JPH0459797A - Purification of antibody - Google Patents

Abstract

PURPOSE:To easily purify immunoglobulin M (IgM) in high purity and efficiency in a short time by subjecting an IgM-containing specimen to chromatography taking advantage of hydrophobic action. CONSTITUTION:An IgM-containing specimen such as ascites fluid of an animal transplanted with hybridoma, culture liquid of hybridoma and lymphocyte and animal serum is subjected to a chromatography taking advantage of mutual hydrophobic action to effect the adsorption of IgM and foreign proteins in a salt solution to a column and the salt concentration is slowly or quickly lowered to selectively separate IgM from the filler. The filler for chromatography is e.g. a hydrophilic polymer resin and a filler for gel-filtration chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for purifying immunoglobulin M (hereinafter referred to as IgM). More specifically, the present invention relates to a method for efficiently purifying a sample containing IgM by subjecting it to chromatography using hydrophobic action to efficiently, quickly and easily purify the substance with high purity.

(Prior art) IgM currently occupies an important position as a reagent for immunoassays in clinical tests, and on the other hand, IgM is a protein that is highly anticipated in the future as a missile therapeutic agent for cancer, viral infections, etc. It is. Thus, an advanced IgM purification method that can be clinically applied in many fields is particularly desired.

Several methods for purifying IgM have been known. A typical example among these is the gel filtration method, which uses the difference in molecular weight of proteins in a sample solution to separate them. Due to its principle, this method has a large molecular weight (different proteins can be separated very easily, but proteins with the same molecular weight cannot be separated).

The molecular weight of IgM is approximately 900,000, and it can be fractionated by gel filtration. However, large amounts of Ig
It is not possible to separate and purify M. This is because gel filtration has a limit to the amount of sample in one purification process.
Usually only a few milliliters can be processed at a time. In addition, it is not suitable for separation and purification in which tens of milligrams or more of protein is processed multiple times. In order to separate and purify larger amounts of proteins using gel filtration, more expensive and larger equipment and columns must be prepared.

In addition, purification methods using ion exchange chromatography are known. Specifically, a sample solution containing IgM is dialyzed against a buffer solution with a low salt concentration, and a cation exchange resin (resin having sulfopropyl (SP) or carboxymethyl (CM) groups) or anion exchange resin (diethylaminoethyl (DEAE)) is used. , quaternary aminoethyl (QA
In this method, the adsorbed IgM is selectively eluted and separated and purified by adsorbing it on a column of E) (resin having groups) and increasing the salt concentration gradually or to a certain degree. In this method, a large amount of IgM may irreversibly precipitate during dialysis against a buffer solution with a low salt concentration, resulting in a reduction in yield.

Furthermore, many contaminant proteins are also adsorbed onto the column, making it difficult to efficiently separate and purify them.

Furthermore, as a pretreatment for applying to the column, dialysis against a starting buffer is complicated and requires a long period of time.

On the other hand, proteins with affinity for IgM and
A purification method that takes advantage of the antigen specificity of M has also been devised. Antibodies (polyclonal antibodies, monoclonal antibodies) specific to the constant region of IgM or the antigen itself are immobilized on an insoluble support, and after specifically and reversibly adsorbing IgM, dioxane ( 10%), propionic acid (pH-2.5, IM), hydrochloric acid (pH-2.5, IM), etc.
-2,5), ethylene glycol (5), etc.
0%), chaotropic ion (1-, CF3 COO
-3CN-, CC13COO-pH-7.0~8.0°3M) etc., or by reducing the interaction of IgM with antibodies or antigens against it, or with urea (pH-7.0~8.0°3M), etc.
-7,0,8M), guanidine hydrochloride (pH-3,1,
IgM is released and eluted using a protein denaturing agent such as 6M). The optimal conditions for elution are determined by the binding strength of the antigen-antibody reaction and the stability of the antibody.
A decrease in antibody activity and yield is inevitable. Furthermore, the carrier deteriorates quickly, which is disadvantageous in terms of reproducibility and cost.

Naturally, it is disadvantageous even when stepping up to large-scale purification.

Furthermore, since IgM hardly adsorbs to relatively inexpensive combination protein A and protein G, which are currently widely used for purifying antibodies (IgG class), they cannot be used.

