JP6630036B2 - Method for purifying target substance and carrier for mixed mode - Google Patents

Description

  The present invention relates to a method for purifying a target substance and a mixed mode carrier.

  Monoclonal antibodies, which are the main components of antibody drugs, are expressed as recombinant proteins in cultures, mainly using cultured mammalian cells, and purified to high purity by several steps of chromatography and membrane processes before being formulated. ing. Aggregates (multimers of two or more) formed or remaining in the steps of culturing, purifying, and formulating are the main causes of side effects, and their reduction is an important issue in antibody drug production. For this reason, in each of the steps of culturing, purifying, and formulating, attempts have been made to suppress the formation of aggregates and remove them by performing complicated management and using additives. In particular, in the purification step, in addition to suppressing the formation of aggregates, its removal is important. Therefore, in the purification process, development of a simple and efficient aggregate removal technique has been demanded.

  In the step of purifying an antibody drug, a platform for a purification technique by a combination of specific unit operations has been developed, and in the purification step, a protein A carrier on which protein A is immobilized as an affinity ligand is widely used. A method is generally used in which antibodies are adsorbed on a protein A carrier under neutral conditions and the antibodies are eluted under acidic conditions.However, during this elution process, the antibodies exposed to acidic conditions tend to denature and coagulate. Aggregates are easily formed. Generally, impurities such as aggregates are removed by a combination of ion exchange chromatography, hydrophobic interaction chromatography, and the like after purification using a protein A carrier. However, when the content of aggregates is large after purification using the protein A carrier, aggregate formation in the purification using the protein A carrier may be reduced in the subsequent impurity removal step, since this may lead to a decrease in the yield of the target antibody monomer. In addition to attempts to suppress them, further attempts have been made to remove aggregates in the purification step (Patent Documents 1 to 5).

International Patent Publication No. WO 2008/085988 JP 2010-507585 A International Publication No. 2010/014933 A International Publication No. 2010/141039 International Publication No. WO 2014/034457

  The problem to be solved by the present invention is to provide a method for purifying a target which is excellent in HCP removability and excellent in the ability to separate an antibody monomer from an aggregate.

The problem to be solved by the present invention has been solved by the following means.
<1> Step A of contacting a mixed mode carrier having a group containing an acidic group and an affinity ligand on a synthetic polymer support with a target that can be captured by the affinity ligand, and the affinity ligand and the target A dissociation solution for dissociating the target, and a step B for dissociating the target by contacting the mixed mode carrier capturing the target in the step A, wherein the step B dissociates the target from the affinity ligand. The step of dissociating by at least one of a stepwise method using two or more kinds of salt concentrations in which the ionic strength increases stepwise, or a gradient method in which the salt concentration increases gradually. A method for purifying a target substance.
<2> the mix mode carrier, as the acidic group, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 _{OH, -S (= O) 2} O - M +, -S (= O) 2 O -, -O-P (= O) (OH) 2, -O-P (= O) (O - M + ^{_{) 2, -O-P (=}} O) (O -) 2, -O-P (= O) (OH) (O - M +), and -O-P (= O) ( OH) (O - ) (Wherein M ⁺ represents a counter ion, respectively).
<3> The purification method according to <1> or <2>, wherein the group having an acidic group has a pKa of −3.0 to 5.0.
<4> The purification method according to any one of <1> to <3>, wherein the content of the group containing an acidic group is 0.05 μmol or more per 1 m ² of surface area of the mixed mode carrier.
<5> The purification method according to any one of <1> to <4>, wherein the affinity ligand is a protein, a nucleic acid, or a chelate compound.
<6> The purification method according to any one of <1> to <5>, wherein the affinity ligand is protein A, protein G, protein L, or an analog thereof.
<7> The method according to any one of <1> to <6>, wherein the affinity ligand is bonded to the surface of the synthetic polymer support via a group formed by opening a cyclic ether group.
<8> The synthetic polymer support contains (M-1) a structural unit derived from an epoxy group-containing monovinyl monomer in an amount of more than 20 parts by mass and not more than 99.5 parts by mass, and ( M-2) Any of <1> to <7>, which is a solid-phase support containing a copolymer containing a structural unit derived from a polyvinyl monomer in an amount of 0.5 to 80 parts by mass based on all structural units. Purification method.
<9> The purification method according to any one of <1> to <8>, wherein the synthetic polymer support is porous.
<10> The purification method according to <1> to <9>, wherein the synthetic polymer support is in the form of particles, monolith, plate, fiber, or film.
<11> A mixed mode carrier comprising a synthetic polymer support having a group containing an acidic group and an affinity ligand.

  According to the present invention, it was possible to provide a method for purifying a target which is excellent in HCP removability and excellent in the ability to separate an antibody monomer and an aggregate.

  The method for purifying a target of the present invention comprises: a mixed mode carrier having a group containing an acidic group and an affinity ligand on a synthetic polymer support; and a step A of bringing the affinity ligand into contact with a target that can be captured, A dissociation solution for dissociating the affinity ligand and the target, and a step B for dissociating the target by contacting the mixed mode carrier capturing the target in step A, wherein the step B comprises: Under the condition that the target substance dissociates from the affinity ligand, dissociation is performed in a stepwise manner using two or more kinds of salt concentrations in which the ionic strength increases stepwise, or in a gradient manner in which the salt concentration is increased in a gradient manner. The process is characterized in that: Hereinafter, the mixed mode carrier of the present invention will be described first, and then the method of purifying the target of the present invention (hereinafter, also referred to as the purification method of the present invention) will be described. In addition, in this specification, description of ab etc. which shows a numerical range is synonymous with a or more and b or less, and includes a and b in a numerical range.

(Mix mode carrier)
The mixed mode carrier of the present invention has a group containing an acidic group and an affinity ligand on a synthetic polymer support. In the purification method of the present invention, the group containing an acidic group functions as a cation exchange group. The mixed mode carrier of the present invention is an affinity separation matrix for mixed mode in which an affinity ligand and a group containing an acidic group are both immobilized via a covalent bond on a water-insoluble synthetic polymer support. By combining the separation functions, excellent separation properties (for example, monomer selectivity and aggregate removal property) can be exhibited from the concerted action of each separation function. In particular, the mixed mode carrier of the present invention is characterized by using a synthetic polymer support whose hydrophobic interaction with an aggregate is stronger than a polysaccharide-based support, and by adopting such a configuration, By exhibiting not only the action of affinity purification and the action of ion exchange groups but also hydrophobic interaction, it is possible to exhibit higher separation performance than polysaccharide-based supports.

<Synthetic polymer support>
The synthetic polymer support that can be used in the present invention is not particularly limited as long as it is a water-insoluble substrate and can fix an affinity ligand and a group containing an acidic group. A) acrylates, poly (meth) acrylamides, polystyrenes, ethylene-maleic anhydride copolymers, and the like. Among these, from the viewpoint of the pressure resistance of the synthetic polymer support, a support composed of a synthetic polymer selected from polyvinyl alcohols, poly (meth) acrylates and poly (meth) acrylamides is more preferable. Supports containing (meth) acrylates as a main component are more preferred.

  The synthetic polymer support in the present invention is preferably a copolymer of a monofunctional monomer and a polyfunctional monomer. More specifically, the synthetic polymer support is (M-1) an epoxy group-containing monovinyl monomer. The structural unit derived from the monomer exceeds 20 parts by mass and 99.5 parts by mass or less based on the total structural unit, and the structural unit derived from the (M-2) polyvinyl monomer is 0.1 part by mass relative to the total structural unit. More preferably, it is a solid support containing a copolymer containing 5 to 80 parts by mass. Further, the synthetic polymer support may contain a natural polymer or a synthetic polymer in addition to the above copolymer. By using a copolymer in which the content of the above monomers (M-1) and (M-2) is in the above range, not only the pressure resistance but also the hydrophobic interaction with the antibody aggregate can be improved. As a result, a mixed mode carrier excellent in the performance of separating the antibody monomer and the aggregate can be obtained.

((M-1) Structural unit derived from epoxy group-containing monovinyl monomer)
The epoxy group-containing monovinyl monomer is a monomer having one polymerizable vinyl group (a group having an ethylenically unsaturated bond) and one or more epoxy groups in one molecule. The epoxy group-containing monovinyl monomer is a component for introducing an appropriate amount of an epoxy group into the solid phase carrier and obtaining an appropriate amount of ligand binding.

