WO2014061411A1 - 抗体精製用陽イオン交換クロマトグラフィー担体および抗体医薬の製造過程で生産される抗体単量体とその重合体の分離方法 - Google Patents
抗体精製用陽イオン交換クロマトグラフィー担体および抗体医薬の製造過程で生産される抗体単量体とその重合体の分離方法 Download PDFInfo
- Publication number
- WO2014061411A1 WO2014061411A1 PCT/JP2013/075941 JP2013075941W WO2014061411A1 WO 2014061411 A1 WO2014061411 A1 WO 2014061411A1 JP 2013075941 W JP2013075941 W JP 2013075941W WO 2014061411 A1 WO2014061411 A1 WO 2014061411A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cation exchange
- exchange chromatography
- polymer
- antibody
- formula
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
- B01D15/362—Cation-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/08—Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/16—Organic material
- B01J39/17—Organic material containing also inorganic materials, e.g. inert material coated with an ion-exchange resin
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
- B01J39/26—Cation exchangers for chromatographic processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
- C08F222/36—Amides or imides
- C08F222/38—Amides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
Definitions
- the present invention relates to a cation exchange chromatography carrier used in a purification process of an antibody drug, and a method for separating an antibody monomer and a polymer produced in the process of producing the antibody drug.
- antibody polymers which are one of the impurities, are produced in the culture or purification stage and are considered to cause a decrease in antigen recognition ability or cause side effects, and their removal is an important management item in antibody drug production. Yes.
- Non-patent Document 1 Biotechnology and Bioengineering 108, 1494-1508, 2011 (Non-patent Document 1) reported the mechanism of antibody polymer formation and the importance of polymer removal, but antibody polymers are affinity purified by protein A chromatography. There is a problem that it cannot be removed in the process. Therefore, in the steps subsequent to the purification step by protein A chromatography, it is an important issue for antibody drug manufacturers to separate antibodies and their polymers and obtain highly pure antibodies.
- Non-Patent Document 1 proposes separation of an antibody and its polymer by a chromatography carrier having a mixed mode ligand.
- J.H. Chromatography A, 1217, 216-224, 2010 (Non-patent Document 2) and J. Org. Chromatography A, 1216, 902-909, 2009 (Non-patent Document 3) proposes separation of an antibody and its polymer by a hydrophobic (phenyl) chromatography carrier.
- the separation methods using these carriers have many practical problems such as a low antibody adsorption capacity as a target product and use at a high salt concentration.
- Cation exchange chromatography is used for the purpose of antibody polymer removal, host-derived protein removal, and protein A leak removal, and is inexpensive and may have a high track record in the production of many biopharmaceuticals. This is a very important step in the purification process after protein A chromatography. Therefore, it is desirable that impurities such as antibody polymers and host-derived proteins generated in the production process of antibody pharmaceuticals be reduced as much as possible by cation exchange chromatography.
- Patent Document 1 discloses purification of IGF1R (Insulin-like Growth Factor-1 Receptor), The use of a cation exchanger effective for removing impurities such as antibody polymers is disclosed.
- the cation exchange chromatography carrier when used for antibody drug production, has a high antibody adsorption amount from the viewpoint of productivity as well as separation performance from impurities, and can be processed at a high flow rate without compression. Is also required.
- Capto registered trademark
- TOYOPEARL registered trademark
- GigaCap S manufactured by Tosoh Corp.
- Fractogel registered trademark
- SE Hicap Eshmuno (registered trademark) S (Merck), UNOSphere (registered trademark) S (Bio-Rad), POROS (registered trademark) XS (APPLIED BIOSSYSTEMS) are being developed and sold. .
- JP-A-1-310744 discloses a method for producing an ion exchange chromatography carrier having a graft polymer as a ligand.
- cation exchange chromatography and anion exchange chromatography using Fractogel (registered trademark) TSK HW 65 (S) or LiChrosphere (registered trademark) diol as a base carrier and further chromatography using a copolymerized polymer as a ligand.
- a method for producing a graphy carrier is described.
- the separation of the antibody and its polymer has not been studied.
- a cation exchange chromatography carrier having a graft polymer other than the above as a ligand for example, in Japanese Translation of PCT International Publication No. 2011-529508 (Patent Document 3), Fractogel (registered trademark) TSK HW 65 (M) is grafted by polymerization.
- a method of introducing a copolymerized polymer as a ligand is disclosed.
- the dynamic binding capacity of a polyclonal antibody measured in a contact time of 2 minutes compared to the case where only a monomer containing sulfonic acid is graft-polymerized by copolymerizing a monomer containing sulfonic acid and a monomer not containing sulfonic acid Is shown to be higher.
- the publication does not clearly show the relationship between the composition of the copolymer and the dynamic binding capacity.
- the publication does not recognize or examine the importance of the influence of the composition of strong cationic groups and neutral groups of the copolymer on the separation of the antibody and its polymer.
- Patent Document 4 a graft polymer having both an ion-exchange group and a hydrophobic group using a monomer having a hydrophobic interaction is used as a ligand for Fractogel (registered trademark) TSK HW 65 (M).
- Fractogel registered trademark
- TSK HW 65 M
- Patent Document 5 a carrier obtained by binding polyacrylic acid as a ligand to a base carrier made of a methacrylic polymer as a raw material directly binds a carboxymethyl group to the base carrier. It has been shown to be superior to the separation of antibody monomers and their polymers compared to the supported carriers. However, when the carrier is produced, it is necessary to synthesize polyacrylic acid in advance, and there is a possibility that the problem of providing an inexpensive chromatography carrier desired by an antibody drug manufacturer may not be addressed. In addition, other important chromatographic carrier characteristics such as antibody binding capacity have not been studied, and problems remain in terms of practicality.
- the separation performance of the ion exchange chromatography carrier that can be suitably used for the purification of the antibody drug, in particular, the antibody monomer produced in the manufacturing process of the antibody drug and the antibody polymer that is a polymer thereof. Therefore, it is desired to provide a cation exchange chromatography carrier that has a good antibody adsorption property, a high antibody adsorption property, and a high purity of the antibody monomer.
- the present inventors bonded a structure obtained by polymerizing a monomer containing at least one sulfone group with a specific composition on a porous particle as a ligand.
- the inventors have found that an antibody monomer and a polymer thereof can be efficiently separated and can provide a cation exchange chromatography carrier having a high antibody adsorptivity, thereby completing the present invention.
- the present invention relates to a cation exchange chromatography carrier for antibody purification and a method for separating an antibody monomer and its polymer as shown below.
- a base carrier containing porous particles [Wherein R 1 , R 2 and R 3 are each independently a hydrogen atom or methyl, and A 1 and A 2 are —R 4 —SO 3 M, wherein R 4 is C 2 to C 4 alkylene, and M is a hydrogen atom, Na or K. ]
- a cation exchange chromatography carrier for purifying an antibody wherein a polymer containing at least one strong cationic monomer unit represented by the formula is combined in a range of 30 to 100 mol% of the whole monomer, and has an ion exchange capacity of 60
- a cation exchange chromatography carrier that is ⁇ 300 ⁇ mol / ml.
- the polymer is represented by the formula (3): [Wherein, R 1 and R 2 each independently represent a hydrogen atom or methyl, and R 5 and R 6 each independently represent a hydrogen atom, C 1 -C 4 alkyl or C 1 -C 4 alkoxymethyl. ] Or at least one neutral monomer unit represented by formula (4): [Wherein, R 1 and R 2 each independently represent a hydrogen atom or methyl, and A 3 represents a hydrogen atom, Na or K. ]
- the porous particles have a gel distribution coefficient Kav of 0.3 to 0.5 when pure water is used as a mobile phase in standard polyethylene oxide having a weight average molecular weight of 1.5 ⁇ 10 5 Da.
- the cation exchange chromatography carrier according to any one of the above [1] to [12], wherein the cation exchange chromatography carrier is a characteristic porous particle
- [15] A method for separating an antibody monomer and a polymer thereof produced in an antibody drug manufacturing process using the cation exchange chromatography carrier according to any one of [1] to [14] above.
- the cation exchange chromatography carrier of the present invention can be suitably used for purification of antibody drugs. According to a preferred embodiment of the present invention, by using the cation exchange chromatography carrier of the present invention, it is possible to efficiently separate the antibody monomer and its polymer produced in the manufacturing process of the antibody drug. Antibody monomers can be obtained with high purity.
- FIG. 1 is a chromatogram showing the measurement results of the resolution ( ⁇ C) of the monoclonal antibody by the gel of Example 1.
- FIG. 2 is a chromatogram showing the measurement results of the resolution ( ⁇ C) of the monoclonal antibody by the gel of Example 2.
- the cation exchange chromatography carrier for antibody purification of the present invention has, as a ligand, a base carrier containing porous particles represented by formula (1) or (2): [Wherein R 1 , R 2 and R 3 are each independently a hydrogen atom or methyl, and A 1 and A 2 are —R 4 —SO 3 M, wherein R 4 is C 2 to C 4 alkylene, and M is a hydrogen atom, Na or K. ]
- porous particles that are base carriers include strong cationic monomer units, and, if necessary, neutral monomer units and There is no particular limitation as long as it has a functional group (for example, a hydroxyl group or a carbamoyl group) for introducing a weak cationic monomer unit.
- porous particles include, for example, polysaccharides and derivatives thereof having the above functional groups, such as agarose, dextran, starch, cellulose, pullulan, chitin, chitosan, cellulose triacetate, cellulose diacetate; Preferred examples include organic polymers such as acrylamide, polymethacrylamide, polyacrylate, polymethacrylate, polyalkyl vinyl ether, and polyvinyl alcohol. Since these porous particles can ensure mechanical strength, it is preferable to form a crosslinked structure. Among these, in the present invention, it is preferable to use crosslinked cellulose particles in which the skeleton of the cellulose particles is reinforced by a crosslinking reaction.
- the crosslinked cellulose particles used in the present invention are not particularly limited as long as they are usually used as a base carrier for chromatography.
- the cellulose used as a raw material may be crystalline cellulose or amorphous cellulose, but crystalline cellulose is preferred because of its high strength.
- crosslinked cellulose particles examples include porous cellulose gels disclosed in Japanese Patent Application Laid-Open No. 2009-242770.
- the cross-linking method disclosed in the publication that is, a group consisting of a hydrochloride, sulfate, phosphate and borate in an amount of 6 to 20 times the number of moles of cellulose monomer in a suspension of uncrosslinked cellulose particles.
- Crosslinked cellulose particles obtained by the method of cross-linking by continuous dropwise addition or divided addition over the above have high mechanical strength, can be used at a high flow rate, and give a highly productive cation exchange chromatography carrier. be able to.
