WO1983001899A1

WO1983001899A1 - Process for the production of human gamma-interferon

Info

Publication number: WO1983001899A1
Application number: PCT/HU1982/000064
Authority: WO
Inventors: Gyogyszervegyészeti Cyar Egyt
Original assignee: BÉLADI, Ilona; TOTH, Miklós; ROSZTOCZY, István; TOTH, Sándor; ENDRÉSZ, Valéria
Priority date: 1981-12-01
Filing date: 1982-11-30
Publication date: 1983-06-09
Also published as: SU1405691A3; FI832508L; HU184972B; CH658391A5; FI832508A0; DE3249215C2; GB2123007B; FI74978B; AU551964B2; FI74978C; AU1017483A; DE3249215T1; GB8320371D0; GB2123007A; JPS58502032A

Abstract

Process for the production of human gamma-interferon by separating the leukocyte fraction (buffy coat) of blood, removing the erythrocytes, suspending the leukocytes in a suitable nutrient medium, treating the same with a mitogenic agent and separating the liquid containing interferon from the cells which comprises pre-treating the leukocytes with 200-20000 IU/ml. of alpha- or beta-interferon prior to treatment with the mitogenic agent. The advantage of the process is that the gamma-interferon production is increased by 500-1000 %.

Description

Process for the production of human gamma-interferon

Technical Field

The invention relates to a new and improved process for the production of human gamma-interferon.

Background Art

It is known that human leukocytes produce gamma- interferon (immune interferon) under the effect of mitogenic agents. Gamma-interferon differs however in its biological properties from the leukocyte (alpha) and fibroblast (beta) interferons produced as a result of virus infection from leukocytes and formed under the influence of synthetic polynucleotides in cells of fibroblast origin, respectively. Due to its cell division inhibiting and the function of the cells of the immune system influencing effect gεimna-interferon exhibits signific-antly more preferable effects than the alpha- and beta-interferons and consequently gamma-interferon will be more and more widespreadly used in the therapy of tumorous diseases [Nature 294, 6 (1981), Celular Immunology 49, 390 (1980)]. Gamma-interferon can be produced by separating the leukocyte fraction (buffy coat) of blood, removing the erythrocites by gradient centrifuging or preferably by haemolysis carried out by treatment with ammonium chloride, incubating the purified leukocytes, treating the same with suitable aspecific mitogenic agents and finally isolating the gamma-interferon thus produced from the supernatant liquid. According to prior art Concanavalin A [Infect. Immun. 26, 36 (1979)], Phytohaemagglutinin [Proc. Natl. Acad. Sci. USA 78, 1601 (1981)] or Enterotoxin [int. Res. Commun. Sys. Med. Sci. 7, 595 (1979)] can be used as mitogenic agent. According to prior art several attempts were made to increase the gamma interferon production induced by mitagenic agents. According to a process gamma- interferon production is increased by pre-treatment of the leukocytes with 12-0-tetradekanoyl-phorbol-13-acetate [Vilcek et al.: Biochemical Characterization of

Lymphokines - Academia, New York, page 323, (1980)]. The drawback of the said method resides in the fact that 12-0-tetradekanoyl-phorbol-13-acetate is a cancerogenic substance and consequently the gamma-interferon thus produced is evidently unsuitable for use in human therapy. According to an other reference [Nature 292, 842 (1981)] the authors tried to increase the gamma- interferon production by treating the leukocytes with sodium butylate or dexamethason, these attemps were however unsuccessful.

Disclosure of invention

The object of the invention is to increase the gamma-interferon production of leukocytes induced by mitogenic agents and to raise the number of units per ml.

According to the present invention there is provided a process for the production of human gamma-interferon by separating the leukocyte fraction (buffy coat) of blood, removing the erythrocytes, suspending the leukocytes in a suitable nutrient medium, treating the same with a mitogenic agent and separating the liquid containing interferon from the cells which comprises pre-treating the leukocytes with -preferably-200-20000IU/ml.of alpha- or beta-interferon prior to treatment with the mitogenic agent.

