GB2196348A - Synthetic medium for hybridoma and myeloma cell cultivation - Google Patents

Description

GB2196348A 1 SPECIFICATION dium are increasing. A number of recipes for

so-called serum-free culture media have been Synthetic medium for hybridoma and mye- proposed where the blood serum is replaced loma cell cultivation by several defined proteins, mostly by trans 70 ferrin, serum albumin and insulin, possibly by The invention relates to a synthetic medium other materials such as unsaturated fatty for hybridoma and myeloma cell cultivation. acids, amines and low molecular hormones Hybridomas obtained by somatic hybridiza- (see J.Kov6f, F. Frank: Methods in Enzymotion of a myeloma cell and of a lymphocyte logy 121: 277,1986).

synthetizing an antibody are industrially used 75 All proteins actually added into culture me- for manufacture of monoclonal antibodies. My- dia for cultivation of hybridoma and myeloma eloma cells, into which by methods of genetic cells have to be free of toxic admixtures, engineering a gene of a biologically active pro- which increases the economic claims on man tein is introduced, for instance a gene of an ufacture. Another drawback of existing cultiva enzyme, an enzyme inhibitor, a hormone, an 80 tion media is the difficult isolation of products immunoregulator, in order to manufacture said of the life activity of hybridoma and myeloma proteins, are equally industrially employed. The cells from culture media, which separation is increasing need of different monoclonal antiaccomplished by repeated separation of re bodies, particularly for therapeutic purposes quired products of protein character from pro and for immunoaffinity chromatography, where 85 teins contained in the culture medium in a kilogram quantities are required, equally as the large excess.

need of different regulating proteins for thera- The mentioned drawbacks are eliminated by peutic purposes, lead to an endeavour to look application of a culture medium according to for more effective and economically more ad- this invention which comprises in addition to vantageous manufacturing methods of said 90 basic components as are anorganic salts in a materials. concentration of 5000 to 12000 mg/l, monoHybridoma and myeloma cells are actually saccharides in a concentration of 300 to 5000 cultivated by first multiplying in laboratory ves- mg/1, amino acids in a concentration of 200 sels such as plastic dishes and bottles, the to 5000 mg/1, vitamines in a concentration of final cultivation and production of the protein 95 2 to 100 mg/1, substances buffering changes is accomplished in an industrial fermentor. The of pH in a concentration of 200 to 20000 monoclonal antibody or some other protein mg/l, possibly sodium pyruvate, antibiotics, an secreted into the cultivation medium is ob- indicator of changes of pH, also a supplement tained from the culture medium after centrifu- formed by at least one water soluble iron gation or filtration of the cells. The isolation of 100 compound in a concentration 2X 10-5 to the produced protein is the more difficult, the 1 X 10-2 mol/I of culture medium, advantage more protein the original culture medium has ously ferric citrate, iron sulphate, ferric chlo contained. The concentration of the produced ride or potassium ferricyanide.

protein, for instance of the monoclonal anti- It is advantageous in case the culture me- body in the cultivation medium is relatively 105 dium contains in combination with the supple low. ment compounds of biogenic trace elements, The synthetic culture medium contains basic particularly of selenium and zinc, ascorbic acid, culture components and a supplement. Basic steroid substances particularly cortisone and culture components are anorganic salts, for in- amines, advantageously ethanolamine, the stance sodium chloride and sodium phosphate, 110 presence of which increases the effectiveness monosaccha rides, for instance glucose or ga- of the supplement.

lactose, amino acids, for instance tryptophan, It has been found, that an entirely indispen- methionine, threonine and glutamine, vita- sable protein component of the serum-free mines, for instance thiamine and nicotinamine, culture medium is transferrin (see J.Kov6f, F.

substances buffering changes of pH, for in- 115 Fran6k: Methods in Enzymology, 121:

stance H-2-hydroxyethylpiperazine-N'-2-ethane 277,1986). As transferrin acts as a transport sulphonic acid, possibly sodium pyruvate, antisubstance of iron from the surrounding me biotics and an indicator of changes of pH. dium into the cell, its functionning could be These basic cultivation components are insuffi- substituted by the presence of water soluble cients for hybridoma and myeloma cells, the 120 iron compounds of a higher concentration than same as for the animal cells and are therefore have been up to now applied in cultivation supplemented by 10% by volume of a blood media. A number of authors (for instance P.D.

serum, for instance cattle-, horse- or fetal bo- Phillips, V.J. Cristofalo in Exp. Cell Res., vine serum. The blood serum introduces into 134:297, 1981 V. Perez. Infante, J.P. Mather the cultivation medium unknown substances, 125 in Exp. Cell Res., 142: 325, 1982; R. Taelle, some of which are supporting the growth of K. Rhyner, J. Castagnola, D.To, J. Mendel cells, others can inhibit the growth. It is there- sohn, J.Clin, Invest., 75:1051,1985) have fore necessary to test the blood serum prior used instead of transferrin water soluble iron to its application and select suitable lots. Thus compounds in concentration of 1 x 10-6 to the costs for manufacture of the culture me- 130 20x 10-6 mol/l. These concentrations are 2 GB2196348A 2 however insufficient in case of hybridoma and of 1,36 x 106 cells/mi within 4 days. The myeloma cells. It has been found that iron number of cells has at this test after 4 days compounds up to a concentration of 1 X 10-2 of cultivation increased fitytimes.