- the “cellulose monomer” means a glucose unit which is a structural unit of cellulose, and the number of moles of the cellulose monomer (that is, the degree of polymerization) is an amount obtained by subtracting water from one glucose unit, that is, a molecular weight 162 of 1. Calculated as moles from the dry weight of cellulose.
- the shape of the crosslinked cellulose particles is not particularly limited, but is preferably spherical because it has high mechanical strength, excellent gel sedimentation, and a uniform packed bed.
- the sphericity of the crosslinked cellulose particles is preferably 0.8 to 1.0.
- “sphericity” means the short diameter / long diameter of the cellulose particles.
- Spherical cellulose particles can be easily obtained by, for example, dissolving and regenerating crystalline cellulose or cellulose composed of a crystalline region and an amorphous region.
- Examples of the method for producing spherical cellulose include a method via an acetate described in JP-B-55-39565 and JP-B-55-40618, and thiocyan described in JP-B-63-62252.
- Examples include a method of molding from a cellulose solution dissolved in an amide containing lithium chloride.
- the spherical crosslinked cellulose particles can be obtained by crosslinking the spherical cellulose particles.
- the particle size of the porous particles used in the present invention is preferably 10 to 500 ⁇ m, more preferably 30 to 200 ⁇ m, and particularly preferably 50 to 150 ⁇ m.
- the average particle size is preferably 30 to 1000 ⁇ m, more preferably 40 to 200 ⁇ m, and further preferably 50 to 100 ⁇ m.
- the particle size and average particle size of porous particles are measured by irradiating a particle group with laser light and calculating the particle size distribution by calculation from the intensity distribution pattern of diffracted / scattered light emitted from the particle group. Calculation is performed using a laser diffraction / scattering particle size distribution measuring apparatus as a principle.
- a laser diffraction / scattering particle size distribution measuring device LA-950 manufactured by HORIBA, Ltd. can be used as a measuring device.
- volume average particle diameter (M V ) ⁇ (nd 4 ) / ⁇ (nd 3 ) [In the formula, nd represents the value of each particle diameter determined from an optical micrograph, and n represents the number of particles measured. ]
- the porosity of the porous particles used as the base carrier in the present invention can be characterized by the characteristics of the pore size.
- One of the indexes indicating the pore size characteristics is the gel distribution coefficient Kav.
- the pore size affects the flow rate characteristics and the dynamic adsorption capacity of the carrier in relation to the physical strength of the particles and the diffusibility of the target substance to be purified in the particles. Therefore, an optimum design according to the purpose is required.
- the pore size of the porous particles used in the present invention is a gel distribution when pure water is used as a mobile phase in standard polyethylene oxide having a weight average molecular weight of 1.5 ⁇ 10 5 Da.
- the coefficient Kav is preferably in the range of 0.15 to 0.6, more preferably 0.2 to 0.55, and still more preferably 0.3 to 0.5.
- the gel partition coefficient Kav can be obtained from the relationship between the elution volume and the column volume of a standard substance (for example, polyethylene oxide) having a specific molecular weight by the following equation.
- Kav (Ve ⁇ V 0 ) / (Vt ⁇ V 0 ) [In the formula, Ve represents a sample holding capacity (mL), Vt represents an empty column volume (mL), and V 0 represents a blue dextran holding capacity (mL). ]
- the method for measuring the gel distribution coefficient Kav is, for example, L. Fischer Biochemistry Experimental Method 2 “Gel Chromatography” 1st Edition (Tokyo Kagaku Dojin) and the like.
- the gel distribution coefficient Kav of the crosslinked porous cellulose particles used in the present invention can be adjusted, for example, by controlling the dissolution concentration of cellulose at the time of particle formation.
- an arbitrary substituent such as a hydrophobic group such as a phenyl group or a butyl group may be introduced as long as the object and the effect of the present invention are not impaired. Absent.
- a hydrophobic group When a hydrophobic group is introduced into the porous particles, it can be introduced by reacting phenyl glycidyl ether or butyl glycidyl ether in an alkaline solution.
- the porous particle has a formula (1) or (2) as a ligand: [Wherein R 1 , R 2 and R 3 are each independently a hydrogen atom or methyl, and A 1 and A 2 are —R 4 —SO 3 M, wherein R 4 is C 2 to C 4 alkylene, and M is a hydrogen atom, Na or K. ]
- a polymer containing at least one strong cationic monomer unit represented by the formula (1) is bound in the range of 30 to 100 mol% of the whole monomer.
- the C 2 -C 4 alkylene of R 4 is not particularly limited, but preferably includes ethylene, n-propylene, iso-propylene, n-butylene, iso-butylene, sec-butylene and the like.
- the polymer may be composed of only a strong cationic monomer unit represented by the formula (1) and / or (2), and is represented by the formula (1) and / or (2).
- another optional monomer unit such as a neutral monomer or a weak cationic monomer may be further included.
- the strong cationic monomer unit contained in the polymer may be either the monomer unit represented by the formula (1) or (2), or the monomer unit represented by the formula (1) and the formula (2).
- the monomer units represented by the formula (1) or (2) may be used alone or in combination of two or more. Moreover, you may use the other monomer unit arbitrarily contained in the said polymer by 1 type, or 2 or more types.
- the ion exchange capacity of the resulting cation exchange chromatography carrier Is 60 ⁇ mol / ml or more, more preferably 70 ⁇ mol / ml or more, and still more preferably 90 ⁇ mol / ml or more. Further, the ion exchange capacity is 300 ⁇ mol / ml or less, more preferably 250 ⁇ mol / ml or less, and further preferably 150 ⁇ mol / ml or less. When it is within this range, the separation performance between the antibody monomer and its polymer is good.
- the ion exchange capacity is, for example, acid-base titration as described in International Publication No. 2007/027139 pamphlet or reverse as described in Journal of Chromatography A, 1146, 202-215. It can generally be determined by titration.
- the monomer represented by the formula (1) examples include 2-acrylamido-2-methylpropanesulfonic acid, 2-acrylamidoethanesulfonic acid, 2-methacrylamide-2-methylpropanesulfonic acid, and 2-methacrylamideamidoethanesulfone. An acid etc. are mentioned. Of these, 2-acrylamido-2-methylpropanesulfonic acid is preferred.
- the monomer represented by the formula (2) include 3-sulfopropyl methacrylate and 2-sulfoethyl methacrylate. Of these, 3-sulfopropyl methacrylate is preferable.
- the polymer further contains any other monomer unit in addition to the strong cationic monomer unit represented by the formula (1) or (2), the other monomer unit does not impair the object of the present invention. If it is, it will not be restrict
- Other monomer units include, for example, formula (3): [Wherein, R 1 and R 2 each independently represent a hydrogen atom or methyl, and R 5 and R 6 each independently represent a hydrogen atom, C 1 -C 4 alkyl or C 1 -C 4 alkoxymethyl. ] Or at least one neutral monomer unit represented by formula (4): [Wherein, R 1 and R 2 each independently represent a hydrogen atom or methyl, and A 3 represents a hydrogen atom, Na or K. ] A weak cationic monomer unit represented by
- the C 1 -C 4 alkyl is not particularly limited, but preferably includes methyl, ethyl, n-propyl, iso-isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl and the like.
- C 1 -C 4 alkoxymethyl is not particularly limited as long as it is alkoxymethyl containing alkyl having 1 to 4 carbon atoms, but methoxymethyl, ethoxymethyl, n-propoxymethyl, iso-propoxymethyl, n-butoxymethyl , Iso-butoxymethyl, sec-butoxymethyl, tert-butoxymethyl and the like are preferable.
- the polymer may contain both a neutral monomer unit and a weak cationic monomer unit.
- neutral monomer represented by the formula (3) examples include N, N-dimethylacrylamide, N, N-diethylacrylamide, N-tert-butylacrylamide, N-iso-propylacrylamide, acrylamide, N, N- Examples include dimethylmethacrylamide, N-ethylmethacrylamide, methacrylamide, N- (methoxymethyl) methacrylamide, N- (iso-butoxymethyl) methacrylamide and the like.
- the weak cationic monomer represented by the formula (4) include acrylic acid, methacrylic acid, sodium acrylate, potassium acrylate, sodium methacrylate, and potassium methacrylate.
- the polymer used as a ligand in the cation exchange chromatography carrier of the present invention contains a neutral monomer unit and / or a weak cationic monomer unit.
- a cation exchange chromatography carrier in which a copolymer containing a strong cationic monomer unit represented by the formula (1) and a neutral monomer unit represented by the formula (3) is bound to a base carrier as a ligand,
- This cation exchange chromatography carrier is preferable because it has an excellent separation performance between the antibody monomer and its polymer and can efficiently separate and purify the antibody monomer.
- separation performance is particularly excellent when a copolymer of 2-acrylamido-2-methylpropanesulfonic acid and N, N-dimethylacrylamide is used as a ligand.
- a cation exchange chromatography carrier in which a copolymer containing a strong cationic monomer unit represented by the formula (1) and a weak cationic monomer unit represented by the formula (4) is bound to a base carrier as a ligand is:
- the weak cationic monomer can also function as a ligand for a weak cation exchanger generally used in the field of protein purification, and due to the effect of the weak cation group, the cation exchange chromatography carrier of the present invention includes a host. It can also be expected to remove impurities derived from it.
- the polymer is a copolymer having a strong cationic monomer unit represented by the formula (1) and / or (2) and a neutral monomer unit represented by the formula (3)
- a copolymer is obtained.
- the ratio of the strong cationic monomer unit to the whole constituting monomer is 30 mol% or more, preferably 40 mol% or more, and more preferably 50 mol% or more.
- the ratio of a strong cationic monomer unit is less than 100 mol%.
- the ion exchange capacity is 60 ⁇ mol / ml or more, preferably 70 ⁇ mol / ml or more, more preferably 90 ⁇ mol / ml or more, and particularly preferably 100 ⁇ mol / ml or more.
- the ion exchange capacity is 300 ⁇ mol / ml or less, preferably 250 ⁇ mol / ml or less, more preferably 210 ⁇ mol / ml or less, and particularly preferably 150 ⁇ mol / ml or less.
- the proportion of strong cationic monomer units is 40 mol% or more and less than 100 mol%, and the ion exchange capacity is 60 to 150 ⁇ mol / ml.
- the polymer is a copolymer having a strong cationic monomer unit represented by the formula (1) and / or (2) and a weak cationic monomer unit represented by the formula (4)
- the copolymer The ratio of the strong cationic monomer unit to the whole monomer constituting is 30 mol% or more, preferably 40 mol% or more, and more preferably 50 mol% or more. Moreover, the ratio of a strong cationic monomer unit is less than 100 mol%. Further, considering the dynamic adsorption capacity, the ion exchange capacity is 60 ⁇ mol / ml or more, preferably 70 ⁇ mol / ml or more, and more preferably 90 ⁇ mol / ml or more.