When trying to obtain high-purity IgM in large quantities, using the latter purification method has a large cost problem as described above, so the former method, ion exchange chromatography, is usually used for mass collection A non-specific purification method is used.

However, when performing the former purification method using ion exchange chromatography, it is necessary to perform a pretreatment of dialysis against a starting buffer solution with a low salt concentration and desalting. Therefore, the protein sample is exposed to a dilute solution, a condition that is extremely unstable for proteins. Depending on the sample, it may precipitate irreversibly.
The disadvantage may arise that the starting buffer for chromatography must be started at a high salt concentration. Furthermore, in that case, when elution is performed by applying a salt concentration gradient, it is often observed that the eluted peak becomes broad, and contaminating proteins may be forced to be mixed in. Concentration operations are difficult in culture medium in which IgM-producing hybridomas are cultured.

In particular, it is difficult to remove added pH indicators such as phenol red, and even simple and effective salting-out methods using ammonium sulfate and ultrafiltration membrane methods cannot sufficiently remove them. This has a great influence on the separation and purification of IgM. It has also been observed that pH indicators, once adsorbed to the filler, are difficult to elute and often accelerate the deterioration of the filler.

In the near future, it is expected that mass culturing methods for human IgM or its chimeric antibodies will be established, and IgM will be applied therapeutically, especially as a drug or drug carrier for neoplastic organisms such as cancer and virus-infected cells. However,
At that time, it is clear that refining methods will play a very important role in the manufacturing process, and the development of such methods is essential.

Recently, high-performance liquid chromatography (-(High-Performance Liquid
Purification is increasingly performed using idChromatography (hereinafter referred to as HPLC). The reason is,
This is because this method allows for very rapid and efficient purification in large quantities. Unfortunately, however, this equipment and the special filler itself are very expensive, and moreover, the method of controlling the equipment requires skill. Furthermore, when using such devices and instruments, a step is required to remove impurities such as insoluble matter and lipids from the sample in advance in order to prevent deterioration of the filler as much as possible. Specifically, pretreatments include centrifugation, salting out, dialysis, and ultrafiltration. Maintenance of the equipment itself is also essential. Considering many considerations, medium-performance liquid chromatography packings are generally superior to HPLC packings in terms of cost, handling, and maintenance of packing materials as consumable materials.

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for purifying IgM that is of great utility. Specifically, IgM and contaminant proteins are adsorbed onto a column in a salt solution using chromatography that utilizes hydrophobic interactions, and the salt concentration is gradually or all at once lowered to selectively separate and elute IgM from the packing material. By doing so, the present invention provides a method for purifying IgM efficiently and in large quantities with high purity and in a shorter time without pretreatment using simpler operations than conventional methods.

(Means for Solving the Problems) The present inventors have arrived at the present invention as a result of intensive studies regarding the above problems.

That is, the present invention uses a sample solution containing IgM and a salt to adsorb the target IgM in the salt solution using chromatography that utilizes hydrophobic interaction, and then selectively desorbs and separates the IgM by lowering the salt concentration. Ig characterized by elution
This is a method for purifying M.

The details will be explained below.

Examples of the IgM to be purified include ascites fluid from an animal transplanted with a hybridoma, a culture solution of hybridomas and lymphocytes, and animal serum. IgM may be a polyclonal antibody or a monoclonal antibody.

The IgM constant region is preferably derived from a mammal, such as human, mouse, rat, goat, cow, horse,
Examples include sheep.

The salt contained in the sample solution and its concentration may be used as normally used in hydrophobic chromatography. For example, ammonium sulfate, potassium phosphate, sodium phosphate, etc. are used.

Chromatography using hydrophobic interactions includes:
There is no particular limitation, and it depends on the hydrophobicity of the IgM sample, but it is preferable to use one with relatively weak hydrophobicity. For example, hydrophilic polymer resins may be used, such as TSK Gel Toyopearl HW-65 (manufactured by Tosoh ■). Moreover, a packing material used in gel filtration chromatography can also be used.

In the IgM purification procedure, first, a salt-containing buffer solution or salt is directly added to IgM. This may be directly adsorbed in chromatography using hydrophobic interaction, and then the salt concentration may be lowered to selectively desorb IgM, followed by separation and elution. The salt concentration may be reduced gradually or all at once. Adsorption/separation/elution can be performed by batch method, column method, stepwise method, gradient method, etc.

