JP2011001336A - Method for producing new protein a immobilization carrier - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a protein A immobilization carrier preventing protein A from leaking in a large amount in purifying an IgG antibody, the protein A being contained in the protein A immobilization carrier that adsorbs antibodies in antibody affinity chromatography.SOLUTION: A carrier 1 and an epoxy group-containing compound containing a terminal epoxy group 2 as a linker precursor 10 are reacted so that the amount of the epoxy group-containing compound per specific surface area of the carrier is 1.5 μmol/mor more and then reacted with a diamine to form a linker 20 and further added with the protein A to produce the protein A immobilization carrier. The protein A immobilization carrier reduces the amount of the protein A that leaks in purifying an IgG antibody, and improves resistance to alkali CIP (cleaning in place).

Description

  The present invention belongs to the technical field of affinity chromatography for adsorbing and separating a specific protein, and particularly relates to a novel method for producing a protein A-immobilized carrier.

  In recent years, it has been important in the pharmaceutical field and the medical field to purify or remove specific proteins with high accuracy. For example, when a chemical having a specific function is purified, it is necessary to purify a specific protein with high accuracy.

  Affinity chromatography is widely used as a means for purifying or removing specific proteins. In affinity chromatography, a specific substance is separated from a mixture by specifically adsorbing the specific substance to the ligand using an antibody affinity carrier in which a substance (ligand) having affinity for the specific substance is fixed to an insoluble carrier. Further, the adsorbed specific substance is eluted by lowering the affinity with the ligand and can be purified by collecting it.

  In affinity chromatography, protein A having a specific binding property is widely used as a ligand. Protein A is a protein derived from the gram-positive cocci staphylococcus staphylococcus (staphylococcus), has a property of specifically binding to the Fc region of IgG derived from various animals, and is widely used for IgG purification. ing. A technique for immobilizing protein A on an insoluble carrier and specifically separating and purifying IgG by affinity chromatography using the insoluble carrier is widely known.

  In antibody affinity carriers used for affinity chromatography, a structure called a linker is generally interposed between an insoluble carrier and a ligand, one end of the linker is bound to the carrier, and the other end of the linker is bound to the ligand. Thus, the ligand is immobilized on an insoluble carrier.

  Until now, a carrier that binds a linker and a ligand using various bonds has been developed. Recently, the immobilized protein A is oriented on the carrier and bound to the carrier at a single point, and the amount of the bound protein A A protein A-immobilized carrier has been developed that eliminates the problem of apparent apparent decrease in activity.

  Some of these carriers use a linker containing a primary amino group and are immobilized by an amide bond with a carboxy terminus of a protein or peptide capable of binding to an antibody molecule to form an antibody affinity carrier. Among them, a protein A-immobilized carrier in which an aminopropyl group is introduced into glass beads using 3-aminopropyltrimethoxysilane is also exemplified (for example, see Patent Document 1).

Japanese Patent Laid-Open No. 2005-112827

As described above, the conventional protein A-immobilized carrier is generally a carrier in which protein A is supported on a carrier such as silica gel or glass beads via an aminopropyl group.
However, protein A immobilized via the aminopropyl group leaks in large quantities when purifying IgG antibodies. For this reason, the extra work of removing the leaked protein A occurs, and there is a problem that the working efficiency in the next purification step is lowered. Moreover, it has the subject that the tolerance with respect to alkali CIP (Cleaning In Place, stationary cleaning) is also scarce.

  The present invention has been made to solve the above-described problems. When a protein A-immobilized carrier that specifically adsorbs an antibody purifies an IgG antibody, it reduces protein A leakage and improves resistance to alkaline CIP. It is an object to provide a method for producing a protein A-immobilized carrier.

The present inventor has found that the above object can be achieved by using a linker having a terminal structure completely different from that of the conventional one, and has derived the present invention. Thus, according to the present invention, the support and the epoxy group-containing compound are reacted so that the amount of the epoxy group-containing compound per specific surface area of the support is 1.5 μmol / m ² or more, and then the diamine is reacted. Provided is a method for producing a protein A-immobilized carrier which is a linker and further carries protein A.

  The protein A-immobilized carrier obtained by the production method of the present invention can reduce leakage of protein A and improve resistance to alkaline CIP even when an IgG antibody is purified.

