JP3375140B2 - Method for producing medium for producing monoclonal antibody and kit therefor - Google Patents
Method for producing medium for producing monoclonal antibody and kit thereforInfo
- Publication number
- JP3375140B2 JP3375140B2 JP06927191A JP6927191A JP3375140B2 JP 3375140 B2 JP3375140 B2 JP 3375140B2 JP 06927191 A JP06927191 A JP 06927191A JP 6927191 A JP6927191 A JP 6927191A JP 3375140 B2 JP3375140 B2 JP 3375140B2
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- JP
- Japan
- Prior art keywords
- medium
- glutamine
- fructose
- human
- producing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、抗体生産細胞によるモ
ノクローナル抗体生産を増強させる培地の作製法及びそ
のキットに関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a medium for enhancing the production of monoclonal antibodies by antibody-producing cells and a kit thereof.
【０００２】[0002]
【従来の技術】従来、抗体生産細胞を培養してモノクロ
ーナル抗体を生産する場合には、培地成分として、ブド
ウ糖、アミノ酸、ビタミン、ヌクレオチド前駆体、脂肪
酸又は脂肪酸エステル、ポリアミン、ピルビン酸ナトリ
ウム、無機塩等を含有する市販の動物細胞用合成培地を
１種類或いは数種類混合したものを基礎培地とし、これ
に成長因子として牛胎児血清或いは他の細胞増殖因子等
を添加したものが用いられていた。2. Description of the Related Art Conventionally, when culturing antibody-producing cells to produce a monoclonal antibody, glucose, amino acids, vitamins, nucleotide precursors, fatty acids or fatty acid esters, polyamines, sodium pyruvate, inorganic salts are used as medium components. As a basal medium, a mixture of one or several commercially available synthetic mediums for animal cells containing, etc. was used as a basal medium to which fetal bovine serum or other cell growth factors were added as growth factors.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、上記従
来の培地では、モノクローナル抗体の生産量が少なく、
工業的に十分な量を得ることが困難であった。特にヒト
−ヒトハイブリドーマを用いてヒト型モノクローナル抗
体を生産する場合には、生産量が極端に少なくその利用
には限界があった。このような状況において、モノクロ
ーナル抗体の生産を増強させることができる培地の作製
法の開発が強く望まれていた。However, in the above-mentioned conventional medium, the amount of monoclonal antibody produced is small,
It was difficult to obtain a sufficient amount industrially. In particular, when a human-human hybridoma is used to produce a human-type monoclonal antibody, the production amount is extremely small, and its use is limited. Under such circumstances, it has been strongly desired to develop a method for producing a medium that can enhance the production of monoclonal antibodies.
【０００４】本発明の目的は、ヒト型モノクローナル抗
体の生産を増強させることができる培地の作製方法及び
それに用いる培地作製キットを提供することにある。An object of the present invention is to provide a method for producing a medium capable of enhancing the production of human type monoclonal antibody and a medium producing kit used therefor.
【０００５】[0005]
【課題を解決するための手段】本発明者らは、ヒト型モ
ノクローナル抗体の生産を上昇させる培地の作製法につ
いて鋭意研究した結果、培地中における果糖及びグルタ
ミンの濃度を適度に調節することによって、ヒト型モノ
クローナル抗体の生産を増強できることを見出し、本発
明を完成するに至った。[Means for Solving the Problems] As a result of earnest studies on a method for producing a medium that increases the production of human-type monoclonal antibodies, the present inventors have found that the concentrations of fructose and glutamine in the medium were appropriately adjusted. It was found that the production of human type monoclonal antibody can be enhanced by adjusting the amount, and the present invention has been completed.
【０００６】本発明のモノクローナル抗体生産用培地の
作製法は、ヒト−ヒトハイブリドーマを用いてヒト型モ
ノクローナル抗体を生産するのに用いる培地の作製法に
おいて、糖質及びグルタミンを含有しない培地組成に、
果糖及びグルタミンを種々の濃度で添加して、果糖及び
グルタミンの含有量が異なる複数の培地を作製し、それ
ぞれの培地を用いて抗体生産細胞を培養し、モノクロー
ナル抗体生産が増強される果糖濃度及びグルタミン濃度
を求めることを特徴とする。[0006] Preparation method of monoclonal antibody production medium of the present invention, human - in the production method of the medium used to produce human type motor <br/> monoclonal antibodies using human hybridomas, containing carbohydrates and glutamine Not to the medium composition,
By adding fructose and glutamine at various concentrations, to produce a plurality of media content is different fructose and glutamine, with each of the medium and cultured antibody-producing cells, fructose concentrations monoclonal antibody production is enhanced and It is characterized in that the glutamine concentration is obtained.
