DE2946275A1 - Antiviral and antitumour human interferon prodn. - by suspension culture of akuba, RN-2, JHTC 33, P3HR-1 or NC-37 cells treated with stimulator substances and viral inducers - Google Patents

Abstract

In a new process for the prodn. of human interferon, Akuba, RN-2, JHTC33, P3HR-1 or NC-37 cells cultivated in suspension are treated with one or more stimulator substances and interferon- prodn. is induced by means of viral inducers e.g. Sendai virus, and the interferon thus produced is sepd. off and/or purified. Pref. stimulator substances are short-chain fatty acids (e.g. n-butyric acid), pref. used in a final concn. of 0.5-5 mmol/litre. Prodn. of human interferon for the prophylaxis and treatment of viral infections or for cancer therapy. Addn. of stimulator substances to cultures of the specified cell types gives high interferon yields (up to 100,000 IU/ml) and high potency (1,000,000 IU/mg protein) without the need for additional purificn. steps.

Description

Improved process for making human inter-

feron from lymphoblastoid cells The production of umaninterferon from cells of human origin has due to the medical importance of interferons in the treatment of acute and chronic viral infections, as well as in cancer therapy received worldwide attention. The quantities of interferon required for this can be used however, not made available by the previously known manufacturing processes because only low concentrations of iluman interferon can be achieved.

The production of the interferons takes place in the previously known processes by treating the cells with an interferon inducer (e.g. virus or nucleic acid) and subsequent incubation of the treated cells in cell culture medium. The interferon yield can be increased here if cells are tight before induction with small Interferon, the so-called priming (see Stewart et al. In J. Virol. 7, 792 (1971)), be treated.

Since interferons are species-specific, they can be used to produce human interferons only use human cells. Come for it both cells of the fibroblast type, which are cultivated on surfaces, as well as cells of hematopoietic origin which are cultured in suspension can. The latter group includes human lymphoblastoid cells, which have the advantage of indefinite time in suspension culture with a minimum to be propagated in technical manipulation. Proved particularly suitable the cell line Namalwa (Strander l!., Mogensen, K.E. and Cantell, K .: Production of human lymphoblastoid interferon in Journal of Clinical Microbiology 1, 116 (1975)). These cells produce after induction with Sendai Virus or Newcastle Disease Virus 102-104 international units (IU) inferon / ml.

It is also known (see EU-OS 0.000.520) that by pretreatment of Namalwa cells with short chain fatty acids prior to priming and virus induction themselves increases the production of interferon and reaches values of up to 6 x 104 IU / ml.

Surprisingly, it has now been found that by adding one or several stimulator substances, such as a short-chain fatty acid, to very specific ones human lymphoblastoid cells, namely to Akuba-, RN-2-, JHTC33-, P3HR-1- and NC-37 cells, interferon production can be increased significantly more than with Namalwa cells and that interferon levels of 10 IU / ml can be produced, being the purity of such a preparation with respect to total protein of the harvest solution is 106 IU / mg protein without the use of further purification steps.

In particular, a saturated, aliphatic substance has proven to be the stimulator substance Carboxylic acids, e.g. an alkanoic acid with 3-6 carbon atoms, such as propionic acid, n-Butyric acid, isobutyric acid, valeric acid, caproic acid and their salts with inorganic ones or organic bases, but especially their alkali salts, and as lymphoblastic bases Cell line in particular the line NC-37 proven, which by Durr, F.E., Monroe, J.H., Schmitter, K., Traul, K.A., and Hirsaut (Y. International Journal of Cancer 6, 436 (1970)) from peripheral blood cells of an Fpstein-Barr virus (EBV) seroactive, healthy donor, has been isolated and used by Associated Biomedical Systems Inc., Buffalo (USA). Furthermore, the Akuba-, RN-2-, JHTC33- and P3HR-1 cell line known from the literature.

NC-37 cells are grown in a growth medium, preferably the medium RPMI 1640 (from Gibco, USA or from Flow, Great Britain) cultivated, which as Contains additives 1-10% by volume, but preferably 2-6% by volume of pre-purified human serum. As an alternative or in addition to human serum, serum or serum fractions can also be used here other species can be used, e.g., fetal bovine serum at one concentration from 2-15% by volume. Lactalbumin hydrolyzate and antibiotics can be used as additional additives - be added to the growth medium.

