WO2005095582A2

WO2005095582A2 - Novel cell culture medium supplements

Info

Publication number: WO2005095582A2
Application number: PCT/EP2005/002228
Authority: WO
Inventors: Karlheinz Landauer; Günter Waxenecker
Original assignee: Igeneon Krebs-Immuntherapie Forschungs-Und Entwicklungs-Ag
Priority date: 2004-03-05
Filing date: 2005-03-03
Publication date: 2005-10-13
Also published as: AT413700B; ATA3832004A; WO2005095582A3

Abstract

Animal cell culture medium supplement for the cultivation of animal cells whereby the medium supplement is acetone and a method for the cultivation of cells.

Description

Novel cell culture medium supplements

The invention refers to a novel animal cell culture medium supplement which can increase growth rate and yield of in-vitro cultivated cells. Furthermore the invention refers to the use of the culture medium supplement for the cultivation of cells and for the increase of production of proteins expressed by the cells.

The requirements on the media for the production of proteins with mammalian cells are manifold. Cell culture media must provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in-vitro environment. Characteristics and compositions of the cell culture media may vary depending on the particular cellular requirements. Media formulations have been used to cultivate a number of cell types including animal, plant and bacterial cells. Cells cultivated in culture media catabolize available nutrients and produce useful biological substances such as monoclonal antibodies, hormones, growth factors, viruses and the like. Such products have therapeutic applications, and, based on recombinant DNA technology, cells can be engineered to produce large quantities of these products. Thus, the ability to cultivate cells in vitro is not only important for the study of cell physiology, but is also necessary for the production of useful substances which may otherwise be impossible or only obtainable by cost-effective means.

There are various media and cell culture systems available. Culture media can be provided as liquid solutions but also as dry powder media. For example, WO 98/36051 describes dry powder cells and nutritive media powder. These powder media can be transferred into moistened powder by adding defined and controlled solvents. These solvents can be water, serum or organic solvents.

US 5,328,844 describes a strictly defined medium composition, COF 1769, for the cultivation of mammalian cells, containing a defined amino acid composition, antioxidants and protective agents.

WO 89/01028 describes serum free media for the growth of insect cells consisting of a basal medium, lipid, peptone components and protective agents to stabilize the cells from damage and death in agitated and sparged insect culture. Lots of different agents, as amino acids, trace elements, serum, vitamins, lipids and hydrolysates (e.g. soybean hydrolysate) are used in media for cell cultivation. The energy source of in-vitro cultured cells is mainly glucose and L-glutamine. Glucose is degraded via glycolysis producing pyruvate and NADH. Pyruvate is normally transferred into the tri-carbonic acid cycle (TCC) and either used as energy source for ATP production (catabolic reactions) or as C2-fragment for amino acid production in anapleurotic pathways. The electrons of NADH + H⁺ are transferred into the mitochondria via active shuttle systems for recycling and ATP production.

The metabolism in in-vitro cultured cells is slightly different. The pyruvate decarboxylase is not working efficiently enough, thus acetyl-CoA cannot be produced sufficiently to feed the TCC with C2-bodies (Elias C.B. et al., 2003, Biotechnol. Progress 19:90-97). Secondly, the shuttle system to transfer the electrons of the produced NADH into the mitochondria is inhibited, thus the cell has to recycle NADH to NAD by reducing it on the large quantity of pyruvate producing lactate. This leads to a couple of problems for the cell. The degradation of glucose to lactate decreases the pH and increases the osmolality of the culture medium, which induces stress to the cell. The efficiency of glucose consumption differs from 30% up 80% depending on the cell line and culture conditions. As there are not enough C2-bodies to maintain the TCC, another agent has to be added to the medium to support the catabolism. L-glutamine is used as further supplement in concentrations of 2 - 10 mM. Glutamine is transferred to the TCC and used for anaplerotic and catabolic pathways. Glutamine is degraded to glutamate and thereby producing ammonia, which is toxic. The rate of degradation can be influenced by pH and ionic conditions but in all cell culture media, formation of these breakdown products often cannot be avoided (Tritsch et al., 1962, Exp.Cell Res. 28:360-364).

