JPH11127890A - Production of glycoprotein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a glycoprotein predominant in a glycoprotein bearing sugar chain of specific structure, esp. free from galactose residue on the terminal, such as immunoglobulin, in particular, immunoglobulin G, through culturing glycoprotein-productive cells. SOLUTION: This method comprises producing the aimed glycoprotein by culturing glycoprotein-productive cells in a medium incorporated with a galactose residue rearrangement-controlling substance, e.g. N-acetylglucosamine. In this production, sugar chain synthesis activity is controlled to control the structure of the sugar chain in the aimed glycoprotein, thus enabling both the sugar chain and glycoprotein each having the aimed type and structure to be obtained more uniformly and in large quantities.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a glycoprotein containing a glycoprotein having a target sugar chain structure in a high ratio. More specifically, in order to produce a glycoprotein having an increased ratio of a glycoprotein having no galactose residue in the sugar chain, the glycoprotein is added to a medium containing a substance that controls the transfer of the galactose residue to the sugar chain. A production method characterized by culturing cells that produce E. coli.

[0002]

2. Description of the Related Art With the recent development of sugar chain structure analysis methods, discovery of sugar chain biosynthesis inhibitors, and development of large-scale preparation of glycoproteins by genetic engineering and cell engineering, etc. However, various structures and functions of sugar chains of glycoproteins, which have been largely unknown, have been elucidated. As a result, it is becoming known that, for example, a sugar chain in which a sugar chain portion of a glycoprotein is bonded to an asparagine residue of the protein portion (hereinafter, an Asn-linked sugar chain) has an important function for a living body. (Makoto Takeuchi & Yoko Takeuchi, Bioscience and Industry, 47 : 44-47 (1989)).

[0003] That is, an Asn-linked sugar chain contains a branched pentasaccharide structure (trimannosyl core) as shown in FIG. 1 as a common core structure. Originally, monosaccharides can have a variety of bonding modes and can have a very large number of sugar chains, and in fact, Asn-linked sugar chains with extremely diverse structures have been confirmed (Sato) Takeshi & Kiyoshi Furukawa, Protein Nucleic Acid Enzyme, 37 : 2082-2087 (1992)). One example is a sugar chain of immunoglobulin G (IgG). IgG is one of glycoproteins having one pair of such an Asn-linked sugar chain at an asparagine residue (Asn297) at position 297 from the N-terminal of the Fc region of the H chain. And Ig
The Gn Asn-linked sugar chain has, for example, the basic structure as shown in FIG. 2 in a mouse and the basic structure as shown in FIG. 3 in a human. That is, 5 of Asn-linked sugar chains
Although the sugar structure (trimannosyl core) is common, differences are found especially in the presence or absence of sugars at the non-reducing end (galactose residue (Gal) and N-acetylglucosamine residue (GlcNAc)).

[0004] In general, the structure of the sugar chain portion of a glycoprotein such as IgG is basically not single, but often differs depending on the kind of animal species from which the glycoprotein-producing cells are derived. Further, even if glycoproteins are produced in the same producing cell, they are basically in a heterogeneous state from the viewpoint of the sugar chain structure. In particular, those having a sugar chain to which a sugar (Gal, GlcNAc, fucose (Fuc), sialic acid (Sia), etc.) is provided and those having no sugar chain are provided at the non-reducing end of the sugar chain. This is due to a mixture of the above. It has been suggested that the presence or absence of Gal at the non-reducing terminal of such an Asn-linked sugar chain of IgG has a correlation with various disease states. One example is an abnormality of IgG sugar chain in rheumatoid arthritis (Naoyuki Tsuchiya). , Protein nucleic acid enzyme, 37 : 1935-1939 (1992)).

[0005] In patients with this disease, IgG in which two residues per Asn-linked sugar chain are deficient in both Gals [IgG is referred to as agalactosyl IgG, hereinafter referred to as Gal (0) IgG, abundant in serum. That is, the ratio of Gal (0) IgG is about 10% to the total amount of IgG in serum in a healthy person, whereas this ratio is about 40% in rheumatoid arthritis patients, and Gal (0) IgG
It has been speculated that there is a correlation between the increase in rheumatoid arthritis and the development of rheumatoid arthritis.

[0006] In addition, an increase in Gal (0) IgG has been observed in multiple myeloma and Castleman's disease (Tetsuo Nishiura et al., Protein Nucleic Acid Enzyme, 37 : 1945-1950 (1992)). It is known that Gal of an IgG sugar chain is also reduced in sexual dystrophy (Ito, K. et al., J. Cli
n. Biochem. Nutr., 14 : 61-69 (1993)), suggesting that there is a correlation between various disease states and the presence or absence of Gal in the sugar chain.