The cells thus cultured are in suspension at a density from 0.2 - 5 x 106 cells / ml, but preferably at a density of 0.4 - 1 x 106 Cells / ml with a stimulator compound, e.g. an alkali salt of n-butyric acid, added in a final concentration of expediently 1 mmol / l and expediently incubated at a temperature of 33-390C. After a period of 6-72 hours, but preferably from 24-48 hours, the cells are collected by centrifugation and expediently at a density of 1-5 × 10 7 cells / ml in medium with or suspended without stimulator substance. This suspension can now optionally be a "Priming" with interferon are subjected: For this purpose, the cell suspension with 1-1000 IU / ml human interferon, but preferably 100-500 IU / ml human interferon, added and incubated at 37 ° C. The addition of the interferon 5 minutes before the addition of the interferon inductor has proven to be sufficient. The cell suspension, with or without zPrlmingZ becomes hemagglutinating, for example, with Sendai virus at a concentration of 29-213 Units HAU / ml, preferred but with 210-211 HAU / ml, as an interferon inducer infected and incubated at 33-39 ° C. After about 1-3 hours they become infected Cells collected by centrifugation and placed at a density of 1-5 x 106 cells / ml in a serum-free cell culture medium, e.g. in wMinlmum Essential Medium Eagle " (Gibco, USA or Flow, Great Britain), for a period of 10-24 hours, but preferably 18-20 hours, at 33-39 ° C and a pH value of the tissue culture from 6.7 to 8.0, but preferably from 7.3, incubated. The obtained solution of the Crude lymphoblastoid interferon is separated from the NC-37 cells by centrifugation and for inactivating residual inducer viruses by adding mineral acid, for example adjusted by hydrochloric acid to a pH value of preferably 2.0 and several days e.g. incubated for 1-5 days at 0-40C.

The lymphoblastoid solution obtained in this way can be used for further purification Raw interferon can be subjected to purification steps known from the literature at will.

The following examples are intended to explain the invention in more detail. but not to limit the scope of the invention: Preliminary remark: NC-37 cells are in suspension culture in shake cultures, roll cultures or in a fermentor increased. Here, the medium RPMI 1640 is used as the growth medium, the 0.1 Vol% lactalbumin hydrolyzate, 2-4 vol% pre-purified human serum and additionally or alternatively 2-5% fetal bovine serum is added. The growth medium also contains Antibiotics; either penicillin-streptomycin or gentamicin. The pre-cleaned Human serum is made by precipitating some of the serum proteins with 6% polyethylene glycol 6000 (Inglot et al. Acta Virol. 19, 250 (1975)). Alternatively, as Growth medium the minimum Essential Medium Eagle "with 5-10% fetal Bovine serum and added antibiotics are used. The Sendai virus is embryonated Chicken eggs propagated, cleaning is carried out by high-speed centrifugation and subsequent suspension in growth medium by means of ultrasound treatment. The Sendai virus is stored at -70 ° C.

Example 1 NC-37 cells at an exponential growth stage in roller bottle cultures at a density of 4 x 105 cells / ml in 2000 ml RPMI 1640 Growth medium cultured for 24 hours at 370C, and then in the presence of 1 mmol / l sodium n-butyrate was incubated in the growth medium for a further 40 hours at 370C. The cells were collected by centrifugation (700 x g, 15 minutes) and at a Density of 20 x 106 cells / ml in 66 ml growth medium at 37 ° C with 2 ml Sendai Virus (HAU = 214 / ml) shaken at 370C for two hours. The induced in this way Cells were collected by centrifugation and at a density of 2.0 x 106 Cells / ml in 660 ml serum-free Eagle's-Minimum Essential Medium (with Earle salts) incubated at 370C for 20 hours at pH 7.3 in roller bottles. The roll speed was 0.5 revolutions / minute. The crude solution (= cell supernatant) was centrifuged off of the cells (200 x g, 15 minutes) at a maximum temperature of 10 ° C with 5N hydrochloric acid adjusted to pH 2 and stored for 4 days at 40C to inactivate the Sendai virus.

The same experimental approach, but without the addition of sodium n butyrate, was used to determine the increase in interferon production by stimulator substances in NC-37 cells.