However, parts of ammonia can be bound via a transamination of pyruvate producing L-alanin, which further increases the osmolality and decreases the amount of C2-bodies for the TCC. Therefore different substances, as glutamate or aspartate instead of L-glutamine and mannose, galactose or other carbohydrates are used to overcome these problems. Nevertheless, techniques to prevent ammonia accumulation or remove ammonia selectively have been relatively unsuccessful to date (Newland M et al., 1990, Cytotechnology 3:215- 229).

So there is still a need to provide animal cells cultivated in vitro with supplements to increase the growth rate of cells and the yield of proteins expressed by these cells. There is also a need in cell culture supplements that avoid accumulation of breakdown products which have negative effects on cell growth, cell productivity and product quality (Elias C.B., 2003, Biotechnol. Progress, 19, 90-97).

Brief summary of the invention:

It was an object of the invention to provide an animal cell culture medium supplements that can be added to cell culture media in order to provide the cells with energy sources that can be used by the cells yet resulting in increased yield. The object of the invention is solved by providing a cell culture supplement and its use as disclosed in the claims.

According to the invention new cell culture supplements are provided which can be used either alone or in combination. It was surprisingly found that the addition of acetone either alone or in combination with β-hydroxyethylstarch (HES) can activate cells resulting in higher growth rates and/or increased specific productivities. Growth rates and/or specific productivity of the animal cells can be increased leading to higher yields of recombinant proteins expressed by the cells.

The potency of the expressed proteins is equal, preferably better compared to the proteins expressed by cells cultivated in standard media. Depending on the cell line and on the proposed fermentation process, yield increased at least 20% compared to cells cultivated in media not supplemented according to the invention, In-vitro cultivated cells can metabolize acetone and increase their yield of expressed proteins up to at least 20%, preferably at least 50%, preferably at least 100%, preferably at least 150%, preferably at least 250% depending on the fermentation mode and the used cell line.

HES is capable to increase the yield of a bioprocess up to 20%, preferably up to

50%, preferably up to 100%, preferably at least 150%, preferably at least 250% depending on the fermentation mode and the used cell line.

A mixture of HES and acetone can activate mammalian cells in order to increase the specific productivity by two fold.

Both substances, either acetone alone or as a mixture with HES, can positively interfere, possibly via reducing the osmotic pressure, with the quality of formed proteins and enhance the metabolism of mammalian cells.

The invention provides the use of acetone or acetone and HES as cell culture supplements for the cultivation of cells.

The supplements according to the invention can easily be removed from the cells or the expressed proteins by any purification means known by the skilled person.

Fortunately, these supplements, when added to the cultivation media, are not toxic to the cells, so even high concentrations of the supplements are tolerable to the cells.

The invention also provides methods for the cultivation of cells either adherently or in suspension using a medium containing the media supplement according to the invention. The invention also provides a method for the production of proteins by cultivation of host cells in a medium containing the supplement. Especially the production of antibodies, derivatives or fragments thereof is provided. The invention also provides a method for obtaining an antibody or antibody derivative or fragment thereof or recombinant proteins by a cell culture which is cultivated in a medium comprising a cell culture supplement according to the invention, comprising the steps of: culturing the cells which produce antibodies or derivatives or fragments thereof in the presence of the supplement according to the invention, continuing said culture until said antibodies or derivatives or fragments accumulate, and isolating said antibodies or derivatives or fragments thereof.

Moreover, the invention further provides a culture of cells cultivated in a cell culture medium supplemented by acetone in a concentration of 1 μM to 20mM, preferably 5μM to 10mM, preferably 10μM to 1mM preferably 10μM to 100μM and optionally together with HES, wherein HES is in a concentration of 0.01 to 10 weight%, preferably 0.1 to 5 weight%, preferably 0.2 to 2 weight%.

Detailed description of the invention:

The term "animal cell culture medium" which can be supplemented by acetone and/or HES can be any medium known from the state of the art. The basal medium can be a synthetic medium such as DMEM/HAM^'s F12, Medium 199, Hybridoma Express or RPMI, IMDM, Ex-Cell SP2/0, Ex-Cell 325-PF or combinations thereof, and others that are known from the literature or are commercially available. The basal medium can comprise a number of ingredients, including amino acids, vitamins, organic or inorganic salts, sources of carbohydrate, each ingredient being present in an amount which supports the cultivation of a cell in vitro.