【０００７】本発明のモノクローナル抗体生産用培地作
製キットは、ヒト−ヒトハイブリドーマを用いてヒト型
モノクローナル抗体を生産するのに用いる培地作製キッ
トにおいて、糖質及びグルタミンを含有しない培地と、
果糖と、グルタミンとを組合せたことを特徴とする。The culture medium preparation kit for producing a monoclonal antibody of the present invention is a human type using a human-human hybridoma.
The medium preparation kit used to produce the monoclonal antibody
In the medium, a medium containing no sugar and glutamine,
It is characterized by a combination of fructose and glutamine.
【０００８】以下、本発明について更に詳細に説明す
る。
抗体生産細胞
抗体生産細胞としては、ヒト−ヒトハイブリドーマを使
用する。 The present invention will be described in more detail below. Antibody-producing cells Human-human hybridomas are used as antibody-producing cells.
To use.
【０００９】果糖
及びグルタミンの含有量を種々組み合わせた培地
糖質としては果糖を利用するが、ブドウ糖、ガラクトー
ス、マンノース等と組合せてもよい。 [0009] Fructose is used as a medium sugar containing various combinations of fructose and glutamine contents.
It may be combined with sugar, mannose and the like.
【００１０】培地の作製法としては、基本合成培地とし
て知られているＲＤＦ培地(ＲＰＭＩ１６４０培地、Ｄ
ＭＥ培地及びハムＦ−１２培地を２：１：１の割合で混
合した培地）の含有成分のうち、糖質であるブドウ糖及
びグルタミンを含有しない培地を調製し、これに新たに
糖質及びグルタミンを添加する方法が例示できるが、基
本合成培地としてはダルベッコＭＥＭ培地（ＤＭＥ）、
ハムＦ−１２培地、Ｅ−ＲＤＦ培地（極東製薬工業株式
会社より販売）等の含有成分も利用することができる。As a method for preparing the medium, RDF medium (RPMI1640 medium, D
ME medium and Ham's F-12 medium were mixed at a ratio of 2: 1: 1) to prepare a medium not containing glucose and glutamine which are sugars, and newly added sugar and glutamine to the medium. The method of adding the can be exemplified, but as a basic synthetic medium, Dulbecco's MEM medium (DME),
Ingredients such as Ham's F-12 medium and E-RDF medium (sold by Kyokuto Pharmaceutical Co., Ltd.) can also be used.
【００１１】上記の基本培地に添加する果糖及びグルタ
ミンの量は、実際上可能な範囲内において任意に設定で
きるが、果糖については０．５〜４．５ｇ／Ｌ、グルタ
ミンについては０〜１，０００ｍｇ／Ｌの範囲で設定す
ることが好ましい。The amounts of fructose and glutamine added to the above basal medium can be arbitrarily set within a practically possible range, but 0.5 to 4.5 g / L for fructose and 0 to 1 for glutamine. It is preferable to set in the range of 000 mg / L.
【００１２】また、本発明に用いる培地は、血清を添加
しない無血清培地でもよく、血清培地であってもよい。
血清培地としては、例えば１０％牛胎児血清を添加した
培地が挙げられるが、モノクローナル抗体生産用培地と
しては、抗体の精製が容易であること、培地が安価であ
ること等の理由から無血清培地を用いるのが望ましい。
無血清培地への添加物としては、血清アルブミン、イン
シュリン、トランスフェリン、エタノールアミン、亜セ
レン酸ナトリウム等が挙げられる。The medium used in the present invention may be a serum-free medium to which serum is not added, or a serum medium.
The serum medium includes, for example, a medium supplemented with 10% fetal bovine serum, but the monoclonal antibody-producing medium is a serum-free medium because of the fact that the antibody can be easily purified and the medium is inexpensive. It is preferable to use.
Examples of additives to the serum-free medium include serum albumin, insulin, transferrin, ethanolamine, sodium selenite and the like.
【００１３】培養法
抗体生産細胞の培養には、例えばペトリ皿、スピンナー
フラスコ、潅流型連続培養装置、又はホローファイバー
型の培養装置が利用でき、例えばペトリ皿を用いた場合
には、５％ＣＯ2−９５％空気の雰囲気下で、３７℃に
て数日間培養すればよい。Culture Method For culturing antibody-producing cells, for example, a Petri dish, a spinner flask, a perfusion type continuous culture apparatus, or a hollow fiber type culture apparatus can be used. For example, when a Petri dish is used, 5% CO 2 It may be cultured at 37 ° C for several days in an atmosphere of 2-95% air.