In particular, IgM in ascites can be purified very simply by simply subjecting the filter-cut material to chromatography directly, and no other pretreatment is required.

(Effects of the Invention) As is clear from the above explanation, according to the present invention, IgM can be purified in a shorter time with simpler operations than in the conventional method, efficiently and in large quantities. It is possible to do so. IgM is more hydrophobic than IgG and the like, so the method of the present invention, which is not suitable for IgG and the like, is also preferably used for purifying IgM.

(Example) Hereinafter, the conditions and the like will be described in detail by giving examples. However, the present invention is not limited to these examples.

[Example 1] Separation and purification of IgM from hybridoma culture supernatant (1) Monoclonal antibody against human myoglobin (
IgM) producing hybridoma 104 cells/
ml of DMEM (Dulbeceo's modifi) containing 10% (v/v) fetal bovine serum (Fe2)
'ied Eagle smedium; manufactured by Gibco) (hereinafter abbreviated as 10% PC8-DMEM), implanted in 100 ml, 37°C, 5% carbon dioxide gas concentration, 9
Culture was started under conditions of 5% humidity. Ten days after the start of culture, the medium was centrifuged at 1,000 rpm for 5 minutes at 25°C to obtain a medium from which hybridomas had been removed as a supernatant. The same operation was performed again to remove the precipitate, and then 13.2 g of ammonium sulfate was added to make the final ammonium sulfate concentration in the medium IM. This medium was supplemented with phosphate buffered saline containing 1M ammonium sulfate (0.85% NaCl).
TSKgel Toyopearl HW - equilibrated with 01% phosphate buffer, pH 7.2 (hereinafter PBS)
4 milliliters of 65 (Fine) (manufactured by Toso Corporation) was added. After standing for 2 hours at room temperature, the culture medium was pumped using a peristaltic pump using an Econo pump at a flow rate of 0.21/1n.
Pour into a column (manufactured by Bio-Rad) and add 100
The filler was washed with 1 part of 1M ammonium sulfate in PBS. Immediately after washing, IgM was selectively eluted in one step with PBS containing 0.7 M ammonium sulfate as an eluent. The eluate was collected in 2 ml portions into a fraction collector, and the absorbance of each solution was measured at a detection wavelength of 280 nm.

The results are shown in Figure 1. Under these conditions, the IgM peak appeared from the i-grain fraction after changing the eluent (first
Figure; Peak 1). This peak was separated into a purified 1 gM fraction. This completes the purification operation.

(2) The purified 1 gM fraction was subjected to 12% 5DS-polyacrylamide gel electrophoresis under mercabutoethanol reduction and gel filtration HPLC using TSKge I G40 (LO8WxL). Figure 3 shows the HPLC results in lane 2 (molecular weight marker in lane, for low molecules, manufactured by Pharmacia).As is clear from Figure 2.3, it was confirmed that the IgM was homogeneous. .

(3) Enzyme immunoassay of purified 1 gM antibody titer (E
nzyme Linked Iavuno 5orbe
Measurement by nt As5ay, ELISA) 0.1 M in each well of an untreated microtiter plate (96-well Nunc plate, manufactured by Intermed)
1 gg dissolved in sodium carbonate buffer (pH 9,6)
Add 50 μl of a solution of human myoglobin at 4
- Incubated overnight. Remove the solution from each well;
After washing three times with a solution containing 0.04% tween-20 in PBS (hereinafter referred to as PBS-T),
% bovine serum albumin (BSA) dissolved in PBS
-T solution (300 μm) was added to each well, subjected to blocking treatment at 4° C., and stored as it was. After returning the microtiter plate to room temperature and washing it with PBS-T solution,
50μ of purified 1gM sample diluted with PBS so that the UV absorbance at 80nm is 0.005 is added to each well.
m added. In this state, it was left at 37°C for 90 minutes.

Next, peroxidase-labeled goat anti-mouse IgM (H chain specific; manufactured by Tabo) antibody was diluted 5,000 times with PBS-T solution, and 50 μm of the antibody was added to each well.

