Immunoglobulin purification method and immunoglobulin purification device, and immunoglobulin production method and immunoglobulin production device

Abstract

The present invention provides an immunoglobulin purification method that does not inactivate antibody properties and has a high recovery rate of immunoglobulins. The immunoglobulin purification method includes an adsorption step and a desorption step. The adsorption step is a step of adsorbing immunoglobulins on porous zirconia particles in a neutral buffer. The desorption step is a step of desorbing immunoglobulins adsorbed on porous zirconia particles from the porous zirconia particles using a neutral desorption solution.

Description

Technical Field

[0001] The present invention relates to an immunoglobulin purification method and an immunoglobulin purification device, and an immunoglobulin production method and an immunoglobulin production device. Background Art

[0002] Immunoglobulin is useful as a therapeutic drug and in vitro diagnostic drug, and it is predicted that the demand will increase in the future. In order to be used for such purposes, it is necessary to develop a technology that can purify immunoglobulin with high purity. In the purification of immunoglobulin, affinity chromatography using protein A is generally used (refer to, for example, patent document 1). Protein A is a protein separated from the bacterial membrane of Staphylococcus aureus that shows binding properties to IgG. Protein A has the property of specifically binding to immunoglobulins from various animals, and the binding amount of immunoglobulins per unit protein is large, so affinity chromatography using a carrier immobilized with the protein A is used in industrial-scale antibody purification processes.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2017-47365 Summary of the invention

[0006] Problems to be solved by the invention

[0007] However, in affinity chromatography using protein A, an acidic solution of pH 4 or less is used as a method for eluting adsorbed immunoglobulins, and therefore, there is a possibility that the higher-order structure of the antibody changes, and further progresses to association or aggregation.

[0008] Furthermore, immunoglobulin aggregates also appear during the cell culture stage, and impurities including these immunoglobulin aggregates (aggregates) are difficult to remove by affinity chromatography using protein A.

[0009] Therefore, in order to solve the above-mentioned problems, a method of combining purification by affinity chromatography using protein A with ion exchange chromatography and hydrophobic interaction chromatography is generally used.

[0010] However, in the conventional ion exchange chromatography, only the monomer of immunoglobulin can be separated in a narrow range of conditions, so there is a problem of low recovery rate of immunoglobulin. In addition, in hydrophobic interaction chromatography, the recovery rate of immunoglobulin is low and a long time is required, so there is a problem of high purification cost of immunoglobulin.

[0011] The present invention has been made in view of the above actual situation, and aims to provide an immunoglobulin purification method and immunoglobulin purification device, as well as an immunoglobulin production method and immunoglobulin production device that do not inactivate antibody properties and have a high recovery rate of immunoglobulins. The present invention can be achieved in the following aspects.

[0012] Means used to solve problems

[0013] [1] A method for purifying immunoglobulin, characterized by comprising:

[0014] an adsorption step of allowing the immunoglobulin to be adsorbed on the porous zirconium oxide particles in a neutral buffer; and

[0015] A desorption step of desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a neutral desorption liquid.

[0016] [2] The immunoglobulin purification method as described in [1] is characterized in that the desorption liquid is a phosphate buffer.

[0017] [3] The method for purifying immunoglobulins according to [1] or [2], wherein the desorption solution contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof.

[0018]

[0019] (In general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0020] [4] An immunoglobulin purification device, characterized in that it comprises:

[0021] allowing immunoglobulin to adsorb onto the adsorption portion of porous zirconia particles in a neutral buffer; and

[0022] A desorption section for desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a neutral desorption liquid.

[0023] [5] The immunoglobulin purification device as described in [4] is characterized in that the desorption liquid is a phosphate buffer solution.

[0024] [6] The immunoglobulin purification device according to [4] or [5], characterized in that the desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof.

[0025]

[0026] (In general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0027] [7] A method for producing an immunoglobulin, comprising:

[0028] an adsorption step of allowing the immunoglobulin to be adsorbed on the porous zirconium oxide particles in a neutral buffer; and

[0029] A desorption step of desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a neutral desorption liquid.

[0030] [8] The method for producing immunoglobulin according to [7], wherein the desorption solution is a phosphate buffer solution.

[0031] [9] The method for producing immunoglobulin according to [7] or [8], wherein the desorption solution contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof.

[0032]

[0033] (In general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0034]

[10] An immunoglobulin production device, characterized by comprising:

[0035] allowing immunoglobulin to adsorb onto the adsorption portion of porous zirconia particles in a neutral buffer; and

[0036] A desorption section for desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a neutral desorption liquid.

[0037]

[11] The immunoglobulin production device as described in

[10] is characterized in that the above-mentioned desorption liquid is a phosphate buffer solution.

[0038]

[12] The immunoglobulin production apparatus as described in

[10] or

[11] is characterized in that the desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof.

[0039]

[0040] (In general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0041] Effects of the Invention

[0042] The immunoglobulin purification method of the present invention adsorbs immunoglobulins to porous zirconia particles in a neutral buffer and desorbs immunoglobulins from the porous zirconia particles using a neutral desorption solution, so that the antibody properties of the immunoglobulins are not inactivated. In addition, the immunoglobulin purification method of the present invention has a high recovery rate of immunoglobulins.

