JP2696001B2 - Medium for recombinant protein production - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium for culturing animal cells that continuously produce a desired recombinant protein by genetic recombination technology, and which is substantially free of protein components. Related to serum medium. More specifically,
The present invention relates to a low or protein-free medium substantially free of serum or plasma proteins for culturing recombinant animal blood coagulation factor VIII-producing transformed animal cells to produce a desired recombinant blood coagulation factor VIII.

[0002]

2. Description of the Related Art At present, various bioactive substances are produced as recombinant proteins in various hosts such as Escherichia coli by genetic recombination techniques. Among them, when a post-translational modification process such as addition of a sugar chain or construction of a higher-order structure is essential for the expression of the biological activity of the product, an animal cell is selected as a host. However, conventionally, there have been some technical problems in obtaining a large amount of a physiologically active substance secreted into the culture supernatant of animal cells.

[0003] In culturing animal cells, it has generally been necessary to add serum such as fetal calf serum (FCS).
Serum is very expensive and may be contaminated with mycoplasma or virus, so that the serum used has to be assayed in advance. In addition, serum contains various heterologous protein components, and therefore, it has not always been easy to purify the target substance from the culture supernatant. Therefore, as a substitute for the serum medium, a medium supplemented with only protein components having obvious properties such as insulin, transferrin, and serum albumin has been developed (D.Bames, GHSato, Cell, Vol. 22, 649).
P. 1980).

[0004] Further, technical ideas for replacing the above-mentioned protein components with other compounds have been increased, and iron-chelating agents such as ethylenediaminetetraacetic acid iron complex and iron gluconate have been already developed as transferrin transferrins. (JP-A-63-141584; Proceedings of the 8th Symposium on Next-Generation Industrial Technology / Biotechnology). Also,
As an alternative to albumin, an inclusion compound of α-cyclodextrin with an unsaturated fatty acid and a fat-soluble vitamin is known (JP-A-56-81600). It has also been reported that serum-free culture of animal cells is possible by addition of a microemulsion of Pluronic F-68 and a fat-soluble factor (International Patent Application WO 90/03429).

[0005] However, the low or protein-free medium disclosed in these reports has been developed using only cell growth as an indicator, and when a recombinant protein is produced using transgenic animal cells, the desired culture medium is required. The effect from the viewpoint of the production efficiency of the recombinant protein was never satisfactory.

[0006] Under such circumstances, the present inventors have actively studied the medium for mass production of recombinant protein by animal cells, and used for example in replacement therapy for hemophilia A patients. For example, when 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 the serum medium, A patent application has already been filed (Japanese Patent Application No. 63-157570).

[0007] However, the addition of protein components such as serum albumin makes purification of the desired product very difficult. In addition, serum albumin is valuable and expensive, so that it is practically difficult to add serum albumin from the viewpoint of economic efficiency when it is intended for mass production.

The inventors of the present invention have conducted intensive studies to solve these problems, and as a result, cultured animal cells producing a desired recombinant protein, particularly transformed animal cells producing a recombinant blood coagulation factor VIII. Culture system, ethylene oxide polypropylene glycol (Pluronic F-68; Pl
pluronic surfactants represented by uronic F-68),
Alternatively, a nonionic surfactant including sorbitan surfactant such as sorbitol monolaurate (Tween 20) and sorbitol monooleate (Tween 80), and cyclodextrin represented by dimethyl-α-cyclodextrin are added. This has led to the development of a low or protein-free medium that substantially no longer requires the addition of protein components such as serum albumin, and has completed the present invention.

[0009]

Means for Solving the Problems In the present invention, the present inventors have determined that a blood coagulation preparation prepared by a genetic recombination technique is used.
When culturing transformed Chinese hamster ovary (CHO) cells that continuously produce factor VIII, a medium containing a pluronic or sorbitan nonionic surfactant and cyclodextrin exerts a surprising effect,
In terms of cell growth and blood coagulation factor VIII-producing ability, it was clarified that the serum albumin-containing medium could be an alternative to the conventional serum-free medium.

The pluronic nonionic surfactants used in the present invention include sorbitan nonionic surfactants such as Pluronic F-61, Pluronic F-68, Pluronic F-71 and Pluronic F-108. As the sorbitol, sorbitol monolaurate (Tween20), sorbitol monooleate (Tween80) and the like are suitably selected.