[0007] It has been suggested that in myotonic dystrophy, the translocation of IgG into blood vessels, that is, into body tissues, is increased (Suzumura, A. et al., Acta Neurol.
Scand., 74 : 132-139 (1986); Hirase, T. & Araki, S.,
Brain & Development, 6 : 10-16 (1984)), it is estimated that an increase in Gal (0) IgG enhances the translocation of IgG to body tissues.

[0008] Immunoglobulin preparations containing IgG are one of the important drugs for various conditions associated with immune diseases. And, as described above, IgG is G at the sugar chain terminal.
The transferability from blood vessels differs depending on the presence or absence of al. For this reason, when administering an immunoglobulin preparation, it is necessary to consider the presence or absence of Gal depending on the site where it is desired to act. That is, when IgG is mainly desired to act in blood vessels (in the case of treatment of sepsis, hepatitis, etc.), IgG with Gal is advantageous, and when it is necessary to transfer from blood vessels to various body tissues (pneumonia, pneumonia, For treatment of meningitis, encephalitis, etc.), Gal (0) IgG may be more advantageous. Therefore, there is a need to control the degree of addition of Gal in the IgG sugar chain.

Furthermore, immunoglobulin preparations are generally prepared by separating and purifying human-derived blood.
Since these are protein preparations, they cannot be sterilized at high temperature and high pressure. Therefore, administration of blood products derived from humans may cause secondary infection of other serious illnesses in some cases, and thus immunoglobulins prepared by production means that can minimize such a possibility as much as possible. There is a need for formulation development. Direct use of human blood, such as a method of tissue-culturing human immunoglobulin-producing cells produced by genetic engineering or cell engineering means, or a method of producing using non-human living organisms as an example of such means A production method that does not require it is required, and technology development for that purpose is being actively promoted.

However, the sugar chain synthesizing activity in IgG-producing cells and living organisms other than humans produced by these methods often differs from the natural sugar chain synthesizing activity in humans. Therefore, those having a sugar chain structure different from immunoglobulins produced in humans will be produced,
In some cases, the biological and / or physiological activities that should be exerted in humans are not exerted. Therefore, there is a need to control the production of human immunoglobulins so that they can synthesize the essential sugar chains.

Furthermore, the difference in sugar chain structure such as the presence or absence of Gal at the non-reducing end may cause a difference in the transferability of IgG to body tissues, etc.
There is a need for the development of a sugar chain engineering technique that freely controls the sugar chain structure. However, at present, a practical method for controlling the sugar chain structure has not been established. Until now, various methods have been tried as methods for obtaining a target sugar chain or a glycoprotein having the same.

That is, after obtaining a glycoprotein, the glycoprotein is treated with various enzymes to directly modify and modify the sugar chain (Ta method).
keuchi, M. et al., J. Biol. Chem., 265: 12127-12130.
(1990)), a method of expressing a desired glycoprotein by introducing a gene encoding a glycoprotein into a host cell capable of synthesizing a desired sugar chain by genetic engineering (Kingsley, DM et al., C
ell, 44 : 749-759 (1986)), a method of modifying the gene encoding a protein to restrict the sugar chain itself (William
s, AM & Enns, CA, J. Biol. Chem., 266 : 17648-
17654 (1991)), a method of changing the sugar chain structure by spatial effects caused by modifying amino acids near the sugar chain (Yuichiro Hayashi et al., Biochemistry, 62 : 690 (1990)), mutating glycoprotein producing cells To obtain the target glycoproteins that cannot bind to specific sugars in the sugar chain and produce the desired glycoprotein (Takahiko Hara & Masao Kawakita, Protein Nucleic Acid Enzyme, 36 : 2200-2202
(1991)), a method of producing and producing a cell line in which glycoprotein-producing cells are fused with cells having the desired sugar chain-synthesizing activity (JP-A-8-336389). A method that utilizes differences (Goochee, CF & Monic
a, T., BIO / TECHNOLOGY, 8: 421-427 (1990)).

However, even with these methods, it is very difficult to obtain a glycoprotein having a target sugar chain.
Moreover, even if it can be obtained, it requires a great effort involving trial and error. In addition, it has disadvantages such as complicated operation and expensive reagents to be used. Theoretically, if a glycoprotein having the target sugar chain was obtained even in a small amount, if the same type of glycoprotein having another different sugar chain could be separated and removed by some method, only the target sugar chain would be obtained. It is thought that it is possible to purify a glycoprotein having

However, at present, there is no practical means for separating and purifying glycoproteins based on such differences in sugar chains without impairing their activities. Therefore, it is required as a practical means to develop a means for producing a glycoprotein having a target sugar chain at a ratio as high as possible.

