JPH07203958A - Chinese hamster ovarian cell variant strain - Google Patents

Abstract

PURPOSE:To obtain the variant strain useful as a host for producing a physiologically active substance (a glycoprotein, etc.) without any H-D antigenicity by mutating a Chinese hamster ovarian(CHO) cell and reducing the productivity of N-glycolylneuraminic acid. CONSTITUTION:This variant strain is obtained by mutating a CHO cell and selecting a strain having a reduced productivity of N-glycolylneuraminic acid (NeuGc). UV rays, X-rays, etc., are used as a physical mutagen and an alkylating agent, especially ethylmethanesulfonate is preferred as a chemical mutagen. The resultant 1A51 strain (FERM P-14066) obtained by the mutation has about 5/14 productivity of NeuGc and <=1/5 hydroxylase activity of cytidine 5'-monophosphate-N-acetylneuraminic acid (CMP-NeuAc) as compared with those of CHO-K1 cell which is a parent strain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a mutant strain of Chinese hamster ovary (hereinafter abbreviated as CHO) cell, and more specifically, it is useful as a host cell for gene recombination technology.
A mutant strain of HO cells and use thereof.

[0002]

2. Description of the Related Art Many attempts have been made to produce a large amount of human-derived physiologically active substances that are present in the living body in a large amount by utilizing gene recombination technology and apply them to pharmaceutical products. In such gene recombination technology, Escherichia coli is most widely used as a host cell into which a recombinant vector is introduced, from the viewpoints of handleability, growth ability, easiness of culture means, and the like. However, human-derived physiologically active substances are often glycoproteins, whereas when expressed in microorganisms such as Escherichia coli, sugar chains are often not attached. Therefore, in recent years, for the purpose of obtaining a target substance in a form closer to that of a human physiologically active substance, production is being carried out using mammalian cells instead of E. coli as host cells. CHO cells are widely used as the host cells derived from the mammal.

[0003]

However, since the sugar chain structure has species specificity, the use of non-human cells such as CHO cells results in the addition of sugar chains different from the original human chains. It has been pointed out that the product may have antigenicity to humans [Hokk
e, C.I. H. Et al: FEBS Letters, 275.
9-14 (1990)].

CHO cells are no exception, and humans and CHO
Sialic acid composition differs from cells. That is, although humans usually do not have N-glycolylneuraminic acid,
Since O cells have N-glycolylneuraminic acid, the glycoprotein produced by using CHO cells is endowed with a glycoconjugate containing N-glycolylneuraminic acid. . This complex sugar containing N-glycolylneuraminic acid has strong antigenicity to humans,
Since ancient times, H-D antigen (Hanganutziu-Dei)
cher antigen). CHO like this
Glycoproteins produced using cells have the drawback that they are highly likely to have antigenicity derived from the HD antigen.

Therefore, an object of the present invention is to produce a novel CHO cell mutant strain which can be used for producing a physiologically active substance derived from the HD antigen and which does not generate antigenicity, and a glycoprotein which uses the mutant strain as a host cell. To provide the law.

[0006]

Therefore, the inventors of the present invention CH
O cells were subjected to mutagenesis treatment, and the mutated strains obtained were examined for sialic acid composition, sugar-related enzyme activity, etc.
-Successful collection of a novel mutant strain having a reduced ability to produce glycolylneuraminic acid, and completed the present invention.

That is, the present invention relates to a CHO cell mutant having a reduced N-glycolylneuraminic acid productivity.

In the present invention, a recombinant vector having a gene encoding a glycoprotein is introduced into this CHO cell mutant strain having a reduced N-glycolylnotemine acid productivity, and the resulting transformant is cultured. The present invention relates to a method for producing a glycoprotein characterized by the following.

The mutant strain of the present invention has the same properties as ordinary CHO cells except that the productivity of N-glycolylneuraminic acid (hereinafter referred to as NeuGc) is lower than that of the unmutated strain. Is. The NeuGc productivity of the mutant strain of the present invention is preferably 1/2 or less that of the non-mutant strain. The decrease in NeuGc productivity of the mutant strain of the present invention is caused by cytidine-
The activity of 5'-monophosphorylated N-acetylneuraminic acid hydroxylase was lower than that of the unmutated strain, and cytidine-5'-monophosphorylated N-acetylneuraminic acid (CM
P-NeuAc) to cytidine-5'-monophosphorylated N
-It occurs because the conversion to glycolylneuraminic acid (CMP-NeuGc) does not occur, and it is preferable that the hydroxylase activity is reduced to 1/5 or less as compared with the unmutated strain.

