FI74978C - Process for producing human gamma interferon. - Google Patents

Description

74978 \

This invention relates to a new and improved process for the preparation of human interferon gamma.

5 It is known that human leukocytes produce interferon-gamma (immune interferon) by mitogenic agents. However, interferon-gamma differs in biological properties from leukocyte (alpha) and fibroblast (beta) interferons, which are produced by leukocytes as a result of viral infection, and which are formed in cells of fibroblast origin by synthetic polynucleotides, respectively. Because interferon-gamma has the ability to inhibit cell division and affect the function of cells in the immune system, it has significantly more desirable properties than interferons alpha and beta, and it follows that interferon-gamma will be used on an ever-expanding scale to treat cancers (Nature 294, 6 (1981)). ), Cellular Immunology 49, 390 (1980).

Interferon-gamma can be produced by separating the blood leukocyte fraction (leukocyte layer), removing erythrocytes by gradient centrifugation or, preferably, by hemolysis with ammonium chloride treatment, incubating the purified leukocytes from the above supernatant, and treating them with appropriate endogenous mitogenic agents. . According to the prior art, concana line A (Infect. Immun. 26, 36 (1979)), phytohemagglutinin (Proc. Natl. Acad. Sci. USA 78, 16OI (1981)) or enterotoxin ( Int. Res. Commun. Sys. Med. Sci. 7, 595 (1979)).

Several attempts have been made to increase the production of interferon-gamma by mitogenic agents. According to one method, the production of interferon-gamma is increased by pretreatment with leukocytes 12-o-tetra- _____. τ ~ 74978 with decanoyl phorbol 13-acetate (Vilcek et al .: Biochemical Characterization on Lymphokines - Academia,

New York, page 323, (1980)). The disadvantage of said method is that 12-o-tetradecanoyl-phorbol-13-5 acetate is a carcinogenic substance, and thus the interferon-gamma thus produced is apparently not suitable for use in the treatment of humans. According to another reference (Nature 292, 842 (1981)), the authors tried to increase interferon-gamma production by treating leukocytes with sodium 10 butylate or dexamethasone, but these attempts failed.

The object of the invention is to increase the production of leukocytes by interferon gamma caused by mitogenic substances, and to increase the number of units per milliliter.

According to the present invention, there is provided a method of producing human interferon-gamma, comprising separating a blood leukocyte fraction (white cell layer), removing erythrocytes, suspending the leukocytes in a suitable medium, treating them with a mitogenic agent and separating the interferon-containing solution from the cells. is characterized in that the leukocytes are pretreated with interferon alpha or beta, preferably at 25,200 to 20,000 IU / ml, before being treated with the Mito genetic agent.

The present invention is based on the finding that the production of interferon-gamma can be significantly increased - by about 500 to 1000% - by subjecting leukocytes to alpha or beta interferon pretreatment before being treated with a mitogenic agent.

Alpha- or beta-interferon is used in the pretreatment in amounts of 200 to 20,000 IU / ml, preferably 500 to 5,000 IU / ml. (IU = International Unit). According to a preferred embodiment of our method, said treatment is carried out using alpha-3 74978 Feron in an amount of 1000 to 2000, in particular in an amount of 1500 IU / ml.

According to another preferred embodiment of our method, said treatment is carried out using interferon beta in an amount of 2000 to 3000, in particular in an amount of 2500 IU / ml.

The pretreatment is preferably carried out 1 to 12 hours, in particular 2 to 8 hours, and particularly preferably 4 hours before the treatment with the mitogenic agent.

The alpha or beta interferon treatment is preferably carried out for 1 to 12 hours, especially for about 4 hours. The pretreatment temperature is preferably 35-39 ° C, especially 37 ° C.

Preferably, the interferon is removed after alpha or beta-15 treatment and before the mitogenic agent is added. This step can advantageously be performed by washing the cells, especially with Hanks' solution. The interferon used in the pretreatment can also be left in the cell culture medium if it is continued. In this case, the interferon-gamma 20 produced must be isolated from the interferon alpha or beta used in the pretreatment step.

The isolation can be performed according to known methods, e.g. by glass chromatography.

Tissue growth media containing various amino acids and vitamins can be used as the growth medium in the method 25 of the present invention, (e.g., the following: Eagle-type MEM, RPMI 1640, Dulbecco-type MEM, Glasgow-modified MEM, etc.). It is preferred to use a medium having the following composition; the advantage of this medium 30 is that it is cheap and simple and can be used well in an autoclave.