[0043] In the immunoglobulin purification method of the present invention, when the desorption liquid is a phosphate buffer, the recovery rate of the immunoglobulin is high.

[0044] In the method for purifying immunoglobulin of the present invention, when the desorption liquid contains at least one selected from the group consisting of compounds represented by the general formula (1) and salts thereof, the recovery rate of immunoglobulin is high.

[0045] The immunoglobulin purification device of the present invention adsorbs immunoglobulins to porous zirconia particles in a neutral buffer and desorbs immunoglobulins from the porous zirconia particles using a neutral desorption solution, so that the antibody properties of the immunoglobulins are not inactivated. In addition, the immunoglobulin purification device of the present invention has a high recovery rate of immunoglobulins.

[0046] In the immunoglobulin purification device of the present invention, when the desorption liquid is a phosphate buffer solution, the recovery rate of the immunoglobulin is high.

[0047] In the immunoglobulin purification apparatus of the present invention, when the desorption liquid contains at least one selected from the group consisting of the compound represented by the general formula (1) and a salt thereof, the recovery rate of the immunoglobulin is high.

[0048] The immunoglobulin production method of the present invention adsorbs immunoglobulin to porous zirconia particles in a neutral buffer and desorbs immunoglobulin from the porous zirconia particles using a neutral desorption solution, so the antibody properties of the immunoglobulin are not inactivated. In addition, the immunoglobulin production method of the present invention has high efficiency in immunoglobulin production.

[0049] In the method for producing immunoglobulin of the present invention, when the desorption liquid is a phosphate buffer solution, the production efficiency of immunoglobulin is high.

[0050] In the method for producing immunoglobulin of the present invention, when the desorption solution contains at least one selected from the group consisting of compounds represented by the general formula (1) and salts thereof, the production efficiency of immunoglobulin is high.

[0051] The immunoglobulin production device of the present invention adsorbs immunoglobulin to porous zirconia particles in a neutral buffer and desorbs immunoglobulin from the porous zirconia particles using a neutral desorption solution, so the antibody properties of the immunoglobulin are not inactivated. In addition, the immunoglobulin purification device of the present invention has high immunoglobulin production efficiency.

[0052] In the immunoglobulin production apparatus of the present invention, when the desorption liquid is a phosphate buffer solution, the production efficiency of the immunoglobulin is high.

[0053] In the immunoglobulin production apparatus of the present invention, when the desorption liquid contains at least one selected from the group consisting of the compound represented by the general formula (1) and a salt thereof, the production efficiency of the immunoglobulin is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a schematic diagram showing the presumed supported structure of ethylenediaminetetramethylenephosphonic acid (EDTPA).

[0055] Figure 2 is a block diagram illustrating an immunoglobulin purification apparatus.

[0056] Figure 3 This is a block diagram illustrating an immunoglobulin production apparatus. DETAILED DESCRIPTION

[0057] The present invention is described in detail below. It should be noted that in this specification, when the numerical range is described using "to", unless otherwise stated, the lower limit and the upper limit are included. For example, in the case of a description such as "10 to 20", any one of "10" as the lower limit and "20" as the upper limit is included. That is, "10 to 20" has the same meaning as "10 or more and 20 or less".

[0058] 1. Immunoglobulin purification method

[0059] The immunoglobulin purification method of the present invention comprises an adsorption step and a desorption step.

[0060] (1) Adsorption process

[0061] The adsorption step is a step of adsorbing the immunoglobulin onto the porous zirconium oxide particles in a neutral buffer.

[0062] (1.1) Neutral buffer

[0063] The neutral buffer is preferably a buffer of pH 6.1 or more and pH 8.0 or less, more preferably a buffer of pH 6.5 or more and pH 7.5 or less, and further preferably a buffer of pH 6.6 or more and pH 7.4 or less. The buffering agent constituting the buffer can be appropriately selected from the buffering agents commonly used by those skilled in the art according to the pH used, and examples thereof include phosphate, acetate, carbonate, and Tris (tris (hydroxymethyl) aminomethane). The concentration of the neutral buffer is not particularly limited, and is preferably 0.1 mM or more and 200 mM or less, more preferably 1 mM or more and 100 mM or less, and further preferably 5 mM or more and 20 mM or less.

[0064] From the viewpoint of maintaining the antibody properties of immunoglobulin, the neutral buffer is particularly preferably phosphate-buffered saline (PBS).

[0065] The temperature of the neutral buffer solution is not particularly limited, but is usually used at 4° C. or higher and 50° C. or lower.

[0066] (1.2) Immunoglobulin

[0067] Immunoglobulin may be any one of IgG, IgA, IgM, IgD, and IgE. IgG, IgD, and IgE exist in the form of monomers, IgA exists in the form of monomers or dimers, and IgM exists in the form of pentamers or hexamers, and the size is about 10 nm to about 30 nm.

[0068] (1.3) Porous zirconia particles

[0069] The porous zirconia particles are not particularly limited, but are preferably the following porous zirconia particles. That is, the porous zirconia particles preferably have D50, D90, and total pore volume within the following ranges.

[0070] It should be noted that the porous zirconia particles may be in the form of an aggregate in which two or more particles are aggregated, in addition to the form in which each particle is independent of each other.

