JPS6251114B2 - - Google Patents

Description

[Detailed description of the invention]

The production of human interferon from human-derived cells has attracted worldwide attention. This is because interferons have therapeutic potential for acute and chronic infections in conjunction with the treatment of cancer. Currently, established methods cannot provide the required amounts of interferon, as conventional methods of derivation from tissue culture cells produce only low concentrations of interferon. Interferon production by previously reported methods is usually carried out by adding an interferon inducer, such as a viral inducer or a nucleic acid-based inducer, to the culture medium of human cells. After this induction period, the cells are removed from the interferon-containing cell supernatant, and the remaining inducer is inactivated by adding mineral acid and treating for several days. The crude interferon thus obtained can then be concentrated and purified by known methods. Please refer to DE-OS2724918. The human cells commonly used to induce interferons are G. Klein's permanent cell lines such as Nomalwa cells.
et al.: Intern.J.Cancer 10 , 44-57, 1972)
or human fibroblasts, in this case diploids with a limited lifespan or heteroploids with unlimited division potential. The viruses used for induction are usually paramyxoviruses such as Hematopoietic Virus Japan (HVJ = Sendai virus) or New Katusuru disease virus;
tongue) virus. Nucleic acid-based inducers are usually double helical ribonucleic acids and poly(I:C), used alone or in combination with metabolic inhibitors and/or polycations. Surprisingly, according to the invention, the addition of a group of so-called "stimulants" or mixtures of stimulants to a cell culture medium increases the number of times (from 4 to 60 They found that the amount of interferon produced was 100%. The stimulant is preferably added before inducing interferon with an interferon-inducing factor. The increase in interferon according to the present invention is based on the increase in the percentage of interferon-producing cells and the increase in the yield of interferon per cell. Stimulating substances include substances that, when added to a cell culture, increase the proportion of interferon-producing cells. These stimulants are defined as substances that, when added to the culture of cells cultured in medium with optimal nutritional conditions, cause cells to transcend from the logarithmic phase of development to the resting phase. This resting phase is the cell's
In terms of DNA (deoxyribonucleic acid) content, it is a post-mitotic stage (similar to the Gl phase of the cell cycle) without DNA synthesis. Substances which have been found to be particularly useful as stimulants according to the invention include N.
N′-diacetylated diamines, especially α·ω-derivatives such as penta-methylenebisacetamide, hexamethylenebisacetamide or heptamethylenebisacetamide, organic sulfur compounds such as sulfoxides and dimethylsulfoxide, or 11β- Glucocorticoids substituted in positions with fluorine, chlorine or bromine atoms, or hydroxy or hydroxymethyl groups, such as hydrocortisone, dexamethazone, prednisolone, aldosterone, triamcinolone, cortexolone ( cortexolone), or mixtures thereof, or mixtures of the above-mentioned stimulants and saturated aliphatic carboxylic acids or salts thereof. The final concentration of these irritants is between 0.01 micromol/liter and 300 mmol/liter. However, the final concentration for use as a stimulant is 1 to 10 mmol/liter for diacetylated diamines and for organic sulfur compounds such as dimethyl sulfoxide.
50 to 300 mmol/liter, and 0.01 to 10 micromol/liter for glucocorticoids. Irritant substances that have been found to be particularly potent are hexamethylene bisacetamide, dimethyl sulfoxide, triamcinolone, hydrocoach and prednisolone. An advantageous method according to the invention is as follows. Namalwa cells are grown in a growth medium, preferably a medium
RPMI1640 (Gibco, USA or
Flow Inc., USA, GB). This medium consists of tryptic phosphate broth (10 to
20% by volume, but preferably 20% by volume), partially purified human serum (1 to 20% by volume, but advantageously 2 to 6% by volume). Alternatively, human serum may be replaced with serum or serum fractions of other species, such as fetal bovine serum at 5 to 15% by volume. Cell growth in suspension ranges from 0.2 to 5 x ¹⁰⁶ cells/
To such a medium maintained at a density of cc, preferably 0.5 to 1×10 ⁶ cells/cc, a stimulant is added, preferably at a concentration of 1 mmol/ml. The cells are incubated in these conditions at a preferred temperature of 35 to 37 degrees Celsius for 6 to 72 hours. 24 to 48 hours is advantageous. After this stimulation period, the cells are harvested, preferably by centrifugation, and resuspended in approximately 50×10 ⁶ cells/ml of medium with or without the addition of stimulants. This suspension contains an interferon-inducing substance called HVJ (Sendai virus).
and incubate for about 1 to 3 hours. The thus treated cells were collected by centrifugation and placed in serum-free cell culture medium (Gibco, USA or Flow, GB, USA), for example.
Resuspend in Eagle's Minimal essential medium,
