JPH04634B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying an antigenic substance corresponding to an enzyme-treated antibody using affinity chromatography using the antibody as a ligand. More specifically, the present invention uses the mechanisms of antigens and antibodies to enzymatically treat immune antibodies specific to the antigen substance to be purified, thereby producing modified purified antibodies with extremely high specificity. , relates to a method for producing an antigenic substance from a crude antigenic substance with high purity and high recovery rate by immobilizing it on an insoluble carrier and using this immobilized carrier with high affinity.

For example, urokinase is a fibrinolytic enzyme that activates plasminogen present in the blood into plasmin. Therefore, by injecting this into the human body, it dissolves blood clots that occur in blood vessels, thereby preventing cerebral thrombosis,
In addition to being effective in the treatment of other thromboses, the therapeutic effect is increased when used in combination as an anticancer agent, and its usefulness is expanding.

In particular, urokinase collected from human urine and purified is currently widely used as a medicine because it has almost no side effects.

Traditionally, urokinase was purified using silica gel,
Hyflo Super Cell, zeolite acrylonitrile, barium sulfate, or various ion exchangers and gel filtration are used, as well as immunoadsorbents in which anti-urokinase antibodies are immobilized using the corresponding antibodies.

Hepatitis B surface antigen (HBsAg) can also be highly purified and inactivated and used as a hepatitis B vaccine.

HBsAg purification methods include ammonium sulfate, Aerosil,
Methods using an ion exchange resin, gel filtration, ultracentrifugation, and an immunoadsorbent immobilized with an anti-HBsAg antibody using the corresponding antibody are used.

Therefore, the present inventors have investigated various purification methods that are better than these known purification methods, and have found that they have a purification efficiency that is superior to any known purification method, are easy to operate, Furthermore, we have discovered a method for purifying crude antigenic substances that can be used repeatedly.

In other words, antibodies generally have other contaminant proteins, sugars, etc. bound to them, and even if antibodies are purified using normal purification methods, such as anion exchangers, these contaminant proteins, sugars, and other impurities cannot be cleaved. , cannot be disassembled.

However, these impurities can be removed by treating antibodies with a combination of specific enzymes under appropriate conditions, and the treated antibodies can be used as adsorbent ligands in affinity chromatography to counteract the antibodies. It has been found that when used to purify antigenic substances, the antigenic substances can be recovered in high yield and with high purity.

The present invention was completed based on this new knowledge, and provides an antibody treated with at least a proteolytic enzyme selected from pepsin, trypsin, and plasmin, and at least one glycolytic enzyme selected from amylase and arginase. The present invention relates to a method for purifying an antigenic substance corresponding to the antibody by adsorbing an immobilized carrier.

According to the present invention, antigenic substances can be obtained with high purity and high yield, and the operation is simple. In addition, the present invention improves the functionality of an insoluble carrier on which immune antibodies are immobilized during large-scale production, and improves biological stability ( (prevention of spoilage, generation of pyrogenic substances, etc.) will be improved.

The antigenic substances to be purified in the present invention include:
There are no particular limitations, as long as it can be used to immunize animals and produce antibodies, such as urokinase,
Examples include HBsAg, kallikrein, and interferon.

The antibody used in the present invention is an antibody corresponding to the above-mentioned antigen, and when subjected to enzyme treatment, it does not necessarily have to be a highly purified product, and may be, for example, plasma or serum from an immunized animal. Furthermore, monoclonal antibodies are also preferably used.

The enzymes used in the enzyme treatment are proteases such as pepsin, trypsin, and plasmin, and glycolytic enzymes such as amylase and arginase, and the treatment may be performed by combining at least one protease and glycolytic enzyme.

Enzyme treatment is carried out under individually suitable conditions, such as the following conditions.

For example, 0.1 μg to 1 mg of antibody depending on antibody purity
For pepsin, trypsin, and plasmin, use 0.1 to 100 μg/ml and treat at PH2 to 4.5 at 25 to 37°C for 10 to 30 hours, and for β-amylase, use 0.1 μg to 100 μg. At PH5.5-8.5,
Treat at 25-37℃ for 10-30 hours, add 0.1μg-100μg of arginase, and incubate at pH7.5-10 for 25 hours.
Perform processing for 10-30 h at ~37 °C.

After this enzyme treatment, it is generally concentrated by dialysis, concentration and/or ultrafiltration, and gel filtration is performed.
E Measure the high part of ₂₈₀ nm using the HAI method for anti-urokinase using the hemagglutination method and pool.

As a carrier for immobilizing the antibody thus obtained, any conventionally known immobilization carrier can be suitably used. Specific examples include purified agar gel, cellulose derivatives, dextran polymers, gels of polymers such as polyacrylamide, and glass particles.

The antibody can be immobilized on a carrier by a conventionally known method [Cuatrecasas et al.'s method (P. Cuaftrecasas, J.
Biol.Chem., 245 , 3059 (1970)] or a method similar thereto.

According to this method, a pure product with extremely high specific activity can be obtained with a recovery rate of 85% or more, and its specificity is very good. Urokinase was recovered.

Similarly, when HBsAg was used after enzyme treatment, high recovery and high purity recovery were obtained.

Examples are shown below, but the present invention is not limited thereto.

Example 1 Specific activity of urokinase purified by a known method,
80,000 units/E 100,000 to 200,000 units/ml of ₂₈₀ nm or more and Freunds complete adjuvant are evenly mixed.

Inject 1 ml of this subcutaneously or intradermally into a 3-4 kg domestic rabbit, then boost 0.5 ml in the same manner every 4-6 weeks, and then measure the anti-urokinase titer using the hemagglutination assay (HAI method). When the level rose, 50ml of blood was collected from the ear vein of the rabbit at a time.

First, 12 g of ammonium sulfate was added to 100 ml of this antiserum, and 13 g was added to the centrifuged supernatant at pH 7.2 to obtain a precipitate by centrifugation.

This precipitate was dissolved at pH 8.0E in a 0.04M phosphate buffer containing 0.03M sodium chloride to an absorbance of around 2 at ₂₈₀ nm.

This solution was then equilibrated with the same buffer.
DEAE-cellulose 70ml (diameter 3cm, height 10cm)
HAI the anti-urokinase moiety by passing it through the column
Approximately 1 g of a highly purified anti-urokinase fraction was obtained as measured by E ₂₈₀ nm.

Per 40 mg of this anti-urokinase fraction, PH3.5
The reaction was carried out at 37°C for 18 hours with pepsin at 1 μg/final concentration, and then the pH was adjusted to 6.5 with 1N-NaOH, and β-amylase was adjusted to 5 μg/final concentration at 25°C.
℃ for 12 hours, and finally at pH 9.2, add 1 μg/final concentration of arginase. After treatment at 25℃ for 18 hours, leave at room temperature for 1 hour at PH 10, add 0.3M NaCl, and add 0.01M of arginase. The product was dialyzed and concentrated using a phosphate buffer pH 8.5, gel-filtered using the same buffer as Sephacryl S-500, and an anti-urokinase fraction equivalent to about 0.4 g was obtained using the PHA method.

E ₂₈₀ nm of this enzyme-treated solution was diluted with 0.1M sodium carbonate buffer PH9 to around 5 to give an approximately equal amount of E 280 nm.
CNBr-activated Sepharose 4B (manufactured by Phalasia) was added, and the mixture was gently stirred at 4°C for 24 hours.

During this time, the initial HAI value changed from 1:2000 to 1:
The mixed liquid decreased to around 2, indicating that the coupling progressed smoothly.

After the reaction is complete, add 0.02M borate buffer with 0.15M sodium chloride.
After thorough washing at PH8, enzyme-treated modified anti-urokinase-immobilized Sepharose 4B was obtained.

Approximately 300,000 units of crude urokinase with a specific activity of 3,000 units/E ₂₈₀ nm can be adsorbed per ml of this product, and after washing with the same buffer, 0.5M sodium chloride was added.
Elute with 0.17M glycine buffer and determine specific activity.
More than 85000 units/E ₂₈₀ nm and a recovery rate of more than 95% were obtained.

Similarly, about 200,000 units of tissue culture urokinase with a specific activity of 1000 units/E ₂₈₀ nm were adsorbed, and the specific activity was 1000 units/E 280 nm.
A recovery rate of 95% or more was obtained at 100,000 units/E ₂₈₀ nm.

In addition, purification using anti-urokinase-immobilized Sepharose 4B using an antibody without enzyme treatment has poor adsorption rate, especially the increase in specific activity of 500,000 units/
E did not exceed ₂₈₀ nm, and the recovery rate was approximately 80%.

Example 2 Using the enzyme-treated modified anti-urokinase immobilized Sepharose 4B prepared in Example 1, approximately 1 ml of the tissue culture urokinase culture solution was added per 1 ml of this product.
It was possible to adsorb 10,000 units, and a specific activity of 90,000 units/E ₂₈₀ nm or more was obtained with a recovery rate of 95%.

In addition, purification using anti-urokinase-immobilized Sepharose 4B using an antibody without enzyme treatment has poor adsorption rate, and in particular, the increase in specific activity is 75,000 units/E ₂₈₀ nm.
The recovery rate was 75%.

Example 3 HBsAg positive plasma was purified by a known method using reverse passive hemagglutination (RPHA) with a purity of 1:4000 and a specific activity of E ₂₈₀ nm of 1:25000.
This 0.1 mixed with Freunds complete adjuvant
Inject 250 to 300 g of guinea pigs subcutaneously or intradermally with 0.1 ml each for 5 weeks, and then measure the anti-HBs titer using the hemagglutination method (PHA method).
When the value rose, the entire amount of blood was collected.

Thereafter, the product was purified in the same manner as in Example 1 to obtain enzyme-treated modified anti-HBs-immobilized Sepharose.

Per ml of this product, specific activity per E ₂₈₀ nm
HBsAg at 1:3000 in RPHA and 1:3000 in titer.
240,000, and after washing and elution, the specific activity was more than 1:32,000 per E ₂₈₀ nm, and the recovery rate was
It showed over 96%.

In addition, in purification using anti-HBs-immobilized Sepharose 4B using an antibody without enzyme treatment, the adsorption rate was poor, especially the increase in specific activity did not exceed 1:8000, and the recovery rate was about 75%.

Example 4 A culture solution of an anti-HBs monoclonal antibody purified by a known method using the HAI method with a specific activity of 1:55,000 per E ₂₈₀ nm was purified using the method of Example 1, and anti-HBs-immobilized Sepharose 4B was purified using the HAI method. Prepared.

1ml of this product can adsorb HBsAg with a titer of 1:300000 per E ₂₈₀ nm as a specific activity, and after washing and elution, the specific activity is 1:300000.
The recovery rate was 95% or more with an E of 1:35000 or more per ₂₈₀ nm.

In addition, in purification using anti-HBs-immobilized Sephaprse 4B using an antibody without enzyme treatment, the adsorption rate was poor, and in particular, the increase in specific activity did not exceed 1:12000.
The recovery rate was also around 73%.

Claims (1)

[Claims] 1. A carrier on which an antibody treated with at least one proteolytic enzyme selected from pepsin, trypsin, and plasmin and at least one glycolytic enzyme selected from amylase and arginase is immobilized is used as an adsorbent. A method for purifying an antigenic substance corresponding to the antibody, characterized in that: 2. The purification method according to claim 1, wherein the antigenic substance is urokinase or hepatitis B surface antigen (HBsAg).