[0015]

SUMMARY OF THE INVENTION The present invention provides a method for efficiently producing a glycoprotein containing a glycoprotein having a target sugar chain at a high ratio. More specifically, in culturing glycoprotein-producing cells to produce glycoproteins,
In the production of immunoglobulins, particularly, glycoproteins such as immunoglobulin G, sugar chains having a specific structure,
In particular, the present invention provides a method for efficiently producing a glycoprotein containing a glycoprotein having a sugar chain having no galactose residue at the sugar chain terminal in a high ratio.

[0016]

Means for Solving the Problems In the course of research on the sugar chain structure of a glycoprotein, the present inventors have studied a specific method for controlling the biosynthesis of a sugar chain in the production of a glycoprotein by culturing glycoprotein-producing cells. We discovered that adding a substance to a culture medium and culturing it enabled efficient production of glycoproteins containing a high proportion of the target sugar chains, and succeeded in developing a simple and innovative method. did. As a result, it has become possible to provide a glycoprotein containing a high ratio of a glycoprotein having a target sugar chain structure having high biological and / or physiological activity.

The glycoprotein according to the present invention is a general term for a group of substances in which a sugar chain is covalently bonded to a protein, and is distinguished from a proteoglycan having a sugar chain having a repeating disaccharide structure. It refers to a complex glycoprotein in which the sugar chain portion is composed of 2 to 6 types of monosaccharides, does not have a certain repeating structure, and is covalently bonded to a protein.

Such glycoproteins include those having biological and / or physiological activities that exist widely in the living world. In such a protein, among the amino acid residues to which a sugar chain such as an asparagine residue (Asn) or a serine residue (Ser) or a threonine residue (Thr) in the amino acid sequence of the protein may bind, It indicates a protein in which a sugar chain is bound to at least one site, and a glycoprotein in which a sugar chain having a galactose residue (Gal) in the sugar chain is covalently bound. In the sugar chains of these glycoproteins, it is common that a galactose residue (Gal) is β-1,4 linked to N-acetylglucosamine (GlcNAc). Methods are provided that allow for control of binding in the chain.

Representative examples of such glycoproteins include immunoglobulins (Ig) G, M, E and A, ceruloplasmin, serum proteins such as prothrombin, transferrin, fibrinogen, glycophorin, rhodopsin, and the like.
Membrane proteins such as myelin A1 protein, α-amylase, β
-Various enzymes such as glucuronidase and RNAse, various cytokines such as tissue plasminogen activator (t-PA), erythropoietin, interferon-γ, and interleukin-2. When the glycoprotein is an immunoglobulin, it may be either a κ chain-bearing antibody or a λ chain-bearing antibody.

[0020] The sugar chains of glycoproteins are roughly classified into two types depending on the mode of binding between the sugar chains and the protein. One is an N-glycoside-linked sugar chain or an asparagine-linked sugar chain (Asn-linked sugar chain) in which N-acetylglucosamine is N-β-glycoside-linked to Asn having an amino acid sequence of Asn-X-Ser / Thr in the polypeptide. Sugar chains), which are often found in serum glycoproteins, and thus are also called serotype sugar chains.
First, N-acetylgalactosamine is added to Ser or Thr by O-α
O-glucoside-linked sugar chains or glycosides are commonly found in mucin, which is a mucous glycoprotein,
It is also called a mucin-type sugar chain.

It is sufficient that each sugar chain binds to an amino acid in a protein at the above-mentioned binding site, and the position of the binding site in the amino acid sequence depends on the biological activity and / or physiological activity of the glycoprotein. As long as activity can be retained
There is no particular limitation. Therefore, the number of sugar chains per glycoprotein molecule may be one, or two or more.

The sugars constituting the sugar chains include N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GlcNAc).
alNAc), D-mannose (Man), D-galactose (Gal), L-
In plants, D-xylose (Xyl) and D-arabinose (Ara) are included in addition to fucose (Fuc) and sialic acid (Sia). These sugars may be linked in any order.

The cells producing glycoproteins that can be used in the present invention are not particularly limited as long as they are derived from eukaryotes. For example, they may be primary cultured cells derived from animal organisms or cell lines thereof. There may be. Such cells may be either adherent cells or floating cells, and commercially available cells may be purchased, modified and used. Also,
Such cells accumulate glycoproteins inside cells,
Any of those expressed on the membrane surface or secreted extracellularly may be used.

Specifically, for example, antibody producing cells represented by human B cells, human T cells producing various cytokines such as interferon (IFN), interleukin (IL), t-PA, CHO Cells and mammalian cell lines such as human myeloma cells (myeloma) can be mentioned. Cells producing the above-mentioned target glycoprotein may be those produced by cell fusion or genetic engineering, and are not particularly limited.

However, it is preferable to use cells capable of producing antibodies, or cell lines in which these cells can be passaged in vitro, and include various cells derived from mice, rats, hamsters, humans, and the like. Can be Specifically, for example, mouse anti-progesterone antibody IgG2b (κ) producing cells 7
The D7.2.3 strain (this cell strain has been deposited with the National Institute of Bioscience and Biotechnology, Ministry of International Trade and Industry under the deposit number FERM BP-6150) and the like are preferably used.