It can be either a serum free or a serum containing medium. If required, a nonionic surfactant such as polyethylene glycol or polypropylene glycol (PLURONIC F-61 ; PLURONIC F-68, SYNPERONIC F-68, PLURONIC F- 71 or PLURONIC F-108) can be added to the medium as a defoaming agent. These agents are well known to protect cells from the negative effect of aeration. The term "animal cell culture" refers to animal cells grown in suspension in roller bottles, flasks, spinners etc. Thebioprocess can be a batch-culutre, Fed-Batch- culture, a continuous or perfusion cultivation. Large scale approaches, such as bioreactors including stirred tank reactors or airlift reactors, or including cells growing attached to microcarriers in stirred bioreactors, fluidized bed reactors or airlift reactors, are also included.

The term "yield" can be defined as overall productivity of a bioprocess. The term "bioprocess" can be defined as a method to produce a biologically derived product.

The term "cultivation" refers to the maintenance of the cells in vitro under conditions permissive for growth and continued viability. Mammalian cells are typically cultivated in a cell incubator at a temperature between 32 and 38°C, in particular at about 37°C. The optimal pH range is about 6.7 to 7.6, preferably between 6.9 to 7.2.

The acetone used as medium supplement can be any quality of acetone, yet "pro analysi"-grade is preferred. Acetone can be added to the cell culture medium in a concentration of 1 μM to 20mM, preferably 5μM to 10mM, preferably 10μM to

1 mM preferably 10μM to 100μM. Using the cell culture medium supplement in these concentrations resulted in increased cell growth and / or high yield.

The HES used as medium supplement is preferably of high purity. HES can be added to the cell culture medium in a concentration of 0.01 to 10 weight percent

(weight%), preferably 0.1 to 5 weight%, preferably 0.2 to 2 weight%. Using the cell culture medium supplement in these concentrations resulted in increased cell growth and / or high yield.

In case acetone and HES are used as combined supplements, the amount of acetone and HES are the same as described above.

The cell culture supplement/supplements according to the invention can be added at any time during cultivation. It can be added to the medium before inoculation of the cells or during the cultivation process. The supplement can also be added continuously to keep a constant concentration of the supplement in the medium.

The animal cells cultivated in a medium supplemented by acetone or acetone and

HES can be any animal cells that can grow in vitro in synthetic media. Within the scope of the invention, the term "cells" means the cultivation of individual cells, tissues, organs, insect cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, stem cells and/or genetically engineered cells, such as recombinant cells expressing a heterologous protein or polypeptide.

The cells used according to the invention are preferably animal cells, more preferably mammalian cells. These can be for example BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells,

293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0. perC6 (human retina cells) or derivatives thereof.

The proteins expressed by these cells can be any proteins known in the art.

Preferably the proteins are recombinant proteins, hormones, viruses, antibodies or derivatives or fragments thereof like. Erythropoietin, insulin or coagulation factors can be exemplary listed as these proteins.

The term "antibody" relates to antibodies of all possible types, in particular to polyclonal and monoclonal antibodies or antibodies produced by chemical, biochemical or gene technology methods. Methods for producing such molecules are known to the person skilled in the art. The antibodies can also be used as carrier molecules for antigenic structures.

Antibody derivatives and fragments can be single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or di-, oligo- or multimers thereof. Also included are peptidomime tics or peptides derived from antibodies e.g. they comprise one or several CDR regions. Further included are human monoclonal antibodies and peptide sequences which, based on a structure activity connection, are produced through an artificial modeling process

(Greer J. et al.. J. Med. Chem. 1994, 37:1035-1054).

The term "epitope" defines any region of a molecule that can be recognised by specific antibody or that provoke the formation of those specific antibodies.

Epitopes may be either conformational epitopes or linear epitopes.