The table below contains the increase in interferon yield found in NC-37 cells after the addition of 1 mmol / l sodium n-butyrate. Concentration sodium-n- butyrate (mmol / 1) 0 1 Volume of the NC-37 cell suspension after induction (ml) 660 660 Interferon production (interna- tional units IU / ml) 30 44 200 Production increase (rector) 1 1 470 Total protein content in the raw interferon preparations (mg / ml) 0.037 0.037 specific activity of the raw interferons (IU interferon / mg Protein) 810 1,215,240 Example 2 To compare the interferon products in NC-37 cells with those in Namalwa cells and without the addition of stimulator substances, the following example was carried out: th parallel cultures were NC-37 cells and Namalwa cells at a density of 0.25 x 106 cells / ml (total volume of 500 ml each) in Minimum Essential Medium Eagle, which was completed with 10% by volume of fetal cervical serum, either with 1 mmol /! Sodium n-butyrate is added or, if left untreated, it is incubated for 26 hours at 370C. The cells were collected by centrifugation (700 × g, 15 minutes) and incubated at a density of 5 × 10 7 cells / ml at 37 ° C. for 2 hours in large culture flasks with growth medium which contained 210 HAU Sendai virus / ml. The cells induced in this way were collected by centrifugation and incubated at a density of 5 × 10 6 cells / ml in serum-free Minimum Essential Medium Eagle at 370 ° C. for 20 hours at pH = 7.3. After the cells had been centrifuged off, the crude solutions were treated analogously to Example 1.

The result of Example 2 is summarized in the table below and shows, in direct comparison, the superiority of NC-37 cells over Namalwa cells for the production of lymphoblastoid human interferon: both the increase in interferon production by sodium n-butyrate treatment and the absolute yield Interferon is higher in NC-37 cells than in Namalwa cells. Concentration Na interferon production increase factor trium-n-butyrate (IU / 106 cells): the production of interferon m moles / 1 Namalwa NC-37 Namalwa NC-37 0 280 16 1 1 7 7 270 # 21 000 26 1 300 Example 3 To determine the WPriming "effect on interferon production in NC-37 cells, the following example was carried out: NC-37 cells were cultured as in Example 1 and at a cell density of 0.55 × 10 6 cells / ml (1000 ml) with 1 mmol / l sodium n-butyrate were incubated for 48 hours at 370 ° C. The cells were harvested by centrifugation (700 × g, 15 minutes), resuspended in 22 ml growth medium and divided into two aliquots of 11 ml Human interferon was added and the mixture was shaken for 5 minutes at 37 ° C. ("priming"), the second aliquot was left untreated.

0.35 ml Sendai Virus Suspension (214 HAU / ml) was added and incubated for 2 hours at 370C with shaking. The induced in this way Cells were collected by centrifugation and at a density of 2.0 x 106 Cells / ml in serum-free Minimum Essential Medium Eagle at 370C for 18 hours at pH = 7.3 incubated. The foal solutions (after centrifuging the cells at 2000 x g, 15 minutes) were treated as in Example 1.

The results are summarized in the table below and show that by "priming" with interferon in NC-37 cells an increase in interferon production to 105 IU / ml crude solution could be achieved: Effect of "priming" with numaninterferon on interferon output in NC-37 cells: "Priming" with IU interferon / ml interferon production in NC-37 Cells IU / ml IU / mg protein Raw solution in the raw solution 0 67 900 622 500 500 97 000 970 000

Claims (10)

Claims 1. Process for the production of human interferon, characterized in that Akuba-, RN-2-, JIsTC33-, P3EIR-1- or NC-37 cells treated with one or more stimulator substances and the Interferon production is stimulated by viral inducers and the so formed Interferon is separated and / or purified. 2. The method according to claim 1, characterized in that the stimulator substances one or more short-chain fatty acids and their salts and as lynphoblastoids Cell line the NC-37 line is used. 3. The method according to claims 1 and 2, characterized in that that the lymphoblastoid cells used before induction with a "priming" substance be treated. 4. Process according to claims 1-3, characterized in that that as stimulator substances alkanoic acids with 3 to 6 carbon atoms and their Salts are used. 5. Process according to claims 1-4, characterized in that for interferon production the human, lymphoblastoid cell line NC-37, as a stimulator substance n-butyric acid and liumaninterferon is used as the "priming" substance. 6. The method according to claim 1-5, characterized in that the final concentration the stimulator substance is 0.5-5 mmol / l. 7. The method according to claims 1-6, characterized in that Human interferon added as a "priming" substance before and / or during virus induction will. 8. The method according to claims 1-7, characterized in that a viral inducer is used as the interferon inducer. 9. Human interferon produced according to claims 1-8. 10. Use of the interferon prepared according to claims 1-8 to combat viral infections, both prophylactically and therapeutically, or for cancer therapy.