The present invention is based on the recognition that the gamma-interferon production can be significantly increased - by about 500-1000 % - by subjecting the leukocytes to pre-treatment with alpha- or beta-interferon prior to the treatment with the mitogenic agent. The alpha- or beta-interferon is used for the pre-treatment in an amount of 200-20000 IU/ml., preferably 500-5000 IU/ml. (IU = International Unit).

According to a preferred embodiment of cur process the said treatment is carried out by using 1000-2000 - particularly 1500 - IU/ml. of alpha-interferon. According to an other preferred embodiment of our process the said treatment is carried out by using 2000-3000 - particularly 2500 - IU/ml. of beta- interferon.

Pre-treatment is preferably carried out 1-12 hours, particularly 2-8 hours, particularly preferably 4 hours prior to the treatment with the mitagenic agent.

The pre-treatment with alpha- or beta-interferon is preferably carried out for about 1-12 hours, particularly for about 4 hours. The temperature of the said pre-treatment is preferably 35~39 °C, particularly 37 °C.

Interferon is preferably removed after the pretreatment with alpha- or beta-interferon and before the addition of the mitagenic agent. This step can be preferably carried out by washing the cells, particularly with a Hanks-solution. One may also proceed by leaving the interferon used for pre-treatment in the nutrient medium of the cells. In this case the gamma-interferon produced must be separated from the alpha- or beta- interferon used in the pre-treatment step. The separation can be carried out by methods known per se, e.g. by glass chromatography.

As nutrient medium in the process of the present invention tissue culture nutrient media containing various amino acids and vitamines can be used (e.g. Eagle-type MEM, RPMI 1640, Dulbecco type MEM, Glasgow modified MEM etc.). It is preferred to use a nutrient medium having the following composition; the advantage of the latter nutrient medium is that it is inexpensive, simple and readily applicable in an autoclave.

Component Amount

Calcium chloride 175-350 mg./l.

Potassium chloride 300-500 mg./l.

Magnesium sulfate or an equivalent amount of magnesium chloride 175-500 mg./l.

Sodium chloride 5000-7000 mg./l.

Sodium hydrogen carbonate 200-3500 mg./l.

Sodium dihydrogen phosphate 30-150 mg./l. Glucose 500-5500 mg./l.

Ferric nitrate 0-0.2 mg./l.

Serum 0.5-10 mg./l.

Gamma-interferon production is carried out in the presence of various animal or human blood-serum or gamma-globuline free plasma (0.5-10 %) .

According to the process of the present invention leukocytes (buffy coat) obtained from anticoagulated human blood stored for not more than 72 hours at 0-8°C are used as starting material. An ACD solution (a solution containing citric acid and dextrose) or an ACD solution completed with, various bases (e.g. adenine or guanine) can be used as anticoagulant. The collected leukocytes can beremoved by gradient centrifuging (e.g.with the aid of Ficoll or Percoll - manufacturer: Pharmacia Sweden) or preferably by haemolysis carried out with ammonium chloride. One may proceed preferably by admixing the concentrated leukocyte suspension with a 0.5-1% - preferably 0.83 % - ammonium, chloride solution at a temperature of 0-10 ºC in a volume/volume ratio of 1 : 3-20, preferably 1 : 5. Tfce suspension is incubated for 5-20 minutes - preferably for about 10 minutes - at 0-8 °C under stirring or without the same. The leukocytes are sep.arated from the disintegrated erythrocytes e.g. by centrifuging. One may proceed preferably by repeating the ammonium chloride treatment by using 10 parts by volume of the ammonium chloride solution per 1 part by weight of cell suspension. The purified leukocytes are suspended in a suitable tissue culture nutrient medium (e.g. Eagle-type MEM, EPMI 1640, Dulbecco-type MEM, Glasgow modified. MEM, etc.) or a cheap nutrient medium disclosed above (containing no amino acids and vitamines ) and the cell number is adjusted to a value between 10⁶ and 10⁸ cells/ml.

According to an advantageous form of realization of the process the cheap nutrient medium disclosed above and containing no amino. acids and vitamines is used both for re-suspension of the cells and adjusting the cell number. The nutrient medium or nutrient solution used is completed with an animal or human blood serum or preferably with human gamma-globuline free plasma (0.5-10 %) . It is preferred to add an antibiotic to the nutrient medium or nutrient solution; for this purpose advantageously neomycin can be used in a concentration of about 10-50 μg./ml., particularly

25 μg./ml.

