JPH1059999A - Separation and purification of antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently separate and purify an antibody at low cost without impairing its physiological activities by bringing an antibody-contg. liquid into contact with a separatory agent consisting of a metal orthophosphate to effect adsorption of the antibody which, in turn, is eliminated following separating the separatory agent. SOLUTION: First, an antibody-contg. liquid is brought into contact with a separatory agent as porous particles each 0.5-150μm in average size consisting of an orthophosphoric acid salt of a metal such as aluminum, an alkaline earth metal, transition metal 21-30 in atomic number, transition metal 39-47 in atomic number or rare earth metal to effect adsorption of the antibody to the separatory agent. Subsequently, the separatory agent is separated from the liquid, and the antibody adsorbed to the separatory agent is then eliminated by the use of a solution adjusted to pH6-8, thus the antibody classified in a group of various immunoglobulins and hopeful of application to preventives and diagnostics for viral infectious diseases, cosmetics, etc., is isolated and purified efficiently, easily at low cost without impairing its physiological activities.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating and purifying an antibody.

[0002]

BACKGROUND ART Antibodies are expected to be applied to diagnostics, therapeutics, cosmetics, and the like for cancer and immunological diseases, but in order to be widely used in such applications, antibodies must be produced in large quantities at low cost. The development of technologies that can be produced is essential. Since antibodies are difficult to synthesize chemically, antibodies produced by living organisms must be used. Currently, methods of producing antibodies by living organisms include immunizing animals such as mice, rabbits and goats, extracting their blood, extracting and purifying antibodies from serum components, and antibodies produced in the blood of immunized birds A method of extracting and purifying an antibody from egg yolk by utilizing the phenomenon that is concentrated and transferred into egg yolk is generally used.

[0003] In order to purify immunoglobulin from animal ascites or blood, methods such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, and ammonium sulfate salting out are known.
Appropriate methods are used according to the characteristics of each purified product. For the separation and purification of IgG antibodies, group-specific affinity chromatography using protein A or protein G is often used. However, this method often requires strongly acidic elution conditions, and therefore the physiological There is a problem that the problem that the activity is impaired easily occurs.
Further, depending on the type of animal producing the antibody, even IgG may not be able to be separated and purified because it does not show affinity for protein A or protein G. Furthermore, protein A and protein G have no affinity for antibodies of other immunoglobulin classes such as IgA and IgM, and
There is a drawback in that the applicable range is narrow, for example, antibodies other than the G antibody cannot be separated and purified.

In recent years, a method has been put to practical use in which the yolk of birds such as chickens is used instead of using animal blood. In general, the isolation and purification of IgG antibodies derived from avian yolks (which are called IgMs (Immunoglobulin Yolk) to distinguish them from IgG antibodies in the blood) involve lipoproteins bound to lipids in the yolks and containing IgY. The method includes a pre-process of separating the liquid and a post-process of separating and purifying only IgY from the IgY-containing liquid. The technology used in the pre-process includes a method using a natural polysaccharide such as carrageenan or a secondary alcohol, There is a method of degreasing. As a technique used in the post-process, affinity chromatography has various problems as described above, and thus, a method combining ion exchange chromatography with salting out or desalting is generally used (for example, J . Fichtali et al, Journal
of Food Science, 58 (6), 1282-1285 (1993)). However, these methods used in the post-process require a plurality of processing steps and take a long time, and thus have many problems such as an increase in cost.

[0005] In order to widely use antibodies as diagnostics and therapeutics for immunological diseases and the like, it is necessary to mass-produce antibodies at low cost. There is a need for the development of technology that can solve such problems.

[0006]

SUMMARY OF THE INVENTION In view of the above-mentioned background art, an object of the present invention is to efficiently, simply and inexpensively separate antibodies of various immunoglobulin classes without impairing their physiological activities. An object of the present invention is to provide a separation and purification method that can be purified and a separation agent used in the separation and purification.

[0007]

Means for Solving the Problems The present inventors have previously reported that metal salts of pyrophosphate and metaphosphate are suitable as stationary phase materials for chromatographically separating and purifying substances such as proteins, enzymes and nucleic acids. (Japanese Patent Application No. 5-229703). In the course of further research, the use of a separating agent consisting of orthophosphate of a specific metal has surprisingly improved the use of IgG, Ig,
Antibodies of various immunoglobulin classes such as M, IgY,
It has been found that separation and purification can be carried out under mild conditions without impairing the physiological activity, and the above-mentioned problems can be solved all at once. Thus, the present invention has been completed.