- the ion exchange capacity is 300 ⁇ mol / ml or less, preferably 250 ⁇ mol / ml or less.
- the proportion of the strong cationic monomer unit is 50 mol% or more and less than 100 mol%, and the ion exchange capacity is 90 to 250 ⁇ mol / ml.
- the ratio (S density) of the strong cationic monomer unit is calculated by calculating the molar ratio of S to N or Na contained in the monomer unit using the measured S content, N content or Na content. You can get it.
- the S content, N content or Na content can be measured using, for example, an emission spectroscopic analysis method, an elemental analysis method or an atomic absorption analysis method.
- the cation exchange chromatography carrier of the present invention is produced by introducing a polymer into a base carrier containing porous particles by a graft polymerization reaction.
- the graft polymerization reaction proceeds to a hydroxyl group based on a radical generated by the action of Ce (IV) under acidic conditions.
- a graft polymer can be formed on the base carrier by the reaction of the radical serving as the base point with the monomer present in the system.
- the reaction is preferably carried out when the pH of the solution is in the range of 0-4.
- the Ce (IV) salt is preferably in the range of 0.0001 to 1 mol / ml, more preferably in the range of 0.01 to 0.1 mol / ml, as the molar concentration in the reaction solution.
- the molar ratio of the charged amount of the strong cationic monomer used in the reaction and the other monomer such as the neutral monomer and the weak cationic monomer is preferably in the range of 99: 1 to 40:60, more preferably 70: It is in the range of 30 to 45:55.
- the cation exchange chromatography carrier for antibody purification of the present invention is excellent in the separation performance between the antibody monomer produced in the antibody drug production process and its polymer, and therefore, purification of biopharmaceuticals and the like. It can be suitably used in the process.
- the column is packed with the cation exchange chromatography carrier of the present invention, and a liquid containing the target product and impurities is poured into the column for the purpose of adsorbing only the target product or impurities.
- the salt concentration By increasing the salt concentration in a stepwise or continuous manner, it can be used for the purpose of separation utilizing the difference in affinity to the chromatographic support.
- antibodies used in the present invention include monoclonal antibodies and polyclonal antibodies.
- Examples of the type of antibody include mouse antibodies, llama antibodies, chimeric antibodies, humanized antibodies, human antibodies or antibodies in which Fc regions thereof are modified, and molecular types include, for example, IgG, IgM, IgA IgD, IgE, Fab, Fc, Fc-fusion protein, VH, VL, VHH, Fab′2, scFv, scFab, scDb or scDbFc.
- the antibodies used in the present invention also include monoclonal antibodies or polyclonal antibodies obtained by positively denaturing a part of these monoclonal antibodies or polyclonal antibodies.
- Examples of the method for denaturing a monoclonal antibody or a polyclonal antibody include the methods described in Journal of PHARMACEUTICAL SCIENCES, 2011, 100, 2104-2119.
- an antibody monomer is a molecule composed of one molecule of the antibody.
- An antibody polymer is a molecule in which two or more antibody monomers are polymerized by covalent bonding or non-covalent bonding, and examples thereof include dimers, trimers, multimers, aggregates, and aggregates. It is done.
- the target antibody monomer and its polymer can be separated by performing cation exchange chromatography using the cation exchange chromatography carrier of the present invention.
- cation exchange chromatography using the cation exchange chromatography carrier of the present invention may be used for separation of antibody monomers and impurities produced in the antibody drug manufacturing process.
- impurities include cultured cell-derived proteins, nucleic acids, viruses, protein A leaks, antibody degradation products, denaturation, removal of sugar chain components, modification of target antibodies that have undergone oxidation or deamidation, etc. And those produced by processes or other chromatographic processes.
- Examples of the antibody-containing aqueous solution to be used for the cation exchange chromatography include a composition obtained from a living body such as plasma, serum, milk or urine, an antibody obtained using a gene recombination technique or a cell fusion technique. Examples thereof include a culture solution of cells to be produced or fungi such as E. coli, or a composition obtained from a transgenic non-human animal, plant or insect.
- Examples of cells that produce antibodies include transformed cells in which a gene encoding a desired antibody is incorporated into a host cell.
- Examples of host cells include cell lines such as animal cells, plant cells, and yeast cells. Specifically, for example, Chinese hamster ovary cells (CHO cells), mouse myeloma cells NS0 cells, SP2 / 0 cells, rat myeloma cells YB2 / 0 cells, IR983F cells, Syrian hamster kidney-derived cells BHK Cells, human myeloma cells such as Namalva cells, embryonic stem cells, or fertilized egg cells.
- CHO cells Chinese hamster ovary cells
- mouse myeloma cells NS0 cells such as mouse myeloma cells NS0 cells, SP2 / 0 cells, rat myeloma cells YB2 / 0 cells, IR983F cells, Syrian hamster kidney-derived cells BHK Cells
- human myeloma cells such
- any medium suitable for culturing each cell can be used as a medium for culturing antibody-producing cells.
- a medium for culturing animal cells can be used for normal animal cell culture.
- a medium is used.
- any medium such as a serum-containing medium, a medium not containing animal-derived components such as serum albumin or serum fraction, a serum-free medium, or a protein-free medium can be used, but preferably a serum-free medium or a protein-free medium Is used.
- physiologically active substances or nutrient factors necessary for the growth of cells that produce antibodies can be added as necessary. These additives are preliminarily contained in the medium before culturing, or appropriately added to the culture medium as an additional medium or an added solution during culturing.
- the additional supply method may be in any form such as one solution or a mixed solution of two or more kinds, and the addition method may be either continuous or intermittent.
- transgenic non-human animals, plants or insects that produce antibodies include non-human animals, plants or insects in which a gene encoding a protein is incorporated into cells.
- non-human animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, sheep, pigs, goats, cows or monkeys.
- the plant include tobacco, potato, tomato, carrot, soy bean, rape, alfalfa, rice, wheat, barley or corn.
- the antibody-containing aqueous solution to be used for the cation exchange chromatography is obtained from a living body such as plasma or urine containing the antibody as described above, or an antibody-containing aqueous solution obtained in a purification step. Is also included. Specific examples include a cell removal solution, a precipitate removal solution, an alcohol fraction solution, a salting-out fraction solution, and a chromatography eluate.
- the antibody-containing aqueous solution may be subjected to the purification method of the present invention after removing insoluble matters such as particles in advance and removing the insoluble matters. Examples of methods for removing insoluble matters such as particles include centrifugation, crossflow filtration (tangential flow filtration), depth filter filtration, membrane filter filtration, dialysis, or a combination of these methods. Method.
- the pH, conductivity, buffer solution, salt concentration, additive, antibody concentration or cation exchange chromatography of the antibody-containing aqueous solution can be used.
- the antibody loading amount per unit volume of the graphic carrier is adjusted to suitable conditions and then subjected to the cation exchange chromatography.
- Examples of methods for adjusting the pH, conductivity, buffer solution, salt concentration, additives, antibody concentration or antibody loading per unit volume of the cation exchange chromatography carrier of the antibody-containing aqueous solution include an ultrafiltration membrane. Examples include the ultrafiltration method used.
- the cation exchange chromatography is performed in an adsorption mode or a non-adsorption mode depending on the purpose.
- the adsorption mode in the cation exchange chromatography means that the antibody-containing aqueous solution to be subjected to the cation exchange chromatography is brought into contact with the carrier, the target antibody is adsorbed on the carrier, and then washed as necessary. After that, it means that the adsorbed fraction is recovered by eluting with a buffer solution in which pH, electric conductivity, buffer component, salt concentration, additive or the like is changed.
- the non-adsorption mode in the cation exchange chromatography means that an antibody-containing aqueous solution to be subjected to the cation exchange chromatography is brought into contact with the carrier, and a non-adsorbed fraction on the carrier is recovered. At this time, by collecting an appropriate fraction, the desired antibody monomer and the compound to be separated can be separated.
- suitable conditions are selected for pH, conductivity, buffer components, salt concentration, additives, and the like.
- the difference in affinity between the target antibody monomer and the compound to be separated is used for the chromatographic carrier.
- the carrier structure ligand species, ligand density, ligand distribution, particle size, pore size, base matrix composition, etc.
- the physicochemical properties isoelectric point, charge, hydrophobicity
- the elution method in the adsorption mode includes a one-step elution method in which a buffer solution having a specific salt concentration or pH is used so that the affinity between the target antibody monomer and the carrier decreases, A stepwise method in which the target antibody monomer is eluted by changing the concentration or pH or a gradient method in which the target antibody monomer is eluted by continuously changing the salt concentration or pH can be mentioned.
- the components of the antibody-containing aqueous solution and the buffer used for washing or elution are not particularly limited as long as they have a buffering capacity.
- a buffering capacity 1 to 300 mmol / L phosphate, citrate, acetate, Examples thereof include succinate, maleate, borate, Tris (base), HEPES, MES, PIPES, MOPS, TES, and Tricine.
- the above salts can also be used in combination with other salts such as sodium chloride, potassium chloride, calcium chloride, sodium citrate, sodium sulfate or ammonium sulfate.
- the buffer component is used in combination with, for example, amino acids such as glycine, alanine, arginine, serine, threonine, glutamic acid, aspartic acid or histidine, sugars such as glucose, sucrose, lactose, sialic acid, or derivatives thereof. You can also.
- amino acids such as glycine, alanine, arginine, serine, threonine, glutamic acid, aspartic acid or histidine
- sugars such as glucose, sucrose, lactose, sialic acid, or derivatives thereof. You can also.
- the pH of the antibody-containing aqueous solution and the buffer used for washing or elution is preferably in the range of 2 to 9, and more preferably in the range of 3 to 8.
- the linear velocity of the antibody-containing aqueous solution and the buffer used for washing or elution is preferably in the range of 50 to 1000 cm / h.
- the antibody loading per unit volume of the cation exchange chromatography carrier is preferably 10 to 200 g / L, more preferably 60 to 150 g / L.
- cation exchange chromatography In the production of antibody drugs, other purification methods may be combined with cation exchange chromatography using the cation exchange chromatography carrier of the present invention. Any purification method combined with the cation exchange chromatography can be used as long as it is suitable for the production of pharmaceuticals. For example, chromatography, activated carbon treatment, alcohol fractionation, precipitate removal, salting out, buffer solution Examples include exchange, concentration, dilution, filtration, virus inactivation, and virus removal.
- the purification method combined with the cation exchange chromatography may combine a plurality of types and numbers. Moreover, the purification method combined with these cation exchange chromatography can be implemented regardless of before and after the cation exchange chromatography.
