Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Reexamination Certificate
2000-11-17
2001-10-30
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
C435S068100, C435S404000
Reexamination Certificate
active
06309862
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to nutritive mediums for animal cells and specifically to a nutritive medium for human leukocytes.
BACKGROUND OF THE INVENTION
Usually animal cells are cultured in media, containing all necessary amino acids, vitamins, an energy source e.g. glucose and a balanced salt solution. The media can also contain trace amounts of different hormones such as insulin. The different components in the media can be altered depending on cell type and also the amount of the different components can be altered depending on the intended use. Some of these media are commercially available such as EMEM (Eagle's Minimum Essential Medium) which is well suited for a broad spectrum of mammalian cells, RPMI 1640, which was originally formulated for suspension cultures or monolayer cultures of human leukaemic cells, and DMEM (Dulbecco's Modified Eagle's Media) also well suited for broad spectrum of mammalian cells. In many cases, sera such as bovine serum and the like are added thereto. The addition of for example bovine serum is often necessary for accomplishing the desired growth and viability of the cultured cells.
Often, desired products are secreted from the cells and following a purification procedure the desired product secreted from the cells may be obtained in a sufficiently pure form. The purity of the end-products are always depending of the purity of the starting material. By using a purer, less complex starting material, in this case the medium, the purified end product will then become purer compared to using a more complex medium.
PRIOR ART
One way to improve the purity of the end product is to use a more simplified media which has been described in inter alia U.S. Pat. No. 4,696,899. This patent describes the manufacture of interferon-alpha and interferon-gamma from leukocytes using a simplified media.
The use of methionine as a component in serum free media for culturing animal cells is disclosed in EP0 501 435, which teaches the addition of methionine in an amount of 8.0 to 14.0 mg/l. Both higher and lower concentrations are rejected as lacking the desired effect.
The use of methionine in the formulation of polypeptide products for pharmaceutical or therapeutical use is known. Methionine is added to inhibit the oxidation of methionine residues with such polypeptides. According to U.S. Pat. No. 5,272,135 me thionine is added to a recombinant human epidermal growth factor (rhEGF) formulation in amounts ranging from 0.01 to0.3% (w/v) or in a ratio of 10:1-100:1 to the methionine residues within the protein.
Further U.S. Pat. No. 5,358,708 describes the addition of methionine for the stabilisation of interferon formulations in an amount of 2 mg/ml or in a ratio of methionine to protein component of 10:1-100:1.
SUMMARY OF THE INVENTION
The present inventors have surprisingly shown that a nutritive medium with a highly reduced number of ingredients but with the addition of methionine in amounts exceeding any previously reported amounts allows for an unchanged or even slightly increased yield of interferon together with a pronounced improvement in end product homogeneity, stability and a striking simplification of the preparation of the media resulting in economical benefits.
According to the invention there is provided a nutrient medium for protein producing cells. Said medium consists essentially of the following components: an aqueous physiological saline solution containing Ca
2+
, K
+
, Mg
2+
and Na
+
, an energy source, a pH buffer and methionine in an amount of 0.015-2.0 g/liter, and optionally antibiotics.
Preferred nutrient media have the following compositions:
A nutrient medium as described, characterized in that the medium has the following composition (g/l):
CaCl
2
× 2H
2
O
0.13-0.35
KCl
0.2-0.5
MgCl
2
× 6H
2
O
0-0.5
NaCl
5.0-8.0
NaH
2
PO
4
× 2H
2
O
0-0.15
Glucose
1.0-7.0
Tricine
1.5-3.0
NaHCO
3
0.5-3.0
Neomycin 10%
0-0.25
Methionine
0.015-2.0, and
Agamma-plasma
1.0-10 % (v/v).
A nutrient medium as described, characterized in that the medium has the following composition (g/l):
CaCl
2
× 2H
2
O
0.13-0.2
KCl
0.2-0.4
MgCl
2
× 6H
2
O
0-0.2
NaCl
6.8-8.0
NaH
2
PO
4
× 2H
2
O
0-0.105
Glucose
1.0-5.0
Tricine
1.5-3.0
NaHCO
3
0.75-1.0
Neomycin 10%
0-0.25
Methionine
0.015-2.0, and
Agamma-plasma
3.0-5 % (v/v).
A nutrient medium as described above but with a methionine content of 0.050-2.0 g/l.
A nutrient medium as described above but with a methionine content of 0.050-0.100 g/l.
A nutrient medium as described, characterized in that the medium has the following composition (g/l):
CaCl
2
× 2H
2
O
0.2
KCl
0.4
MgCl
2
× 6H
2
O
0.2
NaCl
6.8
NaH
2
PO
4
× 2H
2
O
0.105
Glucose
5.0
Tricine
1.5
NaHCO
3
0.75
Neomycin 10%
0.25
Methionine
0.75, and
Agamma-plasma
4% (v/v).
Another aspect of the invention is use of the nutrient medium for animal cells or human leukocytes. In a preferred embodiment the medium is used in a production process for interferon.
In comparison to the conventional and well known Eagle's Minimal Essential Medium (EMEM) the inventive medium totally lacks the amino acid stock solution, the vitamin solution and folic acid solution, constituting integrated parts of EMEM.
On the other hand, compared to the so called Cantell medium, first disclosed by Cantell et al. in 1981 (Methods in Enzymology, vol. 78, 1981) and commonly used for the incubation of human leukocytes, the inventive medium contains MgCl
2
, NaH
2
PO
4
but lacks L-glutamine. In their original work, Cantell et al. report that experiments aimed at identifying those ingredients in EMEM needed for optimum yields of interferon give conflicting results (see Example 2).
With this background, it is even more surprising that the inventive medium not only allows for the production of interferon with unreduced yields in comparison with EMEM. It also produces a marked improvement in yield in comparison with other simplified media. In addition, the less complex medium facilitates purification of the desired product and gives pronounced process economical benefits as the medium is easier to prepare and contains fewer media components to order, register and analyse etc. Moreover, the inventive medium, gives a more homogenous and more stable product with maintained yield.
The invention is also applicable on other types of cells since the invention is of a general nature. For growing cells, this is not the ideal medium but in a stationary phase or protein producing phase this medium gives many advantages as mentioned above. Thus, the invention can be applied on other protein-producing cells/cell-lines.
The main components in the inventive medium are CaCl
2
, a pH-buffer, an energy source, KCl, NaCl and methionine. The exact amount of each component is depending on the cell concentration used. A high cell concentration implies that more of or most of the components are needed, except for NaCl which has to be decreased in order to establish a physiological medium. Tricine is often used in cell cultures together with NaHCO
3
in order to keep the pH in an acceptable range. In the inventive process this range is between 7.0-8.0, most preferred 7.5, otherwise the viability and interferon yield will decrease. Other kinds of pH-buffers may also be used as long as they are non-toxic for the leukocytes. Ca
2+
has to be present in the medium, otherwise the interferon yield will become very low. Glucose is the cheapest energy source and it is also rapidly metabolized by the leukocytes. Therefore, it is well suited for these kinds of processes but other monosaccharides or disaccharides may function as well. Surprisingly we found that methionine had a very positive effect on the product when it is present in the medium. It seems to minimize the oxidation of the interferon-alpha proteins dur
Jarekrans Mats
Olovsson Hans
Bionative AB
Burns Doane Swecker & Mathis L.L.P.
Horlick Kenneth R.
Spiegler Alexander H.
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