Examples of the epoxy group-containing monovinyl monomer include glycidyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth) acrylate, and α- (meth) acryl-ω- Hydroxy group-free (meth) acrylates such as glycidyl polyethylene glycol; hydroxy-containing (meth) acrylates such as glycerin mono (meth) acrylate glycidyl ether; aromatic monovinyl compounds such as vinylbenzyl glycidyl ether; , Allyl glycidyl ether, 3,4-epoxy-1-butene, 3,4-epoxy-3-methyl-1-butene and the like. One of these may be used alone, or two or more may be used in combination. Among these, as the epoxy group-containing monovinyl monomer, epoxy group-containing (meth) acrylates and epoxy group-containing aromatic monovinyl compounds are preferable, and epoxy group-containing (meth) acrylates are more preferable, and glycidyl. (Meth) acrylate and 4-hydroxybutyl (meth) acrylate glycidyl ether are more preferred, and glycidyl (meth) acrylate is particularly preferred. When the solid support is formed into particles by normal phase suspension polymerization, the epoxy group-containing monovinyl monomer is preferably a water-insoluble one because the amount of ring-opening epoxy groups can be easily controlled. Here, the term "water-insoluble" means that 10 g or more is not dissolved in 100 mL of water at room temperature (25 ° C.).
The epoxy group-containing monovinyl monomer may be a commercially available product, or may be synthesized and used according to a known method.

  The content of the structural unit derived from the epoxy group-containing monovinyl monomer is preferably more than 20 parts by mass and not more than 99.5 parts by mass with respect to all the structural units. Within the above range, a synthetic polymer support having excellent hydrophilicity and excellent mechanical strength can be obtained. The content of the structural unit derived from the epoxy group-containing monovinyl monomer is preferably at least 25 parts by mass, more preferably at least 30 parts by mass, still more preferably at least 40 parts by mass, particularly preferably at least 40 parts by mass, based on all structural units. It is at least 50 parts by mass, and preferably at most 95 parts by mass, more preferably at most 90 parts by mass, particularly preferably at most 80 parts by mass, based on all structural units.

((M-2) Structural unit derived from polyvinyl monomer)
The polyvinyl monomer is a vinyl monomer having two or more polymerizable vinyl groups (a group having an ethylenically unsaturated bond) in one molecule. Hereinafter, the polyvinyl monomer will be described by dividing it into a hydroxy group-free polyvinyl monomer and a hydroxy group-containing polyvinyl monomer. In addition, a polyvinyl monomer can be used individually by 1 type or in combination of 2 or more types.

Examples of the hydroxy group-free polyvinyl monomer include, for example, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, 1,4-butane (Meth) acrylic esters such as diol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate, trimethylolpropane tri (meth) acrylate, and pentaerythritol tetra (meth) acrylate; fragrance such as divinylbenzene Group polyvinyl compounds; allyl compounds such as butadiene, diallyl isocyanurate and triallyl isocyanurate; and the like, and one kind can be used alone or two or more kinds can be used in combination. Among these, (meth) acrylic acid esters and aromatic polyvinyl compounds are preferable as the hydroxy group-free polyvinyl monomer, and (meth) acrylic acid esters are more preferable.
The number of polymerizable vinyl groups contained in the hydroxy group-free polyvinyl monomer is preferably 2 to 5, more preferably 2 or 3, in one molecule.

As the hydroxy group-containing polyvinyl monomer, (meth) acrylic acid esters of polyhydric alcohols, (meth) acrylic acid esters having two or more substituents of various sugars, and (meth) acrylamides of polyhydric alcohols are preferable.
Examples of the polyhydric alcohol (meth) acrylates include glycerin di (meth) acrylate, trimethylolethanedi (meth) acrylate, trimethylolpropanedi (meth) acrylate, butanetrioldi (meth) acrylate, and pentaerythritol diacrylate. (Meth) acrylate, pentaerythritol tri (meth) acrylate, dipentaerythritol di (meth) acrylate, dipentaerythritol tri (meth) acrylate, dipentaerythritol tetra (meth) acrylate, dipentaerythritol penta (meth) acrylate, inositol Examples thereof include di (meth) acrylate, inositol tri (meth) acrylate, and inositol tetra (meth) acrylate.
Examples of di- or more substituted (meth) acrylates of various sugars include, for example, glucose di (meth) acrylate, glucose tri (meth) acrylate, glucose tetra (meth) acrylate, mannitol di (meth) acrylate, mannitol tri ( (Meth) acrylate, mannitol tetra (meth) acrylate, mannitol penta (meth) acrylate, and the like.
Furthermore, examples of the hydroxy group-containing polyvinyl monomer include, in addition to those exemplified above, dehydration condensation products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth) acrylic acid. it can.
The number of polymerizable vinyl groups contained in the hydroxy group-containing polyvinyl monomer is preferably 2 to 5, more preferably 2 or 3, and particularly preferably 3 in one molecule.

  Among these, as the polyvinyl monomer, good porosity and mechanical strength can be obtained with the use of a relatively small amount, and from the viewpoint that the amount of the epoxy group-containing monovinyl monomer is less restricted, Hydroxy group-free polyvinyl monomers are preferred, and trimethylolpropane tri (meth) acrylate, ethylene glycol di (meth) acrylate, and polyethylene glycol di (meth) acrylate are more preferred. Trimethylolpropane tri (meth) acrylate, polyethylene glycol Di (meth) acrylate is particularly preferred.

The content of the structural unit derived from the polyvinyl monomer is preferably from 0.5 to 80 parts by mass based on all the structural units. By setting the content of the structural unit derived from the polyvinyl monomer to 0.5 parts by mass or more, the mechanical strength of the carrier increases. When the content is 80 parts by mass or less, the hydrophilicity is increased, the effect of reducing HCP is improved, and the activity of the affinity ligand is improved.
The content of the structural unit derived from the polyvinyl monomer is preferably at least 10 parts by mass, more preferably at least 15 parts by mass, still more preferably at least 20 parts by mass, particularly preferably at least 25 parts by mass with respect to all the structural units. In addition, based on all structural units, preferably 70 parts by mass or less, more preferably 65 parts by mass or less, further preferably 60 parts by mass or less, further preferably 50 parts by mass or less, particularly preferably 40 parts by mass or less. is there.

((M-3) Structural unit derived from monovinyl monomer containing no epoxy group)
The copolymer contained in the synthetic polymer support used in the present invention includes, in addition to the structural unit (M-1) and the structural unit (M-2), (M-3) a monovinyl monomer containing no epoxy group. Those further containing a structural unit derived from a monomer are preferred.

  The monovinyl monomer containing no epoxy group is a vinyl monomer having one polymerizable vinyl group (group having an ethylenically unsaturated bond) in one molecule and not containing an epoxy group. Hereinafter, the monovinyl monomer monomer containing no epoxy group will be described separately for a hydroxy-free monovinyl monomer containing no epoxy group and a hydroxy group-containing monovinyl monomer containing no epoxy group.

  The non-hydroxyl-containing monovinyl monomer containing no epoxy group is preferably a nonionic monomer from the viewpoint of preventing nonspecific adsorption of impurities during purification. Examples of such a nonionic monomer include (meth) acryl such as methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, cyclohexyl (meth) acrylate, and methoxyethyl (meth) acrylate. Acid esters; (meth) acrylamides such as (meth) acrylamide, dimethyl (meth) acrylamide, (meth) acryloylmorpholine, diacetone (meth) acrylamide, and the like; one kind alone or two or more kinds in combination. Can be used. As a nonionic monomer, hydrophobic (meth) acrylates such as 2-ethylhexyl (meth) acrylate and stearyl (meth) acrylate; and aromatic vinyl compounds such as styrene and α-methylstyrene are used. From the viewpoint of suppressing non-specific adsorption of impurities at the time of purification, it is preferable that the monomer exemplified above has an alkyl group or an alkoxyalkyl group having 5 or less carbon atoms, not from such a monomer. Preferred are (meth) acrylic acid alkyl esters and the above (meth) acrylamides.

Examples of the hydroxy group-containing monovinyl monomer containing no epoxy group include glycerol mono (meth) acrylate, trimethylolethane mono (meth) acrylate, trimethylolpropane mono (meth) acrylate, butanetriol mono (meth) acrylate, (Meth) acryl such as pentaerythritol mono (meth) acrylate, dipentaerythritol mono (meth) acrylate, inositol mono (meth) acrylate, hydroxyethyl (meth) acrylate, hydroxypropyl (meth) acrylate, polyethylene glycol (meth) acrylate Acid esters; (meth) acrylamides such as hydroxyethyl (meth) acrylamide, and the like, and one kind can be used alone or two or more kinds can be used in combination.
Further, the number of hydroxy groups contained in the hydroxy group-containing monovinyl monomer containing no epoxy group is preferably from 1 to 5 and more preferably from 1 to 3 in one molecule.

  Among them, as the monovinyl monomer containing no epoxy group, a hydroxy group-containing monovinyl monomer containing no epoxy group is preferable, and glycerol mono (meth) acrylate, hydroxyethyl (meth) acrylate, and hydroxypropyl (meth) acrylate are preferred. Acrylate and hydroxyethyl (meth) acrylamide are more preferred, glycerol mono (meth) acrylate and hydroxyethyl (meth) acrylamide are more preferred, and glycerol mono (meth) acrylate is particularly preferred.