The mutant strain of the present invention is produced by subjecting CHO cells to a mutagenesis treatment and selecting a strain having a decreased NeuGc productivity.

The CHO cells to be used are not particularly limited as long as they are established as a normal culture cell line. For example, CHO-K1 cells, CHO-AA8 cells,
CHO-EM9 cells, CHO-Pro- ⁵ cells, CHO /
dhFr ⁻ cells and the like.

The mutagenesis treatment may be either a physical mutagen or a chemical mutagen. Examples of physical mutagens include ultraviolet rays and X-rays. As chemical mutagens, ethyl methane sulfonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl nitrosourea (EN
U) and other alkylating agents; bromodeoxyuridine (Br)
dUrd), base analogs such as N ⁴ -aminocytidine;
The ICR compound etc. of an intercalator are mentioned. Of these, chemical mutagens, especially alkylating agents, and further EMS
Is preferred.

The mutagenesis treatment of CHO cells is, for example, in the case of induction by a chemical mutagen, 10 ⁶ to 10 ⁹ CH in a logarithmic growth phase in a medium in which CHO cells can grow.
It is carried out by culturing O cells at 25 to 40 ° C. for 0.5 to 30 hours in the presence of a chemical mutagen in an amount such that the viability is 10 to 50%. The medium used here is Ham F-1 containing 5 to 10% serum such as fetal bovine.
2 medium, D-MEM medium, RPMI 1640 medium, serum-free medium and the like are preferable. When EMS is used as a mutagen, it is preferable to carry out mutagenesis by culturing in a medium containing 100 to 1000 μg / ml of EMS at 25 to 40 ° C. for 5 to 30 hours.

In order to screen the mutant strain of the present invention from the CHO cell group subjected to mutation treatment, CMP-Ne, a method for measuring NeuGlc content by direct sialic acid analysis.
Examples include a method for measuring uAc hydroxylase activity, a method for measuring the presence or absence of HD antigenicity, and anti-GM3 (Ne
uAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer)
A method for selecting cells in which the amount of GM3 on the cell surface is reduced by using an antibody, an in situ assay method for glycolipids,
It is preferable to appropriately combine sialic acid analysis and CMP-NeuAc hydroxylase activity measurement. Especially, anti-G
After screening by combining a method of selecting cells having a reduced amount of GM3 on the cell surface using an M3 antibody and an in situ assay of glycolipid, the mutant strain is analyzed by sialic acid or by measuring CMP-NeuAc hydroxylase activity. It is preferable to confirm the properties of

One of the mutant strains of the present invention thus obtained
The example 1A51 strain had a NeuGc content (productivity) of about 5/14 and a CMP-NeuAc hydroxylase activity of 1/5 or less as compared with the parental CHO-K1 cells. The 1A51 strain has been deposited as FERM P-14066 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.

Since the mutant strain of the present invention has reduced NeuGc productivity, it can be used as a host cell in gene recombination technology to produce a glycoprotein having a low NeuGc content. That is, by introducing a recombinant vector having a gene encoding a glycoprotein into the mutant strain of the present invention, and culturing the obtained transformant, HG having a low NeuGc content can be obtained.
It is possible to produce a glycoprotein that does not contain -D antigen.
Here, since the mutant strain of the present invention has the same properties as ordinary CHO cells except that NeuGc productivity is low,
When the mutant strain is used as a host cell, the same method as that using a conventional CHO cell can be adopted as a method for introducing a foreign gene, a culture method, a method for separating a produced glycoprotein, and the like.

As the target glycoprotein, a human-derived glycoprotein is preferable, and examples thereof include erythropoietin, tissue-type plasminogen activator, granulocyte colony-stimulating factor, urokinase, hepatitis B vaccine and the like. In addition, the vector into which the gene encoding this glycoprotein is incorporated is not particularly limited as long as it is a vector that can express the gene in CHO cells, but general stable expression vectors pcD2, pL2neoSR
αIII, pMIKHygB, pMKITNeo, pSV
2bsr, pRC / CMV, pRC / RSC, pcDN
A3, pMAM-neo and the like, and pAdD26SVp (A), p91023 (B), pS are preferable vectors for high expression using dhfr-cells.
VMdhfr, pSV2dhfr and the like can be mentioned. To introduce such a recombinant vector into the mutant strain of the present invention, for example, the competent cell method, calcium phosphate coprecipitation method, electroporation method, DEAE dextran method, lipofectin method, etc. may be used.