[0071] (1.3.1) D50, D90 and total pore volume

[0072] The porous zirconium oxide particles preferably have a pore diameter D50 of 3.20 nm or more and 6.50 nm or less in the pore size distribution measured by the BET method, where the cumulative pore volume accounts for 50% of the total pore volume, and preferably have a pore diameter D90 of 10.50 nm or more and 100.00 nm or less. The pore diameter D50 is more preferably 3.35 nm or more and 6.30 nm or less, and further preferably 3.50 nm or more and 5.00 nm or less. The pore diameter D90 is more preferably 10.80 nm or more and 50.00 nm or less, and further preferably 11.00 nm or more and 30.00 nm or less.

[0073] The porous zirconium oxide particles preferably have a total pore volume greater than 0.10 cm 3 / g. The total pore volume is more preferably greater than 0.15 cm 3 / g, more preferably greater than 0.30cm 3 It should be noted that the upper limit of the total pore volume is not particularly limited, but is usually 10.00 cm 3 / g.

[0074] When D50, D90 and the total pore volume are within the above range, the selectivity of the adsorbed immunoglobulin becomes higher. It should be noted that by making D90 less than 100.00nm, it is easy to selectively adsorb non-aggregates of immunoglobulins. The size of non-aggregates of immunoglobulins is about 10nm to about 30nm, while the size of aggregates of immunoglobulins is about 100nm. Therefore, by making D90 less than 100.00nm, adsorption of aggregates of immunoglobulins can be avoided, and it is easy to adsorb only non-aggregates of immunoglobulins.

[0075] The pore size distribution and the pore volume can be measured, for example, using a pore distribution measuring device (Micromeritics automatic specific surface area / pore distribution measuring device (TriStarII, manufactured by Shimadzu Corporation)).

[0076] Here, the calculation method of D50 and D90 is described.

[0077] First, the calculation method of D50 is explained. From the pore size distribution data, the cumulative pore volume (X (%)) and the pore diameter (Y (nm)) of the two points A and B that are closest to the cumulative pore volume 50% and sandwich the cumulative pore volume 50% are read. Specifically, read A (Xa (%), Ya (nm)), B (Xb (%), Yb (nm)) (where Xa>Xb, Ya>Yb). Then, use these values ​​to calculate D50 using the following formula (1).

[0078] Formula (1)

[0079] D50=log(Xb)+((log(Xa)-log(Xb))*[(50-(Yb)) / ((Ya)-(Yb))]

[0080] D90 is calculated in the same way. That is, first, the cumulative pore volume (X (%)) and the pore diameter (Y (nm)) of the two points C and D sandwiching the cumulative pore volume 90% and closest to the cumulative pore volume 90% are read from the pore size distribution data. Specifically, C (Xc (%), Yc (nm)), D (Xd (%), Yd (nm)) (where Xc>Xd, Yc>Yd) are read. Then, these values ​​are used to calculate D90 by the following formula (2).

[0081] Formula (2)

[0082] D90=log(Xd)+((log(Xc)-log(Xd))*[(90-(Yd)) / ((Yc)-(Yd))]

[0083] (1.3.2) Particle size

[0084] The particle size of the porous zirconia particles is not particularly limited, and the primary particles are usually 10 nm to 100 nm, preferably 10 nm to 50 nm, and more preferably 10 nm to 30 nm. The primary particles form aggregates to form secondary particles with a size of 50 nm to 1000 nm. When the primary particle size is within this range, the specific surface area of ​​the porous zirconia particles is greatly increased, so the adsorption amount of immunoglobulins tends to increase.

[0085] (1.3.3) Porous zirconium oxide particles loaded with chelating agents

[0086] The porous zirconia particles may be loaded with a chelating agent on the surface. By loading the chelating agent, the selectivity of immunoglobulin adsorption is further improved.

[0087] The chelating agent is not particularly limited. The chelating agent is preferably at least one selected from the group consisting of a compound represented by the following general formula (2), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DETPA), diethylenetriaminepentamethylenephosphonic acid (DETPPA) and salts thereof. It should be noted that, as the salt, for example, a salt of an alkali metal (such as sodium) is appropriately exemplified.

[0088]

[0089] (In general formula (2), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0090] As R in the general formula (2) 1 Examples of the alkylene group having 1 to 10 carbon atoms include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a hexamethylene group, and an isobutylene group.

[0091] In addition, as R 2 ~R 5 Examples of the alkylene group having 1 to 10 carbon atoms include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a hexamethylene group, and an isobutylene group.

[0092] From the viewpoint of further improving the selectivity for immunoglobulin, the chelating agent is preferably a compound represented by the general formula (2). Among the compounds represented by the general formula (2), ethylenediaminetetramethylenephosphonic acid (EDTPA; N,N,N',N'-Ethylenediaminetetrakis (methylenephosphonic Acid)) is particularly preferred.

[0093] The amount of the chelating agent supported is not particularly limited. From the viewpoint of further improving the selectivity for immunoglobulin, the amount of the chelating agent supported is preferably 0.01 μg to 10 μg, more preferably 0.02 μg to 5 μg, and even more preferably 0.05 μg to 3 μg per 1 mg of zirconium oxide.

[0094] It should be noted that the loading amount of the chelating agent can be calculated from the weight loss of TG-DTA (thermogravimetric differential thermal analysis).