  The content of the structural unit derived from the monovinyl monomer containing no epoxy group is preferably 40 parts by mass or less based on all the structural units. By setting the content to 40 parts by mass or less, in the case of a hydroxy group-containing monovinyl monomer, the hydrophilicity of the porous particles is improved, aggregation is suppressed, the activity of the affinity ligand is increased, and the dynamics of the target substance are increased. The amount of target binding is improved. Also, the mechanical strength is improved. The content of the above structural unit is more preferably 35 parts by mass or less, further preferably 30 parts by mass or less, and particularly preferably 20 parts by mass or less, based on all structural units. The content of the structural unit derived from the monovinyl monomer containing no epoxy group may be 0 parts by mass, but when the structural unit is contained, the content is preferably 5 parts by mass or more based on all the structural units.

  Preferred combinations of the contents of the structural units (M-1) to (M-3) are as follows: structural unit (M-1): 40 to 80 parts by mass with respect to all structural units, structural unit (M-2): total 20 to 60 parts by mass with respect to the structural unit, structural unit (M-3): 0 to 20 parts by mass with respect to all structural units.

The synthetic polymer support used in the present invention desirably has a large surface area from the viewpoint of the processing capacity per unit time, and is preferably porous having a large number of pores of an appropriate size.
The form of the synthetic polymer support may be any of a particle form (bead form), a monolith form, a plate form, a fiber form, a film form (including a hollow fiber), and any form can be selected. In addition, considering that the synthetic polymer support is a water-insoluble carrier, porous beads, a monolith, or a membrane is preferable, and in particular, the antibody affinity ligand and the cation exchange group arranged on the water-insoluble carrier are concerted. In order to function effectively, porous beads (porous beads) are preferred because the physical distance is short and a certain residence time is obtained so that the function of the separation matrix can be effectively exhibited.

<Group containing acidic group>
In the present invention, the group containing an acidic group functions as a cation exchange group under conditions that dissociate a target such as an antibody from the affinity ligand, and can capture the target, and is counteracted by a counter ion such as sodium ion or potassium ion. It suffices that the target substance can be eluted (desorbed) in the order of the monomer and the aggregate in an order of ionic strength. In the mixed mode carrier of the present invention, a group containing an acidic group is bonded to the surface of the synthetic polymer support. Thereby, excellent antifouling property is obtained.

As the acidic group, specifically, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S ( _{^{= O) 2 O - M +}} , -S (= O) 2 O -, -O-P (= O) (OH) 2, -O-P (= O) (O - M +) 2, -O ^{-P (= O) (O -} ) 2, -O-P (= O) (OH) (O - M +), - O-P (= O) (OH) (O -) 1 monovalent etc. Acidic groups; such as -O- (O = P-OH) -O-, -O- (O = P-O ^- M ⁺ )-O-, -O- (O = P-O ^- ) ^- O- A divalent acidic group is exemplified. The mixed mode carrier may have one or more of these on a synthetic polymer support.
In addition, the above-mentioned monovalent acidic group means one acidic group with a bond, and the divalent acidic group means two acidic groups with a bond. M ⁺ represents a counter ion. Examples of the counter ion include an alkali metal ion such as a sodium ion and a potassium ion; an alkaline earth metal ion such as a magnesium ion and a calcium ion; an ammonium ion; and an organic ammonium ion.
Among these acidic groups, the above-mentioned monovalent acidic groups are preferred. From the viewpoint of antifouling property, -C (= O) OH, -C (= O) O - M +, -C (= O) O -, -S (= O) 2 OH, -S ( _{^{= O) 2 O - M +}} , -S (= O) 2 O - it is more preferred.
As the acidic group, it is preferable to avoid the formation of a local acidic environment in the pH range where the target substance is eluted from the affinity ligand, and it is preferable that the acidic group is a weakly acidic group. For example, when protein A is used as an antibody affinity ligand, it is preferable to use a carboxy group as a cation exchange group.

  The group containing an acidic group may be directly fixed on the synthetic polymer support, or may be fixed via a spacer, a linker, or the like. In addition, as long as it can function as a cation exchanger under acidic pH conditions under which the target substance dissociates from the affinity ligand, the cation exchange group, the spacer or the linker may contain a functional group having other functions, and their molecular shapes Is not particularly limited.

  The group containing an acidic group has a pKa of -3.0 to 4.0 from the viewpoint of dissociating the target substance from the affinity ligand with a small amount of a dissociation solution and efficiently separating, purifying, quantifying or detecting the target substance. Groups within the range of 5.0 are preferred. In particular, when the pKa is in the range of -3.0 to 1.0, even when a dissociation solution generally used as a dissociation solution used when dissociating a target substance is used, the The amount can be kept low, and the target can be collected at a high concentration.

  Examples of the group containing an acidic group include an organic group containing an acidic group, and preferable examples include a monovalent group represented by the following formula (1).

[In the formula (1), R ¹ represents a divalent organic group having 1 to 12 carbon atoms, and X represents an acidic group. ]

The acidic group represented by X in the above formula (1) is the same as the above monovalent acidic group.
The carbon number of the divalent organic group represented by R ¹ is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.
As the divalent organic group represented by R ¹ , a divalent group represented by the following formula (2) is preferable.

[In the formula (2), R ² represents a divalent hydrocarbon group having 1 to 12 carbon atoms, and Y ¹ represents>S,> S = O,> S (＝ O) ₂ ,> NH, or > O, and * indicates the bonding position to X in formula (1). ]

The Y ^1,> S,> NH are preferred,> S is more preferable.

The divalent hydrocarbon group represented by R ² may be linear or branched. The carbon number of the divalent hydrocarbon group is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4. Further, the divalent hydrocarbon group is preferably a divalent aliphatic hydrocarbon group, and more preferably an alkanediyl group. Specific examples of the alkanediyl group include methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, and propane-1,2. -Diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group and the like. No.

From the viewpoint of antifouling property, the content of the group containing an acidic group is preferably 0.05 μmol or more, more preferably 0.5 to 30 μmol, and more preferably 1.0 to 10 μmol, per 1 m ² of the surface area of the mixed mode carrier. More preferably, 2.0 to 5.0 μmol is particularly preferred. The content of the group containing an acidic group may be measured by the same method as in Examples described later.

<Affinity ligand>
The affinity ligand in the present invention selectively collects (binds) a target (target) molecule from a certain set of molecules based on the affinity between specific molecules represented by the binding between an antigen and an antibody. Indicates a substance. The mixed mode carrier of the present invention is preferably a carrier in which an affinity ligand is bound to the surface of a synthetic polymer support without a group containing an acidic group. As a result, multipoint binding of the affinity ligand is suppressed, and the three-dimensional structure of the affinity ligand is maintained, so that the activity of the affinity ligand is maintained and a decrease in affinity for the target can be prevented.

  In the present invention, the affinity ligand may be any as long as it specifically binds to a target substance, and examples thereof include proteins, nucleic acids, peptides, enzymes, chelating compounds, receptors, aptamers, antibodies, antigens, vitamins, and metal ions. No. Among such affinity ligands, proteins, nucleic acids, and chelating compounds are preferred. Above all, in the case of an antibody affinity ligand having a purified target as an antibody, an immunoglobulin binding protein is more preferable.

  The antibody affinity ligand which can be used in the present invention is not particularly limited as long as it has a characteristic capable of specifically binding to an antibody or an Fc-containing molecule which is a constant region of the antibody as a target substance. Sex ligands or chemically synthesized ligands (synthetic compounds) are preferred. From the viewpoint of specificity for the target substance, peptide or protein ligands are more preferable, and among them, the antibody affinity ligands are protein A, protein G, protein L, protein H, protein D, protein Arp, protein FcγR, and antibody binding synthesis. Further preferred are peptide ligands and analogs thereof. As the antibody affinity ligand, protein A, protein G, protein L and their related substances are more preferred, and protein A and its related substances are most preferred. The antibody affinity ligand is not particularly limited as long as it has a target molecule binding domain (monomeric peptide or protein, single domain), but a multimeric peptide or protein (multiple domain) in which two or more domains are linked is included. preferable. The number of the domains is more preferably 2 to 10, more preferably 2 to 8, and still more preferably 2 to 6. In particular, a multimeric protein in which 3 to 6 domains are linked is preferable. These multimeric proteins may be homodimers, such as homodimers and homotrimers, which are conjugates of a single target molecule binding domain, or, if the target is the same, multiple types of target molecule binding domains. It may be a heteropolymer such as a heterodimer or heterotrimer which is a linked body.

  As a method for linking the target molecule binding domain of the antibody affinity ligand, a method that does not destabilize the three-dimensional structure of the multimeric protein is preferable. For example, a linking method via a terminal amino acid of the domain sequence, an amino acid residue of the domain sequence Or a method of linking with amino acid residues other than one or more domain sequences, but is not limited to these methods.