mol/1 are not toxic for hybridoma and mye- Cells of myeloma FO and of hybridomas lorna cells and on the other side concentra- 70 P1-V-01, CMH-02 and T3-03 have been sepa tions of iron compounds higher than 2 X 10-5 rately inocculated to volumes of 1 mi of a mol/1 enable growth of hybridoma and mye- protein-free culture medium of the mentioned lorna cells in a protein-free culture medium. composition contained in wells of a plastic Iron compounds capable to replace transferrin multi-dish with 24 wells. After the first day of are for instance ferric citrate, iron sulphate, 75 cultivation at mentioned conditions the number ferric chloride and potassium ferricyanide. Ef- of cells in a well has been determined and fective concentrations are within limits of after next 24 hours this number has been 2 x 10-5 up to 1 X 10-2 rnol/1. checked again. Thus the mean doubling- time Advantages of the cultivation medium ac- in the exponential growth phase for each hy- cording to this invention is the easy isolation 80 bridoma or myeloma has been determined.

of the protein separated by cells, for instance The mean doubling-time for the myeloma FO of monoclonal antibodies, as the protein se- has been found to be 15,6 h, for the hybri creted by cells is the sole protein which is doma PLV-01 14,8 h, for the hybridoma CMW after cultivation present in the culture medium. 02 14,6 h and for the hybridoma T3-03 11,8 The advantages of the culture medium ac- 85 h.

cording to this invention will be more clearly Cells of the hybridoma PLV01 and of the shown on hand of following examples. hybridoma PGG-05 have been inocculated se parately in a number of 60 x 103 cells to vol Example 1 umes of 1 mi of a protein-free culture medium A basic culture medium which in addition to 90 of the mentioned composition situated in wells the medium RPM1 1640 contained L-glutamine of a plastic multi-dish with 24 wells. For com 1300 ug/mi, sodium pyruvate/ 110 ug/mi/, N- parison cells of the same hybridomas flave 2-hydroxyethylpiperazine-N-2-ethanesulphonic been inocculated into a serum-free culture me acid/ 1,5 x 10-2 Mol/1, penicillin/ 100 units/mi/, dium containing 5 mg/1 of trasnferrin and 10 streptomycin/ 100 ug/mi/ and gentamycin/40 95 mg/l of insulin / SFH medium without lineloic ug/mi/ has been used in order to prepare a acid and albumin, see: J.KovdY and F. Fran6k:

chemically defined protein-free cultivation me- Methods in Enzymology, 121: 277, 1986/.

d[um. The protein-free cultivation medium con- After 4 days of cultivation at above men tained thereafter as substituted of a serum the tioned conditions the number of cells and the following additional materials: ethanolamine 100 concentration of monoclonal antibodies in the /2 x 10-5 mol/1/, ascorbic acid /2 x 10-5 culture medium have been determined using mol/1/, hydrocortisone /5 x 10-9 moi/l/, cad- an enzymoimmunologic test based on the de mium sulphate /CdS0,8/3 H20, 5 x 108 termination of the peroxidase activity. In case mol/1, cobalt chloride, /CoC1,6 H20, 1 X 10 of a growth of the hybridoma PLV-01 in the mol/1, copper sulphate (CUS04.5 H20, 1 X 10-l 105 protein-free medium from an inocculum of moill, ammonium molybdate /NH, Mo,0,,4 60x 103 cells, the concentration of the mono H20, 5.10-10 mol/1, manganese dichloride clonal antibody expressed in relative units of /MnC12.4 H20, 5 x 10- 10 moll], nickel sulphate absorbance had the value of A,,,,=0,298. In /NiSO,.6 H,O, 2,5 x 10-10 mol/1, sodium selen- the comparative experiment carried out in an ite/ /Na2SeO3, 4 x 10-11 moll], sodium silicate 110 SFH medium values of 706 x 103 cells and (Na2SiO,, 2 X 10-7 rnol/1, stannous chloride A4110=0,310 have been obtained. In case of a 1 /SnC1,2 H20, 2,5 x 10-10 mol/1, ammonium growth of the hybridoma PGG-05 in a protein vanadate / W4V03, 2,5 x 10-0 moill, zinc sul- free culture medium for 675x 103 cells, the phate /ZnS0,7 H20, 1 X 10-6 moill and ferric concentration of the monoclonal antibody ex citrate, 5 x 10-4 Mol/1. 115 pressed in relative units of absorbance had Into 6 mi of the protein-free culture medium the value of A = 1,32. In the comparative of the mentioned composition in a Petri dish experiment carried out in an SFH medium (diameter 6 cm) cells of the hybridoma PLVvalues of 731 x 103 cells and A,,,,= 1,26 have 01 have been inocculated in such an amount been obtained.