- Examples of the carrier or membrane used in the chromatography combined with the cation exchange chromatography include an affinity carrier such as a heparin carrier or protein A carrier, a cation exchange carrier, a cation exchange membrane, an anion exchange carrier, an anion exchange membrane, Examples thereof include a gel filtration carrier, a hydrophobic interaction carrier, a reverse phase carrier, a hydroxyapatite carrier, a fluoroapatite carrier, a sulfated cellulose carrier, a sulfated agarose carrier, and a mixed mode (multimodal) carrier.
- the indexes indicating the separation characteristics of the antibody monomer and its polymer for example, the degree of separation ⁇ Cp using a monoclonal antibody or the degree of separation ⁇ Cp using a polyclonal antibody Is mentioned.
- the resolution ⁇ C or ⁇ Cp is obtained by adding a solution containing a monoclonal antibody or a polyclonal antibody containing a polymer to a column packed with a cation exchange chromatography carrier, monitoring the peak of the monomer or polymer in the eluate, It can be determined as the difference in electrical conductivity value corresponding to the peak top of the obtained monomer or polymer. The greater the value of ⁇ C or ⁇ Cp, the higher the separation between the antibody monomer and its polymer.
- 10% dynamic binding capacity can be mentioned.
- the 10% dynamic binding capacity is obtained by adding a solution containing an antibody having a known concentration to a column packed with a cation exchange chromatography carrier, monitoring the absorbance of the eluate, and leaking 10% of the absorbance of the added sample. Calculated from the amount of added protein at the time it was seen. The greater the 10% dynamic binding capacity value, the higher the antibody adsorption.
- the average particle size analyzed using a laser diffraction / scattering type particle size distribution measuring apparatus LA-950 manufactured by Horiba, Ltd. was 93 ⁇ m.
- the gel distribution coefficient Kav was 0.27 when pure water was used as a mobile phase in standard polyethylene oxide having a weight average molecular weight of 1.5 ⁇ 10 5 Da.
- Example 1 In a 500 ml separable flask, 64.4 g of pure water and 8.7 g of 2-acrylamido-2-methylpropanesulfonic acid were added and dissolved. Next, 3.4 g of 48.7% (w / w) sodium hydroxide aqueous solution was added for neutralization. Next, 80.0 g of crosslinked 6% cellulose particles of Reference Example 1 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state.
- the obtained wet gel was stored with excess water removed.
- the ion exchange capacity at this time was 110 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody was 61 mg / ml
- the S content was 3.1% (w / w)
- ⁇ C was 6.6 mS / cm
- ⁇ Cp was 26 mS / ml. cm.
- Example 2 In a 500 ml separable flask, 64.4 g of pure water and 8.7 g of 2-acrylamido-2-methylpropanesulfonic acid were added and dissolved. Next, 3.4 g of 48.7% (w / w) sodium hydroxide aqueous solution was added for neutralization. Next, 2.3 g of N, N-dimethylacrylamide was added. Next, 80.0 g of crosslinked 6% cellulose particles of Reference Example 1 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state.
- Example 3 A wet gel was obtained in the same manner as in Example 2 except that the amount of N, N-dimethylacrylamide charged was changed to 4.5 g.
- the ion exchange capacity at this time is 130 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody is 119 mg / ml
- the S content is 2.6% (w / w)
- the N content is 2.9% (w / w).
- ⁇ Cp was 18 mS / cm.
- Example 4 In a 500 ml separable flask, 50.6 g of pure water and 3.0 g of 2-acrylamido-2-methylpropanesulfonic acid were added and dissolved. Next, the solution was neutralized by adding 1.2 g of a 48.7% (w / w) aqueous sodium hydroxide solution. Next, 0.62 g of N, N-dimethylacrylamide was added. Next, 60.0 g of crosslinked 6% cellulose particles of Reference Example 1 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state.
- the obtained wet gel was stored with excess water removed.
- the ion exchange capacity at this time is 66 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody is 73 mg / ml
- the S content is 1.7% (w / w)
- the N content is 1.2%
- ⁇ C is 6. 8 mS / cm and ⁇ Cp were 22 mS / cm.
- Example 5 The amount of 2-acrylamido-2-methylpropanesulfonic acid charged to 12.2 g, the amount of 48.7% (w / w) sodium hydroxide aqueous solution to 4.85 g, and the amount of N, N-dimethylacrylamide charged to A wet gel was obtained in the same manner as in Example 4 except that the amount was changed to 6.4 g.
- the ion exchange capacity at this time is 210 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody is 101 mg / ml
- the S content is 3.8% (w / w)
- the N content is 4.2% (w / w).
- ⁇ Cp was 17 mS / cm.
- the ion exchange capacity was 172 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody was 93 mg / ml
- the S content was 3.2% (w / w)
- the N content was 3.5% (w / w).
- ⁇ C was 5.8 mS / cm
- ⁇ Cp was 18 mS / cm.
- Example A A wet gel was obtained in the same manner as in Example 2 except that the amount of N, N-dimethylacrylamide charged was changed to 10.1 g.
- the ion exchange capacity at this time is 130 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody is 125 mg / ml
- the S content is 2.3% (w / w)
- the N content is 4.1% (w / w).
- ⁇ Cp was 13 mS / cm.
- Example 7 The amount of 2-acrylamido-2-methylpropanesulfonic acid charged to 2.02 g, the amount of 48.7% (w / w) aqueous sodium hydroxide to 0.82 g, and the amount of acrylic acid charged to 0.39 g A wet gel was obtained in the same manner as in Comparative Example D except that The ion exchange capacity at this time was 97 ⁇ mol / ml, the 10% dynamic binding capacity of the polyclonal antibody was 77 mg / ml, the S content was 1.5% (w / w), and the Na content was 2.0% (w / w). , ⁇ C was 6.5 mS / cm, and ⁇ Cp was 22 mS / cm.
- Example 8> The amount of 2-acrylamido-2-methylpropanesulfonic acid charged to 4.07 g, the amount of 48.7% (w / w) aqueous sodium hydroxide to 1.62 g, and the amount of acrylic acid charged to 1.56 g A wet gel was obtained in the same manner as in Comparative Example D except that the change was made.
- the ion exchange capacity at this time was 246 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody was 70 mg / ml
- the S content was 2.3% (w / w)
- the Na content was 4.8% (w / w).
- ⁇ C was 4.7 mS / cm
- ⁇ Cp was 19 mS / cm.
- Example 9 In a 500 ml separable flask, 32.7 g of pure water and 2.16 g of 2-acrylamido-2-methylpropanesulfonic acid were added and dissolved. Next, 0.86 g of 48.7% (w / w) aqueous sodium hydroxide solution was added for neutralization. Next, 40.0 g of crosslinked 6% cellulose particles of Reference Example 1 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state.
- Example 10 Example 1 except that the amount of 2-acrylamido-2-methylpropanesulfonic acid charged was changed to 4.07 g and the amount of 48.7% (w / w) sodium hydroxide aqueous solution was changed to 1.62 g. Similarly, a wet gel was obtained. The ion exchange capacity at this time was 240 ⁇ mol / ml, the 10% dynamic binding capacity of the polyclonal antibody was 38 mg / ml, the S content was 5.1% (w / w), and ⁇ Cp was 25 mS / cm.
- Example 9 except that the amount of 2-acrylamido-2-methylpropanesulfonic acid charged was changed to 19.4 g and the amount of 48.7% (w / w) sodium hydroxide aqueous solution was changed to 7.70 g. Similarly, a wet gel was obtained.
- the ion exchange capacity at this time was 404 ⁇ mol / ml
- the 10% dynamic binding capacity of the polyclonal antibody was 26 mg / ml
- the S content was 7.4% (w / w)
- ⁇ C was 7.9 mS / cm
- ⁇ Cp was 25 mS / ml. cm.
- Example 11 In a 500 ml separable flask, 66.4 g of pure water and 7.02 g of 3-sulfopropyl methacrylate potassium salt were added and dissolved. Next, 80.0 g of crosslinked 6% cellulose particles of Reference Example 1 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state. After 1 hour, a solution prepared by dissolving 5.20 g of ammonium cerium nitrate in 18.1 ml of 0.17 mol / ml nitric acid was slowly added into the separable flask from the dropping funnel. After completion of dropping, the temperature was raised to 40 ° C. and stirred for 22 hours.
- Example 12 In a 500 ml separable flask, 91.5 g of pure water and 4.20 g of 2-acrylamido-2-methylpropanesulfonic acid were added and dissolved. Then, 1.67 g of 48.7% (w / w) sodium hydroxide aqueous solution was added for neutralization. Next, 50.0 g of crosslinked 10% cellulose particles of Reference Example 2 were added to prepare a slurry. It stirred for 1 hour, blowing nitrogen in a separable flask in this state.
- the column was connected to the system and equilibrated with a buffer at a flow rate of 1 mL / min until the UV (ultraviolet light absorbance, 280 nm), electric conductivity, and pH of the column effluent became constant. Thereafter, the baseline UV was reduced to zero.
- the antibody solution was allowed to flow through the column at a flow rate of 0.5 mL / min. The UV of the column effluent was monitored, and the flow of the antibody solution was stopped when the UV of the column effluent reached 10% of the previously measured UV of the antibody solution.
- the 10% dynamic binding capacity was determined by the following formula. This analysis was performed in a room at 25 ° C.
- the dead volume is a volume (ml) obtained by adding the system piping volume and the column void volume.
- FIGS. 1 and 2 show the analysis results (chromatograms) of the monoclonal antibodies using the gels of Example 1 and Example 2 obtained by measuring the degree of separation ( ⁇ C), respectively.
- the ion exchange capacity (IEC) per ml of each gel is: (0.01 ⁇ 40 / 1000-0.1 ⁇ A / 1000) ⁇ 1000000 ( ⁇ mol / ml) Can be obtained.
- the amount of 0.01 mol / L sodium hydroxide solution was changed from 40 ml to 60 ml, and a solution was prepared in the same manner as described above.
- the ion exchange capacity of 1 ml of the gel obtained in Comparative Example E is given by the following formula: (0.01 ⁇ 60 / 1000-0.1 ⁇ A / 1000) ⁇ 1000000 ( ⁇ mol / ml) Can be obtained.
- Table 1 shows the influence of neutral monomer introduction on the dynamic adsorption capacity and separation characteristics of the cation exchange chromatography carrier of the present invention.
- Table 2 shows the effect of weak cation monomer introduction on the dynamic adsorption capacity and separation characteristics of the cation exchange chromatography carrier of the present invention.
- Table 3 shows the influence of the amount of strong cation monomer introduced, the difference in structure of the strong cation monomer, and the difference in Kav of crosslinked cellulose particles on the dynamic adsorption capacity and separation characteristics of the cation exchange chromatography carrier of the present invention.
