JP2945181B2 - Method for purifying immunoglobulin M - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for purifying immunoglobulin M by chromatography capable of efficiently purifying immunoglobulin M at high speed and high purity.

[0002]

2. Description of the Related Art Various methods have been developed to purify useful antibodies from a sample. Among these methods, immunoglobulin M is purified using a gel using a soft gel such as agarose or acrylamide. Filtration methods are known. However, in the case of this method, there is a problem that the liquid to be treated must be flowed at a low speed because the gel strength is significantly reduced during the treatment, and the purification treatment requires a long time. Furthermore, in order to purify high-purity immunoglobulin M, it is necessary to combine an ion exchange treatment step and at least two steps of gel filtration treatment steps for coarse / fine purification,
There is a problem that the treatment is complicated and inefficient, and the recovery rate is significantly reduced. Therefore, Japanese Patent Application Laid-Open
As disclosed in JP-180900, a method for efficiently purifying immunoglobulin M by high performance liquid gel filtration chromatography based on hydrophilic silica gel has been proposed, but the purification rate is reduced due to gel filtration treatment. There was a problem that the purification process was slow and did not have a specific adsorption action, and high-purity and efficient purification could not be performed.

[0003]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, and allows a liquid to be treated to flow at a flow rate several times higher than that of a conventional liquid, thereby achieving high-speed and large-scale purification. And a method for purifying immunoglobulin M that can specifically and surely adsorb and separate only immunoglobulin M from the liquid to be treated and can be efficiently purified at an extremely high recovery rate. It was completed for the purpose.

[0004]

Means for Solving the Problems A method for purifying immunoglobulin M of the present invention, which has been made to solve the above problems, comprises:
In the method for purifying immunoglobulin M by chromatography, after removing nucleic acids in the liquid to be treated, purifying by affinity chromatography using protamine as a stationary phase on a high-speed affinity carrier based on silica gel or a porous polymer. It is characterized by the following.

Hereinafter, the present invention will be described in detail with reference to the drawings. The drawings are schematic diagrams showing the principle of affinity chromatography. In the figure, reference numeral 1 denotes affinity chromatography, reference numeral 2 denotes a column constituting the affinity chromatography 1, for example, a column made of stainless steel or plastics, and reference numeral 3 denotes an inside of the column 2. The high-speed affinity carrier 4 made of filled porous silica gel or high-molecular polymer is protamine for adsorbing immunoglobulin M formed on the surface of the high-speed affinity carrier as a stationary phase.

In the present invention, for example, a cell culture supernatant containing immunoglobulin M at a low concentration is used as a liquid to be treated, and the liquid to be treated is first treated with a nucleic acid removing agent as a step prior to being subjected to chromatography. The nucleic acid in the liquid released from the dead cells by the addition or the treatment by the addition of the ion exchange gel is removed. This nucleic acid removal treatment is for improving the adsorbing effect of immunoglobulin M by protamine, which will be described later. When the nucleic acid and immunoglobulin M are mixed in the liquid to be treated in the chromatographic treatment, protamine is removed. The present invention is based on the finding of the present inventor that any of these is adsorbed and the nucleic acid having a strong adsorbing power is replaced during the treatment with the immunoglobulin M once adsorbed, resulting in a decrease in the recovery rate. In addition, if this nucleic acid is mixed, the antigen-binding activity of immunoglobulin M is reduced, so that it is removed in the step of ion exchange chromatography in a method other than the present invention.

Next, the liquid from which the nucleic acid has been removed is subjected to a purification treatment by affinity chromatography.
This step will be described with reference to FIG. 1. In the liquid to be treated introduced into the affinity chromatography 1, only immunoglobulin M is selectively adsorbed by protamine 4,
The remaining liquid will be discharged from the lower part. In this case, since the nucleic acid has already been removed from the liquid to be treated in the previous step, protamine 4 reliably and efficiently adsorbs only immunoglobulin M, and the above-mentioned protamine 4 is adsorbed on the surface of the high-speed affinity carrier 3. Since it is formed as a stationary phase in the layer, even if the liquid to be treated is flowed at a high speed and in a large amount, the immobilization of the immunoglobulin M can be surely performed without deterioration of the protamine 4 stationary phase.
In addition, at this time, when an inert inorganic salt such as sodium chloride is added to the liquid to be treated, even if a large amount of contaminating proteins such as albumin is contained in the liquid to be treated, protein By reducing the ion-ion interaction force, specific adsorption of only immunoglobulin M to protamine 4 can be performed, and more efficient treatment can be performed. The concentration of the above-mentioned inert inorganic salt is preferably at least 0.2 mol or more in order to exert a sufficient effect.