  As the antibody affinity ligand, a fusion protein obtained by fusing a multimeric protein as one component and another protein having a different function can be preferably used. Examples of the fusion protein include proteins fused with albumin and GST (glutathione S-transferase), nucleic acids such as DNA aptamers, drugs such as antibiotics, and proteins fused with macromolecules such as PEG (polyethylene glycol). However, the present invention is not limited to these.

  From the viewpoint of dynamic binding capacity, the affinity ligand binding amount is preferably 10 to 200 mg, more preferably 25 to 100 mg, still more preferably 30 to 100 mg, and particularly preferably 50 to 100 mg per 1 g of the dry weight of the carrier. .

  As a method for fixing the affinity ligand to the synthetic polymer support, a general method can be used. For example, the amino group of the affinity ligand may be bound to the synthetic polymer support via a formyl group introduced on the carrier, and the amino group of the affinity ligand is an activated carboxy group on the synthetic polymer support. And the amino group of the affinity ligand may be bound to the carrier via an epoxy group on the synthetic polymer support.

  The functional group introduced into the synthetic polymer support for immobilizing the affinity ligand is not particularly limited as long as it is a functional group capable of forming a covalent bond with the affinity ligand. For example, an epoxy group (epichlorohydrin), Hydroxy group, aldehyde group or activating sulfonic acid group (eg, N-hydroxysuccinimide (NHS) ester, carbonyldiimidazole) activated with cyanogen bromide, N, N-disuccinimidyl carbonate (DSC), etc. Reactive functional groups (r-activating groups) such as (CDI) activated esters) and the like (Hermanson GT, et al., "Immobilized AffinityLigandTechniques, Academic Press", 1992; U.S. Patent No. 5,874, No. 65; U.S. Patent No. 3,932,557; U.S. Patent No. 4,772,653; U.S. Patent No. 4,210,723; U.S. Patent No. 5,250,6123; WO 2004/074741). These include those in which the affinity ligand is directly covalently attached to the synthetic polymer support, and those in which a linear, branched or cyclic linker or spacer is used. When activating a synthetic polymer support having an affinity ligand introduced thereinto, an activation method that does not directly react with the affinity ligand is preferable.

  Among the affinity ligands, a method of immobilizing a proteinaceous ligand on a carrier can be a method of reacting a part of a protein functional group with a part of a carrier functional group. The main functional groups (active groups) on the protein side that can be used for the reaction include N-terminal amino acids or amino groups of lysine (Lys) side chains; thiol groups of cysteine (Cys) side chains; C-terminal amino acids and glutamic acid (GIu). A) a carboxy group on the side chain or aspartic acid (Asp) side chain, but is not limited thereto.

  As a method for immobilizing a protein antibody affinity ligand on a synthetic polymer support by controlling the orientation of the ligand, a method utilizing protein A having a cysteine at the C-terminus has been proposed (US Pat. 399,750, Ljungquist C. et al., REur. J. Biochem. ", 1989, 186, 557-561).

  As an immobilization technique using a linker, other than a method of securing a distance between the synthetic polymer support and the affinity ligand and eliminating steric hindrance to improve performance, a functional group (for example, (Charged amine) is applied and formed. It has been studied to improve the separation performance by effectively accumulating the antibody affinity ligand on the linker or the spacer part when immobilizing the antibody affinity ligand and improving the immobilization yield. For example, there is a technique for immobilizing a proteinaceous ligand on a carrier derivatized with an NHS-activated carboxylic acid as a part of a linker arm (US Pat. No. 5,260,373 and JP-A-2010-133733). This may be used.

  In addition, after using the associative group on the carrier separately from the linker and the spacer, the affinity ligand is accumulated on the support, and then the antibody affinity is formed on the support without forming a covalent bond between the associative group and the affinity ligand. A method for individually fixing ligands has also been proposed (Japanese Patent Application Laid-Open No. 2011-256176), and this may be used.

<Group containing hydroxy group>
In the mixed mode carrier of the present invention, a group containing a hydroxy group may be bonded to the surface of the synthetic polymer support. When a group containing a hydroxy group in addition to the acidic group is bound on the surface of the support, more excellent antifouling properties can be exhibited.

  Examples of the group containing a hydroxy group include an organic group containing a hydroxy group, and preferred examples include a monovalent group represented by the following formula (3), and more preferably the following formula (4) ) Is a monovalent group.

[In the formula (3), R ³ represents a divalent or trivalent organic group having 1 to 12 carbon atoms, and n represents 1 or 2. ]

[In the formula (4), R ⁴ represents a divalent or trivalent hydrocarbon group having 1 to 12 carbon atoms, and Y ² represents>S,> S = O,> S (= O) ₂ ,> NH represents> or O, and n represents 1 or 2. ]

In the formula (3), the carbon number of the divalent or trivalent organic group represented by R ³ is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.

In the formula (4), the Y ^2,> S are preferred.

The divalent or trivalent hydrocarbon group represented by R ⁴ may be linear or branched. The carbon number of the divalent or trivalent hydrocarbon group is preferably 1 to 8, more preferably 1 to 6, and still more preferably 1 to 4.
The divalent or trivalent hydrocarbon group is preferably a divalent or trivalent aliphatic hydrocarbon group, and more preferably an alkanediyl group or an alkanetriyl group.
Specific examples of the alkanediyl group include a methane-1,1-diyl group, an ethane-1,1-diyl group, an ethane-1,2-diyl group, a propane-1,1-diyl group, a propane-1, 2-diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group, etc. Is mentioned.
Specific examples of the alkanetriyl group include a methane-1,1,1-triyl group, an ethane-1,1,2-triyl group, a propane-1,2,3-triyl group, and propane-1,2,2,3. And a 2-triyl group.

  The molar ratio [(α) :( β)] between the content (α) of the group containing an acidic group and the content (β) of the group containing a hydroxy group is not particularly limited, but is preferably 100: 0 to 5: 95, more preferably 99: 1 to 25:75, still more preferably 90:10 to 50:50, still more preferably 85:15 to 55:45, particularly preferably 85:15 to 60:40. And excellent antifouling properties can be obtained.

  In the carrier of the present invention, the affinity ligand, a group containing an acidic group, a group containing a hydroxy group, from the viewpoint of the stability of the carrier, a group formed by ring opening of a cyclic ether group, a group formed by reducing a formyl group, Those in which a reactive group is covalently bonded to the surface of a synthetic polymer support via a group obtained by ring opening, reduction or nucleophilic substitution, such as a group obtained by nucleophilic substitution of a tosyl group. It is more preferable that the cyclic ether group is bonded to the surface of the synthetic polymer support via a ring-opened group. A ring-opened epoxy group is preferable as the group formed by ring opening of the cyclic ether group.

  Here, as the “cyclic ether group”, a cyclic ether group having 3 to 7 atoms constituting a ring is preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include the cyclic ether groups represented by the following formulas (5) to (10), and the cyclic ether group represented by the formula (5) is more preferable.

[Wherein, R ^{5 to} R ⁸ each independently represent a hydrogen atom or an alkyl group, and * indicates a bonding position. ]

The carbon number of the alkyl group represented by R ^{5 to} R ⁸ is preferably 1 to 4, more preferably 1 or 2. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group and a tert-butyl group.
Further, as R ^{5 to} R ⁸ , a hydrogen atom is preferable.

(Method of manufacturing carrier)
The mixed mode carrier of the present invention can be produced, for example, by a method including the following steps 1 and 2. According to such a method, the mixed mode carrier of the present invention can be obtained simply and easily.
(Step 1) Part of the reactive group of a synthetic polymer support (hereinafter, also simply referred to as a raw material support) having a reactive group (hereinafter, also simply referred to as a reactive group) capable of binding to an affinity ligand on its surface. (Step 2) a step of reacting the remaining reactive group with a compound having a functional group capable of reacting with the reactive group and an acidic group

<Step 1>
Step 1 is a step of binding an affinity ligand to a part of the reactive group of the raw material support. The method of binding the affinity ligand to the reactive group is not particularly limited, and may be performed by a conventional coupling method using, for example, an amino group, a carboxy group, or a thiol group present in the affinity ligand. . The coupling method includes (i) activating the raw material support using cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, etc., and coupling the affinity ligand with the affinity ligand. (Ii) adding a condensing reagent such as carbodiimide or a reagent having a plurality of reactive groups in a molecule such as glutaraldehyde to a system in which a raw material support and an affinity ligand are present; A method of fixing by condensation and crosslinking, (iii) a method of introducing a reactive group into the raw material support in advance and performing a coupling reaction without activating the raw material support and fixing the same. .

