FIELD OF THE INVENTION

The present invention relates to biotechnological production processes. More specifically, the present invention relates to improved culture media for use in biotechnological production processes, processes employing such improved media, and to products obtained from the processes using the improved culture media.

BACKGROUND OF THE INVENTION

Biotechnological processes are widely used for the production of biological products. These processes typically involve the cultivation of cells in a culture medium under conditions permissible to the growth and product formation by the cultivated cells. Cells useful in biotechnological production are bacterial cells, fungal cells, yeast cells, and cells of animal or plant origin. Animal cell culture has long been used for the production of biological products, such as therapeutic proteins, polypeptides, peptides or other biological molecules, such as therapeutic polysaccharides. In such cell culture processes, animal cells, normally genetically modified to produce a desired product, are cultivated in a liquid, solid or semi solid culture medium for cell proliferation and product formation. One significant advantage of cell culture is the fact that animal cells and plant cells are able to perform post-translational modifications of the primary product, such as folding and post-translational modification of a polypeptide.

In biotechnological production processes, in particular in animal cell cultures, control and optimization of the cell culture conditions is critical for cell proliferation and product formation. One decisive factor is medium composition. The concentration and quality of the final cell culture product depends heavily on the medium composition. Animal and plant cell cultures are particularly demanding in terms of the required nutrient composition and culture conditions. Required nutrients not only include basic sources for carbon, nitrogen and energy, such as sugar and ammonia, but also more complex nutrients, such as essential amino acids, and vitamins may be required. For this reason, supplementation of animal cell culture media with complex nutrient compositions, such as serum, has been used to provide the animal cells with a broad variety of nutrients. However, regulatory and safety concerns, as well as problems with the heterogeneity of the available sources of sera has led the industry to strive towards eliminating serum and other non-defined media from industrial cell culture processes. Cell cultures grown in serum-free media, however, often show nutritional deficiencies. For this reason, much effort has been given to identify those nutritional components of complex media, as well as their optimal concentrations, which are required for satisfactory growth and protein formation in cell cultures.

The supply of cell cultures with amino acids is known to have a significant effect on growth rate and production. Glutamine is routinely used in cell culture media, since it is an important source of carbon, nitrogen and energy for the cultured cells. It was demonstrated that the amount of glutamine necessary for optimal growth of animal cell cultures is 3 to 10 times greater than the amount of other amino acids (Eagle et al., Science 130:432-37). However, glutamine as a nutrient is unstable when dissolved in water at elevated temperatures, such as heat sterilizing conditions, because pyroglutamate and ammonia are formed under heat (Roth et al. 1988, In Vitro Cellular & Developmental Biology 24(7): 696-98). For this reason, glutamate is often used in cell culture media instead of glutamine (Cell Culture Technology for Pharmaceutical and Cell-based Therapies, 52, Sadettin et al., Eds., Taylor and Francis Group, 2006). Other groups have tried to avoid the formation of unwanted substances, such as pyroglutamate and ammonia, by adding glutamine-containing dipeptides, such as alanyl-glutamine or glycyl-glutamine, to the cell culture medium (Roth et al. (1988), In Vitro Cellular & Developmental Biology 24(7): 696-98). Yet other groups have proposed acylating dipeptides in order to make them more stable under heat sterilization conditions. U.S. Pat. No. 5,534,538 describes N-acyl dipeptides and their use in heat sterilized enteral or parenteral nutrition products.

EP 0220379 and DE3538310 disclose the use of glutamine-containing dipeptides and tripeptides in cell culture media. Glutamine is added in form of dipeptides, in order to avoid problems stemming from the instability of glutamine under elevated temperatures. The glutamine-containing dipeptides are used as a temperature insensitive glutamine source.

JP61271985 discloses a culture medium useful for animal cells including the dipeptides Xxx-L-glutamine, wherein Xxx is glycine, D/L-alanine, L-aspartic acid, L-glutamic acid, L-valine, L-leucine, L-serine, L-lysine or L-phenylalanine.

While cell culture media described in the prior art provide satisfactory performance and properties for certain cell culture processes, there is still a need for cell culture media promoting improved product formation in biotechnological production processes.

THE PRESENT INVENTION

In the context of the present invention, it was found that the following cell culture media increase the productivity of biotechnological production processes:

Cell culture media comprising L-glutamine from a set of N-acylated dipeptides Acyl-X-Q and L-glutamine from a set of other glutamine-sources Qsource in a defined molar ratio R=n(Acyl-X-Q)/n(Qsource);

wherein X is defined as an L-amino acid;

wherein Q is defined as L-glutamine attached via an amide bond to L-amino acid X;

wherein Acyl is defined as a C1-C7-acyl moiety attached via an amide bond to the amino-terminus of L-amino acid X;

wherein R is defined to be in the range of 0,03 to 20;

wherein n(Acyl-X-Q) is the total amount of substance of L-glutamine contained in the set of N-acylated dipeptides Acyl-X-Q in the culture media; and

wherein n(Qsource) is the total amount of substance of L-glutamine contained in the set of other glutamine sources Qsource in the culture media.

The constituents of the set of other L-glutamine sources Qsource are selected from the following: free L-glutamine, dipeptides Y-Q, or mixtures thereof, wherein Y is defined as one of the 20 genetically encoded L-amino acids, wherein Q is defined as L-glutamine attached via an amide bond to L-amino acid Y, wherein the amide bond connecting Y and Q in dipeptide Y-Q is a regular backbone amide bond involving the carboxy terminus of amino acid Y and the amino terminus of glutamine Q.

In a first aspect the present invention relates to above disclosed culture media. The present invention, further, relates to the use of such cell culture media for culturing cells as well as to processes for the manufacture of cell culture products, comprising the step: Contacting cells with a cell culture medium as disclosed herein.