Next, in order to dissociate the immunoglobulin M adsorbed on the protamine 4, the inside of the column 2 is washed with a washing solution, and the eluate is put into the column. to recover. In this case, the eluate has a pH of 7.2 to 7.2.
When a dioxane or ethylene glycol solution of 8.0 is used, it is possible to effectively prevent aggregation and precipitation of immunoglobulin M and to efficiently recover the immunoglobulin M. Thereafter, the collected immunoglobulin M is subjected to weight filtration treatment by gel filtration chromatography or the like, and high-purity immunoglobulin M is isolated to complete the purification treatment.

[0009]

Example 1 A nucleic acid removing agent was added to a serum-free culture of a human monoclonal antibody-producing strain, stirred for 15 minutes, left on an ice bath for 10 minutes, and then centrifuged at 3000 rpm for 10 minutes to remove the nucleic acid. And the precipitate was removed. The obtained liquid to be treated is washed with a phosphate-buffered saline (PBS) at pH 7.6, and then subjected to affinity chromatography (trade name “HiPAC”: Chromatochem, USA), which has been equilibrated, for 24 hours.
Inject at a linear flow rate of cm / min and wash with PBS.
Elution of immunoglobulin M was performed with a 10% dioxane solution of 7.6. The amount of immunoglobulin M antibody in the purified eluate was measured by enzyme-linked immunosorbent assay (ELISA). As a result, the recovery was 82%, and the final recovery obtained by further performing gel filtration chromatography. Was 74%, which was more than twice as high as the conventional one. In addition, as a result of performing SDS-polyacrylamide gel electrophoresis on the recovered immunoglobulin M, it was confirmed that only a single band of immunoglobulin M was detected and that it was of high purity.
Further, the time required for the purification treatment was significantly reduced to one day as compared with the conventional one week.

Example 2 Culture of Mouse Monoclonal Antibody Producing Strain
Nucleic acid in supernatant is removed by ion adsorption on ion exchange gel
did. This mouse immunoglobulin M was concentrated at 30 μg / ml.
500 ml of culture supernatant containing immobilized protamine
At a flow rate of 382 cm / hr directly to a high-speed affinity column
Loaded. After loading the culture supernatant, mouse-type immunoglobulin
Add 0.2M sodium chloride to proteins other than M
20 mM phosphate buffer (pH 6.8) at 1084 cm / hr
The solution was washed away by passing the solution at the same flow rate. Column
Mouse immunoglobulin selectively adsorbed on rotamine
M is 77 mM phosphoric acid-67 mM citrate buffer (p
H4) to elute at a flow rate of 361 cm / hr. Eluate is 1 mole
Immediately return to neutral with Tris-Hcl (pH 8), and then
The mouse immunoglobulin M concentration was measured by a standard method. Get
Mouse immunoglobulin M fraction
12.9mg der Te is, the recovery rate was 86%. In addition, the purity of the mouse immunoglobulin M was confirmed by SDS-polyacrylamide gel electrophoresis and gel three-pass method. As a result, the purity was 95% or more. Furthermore, the time required for the purification treatment was 3 hours, and a purified product concentrated to a high concentration of the order of mg / ml was obtained in a short time.

[0010]

As is apparent from the above description, the present invention allows the liquid to be treated to flow at a flow rate several times higher than that of the conventional liquid, and can perform the purification treatment at a high speed and in a large amount. It can specifically and surely adsorb and separate only immunoglobulin M from the liquid to be treated, and can efficiently purify it with an extremely high recovery rate. Further, since the purification treatment time is short, the activity is high. It also has the advantage that immunoglobulin M can be obtained. Therefore, the present invention provides a method for purifying immunoglobulin M that has eliminated the conventional problems.
The contribution to industrial development is extremely large.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the principle of affinity chromatography.

FIG. 2 Culture supernatant of mouse monoclonal antibody producing strain
From the mouse using a protamine-immobilized affinity column.
FIG. 5 is a graph showing a process of purifying S-type immunoglobulin M;
is there.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Affinity chromatography 2 Column 3 High-speed affinity carrier 4 Protamine

Continued on the front page (58) Fields surveyed (Int. Cl. ⁶ , DB name) C07K 16/00 C07K 1/22 C12P 21/08 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. In a method for purifying immunoglobulin M by chromatography, after removing nucleic acids in a liquid to be treated, affinity chromatography using protamine as a stationary phase on a high-speed affinity carrier based on silica gel or a porous polymer. A method for purifying immunoglobulin M, which comprises purifying with immunoglobulin M. 2. The method for purifying immunoglobulin M according to claim 1, wherein an inert inorganic salt is added to the liquid from which the nucleic acid has been removed, and a high-speed affinity carrier based on silica gel is used. 3. Immunoglobulin M adsorbed on protamine
3. The method for purifying immunoglobulin M according to claim 1 or 2, wherein dissociation is carried out with a dioxane or ethylene glycol solution.