Preferred epitopes or antigenic structures are derived from antigens specific for epithelial tumors, and preferably frequently expressed in breast cancer, gastrointestinal, colorectal, rectal, prostate, pancreatic, and ovary and lung cancer, either being small cell lung cancer (SCLC) or non small cell lung cancer

(NSCLC). The preferred epitopes especially induce humoral immune response and the formation of specific antibodies in vivo. The antibodies preferably also induce T cell specific response.

Among the preferred epitopes are protein epitopes that are expressed on malignant cells of solid tumors, e.g. TAG-72, MUC1 , Folate Binding Protein A-33,

CA125, HER-2/neu, EGF-receptors, PSA, MART etc. Moreover, T cell epitope peptides or mimotopes of such T cell epitopes may be presented by the antibody according to the invention.

Preferred carbohydrate epitopes or antigenic structures are derived from tumor associated aberrant carbohydrate structures, such as Lewis antigens, e.g. Lewis x-, Lewis b- und Lewis y-structures, also sialylated Lewis x structures, GloboH- structures, KH1 , Tn-antigen, TF-antigen and alpha-1-3-galactosyl-epitope.

The preferred TAA targets or epitopes are selected from trie group of determinants derived from the group of antigens consisting of peptides or proteins, such as EpCAM, HER2-9, NCAM, CEA, TOL9-re ceptors and T cell peptides, carbohydrates, such as aberrant glycosylation patterns, Lewis Y, Sialyl-

Tn, Globo H, and glycolipids, such as GD2, GD3 und GM2 .

For example, these antibodies can be antibodies according WO 93/24647, EP 0

285 059, EP 0 528 767, WO 04/005359, WO 03/097663 or A599/2003.

It has been shown that the use of the supplements accordi ng to the invention did lead to increased production rates and high yield of the expressed proteins compared to proteins expressed by cells that are cultivated in standard media without the inventive supplements. Especially the expression of antibodies, derivatives or fragments thereof did result in highly increased yields when using the supplements according to the invention.

Brief description of the drawings:

Figure 1 shows, as control, the roller bottle batch culture viable population curve, viability curve and volumetric monoclonal antibody titer curve. Cells E4 were propagated as free suspension in 850cm² roller bottles agitated with 2 rpm in 450 mL working volume. The test was performed in a double experiment; sampling was done on a one to three day basis. The used medium was Hybridoma Express supplemented with 4mM L-GIutamine and 0.1 % Pluronic F68. The specific growth rate in the exponential phase was 0.20 d-1 (R²=0.99). The specific productivity was qMAb=13.5 pg cell-day^"1, R²=0.99.. Figure 2: Roller bottle batch culture viable population curve, viability curve and volumetric monoclonal antibody titer curve. Cells E4 were propagated as free suspension in 850cm² roller bottles agitated with 2 rpm in 450 mL working volume. Test was performed in a double experiment; sampling was on done a one to three day basis. The used medium was Hybridoma Express supplemented with 4mM L-GIutamine, 0.1 % Pluronic F68 and 0.2% HES. The specific growth rate in the exponential phase was 0.29 d^"1 (R²=0.97). The specific productivity was qMAb=8.5 pg cell-day^"1, R²=0.99.

Figure 3: Roller bottle batch culture viable population curve, viability curve and volumetric monoclonal antibody titer curve are shown. The cells E4 were propagated as free suspension in 850cm² roller bottles agitated with 2 rpm in 450 mL working volume. The test was performed in a double experiment; sampling was done on a one to three day basis. The used medium was Hybridoma Express supplemented with 4mM L-GIutamine, 0.1 % Pluronic F68 and 10μM acetone. The specific growth rate in the exponential phase was 0.24 d^"1 (R²=0.97). The specific productivity was qMAb=10.8 pg cell-day^"1, R²=0.99. Figure 4: Roller bottle batch culture viable population curve, viability curve and volumetric monoclonal antibody titer curve. Cells E4 were propagated as free suspension in 850cm² roller bottles agitated with 2 rpm in 450 mL working volume. Test was performed in a double experiment; sampling was done on a one to three day basis. The used medium was Hybridoma Express supplemented with 4mM L-GIutamine, 0.1% Pluronic F68 and 10μM acetone. The specific growth rate in the exponential phase was 0.19 d^"1 (R²=0.97). The specific productivity was qMAb=10.0 pg cell-day^"1, R²=0.99.

