CS262822B1 - Synthetic medium for the cultivation of myelomic cells - Google Patents

Abstract

Synthetic medium for cultivation of hybridoma and myeloma cells comprising anorganic salts, monosaccharides, amino acids, vitamins, substances buffering changes of pH, possibly also sodium pyruvate, antibiotics, indicators of pH and a supplement formed by at least one water soluble iron compound in a concentration of 2x10<-5> to 1x10<-2> mol/l. The effectiveness of the supplement can be increased by the presence of compounds biogenic trace elements, ascorbic acid, steroid substances and of amines. The iron compound is preferably, iron sulphate, ferric citrate or chloride or potassium ferricyanide. The medium may also contain, hydrocortisone, zinc, selenium or ethanolamine.

Description

(54)

Synthetic medium for cultivation of hybridomas and mye lord cells

Synthetic medium for the cultivation of hybridomas and myeloma cells based on inorganic salts, monoaaccharides, amino acids, vitamins and pH-buffering agents or sodium joyroate, antibiotics and pH indicator, contains water-soluble iron compounds in a concentration of 2 x 10 ^-5 to 1 x 10-2 mol / l · Furthermore, this culture medium may contain compounds of trace biogenic elements at a concentration of 0,002 to 2 mg / l> ascorbic acid at a concentration of 1 to 100 mg / l, steroids at a concentration of 0,0001 to 0,01 mg And amines at a concentration of 0.1 to 15 mg / l.

262 822

The invention relates to synthetic media or to the cultivation of hybridomas and myeloma cells.

Hybridomas constructed by somatic hybridization of myeloma cell and antibody synthesizing lymphocyte are industrially used to produce monoclonal antibodies. Myeloma cells into which a gene for a biologically active protein, such as an enzyme, an enzyme inhibitor, a hormone, an immunoregulator, is introduced by genetic engineering methods, are also industrially used to produce these proteins. The increasing need for various monoclonal antibodies, in particular for therapeutic and immunoaffinity chromatography purposes, where kilogram amounts are desired, as well as the need for various regulatory proteins for therapeutic use, has led to the search for more efficient and economically advantageous methods for producing these substances. The composition of the culture medium, the standard properties and the cost of its components are the most important factors here.

To date, hybridomas and myeloma cells have been cultured by first multiplying the cells in laboratory vessels such as plastic dishes and bottles, and culturing and producing protein in an industrial fermenter. The monoclonal antibody or other protein secreted into the culture medium is recovered from the culture medium after centrifugation or filtration of the cells. Isolation of the protein produced is the more difficult and costly the more protein the original culture medium contained. The concentration of the protein produced, for example the monoclonal antibody, in the culture medium is relatively low. The economic advantage of production is limited by the price of the components of the culture medium. Hybridoma and myeloma cell culture media include inorganic salts such as sodium chloride and sodium phosphate, monosaccharides such as glucose or galactose, amino acids such as tryptophan, methionine, threonine and glutamine, vitamins such as thiamine and nicotinamide, pH buffering agents such as N -2-hydroxyethylpiperazine-N * -2-ethanesulfonic acid, optionally sodium pyruvate, antibiotics and a pH change indicator. The basic culture media thus composed are insufficient for hybridomas and myeloma cells as well as for other animal cells and are therefore supplemented with 10 vol.

262 S22 percent of blood serum, for example bovine, horse or fetal calf serum. Blood serum introduces unknown substances into the culture medium, some of which promote cell growth, others may inhibit growth. Blood sera should therefore be tested and selected before use. This increases the cost of producing the culture medium. As there was a lack of suitable / growth promoting blood sera, recipes of culture media, so-called serum free, have been developed in which blood serum is replaced by several defined proteins, most commonly transferrin, serum albumin, insulin and possibly other substances such as unsaturated fatty acids, amines and low-molecular hormones (Kovar J., Franek F., Methods in Enzymology 121: 277, 1986). Proteins added to the culture medium must be purified to a high degree in order to be free from toxic impurities and therefore their cost is relatively high. A further disadvantage of existing culture media is that it is difficult to isolate the monoclonal antibody or other protein secreted by the cells during cultivation, which must be done by separating the desired protein several times from the other proteins present in a multiple excess.

