IRON CHELATE CULTURE MEDIUM ADDITIVE

Abstract: A culture medium additive comprises an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate. The additive is a suitable iron source for serum-free or protein-free culture media.

Description: Iron c elate culture medium additive. FIELD OF INVENTION The present invention relates to an iron supplement for culture media, primarily serum-free or protein-free media, for growing mammalian cells, and a culture medium containing said iron supplement. BACKGROUND OF THE INVENTION Until fairly recently, conventional media for growing mammalian cells contained serum as an important source of growth factors in the requisite concentrations for the growth and natural multiplication of the cells. The presence of serum or specific added proteins in culture media, however, suffers from the disadvantage that the purification of the desired protein product from the mammalian culture is made more difficult and that there is an increased risk of contamination by infectious agents. It is therefore an important aim in the field of mammalian cell culture to develop media in which the components in serum necessary for cell growth have been replaced with non- proteinaceous substances serving the same purpose. Serum-free or protein-free media have therefore become increasingly important for the cultivation of mammalian cells in the production of biological materials (e.g. monoclonal antibodies, natural or recombinant pharmaceuticals, or the like) . Most serum-free media are based on a commercially available basal medium (e.g. MEM, Ham, RPMI) supplemented with insulin, transferrin, selenium, growth factors, and some protein and lipid sources \Hamilton et al. , In Vitro 13: 537-547, 1977; Ham et al. , Methods Enzymol. 58: 44-93, 1979; Maciag et al.. Cell Biol. Int. Rep. 4: 43-50, 1980; Barnes, BioTechnology 5: 534- 540, 1987; Fiorentini et al. , Am. Biotech. Lab. 8: 35-37, 1990; Bjare, J. Biotech. 1.5: 147-154, 1990; Hewlett, Cytotechnology 5: 3-14, 1991]. SUMMARY OF THE INVENTION It has now been found possible to replace transferrin as the iron source in serum-free media by a non-protein chelate of citrate and an iron salt. Accordingly, the present invention relates to a culture medium additive comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate. Iron chelates for serum-free media have previously been proposed, e.g. in EP 274 445 describing a culture medium additive containing an iron-EDTA/citric acid chelate and aurin tricarboxylic acid. The iron chelate additive of the present invention has the advantage over the one proposed in EP 274 445 that it is composed of inexpensive constituents, and that it contains fewer constituents which might be a εource of contamination. In another aspect, the present invention relates to a culture medium for growing mammalian cells, the medium comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate. DETAILED DISCLOSURE OF THE INVENTION To avoid iron precipitation and potential toxic effects of the iron on the cultured cells, the citrate chelator should be mixed with the iron salt so as to generate an equilibrium prior to the addition to the culture medium. This equilibrium may for instance be formed in a concentrated stock solution and, and the process speeded up by stirring, autoclaving, etc. In the preparation of the iron additive, the requisite equilibrium is most conveniently reached when the alkali metal or alkaline earth metal citrate is present in a molar excess relative to the iron salt, in particular a ratio of the citrate to the iron salt of more than 1:1 and less than 500:1. Suitable iron salts for inclusion in the additive of the invention may be selected from the group consisting of FeCl 2 , FeCl 3 , FeS0 4 , Fe 3 (P0 4 ) 2 , Fe(N0 3 ) 3 and Fel 2 . Examples of suitable alkali metal or alkaline earth metal citrates for inclusion in the additive of the invention are Na-citrate, K-citrate or Mg- citrate. In a particularly preferred embodiment, the iron salt included in the additive is FeCl 2 or FeCl 3 , and the citrate is Na-citrate. In this case, a preferred molar ratio of Na-citrate to FeCl 2 /FeCl 3 is between 2:1 and 200:1. The culture medium in which the additive iε intended to be included is preferably a medium for growing mammalian cells, the additive of the invention constituting an inexpensive iron source which mammalian cells have surprisingly been able to utilise. Thus, the medium may for instance be a low-serum medium or, preferably, a serum-free or protein-free medium in which it is important to provide a non-protein iron supplement. Although it has previouεly been deεcribed that the freshwater ciliate Tetrahy ena thermophila is able to utilise pre-chelated iron citrate as the only iron source (cf. P.B. Suhr-Jessen and L. Raεmusεen, Exp. Cell Res. 139. 1982, pp. 457-460; L. Ras ussen et al., J. Cell. Phys. 122. 1985, pp. 155-158), it haε not been suggeεted that mammalian cellε may alεo utilise a citrate/iron chloride chelate as the iron source in serum- free media. Biologically speaking, it is quite surprising that mammalian cells which exist in an environment enriched in nutrient components and under conditions of considerable osmotic pressure are able to assimilate nutrients in a similar way as a primitive freshwater organism specialized in surviving in a nutrient-poor environment. The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed. EXAMPLE 1. BHK cells Adherent BHK cells cultivated in coated T-flaskε containing a serum-free nutrient medium for BHK cells (as described by Maciag et al. 1980, ibid) with transferrin as the only iron source (SFNMT) , were concomitantly inoculated into a series of coated T-flaskε containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock solution of Na-citrate and iron chloride. Experiments 1 to 3 had different durations and the experimental citrate concentration was 2 mM, 2 mM, and 5 mM (final cone), respectively. Parallel control cultures were cultivated in SFNMT. Each cell culture was independently treated with respect to replacement of used medium with freεh serum-free medium of the identical kind or εub-cultivation into new T-flask containing fresh serum-free medium of the identical kind. At the end of the experiment, the total number of doublings in each medium was calculated: Citrate and iron chloride was not added to SFNMT EXAMPLE 2. BHK cells BHK cells were inoculated into spinner flasks containing SFNM- for BHK cells (see example 1) supplemented with a chelated citrate-iron stock solution resulting in 2 mM Citrate and 100 μM FeCl 3 (final cone.) . Following a few hours where cells were allowed to adhere to coated microcarrierε, cells spread, propagated and remained esεentially confluent and healthy for more than two weeks when the experiment was terminated. EXAMPLE 3. CHO cells Adherent CHO cells cultivated in coated T-flasks containing a serum-free nutrient medium for CHO cells (as described by Ham et al. 1979, ibid.) with transferrin as the only iron source (SFNMT) , were concomitantly inoculated into a series of coated T-flaskε containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock solution of Na-citrate and iron chloride. Experiments 1 and 2 had different durations and the experimental citrate concentration was 2 mM (final cone.) . Parallel control cultures were cultivated in SFNMT. Each cell culture was independently treated with respect to replacement of uεed medium with fresh serum-free medium of the identical kind or sub-cultivation into new T-flask containing fresh serum-free medium of the identical kind. At the end of the experiment, the total number of doublings in each medium was calculated: Citrate and iron chloride was not added to SFNMT EXAMPLE 4. CHO cells CHO cells were inoculated into two spinner flasks containing SFNM- for CHO cells (see example 3) supplemented with chelated citrate-iron chloride stock solutions resulting in 2 mM Citrate and 100 and 300 μM FeCl 3 (final cone), reεpectively. After a few hourε where cells were allowed to adhere to coated micro carriers, cells spread, propagated and remained essentially confluent and healthy for more than two weeks when the experiment was terminated. EXAMPLE 5. MYELOMA cells SP2/0 myeloma cells cultivated in suspension culture in T- flaskε containing an RPMI baεed serum-free nutrient medium (Shacter 1989, TIBTECH, 7, 248-253) with transferrin as the only iron source (SFNMT) , were concomittantly inoculated into a series of T-flasks containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock εolution of Na-citrate and iron chloride. Each cell culture was independently treated with respect to replacement of used medium with fresh serum-free medium of the identical kind or sub-cultivation into new T-flask containing freεh serum-free medium of the identical kind. At the end of the experiment, the total number of doublings in each medium waε calculated: Citrate and iron chloride was not added to SFMNT EXAMPLE 6. HYBRIDOMA cells SP2/0 based hybridoma cells cultivated in suεpenεion culture in T-flaεks containing an RPMI based serum-free nutrient medium for hybridoma cells (Shacter 1989, TIBTECH, 7, 248-253) with transferrin as the only iron source (SFNMT) , were concomittantly inoculated into a series of T-flasks containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock εolution of Na-citrate and iron chloride. Each cell culture waε independently treated with reεpect to replacement of uεed medium with fresh serum-free medium of the identical kind or sub-cultivation into new T-flask containing fresh serum-free medium of the identical kind. At the end of the experiment, the total number of doublingε in each medium was calculated: Citrate and iron chloride was not added to SFNMT