Figure 5: The cumulative cell numbers of living E4 hybridoma cells in serum-free medium per day. The test was performed in replicates. The line is the linear regression of all measured time points. The highest cumulative cell numbers were achieved in HES containing medium after 10 days of cultivation. Cells cultivated in the acetone medium and the HES + acetone medium performed nearly equal, while the standard medium reached only 75% of the amount. Trie slope of the standard medium is less steep than the slope of the other tested media. This indicates a lower overall growth rate of the cells.

Figure 6: The cumulative cell numbers of living SP2/0-311 hybridoma cells in serum-containing medium per day. In behalf of the overall growth rate (slope) no significant differences could be observed.

Figure 7: The specific productivity of monoclonal antibody by recombinant SP2/0 cell line determined from the linear relationship between product titer and cumulative cell-days. The cells cultivated in medium containing both new supplements reached the highest titer. The slopes of both HES containing media are the steepest suggesting HES as the best media supplement for this cell line. Figure 8: The cumulative cell numbers of living CHO-E5 cells in serum-free medium per day. Regarding the overall growth rate (slope), no significant differences were observed.

Figure 9: The specific productivity of monoclonal antibody produced by CHO-E5 cells determined from the linear relationship between product titer and cumulative cell-days. The HES supplemented medium revealed the steepest slope, followed by medium containing acetone. The slopes of the control and the medium with the mixture of both novel supplements were significantly lower.

Having generally described the invention, the same will be further understood by reference to the following examples, which are provided therein for purposes of illustration and are not limiting in any manner.

Methods and Materials Examples 1-3:

Cell Culture

The cell lines used were E4, a mouse hybridoma producing anti-idiotype antibody as surrogate of the Lewis Y carbohydrate antigen and 311 a recombinant SP2/0 producing a humanized anti-Lewis Y antibody and a recombinant SP2/0 producing anti-Lewis Y antibody and a recombinant CHO-E5 producing rmAB17- 1A.

The basal serum-free medium for E4 was Hybridoma Express (PAA, Linz) supplemented with 4 mM L-glutamine (Sigma Aldrich Corp., St. Louis MO, USA) and 0.1 % Pluronic F68 (Sigma, St. Louis MO). For cultivation of SP2/0 311 cell line RPMI 1640 (Gibco) supplemented with 10% Fetal Calf Seru m (Gibco) and 2 mM L-Glutamin was used. The serum-free medium for the SP2/0 311 cells was a mixture of Hybridoma Express (75%) and RPMI 1640 (25%) supplemented with 6 mM L-GIutamine. The CHO cells were cultivated in EX-Cell 325 PF CHO protein- free medium (JRH Biosciences, UK).

The various media were supplemented with either 10 μ or 100 μM acetone

(Merck, Germany), 0.2% Hydroxyethylstarch (Fresenius Kabi, Austria) or both addititives with the concentrations of 10 μM acetone an d 0.2weight% HES.

Cell culture was performed at 37°C in standard grade polystyrene cell culture flasks (Nunc, Roskilde, Denmark) in an incubator with T% CO₂.

All serum free cultivation experiments were done in parallel and in duplicates, indicated in Figures 1 to 4 as "cell nr. 1" and "cell nr. 2".

Cell Counting

Cell number of the supernatant was determined by the trypan blue dye exclusion method using a hemocytometer and following standard protocol (0.2% trypan blue).

Specific productivity was calculated as described elsewhere (Dutton R.L. et al.,

1998, Cytotechnology, 26: 139-152). The growth rate was calculated by linear regression of the log of cell densities versus time of the logarithmic phase of the culture.

Batch Culture

Tests were performed in 850 cm³ standard roller bottles (Corning, Wiesbaden,

Germany) with 450 mL medium. Inoculation density was 1.5 0⁵ cells mL^"1.

Sampling was performed on a one to three day basis. In order to reduce the host cell protein level in the supernatant the experiments we re terminated at a viability score below 40%.