Cyclodextrin is an unmodified α-
Cyclodextrin (α-CD) can be preferably used, but 2,6-dimethyl-α-cyclodextrin (DM-α-CD)
Is one of the best examples. DM-α- in the present invention
The CD is a dextrin in which six (α) glucose units in which the hydroxyl groups at the 2- and 6-positions of the glucose residue are methylated are cyclic.

[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.
~ 1.0%, preferably in the range of 0.01-0.1% pluronic surfactant, and 0.001-0.3%, preferably 0.01-0.
Α-CD in the range of 1%, or 0.001-0.006%, preferably
A medium containing a sorbitan surfactant in the range of 0.002 to 0.003% and α-CD in the same concentration range as described above is preferable.

Generally, Chinese hamster ovary cells (C) are used as animal cells for producing proteins having physiological activities.
HO cells), C-127 cells, Namalwa cells and the like are used, and there are no particular restrictions on the subject of the present invention. However,
The most frequently used CHO cells are used as an example of a suitable target cell.

In an embodiment of the present invention, the medium of the present 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 cultured by replacing the above-mentioned additives with a medium containing an appropriate concentration of the above-mentioned additives. . The nonionic surfactant and cyclodextrin may be added to the medium, and then sterilized by filtration.However, after the high-concentration solution is steam-heat sterilized, a predetermined amount may be added to the medium. It is possible. In the examples, the ASF medium 104 was used as a basal medium, but is not limited thereto.
A synthetic medium containing insulin, transferrin, ethanolamine, selenite and the like can be suitably used.

The present inventors have previously shown that the ability of cultured cells to produce proteins can be enhanced by the addition of butyrate and lithium salt to the medium, and have already filed a patent application (Japanese Patent Application No. Hei. -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 a factor of 2 or more compared to the conventional culture in serum albumin-containing medium. Was confirmed, and it was confirmed that the effect of the present invention could be further increased.

Hereinafter, in order to further clarify the features of the present invention, the present invention will be described in detail with reference to Examples. However, these Examples illustrate specific examples of the present invention. It is not limited to the embodiment.

[0017]Example 1  Pluronic F-68 supplemented medium  Based on ASF medium 104 (Ajinomoto Co., Inc.) containing 0.1% DM-α-CD
As a culture medium, a predetermined concentration of Pluronic F-68 (GI
(BCO) was added. Cells are transformed using genetic engineering technology
Prepared blood coagulation factor VIII-producing Chinese hams
Ovarian cells (Japanese Patent Application No. 63-85454, Deposit No.
No. 73) and use this as a culture dish (Falcon
). At this time, the growth medium is 10% fetal bovine blood.
ASF medium 104 containing clear water, bottom area 9.6 cm^TwoNo well
5 × 10^FiveCells were seeded at a cell density. Cells grow well
And remove the growth medium by suction to remove
 The medium was replaced with a basal medium containing F-68. Medium capacity
The volume was 3 ml per well. Culture is CO_TwoIn the incubator
In CO_TwoThe test was performed at a concentration of 5% and a temperature of 37 ° C. DM-α-CD included
The medium without serum albumin was compared with the control. blood
Serum albumin is a 20% human serum albumin preparation
Was used. DM-α-CD is also available from Cosmo Bio
Was used.

The number of cells was measured using a hemocytometer on living cells other than dead cells stained with trypan blue. Factor VIII activity is determined by standard blood coagulation assays (Hardisty et al., Thrombosis et Diathesis Haemo
logica 72, p.215 (1962)), the ability of factor VIII-deficient plasma to reduce delayed partial thromboplastin time. At this time, the factor standard plasma for clotting factor quantification (Dade) was used as a standard, and this was set to 1 unit (international unit, IU) / ml. Table 1 shows the results. Table 1

[Table 1]

From these results, in addition to 0.1% DM-α-CD, a surfactant such as Pluronic F-68 was added in an amount of 0.005 to 1.
By adding in a concentration range of up to 0%, recombinant recombinant VII
It was confirmed that the ability to produce factor I was increased.

[0020]Example 2  Tween-80 supplemented medium  Except that Tween80 was used instead of Pluronic F-68
The same experimental procedure as in Example 1 was followed. Table 2 shows the results
Shown inTable 2

[Table 2]

From these results, it was revealed that the addition of Tween 80 in a concentration of 0.001 to 0.006% in addition to 0.1% of DM-α-CD increases the ability to produce recombinant factor VIII. .