After incubating at room temperature for 90 minutes, the solution was removed and washed three times with PBS-T solution. Besides, 0.3
mg/ml of 2.2-azinodi-(3-ethylbenzthiazoline sulfate)-diammonium salt (ABTS) and 0
．． Add 100 μl of a substrate solution consisting of 0.1 M citric acid buffer (pH 4.1) containing 0.1% hydrogen peroxide (H2O2) to each well, allow a color reaction at room temperature for 30 minutes, and then add 200 mM oxalic acid solution. The enzyme reaction was stopped by adding 100 μm of .

The absorption intensity of the above microtiter plate was measured for each well at a wavelength of 415 nm and a reference wavelength of 492 nm using an automatic microtiter plate reader (manufactured by Tosoh Corporation).
The results of measurement using MPR-A4 (trade name) are shown in FIG. As is clear from the figure, the affinity of the antibody for the antigen was maintained even after purification according to the present invention.

[Example 2] Separation and purification of IgM from mouse ascites (1) 106 cells of hybridoma producing monoclonal antibody (IgM) against human carcinoembryonic antigen (CEA)
The cells were transplanted into the abdominal cavity of Ba1b/C mice (male) at a concentration of 10 s/mouse, and 10 ml of the obtained ascites fluid was filtered through a 0.8 μm membrane filter. 1.4 mg of ammonium sulfate was added to the ascites fluid, and TSKgel was further equilibrated with PBS containing 1M ammonium sulfate.
) 10 ml of Yopal HW-65 (Fine) (manufactured by Tosoh Corporation) was added, and the mixture was allowed to stand at room temperature for 2 hours.

Next, this ascites fluid was poured into an Econo column (manufactured by Bio-Rad) using a peristaltic pump at a flow rate of 0.2 ml/min, and the packing material was further washed with PBS containing 1M ammonium sulfate 2001. Immediately after washing, the ammonium sulfate concentration was decreased over 863 hours with a linear gradient from ammonium sulfate concentration IM to 0.7 M PBS.
IgM was selectively eluted from the packing material. The flow rate of the eluent at this time was 0.4 ml/win, and the eluate was fractionated in 21 fractions into a fraction collector, and the absorbance of each solution was measured at a detection wavelength of 280 nm. The results are shown in Figure 5. The fraction from 80 minutes to 375 minutes of this peak was collected as a purified 1 gM fraction. This completes the purification operation.

(2) The purified 1 gM fractions were each subjected to 12% 5DS-polyacrylamide gel electrophoresis under mercaptoethanol reduction and gel filtration HPLC using TSKge IO20005WXL.

The electrophoresis results are shown in lane 3 of Figure 2, and the HPLC results are shown in Figure 6. In addition, the results of electrophoresis of the pass-through fraction by HPLC are shown in lane 5 of FIG. As is clear from Figure 2.6, it was confirmed that the IgM was homogeneous with a purity of 90% or more.

(3) Measurement of antibody titer of purified tJ11gM by El and ISA. O,
3 dissolved in 1M sodium carbonate buffer (pH 9,6)
, add 50 μm of a solution of CEA at czg/ml and incubate at 4°C.
-Incubated at night.

After removing the solution from each well and washing three times with PBS-T, 0.1% BSA was dissolved and 300 μm of 7': PBS-T solution was added to each well, and the well was subjected to blocking treatment at 4°C and left as is. saved. After returning the microtiter plate to room temperature and washing it with PBS-T solution,
50 μl of a purified 1 gM sample diluted with PBS so that the UV absorbance of m was 0.005 was added to each well. In this state, it was left at 37°C for 90 minutes. Next, peroxidase-labeled goat anti-mouse IgM (H chain specific; manufactured by Tabo) antibody was diluted 5000 times with PBS-T solution.
A 50 μm thick layer was added to each well.

After incubating at room temperature for 90 minutes, the solution was removed and washed three times with PBS-T solution. Besides, 0.3
mg/ml ABTS and 0.01% hydrogen peroxide (H2
O, 1M citrate buffer (pH 4,1
100 μm of a substrate solution consisting of ) was added to each well, and the color reaction was allowed to occur for 30 minutes at room temperature, and then 100 μm of a 200 mM oxalic acid solution was added to stop the enzyme reaction. For each well of the above microtiter plate, wavelength 415
nm, and absorbance intensity at a reference wavelength of 492 nm was measured using an automatic microtiter plate reader (manufactured by Tosoh Corporation, MPR-
The results of measurements on A4 (trade name) are shown in FIG. As is clear from the figure, the affinity of the antibody for the antigen was maintained even after purification according to the present invention.