HPLC-SEC-Analysis

Antibody concentration of serum-free cell culture supernatant was analyzed by

HPLC analysis using a ZORBAX G-250 (Agilent-technologies) column in a

Dionex HPLC system. To disintegrate potential aggregates, 220 mM NaH₂PO with 10% CH₃CN was used as running buffer. Effluent was monitored online at

214 nm. Product concentration was calculated by peak integration and normalization on a standardized IgG solution.

ELISA - Analysis

The titer of serum containing cell culture supernatant was measured by ELISA following standard procedures. Photometric extinction of the developed color reaction was measured at 492 nm (reference wavelength 620 nm). All tests were performed in duplicates.

IEF - Analysis

Cell culture supernatants and an IEF Marker (SERVA L iquid Mix 3-10) were loaded anodically onto a SERVALYT Precotes IEF flatbed gel (15-20μl) using filter paper pieces. Separation of isoforms according to their isoelectric points was performed by electrophoresis at 4°C using a Pharmacia Multi or II electrophoresis chamber and a Pharmacia EPS 3501 XL power supply. Electrophoresis conditions were 2000V, 6mA and 12W for a time period of 2.5 hours. After electrophoresis the gel was fixed with a fixing solution containing TCA and stained with Comassie Blue.

Results:

Both new supplements were first investigated in standard T-flask with E4 cells in serum-free medium. The observed parameter was volumetric productivity. The tested concentrations of acetone were 1 μM, 10 μM, 100 μM and 1 mM. A concentration of 0.2% for HES was chosen. In this example, e÷xcept of the 1 μM acetone containing medium, all other tests showed some positive effect on the produced amount of antibody (data not shown). The next step was to test also a mixture of both substances. The chosen concentrations were 10 μM acetone and 0.2% HES. Also this test revealed a positive effect on the cells (data not shown).

The next step of the development was to perform a roller bottle culture in batch mode with the same cells and supplement concentrations.

Both new supplements increased the growth rate and maximal cell density of hybridoma E4 cells (Figure 5) in comparison to the basal medium (Figure 1 ).

Using a mixture of the novel supplements in the media resulted in both, a slightly reduced growth rate and a decreased specific productivity.

The volumetric productivity in a proposed fed-batch bioprocess with medium containing HES increases 190% (Figure 2) and 210% for acetone (Figure 3). The combination of both supplements did not show this effect (Figure 4).

The recombinant SP2/0 311 cell line cultivated in serurn-containing media displayed an increase of the specific productivity for HES of 160%. Acetone increased the specific productivity by 120% (Figure 7). The mixture of both supplements showed the best result with an increase of 21 0% (Table 1 ). The growth rate in all tested media was nearly identical (Figure 6).

In this case the best bioprocess to optimize the productivity is a perfusion process. Due to the increased specific productivity the yield could be doubled. In serum-free media the supplements did not show this enormous effect on the cell line, but an increase of the specific productivity could be observed (Table 1 ).

Table 1 : Population growth and productivity batch culture parameters of cell lines (SP2/0 hybridoma, recombinant SP2/0 and CHO). The recombinant CHO-E5 cell line cultivated in media containing the novel supplements displayed an increased specific productivity of 350% for HES (Figure 9) and 220% for acetone. The combination of both supplements did not show this effect. The growth rates of the cells cultured in all tested media were similar (Figure 8). Therefore an increase of the volumetric productivity for HES (270%) and acetone (250%) could be shown.

The quality of the produced monoclonal antibody of both cell lines, analyzed by ELISA and IEF, was not disturbed by the increased productivities (Suriyasathapom, W., A.J. et ai. 1999. Vet Immunol Immunopathol 68:177-186). The measurement of the CDC (complement-dependent cytotoxicity) of 311 monoclonal antibody produced in HES containing medium revealed a slightly positive influence of the culture conditions on the potency of the antibody in this test (data not shown).

Osmotic stress, especially hyperosmotic stress, negatively influences the glycosylation of recombinant proteins (Schmelzer, A.E. and W.M. Miller, 2002, Biotechnology Progress 18:346-353.). The novel media supplements are capable to reduce the osmolality of medium either containing serum or not, thus the glycosylation pattern can be positively influenced by simultaneously raising the available amount of energy sources.