【００１４】培養終了後、培養液上清中の抗体濃度を、
例えば通常の酵素抗体法（イムノケミストリー(Immunoc
hemistory), 8, 871(1971)参照）で測定し、抗体生産量
が最も多い培地の糖質濃度及びグルタミン濃度を求め
る。そして、この濃度になるように糖質及びグルタミン
を添加して培地を作製すれば、モノクローナル抗体の生
産を増強させることができる培地が得られる。After completion of the culture, the antibody concentration in the culture supernatant was
For example, the usual enzyme-antibody method (immunochemistry (Immunoc
hemistory), 8, 871 (1971)) to determine the sugar concentration and glutamine concentration in the medium with the highest antibody production. Then, by adding a sugar and glutamine to this concentration to prepare a medium, a medium capable of enhancing the production of monoclonal antibodies can be obtained.
【００１５】
モノクローナル抗体生産用培地作製キット
本発明によるモノクローナル抗体生産用培地作製キット
は、上述のモノクローナル抗体生産用培地の作製を容易
に行なうためのものである。Monoclonal Antibody Production Medium Production Kit The monoclonal antibody production medium production kit according to the present invention is for easily producing the above-mentioned monoclonal antibody production medium.
【００１６】すなわち、このキットは、（ａ）糖質及び
グルタミンを含有しない培地、(ｂ)果糖を必須とし、必
要に応じてブドウ糖、ガラクトース、マンノース等を含
む糖質、（ｃ）グルタミンから構成され、（ａ）に、
（ｂ）の果糖、又は果糖と他の糖質の組合せ、及び
（ｃ）を添加することのみで果糖及びグルタミンの含有
量を種々組み合わせた培地を作製することができ、これ
を用いて抗体生産細胞を培養することによって、モノク
ローナル抗体生産を増強させる糖質濃度及びグルタミン
濃度を決定することができる。That is, this kit requires (a) a medium containing no carbohydrate and glutamine, and (b) fructose as an essential component.
Contains glucose, galactose, mannose, etc. as needed.
It consists of sugar, (c) glutamine, and in (a),
It is possible to prepare a medium in which the contents of fructose and glutamine are variously combined only by adding fructose of (b) or a combination of fructose and another sugar , and (c), and using this, antibody production By culturing the cells, it is possible to determine the sugar concentration and glutamine concentration that enhance the production of the monoclonal antibody.
【００１７】上記キットは、糖質及びグルタミンを含有
しない培地と、果糖と、グルタミンとを備えたものであ
ればよく、また、糖質は果糖だけであってもよいが、そ
の他にブドウ糖、ガラクトース、マンノース等を含んで
いてもよい。なお、このキットの構成物は、水溶液、乾
燥粉末等の各種の製品形態で提供することができる。[0017] The kit comprises a culture medium containing no carbohydrates and glutamine, as long as it includes a fructose, a glutamine, also carbohydrates may only fructose, but its
In addition to glucose, galactose, mannose, etc.
You may stay . The components of this kit can be provided in various product forms such as an aqueous solution and dry powder.
【００１８】[0018]
【作用】本発明では、果糖濃度及びグルタミン濃度を種
々変えた培地で、抗体生産細胞を培養し、培養液中の抗
体量を測定することにより、抗体生産が最も増大する果
糖濃度及びグルタミン濃度を求めることができる。そし
て、そのような濃度になるように果糖及びグルタミンを
添加した培地を用いることにより、モノクローナル抗体
の生産量を増大させることができる。According to the present invention, in a medium which variously changed the fructose concentration and glutamine concentrations were cultured antibody-producing cells, by measuring the amount of antibody in the culture solution, fruit of antibody production most increased
The sugar concentration and glutamine concentration can be determined. Then, by using a medium in which fructose and glutamine are added so as to have such a concentration, the production amount of the monoclonal antibody can be increased.
【００１９】また、本発明の培地作製キットを用いれ
ば、糖質及びグルタミンを含有しない培地に、果糖及び
グルタミンを添加するだけで、果糖及びグルタミンの濃
度が異なる複数種類の培地を容易に作製することができ
る。Further, by using the medium preparation kit of the present invention, a plurality of types of medium having different concentrations of fructose and glutamine can be easily prepared only by adding fructose and glutamine to a medium containing neither sugar nor glutamine. be able to.
