JP3178080B2 - Antibody separation and purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel separation and purification method capable of obtaining a high-purity antibody from an antibody-containing solution by a simple operation.

[0002]

2. Description of the Related Art Antibodies are protein components that control the immune reaction, and in recent years, their utility has been increasing in use as pharmaceuticals, diagnostic agents, or materials for separating and purifying corresponding antigen proteins. Antibodies can be obtained from the blood of the immunized animal, from a cell culture of cells having antibody-producing ability, or from an ascites culture of the animal. However, blood or culture solution containing those antibodies contains proteins other than antibodies or complicated contaminants derived from the raw material solution used for cell culture, and it is necessary to separate and purify antibodies from those impurity components. In addition, complicated and time-consuming operations are usually required. That is, a large amount of ammonium sulfate, sodium sulfate, polyethylene glycol, or the like is added, a precipitate is collected from a concentrated solution thereof, and then subjected to gel filtration chromatography, and further to anion exchange resin chromatography such as DEAE cellulose. Techniques for processing by immobilized column or antigen-immobilized column chromatography have been commonly used. As described above, since the conventional method involves many steps, many materials and complicated operations are required.In addition, the method using the protein A-immobilized column and the antigen-immobilized column requires expensive materials, When desorbing the adsorbed antibody, there is a problem that the biological activity of the antibody may be lost.

[0003] As a method for separating and purifying an antibody from a liquid containing the antibody by a simple operation, chromatography using hydroxyapatite as a filler is known. That is, Stanker et al. Separated a monoclonal antibody with high purity by chromatography using hydroxyapatite aggregates as a filler [Journal of Immunological Methods, Vol. 76, pp. 157-169, (1985)
The present inventors have previously proposed hydroxyapatite for separation which is effective for separation and purification of biological macromolecules such as proteins and nucleic acids (JP-A-62-176906, US Pat. No. 5,073). , 357). However, since hydroxyapatite alone has a generally low adsorption capacity, a large amount of hydroxyapatite is required to separate and purify an antibody from a serum or a solution containing a large amount of contaminants such as a hybridoma culture solution or ascites. On an industrial scale, this can be a major obstacle.

Another method for separating and purifying an antibody from a solution containing the antibody by a simple operation is chromatography using an ion exchanger having a polymer in which a weak anion exchange group and a weak cation exchange group are bonded as a packing material. Are known. The ion exchanger has a property of mainly adsorbing the antibody protein and allowing most of the other proteins to pass through, and has a property of easily concentrating and separating the antibody protein. If the antibody is separated from the liquid,
In fields where highly contaminating components still remain and high-purity antibodies are required, it is desired to further increase the purity of the antibodies.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a method for separating and purifying an antibody capable of efficiently obtaining a high-purity antibody from an antibody-containing solution while maintaining the activity of the obtained antibody by a simple operation. It is an object to provide