  When using porous particles as the raw material support, the porous particles may be synthesized by known seed polymerization, suspension polymerization, or the like. Upon polymerization, a polymerization initiator, a porogen, an aqueous medium, a dispersion stabilizer, a surfactant, a polymerization regulator, a polymerization inhibitor, seed particles, and the like are used as necessary, in addition to the monomer. As the monomer, a monomer other than the monomer may be used, if necessary, in addition to the monomer having a reactive group. All of these monomers include an ethylenically unsaturated monomer. Specific examples include one or more monomers selected from styrene monomers, vinyl ketone monomers, (meth) acrylonitrile monomers, (meth) acrylate monomers, and (meth) acrylamide monomers.

  Further, as the reactive group included in the raw material support, a cyclic ether group is preferable from the viewpoint that a coupling reaction can be stably performed even in an aqueous system, and a cyclic ether group having 3 to 7 atoms constituting a ring is more preferable. preferable. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include the cyclic ether groups represented by the above formulas (5) to (10).

The affinity ligand bound to the raw material support is the same as described above.
The total amount of the affinity ligand used is usually about 50 to 300 mg, preferably 120 to 180 mg, per 1 g of the raw material support.

Step 1 is preferably performed in the presence of a buffer to which a salt has been added. Examples of the type of salt include trisodium citrate, sodium sulfate, and the like, and examples of the buffer include a sodium phosphate buffer, a potassium phosphate buffer, and a borate buffer. The total amount of the buffer used is usually about 20 to 80 times by mass, preferably 35 to 45 times by mass, based on the raw material support.
The reaction time of Step 1 is not particularly limited, but is usually about 0.5 to 72 hours, and the reaction temperature is usually about 1 to 60 ° C.

<Step 2>
Step 2 is a step of reacting the remaining reactive group with a compound having a functional group capable of reacting with the reactive group and an acidic group (hereinafter also referred to as an acidic group-containing compound).

Examples of the acidic group-containing compound include mercaptoacetic acid (pKa = 3.5-4.0), 3-mercaptopropionic acid (pKa = 3.5-4.0), and 4-mercaptobutanoic acid (pKa = 3.5-4.0). 4.0) and the like; amino acids such as glycine (pKa = 2.0-2.5) and β-alanine (pKa = 2.0-2.5); 2-mercaptoethanesulfonic acid (pKa = -2.5 to -2.0), mercaptosulfonic acid such as 3-mercapto-1-propanesulfonic acid (pKa = -2.5 to -2.0); aminomethanesulfonic acid (pKa = -2.0) To -1.0), 2-aminoethanesulfonic acid (pKa = -2.0 to -1.0), 3-amino-1-propanesulfonic acid (pKa = -2.3 to -1.3) and the like Amino group-containing sulfonic acids; and salts thereof. . Examples of the salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic ammonium salt and the like.
The total amount of the acidic group-containing compound used is usually 0.1 to 40 molar equivalents relative to 1 mol of the remaining reactive group, but from the viewpoint of preventing multipoint binding of the affinity ligand, an excess amount of 2 to 40 molar equivalents. It is preferable to block the residual reactive group by the above.

  When a group containing a hydroxy group is introduced, an alcohol containing a mercapto group such as mercaptoethanol and thioglycerol may be added in combination with an acidic group-containing compound.

The reaction time of step 2 is not particularly limited, but is usually about 0.5 to 72 hours, preferably 1 to 48 hours. The reaction temperature may be appropriately selected below the boiling point of the solvent, but is usually about 2 to 100 ° C.
Steps 1 and 2 are preferably performed in order from step 1 to step 2 from the viewpoint of efficiency and cost.

  The mixed mode carrier of the present invention obtained as described above has a high dynamic binding capacity for a target substance, and even when using a synthetic polymer support in which non-specific adsorption is likely to occur, it has an antifouling property. Excellent. Therefore, the carrier for mixed mode of the present invention has excellent protection against biologically-derived impurities such as HCP (Host Cell Protein) and DNA contained in host cells without depending on the composition of the synthetic polymer support. Shows fouling, has extremely high separation selectivity, and has a hydrophobic interaction with antibody aggregates rather than a polysaccharide-based support, so that a carrier with better separation performance can be obtained. .

(Target method for purification)
The method for purifying a target of the present invention comprises: a mixed mode carrier having a group containing an acidic group and an affinity ligand on a synthetic polymer support; and a step A of bringing the affinity ligand into contact with a target that can be captured, A dissociation solution for dissociating the affinity ligand and the target, and a step B for dissociating the target by contacting the mixed mode carrier capturing the target in step A, wherein the step B comprises: Under the condition that the target substance dissociates from the affinity ligand, dissociation is performed in a stepwise manner using two or more kinds of salt concentrations in which the ionic strength increases stepwise, or in a gradient manner in which the salt concentration is increased in a gradient manner. A step of performing In step B, both the stepwise method and the gradient method may be combined.
In the step A, the above-mentioned mixed mode carrier may be used, and there is no particular limitation. The target substance is not particularly limited, but when an antibody affinity ligand such as protein A is used as the affinity ligand, an antibody is exemplified as the target substance.

  When using the mixed mode carrier of the present invention, both the ligand having an acidic group and the affinity ligand, the separation function of the antibody monomer and aggregates function in concert to provide excellent separation characteristics. In addition to the above, it is possible to shorten the two-step chromatography operation of the affinity purification step and the purification step using an ion-exchange group to one step, and it can be expected that the type and amount of the buffer solution to be used and the working time can be reduced.

  In addition, the purification method using the mixed mode carrier of the present invention employs a narrow pH range (preferably pH 3 to 4, more preferably pH 3.1 to 3.9, still more preferably pH 3.0) in which the target substance dissociates from the affinity ligand. Under the conditions of 2 to 3.8), two or more salt concentrations at which the ionic strength (preferably 10 to 500 mM, more preferably 15 to 400 mM, and still more preferably 20 to 350 mM) increases stepwise are used. It is possible to obtain an eluted fraction having a high monomer content by setting the "wise method" or the "gradient method" in which the salt concentration is increased in a gradient in the above range.

  In particular, in the purification of monoclonal antibodies, since the dissociation pH is far away from the isoelectric point of the target, there is no large difference in the width of the dissociated ionic strength for each antibody, and setting of the use conditions of various targets in a narrow range. Can be expected. Further, as the affinity ligand, an antibody affinity ligand is preferable, and the dissociation pH range can be set to be narrower. Further, the use of alkali CIP washing enables effective washing, so that from the viewpoint of stable process construction. The use of modified protein A is more preferred.

Regarding the method of using the mixed mode carrier of the present invention, when a target such as an antibody is adsorbed near neutrality, it is preferable to add a counter ion of a cation exchange group at a certain concentration or more. Under such conditions, the cation exchanger function does not act, and even if it acts, non-specific adsorbed substances derived from the cation exchange group can be removed by washing with a higher ionic strength.
On the other hand, the ionic strength does not inhibit the adsorption of the antibody affinity ligand, and the target can be adsorbed with high specificity.In addition, the use of a high ionic strength washing solution allows non-specification of the substrate, linker, spacer, ligand and target. The molecules adsorbed on the surface can be effectively removed by washing.

  Usually, a culture supernatant in which a recombinant monoclonal antibody has been expressed has an ionic strength close to that of a body fluid such as a human, so that it can maintain high specificity even when directly applied to the separation matrix of the present invention. Impurities can be further reduced by the washing liquid.

  The function of the mixed mode affinity separation matrix prepared according to the present invention can be controlled by the ratio of the antibody affinity ligand to the cation exchange group. When the binding capacity of the antibody affinity ligand is larger than the binding capacity of the cation exchange group, the antibody tends to be dissociated from the carrier even at a low ionic strength during acidic elution, and the binding capacity of the antibody affinity ligand becomes larger than that of the cation exchange group. When the binding capacity is about the same or lower, the antibody dissociated from the antibody affinity ligand is strongly retained by the cation exchange group at a low ionic strength, and the antibody tends to be hardly dissociated. It is necessary to set the dissociated ionic strength higher. In any case, the recovery rate and the monomer ratio can be controlled by adjusting the ionic strength.

  Although there is no particular limitation on the ratio of the antibody binding capacity based on the antibody affinity ligand to the antibody binding capacity based on the cation exchange group, the target (especially antibody, more preferably human IgG or humanized The dynamic binding capacity of a target (especially an antibody, more preferably an IgG such as a human IgG or a humanized monoclonal antibody) by a cation exchange group under the dissociation pH conditions of the dissociation of an IgG such as a monoclonal antibody depends on the antibody adsorption conditions. It is preferably not more than twice the dynamic binding capacity of a target (especially an antibody, more preferably an IgG such as a human IgG or a humanized monoclonal antibody) by an antibody affinity ligand under (neutral conditions), etc. It is more preferably at most 1 times, particularly preferably at most 1/5. The lower limit may be, for example, 1/100 times or more, or 1/50 times or more. When the amount of antibody bound by the cation exchange group is small, the dissociated ionic strength can be set low, and there is a tendency that treatment such as desalting is not required in the subsequent antibody purification process. The range of strength setting is narrow and process development is easy.