Schematic diagram illustrating a linker in a protein A-immobilized carrier obtained by the present invention Experimental results on affinity performance of protein A immobilized carrier according to the present invention Experimental results on protein A leakage and alkali elution of protein A immobilized carrier according to the present invention Results of alkaline CIP durability test of protein A-immobilized carrier according to the present invention

In the method for producing a protein A-immobilized carrier of the present invention, the carrier and the epoxy group-containing compound are reacted so that the amount of the epoxy group-containing compound per specific surface area of the carrier is 1.5 μmol / m ² or more. In this method, diamine is reacted and protein A is further reacted to be immobilized.

  FIG. 1 is a schematic diagram for explaining steps before protein A immobilization (ie, steps until a linker is formed on a carrier) in the method for producing a protein A-immobilized carrier of the present invention. The left side of FIG. 1 shows a state after the epoxy group-containing compound is reacted with the carrier. In this state, the linker precursor 10 has the terminal epoxy group 2 at the terminal. Subsequently, diamine is allowed to react with the terminal epoxy group at two sites. As a result, as shown on the right side of FIG. 1, the two amino groups contained in the diamine are formed by opening the two terminal epoxy groups 2 to crosslink, and the terminal epoxy group 2 and the diamine are linear. It is considered that a linker 20 having a linear portion 4 having an amino group at the terminal portion is formed. FIG. 1 shows a case where 1,6-hexylenediamine (HDA) is added to the terminal epoxy group 2 in the linker precursor 10 to produce a linker 20 having a plurality of crosslinking sites 3 and linear sites 4. .

  Thus, the linker 20 includes the crosslinking site 3 and the linear site 4, and it is considered that an amino group is present at the terminal portion of the linear site 4. It is considered that the amino group present at the terminal portion of the linear site can be bound by performing an amide bond with the carboxyl group at the terminal portion of protein A as a ligand.

  The carrier 1 is not particularly limited as long as it is mainly composed of conventionally used inorganic materials. For example, a metal oxide can be used. Examples of the metal oxide include silica, alumina, zirconium oxide, glass and the like, and silica is preferable. The carrier 1 is preferably porous. Thus, silica gel is particularly preferable as the carrier 1.

As silica gel, various conventionally known ones can be used. The pore size needs to be larger than the size of the target protein to be separated and purified. The average pore diameter (D, nm), specific surface area (S, cm ² / g), and pore volume (V, cm ³ / g) of the particles have the correlation shown in the following formula (1).

D = 4000 × V / S (1)
Therefore, in order to increase the average pore diameter, it is necessary to increase the pore volume or decrease the specific surface area. However, increasing the pore volume is not preferable because the particle strength generally decreases. Further, if the specific surface area is reduced, the area to which the ligand is added is reduced, the amount of the ligand is reduced, and the ability to adsorb the target protein is reduced, which is not preferable. It is necessary to optimize these physical properties according to the purpose, and generally those having an average pore diameter of 30 to 500 nm, preferably 70 to 300 nm are used. The pore volume is 0.5 to 2.5 cm ³ / g, preferably 0.7 to 2.0 cm ³ / g. A known method can be used to measure these physical properties of the pores. For example, it can be measured by a mercury intrusion method.

  The smaller the average agglomerated particle size of the silica gel, the better the separation characteristics. However, the pressure loss when using the column increases. Therefore, the one with an appropriate value is adopted in consideration of the specifications of the apparatus to be used. Generally, it is 5-500 micrometers, 7-200 micrometers is preferable and 10-100 micrometers is especially preferable. Also, the more uniform the particle size distribution, the better the separation performance.

Cumulative from the smallest particle size, the particle size at which the cumulative amount by mass or volume is 10% of the total particle amount is 10% diameter (D10), and the particle size at 90% is 90% diameter (D90). In this case, the value of D90 / D10 is 3 or less, preferably 2 or less, more preferably 1.6 or less. The average particle size and particle size distribution can be measured by a known method, for example, by an apparatus using a principle such as an electric resistance method or a dynamic light scattering method.
The shape of the particles is not particularly limited. A so-called crushed material can also be used, but a spherical material can be suitably used from the viewpoint of packing properties in the column and suppression of pressure loss during use.

  As the silica gel, those composed of silicon oxide having a purity of usually 99% or more, preferably 99.9% or more, more preferably 99.99% or more are used. From the standpoint of improving alkali resistance, a compound combined with an organic substance or a surface-coated one can be used.