The glycoprotein of interest is produced by culturing the obtained transformant cells and extracting and separating from the cultured cells and / or the culture solution. For culturing the transformant cells, various natural media and synthetic media are used, but NeuGc-free media are preferred. The medium preferably contains carbonic acid sources such as sugars, alcohols and organic acid salts; nitrogen sources such as protein mixtures, amino acids and ammonium salts; and inorganic salts. Furthermore, it is desired to add vitamins and antibiotics corresponding to the selectable marker gene. If the vector can control the expression, it is necessary to add an operation for inducing gene expression during the culture. After culturing, centrifugation is performed to separate the culture solution and the cultured cells. When glycoproteins accumulate in cultured cells, for example, freeze-thaw, sonication, French press, enzyme treatment, homogenizer, etc. are used to disrupt the cells, and then, for example, EDTA, surfactant, urea, guanidine hydrochloride. It is necessary to solubilize the glycoprotein using, for example.

The purified glycoprotein can be obtained by subjecting the obtained culture solution or culture cell extract containing the glycoprotein to various column chromatography. As column chromatography, ion exchange chromatography, affinity chromatography, gel filtration chromatography and the like can be used alone or in combination.

The glycoprotein thus obtained is HD
It contains no antigen and is useful as a drug, diagnostic agent, etc.

[0021]

The present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example (1) Induction of Mutation in CHO-K1 Cells Mutagenesis was performed by using a medium containing CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) containing 400 μg / ml of EMS (ethyl methanesulfonic acid) [10]. % Fetal bovine serum-containing Ham F-12 medium (Dainippon Pharmaceutical Co., Ltd.)] at 33 ° C. for 18 hours.

(2) Selection of cells with low GM3 expression level by complement-dependent cytotoxicity reaction using anti-GM3 antibody CHO cells mutagenized in (1) were placed on a flask and 10% fetal bovine. Cultured in serum-containing Ham's F-12 medium at 37 ° C. in the presence of 5% CO ₂ (culture under these conditions is hereinafter simply referred to as “culture”), and the cells were detached by trypsin-EDTA treatment, Buffer solution A [20 mM
After washing with HEPES (pH 7.3) and Ham's F-12 medium containing 10% fetal bovine serum, the same applies hereinafter], the mixture was centrifuged at 90 × g for 2 minutes. After removing the supernatant, the cell pellet was treated with anti-GM3 monoclonal antibody (M2590, Cosmo Bio Inc., 1
mg / ml) 150 μl and rabbit complement (Dainippon Pharmaceutical Co., Ltd.) 150
μl was added, and the mixture was reacted at 37 ° C. for 1.5 hours. After the reaction, the plate was washed once with a medium and cultured at 37 ° C. in the presence of 5% CO ₂ .
By performing the above operation twice and culturing in a 100 mm dish, about 1000 cell lines considered to have a low GM3 expression level from 2 million mutagenized CHO cells.
A colony of strains was obtained.

(3) G on cell surface by indirect fluorescent antibody method
Detection of M3 The cells obtained in (2) were cultured for 2 to 3 days on a 35 mm dish or a lavout chain. After removing the medium, the anti-GM3 monoclonal antibody (10 μg / m 2
l) and reacted at 0 ° C. for 30 minutes. The cells were washed 4 times with buffer A and reacted with biotinylated anti-mouse IgM antibody (Funakoshi Co., Ltd., 2.5 μg / ml) in buffer A for 30 minutes at 0 ° C. The cells are washed 4 times with buffer A and SA in buffer A
-FITC (streptavidin-fluorescein isothiocyanate) conjugate (Cosmo Bio, 2.8 μ)
(g / ml) at 0 ° C. for 30 minutes. 4 cells with PBS
After washing twice, FITC fluorescence was detected using ACAS570 (Meridian) in PBS. As a result,
Expression of GM3 on the cell surface was decreased in 11 out of about 1000 strains obtained in (2). When the 11 strains were cultured by the limiting dilution method and the amount of GM3 on the cell surface was measured again by the above-mentioned indirect fluorescent antibody method, the expression of GM3 was decreased in 9 strains.

(4) In-situ of enzyme of glycolipid synthesis system
u assay (4) out of 9 obtained in (3)
The sugar synthesis of itu was examined by the following method. The cells cultured on the flask were peeled off by trypsin-EDTA treatment and washed with PBS three times. The cell membrane was damaged by freezing and thawing to facilitate penetration of the sugar nucleotide substrate into the cells, and then a reaction solution containing UDP- [ ¹⁴ C] galactose was added, and the reaction was carried out at 37 ° C. for 30 minutes with shaking.