For culturing the cells, various known methods can be used as long as they do not inhibit the growth of the cells and the production of glycoprotein. For example, any method of batch culture in which the medium is replaced for each culture and continuous culture in which the medium is continuously supplied may be used. In addition, among various culture methods such as suspension culture using roller bottles or tanks, adhesion culture in which cells are adhered to styrene microbead surfaces, and static culture using culture flasks, the properties of cells to be cultured are described. Culture is performed by appropriately selecting the culture method as described above in accordance with the conditions.

In the case of culturing by a batch method, the culturing may be carried out until the cells are sufficiently grown and the glycoprotein is sufficiently produced, and usually about 3 days to 1 month, preferably 5 days to 1 month.
It is 10 days, more preferably about 10 days. In order to exhibit higher production performance, the cells are preferably pre-cultured or conditioned for about two to three days to one week. Here, the conditioned culture refers to, for example, adapting cells cultured in a serum-containing medium to a serum-free medium.

The culture conditions are as follows: after inoculating cells producing glycoprotein in an appropriate amount as described below in an appropriate medium,
The cells are cultured at an appropriate temperature while controlling the aeration state and maintaining the pH of the medium at a predetermined state. 35-39 ° C, 5-10
It is preferable to culture using a% CO ₂ incubator. More preferably, a 5-10% CO ₂ incubator at 37 ° C. is used.

As the basic medium that can be used for culturing the antibody-producing cells used in the present invention, various commercially available cell culture media can be used. Such media include, for example, Eagle's MEM (MEM), DMEM, RPMI1640, Ma
Various media such as cCoy5A (manufactured by Dainippon Pharmaceutical) and NYSF404 (manufactured by Nippon Suisan) can be used, and may be appropriately selected and used depending on the properties of the cells to be cultured, the purpose of culturing, etc. NYSF404 is preferred.

In culturing antibody-producing cells using such a medium, when MEM or the like containing no antibiotic is used among the above-mentioned media, for example, an antibiotic such as penicillin or streptomycin is used. 50 each
Addition in an amount of about 50 μg / mL per unit / mL is suitable for preventing contamination due to mold and the like. Serum may or may not be added, but if added, it is usually in the range of 5 to 25% (v / v). It is preferable to use as a serum-free medium without adding serum, because the IgG produced by the cells can be easily purified.

In the present invention, at the time of culturing the antibody-producing cells, a substance for controlling the binding of galactose residues is added to the above-mentioned basic medium. Such substances include:
Specifically, it is preferable to use N-acetylglucosamine or glucosamine, since a glycoprotein having a high ratio of a glycoprotein having a sugar chain having no galactose residue can be obtained.

The amount of such a substance to be added is specifically 0.1.
2 mM or more, more preferably, 2 mM or more when N-acetylglucosamine is added, and 0.5 mM or more when glucosamine is added, the ratio of glycoprotein having no terminal galactose residue in the sugar chain. Can be easily and efficiently produced. In addition,
Even if the amount of addition of these substances is increased, the degree of the increase in the ratio of glycoprotein having no galactose residue gradually decreases, and reaches a plateau at about 10 mM, so that it should be added at a concentration of 10 mM or less. Is preferred.

Glycoprotein can be recovered from the culture obtained as described above by a conventional purification and recovery method. That is, for example, when the glycoprotein is secreted and produced extracellularly, the culture solution is recovered by a method of appropriately changing the culture solution. When the glycoprotein is accumulated in the cells, the cells are collected by filtering or centrifuging the culture solution.

When the glycoprotein is accumulated in cells, prior to purification, a chemical method using a surfactant, a chelating compound, a certain anionic compound, an alkaline agent, or the like, an enzyme, etc. Glycoproteins are released from the membrane or cells are disrupted by biochemical means such as by using or physical means such as sonication. The cell debris is removed, for example, by centrifugation, and the glycoprotein contained in the centrifugation supernatant is recovered.

Examples of the surfactant include an anionic surfactant such as sodium dodecyl sulfate, a cationic surfactant such as cetyltrimethylammonium bromide, and dodecyl
-N-betaine and other amphoteric surfactants, non-ionic surfactants such as polyoxyethylene-p-tert-octylphenol and sodium cholate can be used, and the enzymes include trypsin, nagase and the like. Can be used.

Purification of the glycoprotein can be carried out by combining various methods depending on the molecular weight and properties of the target glycoprotein. For example, the above-mentioned culture supernatant or centrifugal supernatant can be subjected to ultrafiltration, adsorbed on an affinity column, and the desired fraction can be eluted with an eluent having an appropriate salt concentration, and this fraction can be obtained by concentration. . Depending on the properties of the glycoprotein, an ion exchange column, a column for adsorption chromatography, a column for hydrophobic chromatography, or the like may be further used in combination.