  In the present invention, the carrier is reacted with an epoxy group-containing compound. By this operation, the epoxy group-containing compound is immobilized on the surface of the carrier, and the linker precursor 10 is formed. The linker precursor 10 has an epoxy group at the terminal.

  As the epoxy group-containing compound, a silane coupling agent having an epoxy group is preferably used. As the epoxy group-containing silane coupling agent, γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropylmethyldiethoxysilane, β- (3,4-epoxycyclohexyl) ethyltrimethoxysilane and the like can be used.

  As a method of reacting the carrier and the epoxy group-containing compound, a known method can be used. For example, a method of heating the carrier and the epoxy group-containing compound in a solvent can be employed. The reaction temperature is about 30 to 400 ° C, preferably 100 to 300 ° C. The reaction time is about 0.5 to 40 hours, preferably 3 to 20 hours.

  Any solvent can be used without particular limitation as long as it does not react with the epoxy group-containing compound and is stable at the reaction temperature. From the viewpoints of solubility, boiling point, and affinity with other solvents (that is, removability during washing), usually benzene, toluene, xylene, octane, isooctane, tetrachloroethylene, chlorobenzene, bromobenzene Etc. are preferably used. The reaction operation is desirably performed under reflux of the solvent used.

In general, the amount of the epoxy group-containing compound immobilized is preferably as large as possible, that is, the amount that covers the entire surface of the carrier densely, from the viewpoint of improving the alkali resistance of the carrier. Specifically, the reaction is performed so that the amount of the epoxy group-containing compound per specific surface area of the support (the value obtained by dividing the amount of the epoxy group-containing compound immobilized by the specific surface area of the support) is 1.5 μmol / m ² or more. . Immobilization amount of the epoxy group-containing compound is preferably ^{1.5~50μmol / m 2, 2.5~50μmol / m} 2 is particularly preferred.

  The amount of the epoxy group-containing compound immobilized is determined based on a known method. For example, the amount of carbon contained in one molecule of the epoxy group-containing compound and the specific surface area of the silica gel used as the carrier based on the carbon content measured by elemental analysis on the silica gel after immobilizing the epoxy group-containing compound Can be used to calculate the amount of immobilization of the epoxy group-containing compound.

  Next, the diamine is reacted. The diamine is presumed to react with the terminal epoxy group 2 of the linker precursor 10 to form the cross-linked site 3 and the linear site 4.

  In principle, various diamines can be used as the diamine, but a compound having an amino group at both ends of an alkylene group or alkenylene group having 2 to 10 carbon atoms (preferably 2 to 6 carbon atoms) is preferable.

Specifically, a compound represented by the formula NH ₂ — (CH ₂ ) _n —NH ₂ (n is an integer of 2 to 10) is preferable, and a compound in the case where n is 2 (1,2-ethylenediamine, hereinafter EDA) or a compound in which n is 6 (1,6-hexylenediamine, hereinafter also referred to as HDA) is particularly preferable.

  A known method can be used to react the diamine with the terminal epoxy group 2. For example, a method of heating the carrier on which the linker precursor 10 is formed and the diamine in a solvent can be employed. The reaction temperature is about 30 to 200 ° C, preferably 30 to 100 ° C. The reaction time is about 0.5 to 50 hours, preferably 0.5 to 20 hours.

  As the reaction solvent, a solvent that does not react with the terminal epoxy group 2 and dissolves the diamine is preferable. For example, toluene, isopropyl alcohol, methanol, water, sodium bicarbonate aqueous solution and the like are suitable. In order to form the linear part 4, it is preferable to use 1 equivalent or more of the amount of diamine with respect to the terminal epoxy group 2, and it is especially preferable to use 10-200 equivalent.

Immobilization amount of the diamine, from the viewpoint of improving the alkali resistance, as the amount per specific surface area of the support, is preferably set to 0.5 [mu] mol / m ² or more, to 0.8~1.6μmol / m ² Is particularly preferred.

  The amount of diamine immobilized is determined based on a known method. For example, after reacting diamine, the nitrogen content is measured by elemental analysis to determine the amount of nitrogen, and the amount of nitrogen before immobilization obtained in the same manner is subtracted, and this value and nitrogen per molecule of diamine The amount of diamine immobilized can be calculated using the amount and the specific surface area of silica gel used as a carrier.