[0026]

Table 1 The composition of the reaction solution is as follows. 50 mM M
ES [2- (N-morpholino) ethanesulfonic acid (hydrate) buffer, pH 6.4, 1 mM NADH, 5 mM D
TT (dithiothreitol) 5mM MnCl ₂ , 2.5m
M MgCl ₂ , 22.2 μM UDP- [ ¹⁴ C] galactose (Amersham, 12.2 GBq / mmol)

After the reaction was completed, the cell suspension was collected by centrifugation. After washing this precipitate 3 times with PBS, the cells were freeze-dried. From this, labeled glycolipids can be converted to CHC
_{l 3: MeOH (2: 1} ), (1: 1),: and extracted with (1 2), and analyzed by TLC. CH as a developing solvent
Cl ₃ : MeOH: 0.02% CaCl ₂ .2H ₂ O = 6
0: 35: 8 was used. Radioisotope detection is
The measurement was performed using a bio-imaging analyzer BAS2000 manufactured by Fuji Photo Film Co., Ltd. Exposure is about 16
I went on time.

The obtained results are shown in FIG. Figure 1 TLC
From the image, the parent strain LacCer (Galβ1 → 4Glc
Two bands corresponding to (β1 → 1Cer) and three bands corresponding to GM3 were observed. In contrast, 1A51
The strain had two bands corresponding to LacCer as in the parent strain, but two bands corresponding to GM3. Then, the two strains above the parent strain GM3 and the 1A51 strain GM3
The two mobilities of the two were in agreement with each other, and these two bands were in agreement with the mobility of the standard GM3 (NeuAc for sialic acid). It is known that when one sialic acid molecular species is present, two bands are generated due to the presence of two Cers. Therefore, it was considered that two types of GM3 having different sialic acid molecular species are present in CHO cells, but one of them is deficient in the 1A51 strain.

To confirm this, i in glycolipid synthesis
The reaction was performed by adding CMP-NeuAc (Sigma) or CMP-NeuGc at the time of n situ assay.
As a result, as shown in FIG. 2, when CMP-NeuAc was added to the reaction solution, only the upper two bands of both the parent strain and the 1A51 strain were present, and the upper two bands were sialic acid as Neu.
It was considered to be GM3 containing Ac. CMP-Neu
When Gc was added to the reaction solution, the lower two bands became stronger, and the lower two bands contained GM containing NeuGc as sialic acid.
It was suggested to be 3. Of these, 1A5
One strain was considered to be a mutant strain with reduced NeuGc productivity.

The CMP-Neu used in the above reaction was used.
Gc was manufactured as follows. That is, CMP-Ne
uGc was synthesized by the method of Herman et al. (J. Biol.
Chem. 260, 8838-8849 (1985))
According to. 67 mM CTP in 200 μl of distilled water
100 μl, 22 mM NeuGc 100 μl, 50
126 μl of mM MnCl ₂ and 75 μm of 50 mM DTT
1, 200 mM Tris-HCl (pH 7.4) 400 μ
1, recombinant CMP-sialic acid synthase (Cosmo Bio Inc.) 20 μl (180 mU) were added, and the mixture was mixed at 37 ° C. for 1 hour.
Reacted for hours. After the reaction, add 9 ml of ethanol and add CM
P-NeuGc was precipitated.

(5) Sialic acid analysis Sialic acid analysis of CHO cells and strain 1A51 was carried out by Hara.
Et al. (Anal. Biochem. 179, 162).
-166 (1989)). The cells cultured on the flask were peeled off by trypsin-EDTA treatment and washed with PBS three times. The cells were suspended in 200 μl of a 2M acetic acid aqueous solution, and the sugar chain was hydrolyzed by treatment at 80 ° C. for 3 hours. After the reaction, the cell debris was removed by centrifugation and 200 μl of DMB (1,2-diamino-4,5-
Methylenedioxybenzene) solution (7 mM DMB, 1.
4M acetic acid, 0.75M 2-mercaptoethanol,
18 mM sodium hydrosulfite) was added and 5
The reaction was carried out at 0 ° C for 2.5 hours, and fluorescent labeling with sialic acid was performed. Quantification of fluorescently labeled sialic acid was performed using HPLC. A Bio-Rad model 2700 system was used with a Hitachi F-1050 fluorescence detector. The excitation wavelength was 373 nm and the emission wavelength was 448 nm.
Column is Tosoh's TSKgel ODS-120T (2
50 × 4.6 mm) and the eluent was acetonitrile: methanol: water = 9: 7: 84. Flow rate is 0.9 ml
/ Min. As a result, the only major sialic acid present in CHO cells was NeuAc and NeuGc,
uGc content is 14.2% in the parent strain and 5% in the 1A51 strain
Met.