In order to further obtain a sugar chain from the glycoprotein obtained as described above, various known methods can be used. For example, a sugar chain can be obtained by a method of cutting out a sugar chain by a hydrazinolysis method or a method of cleaving a sugar chain from a protein portion using an enzyme such as glycopeptidase or N-glycanase. The hydrazine degradation method is excellent in that sugar chains can be cleaved irrespective of the higher-order structure of the protein, and the enzymatic method is excellent in that the operation is very simple and modification of the sugar chains hardly occurs. Any of these two methods may be appropriately adopted according to the purpose, and these two methods can be used in combination.

When only the sugar chain bound to a specific site of the glycoprotein is recovered, the glycoprotein containing the objective sugar chain is prepared in advance by appropriately using a protease such as trypsin, chymotrypsin or lysyl endopeptidase. Is fragmented (glycopeptide fragmentation), the desired glycopeptide fragment is isolated and purified by various chromatography methods purifying the glycoprotein, and the sugar chain and the peptide are cleaved by the same method as described above. Good.

[0039] After the sugar chain is separated from the glycoprotein or glycopeptide fragment by the above method, the sugar chain is recovered by various purifications using various chromatography such as gel filtration, ion exchange chromatography, affinity chromatography and the like. What is necessary is just to implement by combining methods suitably. For the detection of the fraction containing a sugar, a sugar-specific coloring method such as an orcinol sulfate method or a phenol sulfate method, an electrochemical detection method, or the like can be used.

In order to analyze the structure of the sugar chain thus obtained, various known methods can be appropriately employed. However, the sugar chain obtained for improving the detection sensitivity is labeled. High-performance liquid chromatography (HPLC) as described below
It is common to perform analysis by

[0041] As a method of labeling a sugar chain, 2-aminopyridine, fluorescein, and a method of fluorescence labeled with a fluorescent compound such as 1,2-diamino-4,5-methylenedioxybenzene (DMB), ³ H, There is a method such as labeling with a radiolabeled compound such as ¹³ C, but handling is easy and HPLC
The method of labeling with a fluorescent compound is preferable because the separation is easy and the detection is easy, and the method of fluorescent labeling with 2-aminopyridine is particularly preferable.

The method of fluorescent labeling with 2-aminopyridine uses a 2-aminopyridine hydrochloric acid solution developed by Hase et al. As a fluorescent labeling agent and sodium cyanoborohydride as a reducing agent (Hase, S. et al.). ., J. Biochem.,
95: 197-203 (1984)) and a method using a 2-aminopyridine acetic anhydride solution developed by Kondo et al. As a fluorescent labeling agent and a borane-dimethylamine complex as a reducing agent (Kondo,
A. et al., Agric. Biol. Chem., 54 : 2169-2170 (1990))
And the like. Either method can be used, but the method of Hase et al. Is simpler and requires less equipment. An apparatus (manufactured by Takara Shuzo) developed to carry out the method of Kondo et al. Is commercially available, and the method of Kondo et al. May be used by using this apparatus.

Fluorescently labeled sugar chains are chemically stable, and are based on characteristics such as molecular weight, charge, hydrophilicity, hydrophobicity and affinity for various lectins of HPLC.
It is possible to analyze by using As a result, there is an advantage that analysis can be performed with high sensitivity in a short time.

A typical example is a case where sugar chains having different numbers of sialic acids are analyzed using an anion exchange column. Sugar chains with different numbers of sialic acids are applied to an anion exchange column and separated and quantified using the difference in their charges.
Thereafter, sialic acid is removed to obtain a neutral sugar chain. The obtained neutral sugar chains were separated by HPLC using reverse phase chromatography (ODS-silica), and the sugar chains in each of the obtained peaks were further subjected to normal phase chromatography (amide-silica). Separate using HPLC. Two-dimensional sugar chain mapping by Takahashi et al. Using the sugar chain structures of each component obtained in this way (Takahashi Reiko, Biochemical Experimental Method 23 Glycoprotein Glycosylation, Gakkai Shuppan Center (1989)) (Takahashi Reiko & Tomiya Noboru, Protein Nucleic acid enzyme, 3
7 : 2117-2130 (1992)). At this time, to remove sialic acid,
For example, a method of performing acid hydrolysis under acidic conditions and a method of using sialidase derived from microorganisms can be used.

As the eluent used for HPLC, a solvent obtained by adding a water-miscible organic solvent, for example, methanol, acetonitrile, propanol, n-butanol or the like to water or a buffer is preferable. Elution may be performed with an eluent having a constant concentration of an organic solvent or salt, or a gradient may be applied. Further, if necessary, the column temperature can be controlled to be constant by using a column jacket or the like to improve the ability to separate each sugar chain. Once the structure of the sugar chain in each peak in HPLC is identified by such a method, thereafter, when estimating the structure of the sugar chain of the same kind of glycoprotein, for example, it is used for identification. HPLC analysis by reverse phase chromatography is performed under the same conditions as above, and the structure of the sugar chain contained in each peak can be easily identified from the resulting chromatogram.