The drawbacks of the cost of blood serum, protein replacement blood serum and the difficulty of isolating the protein produced are eliminated when the culture medium according to the invention is used which, in addition to inorganic salts, monosaccharides, amino acids, vitamins and pH buffering agents or sodium pyruvate, antibiotics and a pH change indicator comprising water-soluble iron compounds such as ferric citrate, ferrous sulfate, ferric chloride or ferricyanide

-5 -2 potassium, at a concentration of 2 x 10 to 1 x 10 mol / l.

It is preferred that the culture medium contains trace biogenic compounds, ascorbic acid, steroids and amines.

In our earlier work, it has been shown that the only completely indispensable protein component of a serum-free culture medium is transferrin (Kovar J., Franek F., Methods in Enzymology 121: 277, 1986). Because transferrin acts as a carrier

262,622 from the environment to the cell, various water-soluble iron compounds were added to the culture medium in further experiments, even at concentrations higher than ever used in the culture media. A number of authors (e.g. Phillips PD, _A Cristofalo VJ, Exp. Cell Res., 134: 297, 1981); Perez-Infante V, Mather JP, Exp. Cell Res., 142: 325, 1982; Taetle R., Rhyner K , Castagnola J., To D., Mendelsohn J., J. Clin. Invest., 75: 1061, 1985) used water-soluble iron compounds at concentrations of 1 x 1θ ⁶ to 6 x 10 mol / L to replace transferrin. However, these concentrations are insufficient for hybridomas and myeloma cells. Iron compounds have been shown to be non-toxic to hybridomas and myeloma cells up to 1 x 10 mol / l and conversely, concentrations of —5 iron over 2 x 10 mol / l allow the growth of hybridomas and myeloma cells in a culture medium without no protein present. Iron compounds capable of replacing transferrin include, for example, ferric citrate, ferrous sulfate, ferric chloride, and potassium ferricyanide. Effective concentrations ranged from 2 x 10 to 1 x 10 mol / L.

The main advantage bezbílkovinného culture medium is a substantial reduction in the price, which is reflected in the price of the cells you · ⁴ · Loučení proteins, for example monoclonal antibodies. A further advantage of the culture medium of the invention is the easy isolation of the cell-secreted protein, for example a monoclonal antibody, since the cell-secreted protein is the only protein found after cultivation in the culture medium.

Example 1

For the preparation of the chemically defined protein-free culture medium, a basic culture medium was used which, in addition to RPMI 1640, also contained L-glutamine (300 µg / ml), sodium pyruvate (110 µg / ml), N-2-hydroxyethylpiperazine-N * -2- ethanesulfonic acid (1.5 x 10 mol / l), penicillin

262 822 (100 units / ml), streptomycin (100 pg / ml) and gentamycin (40 pg / ml) · The protein-free culture medium then contained the following substances as a serum replacement: ethanolamine (2 x 10 ” ⁵ mol / l), - 5 —9 ascorbic acid (2 x 10 mol / l), hydrocortisone (5 x 10 mol / l), cadmium sulphate (CdSO..8 / 3H _o 0.5 x 1θ mol / l), cobalt (II) chloride СоС1 ₂ »б H ₂ 0, 1 x 10 mol / l), copper sulphate (CuSO 4 .5 H ₂ 0, 1 x 10 mol / l), ammonium molybdate ((NH ₄ ) gMo? 0 ₂₄ • 4 H ₂ 0, 5 x 1θ " ¹⁰ mol / l), manganese chloride (MnCl2.4 H 2 O, 5 x ¹⁰ “ ¹⁰ mol / l), nickel sulphate (NiSO 4 6 6 H ₂ 0, 2.5 x 10 ¹⁰ mol / l) , sodium selenite (Na ₂ SeO ₃ , 4 x 1θ mol / l), sodium silicate (Na ₂ SiO ₃ , 2 x 10 ”ml / l), stannous chloride (SnCl ₂ .2 H ₂ 0,

2.5 x 10 mol mol / l), ammonium vanadate (NH ₄ VO ₃ , 2.5 x 1 ”^ mol / l), zinc sulfate (ZnSO ₄ 7 7 H ₂ 0, ^{1 x} ю ** mol / l) and citrate ferric (5 x 10 4 mol / l).

PLV-01 hybridoma cells were inoculated into 6 ml of protein-free culture medium of the above composition in a plastic Petri dish (6 cm diameter) such that the cell density was 50 x 10 cells / ml. After 4 days of cultivation in a thermostat in a humidified atmosphere of 5 vol% carbon dioxide in air at 37 ° C, cells were expanded to a density of 1.53 x 10θ cells / ml. Thus, the number of cells was approximately thirty-fold during a 4-day culture.