[0095] The loading method of the chelating agent is not clear, but it is presumed that the ligand from the chelating agent is bound to the zirconium atom. For example, when the chelating agent is ethylenediaminetetramethylenephosphonic acid, it is presumed that Figure 1 structure.

[0096] (1.3.4) Method for producing porous zirconia particles

[0097] The method for producing porous zirconium oxide particles is not particularly limited. The porous zirconium oxide particles can be produced, for example, by the following method. Zircon is used as a raw material to obtain a zirconium oxychloride (ZrOCl2·8H2O) solution. Then, Zr(OH)4 particles are produced by hydrolysis reaction, and the particles are sintered to produce porous zirconium oxide particles.

[0098] (1.4) Relationship between the amount of porous zirconia particles and the amount of neutral buffer

[0099] The relationship between the amount of the porous zirconia particles and the amount of the neutral buffer in the adsorption step is not particularly limited. For example, the neutral buffer may be used in a ratio of 10 μL or more and 300 μL or less per 1 mg of the porous zirconia particles.

[0100] (2) Desorption process

[0101] The desorption step is a step of desorbing (eluting) the immunoglobulin adsorbed to the porous zirconia particles from the porous zirconia particles using a neutral desorption solution.

[0102] (2.1) Neutral desorption liquid

[0103] The neutral desorption liquid is preferably a desorption liquid having a pH of 6.0 or higher and a pH of 8.0 or lower, more preferably a desorption liquid having a pH of 6.5 or higher and a pH of 7.5 or lower, and further preferably a desorption liquid having a pH of 6.8 or higher and a pH of 7.2 or lower.

[0104] The desorption liquid is preferably a phosphate buffer. As the phosphate buffer, for example, phosphate buffered saline (PBS) can be appropriately used.

[0105] The concentration of the desorption liquid is not particularly limited and can be appropriately selected according to the type of the desorption liquid.

[0106] For example, when phosphate buffered saline (PBS) is used, from the viewpoint of improving the recovery rate of immunoglobulins, it is preferably 50 mM or more and 200 mM or less, more preferably 60 mM or more and 150 mM or less, and further preferably 70 mM or more and 130 mM or less. It should be noted that, when phosphate buffered saline (PBS) is used as a neutral buffer in the adsorption process and phosphate buffered saline (PBS) is used as a desorption solution in the desorption process, from the viewpoint of improving the recovery rate of immunoglobulins, the concentrations of the two are preferably in the following relationship. That is, the concentration of phosphate buffered saline (PBS) in the desorption process is preferably higher than that of phosphate buffered saline (PBS) in the adsorption process.

[0107] The desorption liquid may contain at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof.

[0108]

[0109] (In general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms. 2 ~R 5 is an alkylene group having 1 to 10 carbon atoms, which may be the same or different.

[0110] As R of the general formula (1) 1 Examples of the alkylene group having 1 to 10 carbon atoms include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a hexamethylene group, and an isobutylene group.

[0111] In addition, as R 2 ~R5 Examples of the alkylene group having 1 to 10 carbon atoms include a methylene group, an ethylene group, a trimethylene group, a tetramethylene group, a hexamethylene group, and an isobutylene group.

[0112] From the viewpoint of improving the recovery rate of immunoglobulins, at least one of the compounds represented by the general formula (1) and their salts is preferably selected from ethylenediaminetetramethylenephosphonic acid (EDTPA; N,N,N',N'-Ethylenediaminetetrakis(methylenephosphonic Acid)) and its salts.

[0113] From the viewpoint of improving the recovery rate of immunoglobulins, the concentration of at least one of the compounds represented by the general formula (1) and their salts (the total concentration in the case of more than two kinds) is preferably 0.1 mM to 10 mM, more preferably 0.5 mM to 8 mM, and even more preferably 2 mM to 6 mM.

[0114] The temperature of the desorption liquid is not particularly limited, but is usually 4° C. or higher and 50° C. or lower.

[0115] (2.2) Relationship between the amount of porous zirconia particles and the amount of desorption liquid

[0116] The relationship between the amount of the porous zirconia particles and the amount of the desorption liquid in the desorption step is not particularly limited. For example, the desorption liquid may be used in a ratio of 10 μL or more and 300 μL or less per 1 mg of the porous zirconia particles.

[0117] 2. Immunoglobulin purification device

[0118] The immunoglobulin purification device 1 of the present invention comprises an adsorption section and a desorption section. Figure 2 , an example of an immunoglobulin purification device 1 having an adsorption-desorption section 3 functioning as an adsorption section and a desorption section is shown. The adsorption-desorption section 3, for example, has a cylindrical section having two openings, in which porous zirconium oxide particles 4 are filled. In addition, the immunoglobulin purification device 1 has an adsorption liquid container 5 for loading a neutral buffer solution (adsorption liquid), a desorption liquid container 7 for loading a neutral desorption liquid, and a recovery container 9 for recovering a liquid discharged from the adsorption-desorption section 3. In the adsorption step (adsorption process), a neutral buffer solution (adsorption liquid) containing immunoglobulins is supplied from the adsorption liquid container 5 to the adsorption-desorption section 3 by a pump, so that the immunoglobulins are adsorbed on the porous zirconium oxide particles 4. In the desorption step (desorption process), a neutral desorption liquid is supplied from the desorption liquid container 7 to the adsorption-desorption section 3 by a pump, so that the immunoglobulins adsorbed on the porous zirconium oxide particles 4 are desorbed (eluted). The switching between the adsorption liquid and the desorption liquid is performed by a switching section 11.