As a specific example, FIG. 3 shows a HPLC separation profile of a mixture of a pyridylamino derivative of an IgG sugar chain of a healthy subject by reverse phase chromatography and a structure of the sugar chain contained in each peak. When HPLC is performed under the same conditions,
Each sugar chain shown here is eluted with the retention time shown in the chromatogram in FIG. 3, so that simple identification is possible.

[0047]

The present invention will be described in more detail with reference to the following examples. The examples shown here illustrate, as an example of glycoprotein, the case where the sugar chains of IgG produced by culturing an IgG producing cell line are controlled, but do not limit the present invention in any way.

(Example 1) Preparation of IgG-producing cells (1) Preparation of IgG-producing cell strain 7D7.2.3 Mouse anti-progesterone antibody IgG2b (κ) -producing cell 7D7.2.3
A strain was created by the following procedure (Sawada, J. et al., J. Ste
android Biochem., 28 : 405-410 (1987)). That is, a complex of 17α-hydroxyprogesterone (hereinafter abbreviated as 17-OHP) and a protein was administered five times (50 μg / mouse / one time administration, Fre
immunization of BALB / c mice (8 weeks old, female, obtained from Shizuoka Experimental Animal Cooperative) five times at 20-day intervals from the first immunization using alternating und complete adjuvant and incomplete adjuvant) I do. The 17-OHP protein complex used here was
-OHP-4-carboxyethylthio-bovine serum albumin.

Three days after the final immunization, thymus was excised from these five BALB / c mice to obtain thymocytes. The thymocytes and P3 / NS1 / 1-Ag4-1 (NS1) cells (stored in the National Institute of Health Laboratory Cell Bank as JCRB0009) using polyethylene glycol 4000 (Sigma) (Galfre, G. et al.) .,
Hybridomas were obtained by cell fusion with Nature, 266: 550-552 (1977)). From the hybridoma obtained as described above, the desired antibody-producing cells were subjected to radioimmunoassay.
(Sawada, J. et al., Molec. Immun., 23: 625-630 (198
It was selected and cloned by the following method using 6)).

That is, the hybridoma obtained by the fusion treatment by the PEG method was used to prepare an HAT medium (RPM1640 medium (GIBCO) supplemented with 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, and 50 μM 2-mercaptoethanol). )
Medium further supplemented with 100 μM hypoxanthine (H), 0.4 μM aminopterin (A), and 16 μM thymidine (T))
Approximately 1 × 10 ⁶ cells were placed in a 24-well plate (No. 3024, manufactured by Costar) containing the cells and cultured, and finally a culture in 432 wells was obtained.

Next, these culture supernatants were subjected to radioimmunoassay (RIA), and cells in 43 wells showing positive were further cultured, and as a result, 35 positive cell lines were obtained. As a result of further selecting these 35 positive strains using RIA and immune cross-activity as indices, 6 positive strains having high activity were selected. In addition, the positive strain was preserved in liquid nitrogen. Among these six strains, 7D7.2.3 strain showed relatively high antibody producing activity (Sawada, J. et al., J. Steroid
Biochem., 28 : 405-410 (1987)). This strain is a mouse anti-17
-Cell line producing OHP antibody IgG2b (κ) (FERM BP-6150)
This strain was used for the test below.