- the cation exchange chromatography carrier for antibody purification of the present invention can efficiently separate the antibody monomer and its polymer produced in the manufacturing process of the antibody drug, it is suitable for the purification of biopharmaceuticals. Can be used.
Abstract
Description
[式中、R1、R2およびR3は、それぞれ独立して、水素原子またはメチルであり、A1およびA2は、-R4-SO3Mであり、ここで、R4はC2~C4のアルキレンであり、Mは、水素原子、NaまたはKである。]
で示される少なくとも1種の強カチオン性モノマー単位をモノマー全体の30~100mol%の範囲で含む重合体が結合してなる抗体精製用陽イオン交換クロマトグラフィー担体であって、そのイオン交換容量が60~300μmol/mlである陽イオン交換クロマトグラフィー担体。
[式中、R1およびR2は、それぞれ独立して、水素原子またはメチルであり、R5およびR6は、それぞれ独立して、水素原子、C1~C4のアルキルまたはC1~C4のアルコキシメチルである。]
で示される少なくとも1種の中性モノマー単位、または、式(4):
[式中、R1およびR2は、それぞれ独立して、水素原子またはメチルであり、A3は、水素原子、NaまたはKである。]
で示される少なくとも1種の弱カチオン性モノマー単位をさらに含む、上記[1]記載の陽イオン交換クロマトグラフィー担体。
本発明の抗体精製用陽イオン交換クロマトグラフィー担体は、多孔性粒子を含むベース担体に、リガンドとして、式(1)または(2):
[式中、R1、R2およびR3は、それぞれ独立して、水素原子またはメチルであり、A1およびA2は、-R4-SO3Mであり、ここで、R4はC2~C4のアルキレンであり、Mは、水素原子、NaまたはKである。]
で示される少なくとも1種の強カチオン性モノマー単位をモノマー全体の30~100mol%の範囲で含む重合体が結合した構造を有しており、そのイオン交換容量が60~300μmol/mlであることを特徴としている。
本発明の陽イオン交換クロマトグラフィー担体において、ベース担体として用いる多孔性粒子としては、強カチオン性モノマー単位、ならびに必要に応じて、中性モノマー単位および弱カチオン性モノマー単位を導入するための官能基(例えば、水酸基またはカルバモイル基など)を有するものであれば特に制限されない。そのような多孔性粒子としては、上記のような官能基を有する、例えば、アガロース、デキストラン、でんぷん、セルロース、プルラン、キチン、キトサン、三酢酸セルロース、二酢酸セルロースなどの多糖類およびその誘導体;ポリアクリルアミド、ポリメタクリルアミド、ポリアクリレート、ポリメタクリレート、ポリアルキルビニルエーテル、ポリビニルアルコールなどの有機重合体などが好ましく挙げられる。これらの多孔性粒子は、機械的強度を確保できることから、架橋構造を形成していることが好ましい。これらの中でも、本発明においては、架橋反応によってセルロース粒子の骨格が補強された架橋セルロース粒子を用いることが好ましい。
体積平均粒子径(MV)=Σ(nd4)/Σ(nd3)
[式中、ndは光学顕微鏡写真から求めたそれぞれの粒子径の値を表し、nは測定した粒子の個数を表す。]
Kav=(Ve-V0)/(Vt-V0)
[式中、Veはサンプルの保持容量(mL)、Vtは空カラム体積(mL)、V0はブルーデキストラン保持容量(mL)を表す。]
本発明の抗体精製用陽イオン交換クロマトグラフィー担体においては、前記多孔性粒子に、リガンドとして、式(1)または(2):
[式中、R1、R2およびR3は、それぞれ独立して、水素原子またはメチルであり、A1およびA2は、-R4-SO3Mであり、ここで、R4はC2~C4のアルキレンであり、Mは、水素原子、NaまたはKである。]
で示される少なくとも1種の強カチオン性モノマー単位をモノマー全体の30~100mol%の範囲で含む重合体が結合している。R4のC2~C4のアルキレンとしては、特に制限されないが、エチレン、n-プロピレン、iso-プロピレン、n-ブチレン、iso-ブチレン、sec-ブチレンなどが好ましく挙げられる。
[式中、R1およびR2は、それぞれ独立して、水素原子またはメチルであり、R5およびR6は、それぞれ独立して、水素原子、C1~C4のアルキルまたはC1~C4のアルコキシメチルである。]
で示される少なくとも1種の中性モノマー単位、または、式(4):
[式中、R1およびR2は、それぞれ独立して、水素原子またはメチルであり、A3は、水素原子、NaまたはKである。]
で示される弱カチオン性モノマー単位が好ましく挙げられる。
さらに動的吸着容量を考慮すると、イオン交換容量は60μmol/ml以上であり、70μmol/ml以上が好ましく、90μmol/ml以上がより好ましく、100μmol/ml以上が特に好ましい。また、イオン交換容量は、300μmol/ml以下であり、250μmol/ml以下が好ましく、210μmol/ml以下がより好ましく、150μmol/ml以下が特に好ましい。
最適な実施形態では、強カチオン性モノマー単位の割合は40mol%以上、100mol%未満であり、且つ、イオン交換容量は60~150μmol/mlである。
さらに動的吸着容量を考慮すると、イオン交換容量は60μmol/ml以上であり、70μmol/ml以上が好ましく、90μmol/ml以上がより好ましい。また、イオン交換容量は、300μmol/ml以下であり、250μmol/ml以下が好ましい。
最適な実施形態では、強カチオン性モノマー単位の割合は50mol%以上、100mol%未満であり、且つ、イオン交換容量は90~250μmol/mlである。
次に、本発明の抗体精製用陽イオン交換クロマトグラフィー担体の製造方法について述べる。
本発明の抗体精製用陽イオン交換クロマトグラフィー担体は、抗体医薬製造過程で生産される抗体単量体とその重合体との分離性能が優れているため、バイオ医薬品などの精製工程において好適に使用することができる。具体的にはカラムに本発明の陽イオン交換クロマトグラフィー担体を充填し、そこへ目的物と不純物を含む液を流し目的物あるいは不純物のみを吸着させる目的や目的物と不純物を共に吸着させ溶出時の塩濃度を段階的あるいは連続的に増加させることでクロマトグラフィー担体への親和性の違いを利用して分離する目的に使用することができる。
吸着モードの溶出方法としては、目的の抗体単量体と担体との親和性が低下するような特定の塩濃度またはpHの緩衝液を通液して溶出させる一段階溶出法、段階的に塩濃度またはpHを変化させて目的の抗体単量体を溶出させるステップワイズ法または連続的に塩濃度またはpHを変化させて目的の抗体単量体を溶出させるグラジエント法が挙げられる。
〔6%球状セルロース粒子(含水)の製造〕
(1)100gのチオシアン酸カルシウム60重量%水溶液に6.4gの結晶性セルロース(旭化成ケミカルズ株式会社製、商品名:セオラスPH101)を加え、110~120℃に加熱して溶解した。
(2)この溶液に界面活性剤としてソルビタンモノオレエート6gを添加し、130~140℃に予め加熱したo-ジクロロベンゼン480ml中に滴下し、200~300rpmにて攪拌分散した。
(3)次いで、上記分散液を40℃以下まで冷却し、メタノール190ml中に注ぎ、粒子の懸濁液を得た。
(4)この懸濁液を濾過分別し、粒子をメタノール190mlにて洗浄し、濾過分別した。この洗浄操作を数回行った。
(5)さらに大量の水で洗浄した後、球状セルロース粒子を得た。
(6)次いで、この球状セルロース粒子をJIS標準ふるい規格53μm~125μmのふるいにかけて、所望の粒子径(実粒子サイズ間隔50~150μm、平均粒子径約100μm)にし、目的の6%球状セルロース粒子(含水:セルロース溶解濃度6%)を得た。なお、ここでの平均粒子径は、光学顕微鏡で撮影した画像の粒子径を、ノギスなどを用いて計測して撮影倍率から元の粒子径を求め、それぞれの粒子径の値から、下記の式によって算出して求めた。
体積平均粒子径(MV)=Σ(nd4)/Σ(nd3)
[式中、ndは光学顕微鏡写真から求めたそれぞれの粒子径の値を表し、nは測定した粒子の個数を表す。]
(1)上記で得られた6%球状セルロース粒子(含水)100gに121gの純水を加え、攪拌しながら加温した。30℃に到達したところで45重量%のNaOH水溶液3.3gとNaBH40.5gとを加え、撹拌した。初期アルカリ濃度は0.69%(w/w)であった。
(2)30分後、60gのNa2SO4を反応液に加え、溶解させた。混合物の温度が50℃に到達した時点で、2時間撹拌を継続した。
(3)50℃で混合物の撹拌を継続しながら、45重量%のNaOH水溶液48gと、エピクロロヒドリン50gとをそれぞれ25等分した量を、15分置きにおよそ6時間かけて添加した。
(4)添加終了後、この混合物を温度50℃で16時間反応させた。
(5)この混合物を温度40℃以下に冷却した後、酢酸2.6gを加え、中和した。
(6)反応混合物を濾過してゲルを回収し、純水で濾過洗浄し、目的の架橋6%セルロース粒子を得た。この時、株式会社堀場製作所のレーザ回折/散乱式の粒子径分布測定装置LA-950を用いて分析した平均粒子径は85μmであった。また、重量平均分子量1.5×105Daの標準ポリエチレンオキシドにおいて純水を移動相として使用したときのゲル分配係数Kavは0.38であった。
〔10%球状セルロース粒子(含水)の製造〕
(1)313gのチオシアン酸カルシウム60重量%水溶液に34.8gの結晶性セルロース(旭化成ケミカルズ株式会社製、商品名:セオラスPH301)を加え、110~120℃に加熱して溶解した。
(2)この溶液に界面活性剤としてソルビタンモノオレエート1.83gを添加し、130~140℃に予め加熱したo-ジクロロベンゼン480ml中に滴下し、200~300rpmにて攪拌分散した。
(3)次いで、上記分散液を40℃以下まで冷却し、メタノール533ml中に注ぎ、粒子の懸濁液を得た。
(4)この懸濁液を濾過分別し、粒子をメタノール620mlにて洗浄し、濾過分別した。この洗浄操作を数回行った。
(5)さらに大量の水で洗浄した後、球状セルロース粒子を得た。
(6)次いで、この球状セルロース粒子をJIS標準ふるい規格53μm~125μmのふるいにかけて、所望の粒子径(実粒子サイズ間隔50~150μm、平均粒子径約100μm)にし、目的の10%球状セルロース粒子(含水:セルロース溶解濃度10%)を得た。ここでの平均粒子径は、参考例1の「6%球状セルロース粒子(含水)の製造」において述べた方法と同様の方法で求めた。
(1)上記で得られた10%球状セルロース粒子(含水)100gに199gの純水を加え、攪拌しながら加温した。30℃に到達したところで45重量%のNaOH水溶液4.46gとNaBH40.71gとを加え、撹拌した。初期アルカリ濃度は0.69%(w/w)であった。
(2)30分後、81gのNa2SO4を反応液に加え、溶解させた。混合物の温度が50℃に到達した時点で、2時間撹拌を継続した。
(3)50℃で混合物の撹拌を継続しながら、45重量%のNaOH水溶液62gと、エピクロロヒドリン64gとをそれぞれ25等分した量を、15分置きにおよそ6時間かけて添加した。
(4)添加終了後、この混合物を温度50℃で16時間反応させた。
(5)この混合物を温度40℃以下に冷却した後、酢酸3.3gを加え、中和した。
(6)反応混合物を濾過してゲルを回収し、純水で濾過洗浄し、目的の架橋10%セルロース粒子を得た。この時、株式会社堀場製作所のレーザ回折/散乱式の粒子径分布測定装置LA-950を用いて分析した平均粒子径は93μmであった。また、重量平均分子量1.5×105Daの標準ポリエチレンオキシドにおいて純水を移動相として使用したときのゲル分配係数Kavは0.27であった。
500mlセパラブルフラスコに純水64.4gと2-アクリルアミド-2-メチルプロパンスルホン酸8.7g加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液3.4g加え中和した。次に参考例1の架橋6%セルロース粒子80.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム5.19gを0.17mol/ml硝酸18.1mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを80mlの純水で5回洗浄した。次に1mol/L硫酸120mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液160mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は110μmol/ml、ポリクローナル抗体の10%動的結合容量は61mg/ml、S含量は3.1%(w/w)、ΔCは6.6mS/cm、ΔCpは26mS/cmであった。
500mlセパラブルフラスコに純水64.4gと2-アクリルアミド-2-メチルプロパンスルホン酸8.7g加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液3.4g加え中和した。次にN,N-ジメチルアクリルアミド2.3gを加えた。次に参考例1の架橋6%セルロース粒子80.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム5.19gを0.17mol/ml硝酸に溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを80mlの純水で5回洗浄した。次に1mol/L硫酸120mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液160mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は126μmol/ml、ポリクローナル抗体の10%動的結合容量は114mg/ml、S含量は2.8%(w/w)、N含量は2.1%、ΔCは6.1mS/cm、ΔCpは22mS/cmであった。
