JPH0892298A - Method for purifying mouse igg1 - Google Patents

Abstract

PURPOSE: To purify a mouse IgG1 for diagnosing diseases and pregnancy by culturing a mouse IgG1 producing cell in a bovine serum containing medium, removing bovine IgG by a protein A column, adding a buffer solution to the filtrate and passing the filtrate through the protein A column. CONSTITUTION: A mouse IgG1 producing cell is cultured in a medium containing a bovine serum component to give a supernatant liquid of serum culture, which is used as a solution to be treated. The solution to be treated is passed through a first protein A column, bovine IgG in the solution to be treated is selectively adsorbed and removed under conditions of a low salt concentration/neutral pH. The filtrate having passed through the column is mixed with a buffer solution having a high salt concentration and a high pH. Then the mixed solution is fed to a second protein A column which is equilibrated with a buffer solution having a high salt concentration and a high pH. Mouse IgG1 in the mixed solution is selectively adsorbed and the adsorbed mouse IgG1 is eluted and recovered with an acidic solution to efficiently purify the objective mouse IgG1 useful for diagnosing diseases such as cancers and pregnancy.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying mouse IgG1 using a serum culture supernatant as a stock solution for production.

[0002]

2. Description of the Related Art Monoclonal antibodies, which are one of useful proteins, are used for diagnosis of cancer, pregnancy and the like, and are also used in large quantities for separation and purification and analysis of various physiologically active substances. There are various animal species of antibodies, but mouse IgG is often used for diagnostic purposes. There are four subclasses of mouse IgG, namely mouse IgG1, IgG2a, IgG2b, and IgG3, but mouse IgG1 has the largest abundance ratio, and mouse IgG1 is the mainstream commercialized monoclonal antibody.

Stock solutions of antibody production are roughly classified into two types, mouse ascites fluid and cell culture supernatant, depending on the production method. In the antibody purification from the cell culture supernatant, a crude fraction which also serves as concentration by ammonium sulfate precipitation is used. After that, purification is performed by various chromatography such as ion exchange. In this case, since it is required to purify the antibody with high purity, protein A
In recent years, affinity chromatography in which a ligand having a specific affinity for an antibody such as an antigen or an antigen is immobilized has been widely used.

In this affinity chromatography, the target substance to be purified is specifically adsorbed on a ligand (a substance having an affinity for the target substance) immobilized on an insoluble carrier, and then recovered and purified. The most commonly used ligand for antibody purification is protein A described above. Protein A has the property of specifically binding to the Fc region of IgG derived from various animals, and is used for IgG purification from serum, cell culture supernatant, ascites, and the like.

For the culture of antibody-producing hybridomas, a medium obtained by adding fetal calf serum (FCS) to the basal medium is frequently used. This is because FCS has a cell growth promoting action and increases the antibody production accompanying it. However, FCS contains a large amount of bovine IgG,
Depending on the lot, bovine IgG may be contained in an amount equal to or more than the amount of antibody produced by the hybridoma.

[0006] Therefore, mouse Ig1-producing cell culture supernatants cultured in a medium containing these bovine serum components are used to prepare mouse Ig.
When G1 is to be purified, it is purified by the affinity operation of adsorption, washing and elution using affinity chromatography using protein A as a ligand, and bovine IgG in serum is mixed in a large amount. There was a problem that the useful mouse IgG1 specific activity in the purified product was lowered.

[0007] Both mouse IgG1 and bovine IgG bind to the protein A gel, and bovine IgG is more soluble in mouse Ig.
Since it has stronger binding force than G1 and contains a high concentration,
The adsorption capacity of mouse IgG1 was disturbed by bovine IgG and varied, and the yield of mouse IgG1 was affected, and purification results with good reproducibility could not be obtained. That is, the amount of bovine IgG mixed in the purified product was not constant, which caused variations in the specific activity of the antibody.

Recently, a serum-free medium containing no serum has been developed, but many cells that do not grow in this medium are not used very often. As a medium between serum medium and serum-free medium, Git medium (manufactured by Nippon Pharmaceutical Co., Ltd.), which has extracted growth factors from bovine serum, is commercially available. In this Git medium, bovine IgG is about 0.2 mg. Since it was contained at a high concentration of / ml, there was a similar problem.

[0009]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a method for purifying mouse IgG1 capable of efficiently purifying mouse IgG1 from a culture supernatant containing bovine IgG. It was done by.