that the density of cells has been 50x 10.1 120 cells/m]. After 4 days of cultivation in a ther- Example 2 mostat in a humidifed atmosphere of 5% by Cells of a hybridoma PLV-01 have been in- volume of carbon dioxide in air at a tempera- occulated in a number of JOX 103 cells into ture of 37'C, the cells have multiplied to a 0,1 mi of a protein-free cultivation medium of density of 1,53 x 106 cells/m]. The number of 125 the composition as in example 1 placed in cells has thus in the course of cultivation for wells of a plastic cultivation microplate with 4 days multiplied approximately thirtytimes. 96 wells. For comparison purposes cells of By a similar test with cells of the hybridoma this hybridoma have been inocculated into a CMH-02 the cells from an inoculum of protein-free culture medium where ferric chlo 25 x 103 cellsymi have multiplied to a density 130 ride in a concentration of 5 x 10-4 mol/1 re- 3 GB2196348A 3 placed -the ferric citrate. After 3 days of culti- pound is iron sulphate, ferric citrate, ferric vation at conditions as in example 1 the hybri- chloride, potassium ferricyanide.

doma cells have multiplied to 131 x 103 cells 3. Synthetic medium as in claim 1 or 2 in the culture medium with ferric citrate and to characterised in that it contains compounds of 82x103 cells in the cultivation medium with 70 biogenic trace elements, advantageou ' sly sele- ferric chloride. nium, zinc in a concentration of 0,002 to 2 mg/1.

Example 3 4. Synthetic medium as in one of claims 1 Cells of the hybridoma PLV-01 have been to 3 characterised in that it contains ascorbic inocculated in an amount of '10 X 103 cells to 75 acid in a concentration of 1 to 100 m911.

0,1 mi of basic components mentioned in 5. Synthetic medium as in one of claims 1 example 1. As supplement 5X 10-4 Mol/1 Of to 4 characterised in that it contains steroid potassium ferricyanide have been added into substances, advantageously hydrocortisone in the culture medium. After 3 days of cultivation a concentration of 0,0001 to 0,1 mg/i.

at the temperature of 37% and of 4,5% per 80 6. Synthetic medium as in one of claims 1 volume of carbon dioxide in a humidified at to 5 characterised in that it contains amines, mosphere at pH 7,4 of the medium the cells advantageously ethanolamine in a concentra have multiplied to 62 x 103. tion of 0. 1 to 15 mg/1.

7. Synthetic medium for hybridoma and

Claims (2)

1. Example 4 85 myeloma cell cultivation as claimed in Claim 1

   Cells of the hybridoma PLV-01 have been substantially as described in any one of the inocculated in an amount of 10X 103 cells into examples disclosed herein.

   0,1 m] of basic components mentioned in Pub idhed 1988 at The Patent Office, State House, 66171 High Holborn, example 1. Iron sulphate in a concentration of London WC 1 R 4TP. Further copies may be obtained from 1 X 10-4 mol/1 has been added as supplement The Patent Office, Sales Branch, StMary Cray, Orpington, Kent BR5 3RD.

   Printed by Burgess & Son (Abingdon) Ltd. Con. 1187.

   into the culture medium. After 3 days of culti vation at a temperature of 37T and 4.5% per volume of carbon dioxide in a humidified at mosphere at pH 7,4 of the medium the cells have multiplied to 25 x 103.

   Example 5 Cells of the hybridoma PLV-01 have been inocculated in an amount of 10 X 103 into 0, 1 mi of protein-free culture medium of the com position as in example 1 placed in wells of a plastic cultivation microplate with 96 wells.

   For comparison cells of this hybridoma have been inocculated into a protein-free culture medium containing in addition to the basic medium as in example 1 solely ferric citrate (5 x 10-4 mol/l). After 3 days of cultivation at conditions as in example 1 the hybridoma cells have multiplied to 118 x 103 cells in the protein-free culture medium and to 103 x 103 cells in the protein-free culture medium where solely ferric citrate has been added.

   CLAIMS 1. Synthetic medium for hybridoma and myeloma cell cultivation comprising anorganic salts in a concentration of 5000 to 12000 mg/I, monosaccharides in a concentration of 300 to 5000 mg/1, amino acids in a concen tration of 200 to 5000 mg/1, vitamins in a concentration of 2 to 100 mg/1, substances buffering changes of pH in a concentration of to 20000 mg/1, possibly also sodium py ruvate, antibiotics, an indicator of pH changes and a supplement, characterised in that said supplement is formed by at least one water soluble iron compound in a concentration of 2 x 10-5 to 1 x 10-2 mol/1.
2. 2. Synthetic medium as in claim 1, charac- terised in that the water soluble iron corn-