(2) Production of IgG2b (κ) by 7D7.2.3 strain As a medium for producing IgG2b (κ), a modified serum-free medium NYS
F404 (Nippon Suisan; NaCl 6208mg, KCl 388mg, CaCl
₂ (anhydrous) 97 mg, Ca (NO ₃ ) ₂ (anhydrous) 33.7 mg, MgSO ₄ (anhydrous)
69.0 mg, Na ₂ HPO ₄ (anhydrous) 388.5 mg, NaH ₂ PO ₄ (anhydrous) 5
5.8mg, glucose 1955mg, sodium succinate 48.5m
g, succinic acid 36.4 mg, D-biotin 0.11 mg, calcium D-pantothenate 0.61 mg, choline chloride 26.5 mg, folic acid 0.97 mg,
Myo-inositol 18.0mg, nicotinamide 0.97mg, p-
Aminobenzoic acid 0.485mg, pyridoxal hydrochloride 0.485mg
, Pyridoxine hydrochloride 0.485mg, riboflavin 0.146mg
, Thiamine hydrochloride 0.97 mg, Vitamin B ₁₂ 0.0037 mg, Glutathione (reduced) 0.485 mg, Choline bitartrate 0.873 mg
, Putrescine-2H ₂ O 0.0125 mg, sodium pyruvate 110.0 mg, thymidine 0.0125 mg, hypoxanthine 0.0
25 mg, sodium selenite 0.0017 mg, streptomycin sulfate 100 mg, penicillin G potassium salt 63.3 mg, phenol red 5.0 mg, human transferrin 10.0 mg,
Bovine insulin 10.0 mg, HEPES 3570 mg, sodium bicarbonate 1400.0 mg, L-arginine hydrochloride 76.1 mg, L-arginine 9
7.0 mg, L-asparagine · H ₂ O 42.5mg, L- aspartic acid 9.7 mg, L-cystine 31.5 mg, L-cysteine hydrochloride · H ₂ O
16.7mg, L-glutamic acid 9.7mg, L-glutamine 450.0m
g, L-histidine 27.7 mg, L-hydroxyproline 9.7 mg
, L-isoleucine 49.5mg, L-leucine 49.5mg, L-lysine hydrochloride 54.8mg, L-methionine 14.6mg, L-proline 14.7m
g, L-serine 29.6 mg, L-threonine 48.0 mg, L-valine 47.
0 mg, L-phenylalanine 22.8 mg, L-tryptophan 7.
Medium containing 3mg, L-tyrosine 27.2mg, glycine 9.9mg / L)
Was used.

Conditioned culture of the 7D7.2.3 strain in a tissue culture flask (manufactured by Corning) having a bottom area of 25 cm ² containing 5 ml of this medium (still culture at 37 ° C. for 3 days, in humidified air containing 5% carbon dioxide gas) )did. Thereafter, N-acetylglucosamine or glucosamine.HCl was added to the modified serum-free medium in a stepwise manner at a concentration ranging from 0 to about 4.6 mM (Table 1).
Approximately 1 × 10 ⁶ cells cultured as described above were inoculated in a tissue culture flask having a bottom area of 150 cm ² containing ml, and main culture was performed for 10 days under the same conditions as the conditioned culture, and the IgG2b (κ) was recovered. Produced.

Example 2 Purification of IgG2b (κ) IgG2b (κ) was purified by the method of Kim, H. et al. (Kim, H. et al.,
J. Biol. Chem., 269 : 12345-12350 (1994)). That is, the medium 50 in each flask after the main culture was completed
0 ml was ultrafiltered (MilliporeMinitan ultrafiltration
system) to obtain about 50 ml of IgG2b (κ) concentrate. Thereafter, the concentrated solution was applied to a protein A column (Affi-Gel protocol).
in A column (manufactured by Bio-Rad)), and eluted with 50 mM glycine buffer (pH 2.8) containing 1.5 mM NaCl.
1.5 M Tris-HCl (pH 8.5) was added to pH 7.0, and IgG2b (κ)
About 50 ml of the purified fraction was obtained.

The purified IgG2b (κ) fraction was concentrated with Centriprep 30 (manufactured by Amicon), diluted with a buffer (pH 7.3) containing 10 mM sodium phosphate, 150 mM NaCl, and 3 mM sodium azide. Was repeated to exchange the buffer solution. The final concentration of IgG2b (κ) is about
0.3%. Thereafter, these samples were dialyzed against water and freeze-dried to obtain an IgG2b (κ) fraction.

Example 3 Structural Analysis of IgG2b (κ) Sugar Chain (1) Excision of Sugar Chain To analyze the IgG2b (κ) sugar chain, the above IgG2b (κ) fraction (about 3
mg) was dissolved in a 0.1 M Tris-HCl buffer (pH 8.2) and then adjusted to pH 7-8. 1M CaCl ₂ was added to 0.01 M, and trypsin (Sigma) and chymotrypsin (Wo
1% (w / w) of the amount of the contained protein was added thereto, and the mixture was digested with an enzyme (at 37 ° C. overnight).

After the completion of the reaction, the enzyme reaction was stopped by heating (100 ° C., 10 minutes), and the reaction solution was concentrated to dryness using a centrifugal concentrator, and then concentrated to a 0.5 M citrate-phosphate buffer ( pH 4.0). After adjusting this solution to pH 4 to 6, a 10 milliunit / 500 μl solution of glycopeptidase A (manufactured by Seikagaku Corporation) was added at a ratio of 0.3 milliunit / 100 nmol of sugar chain, and reacted at 37 ° C. overnight to convert the sugar chain. Released.

Next, 1 M Tris-HCl
After adjusting the pH to 7 to 8 by adding a buffer (pH 8.2), pronase (trade name: Actinase E, manufactured by Kaken Pharmaceutical Co., Ltd.) was added at 2% (w / w) of the protein content to decompose the peptide. The centrifuged supernatant was collected. The supernatant was desalted over 1 mL of each of the cation exchange resin and the anion exchange resin which had been equilibrated with ion exchange water in advance. The desalted solution was concentrated to dryness using a centrifugal concentrator to obtain a sugar chain fraction.