That is, the present invention comprises contacting a liquid containing an antibody to be separated and purified, such as serum, ascites, and culture supernatant, and a contaminant component such as serum albumin with a separating agent, and adsorbing the target antibody to the separating agent. Then, after separating the liquid and the separating agent,
The present invention relates to a method for separating and purifying an antibody, wherein a specific metal orthophosphate is used as a separating agent when desorbing and recovering an antibody adsorbed on the separating agent.

The separation of antibodies in the separation and purification method of the present invention mainly differs from that of affinity chromatography which is based on the difference in biological affinity between the biological sample to be separated and the separating agent. It is presumed to be based on the electrostatic interaction between the separated biological sample and the metal orthophosphate. For this reason, there is a feature that the separating agent in the present separation and purification method can be applied regardless of the type of animal that produces the biological sample to be separated. Further, since a solution adjusted to a pH near neutrality can be used as a solution relating to the adsorption and desorption of the antibody, there is no possibility that the antibody is denatured by the separation and purification operation and the physiological activity is impaired. This is a particular advantage as a method for separating and purifying.

The metal of the orthophosphate according to the present invention is
Aluminum, transition from atomic number 21 to 30
Metals, transition metals with atomic numbers 39 to 47, rare earth gold
Metals selected from the genus can be suitably used, particularly preferred
Are aluminum, magnesium, strontium, titanium
Tan, manganese, iron, nickel, copper, zinc, yttrium
, Zirconium, lanthanum, cerium, etc. can be used
You. Examples of the orthophosphate include magnesium and the like.
Orthophosphates of divalent metals such as Mg_Three(PO_Four) _TwoAnd
Orthophosphate of a trivalent metal such as Al, for example, AlPO
_FourAnd orthophosphates of tetravalent metals such as Zr, for example, Z
r_Three(PO_Four)_FourAre suitable as the separating agent of the present invention.
It is. In addition, two or more of the above metals
Orthophosphates, such as divalent metals
(Mg_1/2Fe_1/2)_Three(PO_Four)_Two, (Mg_2/3
Mn_1/3)_Three(PO_Four)_Two, (Zn_2/3Mg_1/3)_Three(PO_Four)_TwoAnd 2
MgPO (PO_Four)_Two, Mg
Zr_Four(PO_Four)₆Orthophosphates such as
Separation by selecting the type and combination of metals
One can control the strength with which the agent retains the antibody.

In the separating agent according to the present invention, the metal salt of orthophosphoric acid functions as a carrier of antibody adsorption. For this purpose, the surface of the separating agent that comes into contact with the sample to be separated is made of a metal salt of orthophosphoric acid. is necessary. On the other hand, since the material inside the material that does not come into contact with the sample to be separated does not participate in the separation mechanism, as long as the surface of the material is only the above metal salt of orthophosphoric acid, the inside of the material is metal or Other materials such as glass and ceramics may be used. Further, from the above-mentioned point, all of the material surface and the inside of the material may be metal salts of orthophosphoric acid. The metal salt of orthophosphoric acid may be crystallographically amorphous or crystalline. In addition, since the porosity of the separating agent is not directly related to the separation mechanism by electrostatic interaction, it may be either a nonporous material or a porous material, but from a practical point of view, It is desirable that the amount of antibody that can be separated and purified by one operation be large in terms of cost. For this purpose, it is preferable to prepare a porous separating agent to increase the surface area, to increase the amount of antibody adsorbed per unit amount of the separating agent, and to have a specific surface area of at least 1 m ² / g or more. Is preferred. In order to increase the surface area, for example, a method of forming a porous agglomerate in which submicron fine particles are aggregated, and forming an aggregate in which the surface of the fine particles or the entire fine particles are made of a metal salt of orthophosphoric acid is used. Such a porous aggregate can be very suitably used as the separating agent material according to the present invention. In that case, in order to impart sufficient mechanical strength to use the aggregate as a separating agent material, as a means for firmly bonding fine particles to each other,
For example, a method of heating the aggregate at a firing temperature of 600 to 1000 ° C. to sinter the fine particles can be used.

The shape of the separating agent is preferably particles when used as a packing material for column chromatography, and in particular, spherical particles are preferable for uniform packing. Alternatively, a column having a pore diameter of 0.1 μm or more and a continuous three-dimensional network can be attached to the column. Also,
It is also possible to purify the antibody according to the present invention by a batch method as a method for separating and purifying the antibody, and in this case, the shape of the separating agent may be in the form of particles or a column, a disk, or a thick film in various shapes according to the implementation status. It can be used as an integral type.