[0006]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems. As a result, first, an ion exchanger having both an anion exchange group and a cation exchange group (hereinafter sometimes referred to as an AC ion exchanger). ) Was used as the packing material, and the resulting antibody-containing effluent was subjected to chromatography using hydroxyapatite as the adsorbent. The invention has been completed. That is, the present invention relates to a method of separating and purifying an antibody from an antibody-containing liquid by liquid chromatography, wherein the antibody-containing liquid is passed through a column filled with an AC ion exchanger to obtain an antibody-containing effluent. And then passing the antibody-containing effluent through a column packed with hydroxyapatite. Hereinafter, the present invention will be described in detail. The separation and purification method of the present invention separates an antibody from a liquid containing an antibody by a liquid chromatography method. Specifically, for example, the antibody can be separated by the following operation. That is, the stationary phase filler is packed in a column, and the antibody-containing liquid is injected into the column, whereby the antibody is adsorbed to the stationary phase filler. Thereafter, by supplying an appropriate mobile phase liquid, the antibodies adsorbed on the stationary phase filler are sequentially eluted from those having a smaller distribution to the stationary phase. In the case of separating an antibody having a large distribution, it is preferable to elute the antibody by changing the composition of the mobile phase liquid to reduce the distribution to the stationary phase. The separation method of the present invention can be sufficiently carried out under normal pressure, but can also be carried out under pressure if necessary. The invention uses AC
The first step is liquid chromatography using an ion exchanger as a packing material. The antibody-containing solution of the present invention has no particular limitation on the type of antibody contained therein.
Any antibody classified into G, IgM, IgA, IgE and IgD may be used, and specific examples include serum containing the antibody, supernatant of mouse ascites fluid, and supernatant of in vitro cell culture. The AC ion exchanger used in the first step of the present invention has a property of selectively adsorbing an antibody protein,
It has the property of passing most of other proteins, and is an ion exchanger having both an anion exchange group and a cation exchange group. The anion exchange group is preferably a weak anion exchange group, and the cation exchange group is preferably a weak cation exchange group.Specifically, examples of the weak anion exchange group include amine-based ion exchange groups. There are ion exchange groups such as carboxylic acids. As the AC ion exchanger in the present invention, an ion exchanger having a polymer in which a weak cation exchange group and a weak cation exchange group are bonded to each other is preferable. As a preferable specific example of the AC ion exchanger, for example, US Pat. No. 5,092,992 No. 5, U.S. Pat.
No. 087,359, U.S. Pat. No. 5,085,779,
U.S. Pat. No. 5,087,359, U.S. Pat.
No. 6,395, U.S. Pat. No. 4,721,573, U.S. Pat. No. 4,606,825, U.S. Pat.
235 and U.S. Pat. No. 4,469,630. Commercially available AC ion exchangers include Bacarboned ABx (trade name, manufactured by JT Baker, USA). It is preferable to equilibrate the AC ion exchanger tower before passing the antibody-containing solution through the AC ion exchanger. For equilibration, for example, an aqueous solution of 25 mM 2-morpholinoethanesulfonic acid (pH 5.6) or the like can be used.

In the conventional antibody purification method, prior to the treatment by chromatography, ammonium sulfate or polyethylene glycol is added to the antibody-containing solution, and a precipitate containing the antibody is separated by so-called salting out, and the precipitate is dissolved. Then, it is generally supplied after dialysis desalting.
This is because the antibody-containing liquid such as serum contains a lot of contaminant components such as proteins other than the antibody, and if this is directly adsorbed to the adsorbent, the expensive adsorbent will be overused and avoided. . In the present invention, there is no problem in adopting this method, but these steps are essentially unnecessary, and in the first step of the present invention, the salting-out step is omitted, and the antibody-containing solution is used. It can be immediately supplied to the AC ion exchanger column. Omitting the salting-out step is particularly true for i
This is advantageous when an n vitro cell culture solution is used as a raw material. That is, the in vitro cell culture has, at least at present, a very low production antibody concentration, at most 0.1 mg / ml, typically 1/10 to 1/1.
In the case of an industrial scale with a capacity of cubic meters, the amount of salt required for salting out is extremely large, and the treatment of an organic waste liquid having a high salt concentration is added as a difficult problem, and the salting out step is made unnecessary. Has great significance.

The conditions for passing the antibody-containing solution may be any of those commonly used for liquid chromatography. Preferably, the concentration of salts contained in the antibody-containing solution is reduced to about 100 mM or less by dilution with water or the like. And p
It is preferable to adjust H in the range of 4.5 to 6.0 and pass the solution through a column filled with an AC ion exchanger (hereinafter abbreviated as an AC ion exchanger column).

Next, the operation proceeds to the operation of developing and eluting the AC ion exchanger column. The developing and eluting operation at this time can be carried out in accordance with the conditions generally used in liquid chromatography.First, components mainly containing proteins other than antibodies are eluted by passing a dilute eluent. Let it. Preferred diluted eluents are aqueous solutions of potassium, sodium and ammonium salts such as phosphoric acid, acetic acid, morpholinoethanesulfonic acid (MES) and morpholinohydroxypropanesulfonic acid (MOPSO), of which MES is particularly preferred. . The preferred pH and concentration of the diluted eluent are pH 4.5 to 6.0 and pH 10 to 1 respectively.
00 mM, more preferably pH 4.8-5.8.
And the concentration is 10-50 mM.