Tho cells are then subjected to treatment with alpha- or beta-interferon. For this purpose inducerfree (virus or synthetic polynucleotide free) crude or purified alpha- or beta-interferon can be used in an amount of 200-20000 IU/ml. The said pre-treatment is carried out at a temperature of about 35-39 ºC, preferably at 37 °C for about 1-12 hours, particularly for 4 hours.

The interferon used in the said pre-treatment step may be removed from the cells, preferably by washing. This step can be accomplished preferably by using a Hanks solution. After, the interferon has been washed out the cell concentration is adjusted to the original value in a nutrient medium or nutrient solution which contains human or animal serum or gamma-globuline free plasma, preferably gamma-globuline free human serum. It is not absolutely necessary to wash out the interferon because the presence of alpha- or beta-interferon does not effect the production of gamma-interferon in an adverse manner. In this case the gamma- interferon produced 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 preferably Concanavalin A, Phytohaemagglutinin, Staphylococcus enterotoxin etc. - referred to above - can be used. According to a preferred embodiment of the process of the present invention Concanavalin A is used as inducer, preferably in a concentration of 2.5-30 μg/ml., particularly 15 /ug./ml. Induction is generally carried out for about 5-48 hours, preferably for 10-12 hours at a temperature of about 35-39 °C, preferably at 37 °C After a certain period of time (about an hour) the inducer can be removed by washing; this step can, however, be omitted because the presence of the inducer does not damage the gamma-interferon production. The inducer is therefore generally not removed.

Thereafter the cells are removed from the super natant layer by centrifuging. The supernatant layer contains the crude gamma-interferon which can be stored at a low temperature (approximately at about -20 °C) or purified by known methods.

Industrial Applicability The advantage of the process of the present invention is that as a result of the pre-treatment

1 carried out with alpha- or beta-interferon-fche production of gamma-interferon is increased by five to ten times. The physicochemical properties (pH 2 sensitivity, stability measured at 56 °C and 37 °C) and the biological activity (antiviral and anticellular effect) of the gamma-interferon produced by the process of the present invention are completely equivalent to and equal with those of gamma-interferon produced by conventional methods.

Modes of Carrying out the Invention

Further details of the present invention are to be found in the following Examples without limiting the scope of the invention to the said Examples.

Example 1

Blood obtained in an ACD solution (aqueous solution containing citric acid and dextrose) is stored at +4 °C for 3 hours. The "buffy coat" fraction is collected by centrifuging and allowed to stand overnight at +4 °C. 1 part by volume of a leukocyte concentrate thus obtained is admixed with 5 parts by weight of a 0.83% aqueous ammonium chloride solution (temperature: +4 ºC). The suspension is allowed to stand at +4 ºC until the erythrocites are dissolved (5-10 minutes) and the leukocytes are isolated by centrifuging. The leukocytes are suspended in a nutrient medium having the following composition:

Component Amount, mg./l.

Calcium chloride 265

Ferric nitrate 0.1 Potassium chloride 400

Magnesium sulfate heptahydrate 200

Sodium chloride 6400

Sodium hydrogen carbonate 2750

Sodium dihydrogen phosphate dihydrate 140 Glucose 4500

The dissolving of the erythrocytes by the above method is repeated except that 10 parts by volume of a

0.83 % aqueous ammonium chloride solution are used.

The leucocytes are suspended in a nutrient solution having the above composition and the cell number is adjusted to the value of 10⁷/ml. The nutrient solution contains 2 mg./ml. of human agamma serum and 25 μg./ml. of neomycin . The cells are treated with an amount of 1500 IU/ml. of pH 2 treated (made free of Sendai virus) concentrated alpha-interferon at 37 °C for 4 hours under constant stirring. Thereafter the alpha-inter- feron used for pre-treatment is removed by washing with. a Hanks-solution twice. The cells are treated with an amount of 15 μg./ml. of Concanavalin A and incubated at 37 ºC for 12 hours under constant stirring. The gamma- interferon content of the supernatant liquid amounts to

50000IU/ml.