When the separating agent is used in the form of particles, it is desirable to select an appropriate size for the particles depending on the use conditions. For example, when used as a packing material for an HPLC column, it is preferable to use particles having an average particle size of 0.5 to 15 μm, particularly preferably 3 to 10 μm in order to realize high-resolution chromatography. It is suitable. In addition, when used as a filler for an open column, it is preferable to use particles having an average particle diameter of about 30 to 150 μm in order to achieve easy passage and sufficient sample load, but depending on the use conditions. Is 150μ
It is also possible to use a separating agent having a large particle size of at least m, for example, by using a separating agent having a particle size of several mm, even if using a large column filled with a large amount of separating agent, high liquid permeation is possible. There is an advantage that the sex can be maintained. Further, regardless of the particle diameter of the separating agent, it is particularly preferable that the shape of the separating agent particles is spherical, in order to facilitate the packing into the column and to enhance the separation performance of the column.

The method for producing the metal salt of orthophosphoric acid is not particularly limited, and various known production methods can be used. For example, to prepare aluminum orthophosphate, a slurry is prepared by mixing and stirring an aqueous solution of aluminum nitrate and an aqueous solution of ammonium phosphate such that the atomic ratio of aluminum to phosphorus is 1: 1 and spray drying at 300 ° C. Then, by baking at 900 ° C., the separating agent of the present invention in which the whole particles are made of aluminum orthophosphate can be obtained.

It is also possible to produce a separating agent in which only the material surface of the separating agent is made of a metal salt of orthophosphoric acid. For example, for use as a material inside a separating agent material, 3
A ceramic porous body such as alumina having pores continuous in a three-dimensional network is prepared using a known technique, and the porous body is immersed in a slurry of a zinc orthophosphate composition, and the slurry is sufficiently filled in the pores of the porous body. After removing the excess slurry by pulling up and then drying and baking, a separating agent in which the surface of the separating agent is coated with zinc orthophosphate can be produced.

In any case, the solution used in the method for separating and purifying an antibody according to the present invention has a p-type which is mild to a biological sample.
Solutions ranging from H6 to 8 can be used. Solution p
Although it is possible to separate and purify the antibody according to the present invention even when H is 6 or less or 8 or more, generally under alkaline conditions, the adsorption of the biological sample to the separating agent becomes weak and the separation tends to be poor. On the other hand, if it is performed under acidic conditions, problems such as the possibility of impairing the biological activity of the biological sample are likely to occur, and it is exceptional compared with separation and purification conditions performed using a solution adjusted to a pH near neutrality. There is very little benefit. Therefore, it is preferable to carry out the separation and purification of the antibody according to the present invention under conditions of pH 6 to 8.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Separation and purification of antibodies using a metal salt of orthophosphoric acid according to the present invention as a separating agent can be carried out as follows.

In separating and purifying the target antibody from a solution containing the target antibody, such as serum, ascites, or culture supernatant,
When column chromatography is used, a separating agent composed of a metal salt of orthophosphoric acid prepared in a particulate form, particularly preferably in a spherical form, is packed in a column, and a sodium phosphate buffer or a potassium phosphate buffer adjusted to pH 6 to 8, Using a sodium chloride solution or a potassium chloride solution adjusted to pH 6 to 8 with sodium phosphate or potassium phosphate as an eluent, the antibody can be purified by a linear gradient method or a stepwise method of eluent concentration. When purifying by a batch method, for example, particles,
An orthophosphate prepared so as to be suitable for use conditions, such as an integral porous body, is placed in an appropriate container, and a solution containing the target antibody is poured into the container and gently stirred to allow the antibody to sufficiently adsorb to the separating agent. Thereafter, the solution is separated from the separating agent to which the antibody is adsorbed by means such as filtration or centrifugation, and then the salt concentration of the mild eluent adjusted to pH 6 to 8 is desorbed for desorption of the target antibody. Control to desorb the target antibody from the separating agent
Can be recovered.

[0019]

The present invention will be specifically described below with reference to examples of the present invention. However, the present invention is not limited to the following examples. Example 1 A slurry prepared by mixing and stirring an aqueous solution of aluminum nitrate and an aqueous solution of ammonium phosphate so as to have an aluminum orthophosphate composition was spray-dried at 200 ° C.
球状 60 μm spherical particles were obtained. The product is further added to 900
C. for 4 hours to obtain porous spherical particles of aluminum orthophosphate.