[0119] In the immunoglobulin purification apparatus 1 , the terms “neutral buffer solution”, “immunoglobulin”, “porous zirconia particles”, and “neutral desorption solution” are as described in “1. Immunoglobulin purification method”.

[0120] 3. Immunoglobulin production method

[0121] The method for producing immunoglobulin of the present invention comprises an adsorption step and a desorption step.

[0122] In addition, in the immunoglobulin production method, the description in "1. Immunoglobulin purification method" is applied directly to the "adsorption step" and "desorption step".

[0123] In the immunoglobulin production method of the present invention, an immunoglobulin with improved purity compared to the immunoglobulin (raw material immunoglobulin) used in the adsorption step can be produced. The raw material immunoglobulin can be obtained by, for example, separation from blood or tissue fluid, or biosynthesis or chemical synthesis, or by a known method through a culturing step. The immunoglobulin production method of the present invention can include a step of obtaining the raw material immunoglobulin.

[0124] 4. Immunoglobulin production device

[0125] The immunoglobulin production apparatus 21 of the present invention comprises an adsorption unit and a desorption unit. Figure 3 , an example of an immunoglobulin production apparatus 21 having an adsorption-desorption section 23 functioning as an adsorption section and a desorption section is shown. The adsorption-desorption section 23, for example, has a cylindrical section having two openings, in which porous zirconium oxide particles 24 are filled. In addition, the immunoglobulin production apparatus 21 has an adsorption liquid container 25 for loading a neutral buffer solution (adsorption liquid), a desorption liquid container 27 for loading a neutral desorption liquid, and a recovery container 29 for recovering the liquid discharged from the adsorption-desorption section 23. In the adsorption step (adsorption process), a neutral buffer solution (adsorption liquid) containing immunoglobulins is supplied from the adsorption liquid container 25 to the adsorption-desorption section 23 by a pump, so that the immunoglobulins are adsorbed on the porous zirconium oxide particles 24. In the desorption step (desorption process), a neutral desorption liquid is supplied from the desorption liquid container 27 to the adsorption-desorption section 23 by a pump, so that the immunoglobulins adsorbed on the porous zirconium oxide particles 24 are desorbed (eluted). The switching between the adsorption liquid and the desorption liquid is performed by a switching section 31.

[0126] In the immunoglobulin production apparatus 21 , the terms “neutral buffer solution,” “immunoglobulin,” “porous zirconia particles,” and “neutral desorption solution” are as described in “1. Immunoglobulin purification method.”

[0127] In the immunoglobulin production device 21 of the present invention, it is possible to produce an immunoglobulin with a higher purity than the immunoglobulin (raw material immunoglobulin) used in the adsorption section. The raw material immunoglobulin can be obtained by, for example, separation from blood or tissue fluid, or by biosynthesis, chemical synthesis, or by a known method through a culture step. The immunoglobulin production device 21 of the present invention can be equipped with a section for obtaining raw material immunoglobulin.

[0128] Example

[0129] The present invention will be described in more detail.

[0130] 1. Experiment A

[0131] (1) Porous zirconia particles

[0132] As the porous zirconia particles, the porous zirconia particles described in Table 1 were used.

[0133] It should be noted that in Test Examples 1 to 9, PBS (phosphate buffered saline) as a neutral buffer was used as the adsorption solution, but in Test Example 10, an acidic HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid) solution was used.

[0134] [Table 1]

[0135]

[0136] In Table 1, "PCS140 (SD)" is porous zirconia particles manufactured by Nippon Electric Industries, Ltd.

[0137] In Table 1, “PCS140(SD)-P(0.00125M)” refers to porous zirconia particles loaded with EDTPA, obtained by treating porous zirconia particles manufactured by Nippon Electric Industries, Ltd. (PCS140(SD), raw material) with a 0.00125M EDTPA solution.

[0138] In Table 1, "PCS140(SD)-P(0.0025M)" refers to porous zirconia particles loaded with EDTPA obtained by treating porous zirconia particles (PCS140(SD), raw material) manufactured by Nippon Electric Industries, Ltd. with a 0.0025M EDTPA solution. These abbreviations are the same in the following Tables 2 to 3.

[0139] It should be noted that the preparation of "PCS140(SD)-P(0.00125M)" was carried out as follows. 250 mg of porous zirconia particles (PCS140(SD)) that had been degassed and dried at 100°C for 2 hours were added with 10 mL of a 0.00125M EDTPA solution, degassed for 15 minutes, and stirred and / or shaken for 17 hours. Then, after further refluxing for 4 hours, the mixture was washed with pure water and freeze-dried to obtain PCS140(SD)-P(0.00125M).

[0140] The preparation of "PCS140(SD)-P(0.0025M)" was also carried out in the same manner as "PCS140(SD)-P(0.00125M)". That is, "PCS140(SD)-P(0.0025M)" was prepared in the same manner as "PCS140(SD)-P(0.00125M)" except that "0.0025M EDTPA solution" was used instead of "0.00125M EDTPA solution".