For the sake of comparison the above Example is carried out except that the leukocytes are not treated with alpha-interferon before the addition of the inducer. The gamma-interferon titer thus obtained amounts only to 6500 IU/ml.

Example 2 One proceeds as described in Example 1 except that as nutrient solution a Glasgow-type MEM nutrient medium is used [Virology 14, 359 (1961)]. The active ingredient content of the crude gamma-interferon thus obtained amounts to 48000 IU/ml. The above process is carried out except that the treatment with alpha-interferon is omitted. The active ingredient content of Mie crude gamma-interferon thus obtained amounts but to 6200 IU/ml.

Example 3 One proceeds as described in Example 1 except that the alpha-interferon used for the pre-treatment is not removed. The treatment with alpha-interferon having been completed the cells are incubated by stirring in the presence of 15 μg./ml. of Concanavalin A at 37 °C for 12 hours under constant stirring. The interferon content of the supernatant liquid amounts to 52000 IU/min.

The product thus obtained, containing alpha- and gamma-interferon, is separated into its components by CPG glass cliromatography. The crude product is applied onto a column filled with CPG glass, the alpha-interferon and the contaminations pass through the column while the gamma-interferon is bound. The gamma-inter- feron is eluted with an aqueous solution containing 20 parts by volume of ethylene glycol, 150 millimoles of sodium chloride and 20 millimoles of phosphate buffer. Example 4 One proceeds as described in Example 1 except that in the pre-treatment step in the place of alpha-inter-feron 2500 IU/ml. of beta-interferon are used. The gamma-interferon content of the crude product thus obtained amounts to 56000 IU/ml.

The above process is repeated except that the pretreatment with beta-interferon is omitted. Thus crude-ganima-interferon having an active ingredient content of 6800 IU/ml. is obtained.

Claims

What we claims is:

1. Process for the production of human gamma- interferon by separating the leukocyte fraction (buffy coat) of blood, removing the erythrocytes, suspending the leukocytes in a suitable nutrient medium, treating the same with a mitogenic agent and separating the liquid containing interferon from the cells which comprises pre-treating the leukocytes with alpha- or beta-interferon prior to treatment with the mitogenic agent.

2. Process according to Claim 1 which comprises carrying out the pre-treatment with an amount of 200-20000, preferably 500-5000 IU/ml. of alpha- or beta-interferon. 3o Process according to Claim 1 or 2 which comprises carrying out pre-treatment with an amount of 1000-2000 IU/ml. - preferably 1500 IU/ml. - of alpha- interferon.

4. Process according to Claim 1 or 2 which comprises carrying out pre-treatment with an amount of

2000-3000 IU/ml. - preferably 2500 IU/ml. - of beta- interferon.

5. Process according to any of Claims 1 to 4 which comprises carrying out pre-treatment 1-12 hours, preferably 2-3 hours, particularly preferably 4 hours prior to the treatment with the mitogenic agent.

6. Process according to any of Claims 1 to 5 which comprises carrying out pre-treatment with alpha- or beta-interferon for 1 to 12 hours, preferably for 4 hours.

7. Process according to any of Claims 1 to 6 which comprises washing out the alpha- or beta-interferon used in the pre-treatment step prior to the treatment with the mitogenic agent.

8. Process according to any of Claims 1 to 7 which comprises using Concanavalin A, Phytohaemagglutinin or Enterotoxin as mitogenic agent.

9. Process according to any of Claims 1 to 8 which comprises removing the erythrocites from the buffy coat fraction by means of haemolysis with ammonium chloride.

10. Process according to any of Claims 1 to 9 which comprises using as nutrient medium an aqueous solution containing 175-350 mg./l. of calcium chloride; 300-500 mg./l. of potassium chloride; 175-500 mg./l. of magnesium sulfate or an equivalent amount of magnesium chloride; 5000-7000 mg./l. of sodium chloride; 200-3500 mg./l. of sodium hydrogen carbonate; 30-150 mg./l of sodium dihydrogen phosphate; 500-5500 mg./l. of glucoseand optionally 0.05-0.2 mg./l. of ferric nitrate and 0.5-10 % of serum.