35 to 39 degrees C, PH of 6.7 to 8.0 preferably 7.3
The cells are incubated for 15 to 24 hours, preferably 18 to 20 hours, with or without stimulants. The suspension thus obtained containing the crude interferon is separated from the cells and cell debris. To inactivate any remaining induced virus,
A mineral acid such as hydrochloric acid is added to the supernatant and cultured at 0 to 4 degrees Celsius for 3 to 5 days. The following purification steps may be applied to further purify, concentrate and stabilize the crude human N. gondii interferon. (a) The resulting solution is neutralized with sodium hydroxide, the interferon is precipitated from the solution by adding a zinc salt such as zinc acetate, and the interferon is made into a lump by centrifugation. The resulting mass is solubilized in cold dilute hydrochloric acid, such as 0.1N hydrochloric acid. (b) The solution is clarified by centrifugation, and an ion exchanger, preferably SP-Sephadex C-25 (Pharmacia Fine
Chemicals Uppsala, Sweden) and stir mechanically for several hours. After the interferon is adsorbed onto the ion exchanger, the remaining zinc is removed by repeated washing with a buffer solution. For example, wash three times with 0.1 mol/liter glycine/hydrochloride buffer at PH 2.5, then wash with 0.1 mol/liter potassium citrate and 0.005 mol/liter disodium ethylenediaminetetraacetate at PH 4.0. Wash three times with buffer. After the washing operation, the ion exchanger including the adsorbed interferon is suspended in a citrate buffer and transferred to a chromatographic column. Interferon elutes from the ion exchanger with moderately basic buffers. Tobeba, 1
0.1 containing mole/liter potassium chloride
Mol/liter potassium phosphate (PH=8.0)
use. (c) The protein-containing fraction of the eluate is collected at low temperature and adjusted to pH 3.5. After clarification by centrifugation, a potassium thiocyanate solution is added to precipitate interferon from the clarified supernatant. The precipitated protein is allowed to precipitate, collected by centrifugation, and added to a small volume of moderately basic buffer.
Dissolve in 0.1 mol/liter potassium phosphate, pH 8, and ultracentrifuge. (d) Purify the supernatant using column chromatography. Preferably Sephadex G-100 with particle size between 40 and 120 micrometers.
(Pharmacia Fine Chemicals, Uppsala,
Sweden) and eluted with phosphate buffered saline containing 0.001 mol/liter disodium ethylenediaminetetraacetate.
Before applying the interferon solution, the column is usually standardized with a protein of known molecular weight. Collect eluates corresponding to proteins with molecular weights ranging from 10,000 to 35,000 Daltons. (e) The collected fractions should be adjusted to pH 3 preferably with hydrochloric acid.
Apply to a small column containing an ion exchanger of SP-Sephadex (see step b), which has been prewashed with 0.1 mol/liter of potassium citrate, PH=3. At this pH, interferon is adsorbed on the ion exchanger, so after washing with citrate buffer, it can be eluted with 0.1 mol/liter of phosphate buffer at pH=8. Fractions containing protein are pooled and dialyzed against phosphate buffered saline. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Preparation Namalwa cells are grown in suspension culture in fermentation vessels. The medium contains RPMI 1640 medium supplemented with 20% by volume tryptose phosphate broth and 2 to 4% by volume fractionated human serum.
To fractionate human serum, serum proteins are partially precipitated with 6% polyethylene glycol 6000.
See (Inglot et al., Acta Virol. 19 , 250-254, 1975). Cells grow from 2 to 7×10 ⁶ cells/ml. Sendai virus (HVJ) is grown in chicken embryos and purified using high-speed centrifugation. Sendai virus is suspended in cell culture by ultrasonication and
Store at 70 degrees C. Example 1 Namalwa cells in logarithmic growth phase are suspended in growth medium at a density of 5×10 ⁵ cells/ml. To this cell suspension, the stimulant is added and treated at a concentration of C mmol/liter at 37°C. During treatment with stimulants, cells
The rate of DNA synthesis will be 2% lower than in control cultures (no stimulant). Cells become quiescent, which corresponds to the Gl phase of the cell cycle. After this treatment, the cells were collected by centrifugation (700 x g, 15 min) and incubated in a roller bottle at 5 x 10 ⁷ cells/ml in 1.7 liters of growth medium with 2 ¹⁰ clot units/ml HVJ. Incubate for an additional 2 hours at 37°C. The cells thus induced were collected by centrifugation and incubated at a density of 5 x 10 ⁶ cells/ml in 17 liters of serum-free Eagle's Minimum essential medium (using Earle's salts) at pH 7.3. Incubate for an additional 30 hours at 35.5°C. At this stage, 20 liter bottles are used with a mechanical stirrer. The cellular material was centrifuged at 2000 x g for 30 minutes to form a clump, and the crude solution (cell supernatant) was brought to pH 2 with 5N hydrochloric acid at a temperature below 10 degrees C, stored at 4 degrees C for 3 to 5 days, and interfermented. Inactivates HVJ used to induce elon. The following table shows the increase in interferon when n-lactate is added to C mmol/liter.