Next, the AC ion exchanger column is developed with a concentrated eluent by a gradient method or a stepwise method. As the concentrated eluent, an aqueous solution of potassium, sodium, or ammonium salt such as sulfuric acid or hydrochloric acid can be used in addition to the salts of the dilute eluent, and the preferable pH is 5.0 to 5.0.
8.0, and the upper limit of the concentration can be set to 1M if necessary. For the effluent of the AC ion exchanger column,
For example, by observing a change in the protein concentration in the effluent by ultraviolet absorption at a wavelength of 280 nm, the effluent of the antibody (hereinafter referred to as the first step effluent) is fractionated,
In the process, it is loaded on a tower filled with hydroxyapatite (hereinafter referred to as a hydroxyapatite tower).

The second step of the present invention is liquid chromatography using hydroxyapatite as a filler. Although it is possible to directly load the effluent of the first step onto the hydroxyapatite column, it is preferable to adjust the pH to 4.
It is loaded after being adjusted to 5 to 8.0, more preferably 5.2 to 7.0. The development of the second step starts with a dilute eluent and ends with a concentrated eluent. Any eluent usable in the first step can be used as the dilute eluent used at this time.

Next, the concentrated eluent is passed by a gradient method or a stepwise method to develop a hydroxyapatite column. At this time, any of the concentrated eluents usable in the first step can be used. In the first step, contaminant components can be separated to obtain an effluent in which the antibody has been considerably concentrated, and after the development of the second step, impurities remaining in a small amount in the first step are further separated and removed. A purified antibody-containing effluent can be obtained. With respect to the effluent of the second step, ultraviolet absorption can be observed in the same manner as in the first step, and an antibody-containing effluent (hereinafter abbreviated as the second step effluent) can be collected. Further, from the effluent of the second step, desalting can be carried out appropriately to obtain a higher-purity antibody sample.

In the present invention, the use of hydroxyapatite described in Japanese Patent Application Laid-Open No. 62-176906 or US Pat. No. 5,073,357 is effective in exhibiting the effects of the present invention to a high degree. It is valid.
This hydroxyapatite aggregate has an average particle size of 60 in which hexagonal columnar or needle-like hydroxyapatite single crystals are aggregated.
It is an aggregate of 凝集 100 μm. It is well known that conventional hydroxyapatite has a plate-like or scale-like shape and liquid permeability tends to be low, and it is necessary to apply pressure to obtain a flow rate sufficient for analysis, and this is currently applied. . However, pressurization requires special equipment and is an obstacle particularly for preparation applications requiring a large amount of processing. In particular, since the AC ion exchanger used in the first step of the present invention is used by appropriately selecting the particle size so that the antibody-containing liquid can be treated at a normal flow rate and a high flow rate, it is used. It is desirable that hydroxyapatite, which is a filler used in the second step, is also used in a balanced manner, whereby the technical value of the present invention is maximized. The hydroxyapatite aggregate has extremely high liquid permeability, and a flow rate corresponding to the first step is sufficiently ensured even at normal pressure, and is particularly effective for large-scale preparation of antibodies.

The hydroxyapatite aggregate preferably has a pore volume of 1 to 5 ml / g, and more preferably 1.5 to 3 ml / g. Here, the pore volume is
Refers to the total volume of the pores contained in the hydroxyapatite aggregate, the mercury contact angle is 130 °, and the surface tension is 4
It is a value measured by a mercury intrusion method under the condition of 84 dyn / cm. If the pore volume is less than 1 ml / g, it is difficult to obtain a sufficient flow rate, it is easy to cause clogging, and the amount of adsorbed antibody is not sufficient. On the other hand, when the pore volume exceeds 5 ml / g, the strength of the aggregate is not sufficient for long-term use.

The preferred average particle size of the hydroxyapatite aggregate is 60 to 100 μm, and the ratio of the microaggregate having a particle size of less than 10 μm in the hydroxyapatite aggregate is preferably 1 vol% or less. Particle size 1
If the ratio of microaggregates less than 0 μm exceeds 1 vol%, these microaggregates enter the pores, easily cause clogging, causing a decrease in separation performance, and further, packing the column with hydroxyapatite aggregates In this case, it may be necessary to remove fine particles present in the supernatant by decantation.

The hydroxyapatite aggregate can be produced, for example, by synthesizing hexagonal columnar or needle-like crystals of monetite, followed by hydroxylation by addition of an alkali, and has a preferred pore volume of 1 to 5 ml / g. In order to obtain an apatite aggregate, it is preferable to select KOH or LiOH as an alkali to be used when a hexagonal columnar or needle-like monetite crystal is subjected to an alkali treatment under heating.