[0010]

Means for Solving the Problems The present invention made to solve the above problems is to use a serum culture supernatant obtained by culturing mouse IgG1-producing cells in a medium containing a bovine serum component as a liquid to be treated. , This treated liquid is passed through the first protein A column, and bovine in the treated liquid under low salt concentration and neutral pH conditions.
The first step of selectively adsorbing IgG and removing it, the second step of mixing the column passage solution with a high salt concentration / high pH buffer solution, and this mixture solution in advance with a high salt concentration / high pH buffer solution The third step of supplying mouse IgG1 in the mixed solution selectively to the second protein A column equilibrated with 1. and washing the inside of the column with the above buffer solution, followed by elution and recovery with an acidic solution It is characterized by comprising four steps.

As described above, in the present invention, the serum culture supernatant (low salt concentration / neutral pH) is first added to PBS which is an ordinary adsorption buffer.
The solution is passed through a first Protein A column that has been pre-equilibrated with (pH 7.4) or the like to selectively bind and remove only bovine IgG. This is because the binding between mouse IgG1 and protein A is PB.
It does not occur under the condition of low salt concentration and neutral pH of S, high salt concentration,
It utilizes the fact that it occurs in a high pH buffer solution. The volume of the first protein A column is determined by measuring the adsorption capacity per 1 ml of gel up to the point of breakthrough of bovine IgG.

The solution containing mouse IgG1 which has passed through the first protein A column in this manner is treated with a high salt concentration / high pH.
It is mixed with the above buffer solution and supplied to a second protein A column which has been equilibrated with a high salt concentration / high pH buffer solution in advance. This second
In the Protein A column, the mouse IgG1 is selectively adsorbed without being affected by bovine IgG. As a result, a highly reproducible purification result with a high mouse IgG1 content in the purified product can be obtained. Mouse Ig with high yield
For recovery of G1, the volume of the second Protein A column shall be determined by measuring the 10% breakthrough adsorption capacity of mouse IgG1.

As a support for immobilizing a ligand, a natural polysaccharide such as agarose has been conventionally used. However, since the gel matrix of these fillers is soft, when the liquid is passed at a high speed, the particles of the filler are deformed by the passing pressure and the separation efficiency is significantly lowered. When the cell culture supernatant (liquid to be treated) is directly passed through the affinity chromatography column for use, it is necessary to remove all substances that are normally adsorbed and prepare for the next separation step. In the present invention, in addition to shortening the time of the adsorption step of the substance to be separated, in order to shorten the time of the washing and regeneration steps, a polymer or silica gel with a large pressure resistance capable of passing at a high flow rate is used as a material. It is preferable to use the above-mentioned packing material for high performance affinity chromatography.

As the adsorbing solution for binding mouse IgG1 to protein A, it is preferable to use a buffer solution containing sodium chloride and ammonium sulfate at high salt concentration and high pH. Here, high salt concentration and high pH mean that the salt concentration is 1 to 4 M, pH
Means 8.5 to 9.5. If this condition is not satisfied, mouse IgG1 will not bind to protein A. The buffer for binding mouse IgG1 may be, for example, 1.5
M glycine, 3M NaCl (pH 8.9) and 1M (NH ₄ ) ₂ SO ₄ (pH
9.0) is used.

FIG. 1 is an explanatory view of the above-mentioned 10% breakthrough adsorption capacity. When the raw material liquid containing the target substance is supplied to the column packed with the affinity packing, the adsorbent is sequentially saturated from the vicinity of the column inlet, and the part where adsorption is advancing in the column (adsorption zone) reaches the lower end of the column. Then, as shown in FIG. 1, the target substance begins to appear in the effluent. In the breakthrough curve showing this situation, the point at which the concentration of the effluent reaches 10% of the inlet concentration is the 10% breakthrough point. The adsorption capacity up to the 10% breakthrough point is defined as the effective adsorption capacity, and by measuring this value by the ultraviolet absorption value at OD280nm or the ELISA method, the sample loading amount per cycle can be determined.

[0016]

According to the present invention, the fact that the binding between mouse IgG1 and protein A is carried out only under the conditions of high salt concentration and high pH is utilized in the first protein A column under low salt concentration and neutral pH conditions. The bovine IgG in the liquid to be treated is selectively adsorbed and removed below, and the column passage liquid is supplied to the second protein A column to select mouse IgG1 in the mixed liquid under high salt concentration and high pH conditions. To be absorbed. Therefore, mouse IgG1 can be efficiently purified without being affected by bovine IgG contained in the liquid to be treated.