  Targets purified by the mixed-mode affinity separation matrix of the present invention include, for example, immunoglobulin G and its analogs (including derivatives), and the like. An Fc fusion protein (Fc-containing molecule) obtained by fusing an Fc region as a constant region with another functional protein or peptide is included. These are used as raw materials for antibody drugs.

  Hereinafter, a detailed description of the purification method using the carrier for mixed mode of the present invention will be given for the case where the target is immunoglobulin G, but the present invention is not limited thereto.

  The method for purifying a target of the present invention includes a step A of contacting a mixed mode carrier having a group containing an acidic group and an affinity ligand on a synthetic polymer support with a target that can be captured by the affinity ligand. . After adjusting the pH of the protein solution containing immunoglobulin G to be around neutral, the solution is passed through a column filled with the mixed mode carrier of the present invention, and the immunoglobulin G is specifically bound via the affinity ligand. The mixture is allowed to adsorb to the mixed mode carrier. For example, when protein A is used as the affinity ligand, the loading pH is preferably 6 or more, more preferably 6.3 or more, and even more preferably 6.5 or more and 8.5 or less. In the purification of immunoglobulin G produced by cultured mammalian cells, it is not particularly necessary to adjust the ionic strength, and the ionic strength can be increased in advance to further suppress nonspecific adsorption.

  In the present invention, it is preferable to further include a washing step. In the washing step, an appropriate amount of a buffer in a condition range in which the affinity ligand functions is passed through to wash the inside of the column. That is, the preferable range of the pH may be the same range (pH near neutrality) as at the time of loading, and for example, is preferably 6 or more. At this time, the immunoglobulin G as the target is adsorbed on the mixed mode carrier of the present invention. At this time, impurities may be effectively removed by optimizing the ionic strength or the composition at a pH near neutrality. It is preferable that the cation exchange group does not function during loading and washing, that is, it is preferable to use a washing solution having a pH around neutral and a ionic strength of a certain level or more. In this process, impurities remaining on the column can be washed non-specifically through the separation matrix and / or immunoglobulin G. The ionic strength is, for example, preferably 0.2 M or more, more preferably 0.5 M or more.

  The purification method of the present invention includes a step B of bringing the dissociation solution for dissociating the affinity ligand and the target substance into contact with the mixed mode carrier capturing the target substance in the step A. Step B includes at least either a stepwise method using two or more kinds of salt concentrations in which the ionic strength increases stepwise under a condition of dissociation from the affinity ligand, or a gradient method using a salt concentration in which the gradient increases. This is the step of dissociation.

In the step B, the column is replaced with a buffer having a low ionic strength near neutrality to prepare for the expression of an ionic strength-dependent dissociation function by a cation exchange group during dissociation.
During dissociation, a combination of acidic pH and ionic strength enables the cation exchange separation mode to function during dissociation from the antibody affinity ligand, and the concerted action of both ligands allows the fraction with high monomer content to be reduced to low ion. It can be collected in a strong elution fraction. As the pH of the dissociation solution, the dissociation pH of immunoglobulin G from the antibody affinity ligand can be applied. Since the pH is determined mainly on the separation conditions determined by the type of the affinity ligand and immunoglobulin G used for preparing the mixed mode carrier, no special condition setting is required.

  When protein A is used as the antibody affinity ligand, the pH is preferably set between 2 and 6. However, in order to avoid acid denaturation of the target, pH 3.0 or more is more preferable, pH 3.3 or more is more preferable, and pH 3.5 or more is particularly preferable. The pH is preferably 5.5 or less, more preferably 5.0 or less. When an alkali-resistant protein A ligand is used, its dissociation pH is generally set at a value between 3.5 and 4.0, but is not limited thereto.

  The dissociated ionic strength depends not only on the introduction ratio of the antibody affinity ligand and the cation exchange group, but also on the loading amount of immunoglobulin G per unit volume. Can be easily set.

  Antibody dissociation from the mixed mode carrier prepared according to the present invention can be applied in either a salt concentration gradient system or a stepwise system, or may be a combination of these, but with the aim of reducing the amount of eluate. In this case, a stepwise method based on ionic strength is preferable. Further, for simplification of the operation, it is preferable to set conditions that can achieve antibody recovery and high monomer content in one step.

  When aggregates remain in the column and do not mix with the eluted fraction even in the combination of ionic strength and acidic pH in the washing step, the ionic strength adjusting step can be omitted.

  The immunoglobulin G purified using the mixed mode carrier prepared according to the present invention shows higher monomer selectivity than the antibody affinity separation matrix based on the single separation mode, and has a higher monomer content in the eluate. .

  By using the mixed mode antibody affinity separation matrix of the present invention, affinity purification with high specificity and the improvement of monomer content which can be achieved mainly by cation exchange chromatography can be performed in a single chromatographic column while maintaining high recovery. Since the operation can be efficiently achieved, the load on the subsequent process can be reduced, which can contribute to an improvement in the yield of the entire process and an increase in the monomer content. That is, according to the present invention, it is possible to contribute to the improvement of productivity and the purification of antibody drug production processes.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. Each analysis condition in the examples is shown below. Example 1 is a reference example.

<Epoxy group content>
The porous particle dispersion (S-3) is accurately measured in a polyethylene bottle so that the number of moles of the epoxy group calculated from the amount of the epoxy group-containing monomer used in the polymerization is 1.00 mmol. 25 mL of a 37.5% by mass aqueous solution and 10 mL of 0.2 N hydrochloric acid were added, and the mixture was stirred at 75 ° C. for 1 hour to open the epoxy group. After cooling, 10 mL of a 0.2 N aqueous sodium hydroxide solution was added. The mixture was then back-titrated with 0.1 N hydrochloric acid while monitoring the pH with a pH meter to quantify the epoxy group content of the porous particles before binding with the affinity ligand.

<Binding amount of acidic group>
Carriers 1 to 5 were accurately measured in terms of solid content in an amount of 0.5 g, and the amount of acid groups bonded was calculated using an electric conductivity meter (794 Basic Titrino manufactured by Metrohm).

(Example 1)
(1) To 360 g of pure water, 0.72 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) was added, and the mixture was heated and stirred to dissolve the polyvinyl alcohol. After cooling, sodium dodecyl sulfate (Emal 10G manufactured by Kao Corporation) was added. 18 g, sodium carbonate 0.36 g and sodium nitrite 0.18 g were added and stirred to prepare an aqueous solution (S-1).
On the other hand, 6.88 g of glycidyl methacrylate (manufactured by Mitsubishi Rayon Co.), 1.37 g of glycerol monomethacrylate (manufactured by NOF CORPORATION), 4.12 g of trimethylolpropane trimethacrylate (manufactured by Sartomer) and polyethylene glycol # 400 dimethacrylate (Shinnakamura) A monomer composition consisting of 1.37 g of 9G) manufactured by Kagaku Co., Ltd. was dissolved in a mixed solution of 20.63 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) and 5.30 g of acetophenone (manufactured by Inoue Perfumery), and A monomer solution (S-2) was prepared.
Next, the whole amount of the aqueous solution (S-1) was put into a 500 mL separable flask, and a thermometer, a stirring blade and a cooling tube were attached, and the flask was set in a hot water bath, and stirring was started under a nitrogen atmosphere. The whole amount of the monomer solution (S-2) was put into a separable flask, heated in a hot water bath, and when the internal temperature reached 85 ° C., 2,2′-azoisobutyronitrile (Wako Pure Chemical Industries, Ltd.) 0.53 g (manufactured by Kogyo Co., Ltd.) was added, and the temperature was maintained at 86 ° C.

  (2) Thereafter, stirring was performed for 3 hours while maintaining the temperature at 86 ° C., and after cooling the reaction solution, the reaction solution was filtered and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. Next, the particles were dispersed in pure water so that the concentration of the particles became 10% by mass, to obtain a porous particle dispersion (S-3). The epoxy group content of the porous particles was 1.57 mmol per 1 g of dry weight of the particles.

  (3) Next, 0.15 g of modified protein A (rSPA manufactured by Repligen) was dispersed in 40 mL of 1.0 M trisodium citrate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A dispersion. To the protein A dispersion, 1 g of the porous particle dispersion (S-3) was added in terms of particle dry weight. This dispersion was shaken and stirred at 25 ° C. for 5 hours to fix protein A to the particles. The particles are referred to as Protein A-fixed particles (S-4).