An example of the synthesis of a hydroxyapatite aggregate is shown below. Synthesis Example First, an acicular hydroxyapatite aggregate was synthesized as follows. That is, 3 equipped with a stirrer and a cooling pipe
l Add 1 liter of pure water to a flask and heat it to 95 ° C.
0.5 mol / l calcium chloride aqueous solution and 0.5 mol / l
Then, 1 l of each aqueous solution of disodium phosphate was added dropwise over 5 hours to obtain an aggregate of needle-like crystals of monetite. Subsequently, at the same temperature, a 1 mol / l potassium hydroxide aqueous solution 3
00 ml was added dropwise over 1 hour to obtain an acicular hydroxyapatite aggregate.

The acicular hydroxyapatite aggregate obtained as described above was evaluated for pore volume, particle size distribution and liquid permeability as follows. (Pore volume) The mercury contact angle is 130 ° and the surface tension is 4
Under the condition of 84 dyn / cm, the pore volume of the hydroxyapatite aggregate was measured by a mercury intrusion method. (Particle size distribution) The particle size distribution was measured using a laser diffraction type particle size distribution analyzer LA-500 manufactured by Horiba Seisakusho, and the average particle size was determined. (Liquid permeability test) A hydroxyapatite aggregate was packed into a glass open column having an inner diameter of 1.25 cm and a length of 40 cm so as to have a bed volume of 15 ml.
A phosphate buffer (pH 6.8) was added to adjust the total volume to 30 ml. The buffer was passed under normal pressure, and the outflow rate (ml / Hr) per unit time was measured. The results of the pore volume, the average particle size, the liquid permeability test and the filling rate are described below. Pore volume: 1.8 ml / g Average particle size: 89 μm (rate of 10 μm or less: 0.3%) Filling rate: 4.8% Liquid permeability: 240 ml

In the antibody separation method of the present invention, the packing ratio of the hydroxyapatite column in the second step is preferably 10% or less, more preferably 2 to 8%, still more preferably 3 to 8%. Increase to 5%. If the filling ratio exceeds 10%, it tends to be difficult to obtain a sufficient flow rate under normal pressure.
Here, the filling rate (A) is a value calculated by the following equation.

The filling rate can be easily controlled by the particle size distribution and pore volume of the hydroxyapatite aggregate. In order to prepare a column having such a low packing ratio, the above hydroxyapatite aggregate is dispersed in a solvent, and a slurry having an appropriate concentration may be packed in the column. As a solvent to be dispersed, a solution used when performing liquid chromatography, for example, 5-1
0 mM phosphate buffer may be used.

As a commercially available product of the hydroxyapatite aggregate, there is Gigapite (trade name, manufactured by Toagosei Chemical Industry Co., Ltd.).

Other separation operations using hydroxyapatite aggregates may be performed according to a general liquid chromatography operation. Preferred conditions include, for example, the following. Flow rate: generally varies depending on the size of the column, but it is appropriate to use 0.5 to 1 ml / min for analysis and 3 to 5 ml / min for fractionation.
cm, 40 cm long glass open column
3 ml / min is appropriate. Amount of mobile phase liquid passed: about 10 times the volume of hydroxyapatite aggregate packed in the column. Composition of mobile phase solution: phosphate buffer, more preferably potassium phosphate buffer. Elution conditions: gradient elution from 10 to 300 mM. Column temperature: room temperature. More preferably, at 4 ° C. to prevent deterioration of the antibody. Loading amount of antibody-containing liquid: about 1/10 of the maximum adsorption amount.