In a similar experiment with CMH-02 hybridoma cells, they multiplied

6 cells from an inoculum of 25 x 10 cells / ml in 4 days to a density of 1.36 x 10 cells / ml. The number of cells in this experiment was approximately 50-fold during a 4-day culture.

FO myeloma cells and PLV-01, CMH-02 and T3-03 hybridomas were separately inoculated into 1 ml volumes of protein-free culture medium of the indicated composition in wells of a 24-well plastic culture plate. After one day of culture under the above conditions, the number of cells in the well was determined and after another 24 hours the number of cells was determined again. Thus, the mean doubling time in the exponential growth phase was determined for each hybridoma or myeloma. A mean doubling time of 15.6 h was found for myeloma FO, 14.8 h for PLV-01, 14.6 h for CMH-02, and 11.8 h for T3-O3.

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PLV-01 hybridoma and PGG-O5 hybridoma cells were inoculated separately at 60 x 10 cells into 1 ml volumes of protein-free culture medium of the indicated composition placed in the wells of a 24-well plastic culture plate. For comparison, cells of the same hybridomas were inoculated into a serum-free culture medium containing transferrin and insulin (SFH medium without linoleic acid and albumin, J. Kovar and F. Franek, Methods in Enzymol. 121: 277, 1986). After 4 days of culture under the above conditions, the number of cells and the concentration of monoclonal antibody in the culture medium were determined using an enzyme-based immunoassay based on the determination of peroxidase activity. When the PLV-01 hybridoma increased in protein-free medium from an inoculum of 60 x 10 cells to 819 x 10 cells, the concentration of monoclonal antibody expressed in relative absorbance units had an ^A value of 49q 98 0.298. In a comparative experiment in SFH medium, 706 x 10 6 cells and 0.310 were achieved. When the PGG-05 hybridoma was increased in a protein-free culture medium to 675 x 10 cells, the monoclonal antibody concentration expressed in relative absorbance units was 1.32. In a comparative experiment in SFH medium, values of 731 x 10 6 cells α were obtained. _from 1.26.

490

Example 2

PLV-01 hybridoma cells were inoculated at 10 x 10 cells into 0.1 ml of protein-free culture medium of the composition shown in Example 1 placed in wells of a 96-well plastic culture plate. For comparison, cells of this hybridoma were inoculated analogously into a protein-free culture medium in which ferric citrate was replaced with ferric chloride (5 x 10 mol / l) or potassium ferrocyanide (5 x 10 mol / l). After 3 days of culture under the conditions of Example 1, hybridoma cells were expanded to 108 x 10 cells in ferric citrate culture medium, to 67 x 10 cells in ferric chloride culture medium, and to 51 x 10 cells in culture medium with potassium ferricyanide.

Example 3 262 822 о

PLV-01 hybridoma cells were inoculated at 10 x 10 cells into 0.1 ml of protein-free culture medium of the composition shown in Example 1 placed in wells of a 96-well plastic culture plate. For comparison, cells of this hybridoma were inoculated analogously into a protein-free culture medium containing, in addition to the basic medium as in Example 1, only ferric citrate (5 x 10 6 mol / l). After 3 days of culture under conditions as in Example 1, hybridoma cells were expanded to 118 x 10 cells in complete protein-free culture medium and to 103 x 10 cells in protein-free culture medium with ferric citrate only.

Claims (2)

REPLACE THE INVENTION Synthetic medium for cultivation of hybridoma and myeloma cells containing water-soluble compounds, iron, inorganic salts at a concentration of 5000 to 12000 mg / l, monosaccharides at a concentration of 300 to 5000 mg / l, amino acids at a concentration of 200 to 5000 mg / l, vitamins at a concentration of 2 to 100 mg / l depressants grains pH in a concentration of from 200 to 20,000 mg / l, or sodium pyruvate, antibiotics, and indicator of pH change _on characterized in that it contains water-soluble iron compound in a concentration of 2 x ΙΟ * 'to 1 x 10 2 mol / l, for example ferrous sulfate, ferric citrate, ferric chloride, potassium ferrocyanide. 2. Synthetic medium according to claim 1 _, characterized in that it contains compounds of trace biogenic elements, for example selenium, zinc, at a concentration of 0.002 to 2 mg / l, ascorbic acid at a concentration of 1 to 100 mg / l, a steroid, for example hydrocortisone at a concentration of 0.0001 to 0.01 mg / l and amines such as ethanolamine at a concentration of 0.1 to 15 mg / l.