N,N-ジメチルアクリルアミドの仕込み量を4.5gに変えたことを除いて実施例2と同様に湿ゲルを得た。このときのイオン交換容量は130μmol/ml、ポリクローナル抗体の10%動的結合容量は119mg/ml、S含量は2.6%(w/w)、N含量は2.9%(w/w)、ΔCpは18mS/cmであった。
500mlセパラブルフラスコに純水50.6gと2-アクリルアミド-2-メチルプロパンスルホン酸3.0g加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液1.2g加え中和した。次にN,N-ジメチルアクリルアミド0.62gを加えた。次に参考例1の架橋6%セルロース粒子60.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム3.7gを0.17mol/ml硝酸13.9mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを60mlの純水で5回洗浄した。次に1mol/L硫酸90mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液120mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は66μmol/ml、ポリクローナル抗体の10%動的結合容量は73mg/ml、S含量は1.7%(w/w)、N含量は1.2%、ΔCは6.8mS/cm、ΔCpは22mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を12.2gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込みを4.85gに、N,N-ジメチルアクリルアミドの仕込み量を6.4gに変えたことを除いて実施例4と同様に湿ゲルを得た。このときのイオン交換容量は210μmol/ml、ポリクローナル抗体の10%動的結合容量は101mg/ml、S含量は3.8%(w/w)、N含量は4.2%(w/w)、ΔCpは17mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を13.1gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込みを5.2gに、N,N-ジメチルアクリルアミドの仕込み量を5.30gに変えたことを除いて実施例2と同様に湿ゲルを得た。このときのイオン交換容量は172μmol/ml、ポリクローナル抗体の10%動的結合容量は93mg/ml、S含量は3.2%(w/w)、N含量は3.5%(w/w)、ΔCは5.8mS/cm、ΔCpは18mS/cmであった。
N,N-ジメチルアクリルアミドの仕込み量を10.1gに変えたことを除いて実施例2と同様に湿ゲルを得た。このときのイオン交換容量は130μmol/ml、ポリクローナル抗体の10%動的結合容量は125mg/ml、S含量は2.3%(w/w)、N含量は4.1%(w/w)、ΔCpは13mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を12.6gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込みを5.0gに、N,N-ジメチルアクリルアミドの仕込み量を23.2gに変えたことを除いて実施例2と同様に湿ゲルを得た。このときのイオン交換容量は102μmol/ml、ポリクローナル抗体の10%動的結合容量は5mg/ml、S含量は2.6%(w/w)、N含量は6.2%(w/w)であった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を18.5gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込みを7.35gに、N,N-ジメチルアクリルアミドの仕込み量を8.49gに変えたことを除いて実施例4と同様に湿ゲルを得た。このときのイオン交換容量は318μmol/ml、ポリクローナル抗体の10%動的結合容量は44mg/ml、S含量は4.6%(w/w)、N含量は4.9%(w/w)、ΔCpは19mS/cmであった。
500mlセパラブルフラスコに純水33.3gと2-アクリルアミド-2-メチルプロパンスルホン酸1.25g加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液0.49g加え中和した。次にアクリル酸0.14gを加えた。次に参考例1の架橋6%セルロース粒子40.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム2.45gを0.17mol/ml硝酸9.2mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを40mlの純水で5回洗浄した。次に1mol/L硫酸60mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液80mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は48μmol/ml、ポリクローナル抗体の10%動的結合容量は45mg/ml、S含量は0.95%(w/w)、Na含量は1.1%、ΔCは6.6mS/cm、ΔCpは23mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を2.02gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込み量を0.82gに、アクリル酸の仕込み量を0.39gに変えたことを除いて比較例Dと同様に湿ゲルを得た。このときのイオン交換容量は97μmol/ml、ポリクローナル抗体の10%動的結合容量は77mg/ml、S含量は1.5%(w/w)、Na含量は2.0%(w/w)、ΔCは6.5mS/cm、ΔCpは22mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を4.07gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込みを1.62gに、アクリル酸の仕込み量を1.56gに変えたことを除いて比較例Dと同様に湿ゲルを得た。このときのイオン交換容量は246μmol/ml、ポリクローナル抗体の10%動的結合容量は70mg/ml、S含量は2.3%(w/w)、Na含量は4.8%(w/w)、ΔCは4.7mS/cm、ΔCpは19mS/cmであった。
500mlセパラブルフラスコに純水32.7gと2-アクリルアミド-2-メチルプロパンスルホン酸2.16gを加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液0.86g加え中和した。次に参考例1の架橋6%セルロース粒子40.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム2.60gを0.17mol/ml硝酸9.1mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを40mlの純水で5回洗浄した。次に1mol/L硫酸60mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液80mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は60μmol/ml、ポリクローナル抗体の10%動的結合容量は53mg/ml、S含量は1.6%(w/w)、ΔCは6.3mS/cm、ΔCpは26mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を4.07gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込み量を1.62gに変えたことを除いて実施例1と同様に湿ゲルを得た。このときのイオン交換容量は240μmol/ml、ポリクローナル抗体の10%動的結合容量は38mg/ml、S含量は5.1%(w/w)、ΔCpは25mS/cmであった。
2-アクリルアミド-2-メチルプロパンスルホン酸の仕込み量を19.4gに、48.7%(w/w)水酸化ナトリウム水溶液の仕込み量を7.70gに変えたことを除いて実施例9と同様に湿ゲルを得た。このときのイオン交換容量は404μmol/ml、ポリクローナル抗体の10%動的結合容量は26mg/ml、S含量は7.4%(w/w)、ΔCは7.9mS/cm、ΔCpは25mS/cmであった。
500mlセパラブルフラスコに純水66.4gと3-スルホプロピルメタクリレートカリウム塩7.02gを加え溶解させた。次に参考例1の架橋6%セルロース粒子80.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム5.20gを0.17mol/ml硝酸18.1mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを80mlの純水で5回洗浄した。次に1mol/L硫酸120mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液160mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は124μmol/ml、ポリクローナル抗体の10%動的結合容量は45mg/ml、ΔCpは28mS/cmであった。
500mlセパラブルフラスコに純水91.5gと2-アクリルアミド-2-メチルプロパンスルホン酸4.20gを加え溶解させた。次に48.7%(w/w)水酸化ナトリウム水溶液1.67g加え中和した。次に参考例2の架橋10%セルロース粒子50.0gを加えスラリーとした。この状態でセパラブルフラスコ内に窒素を吹き込みながら1時間攪拌した。1時間後、硝酸アンモニウムセリウム5.07gを0.17mol/ml硝酸17.7mlに溶解させた溶液を滴下ロートからゆっくりセパラブルフラスコ内に加えた。滴下終了後40℃に昇温し、22時間攪拌した。22時間後攪拌を止めスラリーを吸引ろ過した。得られた湿ゲルを50mlの純水で5回洗浄した。次に1mol/L硫酸75mlで10回洗浄したあと純水で洗浄液が中性になるまで洗浄した。このときの湿ゲルの一部を取り出し別途イオン交換容量の測定に使用した。その後0.5mol/L水酸化ナトリウム水溶液100mlで洗浄した。最後に純水で洗浄液が中性になるまで洗浄した。得られた湿ゲルは余分な水分を取り除いた状態で保存した。このときのイオン交換容量は111μmol/ml、ポリクローナル抗体の10%動的結合容量は38mg/ml、S含量は1.5%(w/w)、ΔCpは24mS/cmであった。
ポリクローナル抗体を用いた10%動的結合容量(DBC)の測定
(1)使用機器および試薬
LCシステム :AKTA explorer 10S(登録商標)
バッファー :酢酸バッファーpH5.0、0.05mol/LのNaCl
ポリクローナル抗体:γ-グロブリン、人血清由来(和光純薬)
カラム :直径5mm、長さ5cm
(2)測定方法
まず抗体をバッファーに溶かし1mg/mlの抗体溶液を作製した。そしてカラムにイオン交換基を付加したゲルを隙間のないよう充填した。次にカラムをシステムに接続しバッファーを用いて、カラム流出液のUV(紫外線吸光度、280nm)と電気伝導度、pHが一定になるまで流速1mL/分で平衡化した。その後、ベースラインのUVをゼロにした。次にカラムに抗体溶液を流速0.5mL/minで流した。カラム流出液のUVをモニターし、カラム流出液のUVが、予め測定しておいた抗体溶液のUVの10%に達した時点で抗体溶液を流すのを止めた。以下の式により10%動的結合容量を求めた。なお、この分析は25℃の部屋で行った。
{抗体溶液濃度(mg/ml)×抗体溶液を流し始めてから終えるまでの時間(min)×流速(ml/min)-デッドボリューム}/カラム体積=10%動的結合容量(mg/ml)
ここで、デッドボリュームは、システム配管体積とカラム空隙体積を足した体積(ml)である。
重合体を含むモノクローナル抗体を用いた分離度(ΔC)の測定
(1)使用機器および試薬
LCシステム :AKTA explorer 10S(登録商標)
Aバッファー :クエン酸バッファーpH5.0
Bバッファー :クエン酸バッファーpH5.0、0.5mol/LのNaCl
カラム :直径5mm、長さ5cm
(2)分離度(ΔC)の測定
ゲルを充填したカラムをシステムに接続しAバッファーを80容量%、Bバッファーを20容量%の混合溶液からなる平衡化緩衝液で平衡化した。1mlのモノクローナル抗体溶液をカラムへアプライした。アプライ終了後、5カラム容量の平衡化緩衝液をカラムに通液した。Bバッファーの割合を30カラム容量かけて20容量%から100容量%まで増加させ、吸着したモノクローナル抗体を溶出した。流速はすべて0.66ml/minで実施した。
(3)分離度(ΔC)の計算方法
(2)の分析によって単量体および重合体のピークが得られた。単量体のピークトップに対応する電気伝導度をC1(mS/cm)、重合体のピークトップに対応する電気伝導度をC2(mS/cm)とし、以下の式よりΔCを求めた。
ΔC(mS/cm)=C2(mS/cm)-C1(mS/cm)
(4)精製結果
本分離度(ΔC)の測定により得られた実施例1および実施例2のゲルを用いたモノクローナル抗体の分析結果(クロマトグラム)をそれぞれ図1および図2に示す。
変性ポリクローナル抗体を用いた分離度(ΔCp)の測定
(1)使用機器および試薬
LCシステム :AKTA explorer 10S(登録商標)
Aバッファー :酢酸バッファーpH5.0、0.05mol/LのNaCl
Bバッファー :酢酸バッファーpH5.0、1.0mol/LのNaCl
変性ポリクローナル抗体:(2)に記載
カラム :直径5mm、長さ5cm
(2)変性ポリクローナル抗体の作製方法