[0022]Example 3  Dimethyl-α-cyclodextrin (DM-α-CD) supplemented medium  Add Pluronic F-68 at a concentration of 0.1% to ASF medium 104
As a basal medium, and add each concentration of DM-α-CD to this.
And compare its effect with that of serum albumin-containing medium.
Was. Otherwise, the experimental procedure was the same as in Example 1. Get
The results obtained are shown in Table 3.Table 3

[Table 3]

As a result, it was confirmed that the addition of DM-α-CD in a concentration range of 0.001 to 0.3% in addition to 0.1% Pluronic F-68 increases the production ability of recombinant factor VIII. Was. No effect was observed at concentrations lower than this range, and toxicity was observed in cultured cells at higher concentrations.

[0024]Example 4  Effect of adding butyrate and lithium salt  As a production medium, DM-α-CD and Pluronic
F-68 was added at a concentration of 0.1% each, and further 1 mM
Sodium butyrate (Wako Pure Chemicals, special grade) and 5 mM acetic acid
Titium (Wako Pure Chemicals, special grade) is added and cultured for 48 hours
Was continued. In addition, sodium butyrate was neutralized beforehand.
Was used. The results are shown in Table 4.Table 4

[Table 4]

As a result, DM-α-CD, Pluronic F-68
The effect of adding butyrate and lithium salt was also observed in the medium supplemented with 0.1% of each, and the production capacity increased more than twice that of the culture in the medium without butyrate and lithium salt or in the medium containing serum albumin. confirmed.

[0026]Example 5  Comparison of cell proliferation with serum albumin-containing medium  0.1% each of Pluronic F-68 and DM-α-CD
Growth of added media and conventional serum albumin-containing media
A comparative study was conducted. At this time, as a basal medium
ASF medium 104 was used.

Blood coagulation factor VIII-producing CHO cells were seeded at 2 × 10 ⁵ cells per dish well and cultured for 7 days while changing the medium every two days. Table 5 shows the results. Table 5

[Table 5]

From these results, Pluronic F-68,
And medium containing 0.1% DM-α-CD, respectively, show almost the same cell growth as the medium containing 1.0% serum albumin,
Even without adding serum albumin, it has become possible to obtain cell growth comparable to conventional serum albumin-supplemented media and accompanying substance production.

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-2-5890 (JP, A) JP-A-62-55076 (JP, A)

Claims (6)

(57) [Claims] 1. A medium for culturing transformed Chinese hamster ovary (CHO) cells that continuously produce blood coagulation factor VIII prepared using a genetic recombination technique, comprising a Pluronic or A low or protein-free medium for producing blood coagulation factor VIII, comprising a sorbitan-based nonionic surfactant and cyclodextrin. 2. The sorbitan-based nonionic surfactant is selected from the group consisting of Pluronic F-61, Pluronic F-68, Pluronic F-71, and Pluronic F-108. The low or protein-free medium for the production of blood coagulation factor VIII according to claim 1, wherein is selected from sorbitol laurate (Tween 20) and sorbitol monooleate (Tween 80). 3. The low or protein-free medium for producing blood coagulation factor VIII according to claim 1, wherein the cyclodextrin is selected from unmodified α-cyclodextrin and methylated α-cyclodextrin. 4. A pluronic nonionic surfactant and cyclodextrin in an amount of 0.005 to 1.0% and 0.001%, respectively.
The low or protein-free medium for the production of blood coagulation factor VIII according to claim 1, which is contained at a concentration of about 0.3 to 0.3%, preferably 0.01 to 0.1% and 0.01 to 0.1%, respectively. 5. A sorbitan-based nonionic surfactant and cyclodextrin are added in an amount of 0.001 to 0.006% and 0.006%, respectively.
The low or protein-free medium for producing blood coagulation factor VIII according to claim 1, which is contained at a concentration of 1 to 0.3%, preferably 0.002 to 0.003%, and 0.01 to 0.1%. 6. The composition according to claim 1, further comprising butyric acid or a salt thereof, and a lithium salt in a concentration of 0.1 to 5.0 mM, 2.0 to 50 mM, preferably 0.5 to 2.0 mM, 2.0 to 5.0 mM, respectively.
6. A low or protein-free medium for producing blood coagulation factor VIII according to any one of 5.