【００２０】[0020]
【実施例】実施例１
（１）培地
糖質及びグルタミンを含有しない培地として、表１に示
す組成の変法ＲＤＦ培地を用い、これにグルタミンを最
終濃度で１７４．５ｍｇ／Ｌ及び３４９ｍｇ／Ｌになる
ように添加した培地を作製し、更にこれらの培地それぞ
れに果糖を０．６８、１．３５、２．０、２．７ｇ／Ｌ
となるように添加した培地を調製して基礎培地とした。
比較対照には、ＲＤＦ培地（糖質としてブドウ糖２．７
ｇ／Ｌ及びグルタミン３４９ｍｇ／Ｌを含有する）を用
いた。また、上述の基礎培地それぞれに表２に記載した
ものを添加した。Example 1 (1) Medium As a medium containing neither carbohydrate nor glutamine, a modified RDF medium having the composition shown in Table 1 was used, and glutamine was added thereto at final concentrations of 174.5 mg / L and 349 mg / L. To each of these media, and fructose was added to each of these media at 0.68, 1.35, 2.0 and 2.7 g / L.
A medium added so as to give a basal medium was prepared.
As a comparative control, RDF medium (glucose 2.7 as sugar was used.
g / L and glutamine 349 mg / L) were used. In addition, each of the above-mentioned basal media was added with the one described in Table 2.
【００２１】[0021]
【表１】 [Table 1]
【００２２】[0022]
【表２】 [Table 2]
【００２３】（２）培養方法及び結果
３５ｍｍペトリ皿に上記培地をそれぞれを注入し、これ
に抗体生産細胞としてＩｇＭ型のヒト型モノクローナル
抗体を生産するヒト−ヒトハイブリドーマＨＢ４Ｃ５
（微工研条寄第１８７９号）を、前記変法ＲＤＦで２回
洗浄した後、８×１０5細胞／ｍｌになるように播種し
た。３７℃の炭酸ガスインキュベーター中で、７日間培
養して培地中に生産された抗体濃度を通常の酵素抗体法
で測定した。この結果を表３に示す。(2) Culture Method and Results Human-human hybridoma HB4C5 which injects each of the above-mentioned medium into a 35 mm Petri dish and produces IgM type human monoclonal antibody as antibody-producing cells.
(Microtechnology Research Institute No. 1879) was washed twice with the modified RDF and then seeded at 8 × 10 5 cells / ml. After culturing for 7 days in a carbon dioxide gas incubator at 37 ° C., the concentration of the antibody produced in the medium was measured by a usual enzyme antibody method. The results are shown in Table 3.
【００２４】[0024]
【表３】 [Table 3]
【００２５】表３に示されるように、培地中のグルタミ
ン濃度及び果糖濃度を変化させることにより、培地中に
生産される抗体濃度が変化し、グルタミン濃度が１７
４．５ｍｇ／Ｌで、果糖濃度が１．３５ｇ／Ｌのときに
抗体生産量が最も増大し、比較対照のＲＤＦ培地に比べ
て生産量が１．５倍になることがわかる。As shown in Table 3, by changing the glutamine concentration and the fructose concentration in the medium, the antibody concentration produced in the medium was changed, and the glutamine concentration was 17%.
It can be seen that at 4.5 mg / L, the antibody production increased most when the fructose concentration was 1.35 g / L, which was 1.5 times that of the comparison control RDF medium.
【００２６】実施例２
糖質及びグルタミンを含有しない培地として、前記変法
ＲＤＦ培地を用い、これにグルタミンを最終濃度で１５
０ｍｇ／Ｌ、１７４．５ｍｇ／Ｌ、３４９ｍｇ／Ｌにな
るように添加した培地を作製し、更にそれぞれの培地に
果糖を１．３５ｇ／Ｌとなるように添加した培地を調製
して基礎培地とした。比較対照には、ＲＤＦ培地（糖質
としてブドウ糖２．７ｇ／Ｌ及びグルタミン３４９ｍｇ
／Ｌを含有する）を用いた。また、上述の基礎培地それ
ぞれに表２に記載したものを添加した。Example 2 The modified RDF medium was used as a medium containing neither sugar nor glutamine, and glutamine was added thereto at a final concentration of 15
0 mg / L, 174.5 mg / L, and 349 mg / L were added to each medium to prepare a medium, and fructose was added to each medium to a concentration of 1.35 g / L to prepare a basal medium. did. As a comparative control, RDF medium (glucose 2.7 g / L and glutamine 349 mg as sugars) was used.
/ L) was used. In addition, each of the above-mentioned basal media was added with the one described in Table 2.