[0023]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. Example 1 [First step] Baker ion A as AC ion exchanger
Bx40 μm (trade name, manufactured by JT Baker, USA) was used, and 16 ml thereof was placed in a tube having an inner diameter of 1.5 cmφ and a height of 9 cm.
Filled. 10mM-KH ₂ PO ₄ a (pH5.5) 2m
After stabilizing by flowing at 1 / min, the ascites supernatant of a mouse inoculated and grown with a human chorionic gonadotropin β subunit (hCG-β) monoclonal antibody (IgG) -producing hybridoma was adjusted to pH 5.5, and 20 ml of the supernatant was 2 ml.
/ Min. Next, the eluate was eluted from 10 mM-KH ₂ PO ₄ (pH 5.5), which is a dilute eluent, to 500 mM-KH ₂ PO ₄ (pH 5.5), which is a concentrated eluent, in a linear gradient up to 50% to 2 ml in 60 minutes. / Min. FIG. 1 shows the state in which the effluent of the tower was traced by ultraviolet absorption (wavelength 280 nm). In FIG. 1, the abscissa indicates the liquid passing time, and the ordinate indicates the absorbance of ultraviolet rays (solid line) and the concentration gradient of the eluent (dotted line) (the notation in FIGS. 2 to 7 is the same as in FIG. 1). Is.). Preliminary experiments show that the hatched portion in FIG. 1 contains antibodies, and this outflow (a) is collected and the process proceeds to the second step. In addition, a small amount of the effluent (a) is used for purity inspection. According to FIG. 1, a large number of ultraviolet absorption peaks are observed in addition to the outflow (a), which indicates that contaminants are separated and removed.

[Second Step] As a hydroxyapatite, 18.5 ml of gigapite (trade name, manufactured by Toagosei Chemical Industry Co., Ltd.) was filled into a tube having an inner diameter of 1.5 cm and a height of 10.5 cm. 10mM-KH ₂ PO ₄ and (pH 5.5) 2 ml
/ Min at a flow rate of 2 ml / min, and then dilute 10 mM-KH ₂ PO ₄ (pH 5.5) from concentrated eluate 5
5 to 100 mM-KH ₂ PO ₄ (pH 7.0) with a linear gradient.
It shifted to 0% at 2 ml / min in 60 minutes. FIG. 2 shows the situation where the effluent of the column was traced by ultraviolet absorption (wavelength 280 nm). The hatched portion contains the antibody, and this effluent is used as (b), and the effluent (b) is used for the purity test in the same manner as the effluent (a) in the first step. According to FIG. 2, the effluent (b)
In addition, a slight ultraviolet absorption peak was observed, indicating that impurities other than the outflow (b) were separated and removed from the outflow (a).

[Purity Inspection] FIG. 8 shows 1% SDS addition 12.5%.
5 is a photograph showing a polyacrylamide gel electrophoresis image, lane M is a molecular weight marker, lane A is an antibody raw material solution, lane 1 is an effluent (a) obtained in the first step, and lane 2 is obtained in the second step. The outflow (b) is shown. Also, 5
The protein appearing around 0K is the heavy chain of antibody IgG, 28K
Proteins appearing nearby indicate light chains of the antibody IgG. As shown in FIG. 8, it is clear that the effluent (a) obtained from the antibody raw material through the first step has been subjected to removal of contaminants and further purified by the second step.

Example 2 [First Step] Similar to Example 1, the baked ABx
The antibody-containing liquid was supplied to a column filled with 40 μm. However,
The eluate was developed and eluted using 25 mM- [2-morpholinoethanesulfonic acid (pH 5.5)] as an equilibration solution and a dilute eluent, and 1 M sodium acetate (pH 7.0) as a concentrated eluent. The trace by ultraviolet absorption (wavelength 280 nm) is as shown in FIG. 3, and the hatched portion was collected and used as the outflow (c) as the raw material liquid in the second step.

[Second Step] The effluent (c) of the first step was received, and the same operation as in the second step of Example 1 was performed to obtain an effluent (d) indicated by the hatched portion in FIG. [Purity test] This is a photograph showing an electrophoresis image of 12.5% polyacrylamide gel with 1% SDS. Lane 3 is the outflow (c) obtained in the first step, and lane 4 is the outflow obtained in the second step. (D) is shown. As shown in FIG. 8, the selection of the eluent in the first step can improve the degree of purification and further purify the product obtained in the second step.

[Confirmation of Antibody Activity] An enzyme-labeled antigen-antibody reaction test (ELISA) was performed on human chorionic gonadotropin β subunit, which is the antigen of the antibody used in the examples. The detection limit of the amount of protein for each sample was 0.125 mg / ml for the antibody raw material solution, 0.063 mg / ml for the effluent (c) passed through the first step, and the effluent passed through the second step. 0.032 for minute (d)
mg / ml. Thus, the antibody obtained by the method of the present invention does not impair its activity,
Further, although not quantitative because of the accuracy of the ELISA method, an improvement in antibody purity is clearly recognized from the first step to the second step.