Table: Example 2 Namalwa cells are cultured in 85 liters of growth medium to a density of 5×10 ⁵ cells/ml. At this stage, the cells are in log phase
DNA synthesis is at its maximum. The stimulant is added as an aqueous solution to the cell culture medium at C mmol/liter and treated at 35.5 to 37 degrees C for time t. During this treatment, DNA synthesis occurs in the untreated culture.
12%, and most of the cells are in the quiescent phase, which corresponds to the Gl phase of the cell cycle. Cells are agglomerated at 700×g by continuous flow centrifugation. The cells thus collected are resuspended in 9 liters of growth medium to a density of 1×10 ⁷ cells/ml. In this medium, the stimulant is C mmol/
Contains in a liter. Also add ²⁸ clotting units/ml HVJ and incubate for 4 hours at 35.5 to 37 degrees Celsius in a mechanically stirred bottle.
The cells induced in this way are collected by centrifugation,
18 liters of serum-free Eagle's minimum
Suspend at 5×10 ⁶ cells/ml in essential medium (Earle's salts) and incubate at 36° C. at pH 7.1 in a 20 liter container with mechanical stirring. Cell material was removed by centrifugation (2000 x g, 30 min).
Cell supernatants are obtained as crude interferon preparations. Add 5N hydrochloric acid to adjust the pH to 2, and keep at 4 degrees Celsius for 3 to 5 days to inactivate the virus inducer.
As shown in the following table, the yield of interferon increases after adding the stimulant hexamethylene bisacetamide to C mmol/liter.