[0017]

EXAMPLES Examples of the present invention will be shown below. Bovine serum (FCS)
Bovine IgG, mouse IgG1 in cell culture supernatant containing 5% of
When the concentration was measured by ELISA, it was 0.12 mg each.
-Bovine IgG / ml, 0.032 mg-Mouse IgG1 / ml. In the first protein A column 1 shown in FIG. 2, a protein A was used as a carrier for perfusion chromatography made of a copolymer of polystyrene and divinylbenzene.
Was loaded with Protein A gel to which was bound.

In this protein A gel, bovine IgG was used.
As a result of measuring the adsorption capacity up to the point of breakthrough, it was 15 mg / ml gel at a flow rate of 60 column volume / hr. Therefore, the amount of protein gel for treating 3 liters of serum culture supernatant (360 mg of bovine IgG) was 24 ml, and the first protein A
The volume of column 1 was 24 ml. The second protein A
For column 2, for protein A gel mouse IgG1
Since the 10% breakthrough adsorption capacity was 10 mg / ml-gel, the capacity was set to 10 ml.

This first protein A column 1 was previously
After equilibration with S buffer (pH 7.4), the serum culture supernatant filtered through a 0.45 μm filter was supplied at a high flow rate of 60 column volume / hr. This allows bovine IgG in serum culture supernatant
Are selectively adsorbed and removed (first step). Second
Protein A column 2 was equilibrated with 1.5 M glycine (pH 8.9) solution containing 3 M NaCl (second step), and 1.5 M glycine was added to the culture supernatant passing through the first protein A column 1.
3M NaCl (pH 8.9) was mixed at a ratio of 1: 2 and continuously supplied to the second protein A column 2 (third step). After the supply, it was confirmed by UV detector 3 that the UV absorption at 280 nm had dropped to the baseline, and then 0.1 M citric acid (pH 6.0) was added to the mouse from the second protein A column 2
IgG1 was eluted and collected (4th step).

The content of bovine IgG in the obtained purified product was 4
%, The mouse IgG1 content was 92%. On the other hand, when using the conventional method, the bovine IgG content in the purified product was 62%,
The mouse IgG1 content was 33%, and it was confirmed that the present invention can suppress the contamination of bovine IgG in the purified product to an extremely low level. Moreover, the specific activity of mouse IgG1 obtained by the present invention was measured by an ELISA method,
It was confirmed to be twice as expensive.

As described above, in this example, the polymer-based affinity filler for perfusion chromatography was used. This packing material has a flow rate (column volume / hr)
Linear velocity (flow rate per unit column cross-sectional area)
There is an advantage that scale-up can be easily performed by increasing the column capacity with the 10% breakthrough adsorption capacity as the standard without being affected by. In contrast, the conventional agarose carrier has a maximum linear velocity of 1 cm / min from the viewpoint of mechanical strength, and this maximum operating linear velocity decreases with scale-up, so scale-up is not easy.

[0022]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, there is an advantage that mouse IgG1 can be efficiently and reproducibly purified from a culture supernatant containing bovine IgG, and further, it can be used on an industrial scale. Its value is enormous because it is easy to scale up.

[Brief description of drawings]

FIG. 1 is a graph illustrating a 10% breakthrough adsorption capacity.

FIG. 2 is a block diagram of an apparatus used in Examples.

[Explanation of symbols]

1 1st protein A column, 2 2nd protein A column, 3 UV detector

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takeshi Majima 2-56, Sudacho, Mizuho-ku, Nagoya-shi, Aichi Prefecture

Claims (1)

[Claims] 1. Mouse IgG1 in a medium containing bovine serum components
Serum culture supernatant obtained by culturing producer cells is used as a liquid to be treated, and the liquid to be treated is passed through the first protein A column, and bovine in the liquid to be treated under low salt concentration and neutral pH conditions. The first step of selectively adsorbing IgG and removing it, the second step of mixing the column passage solution with a high salt concentration / high pH buffer solution, and this mixture solution in advance with a high salt concentration / high pH buffer solution Second equilibrated with
Mouse IgG1 in the mixture supplied to the Protein A column of
A method for purifying mouse IgG1, which comprises a third step of selectively adsorbing C., and a fourth step of washing the inside of the column with the above buffer solution, followed by elution and recovery with an acidic solution.