[Example 3 Separation and purification of IgM from culture supernatant of human-human hybridoma (1) Monoclonal antibody against human adenocarcinoma (IgM
) was inoculated into 100 ml of 10% FC8=DMEM, and culture was started at 37° C., 5% carbon dioxide gas concentration, and 95% humidity. 14 days after the start of culture, the low medium was 1.0
The mixture was centrifuged at 00 rpm for 5 minutes at 25°C to obtain a medium from which hybridomas had been removed as a supernatant.

The same operation was performed again to remove the precipitate, and then 13.2 g of ammonium sulfate was added to make the final ammonium sulfate concentration in the medium IM. To this medium, 5 milliliters of TSKgel Toyopearl HW-65 (Fine) (manufactured by Tosoh Corporation) equilibrated with PBS containing 1M ammonium sulfate was added. After standing overnight at room temperature, the culture medium was transferred to an Econo column (manufactured by Bio-Rad) using a peristaltic pump at a flow rate of 0 and 251/1 inch.
The filler was washed with PBS containing 2001 1M ammonium sulfate. Immediately after washing, the ammonium sulfate concentration was decreased over 8 hours using a linear gradient of ammonium sulfate concentration from IM to OM. The eluate was collected in 2 ml portions into a fraction collector, and the absorbance of each solution was measured at a detection wavelength of 280 nm. The results are shown in FIG. Under these conditions, an IgM peak appeared in the 9th fraction. The 4th to 14th fractions were collected and used as purified IgM fractions. This completes the purification operation.

(2) The fractionated purified 1 gM was subjected to 12% 5DS-polyacrylamide gel electrophoresis under mercaptoethanol reduction and gel filtration HPLC using TSKgel G 4000 SW XL. The electrophoresis results are shown in lane 4 of Figure 2, and the HPLC results are shown in Figure 9. As is clear from Figure 2.9, it was confirmed that the IgM was uniform.

[Brief explanation of drawings]

Figures 1, 5 and 8 show Example 1.2, respectively.
．． 3 is a diagram showing the elution pattern of hydrophobic interaction chromatography in No. 3, in which the horizontal axis shows the fraction number and the vertical axis shows the absorbance at 280 nm and the ammonium sulfate concentration. Figure 2 shows 12% of purified IgM in Example 1.2.3.
The results of 5DS-PAGE are shown in lanes 2.3.4, respectively, lane 1 shows a molecular weight marker (for low molecules: manufactured by Pharmacia), and lane 5 shows the chromatographic fraction of Example 2. . FIG. 3, FIG. 6, and FIG. 9 show examples 1 and 2, respectively.
FIG. 3 shows a chart of purified 1 gM gel filtration HPLC in Example 3. The horizontal axis is elution time, and the vertical axis is absorbance at 280 nm. Figures 4 and 7 show 1 g of purified Example 1.2, respectively.
This shows the antibody titer against the antigen of M. The vertical axis is the absorbance at 415 nm during the color reaction, and the horizontal axis is the antigen concentration (ng
/ n11).

Claims (6)

[Claims] (1) Using a sample solution containing immunoglobulin M and a salt, use chromatography that utilizes hydrophobic interaction to adsorb the target immunoglobulin M in the salt solution, and then selectively select immunoglobulin M by lowering the salt concentration. 1. A method for purifying immunoglobulin M, which comprises separating and eluating immunoglobulin M. (2) The method according to claim 1, wherein the chromatography filler is a hydrophilic polymer resin. (3) The method according to claim 1, wherein the sample solution containing immunoglobulin M is ascites fluid of an animal to which the hybridoma has been transplanted, a culture solution of the hybridoma and lymphocytes, or serum of the animal. (4) The method according to claim 1, wherein the immunoglobulin M contained in the sample solution is a polyclonal antibody or a monoclonal antibody. (5) The method according to claim 1, wherein the constant region of immunoglobulin M contained in the sample solution is derived from a mammal. (6) The method according to claim 1, wherein the salt used is ammonium sulfate, sodium sulfate, potassium phosphate, or sodium phosphate.