[Table] Unit Example 3 Namalwa cells are cultured in 85 liters of growth medium to a density of 1×10 ⁶ cells/ml. Cells enter a logarithmic phase of growth, and DNA synthesis is at its highest rate. 2.5 mmol of propionic acid/
Little and hydrocortisone 1 micromol/
A mixture of stimulants mixed with a small amount of stimulants is added to the neutral solution and treated at 35.5 to 37 degrees C for 48 hours.
The rate of DNA synthesis is 5% lower than the untreated control within 48 hours. Most of the cells enter a resting phase similar to the Gl phase. Cells were centrifuged at 700× in a continuous flow centrifuge.
Sediment at g. The cells thus collected were suspended in 2 liters of growth medium at a density of 4.5 x 10 ⁷ cells/ml, HVJ was added at ²¹⁰ clot units/ml, and incubated at 37°C for 2 hours. Incubate. Cells are then collected and resuspended in 85 liters of serum-free medium to a density of 1×10 ⁶ cells/ml. 35.5 to 37 at PH7.3
Cells cultured in suspension at 10°C for 36 hours are removed by continuous flow centrifugation (2000xg), and the cell suspension is adjusted to pH=2 with 5N hydrochloric acid at a temperature lower than 10°C. Then keep at 4 degrees C for 3 to 5 days. The crude interferon preparation thus obtained contains 22 times more interferon than a control culture not previously treated with stimulants. (Control culture: 1050 IU interferon/10 ⁶ cells; propionic acid + hydrocortisone treated culture: 23200 IU interferon/10 ⁶ cells). Example 4 Analogously to the previous example, Namala cells at a density of 5×10 ⁵ cells/ml are treated with the following stimulants: With this treatment, the interferon yield increases by a factor x compared to untreated control cultures.

[Table] Side dimethyl sulfoki 280 16
Side Example 5 Analogously to the previous example, Namalwa cells at a density of 5×10 ⁵ cells/ml are treated with the following stimulants. The yield of interferon is increased by a factor of X compared to untreated control cultures.

[Table] 〓 〓 〓 〓 〓 〓 〓 〓 〓 〓 〓 〓 〓
〓 〓 〓 〓 〓 〓 〓 〓 〓
Prednisone 10 3.5

Claims (1)

[Scope of Claims] 1. Before incubating the cells of a permanently culturable cell line derived from N. gondii blast cells with a viral interferon-inducing factor, the N. gondii blast cells, which are in the logarithmic growth phase, are removed from their growth phase. are treated under optimal growth conditions with a stimulant that moves them into a resting phase, defined as a phase in which cells correspond to post-mitotic ( _G1 phase) cells in terms of their DNA content, but no DNA synthesis is occurring; This then optionally consists in separating and/or purifying the interferon produced during the incubation in a manner known per se, and as said stimulant a diacetylated diamine compound, an organic sulfoxide compound or the 11β-position. using a glucocorticoid compound in which is substituted with a hydrophilic group, a mixture thereof, or a mixture consisting of these stimulating substances and a saturated aliphatic carboxylic acid or a salt thereof, with the exception that a linear saturated aliphatic carboxylic acid or a salt thereof A method for producing interferon, which excludes the use of interferon as a sole stimulant. 2 The stimulant is an N・N′-diacetylated α・ω-diamine compound having 4 to 8 carbon atoms, dimethyl sulfoxide, or a glucocorticoid compound in which the 11β-position is substituted with a hydroxy group or a hydroxymethyl group, and those or a mixture of these stimulating substances and an alkanoic acid having 3 to 6 carbon atoms or a salt thereof. 3. Irritating substances include pentamethylene bisacetamide, hexamethylene bisacetamide, heptamethylene bisacetamide, dimethyl sulfoxide, prednisolone, dexamethasone, triamcinolone, hydrocortisone, 11-desoxycortisone and prednisolone, and mixtures thereof, or these irritating substances. and propionic acid, n-lactic acid, isolactic acid, n-valeric acid, caproic acid, or their salts with inorganic or organic bases, and the stimulant precedes the addition of the interferon-inducing factor. 2. The method according to claim 1, wherein the cell is added to the cells between 6 and 72 hours, preferably between 24 and 48 hours. 4. The method according to any one of claims 1 to 3, wherein the final concentration of the irritant substance is between 0.01 micromol/liter and 300 mmol/liter. 5 N・having 4 to 8 carbon atoms as the stimulant
According to any one of claims 1 to 3, the N'-diacetylated diamine compound and/or dimethyl sulfoxide are used in a final concentration of 0.1 micromol/liter to 250 mmol/liter. the method of.