  Although the mechanism of improving the alkali resistance by reacting the terminal epoxy group 2 and the diamine in the linker precursor 10 is not clear, some diamines react with the two terminal epoxy groups 2 to form a crosslinked structure 3 It is presumed that this is because the alkali is prevented from entering the carrier.

  In the present invention, the ratio of the crosslinked structure 3 calculated by the following formula (2) is preferably 50% or more and less than 100%, and particularly preferably 70 to 99%. When the ratio of the crosslinked structure 3 is 100%, an amino group capable of binding to protein A does not exist, which is not preferable.

(E ₀ −D _a ) / D _a (2)
Here, E ₀ is terminal epoxy group content before reacting the diamine ^{(μmol / m 2), D} a is a diamine immobilized amount (μmol / m ^2). The amount of terminal epoxy can be measured by a known method. For example, the sample can be obtained by suspending the sample in water, reacting with Na ₂ S ₂ O ₃ , and titrating the liberated NaOH amount with HCl.

  Next, protein A is carried. As protein A, one having a carboxyl terminus as a linking group can be used. Among these, recombinant protein A having a cysteine residue and a peptide chain having a low isoelectric point at the carboxyl terminus (see, for example, JP-A-2004-34595, hereinafter abbreviated as “r-protein A”) is preferably used. it can.

  In the present invention, protein A is supported by an adsorption step in which r-protein A is dissolved in a phosphate buffer at room temperature and adsorbed on the linker, and a cysteine sulfhydryl group of r-protein A is converted into a cyanation solution. An activation step of cyanating and converting to cyanocysteine and activating, and an immobilization reaction in which the r-protein A activated by the activation step is immobilized on the linker 2 with a borate buffer The protein A-immobilized carrier can be formed by including the crystallization step.

  Specifically, in the adsorption step, r-protein A dissolved in a phosphate buffer (pH 7.0) is allowed to act at room temperature for 2 hours or more. At this time, since the peptide site at the end of r-Protein A is negatively charged, r-Protein A is electrostatically adsorbed to the linker (linear structure 4) having a free primary amino group. It is thought to do.

  In the activation step, a 2-nitro-5-thiocyanobenzoic acid (NTCB) reagent as a cyanation solution is added after completion of the adsorption step and allowed to react at room temperature for 4 hours. By this reaction, the sulfhydryl group (SH) of cysteine of r-protein A is cyanated and converted to cyanocysteine.

  In the immobilization step, an immobilization reaction in which r-protein A is immobilized on the linker at room temperature for 24 hours is performed by using a 10 mmol / L borate buffer solution to make it weakly alkaline at pH 9.5. Thus, by making the solution weakly alkaline, an amide bond is formed between the primary amino group of the linker and the carboxyl group of the amino acid residue immediately before the cyanocysteine residue. The polypeptide is cleaved. Thereafter, the polypeptide is washed with a high concentration aqueous KCl solution to remove the cleaved polypeptide.

  The amount of r-protein A immobilized is determined based on a known method. For example, the concentration of the r-protein A solution used in the adsorption step and the concentration of the r-protein A solution obtained by immersing the carrier in the solution to adsorb r-protein A and then separating the carrier. From the difference, the amount immobilized on the carrier can be calculated. The solution concentration can be measured optically.

Hereinafter, the present invention will be described in more detail with reference to examples. However, as will be apparent to those skilled in the art, the present invention is not limited to these examples.
[1] Preparation of protein A-immobilized carrier (Example 1)
Silica gel (manufactured by AGC S-Itech Co., Ltd., MS GEL. SIL D-50-1000AW; specific surface area = 74 m ² / g, average particle diameter = 44.8 μm, average pore diameter = 97.5 nm) (50 g), γ-Glycidoxypropyltrimethoxysilane (34 mL) was heated at 90-95 ° C. for 4.5 hours. At this time, the amount of γ-glycidoxypropyltrimethoxysilane was such that the amount immobilized was 3.0 μmol / m ² with respect to the silica gel. Moreover, when the amount of epoxy groups was measured, it was the quantity used as 2.72 micromol / m < ² >.

Subsequently, 1,6-hexylenediamine (44 g) was added, and the mixture was heated at 60 to 65 ° C. for 2 hours. The amount of diamine immobilized was 1.43 μmol / m ² .
When the ratio of the crosslinked structure represented by the formula (2) was calculated, it was 90%.

