JPH04316484A - Medium for producing recombinant protein - Google Patents

Abstract

PURPOSE:To provide the title medium virtually free from serum protein, for incubating transformed animal cells capable of producing desired recombinant protein prepared by using the gene recombinant technique. CONSTITUTION:The objective low-protein or non-protein medium characterized by containing a nonionic surfactant or cyclodextrin, a medium for incubating transformed animal cells capable of sustainedly producing desired protein prepared by using the gene recombinant technique.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a culture medium for culturing animal cells that continuously produce a desired recombinant protein using genetic recombination technology, and relates to a medium for culturing animal cells that continuously produce a desired recombinant protein using genetic recombination technology. Regarding serum media. More specifically,
The present invention relates to a low or protein-free medium substantially free of serum or plasma proteins for culturing transformed animal cells producing recombinant blood coagulation factor VIII to produce the desired recombinant blood coagulation factor VIII.

0002

BACKGROUND OF THE INVENTION Today, various physiologically active substances are produced as recombinant proteins in various hosts including Escherichia coli by genetic recombination technology. Among these, animal cells are selected as hosts when post-translational modification processes such as addition of sugar chains and construction of higher-order structures are essential for the expression of physiological activity of the product. However, conventionally, there have been several technical problems in obtaining a large amount of physiologically active substances secreted into animal cell culture supernatants.

[0003] In culturing animal cells, it has generally been necessary to add serum such as fetal calf serum (FCS). Since serum is very expensive and may be contaminated with mycoplasma or viruses, it is complicated to test the serum to be used in advance. Furthermore, serum contains various foreign protein components, and therefore it has not always been easy to purify the target substance from the culture supernatant. Therefore, as an alternative to serum media, media containing only protein components with clear properties such as insulin, transferrin, and serum albumin have been developed (D. Bames, GH Sato,
Cell, vol. 22, p. 649, 1980).

[0004]Furthermore, there has been increasing technological thought to replace the above-mentioned protein components with other compounds, and iron chelating agents such as ethylenediaminetetraacetic acid iron complex and iron gluconate have already been developed as substitutes for transferrin. (Japanese Patent Application Laid-Open No. 63-141584; Proceedings of the 8th Next Generation Industrial Infrastructure Technology Symposium on Biotechnology). In addition, as a substitute for albumin, an inclusion compound of α-cyclodextrin, unsaturated fatty acid, and fat-soluble vitamins is known (Japanese Patent Application Laid-open No. 81600/1983). It has also been reported that serum-free culture of animal cells is possible by adding a microemulsion of Pluronic F-68 and a lipophilic factor (International Patent Application WO 90/03429).

[0005] However, the low- or protein-free media disclosed in these reports were developed using only cell proliferation as an indicator, and when producing recombinant proteins using transformed animal cells, it is difficult to obtain the desired protein content. The effects from the viewpoint of production efficiency of engineered proteins were by no means satisfactory.

Under these circumstances, the present inventors have been actively investigating a culture medium for the purpose of mass production of recombinant proteins using animal cells. In cases where the desired protein is unstable, such as human blood coagulation factor VIII, by adding 1% serum albumin to the medium, it is possible to increase the production capacity to about 4 times that of a serum medium. A patent application has already been filed (
Patent application Sho 63-157570).

However, the addition of protein components such as serum albumin makes purification of the desired product very difficult. Moreover, since serum albumin is valuable and expensive, it is practically difficult to add it from an economical point of view when mass production is aimed at.

[0008] The present inventors have conducted extensive research to solve these problems, and as a result, we have found that the culture of animal cells that produce the desired recombinant protein, particularly the transformed animal cells that produce recombinant blood coagulation factor VIII. In the culture system, ethylene oxide polypropylene glycol (Pluronic F
-68; Pluronic surfactants such as Pluronic F-68), or nonionic surfactants including sorbitan surfactants such as sorbitol monolaurate (Tween 20) and sorbitol monooleate (Tween 80) By adding cyclodextrin, typified by I ended up doing it.

[0009]

[Means for Solving the Problems] In the present invention, the present inventors have provided a nonionic surfactant and It has been shown that a medium containing cyclodextrin exhibits surprising effects and can serve as an alternative to serum albumin-containing medium, which outperforms conventional serum-free medium in terms of cell growth and desired protein production.

[0010] Examples of the nonionic surfactants used in the present invention include Pluronic F-61 and Pluronic F-68.
Pluronic surfactants such as , Pluronic F-71 and Pluronic F-108, or sorbitan surfactants such as sorbitol monolaurate (Tween 20) and sorbitol monooleate (Tween 80) are preferably selected.