変性ポリクローナル抗体はJournal of PHARMACEUTICAL SCIENCES、 2011、 100、2104-2119記載の方法に従って作製した。すなわち、2mgのγ-グロブリン、人血清由来(和光純薬)を20mmol/Lリン酸/クエン酸バッファーpH3.5、1mlに溶かした。ブロックヒーターを用いて55℃で15分加熱した。その後、氷浴で冷やした。PD-10(GEヘルスケア)を用い、酢酸バッファーpH5.0、0.05MのNaClにバッファー交換を行った。変性ポリクローナル抗体の生成の確認はゲルろ過クロマトグラフィーを用いて行った。使用するまでは冷蔵で保存した。
(3)分離度(ΔCp)の測定
ゲルを充填したカラムをシステムに接続しAバッファーで平衡化した。サンプルループを用いて1mlの変性ポリクローナル抗体溶液をカラムへアプライした。未吸着部分を洗浄後、Bバッファーの割合を120分かけて0容量%から100容量%まで増加させた。流速はすべて0.5ml/minで実施した。
(4)分離度(ΔCp)の計算方法
(3)の分析によって二つのピークが得られた。前のピークのピークトップの溶出容量に対応する電気伝導度をC1(mS/cm)、後ろのピークのピークトップに対応する電気伝導度をC2(mS/cm)とし、以下の式よりΔCpを求めた。
ΔCp(mS/cm)=C2(mS/cm)-C1(mS/cm)
イオン交換容量の測定
実施例1~12および比較例A~Dにおいては、硫酸洗浄後、純水洗浄したゲル1mlをビーカーに加え、0.01mol/L水酸化ナトリウム溶液40mlとフェノールフタレイン溶液1滴を加えた。この溶液に、0.1mol/L塩酸を溶液の色が透明になるまで加えた。透明になるまで加えた塩酸量をAmlとすると、各ゲル1mlあたりのイオン交換容量(IEC)は、次式:
(0.01×40/1000-0.1×A/1000)×1000000(μmol/ml)
で求めることができる。
比較例Eにおいては、0.01mol/L水酸化ナトリウム溶液の量を40mlから60mlに変更し、その他は上記と同様にして溶液を調製した。比較例Eで得られたゲル1mlのイオン交換容量は、次式:
(0.01×60/1000-0.1×A/1000)×1000000(μmol/ml)
で求めることができる。
S含量の測定
実施例1~10、12、比較例A~Eのゲルを乾燥させICP(INDUCTIVELY COUPLED PLASMA)発光分光分析により乾燥重量あたりのS含量(重量%)を求めた。
N含量の測定
実施例2~6および比較例A~Cのゲルを乾燥させCHN(Carbon Hydrogen Nitrogen)元素分析により乾燥重量あたりのN含量(重量%)を求めた。
Na含量の測定
実施例7、8および比較例Dのゲルを乾燥させ原子吸光分析により乾燥重量あたりのNa含量(重量%)を求めた。
(i)実施例1~6および比較例A~C
測定法4、測定法5で測定したS含量、N含量の値を使用し、以下の式から共重合ポリマー中の強カチオン性モノマー単位の割合(s密度)を求めた。
{(S含量/32)/(N含量/14)}×100(%)
(ii)実施例7、8および比較例D
測定法4、測定法7で測定したS含量、Na含量の値を使用し、以下の式から共重合ポリマー中の強カチオン性モノマー単位の割合(S密度)を求めた。
(S含量/32)/(Na含量/23)×100(%)
なお、上記実施例では、強カチオン性モノマーとして式(1)で示されるモノマー単位を用いているため、便宜上、上式で示されるS密度を求めることで、共重合ポリマー中に含まれる強カチオン性モノマー単位の割合を概算することができる。
Claims (15)
- 前記重合体が、式(1)で示される少なくとも1種の強カチオン性モノマー単位と、式(3)で示される少なくとも1種の中性モノマー単位とを含む、請求項2記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体が、2-アクリルアミド-2-メチルプロパンスルホン酸と、N,N-ジメチルアクリルアミドとの重合体である、請求項3記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体に含まれる強カチオン性モノマー単位の割合が40mol%以上、100mol%未満であり、かつ、イオン交換容量が60~150μmol/mlである、請求項3または4記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体が、式(1)で示される少なくとも1種の強カチオン性モノマー単位と、式(4)で示される少なくとも1種の弱カチオン性モノマー単位とを含む、請求項2記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体が、2-アクリルアミド-2-メチルプロパンスルホン酸と、アクリル酸との重合体である、請求項6記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体に含まれる強カチオン性モノマー単位の割合が50mol%以上、100mol%未満であり、かつ、イオン交換容量が90~250μmol/mlである、請求項6または7記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体が、式(1)で示される少なくとも1種の強カチオン性モノマー単位からなる重合体である、請求項1記載の陽イオン交換クロマトグラフィー担体。
- 前記重合体が、2-アクリルアミド-2-メチルプロパンスルホン酸の重合体である、請求項9記載の陽イオン交換クロマトグラフィー担体。
- イオン交換容量が70~250μmol/mlである、請求項9または10記載の陽イオン交換クロマトグラフィー担体。
- 前記多孔性粒子が架橋セルロース粒子である、請求項1~11のいずれか1項記載の陽イオン交換クロマトグラフィー担体。
- 前記多孔性粒子が、重量平均分子量1.5×105Daの標準ポリエチレンオキシドにおいて純水を移動相として使用したときのゲル分配係数Kavが0.3~0.5であることを特徴とする多孔性粒子である、請求項1~12のいずれか1項記載の陽イオン交換クロマトグラフィー担体。
- 抗体医薬製造過程で生産される抗体単量体とその重合体を分離するためのものである、請求項1~13のいずれか1項記載の陽イオン交換クロマトグラフィー担体。
- 請求項1~14のいずれか1項記載の陽イオン交換クロマトグラフィー担体を用いて、抗体医薬製造過程で生産される抗体単量体とその重合体を分離する方法。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201380052566.2A CN104718450B (zh) | 2012-10-18 | 2013-09-25 | 抗体精制用阳离子交换色谱法载体及抗体医药的制程中生产的抗体单体与其聚合物的分离法 |
US14/435,456 US20150266919A1 (en) | 2012-10-18 | 2013-09-25 | Cation exchange chromatography carrier for refining of antibodies, and method for separation of antibody monomers from polymers thereof produced in antibody drug manufacturing process |
JP2014542009A JP6340317B2 (ja) | 2012-10-18 | 2013-09-25 | 抗体精製用陽イオン交換クロマトグラフィー担体および抗体医薬の製造過程で生産される抗体単量体とその重合体の分離方法 |
EP13846286.6A EP2910943B1 (en) | 2012-10-18 | 2013-09-25 | Cation exchange chromatography carrier for refining of antibodies and its use and method for separation of antibody monomers from aggregates thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012-230396 | 2012-10-18 | ||
JP2012230396 | 2012-10-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2014061411A1 true WO2014061411A1 (ja) | 2014-04-24 |
Family
ID=50487984
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2013/075941 WO2014061411A1 (ja) | 2012-10-18 | 2013-09-25 | 抗体精製用陽イオン交換クロマトグラフィー担体および抗体医薬の製造過程で生産される抗体単量体とその重合体の分離方法 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20150266919A1 (ja) |
EP (1) | EP2910943B1 (ja) |
JP (1) | JP6340317B2 (ja) |
CN (1) | CN104718450B (ja) |
WO (1) | WO2014061411A1 (ja) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017126496A1 (ja) * | 2016-01-22 | 2017-07-27 | 旭化成メディカル株式会社 | タンパク質の精製方法 |
JP2017186555A (ja) * | 2016-03-31 | 2017-10-12 | 株式会社Nbcメッシュテック | 抗ウイルス性を有するポリマー粒子およびその製造方法 |
JP2018167260A (ja) * | 2017-03-29 | 2018-11-01 | 三菱ケミカル株式会社 | イオン交換体の保存方法、生理活性物質の精製装置及び生理活性物質の精製方法 |
US11801505B2 (en) | 2016-09-09 | 2023-10-31 | Asahi Kasei Medical Co., Ltd. | Strong cation exchange chromatographic matrix and method for using same |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3173783B1 (en) * | 2014-07-25 | 2019-02-27 | Asahi Kasei Medical Co., Ltd. | Cation-exchange chromatography support and method for using same |
US20210024573A1 (en) * | 2018-04-03 | 2021-01-28 | Merck Patent Gmbh | Cex chromatography media and low salt elution of target proteins from biopharmaceutical feeds |
CN108760962A (zh) * | 2018-05-04 | 2018-11-06 | 上海药明生物技术有限公司 | Fab或Fab’含量的测定方法及应用 |
Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5539565B2 (ja) | 1976-07-09 | 1980-10-13 | ||
JPS5540618B2 (ja) | 1977-01-11 | 1980-10-18 | ||
JPS5938203A (ja) | 1982-08-25 | 1984-03-02 | Daicel Chem Ind Ltd | アモルフアスセルロ−スの製造方法 |
JPS6362252B2 (ja) | 1978-09-25 | 1988-12-01 | ||
JPH01310744A (ja) | 1988-03-31 | 1989-12-14 | Merck Patent Gmbh | 分離材料 |
JP3663666B2 (ja) | 1995-04-19 | 2005-06-22 | チッソ株式会社 | 球状セルロース及びその製造法 |
WO2007027139A1 (en) | 2005-08-31 | 2007-03-08 | Ge Healthcare Bio-Sciences Ab | Manufacture of chromatography matrices |
WO2007123242A1 (ja) | 2006-04-25 | 2007-11-01 | Tosoh Corporation | IgG精製用分離剤、及びそれを用いたIgG単量体の精製方法 |
JP2009242770A (ja) | 2007-08-31 | 2009-10-22 | Chisso Corp | 多孔性セルロースゲル、その製造方法及びその用途 |
JP2010528271A (ja) | 2007-05-25 | 2010-08-19 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | カチオン交換クロマトグラフィーのためのグラフトコポリマー |
WO2010127069A1 (en) | 2009-04-29 | 2010-11-04 | Schering Corporation | Antibody purification |
US20110301330A1 (en) * | 2009-02-20 | 2011-12-08 | Jnc Corporation | Cellulose gel for purification of immunoglobulin |
JP2011529508A (ja) | 2008-07-30 | 2011-12-08 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | イオン交換クロマトグラフィー用グラフトコポリマー |
