法。
- 前記抗体または抗体由来物質が、免疫グロブリンG、免疫グロブリンG誘導体、または、Fc含有分子である請求項1~8のいずれかに記載の精製方法。
- 前記抗体または抗体由来物質が、Fab、scFv、diabody、または抗原結合部位含有分子である請求項1~9のいずれかに記載の精製方法。
- 前記陽イオン交換基を有する担体2が、カルボキシル基をリガンドとする担体である請求項1~10のいずれかに記載の精製方法。
- 前記カルボキシル基が酸性アミノ酸に由来する請求項11に記載の精製方法。
- 前記抗体または抗体由来物質の溶出pHが5.0未満である請求項1~12のいずれかに記載の精製方法。
- 前記担体1のIgGに対する滞留時間6分での10%DBCが1mg/mL以上100mg/mL以下である請求項1~13のいずれかに記載の精製方法。
- 前記担体2のイオン交換容量が0.001mmol/mL以上0.5mmol/mL以下である請求項1~14のいずれかに記載の精製方法。
- 前記担体1の体積平均粒径が1μm以上1000μm以下であり、前記担体2の体積平均粒径が1μm以上1000μm以下である請求項1~15のいずれかに記載の精製方法。
- pKaが4.0以上であるカルボキシル基含有リガンドを有する陽イオン交換基を有する担体2に抗体または抗体由来物質含有溶液を負荷した後、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する請求項1~16のいずれかに記載の精製方法。
- 前記カルボキシル基含有リガンドを有する陽イオン交換基を有する担体2のIgGに対する滞留時間6分での10%DBCが1mg/mL以上200mg/mL以下である請求項1~17のいずれかに記載の精製方法。
- 前記陽イオン交換基を有する担体2を充填したカラムの前にアフィニティリガンドを有する担体1を充填したカラムを連結して一体型カラムを作製し、中性pH条件で抗体または抗体由来物質含有溶液を前記一体型カラムに負荷した後に、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する請求項2~18のいずれかに記載の精製方法。
- 前記一体型カラムを構成する担体1と担体2の割合が、体積基準で、1/20以上20/1以下である請求項2~19のいずれかに記載の精製方法。
- 前記陽イオン交換基を有する担体2をアフィニティリガンドを有する担体1と共に混合状態で有する混合型カラムを作製し、中性pH条件で抗体又は抗体由来物質含有溶液を負荷してpH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する請求項2~18のいずれかに記載の精製方法。
- 前記混合型カラムを構成する担体1と担体2の割合が、体積基準で、1/20以上20/1以下である請求項2~18、21のいずれかに記載の精製方法。
- 吸着条件下における担体1のIgGに対する滞留時間6分での10%DBCに対する、担体2のIgGに対する滞留時間6分での10%DBCが、1/10倍以上10倍以下である請求項1~22のいずれかに記載の精製方法。
- 請求項1~23のいずれかに記載の精製方法で精製された抗体または抗体由来物質。
- pKaが4.0以上であるカルボキシル基含有リガンドを有する陽イオン交換基を有する担体2に抗体または抗体由来物質含有溶液を負荷した後、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する陽イオン交換基を有する担体の使用方法。
- 前記カルボキシル基含有リガンドが酸性アミノ酸に由来する請求項25に記載の使用方法。
- 前記カルボキシル基含有リガンドを有する陽イオン交換基を有する担体2のIgGに対する滞留時間6分での10%DBCが1mg/mL以上200mg/mL以下である請求項25または26に記載の使用方法。
- 前記陽イオン交換基を有する担体2のイオン交換容量が0.001mmol/mL以上0.5mmol/mL以下である請求項25~27のいずれかに記載の使用方法。
- 前記陽イオン交換基を有する担体2の体積平均粒径が1μm以上1000μm以下である請求項25~28のいずれかに記載の使用方法。
- 前記陽イオン交換基を有する担体2を充填したカラムの前にアフィニティリガンドを有する担体1を充填したカラムを連結して一体型カラムを作製し、中性pH条件で抗体または抗体由来物質含有溶液を前記一体型カラムに負荷した後に、pH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する請求項25~29のいずれかに記載の使用方法。
- 前記陽イオン交換基を有する担体2をアフィニティリガンドを有する担体1と共に混合状態で有する混合型カラムを作製し、中性pH条件で抗体又は抗体由来物質含有溶液を負荷してpH4.0以下の酸性バッファーを使用して抗体または抗体由来物質を溶出する請求項25~29のいずれかに記載の使用方法。
- 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液よりも低イオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する請求項30または31に記載の使用方法。
- 前記一体型カラムまたは混合型カラムを平衡化溶液で平衡化し、前記抗体または抗体由来物質含有溶液を負荷し、この負荷後溶出開始前に平衡化溶液および抗体または抗体由来物質含有溶液と同じまたはより高いイオン強度の、かつ溶出液よりも高いpHの洗浄液を通液し、次いで平衡化溶液および抗体または抗体由来物質含有溶液よりも低イオン強度の、かつ溶出液よりも高いpHの洗浄液を通液する請求項30または31に記載の使用方法。
- 前記抗体または抗体由来物質が免疫グロブリンG、免疫グロブリンG誘導体、Fc含有分子、またはFab、scFv、diabodyもしくは抗原結合部位含有分子である請求項25~33のいずれかに記載の使用方法。
- 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のグラジエントで行う請求項25~34のいずれかに記載の使用方法。
- 前記抗体または抗体由来物質の溶出を酸性pHでかつイオン強度のステップワイズで行う請求項25~34のいずれかに記載の使用方法。
- 請求項25~36のいずれかに記載の使用方法で精製された抗体または抗体由来物質。
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SG11201602061YA SG11201602061YA (en) | 2013-09-17 | 2014-09-16 | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom |
JP2015537927A JPWO2015041218A1 (ja) | 2013-09-17 | 2014-09-16 | 新規抗体精製方法及びそれから得られる抗体(NovelAntibodyPurificationMethodandAntibodyobtainedtherefrom)、並びに陽イオン交換基を用いた新規抗体精製法及びそれから得られる抗体(NovelAntibodyPurificationmethodusingCationExchangerandAntibodyobtainedtherefrom) |
EP14846521.4A EP3048109A4 (en) | 2013-09-17 | 2014-09-16 | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom |
CN201480051360.2A CN105555795A (zh) | 2013-09-17 | 2014-09-16 | 新抗体纯化方法和由该方法得到的抗体、以及使用了阳离子交换基团的新抗体纯化法和由该方法得到的抗体 |
US15/022,890 US10519195B2 (en) | 2013-09-17 | 2014-09-16 | Antibody purification method, antibody obtained therefrom, novel antibody purification method using cation exchanger, and antibody obtained therefrom |
KR1020167009648A KR20160054597A (ko) | 2013-09-17 | 2014-09-16 | 신규 항체 정제 방법 및 그로부터 얻어지는 항체, 및 양이온 교환기를 사용한 신규 항체 정제법 및 그로부터 얻어지는 항체 |
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EP3048109A1 (en) | 2016-07-27 |
KR20160054597A (ko) | 2016-05-16 |
EP3048109A4 (en) | 2017-04-19 |
CN105555795A (zh) | 2016-05-04 |
JP2019034963A (ja) | 2019-03-07 |
JPWO2015041218A1 (ja) | 2017-03-02 |
US10519195B2 (en) | 2019-12-31 |
US20160237113A1 (en) | 2016-08-18 |
SG11201602061YA (en) | 2016-04-28 |
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