[0141] It should be noted that the amount of EDTPA loaded was calculated from the weight loss of TG-DTA (thermogravimetric differential thermal analysis). That is, about 10 mg of porous zirconia particles loaded with EDTPA were weighed, and the weight change from room temperature to 1000°C was measured, and differential thermal analysis (TG-DTA; Thermo plus TG8120, Rigaku) ​​was performed. The result calculated from the weight loss at 200°C to 600°C was that 0.06μg to 2.20μg of EDTPA was loaded per 1 mg of porous zirconia particles.

[0142] (2) Adsorption evaluation

[0143] <Test Examples 1 to 9>

[0144] Using the various porous zirconia particles listed in Table 1, the protein adsorption properties were evaluated.

[0145] The following test was performed on each porous zirconia particle for three proteins, namely, IgG, HAS (human serum albumin), and Trf (transferrin human).

[0146] 500 μL of the adsorption solution was placed in a Spitz tube, and 3 mg of porous zirconia particles were added to the solution. After the porous zirconia particles were fully dispersed, 500 μL of a protein solution (500 μg / 500 μL) was added, and the mixture was stirred at 4°C overnight under light shielding.

[0147] The Spitz tube was centrifuged at 12,000 rpm for 10 minutes to separate the porous zirconia particles. The mass of unfixed protein remaining in the supernatant solution was quantified using a protein quantitative staining solution (BIO-RAD) with an ELISA reader (Infinite F200 PRO, TECAN). The difference between the mass of the initially added protein and the mass of the unadsorbed protein was taken as the mass of the adsorbed protein. The ratio of the mass of the adsorbed protein to the mass of the initially added protein was calculated as the adsorption rate (%).

[0148] <Test Example 10>

[0149] Test Example 10 was carried out by the same operation as in Test Examples 1 to 9, except that 750 μg / 500 μL of the protein solution was used and 500 μL of the solution was added. Test Example 10 was carried out in the same manner as in Test Examples 1 to 9 except for the amount of protein added.

[0150] (3) Test results

[0151] The test results are collectively recorded in Table 1.

[0152] (3.1) Effect of the type of adsorption liquid

[0153] The effect of the type of adsorption solution on the adsorption of proteins was studied.

[0154] Here, the results of Test Examples 1 to 3 and 10 were compared and examined. As for the adsorption rate of IgG, it was found that Test Examples 1 to 3 were higher than Test Example 10. From this result, it was confirmed that the adsorption rate of IgG was improved when a neutral buffer solution was used as the adsorption solution.

[0155] Next, the selectivity of IgG was investigated based on the results of Test Examples 1 and 10. In Test Example 10, it was confirmed that HAS and Trf were adsorbed in addition to IgG. In Test Example 10, the adsorption rate of IgG was 31.7%, the adsorption rate of HAS was 72.7%, and the adsorption rate of Trf was 60.2%. It can be seen that in Test Example 10, the adsorption rate of HAS and the adsorption rate of Trf were high relative to the adsorption rate of IgG, and the selectivity of IgG adsorption was low.

[0156] On the other hand, in Test Example 1, HAS and Trf were adsorbed in addition to IgG, but the adsorption rates of HAS and Trf were low relative to the adsorption rate of IgG, and the selectivity of IgG adsorption was high.

[0157] According to these results, by using a neutral buffer as an adsorption liquid, the selectivity of IgG adsorption can be improved, and IgG can be adsorbed efficiently. In addition, since the adsorbed IgG is desorbed and then recovered, the result is that by using a neutral buffer as an adsorption liquid, IgG is efficiently adsorbed so that the recovery rate of IgG is improved.

[0158] (3.2) Effect of loading chelating agent

[0159] The effect of loading a chelating agent on porous zirconia particles on the adsorption of proteins was studied.

[0160] The results of Test Example 1 using porous zirconia particles without EDTPA were compared with those of Test Examples 2 to 3 using porous zirconia particles loaded with EDTPA. Compared with Test Example 1, in Test Examples 2 to 3, the adsorption rates of HAS and Trf were lower relative to the adsorption rate of IgG, and the adsorption selectivity of IgG was improved. Therefore, by loading the porous zirconia particles with a chelating agent, the selectivity of IgG was further improved, and the recovery rate of IgG was further improved.

[0161] (3.3) Effect of adsorption solution concentration

[0162] The effect of the concentration of the adsorption solution on the adsorption of proteins was studied.

[0163] Here, the results of Test Examples 4 to 6 were compared and examined. As for the adsorption rate of IgG, it was found that Test Example 4, in which the concentration of the adsorption solution was 10 mM, had the highest adsorption rate.

[0164] From these results, it was confirmed that the adsorption rate of IgG increased and the recovery rate of IgG improved by setting the concentration of the adsorption solution to 5 mM or more and 20 mM or less.

[0165] (3.4) Effect of pH of adsorption solution

[0166] The effect of the pH of the adsorption solution on the adsorption of proteins was studied.

[0167] Here, a comparative study was conducted on the results of Test Examples 7 to 9. As for the adsorption rate of IgG, it was found that Test Example 8, in which the pH of the adsorption solution was 7.0, had the highest adsorption rate.

[0168] From these results, it was confirmed that the adsorption rate of IgG increased and the recovery rate of IgG improved by adjusting the pH of the adsorption solution to 6.6 or more and 7.4 or less.