  Next, r-Protein A (IBP “Amano 2” manufactured by Amano Enzyme Co., Ltd., trade name: IBP-2, lot number: IBPG0250301R) was bound to the linker formed on the silica gel surface according to the above method, A immobilization support (1) was prepared. The amount of r-protein modification was estimated to be 5.6 mg / mL-bed by a known method.

(Example 2)
A protein A-immobilized carrier (2) was prepared in the same manner as in Example 1 except that 1,6-hexylenediamine (44 g) was changed to 1,2-ethylenediamine (22 g). The amount of diamine immobilized was 1.55 μmol / m ^2, and the ratio of the crosslinked structure was calculated as 76% from the formula (2). The amount of r-protein modification was estimated to be 5.8 mg / mL-bed by a known method.

(Comparative Example 1)
Instead of using γ-glycidoxypropyltrimethoxysilane, using 3-aminopropylmethoxysilane (12 g) and omitting the step of reacting 1,6-hexylenediamine, the same as in Example 1, An r-protein A-immobilized carrier (hereinafter referred to as AP carrier) immobilized through an aminopropyl group was obtained. The amount of r-protein modification was estimated to be 5.5 mg / mL-bed by a known method.

[2] Evaluation of affinity performance of protein A-immobilized carrier (evaluation of equivalence with AP carrier 1)
For the protein A-immobilized carrier prepared in Examples 1 and 2 and the AP carrier prepared in Comparative Example 1, (1) Dynamic adsorption capacity (Dynamic Binding Capacity, DBC), (2) Static adsorption quantity (Static Binding Capacity, SBC) (3) IgG recovery rate and (4) non-specific adsorption amount were evaluated.

  DBC was evaluated by defining the value calculated from the elution volume at the 10% breakthrough point of the measured breakthrough curve as DBC. The non-specific adsorption amount indicates the ability to avoid adsorption of other proteins other than protein A. Here, lysozyme (Lysozyme), which is an enzyme that hydrolyzes polysaccharides constituting the cell walls of eubacteria, Endopeptidase, trypsin, a kind of serine protease, and bovine serum albumin (BSA) were measured for each protein.

  DBC is a polyclonal human IgG solution (concentration 0.5 mg) in which a protein A-immobilized carrier is packed in a 5 mmφ × 25 mm glass column (GE Healthcare Tricorn 5/20 Column) and dissolved in PBS buffer (pH 7.4). / mL) was sent to this column at a flow rate of 0.6 mL / min and evaluated by obtaining a breakthrough curve. Fig. 2 (a) shows a breakthrough curve. From this measurement result, the protein A-immobilized carrier (1) according to the present invention shows 37 mg / mL-bed, and the AP carrier as a control shows 40 mg / mL-bed. Was shown to hold.

  After that, this column is washed with PBS buffer, and elution buffer (acetic acid buffer, pH 4.5) is fed at a flow rate of 0.6 mL / min to elute IgG, and the fraction (Fraction) which is the obtained droplet. After neutralization, IgG recovery (%) was measured using gel permeation chromatography (GPC). As a result, both showed a high recovery rate of 90% or more, and showed the same performance with respect to the IgG recovery rate.

  The SBC evaluation was performed by the following method. A polyclonal human IgG solution having an initial concentration of 0 to 1.82 mg / mL was added to 0.1 mL-bed of protein A-immobilized carrier, and the mixture was stirred with a rotary mixer overnight at room temperature. After centrifugation, the residual IgG concentration was quantified by measuring the supernatant at 280 nm absorbance, and SBC was calculated using a Langmuir plot. As a result, the protein A-immobilized carrier (1) according to the present invention shows 82 mg / mL-bed, and the AP carrier as a control shows 85 mg / mL-bed, so that the same amount of protein adsorption is maintained. Was shown to do.

  Moreover, the nonspecific adsorption amount was measured by the following method. A protein A-immobilized carrier was packed in a 5 mmφ × 25 mm glass column, and a PBS buffer solution (pH 7.4) was fed to the column at a flow rate of 1.0 mL / min. Subsequently, 20 μL of each protein solution of lysozyme, trypsin, and bovine serum albumin prepared to a concentration of 0.08 mg / mL with PBS was injected, and the non-eluting nonspecific adsorption amount was measured. As a result, the protein A-immobilized carrier (1) according to the present invention and the AP carrier as a control showed equivalent performance for lysozyme, trypsin and bovine serum albumin. The above results are summarized and shown in FIG.