Component Quantity

Calcium chloride 175-350 mg / l 35 Potassium chloride 300-500 mg / l .----- "TT" "74978

Magnesium sulphate or equivalent amount of magnesium chloride 175-500 mg / l

Sodium chloride 5000-7000 mg / l 5 Sodium hydrogen carbonate 200-3500 mg / l

Sodium dihydrogen phosphate 30-150 mg / l

Glucose 5ΟΟ-55ΟΟ mg / l

Iron (III) nitrate 0-0.2 mg / l

Serum 0.5-10 mg / l 10

Production of interferon-gamma is performed in the presence of various animal or human blood sera or gamma-globulin-free plasma (0.5-10%).

According to the method 15 of the present invention, leukocytes (leukocyte layer) obtained from human blood that has been inhibited from coagulation for up to 72 hours at 0-8 ° C are used as a starting material.

As an anticoagulant, an ACD solution (a solution containing citric acid and dex-20 trout) or an ACD solution supplemented with various bases (e.g., adenine or guanine) can be used. The collected leukocytes can be removed by gradient centrifugation (e.g. Ficoll or Percoll products manufactured by the Swedish company Pharmcia), or preferably by hemolysis with ammonium chloride. It is advantageous to proceed by stirring in a concentrated leukocyte suspension a 0.5-1% - preferably $ 0.83 - ammonium chloride solution at a temperature of 0-10 ° C in a volume / volume ratio of 1: 3-20, preferably 1: 5. Incubate for 5-20 minutes - preferably about 10 minutes - at 0-8 ° C with or without agitation. Leukocytes are distinguished from disintegrated erythrocytes, e.g. by centrifugation. Advantageously, the ammonium chloride treatment can be continued using 10 volumes of ammonium chloride solution per part by weight of the cell suspension.

Purified leukocytes are suspended in a suitable 5,74978 medium (suitable as e.g.

Eagle-type MEM, RPMI 16 ^ 0, Dulbecco-type MEM,

Glasgow-modified MEM, etc.) or the inexpensive medium described above (containing no amino acids or vitamin mines), and the number of cells is set at 10 ° to 10 ° cells / ml. In a preferred embodiment of the method, the inexpensive medium described above, which does not contain amino acids or vitamins, is used to both resuspend the cells and set the number of cells. The medium or medium to be used is supplemented with animal or human blood serum or, preferably, gamma globulin-free human plasma (0.5-10%). It is preferred to add the antibiotic to the medium or solution; for this purpose, neo-15 mycin can be advantageously used at a concentration of about 10 to 50 pg / ml, in particular at a concentration of 25 μg / l.

The cells are then subjected to alpha or beta interferon treatment. For this purpose, inducer-free viral or synthetic polynucleotide-free) crude or purified alpha or beta interferon in amounts of 200 to 20,000 IU / ml can be used. Said pretreatment is carried out at a temperature of 35 to 39 ° C, in particular 37 ° C, for about 1 to 12 hours, preferably for one hour.

The interferon used in said pretreatment step can be removed from the cells, preferably by washing.

This step can be preferably performed using Hanks' solution. After the interferon has been washed out of the cell, the cell concentration is reset using human or animal serum or gamma-globulin-free plasma, especially gamma-globulin-free human serum.

It is not absolutely necessary to wash the interferon from the product, since the presence of interferon alpha or beta does not adversely affect the production of interferon-gamma. The gamma interferon 6 74978 produced in this case is separated from the alpha or beta interferon present by known methods.

The cells are then contacted with a suitable interferon inducer. For this purpose, concanavalin A, phytohem eguthin, staphylococcus, enterotoxin, etc. referred to above can be used most suitably. According to a preferred embodiment of the present invention, concanavalin A is used as the inducer, most preferably at a concentration of 2.5-30 μg / ml, preferably 15 μg / ml. The induction is generally carried out for about 5 to 8 hours, more preferably for 10 to 12 hours at a temperature of 35 to 39 ° C, preferably 37 ° C. After a certain time (after about an hour) the inductor can be removed by washing; however, this step can be omitted because the inducer does not interfere with the production of interferon-gamma by its presence. Therefore, the inductor is usually not removed.

The cells are then removed from the supernatant by centrifugation. The solution above the precipitate contains crude gamma interferon, which can be stored at low temperature (about -20 ° C) or purified 2q according to known methods.

An advantage of the present invention is that pretreatment with alpha or beta interferon results in a five- or ten-fold increase in interferon-gamma production. The physicochemical properties (pH 2 sensitivity, Stability measured at 56 ° C and 37 ° C) and its biological activity (antiviral and anticellular activity) of the gamma interferon produced by the method of the present invention are completely equivalent and similar to the properties of jq gamma interferon produced by conventional methods.

Further details of the present invention are set forth in the following examples without limiting the applicability of the invention to said examples.