[0011] Cyclodextrin is also an unmodified α-
Cyclodextrin (α-CD) can be preferably used, and 2,6-dimethyl-α-cyclodextrin (DM-α-CD) is the best example. DM-α-CD in the present invention refers to 2 glucose residues.
Dextrin is a cyclic dextrin consisting of six (α) glucose units with methylated hydroxyl groups at position and 6-position.

[0012] The concentration range of each additive in the present invention should be set so as not to adversely affect the growth and production efficiency of the transformed animal cells to be grown.
Pluronic surfactants ranging from 0.05 to 1.0%, preferably from 0.01 to 0.1%, and from 0.001 to 0.00%.
3% α-CD, preferably in the range of 0.01-0.1%;
or 0.001-0.006%, preferably 0.00
A medium containing a sorbitan surfactant in the range of 2 to 0.003% and α-CD in the same concentration range as above is preferred.

Generally, Chinese hamster ovary cells (
CHO cells), C-127 cells, Namalwa cells, etc., are used, and there are no particular restrictions on the targets of the present invention. However, the most frequently used CHO cells are used as an example of suitable target cells.

In an embodiment of the invention, the medium of the invention containing a nonionic surfactant and cyclodextrin is used as follows. First, target cells are cultured in a medium containing 10% serum for 8 to 24 hours, and then the cells can be grown by replacing the medium with a medium containing the above-mentioned additives at a suitable concentration. . Note that nonionic surfactants and cyclodextrins can also be prepared by adding them to the culture medium and sterilizing them by filtration.
It is also possible to use a high concentration solution by adding a predetermined amount to the culture medium after sterilizing it by steam heating. Furthermore, although ASF medium 104 was used as the basal medium in the examples, the present invention is not limited thereto, and synthetic media containing insulin, transferrin, ethanolamine, selenite, etc. may be suitably used.

The present inventors have previously revealed that the protein production ability of cultured cells is enhanced by the addition of butyrate and lithium salt to the medium, and have already filed a patent application (Patent Application No. -121729). When the above-mentioned butyrate and lithium salt are added to the above-mentioned low or protein-free medium provided in the present invention, the protein production ability is increased by more than twice compared to culture in a conventional serum albumin-containing medium. was observed, and it was confirmed that the effects of the present invention could be further increased.

[0016] Hereinafter, in order to further clarify the characteristics of the present invention, the present invention will be described in detail along with Examples, but these Examples are intended to explain specific examples of the present invention, and the present invention It is not limited to the examples.

Example 1 Pluronic F-68 supplemented medium ASF medium 104 containing 0.1% DM-α-CD (
Ajinomoto Co., Inc.) was used as a basal medium, and a predetermined concentration of Pluronic F-68 (GIBCO) was added thereto. The cells were blood coagulation factor VIII-producing Chinese hamster ovary cells (
Patent application 1986-85454, deposit number: FEIKEN No. 98
No. 73) and culture dish (Fa
(manufactured by lcon). At this time, the growth medium is 1
ASF medium 104 containing 0% fetal bovine serum was used and cells were seeded at a density of 5 x 105 cells per well with a base area of 9.6 cm2. After the cells had grown sufficiently, the growth medium was removed by suction and replaced with a basal medium containing various concentrations of Pluronic F-68. The volume of medium was 3 ml per well. Culture in a CO2 incubator with a CO2 concentration of 5%.
, at a temperature of 37°C. A comparison study was made with a serum albumin-containing medium that does not contain DM-α-CD. As the serum albumin, a 20% human serum albumin preparation (manufactured by Kaketsuken) was used. Moreover, DM-α-CD manufactured by Cosmo Bio was used.

The cell number was measured using a hemocytometer for living cells other than dead cells stained with trypan blue. Factor VIII activity can also be determined using standard blood coagulation assays (Hardisty et al., Thro
mbosis et diathesis Haemo
logica 72, p. 215 (1962)), the ability of factor VIII-deficient plasma to reduce delayed partial thromboplastin time was determined. At this time, factor standard plasma for coagulation factor determination (Dade) was used as the standard, and this was measured at 1 unit (international unit, IU)/
ml. The results are shown in Table 1. Table 1

[Table 1]

From these results, it can be seen that 0.1% DM-α-
It was confirmed that the production ability of recombinant factor VIII was increased by adding a surfactant such as Pluronic F-68 in a concentration range of 0.005 to 1.0% in addition to CD.