(6) Measurement of CMP-NeuAc hydroxylase activity NeuGc is the same as CMP-NeuAc.
CMP of CHO cells and 1A51 strain was examined in order to investigate whether the cause of the decrease in NeuGc content of the 1A51 strain is due to the deficiency of this enzyme since it is caused by hydroxylation by c-hydroxylase. -The NeuAc hydroxylase activity was measured. CMP-Neu of CHO cells
Ac hydroxylase activity was measured by the method of Kawano et al. (Glycoconjugate J. 10, 109-
115 (1993)). The cells cultured on the flask were peeled off by trypsin-EDTA treatment and washed with PBS three times. After the cell membrane was damaged by freeze-thawing, 10 mM Tris-HCl (pH 7.
CMP-Ne was used as a zymogen suspended in 5).
A reaction solution containing uAc was added, and the mixture was reacted at 37 ° C. for 60 minutes while shaking.

[0033]

Table 2 The composition of the reaction solution (50 μl) is as follows. 10 mM Tris-HCl (pH 7.5) 5 μM cytochrome b5 12 μg NADH-dependent cytochrome b5 reductase 40 μM CMP-NeuAc 1 mM DTT 0.7 mM NADH

After completion of the reaction, 200 μl of cold ethanol was added, and the mixture was allowed to stand at 0 ° C. for 15 minutes and then centrifuged at 15,000 rpm for 5 minutes to precipitate the protein. The quantification of CMP-NeuGc of the enzyme reaction product contained in the supernatant was performed using HPLC. Bio-Rad's Model 2700 System with Model 1706 UV / VIS Monitor 2
The absorbance at 71 nm was measured. Column is Tosoh TSKg
Using el ODS-80TM (250 x 4.6 mm),
The eluent was 50 mM ammonium dihydrogen phosphate. The flow rate was 0.5 ml / min. Protein quantification was carried out using a protein assay kit from Bio-Rad with bovine serum albumin as a standard protein.

As a result, as shown in FIG. 3, the CMP-NeuAc hydroxylase activity of the 1A51 strain was 1/5 or less of that of the parent strain. Therefore, 1A51 strain is CMP-NeuA
It is a defective strain of c-hydroxylase, and therefore Ne in the cell is
It was found to be a cell line having a low uGc content (reduced productivity).

[0036]

The CHO cell mutant of the present invention is NeuGc.
Since the productivity is decreased, the glycoprotein produced by the gene recombination technique using this as a host cell is Neu
It contains almost no Gc. Therefore, the mutant strain of the present invention is useful as a host cell for producing a glycoprotein having no HD antigenicity.

[Brief description of drawings]

FIG. 1 shows the results of in situ assay of enzymes of glycolipid synthesis system of CHO-K1 cells (parent strain) and 1A51 strain (TL).
It is a figure which shows C).

FIG. 2 shows the effect of C on the in situ assay of enzymes in the glycolipid synthesis system of CHO-K1 cells (parent strain) and 1A51 strain.
It is a figure which shows the addition effect of MP-NeuAc or CMP-NeuGc.

FIG. 3 C of CHO-K1 cells (parent cell line) and 1A51 cell line
It is a figure which shows MP-NeuAc hydroxylase activity.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area // (C12P 21/00 C12R 1:91)

Claims (5)

[Claims] 1. A Chinese hamster ovary cell mutant having reduced N-glycolylneuraminic acid productivity. 2. The productivity of N-glycolylneuraminic acid is
The mutant strain according to claim 1, which is 1/2 or less of the unmutated strain. 3. The activity of cytidine-5'-monophosphorylated N-acetylneuraminic acid hydroxylase is 1 / n that of the unmutated strain.
The mutant strain according to claim 1 or 2, which is 5 or less. 4. The mutant strain according to claim 1, 2 or 3, which is obtained by subjecting a Chinese hamster ovary cell to a mutagenesis treatment. 5. A method for producing a glycoprotein, which comprises introducing a recombinant vector having a gene encoding a glycoprotein into the mutant strain according to claim 1 and culturing the obtained transformant.