Example 3 The first step was carried out in the same manner as in Example 2. In the second step, 25 mM- [2-morpholinoethanesulfonic acid (pH 5.5)] was used as a dilute eluent. Elution was performed using 1M sodium acetate (pH 7.0) as an eluent. The elution state at this time is shown in FIG. 1% SDS addition for the outflow (e) in the hatched area in FIG.
The 2.5% polyacrylamide gel electrophoresis image is shown in lane 5 in FIG. As is clear from the spectrum of Lane 5, the outflow (e) was purified to an extremely high purity without any other contaminants.

Example 4 Mouse ascites containing the monoclonal antibody IgM derived from the mouse renal basement membrane was purified as in Example 1 as an antibody-containing solution. FIG. 6 shows the state of elution in the first step. The hatched portion in this figure is the fraction containing the antibody IgM, which was collected and used as outflow (f). FIG. 7 shows the state of elution in the second step. The hatched portion contains the antibody IgM, which is collected and effluent (g)
And FIG. 6 shows that contaminant components other than the antibody were removed, and FIG. 7 shows that other contaminant components were further separated and removed. It is clear that the purity of the antibody could be further increased. It is.

[0031]

The present invention essentially consists of only two steps, and is an extremely simple step as compared with the conventional method, and makes it possible to separate and purify antibodies with high purity at a high flow rate even under normal pressure. Since it can be separated and purified under mild conditions and in a few steps, the present invention makes it possible to obtain an antibody in a high yield and to maintain antibody activity. In particular, omitting salting-out in the preparation of antibodies from in vitro cell culture can lead to a reduction in consumption of materials required for separation and purification and wastewater treatment, which will promote future development on an industrial scale. It is expected to be. Further, the fact that the present invention has made it possible to purify antibody IgM means that the protein A-immobilized adsorbent, which has been regarded as an important means for purifying antibodies by the conventional method, can be used.
Since it was weak against M, it provided a new and powerful technology in this field.

[Brief description of the drawings]

FIG. 1 is a chromatogram showing the results of observation of the effluent of the first step in Example 1. The solid line indicates the absorbance of ultraviolet light having a wavelength of 280 nm, and the dotted line indicates the change in the concentration gradient of the eluent.

FIG. 2 is a chromatogram showing the results of observation of the effluent of the second step of Example 1, where the solid and dotted lines have the same meaning as in FIG.

FIG. 3 is a chromatogram showing the results of observation of the effluent of the first step of Example 2, wherein the solid line and the dotted line have the same meaning as in FIG.

FIG. 4 is a chromatogram showing the results of observation of the effluent from the second step in Example 2, wherein the solid line and the dotted line have the same meaning as in FIG.

FIG. 5 is a chromatogram showing the results of observation of the effluent of the second step in Example 3, where the solid and dotted lines have the same meaning as in FIG.

FIG. 6 is a chromatogram showing the results of observation of the effluent of the first step in Example 4, where the solid and dotted lines have the same meaning as in FIG.

FIG. 7 is a chromatogram showing the results of observation of the effluent of the second step in Example 4, where the solid and dotted lines have the same meaning as in FIG.

FIG. 8 is an SDS-polyacrylamide gel electrophoresis image, lane A is an image of the antibody-containing solution used in Examples 1, 2, and 3, and lanes 1, 2, 3, 4, and 5 are effluents, respectively. It is an image of (a), outflow (b), outflow (c), outflow (d), and outflow (e).

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C07K 1/14-1/22 C07K 16/00-16/42 G01N 33/531 BIOSIS (DIALOG)

Claims (2)

(57) [Claims] 1. A method for separating and purifying an antibody from an antibody-containing liquid by liquid chromatography, wherein the antibody-containing liquid is passed through a column packed with an ion exchanger having both an anion exchange group and a cation exchange group. Collecting an antibody-containing effluent, and then passing the antibody-containing effluent through a column filled with hydroxyapatite. 2. The method according to claim 1, wherein the hydroxyapatite is an agglomerate of hydroxyapatite in the shape of a hexagonal column or a needle, and has a pore volume of 1 to 5 ml / g. A method for separating and purifying antibodies.