[0169] 2. Experiment B

[0170] (1) Porous zirconia particles

[0171] As the porous zirconia particles, the EDTPA-supported porous zirconia particles (PCS140(SD)-P(0.0025M)) shown in Table 2 were used.

[0172] [Table 2]

[0173]

[0174] (2) Adsorption and desorption evaluation

[0175] (2.1) Adsorption evaluation

[0176] 500 μL of adsorption solution was placed in a Spitz tube, and 3 mg of porous zirconia particles loaded with EDTPA (PCS140 (SD) -P (0.0025 M)) were added to the solution. After the porous zirconia particles loaded with EDTPA were fully dispersed, 500 μL of IgG solution (500 μg / 500 μL) was added, and the mixture was stirred at 4°C overnight under light shielding.

[0177] The Spitz tube was centrifuged at 14,000 rpm for 5 minutes to separate the porous zirconium oxide particles. The mass of the unfixed IgG remaining in the supernatant solution was quantified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, image analysis software CS Analyzer 4 manufactured by ATTO). The difference between the mass of the initially added IgG and the mass of the unadsorbed IgG was taken as the mass of the adsorbed IgG. The ratio of the mass of the adsorbed IgG to the mass of the initially added IgG was calculated as the adsorption rate (%).

[0178] (2.2) Desorption evaluation

[0179] After the above adsorption evaluation, the desorption of IgG was evaluated using various desorption solutions listed in Table 2.

[0180] Desorption of IgG was performed as follows: After the above-mentioned adsorption evaluation, the supernatant solution was removed, and 500 μL of each desorption solution listed in Table 2 was added to the Spitz tube from which the supernatant solution was removed, and the tube was resuspended to desorb (elute) IgG.

[0181] Next, the Spitz tube was centrifuged at 14,000 rpm for 5 minutes to separate the porous zirconium oxide particles from the sediment. The mass of IgG desorbed from the porous zirconium oxide particles and present in the supernatant solution was quantified by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, image analysis software CS Analyzer 4 manufactured by ATTO). The mass of IgG thus obtained was taken as the mass of the recovered IgG.

[0182] Then, the ratio of the mass of the recovered IgG to the mass of the initially added IgG was calculated as the recovery rate (%). The method for calculating the recovery rate (%) was the same in Test C as well.

[0183] (3) Test results

[0184] The test results are also shown in Table 2.

[0185] It was confirmed that the recovery rates of Test Examples 11 to 16 using a neutral desorption solution were higher than those of Test Example 17 using an acidic desorption solution.

[0186] 3. Experiment C

[0187] (1) Porous zirconia particles

[0188] The porous zirconia particles used were those listed in Table 3. In addition, "Rhinophase-AB" refers to porous zirconia particles manufactured by Zir Chrom Seperations Inc.

[0189] [Table 3]

[0190]

[0191] (2) Adsorption and desorption evaluation

[0192] (2.1) Test Examples 18-19

[0193] Adsorption and desorption evaluation was performed in the same manner as in Test Example 11 except that the porous zirconia particles described in Table 3 were used and 500 μL of goat serum containing IgG (300 μg / 500 μL) was used.

[0194] (2.2) Test Examples 20-21

[0195] The porous zirconia particles listed in Table 3 were used, and a solution of 20 mM MES (2-(N-Morpholino)ethanesulfonic acid), 4 M EDTPA, and 50 mM NaCl was used as the adsorption solution, and a solution of 20 mM MES, 4 M EDTPA, and 2 M NaCl was used as the desorption solution. The adsorption and desorption evaluation was performed in the same manner as in Test Example 11. It should be noted that Test Examples 20-21 are standard purification processes of Rhinophase-AB as the prior art.

[0196] (3) Test results

[0197] The test results are also shown in Table 3.

[0198] First, the results of Test Examples 18 and 20 were compared and studied. In Test Example 18, both the adsorption liquid and the desorption liquid were neutral buffer solutions. On the other hand, in Test Example 20, both the adsorption liquid and the desorption liquid were acidic buffer solutions. Regarding the adsorption rate, the adsorption rate of Test Example 18, in which the adsorption liquid was neutral, was 69.8%, and the adsorption rate of Test Example 20, in which the adsorption liquid was acidic, was 63.8%, and there was no significant difference between the two. However, regarding the recovery rate, the recovery rate of Test Example 18 was 69.8%, and the recovery rate of Test Example 20 was 30.3%, and the recovery rate of Test Example 18 was significantly higher than that of Test Example 20.

[0199] Next, the results of Test Examples 19 and 21 were compared and studied. In Test Example 19, both the adsorption liquid and the desorption liquid were neutral buffer solutions. On the other hand, in Test Example 21, both the adsorption liquid and the desorption liquid were acidic buffer solutions. Regarding the adsorption rate, Test Example 19 was 26.5%, and Test Example 21 was 45.5%, which shows that the adsorption rate of Test Example 21 was high. However, regarding the recovery rate, Test Example 19 was 14.6%, and Test Example 21 was 6.5%, and the recovery rate of Test Example 19 was significantly higher than that of Test Example 21.

[0200] The above results show that when a neutral buffer solution is used as the desorption solution, IgG adsorbed to the porous zirconia particles is easily desorbed, and the recovery rate is improved.