WO2012015379A1 (en) * | 2010-07-29 | 2012-02-02 | Millipore Corporation | Grafting method to improve chromatography media performance |
JP2012032392A (ja) * | 2010-07-28 | 2012-02-16 | Rohm & Haas Co | クロマトグラフィー媒体性能を向上させるグラフト化方法 |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE15378T1 (de) * | 1980-06-27 | 1985-09-15 | Akzo Nv | Mit einer unbeweglichen organischen phase ueberzogenes anorganisches poroeses traegermaterial, anwendung fuer die chromatographie und sein herstellungsverfahren. |
US4329373A (en) * | 1980-07-17 | 1982-05-11 | International Flavors & Fragrances Inc. | Use of carboamidoalkyl norbornanes for augmenting or enhancing the aroma or taste of a foodstuff |
US5445732A (en) * | 1992-06-19 | 1995-08-29 | Sepracor Inc. | Passivated porous polymer supports and methods for the preparation and use of same |
US5906747A (en) * | 1995-11-13 | 1999-05-25 | Biosepra Inc. | Separation of molecules from dilute solutions using composite chromatography media having high dynamic sorptive capacity at high flow rates |
DE60138345D1 (de) * | 2000-08-11 | 2009-05-28 | Rohm & Haas | Verfahren zur Bestimmung chromatographischer Medienparameter in gepackten Betten hydrophobischer Polymerteilchen |
AU2002352016A1 (en) * | 2001-11-21 | 2003-06-10 | Basf Aktiengesellschaft | Crosslinked polyamine coating on superabsorbent hydrogels |
US20050181378A1 (en) * | 2004-02-18 | 2005-08-18 | Applera Corporation | Polyelectrolyte-coated size-exclusion ion-exchange particles |
KR101847405B1 (ko) * | 2010-07-30 | 2018-04-10 | 이엠디 밀리포어 코포레이션 | 크로마토그래피 매질 및 방법 |
CN102603872A (zh) * | 2012-04-11 | 2012-07-25 | 苏州纳微生物科技有限公司 | 单分散聚甲基丙烯酸酯混合型阳离子交换层析介质在万古霉素柱层析纯化中的应用 |
EP2682168A1 (en) * | 2012-07-02 | 2014-01-08 | Millipore Corporation | Purification of biological molecules |
-
2013
- 2013-09-25 JP JP2014542009A patent/JP6340317B2/ja active Active
- 2013-09-25 US US14/435,456 patent/US20150266919A1/en not_active Abandoned
- 2013-09-25 CN CN201380052566.2A patent/CN104718450B/zh active Active
- 2013-09-25 WO PCT/JP2013/075941 patent/WO2014061411A1/ja active Application Filing
- 2013-09-25 EP EP13846286.6A patent/EP2910943B1/en active Active
Patent Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5539565B2 (ja) | 1976-07-09 | 1980-10-13 | ||
JPS5540618B2 (ja) | 1977-01-11 | 1980-10-18 | ||
JPS6362252B2 (ja) | 1978-09-25 | 1988-12-01 | ||
JPS5938203A (ja) | 1982-08-25 | 1984-03-02 | Daicel Chem Ind Ltd | アモルフアスセルロ−スの製造方法 |
JPH01310744A (ja) | 1988-03-31 | 1989-12-14 | Merck Patent Gmbh | 分離材料 |
JP3663666B2 (ja) | 1995-04-19 | 2005-06-22 | チッソ株式会社 | 球状セルロース及びその製造法 |
WO2007027139A1 (en) | 2005-08-31 | 2007-03-08 | Ge Healthcare Bio-Sciences Ab | Manufacture of chromatography matrices |
WO2007123242A1 (ja) | 2006-04-25 | 2007-11-01 | Tosoh Corporation | IgG精製用分離剤、及びそれを用いたIgG単量体の精製方法 |
JP2010528271A (ja) | 2007-05-25 | 2010-08-19 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | カチオン交換クロマトグラフィーのためのグラフトコポリマー |
JP2009242770A (ja) | 2007-08-31 | 2009-10-22 | Chisso Corp | 多孔性セルロースゲル、その製造方法及びその用途 |
JP2011529508A (ja) | 2008-07-30 | 2011-12-08 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング | イオン交換クロマトグラフィー用グラフトコポリマー |
US20110301330A1 (en) * | 2009-02-20 | 2011-12-08 | Jnc Corporation | Cellulose gel for purification of immunoglobulin |
WO2010127069A1 (en) | 2009-04-29 | 2010-11-04 | Schering Corporation | Antibody purification |
JP2012032392A (ja) * | 2010-07-28 | 2012-02-16 | Rohm & Haas Co | クロマトグラフィー媒体性能を向上させるグラフト化方法 |
WO2012015379A1 (en) * | 2010-07-29 | 2012-02-02 | Millipore Corporation | Grafting method to improve chromatography media performance |
Non-Patent Citations (7)
Title |
---|
BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, 2011, pages 1494 - 1508 |
J. CHROMATOGRAPHY A, vol. 1216, 2009, pages 902 - 909 |
J. CHROMATOGRAPHY A, vol. 1217, 2010, pages 216 - 224 |
JOURNAL OF CHROMATOGRAPHY A, vol. 1146, pages 202 - 215 |
JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 100, 2011, pages 2104 - 2119 |
JOURNAL OF POLYMER SCIENCE, 1958, pages 242 - 243 |
L. FISCHER: "Seibutsukagaku Jikkenho (Biochemistry Experimental Method)", vol. 2, article "Gel chromatography" |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2017126496A1 (ja) * | 2016-01-22 | 2017-07-27 | 旭化成メディカル株式会社 | タンパク質の精製方法 |
JPWO2017126496A1 (ja) * | 2016-01-22 | 2018-08-23 | 旭化成メディカル株式会社 | タンパク質の精製方法 |
JP2017186555A (ja) * | 2016-03-31 | 2017-10-12 | 株式会社Nbcメッシュテック | 抗ウイルス性を有するポリマー粒子およびその製造方法 |
US11801505B2 (en) | 2016-09-09 | 2023-10-31 | Asahi Kasei Medical Co., Ltd. | Strong cation exchange chromatographic matrix and method for using same |
JP2018167260A (ja) * | 2017-03-29 | 2018-11-01 | 三菱ケミカル株式会社 | イオン交換体の保存方法、生理活性物質の精製装置及び生理活性物質の精製方法 |
Also Published As
Publication number | Publication date |
---|---|
JPWO2014061411A1 (ja) | 2016-09-05 |
US20150266919A1 (en) | 2015-09-24 |
CN104718450B (zh) | 2018-12-14 |
CN104718450A (zh) | 2015-06-17 |
EP2910943A1 (en) | 2015-08-26 |
EP2910943A4 (en) | 2016-07-13 |
JP6340317B2 (ja) | 2018-06-06 |
EP2910943B1 (en) | 2021-11-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6340317B2 (ja) | 抗体精製用陽イオン交換クロマトグラフィー担体および抗体医薬の製造過程で生産される抗体単量体とその重合体の分離方法 | |
JP6580650B2 (ja) | フロースルー式での生物製剤からのタンパク質凝集体の除去 | |
JP5064225B2 (ja) | 抗体精製法 | |
JP6630036B2 (ja) | 標的物の精製方法、及び、ミックスモード用担体 | |
WO2015041218A1 (ja) | 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) | |
WO2018092691A1 (ja) | 抗体の精製方法 | |
JP2014210797A (ja) | 非グリコシル化タンパク質の精製 | |
JP6769695B2 (ja) | 血漿からの免疫グロブリンの精製 | |
WO2017069254A1 (ja) | アフィニティクロマトグラフィ担体および生体物質の精製方法 | |
WO2015138928A2 (en) | Mixed mode ligands | |
US20130109807A1 (en) | Removal of virucidal agents in mixed mode chromatography | |
WO2012121409A1 (ja) | 温度応答性クロマトグラフィー担体による生理活性物質の精製方法 | |
WO2016093251A1 (ja) | 生理活性物質の精製方法 | |
JP2011036128A (ja) | 抗体製造方法 | |
JP2019194547A (ja) | ウイルス除去方法 | |
JP2019182791A (ja) | 抗体凝集体の低減方法 | |
JP6927259B2 (ja) | 吸着材、カラム、精製装置及び吸着材の製造方法 | |
Şaşmaz | Synthesis and characterization of boronic acid functionalized sorbents for the antibody removal from human plasma |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13846286 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2013846286 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14435456 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2014542009 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |