JPH0478639B2 - - Google Patents

Description

[Detailed Description of the Invention] Interleukin 2 (hereinafter abbreviated as "IL-2") is an immune mediating factor that exists in humans, mice, etc., and is used as an antitumor drug, immunodeficiency treatment drug, and autoimmune drug. Applications such as disease therapeutics, diagnostic agents, and reagents are expected. IL-2 is produced, for example, using established cell lines such as splenocytes, blood lymphocytes, and malignant cell lines derived from T lymphocytes, or produced using other organisms or microorganisms by genetic recombination technology. However, the crude IL-2 obtained in this way generally contains only a low concentration of IL-2 and contains many impurities, so it is difficult to use it for practical use as a medicine. It is essential to purify such IL-2. The following methods have been reported for purifying IL-2. Gallo et al. investigated the production of lymphocytes in human peripheral blood.
After salting out IL-2 and performing ion exchange chromatography (DEAE-Sepharose), gel permeation chromatography was performed twice in a buffer containing 0.1% polyethylene glycol, and then SDS
Purification was performed by polyacrylamide gel electrophoresis using IL-2 with a purity of 808 times and a recovery rate of 0.8.
% (RCGallo et al., J. Immunol.
128 1122 (1982)). However, this method has a low recovery rate, and purification by electrophoresis is not suitable for large-scale preparation. Furthermore, since SDS is used, it is extremely difficult to remove the surfactant sodium medecyl sulfate from the purified sample, making it unsuitable for clinical use. Gallo et al. add polyethylene glycol to the buffer solution during gel filtration, but this is because IL-2, like interferon, is present in very small amounts in the culture supernatant and contains little impure protein. This is to prevent it from adhering to containers, resins, etc. during various operating processes, making it difficult to recover. Various reports have been made regarding recovery methods, but the current situation is that they are difficult. Otzpenheim et al.
IL-2 produced from human peripheral blood, tonsils, and spleen has been purified using DEAE-Sephacel gel filtration and electrophoresis, but the degree of purification is as low as 70 to 120 times. It is not suitable for large-scale preparation (J. Oppenheim et al., J.
Immunol. 128 1620 (1982)) Also, MAS Moore et al., J.
Exp.Med. 156 454 (1982)), human IL-2 produced from lymphocytes collected from human peripheral blood was purified by ion-exchange chromatography (DEAE-cellulose) and gel filtration, and then purified into two types. Affinity chromatography has been attempted, and although it is superior in terms of purity (more than 30,000 times), the IL of the raw material is
Since the specific activity of -2 is low, it is necessary to take into account that the degree of purification is increasing, and the yield is low. In others, S. Gillis et al., J.
Immunol. 124 1954 (1980)), E. Reich et al.
et al., J.Exp.Med. 154 422 (1981)) have reported a purification method that combines ion exchange chromatography, gel permeation chromatography, electrophoresis, etc.; It has disadvantages in properties and is not suitable for large-scale preparation. The present inventors conducted intensive research to establish a method for mass-preparing IL-2 with a good recovery rate, and as a result, they found that (A)
Three or more of the following five types of chromatographic carriers: porous glass booze chromatographic carrier, (B) cation exchange chromatographic carrier, (C) gel filtration chromatographic carrier, (D) hydrophobic chromatographic carrier, and (E) metal chelate chromatographic carrier. By using these in combination, we have achieved the three conditions required for the IL-2 concentration and purification method: a high recovery rate of IL-2, the ability to perform large-scale processing of several dozen or more with relatively simple operations, and a high degree of purification. We have found that there is a method that satisfies the above three conditions, and furthermore, even when three or more of the five types of chromatographic carriers are used in combination, the above three conditions can be better achieved by using specific carriers in combination in a specific order. They found that the conditions were satisfied (compare Examples 1 to 5, 14, and 15 with Examples 6 to 13 below), and completed the present invention. That is, the present invention provides an aqueous solution containing interleukin 2 on (A) a porous glass bead chromatography carrier;
The present invention relates to a method for purifying and concentrating interleukin 2, which is characterized by using (B) a cation exchange chromatography carrier, (C) a gel filtration chromatography carrier, and (D') a reversed phase high performance liquid chromatography carrier in this order. In addition, in this method, between the treatment using (C) gel filtration chromatography carrier and the treatment using (D') reversed phase high performance liquid chromatography carrier, (D) hydrophobic chromatography carrier and/or (E) metal chelate chromatography carrier is added. Treatment using a carrier can also be added. There is no particular restriction on the type of IL-2 that can be subjected to the purification and concentration method of the present invention, and it may be derived from human peripheral blood, spleen, tonsils, human T leukemia cells, monkey, cow, horse, rat, or mouse. , or may be derived from IL-2 culture produced by genetic engineering techniques. The medium for producing IL-2 may be either a serum medium or a serum-free medium, but the method of the present invention is most effective when IL-2 is produced using a serum medium containing more impure proteins. The usage of each of the chromatographic carriers used in the present invention is shown below. A: Porous glass bead chromatography carrier IL-2 solution containing impurities such as proteins is PH
IL-2 is adsorbed by contacting with porous glass beads at a pH of 5 to 10, preferably 6.5 to 8.5. PH
If it is too low, the adsorption efficiency of IL-2 will decrease, and if it is too high, the stability of IL-2 will decrease. The solution containing IL-2 used here is:
For example, it may be a culture supernatant cell extract, a concentrated solution obtained by ultrafiltration or the like, a redissolved protein precipitate obtained by salting out, or a crudely purified IL-2 solution. Note that the ionic strength of the IL-2 solution during adsorption does not have a large effect on adsorption, but
If the NaCl concentration exceeds 1.0M, the amount of non-adsorbed IL-2 will increase, so a NaCl concentration of 0.5M or less is desirable. IL-2 thus adsorbed is eluted, for example, by the following method. That is (1)
1-99% ethylene glycol, glycerin, etc.
Elution is preferably carried out using a buffer containing 20-90%. Here, as the buffer solution, any commonly used buffer solution may be used, and the pH range may vary.
Any range in which IL-2 is stable is sufficient. or (2) elution with an eluent having a pH of 1 to 5, preferably 1.5 to 3.5. Any acid may be used as the eluent as long as it has a pH within this range.
or (3) CNS ^- , I ^- , Br ^- , ClO ₄ ^- , Li ⁺ , Ca ²⁺ ,
Elute using an eluent containing salts such as alkylammonium ions. Here, the concentration of the salts may be 0.1M or more, preferably 0.4 to 1.0M. Note that the pH and acid are the same as the eluent in (1). By the way, examples of porous glass beads include CPG-10 (manufactured by Electronucleonics).
etc. B: Cation exchange chromatography carrier As a cation exchange chromatography carrier, an ion exchange group such as a sulfonic acid group, a phosphoric acid group, a carboxylic acid group, a sulfopropyl group, or a carboxymethyl group is used as a cation exchange chromatography carrier such as silica gel alumina, synthetic polymer, natural polymer, or Many compounds are known that are bonded to natural polymers that have been modified such as crosslinking. These are IL
Any product that can be used in the pH range where -2 is stable can be used for purifying IL-2. Among the cation exchange chromatographic carriers, the usage of a chromatographic carrier having a carboxymethyl group will be described. First, an IL-2 solution containing impurities such as proteins is brought into contact with a carboxymethyl cation exchange chromatography carrier at pH 2 to 8, preferably pH 5 to 7, and IL-2 is adsorbed onto the carrier. At this time, the ionic strength needs to be kept low in order to increase the adsorption efficiency of IL-2 onto the carrier, and although it varies depending on the pH of the buffer used and the type of salt, In some cases, it is better to keep the concentration below about 0.15M. The method to elute the adsorbed IL-2 is as follows:
() Increase in PH, () Increase in ionic strength, ()
Both methods of increasing pH and ionic strength are effective, but when the amount of IL-2 is small, the recovery rate may decrease due to adhesion to carriers, containers, etc. at low ionic strength. It is preferable to elute by increasing . In the case of acetic acid-sodium acetate buffer (PH6.0),
It is preferably 0.3M or more, preferably 0.5M or more. Incidentally, a cation chromatographic carrier suitable for carrying out the present invention is CM Sephadex C-25 (manufactured by Pharmacia). C: Gel perchromatography carrier There are many known gel perchromatography carriers such as cross-linked dextran, porous synthetic polymer, and porous silica gel, and the molecular weight fraction ranges from several thousand to about 100,000. Any material can be used as long as it does not non-specifically adsorb IL-2. The pH of the buffer solution used may be within a range in which IL-2 is stable, but since contaminating proteins may precipitate under acidic conditions, a pH near neutrality is desirable. Regarding ionic strength, in order to prevent adsorption of IL-2 to the carrier and container, when using NaCl, a concentration of 0.5M or higher is desirable. Incidentally, as a gel perchromatography carrier suitable for carrying out the present invention, there is Sephadex G-75 (manufactured by Pharmacia), and Superfine grade, which has a small particle size, is particularly suitable. D': Reverse phase high performance liquid chromatography carrier Examples of the reverse phase high performance liquid chromatography carrier include porous polystyrene carriers and chemically bonded silica gel carriers. By the way,
Porous polystyrene carriers suitable for carrying out the present invention include, for example, Hitachi Gel #3013 (manufactured by Hitachi, Ltd.) (see Example 6), while chemically bonded silica gel carriers include, for example:
Ultrapore RPSC (manufactured by Beckman), Protesil
300 Diphenyl (manufactured by Watmann), Unisil Q
Examples include PH (see Example 11) and Unisil Q CN (see Example 12) (both manufactured by Gascro Kogyo Co., Ltd.). Among these, Examples 7 to 10 and
13, chemically bonded silica gel carriers, especially Ultrapore RPSC and Protesil 300
A higher degree of purification can be obtained by using a product with a large pore size (300 Å in each case) like Diphenyl. When using these carriers, adsorption of IL-2 from a solution containing IL-2 may be performed at any pH range in which IL-2 and the chromatography carrier are stable, but in particular, chemically bonded silica gel carriers may be used. When using a neutral
Preferably in the PH range. Also, IL-2
There is also no particular restriction on the salt concentration of the solution. Furthermore, the IL-2 solution contains an organic solvent that is miscible with water.
It may contain up to 60%, preferably the proportion of organic solvent is 20% or less. adsorbed on carrier
IL-2 can be eluted with an eluent containing 30% or more, preferably 50% or more of a water-miscible organic solvent, preferably methanol, ethanol, propanol or acetonitrile. At this time, as shown in Examples 7 to 13, it is desirable to perform gradient elution in which elution is performed while increasing the content of the organic solvent using a conventional high performance liquid chromatography apparatus. D: Hydrophobic chromatographic carrier Hydrophobic chromatographic carriers include alkyl groups such as propyl, butyl, octyl, and octadecyl groups, allyl groups such as phenyl groups, aroalkyl groups such as phenylmethyl groups and diphenylmethyl groups, or cyanopropyl groups. A number of synthetic polymers such as those in which substituted alkyl groups, etc. are bonded to natural polymers such as cross-linked dextran, synthetic polymers, or silica gel, and porous polystyrene are known. Any of these can be used as long as IL-2 can be adsorbed and desorbed in a stable pH range.
It can be used for purification of IL-2. Using these carriers, IL-2 was extracted from IL-2 solution.
When adsorbing () lower the pH of the solution,
() Increasing the salt concentration in the solution, () Lowering the proportion of organic solvent in the solution or not including it () It is better to use one of () to (), or a combination of two or more. You can get good results. In addition, when IL-2 adsorbed on these carriers is eluted from the carrier, it is necessary to (1) increase the pH of the eluent, (2) lower the salt concentration of the eluent, and (3) reduce the organic Efficient elution and recovery can be achieved by increasing the proportion of the solvent, using any one of (1) to (3), or by combining two or more of them. Incidentally, examples of hydrophobic chromatographic carriers suitable for carrying out the present invention include phenylcepharose CL.
-4B (manufactured by Pharmacia) and Hitachi Gel #3013 (manufactured by Hitachi, Ltd.), and the purification of IL-2 by hydrophobic chromatography using these will be described below. When phenylcepharose CU-4B is used as a chromatographic carrier, the IL-2 solution containing proteins etc. as impurities is heated to a pH of 2 to 8, preferably 6 to 7.
In order to further increase the ionic strength, if NaCl is used, the final concentration is adjusted to 0.5M or higher, preferably 0.75M or higher, and then adsorbed onto the carrier. The adsorbed IL-2 can be eluted from the carrier by lowering the ionic strength, but when using NaCl as the eluent, the concentration is preferably 0.5M or less, preferably 0.25M or less. During this adsorption/desorption, the
When glucose and sucrose are added to a concentration of 0.3 M or more during IL-2 solution, carrier washing solution, and elution, both the recovery rate and purification degree of IL-2 from the carrier are significantly improved. When using Hitachi Gel #3013, a porous polystyrene carrier, as a chromatography carrier, IL
Adsorption of IL-2 from a solution containing -2 onto a carrier may be performed at any pH range in which IL-2 is stable, and there is no particular restriction on the salt concentration of the IL-2 solution. Furthermore, the IL-2 solution may be an aqueous solution or contain up to 60% of a water-miscible organic solvent, preferably the proportion of organic solvent is 20% or less. The IL-2 adsorbed on the carrier is treated with a water-miscible organic solvent, preferably methanol, ethanol, plapanol or acetonitrile.
Elution can be carried out with an eluent containing 30% or more, preferably 50% or more. Furthermore, as hydrophobic chromatographic carriers suitable for carrying out the present invention, there are many carriers collectively referred to as chemically bonded silica gels, such as Ultrapore RPSC
(manufactured by Beckmann), Protesil300 Diphenyl (manufactured by Watmann), UnisilQ PH and Unisil Q
Examples include CN (all manufactured by Gascro Industries). E: Metal chelate chromato carrier Copper chelate resin, zinc chelate resin, etc. are known as metal chelate chromato carriers. For example, copper chelate resins are described in the literature (J.Porath
et al., Nature 258 598 (1975)). An aqueous solution containing IL-2 is adsorbed onto the prepared copper chelate resin at a pH of 5 or higher, preferably 6 or higher. In this case, the ionic strength does not have much influence, but the adsorption efficiency of IL-2 decreases when the NaCl concentration exceeds 1.5M. IL-2 thus adsorbed is eluted by lowering the pH of the eluate to 4.5 or less, preferably 3.5 or less. During IL-2 adsorption and desorption, glucose, sucrose,
By adding urea or the like, the recovery rate and degree of purification are improved. In addition, since copper chelate resin can adsorb a large amount of protein to a small amount of resin, it is useful not only for purification purposes but also for dilute IL-2
It is also effective for concentrating solutions. By combining three or more of the above operations using various chromatographic carriers from A to E, inter alia,
A, B, C, (D and/or E _p as desired)
The order of D and E does not matter. ), and D′ using various chromatographic carriers in this order, IL
Highly purified IL-2 can be obtained with a recovery rate that greatly exceeds that of IL-2. Each chromatographic carrier may be used by filling a column, or may be used by a patch method. Furthermore, a combination of a carrier and an apparatus called high-performance liquid chromatography is particularly excellent as a method for purifying IL-2 to a single level. The elution of IL-2 adsorbed onto the carrier may be carried out using one type of eluent, the so-called step size method in which the composition of two or more eluents is changed stepwise, or the composition of the eluent may be continuously changed. A so-called gradient method may be used in which the temperature is varied. As mentioned above, the order of combination of various chromatographic carriers is A,
B, C, (optionally D and/or E), and
Let it be D′. Of course, pre-treatment and post-treatment for each operation include concentration, dilution, pH adjustment, addition of other substances, dialysis, salting out, centrifugation, heating, cooling, freezing, etc.
Freeze-drying, dissolution, or other routine treatments may be added, the same carrier may be used more than once, or multiple carriers belonging to the same group may be used,
Operations using chromatographic carriers based on principles other than A to E may be added. According to the method of the invention,
(A) Treatment using a porous glass bead chromatography carrier makes it possible to concentrate the culture supernatant volume to 1/100 at once, and (B) treatment using a cation-exchange chromatography carrier makes it possible to further concentrate the culture supernatant by 1/100. one part
(C) Treatment using a gel filtration chromatography carrier further removes high-molecular weight proteins and increases the purity, making it possible to significantly simplify subsequent handling in large-scale processing. )
(D') Treatment using a reversed-phase high-performance liquid chromatography carrier allows for very high purity IL-2 with high recovery rate.
can be obtained. In addition, (D) a hydrophobic chromatographic carrier and/or (E) a metal chelate chromatographic carrier is used between (C) a treatment using a gel filtration chromatographic carrier and (D') a treatment using a reversed phase high performance liquid chromatographic carrier. By adding processing, higher purity can be achieved.
It becomes possible to obtain IL-2 and increase the purification efficiency in the operation of (D'). Incidentally, for example, in the case of human IL-2, this A~
In the example that combines all the operations of E, the purity is 5000.
The recovery rate in the entire operation is 50%, which is greatly improved compared to other methods, and the purified product obtained is 5 × 10 ⁶ ~
It showed a specific activity of 10 ⁷ units/mg. In addition, the measurement of IL-2 activity was performed as follows. Place 100μ of the sample in the first row of a 96-well microtiter plate, and repeat the 2-fold dilution with Dulbecco's modified Eagle's medium (DMEM) containing 5% FEB to create a dilution series of 100μ each on the 96-well microplate. . Then, an activated T lymphocyte cell line prepared according to the method taught by Gillis et al. (Nature, Vol. 268, p. 154 (1977)) was prepared at a cell density of 4 x 10 ³ cells/100μ, and a 100μ cell line was prepared. Add to each well. 37
After 20 hours of incubation in a 5% carbon dioxide incubator at °C, 0.5 μCi of tritiated thymidine was added and pulsed for 4 hours. The cells were harvested according to methods well known in the field and intracellularly cultured. Measure the amount of radiation absorbed. Since the culture supernatant with higher IL-2 activity has a larger amount of tritiated thymidine incorporated into activated T lymphocytes, the amount of IL-2 produced in the culture supernatant can be easily determined. In this case, ConA-stimulated rat spleen cell culture (1 x ¹⁰⁶ splenocytes/ml, ConA 5 μg/ml added48
The amount of IL-2 produced during the culture (time culturing) is defined as 1 unit/ml, and the activity unit is calculated from the relative value. (Gillis et al. Journal of Immunology (J.
Immunol.) Vol. 120, p. 2027 (1978)). Example 1 (Combination of carriers ABCD and E) Stimulated with concanavalin A 10 μg/ml for 24 hours
Culture supernatant containing human IL-2 obtained using ATCC CRL 8129 cells (human T lymphocyte leukemia cells, cultured in RPMI medium with 1% fetal bovine serum) 20
(Protein concentration 0.57 mg/ml, IL-2 specific activity 980 units/
mg) was concentrated using an ultrafiltration device Holofiver HIP5 (manufactured by Amicon) to a volume of 3.5, and then
The pH was adjusted to 7.7 with hydrochloric acid and sodium hydroxide. This IL-2 solution was passed through a column (column size: 44 mm diameter x 70 mm, volume 106 ml) of CPG-10 (pore size 350 Å, 120-200 mesh, manufactured by Electro-Nucleonics), which is a porous glass bead chromatographic carrier. , IL-2 was adsorbed. This column is
It was sufficiently equilibrated in advance with 0.1M Tris-HCl buffer (PH7.7) containing 0.2M NaCl. After that, 400ml of 0.1M Tris-acetate buffer (PH7.7) containing 0.2M NaCl, then 300ml of 0.1M Tris-acetate buffer (PH8.0) was passed through the column to wash the inside of the column, and then 0.75M Contains potassium thiocyanate of
IL- with 350ml of 0.1M Tris-acetate buffer (PH8.0)
2 was eluted. The elution curve is shown in Figure 1. In the figure, the solid line indicates absorbance, and the dotted line indicates IL-2 activity.
Add 1.4 ml of saturated ammonium sulfate to the obtained IL-2 eluent,
After standing overnight at 4℃, the precipitated protein was centrifuged at 6000 rpm for 30 minutes, the supernatant was removed, and 140 ml of
It was dissolved in 0.07M acetic acid-sodium acetate buffer (PH6.0). The obtained IL-2 solution was placed in a commercially available dialysis membrane container and dialyzed in 50 times the volume of the same buffer at 4°C overnight to keep the pH and ionic strength constant. Thereafter, the solution was passed through a column (column size: 32 mm diameter x 120 mm, capacity: 95 ml) of cation exchange chromatography carrier CM Sephadex C-25 (manufactured by Pharmacia) to adsorb IL-2. The column used had been equilibrated in advance with a sufficient amount of 0.07M acetic acid-sodium acetate buffer (PH6.0). After that, the column was washed with 150 ml of 0.07M acetic acid-sodium acetate buffer (PH6.0), and then the IL-2 was washed with 150 ml of 0.5M acetic acid-sodium acetate buffer (PH6.0).
was eluted. The elution curve is shown in Figure 2. The distinction between solid lines and dotted lines is the same as in Figure 1. Solid ammonium sulfate was added to the eluted IL-2 solution to bring the saturation level to 80%, and the mixture was left overnight at 4°C, centrifuged at 6000 rpm for 30 minutes, the supernatant was removed, and 20 ml of 0.05 M phosphate buffer (PH7.0) was added to the eluted IL-2 solution. ) was dissolved in The obtained IL-2 solution was subjected to gel permeation column chromatography. The carrier used was Cephadex G-75 Super Fine (manufactured by Pharmacia), and the column was 44 mmφ x 90 cm. To equilibrate the gel permeation column, use 0.05M phosphate buffer (PH7.0) containing 1.25M sodium chloride.
The same buffer was used during elution. Flow rate is 10ml/
hr. The elution curve of gel permeation column chromatography is shown in FIG. IL-2 activity is determined by molecular weight
Elutes around 15,000 to 20,000. IL- obtained by gel perchromatography
Glucose was added to the two fractions to a concentration of 1.0M, and a column of Phenyl-Sepharose CL-4B (manufactured by Pharmacia) (16 mmφ x 35 mm, capacity 7.0
ml) to adsorb IL-2. Phenylcephalose column was pre-prepared with 1.0M glucose,
A solution equilibrated with 0.05M phosphate buffer (PH7.0) containing 1.25M sodium chloride was used. After adsorption, the column was washed with the same buffer used for column equilibration, and then IL-2 was washed with 60 ml of 0.05 M phosphate buffer (PH 7.0) containing 1.0 M glucose and 0.1 sodium chloride. eluted. The elution curve of phenyl sepharose chromatography is shown in FIG. After adding urea to the obtained IL-2 solution to a concentration of 1.0M, the method of Porus et al. (Nature,
258, 598 (1975)) (column size 10 mmφ x 15 mm, capacity 1.2 ml,
Glucose 1.0M, Urea 1.0M, Sodium Chloride
IL-2 was adsorbed by passing the solution through a 0.05M phosphate buffer containing 0.1M (which had been equilibrated with PH7.0). The column was soaked in 0.05M phosphate buffer (PH) containing 0.1M sodium chloride, 1.0M glucose and 1.0M urea.
7.0) 10ml, washed with 10ml of 0.05M phosphate buffer (PH6.0) containing 1.0M glucose and 1.0M urea, then washed with 10ml of 0.05M phosphate buffer (PH6.0) containing 1.0M glucose and 1.0M urea
0.05M sodium citrate-hydrochloric acid buffer (PH3.0)
IL-2 adsorbed on the column was eluted with 10 ml. The elution curve of copper chelate chromatography is shown in FIG. Furthermore, the human IL obtained in this way
-2 is isoelectric focusing (manufactured by Pharmacia)
Analysis using FBE3000) showed that the isoelectric point was 8.0 to 8.1. Analysis by SDS-polyacrylamide gel electrophoresis (GE-4 manufactured by Pharmacia) revealed that the molecular weight was approximately 17,000. The results of purification are shown in Table 1. [Table] Example 2 (Combination of carriers ACD and E) IL-2-containing culture supernatant 14 obtained in the same manner as in Example 1 (protein concentration 0.53 mg/ml, IL-2 specific activity
Solid ammonium sulfate was added to the solution (1080 units/mg) to dissolve it to a saturation level of 75%, and the mixture was left overnight at 4°C. The precipitated protein was centrifuged at 6000 rpm for 30 minutes and then dissolved in 1500 ml of 0.1M Tris-HCl buffer (PH7.7) containing 0.2M sodium chloride. This IL-2 solution was pre-equilibrated with a 0.1M Tris-HCl buffer (PH7.7) containing 0.2M sodium chloride on a CPG-10 column (volume 85ml).
to adsorb IL-2. After washing with 200 ml of the above equilibration buffer, then with 150 ml of 0.1 M Tris-acetate buffer (PH8.0), 0.1 M Tris-acetate buffer (PH8.0) containing 50% (v/v) ethylene glycol. IL-2 was eluted with 300 ml. Next, saturated ammonium sulfate solution (1) was added to the eluted IL-2 solution, and the mixture was lightly stirred and left overnight at 4°C. After centrifuging the precipitated protein at 5000 rpm for 30 minutes,
It was dissolved in 60 ml of 0.05M phosphate buffer (PH7.0) and subjected to gel permeation column chromatography. As the resin, Cephadex G-75 Superfine was used. The same buffer as in Example 1 was used, and the flow rate was 40 ml/hr (column size 44 mmφ).
×90cm). IL- obtained by gel perchromatography
Glucose was added to the 2 active fractions to a concentration of 1.0M, and a phenylcephalose column (32 mmφ
x 30 mm, volume 24 ml) to adsorb IL-2. Next, the column was washed with equilibration buffer (0.05 phosphate buffer (PH7.0) + 1.25 M NaCl + 1.0 M glucose), and then IL was added with 100 ml of 0.05 M phosphate buffer containing 1.0 M glucose and 0.15 M sodium chloride. -2 was eluted. After adding urea to the obtained IL-2 eluent to a concentration of 1.2M, a copper chelate resin (column size 16 mm) was added.
φ×15mm, capacity 3ml, glucose 1.0M, urea
It was equilibrated with 0.05M phosphate buffer (PH7.0) containing 1.2M and 0.15M sodium chloride. ) and washed with 15 ml each of the above equilibration buffer, 0.1 M acetic acid-sodium acetate buffer (PH5.5) containing 1.0 M glucose and 1.2 M urea, and
in 7 ml of 0.05 M sodium citrate-hydrochloric acid buffer (PH2.8) containing 1.0 M glucose and 1.2 M urea.
IL-2 was eluted. The purification results are shown in Table 2. [Table] Example 3 (combination of single substances A, B, C and D) IL-2-containing culture supernatant 9 obtained in the same manner as in Example 1 (protein concentration 0.49 mg/ml, IL-2 specific activity
720unit/mg) by the method described in Example 1.
The solution was concentrated and passed through a CPG-10 chromatography column (resin capacity: 48 ml) to adsorb and desorb IL-2. The adsorption/desorption conditions are the same as those shown in Example 1. Then, add saturated ammonium sulfate solution to 150 ml of the obtained IL-2 solution.
450 ml was added, salted out overnight at 4℃, and the precipitated protein precipitate was dissolved in 0.07M acetic acid-sodium acetate buffer (PH
6.0) The solution was redissolved in 80 ml and subjected to CM-Sephadex C-25 column chromatography using the method described in Example 1. After the obtained IL-2 solution was again subjected to ammonium sulfate salting out treatment, it was added to Cephadex G-
100 (manufactured by Pharmacia) and subjected to gel permeation chromatography. The IL-2 fraction obtained by the above procedure was divided into two parts and subjected to phenyl-sepharose chromatography using the following two methods. (a) Glucose at a concentration of 1.0 M was added to the IL-2 solution obtained by gel permeation chromatography as described in Example 1, and then purified by the method of Example 1. (b) Phenylcephalose column (resin volume 2.5 ml) was prepared in advance without glucose addition.
A solution containing 1.25M sodium chloride and equilibrated with 0.05M phosphate buffer (PH7.0) was passed to adsorb IL-2. After washing with 10 ml of the above equilibration buffer, an attempt was made to elute IL-2 with 15 ml of 0.05 M phosphate buffer containing 0.1 M sodium chloride.
Since the recovery rate of IL-2 activity was low, the elution buffer (containing 0.1M sodium chloride) was
8 ml of an eluent containing 50% volume of ethylene glycol (0.05 M phosphate buffer) was applied to the column to elute the remaining IL-2, and both active fractions were combined. The above purification results are shown in Table 3. [Front] Su(D)
[Front] Su(D)

Claims (1)

[Claims] 1. An aqueous solution containing interleukin 2 is transferred to (A) a porous glass bead chromatography carrier, (B) a cation exchange chromatography carrier, (C) a gel filtration chromatography carrier, and (D')
A method for purifying and concentrating interleukin 2, which is characterized in that it is treated using a reversed phase high performance liquid chromatography carrier in this order. 2. A treatment using (D) a hydrophobic chromatographic carrier and/or (E) a metal chelate chromatographic carrier is carried out between (C) the treatment using the gel filtration chromatographic carrier and (D') the treatment using the reversed phase high performance liquid chromatographic carrier. A method for purifying and concentrating interleukin-2 as claimed in claim 1.