Example 2 Tween-80 supplemented medium Tween 80 was used instead of Pluronic F-68
The same experimental procedure as in Example 1 was followed except that . The results are shown in Table 2. Table 2

[Table 2]

From these results, 0.1% DM-α-
In addition to the CD, Tween 80 from 0.001 to 0.00
It was revealed that the production ability of recombinant factor VIII was increased by adding it in a concentration range of 6%.

Example 3 Dimethyl-α-cyclodextrin (DM-α-C
D) Supplementary medium ASF medium 104 with Pluronic F-68 added at a concentration of 0.1% is used as a basal medium,
Various concentrations of DM-α-CD were added to this, and the effects were compared with a serum albumin-containing medium. Other than this, the same experimental procedure as in Example 1 was followed. The results obtained are shown in Table 3. Table 3

[Table 3]

As a result, 0.1% Pluronic F-6
In addition to 8, it was observed that the production ability of recombinant factor VIII was increased by adding DM-α-CD in a concentration range of 0.001 to 0.3%. No effect was observed at concentrations lower than this range, and toxicity was observed in cultured cells at higher concentrations.

Example 4 Effect of addition of butyrate and lithium salt As a production medium, DM-α-CD and Pluronic F-68 were added to ASF medium 104 at a concentration of 0.1%, and 1 mM sodium butyrate ( Wako Pure Chemical, special grade) and 5m
M lithium acetate (Wako Pure Chemical, special grade) was added to each, and the culture was continued for 48 hours. Note that the sodium butyrate used was neutralized in advance. The results are shown in Table 4. Table 4

[Table 4]

As a result, the effect of addition of butyrate and lithium salt was observed even in the medium to which DM-α-CD and Pluronic F-68 were added at a concentration of 0.1%, and the effect of the addition of butyrate and lithium salt was observed. Alternatively, it was confirmed that the productivity increased by more than twice that of culture in a medium supplemented with serum albumin.

Example 5 Comparison of cell proliferation with serum albumin-containing medium
A comparative study was conducted on the proliferation performance of a medium to which 0.1% of each of Pluronic F-68 and DM-α-CD was added and a conventional serum albumin-containing medium. At this time, ASF medium 104 was used as the basal medium.

Blood coagulation factor VIII-producing CHO cells were seeded at 2×10 5 cells per dishwell and cultured for 7 days with repeated medium exchange every 2 days. The results are shown in Table 5. Table 5

[Table 5]

From these results, Pluronic F-6
A medium supplemented with 0.1% of 8 and DM-α-CD each exhibited cell proliferation properties almost equivalent to a medium containing 1.0% serum albumin, and even without the addition of serum albumin, it was superior to a conventional serum albumin-containing medium. It has now become possible to obtain cell proliferation and associated material production comparable to that of

Claims (7)

[Claims] Claim 1: A medium for culturing transformed animal cells that continuously produce a desired protein prepared using genetic recombination technology, the medium containing a nonionic surfactant and a cyclodextrin. A low or no protein medium for protein production, characterized by: 2. The low- or protein-free medium for protein production according to claim 1, wherein said cells are transformed animal cells that produce blood coagulation factor VIII prepared using genetic recombination technology. 3. The nonionic surfactant is Pluronic F-61, Pluronic F-68, Pluronic F
-71, Pluronic surfactant selected from Pluronic F-108, or sorbitol laurate (
Tween20), sorbitol monooleate (Tw
The low- or no-protein medium for protein production according to claim 1, which is a sorbitan-based surfactant selected from the following. 4. The cyclodextrin is unmodified α-
The low or protein-free medium for protein production according to claim 1, which is selected from cyclodextrin and methylated α-cyclodextrin. 5. Pluronic surfactant and cyclodextrin at 0.005 to 1.0% and 0.00%, respectively.
1-0.3%, preferably 0.01-0.1% each, 0
．． The low or no protein medium for protein production according to claim 1, containing the medium at a concentration of 0.01 to 0.1%. 6. Sorbitan surfactant and cyclodextrin in amounts of 0.001 to 0.006% and 0.00%, respectively.
001-0.3%, preferably 0.002-0.003
%, low or protein-free medium for protein production according to claim 1, containing the medium at a concentration of 0.01 to 0.1%. 7. Additionally, butyric acid or a salt thereof, and a lithium salt are added at 0.1 to 5.0 mM and 2.0 to 50 mM, respectively.
M, preferably 0.5-2.0mM, 2.0-5.
A low or protein-free medium for protein production according to any one of claims 1 to 6, containing the medium at a concentration of 0mM.