The immunoglobulin class G2a (IgG2
a) 10 m of rat ascites containing the monoclonal antibody of a)
was placed in a test tube, 4.0 g of aluminum orthophosphate spherical particles were added, and the mixture was shaken for 5 minutes to adsorb the IgG2a antibody to the aluminum orthophosphate separating agent. Then
Ascites was poured together with the separating agent adsorbing the antibody into a syringe equipped with a filter having a pore size of 10 μm at the bottom, and IgG2a
Aluminum orthophosphate to which the antibody was adsorbed was separated from ascites. Further, the pH was adjusted to 6.8 to remove contaminant components such as serum albumin adsorbed with the antibody.
20 ml of 0 mM sodium phosphate buffer was injected into the syringe, and finally 5 ml of a 400 mM potassium chloride solution was poured into the syringe, and the eluate flowing out from the bottom of the syringe was collected to obtain about 5 ml of a rat IgG2a antibody solution.
As a result of examining the purity of the IgG2a antibody solution by SDS-PAGE electrophoresis, only two bands derived from the H chain and L chain of the IgG2a antibody were detected. In addition, the recovery rate of the IgG2a antibody by the absorbance measurement method in this purification treatment was almost 100%. Thus, the monoclonal IgG2a antibody could be separated and purified from rat ascites with high purity by the batch method using aluminum orthophosphate. Example 2 A slurry prepared by mixing and stirring zirconium oxynitrate, magnesium nitrate and phosphoric acid so as to have a composition of MgZr ₄ (PO ₄ ) ₆ and further adding methanol was spray pyrolyzed at 700 ° C. The product was calcined at 800 ° C. for 4 hours and classified to obtain porous spherical particles of MgZr ₄ (PO ₄ ) ₆ of ₄ to 10 μm. The 4-10 μm spherical particles were further heat-treated at 800 ° C. for 6 hours, and then filled in a stainless steel tube (8 mmφ × 100 mm).

Using the column thus prepared,
The method of L. Kwan et al. [Journal of Food Science, 1537-1541] was carried out by the linear gradient method (flow rate 1 ml / min, concentration gradient: increased from 10 mM to 50 mM in 50 minutes) of the concentration of an aqueous sodium chloride solution (pH 6.5). (1991))
The IgY-containing liquid obtained by subjecting the egg yolk to a lipid-free treatment was chromatographed on the basis of the above. As a result, first, most of the protein components other than IgY were eluted without being retained on the column, and the sodium chloride concentration was 180 mM.
When the increase was reached, IgY was eluted, and when the concentration of sodium chloride was further increased, other protein components were eluted. The content of IgY in the IgY-containing liquid used in this example was 8% before purification, but became 99% pure after purification, and the recovery rate by the absorbance measurement method was almost 10%.
It was 0%. As described above, the antibody purification method according to the present invention was able to separate and purify IgY from the IgY-containing liquid with high purity without loss of IgY. Example 3 Magnesium nitrate, zinc nitrate and an aqueous solution of ammonium phosphate were prepared so as to obtain an orthophosphate composition in which two metals of magnesium and zinc were combined (mixed in an atomic ratio of 2: 1). A three-dimensionally continuous disk-shaped alumina ceramic foam (15 mmφ × 10 mm) having openings of 500 μm is immersed in the slurry, the alumina foam is pulled up to remove excess slurry, dried, and then dried. At 800 ° C. for 6 hours to obtain a porous magnesium-zinc composite orthophosphate in which the orthophosphate covered the surface of the alumina foam. Next, a syringe was prepared in which the disc-shaped orthophosphate was attached to one end of a plastic tube having an inner diameter of 15 mm x 100 mm.

10 ml of 10-fold diluted mouse serum was injected into the above-mentioned syringe equipped with orthophosphate, and the antibody in the serum was adsorbed to the orthophosphate. Then the pH
10 mM sodium phosphate buffer 4 adjusted to 6.8
0 ml was poured into the syringe to remove contaminant components such as serum albumin adsorbed together with the antibody. Then, 10 ml of a 500 mM sodium chloride solution adjusted to pH 6.8 with sodium phosphate was poured into the syringe, and the mixture was discharged from the lower part of the syringe. The eluate was collected to obtain about 10 ml of a solution containing a mouse IgG antibody. Finally, 10 ml of sodium phosphate buffer adjusted to pH 6.8 was injected, and the eluate flowing out from the lower part of the syringe was collected.
About 10 ml of a solution containing was obtained. Example 4 Lanthanum orthophosphate, cerium orthophosphate, lanthanum nitrate so as to have a composition of yttrium orthophosphate,
A slurry prepared using an aqueous solution of cerium nitrate, yttrium nitrate, and ammonium phosphate was spray-dried in the same manner as in Example 1 to produce spherical particles of 80 to 120 μm,
Subsequently, heat treatment was performed at 900 ° C. to obtain porous orthophosphates of three kinds of metals. The three types of orthophosphate spherical particles (2 g) were separately packed in three BIO-RAD econo columns (inner diameter: 15 mm) to prepare three types of separation columns.

