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Animal protein-free media for cultivation of cells

a technology of protein-free cell culture and animal protein, which is applied in the field of animal protein-free cell culture media, can solve the problems of bse contamination of all serum-derived products, risk of contamination with mycoplasma, and contaminated products derived from them, so as to increase the protein expression per cell, promote cell growth, and increase the yield of desired products

Inactive Publication Date: 2006-05-04
BAXTER INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013] An object of the present invention is to provide an animal protein-free cell culture medium which does not contain any added supplementary proteins derived from an animal source and / or recombinant animal proteins, which allows efficient cell growth and in particular protein production in a continuous quality with respect to the amount of expression per cell. A further object of the present invention is to provide a method for cultivating cells and a method for efficient expression of recombinant proteins which are free of animal proteins.
[0016] Surprisingly, the addition of at least one polyamine, in particular the addition of putrescine, to the animal protein-free cell culture medium provides the advantageous effect not only to promote the cell growth but in particular to increase the protein expression per cell and, in particular, recombinant protein expression per cell.
[0017] Further, the animal protein-free medium according to the present invention allows consistent cell growth and increased yield of desired products, particularly of target proteins such as recombinant proteins, independent of the quality or lot variations of the protein hydrolysate, in particular of the vegetable hydrolysates, in the animal protein-free cell culture medium. The specific supplementation of cell culture media with polyamines and a plant- and / or yeast-derived hydrolysate acts synergistically to increase cell growth, cell specific productivity and final cell density.
[0018] Therefore, the animal protein free medium according to the present invention is more favorable for recombinant protein expression and cell growth rate compared to the media known in the art. Furthermore, the animal protein-free medium according to the present invention allows the reduction of the amount of protein hydrolysate to be added to a given volume of the cell culture medium.

Problems solved by technology

In general, serum or serum-derived substances, such as albumin, transferrin or insulin, may contain unwanted agents that can contaminate the cell cultures and the biological products obtained therefrom.
Moreover, bovine serum and products derived therefrom bear the risk of BSE contamination.
In addition, all serum-derived products can be contaminated by unknown constituents.
In the case of serum or protein additives that are derived from human or other animal sources in cell culture, there are numerous problems (e.g. the varying quality in composition of the different batches and the risk of contamination with mycoplasma, viruses or BSE), particularly if the cells are used for production of drugs or vaccines for human administration.
However, the media known in the state of art are often nutritionally insufficient and / or must be supplemented with animal-derived protein supplements or recombinant versions of proteins, such as insulin, insulin like growth factor or other growth factors.

Method used

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  • Animal protein-free media for cultivation of cells
  • Animal protein-free media for cultivation of cells
  • Animal protein-free media for cultivation of cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

BAV-Medium

[0071] Animal protein free medium was prepared with basal DMEM / HAM's F12 (1:1) medium supplemented with inorganic salts, amino acids, vitamins and other components (Life technologies, 32500 Powder). Also added were L-glutamine (600 mg / L), ascorbic acid (20 μM), ethanol amine (25 μM), Synperonic® (SERVA) (0.25 g / L), sodium selenite (50 nM). Additionally, essential amino acids were supplemented to the cell culture medium. Further, varying concentrations of soy hydrolysate (Quest Technologies, NY or DMV Intl., NY ) in the range of 0.0-1.0% and varying concentrations of polyamines (0-10 mg / L) were added (FIG. 1-9)

example 2

[0072] Cell cultures of recombinant mammalian cells (e.g. CHO-cells stably expressing Factor VIII=GD8 / 6-cells) were grown in suspension in a chemostat culture in 10 I bioreactors. The culture conditions of 37° C., oxygen saturation 20% and pH 7.0 to 7.1 were kept constant. The cultures were supplied with a constant feed of BAV-medium as defined in Example 1 additionally supplemented with soy hydrolysates in the range of 0.1-1.0% and / or addition of putrescine.2HCl in the range of 0-1 mg / L (cf. FIG. 1-5).

[0073] Small scale experiments with GD8 / 6 cells in suspension culture were carried in Techne spinner flasks at 200 ml working volume in batch refeed mode at 37° C., without pH and pO2 control. The cultures were supplied with BAV-medium as defined in Example 1 without supplementation of soy hydrolysate and polyamines, or supplemented with soy hydrolysate in the range of 0.1-0.4% and / or putrescine.2HCl, ornithine.HCl, spermine.4HCl in the range of 0-18 mg / L (equivalent to 0-10 mg / L of ...

example 3

cf. FIG. 1 to 57, and 9

[0074] Cell counts from suspension cells or immobilized cells were determined either by counting with a CASY® cell counter as described by Schärfe et al., (Biotechnologie in LaborPraxis 10: 1096-1103 (1988)) or by citric acid extraction and flourescent staining of the nuclei followed by counting with a NucleoCounter® (Chemometec, DK). The specific growth rate (μ) is calculated from the increase of the cell densities (Xt) and / or the dilution rate (D) of the steady state of chemostat cultures of suspensions cells over a certain time interval (t):

μ=D+In(Xt / X0) / t

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Abstract

The present invention relates to animal protein-free cell culture media comprising polyamines and a plant and / or yeast-derived hydrolysate. The invention also relates to animal protein-free culturing processes, wherein cells can be cultivated, propagated and passaged without adding supplementary animal proteins in the culture medium. These processes are useful in cultivating cells, such as recombinant cells or cells infected with a virus, and for producing biological products by cell culture processes.

Description

FIELD OF THE INVENTION [0001] The present invention relates to animal protein-free cell culture media comprising a polyamine and a plant- and / or yeast-derived hydrolysate. The invention also relates to animal protein-free culturing processes, wherein cells can be cultivated, propagated and passaged without adding supplementary animal proteins in the culture medium. These processes are useful in cultivating cells, such as recombinant cells or cells infected with a virus, and for producing biological products by cell culture processes. BACKGROUND OF THE INVENTION [0002] For cultivation of cells, particularly eukaryotic cells, and more specifically mammalian cells, there is a constant need to use special culture media that make available the growth nutrient substances that are required for efficient growth of the cells and for the production of the proteins or viruses that are desired. For the efficient production of biological products, such as viruses or recombinant proteins, it is i...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N7/00C12N5/02
CPCC12N5/0031C12N2500/46C12N2500/74C12N2500/76C12N2500/99C12N5/0043C12N2500/92C12N5/00C12N5/04C12N1/00C12N5/0682
Inventor GRILLBERGER, LEOPOLDREITER, MANFREDMUNDT, WOLFGANGDORNER, FRIEDRICH
Owner BAXTER INT INC
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