Hydroxyethylstarch can be used as glucose storage. The cells can degrade the polymer as needed to obtain the necessary amount of the primary energy source glucose. The initial amount of glucose can be reduced and the high molecular weight of the biopolymer helps to decrease the osmolality in the medium, while maintaining high glucose levels.

Acetone on the other side opens a third source of energy. The up-take of acetone into the cells is not known, but as it dissolves easily in fatty acids, it might freely access the cells. There NADPH and acetone are co-oxidized by acetone- monooxygenase producing NADP and acetol. Acetol can be metabolized either via acetic acid or via pyruvic acid to acetyl-CoA. The acetyl-CoA can than be degraded in the TCC to CO₂ producing ATP or it can be used to produce other metabolites, as necessary. Preliminary measurements of the concentration of acetone in the media with GC-MS revealed a consumption of the supplement during batch culture.

Claims

1. Animal cell culture medium supplement for the cultivation of cells characterised in that the medium supplement is acetone .

2. Animal cell culture medium supplement according to claim 1 characte rized in that said supplement contains hydroxyethylstarch (HES).

3. Animal cell culture medium supplement according to claim 1 characte rised in that acetone is in a concentration of 1 μM to 20mM, preferably 5μM to 10mM, preferably 10μM to 1mM, preferably 10 to 100μM.

4. Animal cell culture medium supplement according to any one of claim s 2 or 3, characterised in that HES is in a concentration of 0.01 to 10 weight%, preferably 0.1 to 5 weight%, preferably 0.2 to 2 weight%.

5. Animal cell culture medium supplement according to any one of claim s 1 to 4, characterised in that the animal cells are mammalian cells.

6. Animal cell culture medium supplement according to claim 5, characterised in that the mammalian cells are hybridoma cells.

7. Animal cell culture medium supplement according to claim 5, characterised in that the mammalian cells are CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDCK cells, T-FLY cells, BHK cells, SP2/0, NS0 or PerC6 cells or derivatives thereof.

8. Use of an animal cell culture supplement according to any one of claims 1 to 7 for the production of proteins.

9. Use according to claim 8, characterised in that the proteins are for the production of hybridoma derived proteins.

10. Use according to any one of claims 8 or 9 characterised in that the proteins are antibodies or derivatives or fragments thereof.

11. Use according to claim 10, characterised in that the antibodies or deriΛ/atives or fragments thereof are directed against a tumor associated antigen.

12. Use according to claim 11 characterised in that the antibodies or derivatives or fragments thereof are directed against EpCAM, Her2-neu, NCAM, CEA, TOL9- receptors, a T-cell peptide, Lewis Y, SialylTn, Globo H, GD2, GD3 or GIV12.

13. Method for the cultivation of cells comprising the steps of a) contacting said cells with a cell culture medium comprising a supplement according to any one of claims 1 to 7 and b) cultivating said cells under conditions suitable to support cultivation of said cells and production of a protein.

14. Method according to claim 13 characterised in that the cells are cultivated in suspension.

15. Method for increasing the growth rate of cells characterised in that the cells are cultivated in a cell culture medium comprising a supplement according to any one of claims 1 to 7.

16. Method for increasing the yield of cells characterised in that the cells are cultivated in a cell culture medium comprising a supplement according to any one of claims 1 to 7.

17. Method according to claim 16, characterised in that the yield is increased up to at least 20%, preferably at least 50%, preferably at least 100%; preferably at least 150%, preferably at least 250%.

18. Method for obtaining an antibody or antibody derivative or fragment thereof by a cell culture which is cultivated in a medium comprising a cell culture supplement according to any one of claims 1 to 7, comprising the steps of:

(a) culturing the cells which produce antibodies or derivatives or fragments thereof in the continuous presence of the supplement according to any of claims 1 to 7

(b) continuing said culture until said antibodies or derivatives or fragments thereof accumulate; and

(c) isolating said antibodies or derivatives or fragments thereof.