Using the three types of columns, Ig from rat serum diluted 10-fold by the stepwise elution method was used.
G and IgM were separated and purified. 10m for each column
l of rat serum was injected, and the antibody in the serum was adsorbed to the separating agent. Next, 40 ml of a 10 mM sodium phosphate buffer adjusted to pH 6.8 with sodium phosphate was injected to remove contaminants such as serum albumin adsorbed together with the antibody. Next, in order to separate and collect the IgG antibody, 10 ml of a 500 mM sodium chloride eluate adjusted to pH 6.8 with sodium phosphate was injected into the column, and the eluate flowing out from the lower part of the column was collected. About 10 ml of a solution containing was obtained. Finally, 10 ml of 300 mM sodium phosphate buffer adjusted to pH 6.8 with sodium phosphate is injected into the column, and the eluate flowing out from the lower part of the column is collected to obtain about 10 ml of a solution containing rat IgM antibody. Was.

[0024]

As described above, the method for separating and purifying an antibody according to the present invention using a metal salt of orthophosphoric acid as a separating agent is expected to be applied to preventive agents, diagnostic agents, cosmetics and the like for viral infectious diseases. Since antibodies of various immunoglobulin classes can be efficiently, easily and inexpensively separated, it is effective as a method for industrial mass production. Furthermore, since the method of the present invention can use a solution adjusted to a pH near neutrality as a solution for absorbing and desorbing the antibody to the separating agent, the separation and purification conditions are extremely mild for biological samples, and the resulting antibody It exerts an excellent effect that there is no fear of impairing the physiological activity of the protein.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication location C07K 16/06 C07K 16/06 C12P 21/08 C12P 21/08 // A61K 35/16 A61K 35 / 16 (72) Inventor Junji Takeishi 1-7-1 Inari, Soka City, Saitama Prefecture Kanto Chemical Co., Ltd., Central Research Laboratory (72) Inventor Kazunori Yoshino 1-7-1, Inari, Soka City, Saitama Prefecture Kanto Chemical Co., Ltd. Inside

Claims (11)

[Claims] 1. A method comprising the steps of: contacting a liquid containing an antibody with a separating agent to adsorb the antibody to the separating agent; separating the separating agent from the liquid; and separating the antibody from the separating agent. A method for separating and purifying an antibody, comprising using a separating agent comprising a metal orthophosphate. 2. The metal of the orthophosphate is a metal selected from aluminum, alkaline earth metals, transition metals with atomic numbers 21 to 30, transition metals with atomic numbers 39 to 47, and rare earth metals. A method for separating and purifying an antibody according to claim 1. 3. The method of claim 1, wherein the metal of the orthophosphate is a metal selected from magnesium, calcium, strontium, titanium, manganese, iron, nickel, copper, zinc, yttrium, zirconium, lanthanum, and cerium. The method for separating and purifying an antibody according to the above. 4. The method for separating and purifying an antibody according to claim 1, wherein the separating agent is a porous material. 5. The method for separating and purifying an antibody according to claim 4, wherein the porous body is a particle. 6. The particles having an average particle size of 0.5 to 150.
The method for separating and purifying an antibody according to claim 5, wherein the particle diameter is μm. 7. The method for separating and purifying an antibody according to claim 5, wherein the particles are spherical. 8. The method for separating and purifying an antibody according to claim 4, wherein the porous body is a thin-film or plate-like molded product. 9. The method according to claim 1, wherein the adsorption of the antibody to the separating agent and the desorption of the antibody from the separating agent use a solution adjusted to a pH in the range of 6 to 8. The method for separating and purifying an antibody according to any one of the above. 10. The method according to claim 1, wherein the separating agent is used as a packing material for column chromatography.
A method for separating and purifying an antibody according to any one of claims 1 to 9. 11. The method for separating and purifying an antibody according to claim 1, wherein the separating agent is used in a batch method.