Claims: CLAIMS 1. An culture medium additive comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate. 2. An additive according to claim 1, wherein the alkali metal or alkaline earth metal citrate iε present in a molar excess relative to the iron salt 3. An additive according to claim 1 or 2, wherein the iron salt is εelected from the group conεisting of FeCl 2 , FeCl 3 , FeS0 4 , Fe 3 (P0 4 ) 2 , Fe(N0 3 ) 3 and Fel 2 . 4. An additive according to any of claims 1-3, wherein the alkali metal or alkaline earth metal citrate iε selected from the group consisting of Na-citrate, K-citrate and Mg-citrate. 5. An additive according to any of claims 1-4, wherein the molar ratio of alkali metal or alkaline earth metal citrate to iron salt is more than 1:1 and less than 500:1. 6. An additive according to any of claimε 1-5, wherein the culture medium in which it iε included is for growing mammalian cells. 7. An additive according to any of claims 1-6, wherein the culture medium in which it is included iε a serum-free or protein-free medium. 8. An additive according to any of claims 1-7, wherein the iron salt is FeCl 2 or FeCl 3 , and wherein the citrate is Na-citrate. 9. An additive according to claim 8, wherein the molar ratio of Na-citrate to FeCl 2 /FeCl 3 is between 2:1 and 200:1. 10. A culture medium for growing mammalian cells, the medium comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate. 11. A culture medium according to claim 10, wherein the alkali metal or alkaline earth metal citrate is present in a molar excess relative to the iron salt. 12. A culture medium according to claim 10 or 11, wherein the iron salt is selected from the group consisting of FeCl 2 , FeCl 3 , FeS0 4 , Fe 3 (P0 4 ) 2 , Fe(N0 3 ) 3 and Fel 2 . 13. A culture medium according to any of claims 10-12, wherein the alkali metal or alkaline earth metal citrate is selected from the group consisting of Na-citrate, K-citrate and Mg- citrate. 14. A culture medium according to any of claimε 10-13, wherein the molar ratio of alkali metal or alkaline earth metal citrate to iron salt is more than 1:1 and less than 500:1. 15. A culture medium according to any of claims 10-14, which is a serum-free or protein-free medium. 16. A culture medium according to any of claims 10-15, wherein the iron salt is FeCl 2 or FeCl 3 , and wherein the citrate is Na- citrate. 17. A culture medium according to claim 16, wherein the molar ratio of Na-citrate to FeCl 2 /FeCl 3 is between 2:1 and 200:1.

Inventors: SUHR-JESSEN PETER BERNT, SUHR-JESSEN, PETER, BERNT
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: C12N5
IPC codes: C12N5