[3] Evaluation of affinity performance of protein A-immobilized carrier (evaluation of equivalence with AP carrier 2)
Next, regarding the protein A-immobilized carrier, the DBC as the affinity performance was evaluated by changing the residence time of the column (Residence time) according to the liquid feeding speed to the column. This DBC evaluation was performed by packing a protein A-immobilized carrier in a 5 mmφ × 25 mm glass column and feeding a polyclonal human IgG solution (concentration 0.5 mg / mL) to this column.

  As a result, a DBC curve corresponding to the residence time was obtained as shown in FIG. In the figure, a curve a is a measurement result for a conventional AP carrier, and a curve b is a measurement result for a protein A-immobilized carrier (1) according to the present invention.

  From this measurement result, the protein A-immobilized carrier (1) obtained by the production method of the present invention showed a curve close to that of the AP carrier as a control. Was shown to do.

[4] Evaluation of leakage of protein A-immobilized carrier
ELISA (Enzyme-Linked ImmunoSorbent Assay, ELISA) was measured using Assay Designs TiterZyme EIA Kit, and the amount of protein A leak was evaluated. Specifically, 0.1 mol / L glycine HCl buffer (pH 2.0) was used as the elution buffer, and each fraction (Fraction) was neutralized with 1 mol / L sodium carbonate aqueous solution (pH 8.0), and coated. The 96 microwell ELISA measurement was performed. The measurement results are shown in FIG. Whereas the conventional AP carrier showed about 700 ng / mg-IgG, the protein A-immobilized carrier according to the present invention is 1,2-hexylenediamine (HDA) as a diamine, When ethylenediamine (EDA) was used, both showed about 80 ng / mg-IgG, indicating that the amount of protein A leak can be greatly reduced.

[5] Evaluation of alkali resistance of protein A-immobilized carrier (alkali CIP durability test)
Next, an alkaline CIP durability test was conducted in a situation closer to actual industrial use. Here, repeated cleaning with alkali CIP was performed, and the degree of capability deterioration (capacity deterioration) due to the repeated cleaning was confirmed.

  More specifically, protein A-immobilized carrier (1) is packed in a 4.6 mmφ × 10 mm stainless steel column, and a polyclonal human IgG solution (concentration 0.5 mg / mL-bed) is sent to this column at a flow rate of 0.83 mL / min. Then, the column was washed by feeding a PBS buffer solution (pH 7.4) (Wash step). Thereafter, elution with 8.3 mL of citrate buffer (pH 2.2) (Elution step), washing for 20 minutes with 50 mM NaOH aqueous solution (pH 12.7) (alkaline CIP step), PBS buffer (pH 7.4) Equilibration (Conditioning process) was performed. The loading of polyclonal human IgG was repeated once every 10 cycles to evaluate DBC. As the DBC evaluation result, as shown in FIG. 4, the DBC at each cycle number when the first DBC is 100 was calculated as relative DBC (Relative DBC) (%). In the figure, the curve a is the measurement result of the protein A-immobilized carrier (1) according to the present invention, and the curve b is the measurement result of the conventional AP carrier.

  From the figure, in the AP carrier, the relative DBC was reduced to 80% or less by about 20 alkaline CIPs, but in the protein A-immobilized carrier (1) according to the present invention, the relative CBC was about 120 DBC was able to maintain 80%, and it was shown that it has much stronger alkali CIP resistance compared with the past.

  The protein A-immobilized carrier obtained by the production method of the present invention can be applied to protein purification. In addition, it can be applied to the use of separating protein subclasses by changing the pH value of an acidic solution during elution (Elution step).

10 linker precursor 1 carrier 2 terminal epoxy group 3 crosslinking site 4 linear site 20 linker

Claims (4)

Reacting the carrier with the epoxy group-containing compound so that the amount of the epoxy group-containing compound per specific surface area of the carrier is 1.5 μmol / m ² or more, then reacting the diamine, and further supporting protein A. A method for producing a protein A-immobilized carrier.   The production method according to claim 1, wherein the carrier is a carrier made of an inorganic oxide.   The manufacturing method according to claim 1, wherein the epoxy group-containing compound is an epoxy group-containing silane coupling agent.   The method according to any one of claims 1 to 3, wherein the diamine is a compound having an amino group at both ends of an alkylene group having 2 to 10 carbon atoms or an alkenylene group.

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