[0201] Note that, focusing on the adsorption rate and recovery rate of Test Example 18, the adsorption rate was 69.8% and the recovery rate was 69.8%, and it was found that almost all of the IgG adsorbed to the porous zirconia particles was desorbed.

[0202] 4. Effects of the Embodiments

[0203] In the immunoglobulin purification method, in which immunoglobulin is adsorbed on porous zirconia particles in a neutral buffer and then desorbed from the porous zirconia particles using a neutral desorption solution, the recovery rate of immunoglobulin is high. In addition, in the immunoglobulin purification method, since no acid solution or alkaline solution is used, the antibody properties of the immunoglobulin are not inactivated.

[0204] <Other embodiments (modifications)>

[0205] It should be noted that the present invention is not limited to the above-described embodiments and implementation modes, and can be implemented in various forms without departing from the gist of the present invention.

[0206] (1) In the above description, an example of the immunoglobulin purification apparatus 1 and the immunoglobulin production apparatus 21 is described, but the configuration of the immunoglobulin purification apparatus 1 and the immunoglobulin production apparatus 21 can be appropriately changed depending on the types of adsorption liquid and desorption liquid, the scale of the apparatus, etc.

[0207] (2) In the above description, the adsorption / desorption section 3 includes a cylindrical section, but may not include a cylindrical section.

[0208] Industrial Applicability

[0209] The immunoglobulin purification method of the present invention is suitable for purification of immunoglobulins (antibodies).

[0210] The method for producing immunoglobulin of the present invention is suitable for producing immunoglobulin (antibody).

[0211] The immunoglobulin purification device of the present invention is suitable for purification of immunoglobulins (antibodies).

[0212] The immunoglobulin production apparatus of the present invention is suitable for production of immunoglobulins (antibodies).

[0213] Explanation of symbols

[0214] 1…Immunoglobulin purification device

[0215] 3…Adsorption and desorption section

[0216] 4…Porous zirconia particles

[0217] 5…Adsorption liquid container

[0218] 7…Desorption liquid container

[0219] 9…Recycling containers

[0220] 11…Switching unit

[0221] 21…Immunoglobulin production equipment

[0222] 23…Adsorption and desorption section

[0223] 24…Porous zirconia particles

[0224] 25…Absorption liquid container

[0225] 27…Desorption liquid container

[0226] 29…Recycling containers

[0227] 31…Switching unit

Claims

1. A method for purifying immunoglobulin, characterized in that: have: an adsorption step of allowing the immunoglobulin to adsorb onto the porous zirconia particles in a buffer solution at a pH of 6.1 or higher and 8.0 or lower; and A desorption step of desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a desorption liquid having a pH of 6.0 or higher and a pH of 8.0 or lower, wherein the desorption liquid is a phosphate buffer solution.

2. The method for purifying immunoglobulin according to claim 1, characterized in that: The desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof, In the general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms; in addition, R 2 ~R 5 The alkylene groups have 1 to 10 carbon atoms and may be the same or different from each other.

3. An immunoglobulin purification device, characterized in that: have: an adsorption liquid container filled with a buffer solution having a pH of at least 6.1 and at most 8.0; a desorption liquid container filled with a desorption liquid having a pH value of not less than 6.0 and not more than 8.0, wherein the desorption liquid is a phosphate buffer; Adsorbing immunoglobulin to the adsorption portion of the porous zirconia particles in the buffer solution supplied from the adsorption liquid container; and A desorption section for desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using the desorption liquid supplied from the desorption liquid container.

4. The immunoglobulin purification device according to claim 3, characterized in that: The desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof, In the general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms; in addition, R 2 ~R 5 The alkylene groups have 1 to 10 carbon atoms and may be the same or different from each other.

5. A method for producing an immunoglobulin, characterized in that: have: an adsorption step of allowing the immunoglobulin to adsorb onto the porous zirconia particles in a buffer solution at a pH of 6.1 or higher and 8.0 or lower; and A desorption step of desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using a desorption liquid having a pH of 6.0 or higher and a pH of 8.0 or lower, wherein the desorption liquid is a phosphate buffer solution.

6. The method for producing immunoglobulin according to claim 5, wherein: The desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof, In the general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms; in addition, R 2 ~R 5 The alkylene groups have 1 to 10 carbon atoms and may be the same or different from each other.

7. An immunoglobulin production device, characterized in that: have: an adsorption liquid container filled with a buffer solution having a pH of at least 6.1 and at most 8.0; a desorption liquid container filled with a desorption liquid having a pH value of not less than 6.0 and not more than 8.0, wherein the desorption liquid is a phosphate buffer; Adsorbing immunoglobulin to the adsorption portion of the porous zirconia particles in the buffer solution supplied from the adsorption liquid container; and A desorption section for desorbing the immunoglobulin adsorbed on the porous zirconia particles from the porous zirconia particles using the desorption liquid supplied from the desorption liquid container.

8. The immunoglobulin production device according to claim 7, characterized in that The desorption liquid contains at least one selected from the group consisting of compounds represented by the following general formula (1) and salts thereof, In the general formula (1), R 1 is an alkylene group having 1 to 10 carbon atoms; in addition, R 2 ~R 5 The alkylene groups have 1 to 10 carbon atoms and may be the same or different from each other.

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