(2) Fluorescent labeling of sugar chain 1 g of 2-aminopyridine was added to the obtained sugar chain fraction in 56 g.
40 μl of a solution dissolved in 0 μl of concentrated hydrochloric acid was added, and the mixture was treated at 90 ° C. for 10 minutes. Thereafter, 4 μl of an aqueous solution obtained by dissolving 20 mg of sodium cyanoborohydride in 12 μl of water was added to this solution, and reacted at 90 ° C. for 1 hour. Then Sephadex
G-15 column (Pharmacia, φ1cm × 40cm (bed volume 3
0 ml)) and an eluent (10 mM ammonium bicarbonate) were monitored at 280 nm to purify the sugar chain. The 2-aminopyridine-sugar chain fraction was collected, dried, and subjected to the following HPLC analysis.

(3) HPLC Analysis Conditions The HPLC conditions for the 2-aminopyridine-sugar chain fraction are as follows. -Eluent A: 10 mM monosodium phosphate solution (pH 3.8)-Eluent B: Eluent A containing 0.5% n-butanol at a final concentration-Elution conditions: The concentration of eluent B was measured from the start of analysis to 60 minutes・ Radiation gradient from 20% to 50% ・ Column: ODS column HRC-ODS (φ6.0mm × 150mm, manufactured by Shimadzu) ・ Fluorescence detector: SHIMADZU RF-550 (manufactured by Shimadzu) ・ Detection: fluorescence detection (Excitation wavelength: 320nm, fluorescence wavelength: 400n
m) ・ Flow rate: 1ml / min ・ Column temperature: 55 ℃

(4) Analysis after HPLC analysis The chromatogram detected by HPLC was compared with a chromatogram obtained by analyzing a sugar chain identified in advance to estimate the structure of the sugar chain. The ratio of the Gal (0) sugar chain was calculated from the ratio of the peak area of the Gal (0) sugar chain to the peak area of the other entire sugar chains.

(5) Results of analysis of IgG2b (κ) sugar chains Table 1 shows the results obtained as described above.

[0063]

[Table 1]

From the above results, when N-acetylglucosamine or glucosamine / HCl was added to the medium, IgG
In 2b (κ), the ratio of agalactosyl sugar chains without galactose residues in the sugar chains increased. The effect is
Regardless of whether N-acetylglucosamine or glucosamine / HCl was added, it was recognized from a concentration of about 0.2 mM or more. More particularly, when N-acetylglucosamine is added, at a concentration of about 2 mM or more, and in the case of glucosamine / HCl, at a concentration of about 0.5 mM or more, the proportion of those having no galactose residue is as high as 65% or more. Could be increased.

[0065]

According to the present invention, in the production of glycoproteins, the sugar chain synthesizing activity is controlled to control the structure of the glycoprotein sugar chain, and the sugar chains and glycoproteins having the desired type and structure can be obtained. It is possible to obtain uniform and large quantities. That is, it becomes possible to efficiently produce a glycoprotein containing a glycoprotein having a sugar chain having no galactose residue in the sugar chain at a high ratio by a relatively simple method. As a result, when producing glycoproteins as pharmaceuticals,
Of the glycoproteins, it is possible to obtain a glycoprotein having a target sugar chain more uniformly by a relatively simple method,
For example, it is possible to relatively easily obtain an excellent glycoprotein having an increased activity such as transferability to a body tissue.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a trimannosyl core having a pentasaccharide structure common to Asn-linked sugar chains.

FIG. 2 is a schematic diagram showing the structure of an Asn-linked sugar chain of mouse IgG.

FIG. 3 is a diagram showing a separation profile of a mixture of pyridylamino derivatives of IgG sugar chains of a healthy subject by HPLC (reverse phase chromatography) and a structure of sugar chains contained in each peak (Takahashi, N., et al., Biochemistry, 26 : 1137-1144
(1987)).

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 5/06 C12R 1:91)

Claims (5)

[Claims] 1. A method for producing a glycoprotein, comprising culturing a glycoprotein-producing cell in a medium to which a galactose residue transfer regulator is added to produce the glycoprotein. 2. The method for producing a glycoprotein according to claim 1, wherein the glycoprotein is an immunoglobulin. 3. The method for producing a glycoprotein according to claim 1, wherein the glycoprotein is immunoglobulin G. 4. The method for producing a glycoprotein according to claim 1, wherein the galactose residue transfer regulator is N-acetylglucosamine or glucosamine. 5. As a galactose residue transfer regulator, 2
The method for producing a glycoprotein according to any one of claims 1 to 3, wherein the glycoprotein-producing cell is cultured in a medium to which N-acetylglucosamine of 0.5 to 10 mM or glucosamine of 0.5 to 10 mM is added.