(4) After washing the protein A-fixed particles (S-4) with a 0.1 M sodium phosphate buffer (pH 7.6), 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries) /0.1 M sulfuric acid The resultant was dispersed in 40 mL of an aqueous sodium solution (pH 8.3) and shaken and stirred at 25 ° C. for 17 hours to completely open (block) unreacted epoxy groups.
Thereafter, the carrier was washed with a 0.1 M sodium phosphate buffer (pH 7.6), a 0.5 M sodium hydroxide aqueous solution, and a 0.1 M sodium citrate buffer (pH 3.2) to obtain a mixed mode carrier (carrier 1). . The amount of mercaptoacetic acid bound was 2.48 μmol per 1 m ² of the surface area of the carrier.

(Example 2)
The solution used in the epoxy ring-opening step of Example 1 was prepared from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M aqueous sodium sulfate solution (pH 8.3) using 1.0 M 3-mercapto-1-propane. The same operation as in Example 1 was carried out except that sodium sulfonate (manufactured by Wako Pure Chemical Industries, Ltd.) / 0.1 M aqueous sodium sulfate solution (pH 8.3) was used, to obtain a mixed mode carrier (carrier 2). The amount of sodium 3-mercapto-1-propanesulfonate bound was 3.12 μmol per 1 m ² of the surface area of the carrier.

(Comparative Example 1)
The solution used in the epoxy ring-opening step of Example 1 was converted from 1.0 M mercaptoacetic acid (manufactured by Wako Pure Chemical Industries) /0.1 M sodium sulfate aqueous solution (pH 8.3) to 1.0 M thioglycerol (Asahi Chemical Industry Co., Ltd.) Carrier 3 was obtained by performing the same operation as in Example 1 except that the solution was changed to a 0.1M aqueous solution of sodium sulfate (pH 8.3). The binding amount of the acidic group was <0.05 μmol / m ² of the surface area of the carrier.

(Comparative Example 2)
MabSelectSuRe (GE Healthcare Bioscience) replaced with 0.5 M saline was used as a protein A fixed carrier, and a carboxy group was introduced into this.
That is, 4 mL of MabSelectSuRe as a wet volume was placed in a reaction vessel, the slurry volume was adjusted to 5 mL with water, and 1 mL of a 0.25 M sodium citrate (pH 3.5) solution was added thereto. Next, 1 mL of a 0.1 M citric acid / 0.1 M glutamic acid (pH 3.5) solution was added and stirred. Then, 0.5 mL of a 0.8 M sodium periodate solution was added, and the mixture was inverted and stirred at room temperature for 1 hour to introduce an aldehyde group. This carrier slurry was washed five times with 1 M glutamic acid / PBS (pH 7) diluted 100-fold with cold water, and after the collection, the amount of the slurry was adjusted to 5 mL. 5 mL of 1M glutamic acid / PBS (pH 7) was added thereto, and the mixture was inverted and stirred at room temperature for 2 hours. Then, 0.5 mL of an aqueous solution of 1M dimethylamine borane was additionally added, and the mixture was inverted and stirred overnight at room temperature. After centrifugation to settle the carrier and removing the supernatant so that the liquid level became 6 mL, 20 mg of sodium borohydride was directly added, and the mixture was further inverted and stirred at room temperature for 2 hours. The wells were thoroughly washed with water, 0.1 M citric acid, 0.1 M sodium hydroxide, and PBS supplemented with 0.5 M NaCI. In this way, a carboxyl group was introduced into the aldehyde group by a reductive amination method via the amino group of glutamic acid to obtain a mixed mode carrier (carrier 4). The amount of the carboxy group bonded was 2.98 μmol per 1 m ² of the surface area of the carrier.

(Comparative Example 3)
4 mL of Low Density Glyoxal 4 Rapid Run (ABT) replaced with water as 4% agarose beads was placed in a reaction vessel in a wet volume of 4 mL, and the slurry volume was adjusted to 5 mL with water. Thereafter, 1 mL of a 0.25 M sodium citrate (pH 3.5) solution was added. Further, 2 mL of 0.8 M sodium periodate was added, and the mixture was stirred by inversion at room temperature for 0.5 hour to obtain a carrier into which an aldehyde group was introduced. This carrier slurry was sufficiently washed with Dulbecco's PBS (I) (Nichizu) (hereinafter, PBS) as water and a phosphate buffer, and the amount of the slurry was adjusted to 5 mL after collection. Next, 5 mL of a mixed solution (pH 6.8) of 0.1 M sodium phosphate, 1 M sodium citrate, and 0.3 M sodium chloride was added, and after mixing, the liquid volume was adjusted to 5.5 mL. After adding a 5N aqueous sodium hydroxide solution to adjust the pH of the slurry of the carrier to 11.5 to 12, 100 mg of protein A was immediately added, and the mixture was stirred at 2 to 8 ° C for 2.5 hours. After adjusting the pH of the carrier slurry to 7 to 5 using a 1 M citric acid solution, 0.5 mL of 1 M dimethylamine borane was added, and the mixture was inverted and stirred at room temperature overnight. After sufficiently washing with water, 0.1 M citric acid, 0.1 M sodium hydroxide, and PBS, a carrier (carrier 5) in which protein A was immobilized (bound) by covalent bond to agarose was obtained. The binding amount of the acidic group was <0.05 μmol / m ² of the surface area of the carrier.

(Test Example 1 Dynamic binding capacity (DBC) measurement test based on antibody affinity ligand)
For the carriers 1 to 5 prepared in Examples 1 and 2 and Comparative Examples 1 to 3, the dynamic binding capacity of the antibody as a carrier for separating antibodies was measured. Specifically, from the value obtained by dividing the amount of antibody bound to the column by the volume of the carrier in the column until 10% of the loaded IgG other than the fraction such as IgG3 that does not bind to the carrier leaks, the amount of IgG adsorbed per mL of the carrier was determined. The amount was calculated as 10% dynamic binding capacity (DBC). The chromatographic conditions are shown below. The flow rate under load was 0.4 mL / min, and the dynamic binding capacity for 6 minutes of contact time was determined. In addition, the flow rate setting other than the time of load was set to 0.6 mL / min (contact time = 4 minutes).
The chromatography conditions used for 10% DBC measurement based on the antibody affinity ligand are as follows.
Column = ID 0.66 cm x Height 7 cm (Omnifit)
Flow rate = 0.4 mL / min (contact time = 6 minutes) or 0.6 mL / min (contact time = 4 minutes)
Polyclonal antibody (IgG) = Gamma globulin / Nichiaku (human immunoglobulin G) (Nippon Pharmaceutical)
Loading solution = 2.5 mg-IgG / mL (PBS = Dulbecco / Nissui) Equilibration solution = PBS (pH 7.4)
Dissociation solution = 50 mM acetic acid, 0.1 M sodium chloride (pH 3.75) Regeneration solution = 0.1 M acetic acid, 1 M sodium chloride CIP solution = 0.1 M sodium hydroxide, 1 M sodium chloride neutralization / re-equilibration solution = PBS ( pH 7.4)

(Test Example 2 Dynamic Binding Capacity (DBC) Measurement Test Based on Cation Exchange Group)
The antibody binding capacity based on the cation exchange group was measured for the carriers 1 to 5 prepared in Examples 1 and 2 and Comparative Examples 1 to 3. Specifically, as a condition for loading an antibody, a condition in which protein A as an antibody affinity ligand has almost no antibody-capturing ability and an acidic group-containing group introduced as a cation exchange group can function is a 10 mM acetate buffer of pH 3.5. The liquid was used. Since the cation exchange group does not show selectivity for IgG3 and the like unlike the protein A ligand, the amount of antibody bound to the column from the start of loading until 10% of the total loaded IgG leaks was divided by the volume of the carrier in the column. From the values, the amount of IgG adsorbed per 1 mL of the carrier was calculated as 10% DBC. The chromatographic conditions are shown below. The flow rate under load was 0.4 mL / min, and the dynamic binding capacity for 6 minutes of contact time was determined. In addition, the flow rate setting other than the time of load was set to 0.6 mL / min (contact time = 4 minutes).
The chromatography conditions used for 10% DBC measurement based on a cation exchange group are as follows.
Column = ID 0.66 cm x Height 7 cm (Omnifit)
Flow rate = 0.4 mL / min (contact time = 6 minutes) or 0.6 mL / min (contact time = 4 minutes)
Polyclonal antibody (IgG) = Gamma globulin / Nichiyaku (Nippon Pharmaceutical) Loading solution = 2.5 mg-IgG / mL (10 mM acetic acid = pH 3.5) Equilibration solution = 10 mM acetic acid (pH 3.5)
Dissociation solution = 10 mM acetic acid, 0.5 M sodium chloride (pH 3.5) CIP solution = 0.1 M sodium hydroxide, 1 M sodium chloride neutralization / re-equilibration solution = 10 mM acetic acid (pH 3.5)