35 7 74978

Example Blood obtained in an ACD solution (aqueous solution containing citric acid and dextrose) is stored at + 4 ° C for three hours. The leukocyte layer is collected by centrifugation and allowed to stand overnight at + 4 ° C. The leukocyte concentrate thus obtained is mixed with 1 part by volume of 5 parts by weight of 0.83% aqueous ammonium chloride solution (temperature: + 4 ° C). The suspension is allowed to stand at + 4 ° C until the erythrocytes dissolve (5-10 minutes) and the leukocytes are separated by centrifugation. Leukocytes are suspended in 10 media having the following composition:

Component Amount (mg / l)

Calcium chloride 265

Iron (III) nitrate 0.1

Potassium chloride 400 15 Magnesium sulphate heptahydrate 200

Sodium chloride 6400

Sodium bicarbonate 2750

Sodium dihydrogen phosphate dihydrate 140 Glucose 4500 20

The dissolution of the erythrocytes by the above method is repeated with the difference that 10 parts by weight of a 0.83 aqueous solution of ammonium chloride are used. The leukocytes are suspended in a growth solution having the above composition, and the number of cells is set at 10 cells / ml.

The growth solution contains 2 mg / ml human gamma serum and 25 μg / ml neomycin. Cells are treated at 37 ° C for 4 hours with continuous agitation with pH 2 (Sendai virus-free) concentrated alpha interferon at 1500 IU / ml. The interferon alpha used for pretreatment is then removed by washing twice with Hanks' solution. Cells are treated with concanavalin A at 15 μg / ml and incubated at 37 ° C for 12 hours with continuous agitation. The solution of the supernatant above has a gamma-35 interferon concentration of 50,000 U / ml.

8 74978

For comparison, the above example is performed without leukocytes being first treated with alpha interferon before the addition of the inducer. The gamma interferon titer thus obtained is only 6500. U / ml.

5

Example 2

The procedure is as described for Example 1, but the medium is a Glasgow-type MEM medium (Virology 14, 359, (1961)). The amount of crude gamma interferon-10 thus obtained is ^ 8000 U / ml.

The above procedure is repeated but the alpha interferon treatment is omitted. The amount of crude gamma interferon active ingredient thus obtained is only 6200 U / ml.

15 Example 3

The procedure is as described for Example 1, but the alpha interferon used in the pretreatment is not removed. After completion of the alpha interferon treatment, the cells are incubated with constant agitation in the presence of concanavalin A at 37 ° C for 12 hours. The above solution of the precipitate has an interferon concentration of 52,000 U / ml.

The product thus obtained, containing interferon alpha and gamma, is separated into its components by CPG glass chromatography. The crude product is loaded onto a CPG glass-filled column, with alpha interferon and impurities flowing through the column as interferon-gamma binds. Interferon-gamma is eluted with an aqueous solution containing 20 volumes of ethylene glycol, 150 millimoles of sodium chloride and 20 millimoles of phosphate buffer.

30

Example 4

The procedure is as described in Example 1, but in the pretreatment step interferon beta is used instead of interferon beta at 2500 IU / ml. The concentration of interferon gamma-35 thus produced in the crude product is 56,000 U / ml.

The above procedure is repeated with the difference of 9,74978 that beta interferon treatment is omitted. The crude gamma interferon thus obtained has an active ingredient content of 6800 U / ml.

*

Claims (8)

A process for the preparation of human gamma interferon, according to which the process of separating the blood leukocyte fraction (buffy coat), removing the erythrocytes, suspending the leukocytes in a suitable nutrient medium, treating them with a mitogenic agent and separating the solution containing interferon from the cells, characterized by pretreating the leukocytes with alpha or beta interferon before treatment with the mitogenic agent. 2. A process according to claim 1, characterized in that the pretreatment is carried out with an amount of between 1000 and 2000 IU / ml, preferably 1500 IU / ml, alpha interferon. 15 3. Process according to claim 1, characterized in that the pretreatment is carried out with an amount of between 2000 and 3000 IU / ml, preferably 2500 IU / ml, beta-interferon. Method according to any of claims 1-3, characterized in that the pretreatment with alpha or beta interferon is carried out for a period of between 1 and 12 hours, preferably for 4 hours. Process according to any of claims 1 to 4, characterized in that the alpha or beta interferon used in the pretreatment is washed prior to the treatment with the mitogenic agent. Process according to any one of claims 1 to 5, characterized in that, as a mitogenic agent, concanavalin A, phytohemagglutin or enterotoxin are used. 30 Process according to any of claims 1-6, characterized in that the erythrocytes are removed from the buffy coat fraction by hemolysis with ammonium chloride. Process according to any of claims 1-7, characterized in that an aqueous solution containing 175 to 350 mg / 1 calcium chloride, 300 to 500 mg / 1 potassium chloride, 175 to 500 mg / 1 magnesium sulfate or an equivalent is used as a nutrient medium. amount of magnesium chloride