(Test Example 3 Dissociation and Aggregate Separation by Stepwise Method under Acidic Conditions)
1 mL of the human polyclonal antibody was added to the carriers 1 to 5 prepared in Examples 1 and 2 and Comparative Examples 1 to 3 packed in the column (ID 0.66 cm × Height 7 cm) manufactured by Omnifit used in Test Example 1 under neutral conditions. The antibody was eluted with a dissociation solution of various ionic strengths (NaCl 0 mM, 50 mM, 300 mM) under acidic conditions. Each eluate was analyzed by gel filtration chromatography, and the antibody content and the antibody yield (YieId) were determined from the protein peak area value of each eluted fraction (fraction). Further, the ratio of the monomer (monomer) to the aggregate (multimer) and the like was determined from the protein peak analysis, and the monomer content (Monomer content) and the monomer yield of the carrier 5 (MonomerYieId) were calculated.
The monomer yield of the carriers 1 to 5 was determined from the antibody yield and the monomer content by the following formula, with the sum of the area values of the eluted fractions from the carrier 5 being 100%.
[Monomer yield of carriers 1-4 at 0 mM, 50 mM or 300 mM NaCl]
(Monomer yield (%) at each NaCl concentration of carriers 1 to 5) = ｛(antibody yield of carriers 1 to 5 (%)) × (monomer content of carriers 1 to 5 (%)) × 100｝ / {99.4 × 94.5 + 0.6 × 86.3}
In order to prevent the formation of aggregates in the fraction eluted from the affinity separation matrix, arginine was added to each dissociation solution so that the final concentration was 0.05 M or more, and a sodium phosphate solution of pH 5 was used. The pH of each dissociated solution was adjusted to 5 to 6 and subjected to gel filtration chromatography.
Each chromatography condition is as follows.
Column = ID 0.66 cm x Height 7 cm (Omnifit)
Flow rate = 0.6 mL / min (contact time = 4 minutes)
Polyclonal antibody (IgG) = gamma globulin / Nichiyaku (Nippon Pharmaceutical) Loading solution = 2.5 mg-IgG / mL (PBS = Dulbecco / Nissui) Equilibration solution = PBS (pH 7.4) (3 CV, CV = column volume) Load = 6.8mL
Washing after loading = PBS (pH 7.4) (5 CV)
Washing solution before dissociation = 10 mM Tris / HCI (pH 7) (5 CV) Dissociation solution 1 = 10 mM acetic acid (pH 3.5) (8 CV)
Dissociation solution 2 = containing 50 mM sodium chloride, 10 mM acetic acid (pH 3.5) (8 CV)
Dissociation solution 3 = containing 300 mM sodium chloride, 10 mM acetic acid (pH 3.5) (4 CV)
CIP solution = 0.1 M sodium hydroxide, 1 M sodium chloride (4 CV) neutralization / re-equilibration solution = PBS (pH 7.4)
Gel filtration chromatography conditions Column = Superdex2010 / 300GL (IDcmxHeight30cm) (GE Healthcare Bioscience)
Flow rate = 0.5 mL / min Detection wavelength = 214 nm
Load solution = 100 μL / injection (diluted to a range where the absorbance value does not exceed 1)
Eluent = PBS (pH 7.4)
Table 1 shows the evaluation results of the stepwise salt elution and the separation characteristics of the aggregates under acidic conditions of the carriers 1 to 5.

(Test Example 4 HCP measurement test)
70 μL (wet state) of each of the carriers 1 to 5 prepared in Examples 1 and 2 and Comparative Examples 1 to 3 was weighed, and a 1.0 mg / mL monoclonal antibody solution (containing a biosimilar of the monoclonal antibody Trastuzumab) was measured for each antibody. 2.45 mL of CHO cell culture supernatant) was added, and the mixture was shaken with stirring at room temperature for 1.5 hours to capture the antibody on the carrier.
Next, the entire amount of the carrier capturing the antibody was transferred to a spin column (manufactured by Thermo), and the antibody solution was removed by centrifugation (1000 rpm).
Next, 20 mM sodium phosphate / 150 mM NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Next, 20 mM sodium phosphate / 1.0 M NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Next, 20 mM sodium phosphate / 150 mM NaCl buffer (pH 7.5) was added to the spin column, and a total of 840 μL was passed by centrifugation to wash the carrier.
Thereafter, 50 mM citrate buffer (pH 3.2) was added to the spin column, and a total of 700 μL was passed by centrifugation to dissociate the monoclonal antibody captured by the carrier. The antibody concentration in the dissociation solution was calculated from the absorbance using SmartSpec Plus manufactured by BioRAD.
Then, the host cell protein (HCP) concentration was measured using a CHO HCP ELISA kit, 3G manufactured by Cygnus Technologies, and standardized by the antibody concentration. Table 1 shows the results.
×: exceeding 1000 ppm / IgG ○: 500 to 1000 ppm / IgG
◎: less than 500 ppm / IgG

  When the monomer content of Example 1 is compared with the monomer content of Comparative Example 2 when the NaCl concentration is 50 mM, Comparative Example 2 is 99.2%, whereas Comparative Example 2 is 99.2%. The result was 3%, indicating that the mixed mode carrier of the present invention was more excellent in the performance of separating monomers and aggregates. Further, as in Example 2, depending on the type of acidic group, HCP could be reduced as compared with Comparative Example 2 while maintaining the ability to remove aggregates. Each of these carriers separates a monomer and an aggregate by a mixed mode of protein A and an acidic group, but the carrier in the examples uses a synthetic polymer as a support, and the carrier in the comparative example supports a polysaccharide. There is a difference in the body. Since the synthetic polymer has a stronger interaction with the aggregate of the antibody than the polysaccharide, it is expected that the synthetic polymer has advantageously acted on the separation between the monomer and the aggregate. On the other hand, when a synthetic polymer support was used, HCP would normally increase, but HCP could be reduced by introducing an acidic group.

Claims (11)

Having a group and antibody affinity ligand contains an acidic group on a synthetic polymeric support, as the acidic _{group, -S (= O) 2 OH} , -S (= O) 2 O - M + (M + vs shows the ion) and -S (= O) ₂ O ^- has one or more selected from, and wherein the synthetic polymer support is from (meth) acrylate monomers and (meth) acrylamide monomer A step A of contacting an antibody with a mixed mode carrier derived from one or more selected monomers ,
A dissociation solution for dissociating the antibody affinity ligand and the antibody, and a step B of dissociating the antibody by contacting the mixed mode carrier capturing the antibody in the step A,
The step B is a stepwise method using two or more kinds of salt concentrations in which the ionic strength increases stepwise under the condition that the antibody dissociates from the antibody affinity ligand, or a gradient method in which the salt concentration is increased in a gradient manner. Characterized in that it is a step of dissociating at least in any,
A method for purifying antibodies . The purification method according to claim 1 , wherein the group having an acidic group has a pKa of -3.0 to 1.0 . The purification method according to claim 1 , wherein the group containing an acidic group is a monovalent group represented by the formula (1).
[In the formula (1), R ¹ represents a divalent group represented by the formula (2), and X represents an acidic group. ]
[In the formula (2), R ² represents a divalent hydrocarbon group having 1 to 12 carbon atoms, and Y ¹ represents>S,> S = O,> S (＝ O) ₂ ,> NH, or > O, and * indicates the bonding position to X in formula (1). ] Y ¹ is,>S,> S = O , or> S (= O) is _2, the purification method according to claim 3. The content of the group comprising an acidic group, it is the surface area 1 m ² per 0.05μmol more Mixed mode support, the purification method according to any one of claims 1-4. The antibody affinity ligand is a immunoglobulin binding protein, the purification method according to any one of claims 1-5. The purification method according to any one of claims 1 to 6 , wherein the antibody affinity ligand is bonded to the surface of the synthetic polymer support via a group formed by opening a cyclic ether group. The synthetic polymer support contains (M-1) a structural unit derived from an epoxy group-containing monovinyl monomer in an amount of more than 20 parts by mass, not more than 99.5 parts by mass, and (M-2) based on all structural units. A) a solid-phase support containing a copolymer containing a structural unit derived from a polyvinyl monomer in an amount of 0.5 to 80 parts by mass based on all structural units, wherein the epoxy group-containing monovinyl monomer is an epoxy The purification method according to any one of claims 1 to 7 , wherein the method is a group-containing (meth) acrylate-based monovinyl monomer, and the polyvinyl monomer is a (meth) acrylate-based polyvinyl monomer. The purification method according to any one of claims 1 to 8 , wherein the synthetic polymer support is porous. The purification method according to any one of claims 1 to 9 , wherein the synthetic polymer support is in the form of a particle, a monolith, a plate, a fiber, or a film. Having a group containing an acidic group and an antibody affinity ligand on the synthetic polymer support,
As the acid _{group, -S (= O) 2 OH} , -S (= O) 2 O - M + (M + represents a counter ion) and ^{_{-S (= O) 2 O -}} 1 kind or selected from Have two or more,
The synthetic polymeric support, characterized in that is derived from one or more monomers selected from (meth) acrylate monomers and (meth) acrylamide monomer,
Mixed mode carrier.