【００２７】上記の培地をそれぞれ用いて、実施例１と
同様にして、抗体生産細胞であるヒト−ヒトハイブリド
ーマＨＢ４Ｃ５を培養し、培地中に生産された抗体濃度
を通常の酵素抗体法で測定した。この結果を表４に示
す。Human-human hybridoma HB4C5, which is an antibody-producing cell, was cultured in the same manner as in Example 1 using each of the above-mentioned mediums, and the concentration of the antibody produced in the medium was measured by a usual enzyme antibody method. . The results are shown in Table 4.
【００２８】[0028]
【表４】 [Table 4]
【００２９】表４に示されるように、培地中の果糖濃度
を１．３５ｇ／Ｌにした場合、グルタミン濃度が１７
４．５ｍｇ／Ｌのときに、抗体生産量が最も増大するこ
とがわかる。As shown in Table 4, when the fructose concentration in the medium was 1.35 g / L, the glutamine concentration was 17
It can be seen that the amount of antibody produced increases most at 4.5 mg / L.
【００３０】[0030]
【発明の効果】以上説明したように本発明によれば、果
糖濃度及びグルタミン濃度を種々変えた培地で抗体生産
細胞であるヒト−ヒトハイブリドーマを培養することに
より、抗体生産が最も増大する培地中の果糖濃度及びグ
ルタミン濃度を求めることができる。こうして果糖濃
度、グルタミン濃度を設定した培地を用いることによ
り、ヒト型モノクローナル抗体の生産量を増大させるこ
とができる。また、本発明の培地作製用キットを用いる
ことにより、果糖濃度及びグルタミン濃度を種々変えた
培地を容易に作製することができ、上記の作業を能率的
に行なうことができる。According to the present invention as described above, according to the present invention, fruit
By culturing the human-human hybridoma that is an antibody-producing cell in a medium in which the sugar concentration and the glutamine concentration are variously changed, the fructose concentration and the glutamine concentration in the medium in which the antibody production is most increased can be determined. By using the medium in which the fructose concentration and the glutamine concentration are set in this manner, the production amount of human-type monoclonal antibody can be increased. Further, by using the medium preparation kit of the present invention, it is possible to easily prepare a medium in which the fructose concentration and the glutamine concentration are variously changed, and the above-mentioned work can be efficiently performed.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭61−25480（ＪＰ，Ａ) 特開 昭63−267269（ＪＰ，Ａ) 特開 平２−138968（ＪＰ，Ａ) 特開 昭64−34282（ＪＰ，Ａ) 「新生化学実験講座18 細胞培養技 術」日本生化学会編 1990年10月20日 第１版発行 第25〜３ (58)調査した分野(Int.Cl.7，ＤＢ名) C12P 21/08 C12N 5/10 C12N 15/02 ＢＩＯＳＩＳ／ＷＰＩ（ＤＩＡＬＯＧ)─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-61-25480 (JP, A) JP-A-63-267269 (JP, A) JP-A 2-138968 (JP, A) JP-A 64-- 34282 (JP, A) “Newborn Chemistry Experimental Course 18 Cell Culture Technology” edited by the Biochemical Society of Japan October 20, 1990 Issued 1st edition 25-3 (58) Fields investigated (Int.Cl. 7 , DB name) ) C12P 21/08 C12N 5/10 C12N 15/02 BIOSIS / WPI (DIALOG)
Claims (2)
型モノクローナル抗体を生産するのに用いる培地の作製
法において、糖質及びグルタミンを含有しない培地組成
に、果糖及びグルタミンを種々の濃度で添加して、果糖
及びグルタミンの含有量が異なる複数の培地を作製し、
それぞれの培地を用いて抗体生産細胞を培養し、モノク
ローナル抗体生産が増強される果糖濃度及びグルタミン
濃度を求めることを特徴とするモノクローナル抗体生産
用培地の作製法。1. A human using a human-human hybridoma
In the preparation method of the culture medium used to produce the type monoclonal antibody, the medium composition containing no carbohydrates and glutamine, with the addition of fructose and glutamine at various concentrations, different content of fructose <br/> and glutamine Make multiple media,
A method for producing a culture medium for producing a monoclonal antibody, which comprises culturing antibody-producing cells in each culture medium and determining a fructose concentration and a glutamine concentration at which monoclonal antibody production is enhanced.
型モノクローナル抗体を生産するのに用いる培地作製キ
ットにおいて、糖質及びグルタミンを含有しない培地
と、果糖と、グルタミンとを組合せたことを特徴とする
モノクローナル抗体生産用培地作製キット。2. A human using a human-human hybridoma
Culture medium used to produce type I monoclonal antibodies
A kit for preparing a medium for producing a monoclonal antibody, characterized in that a medium containing no carbohydrate and glutamine, fructose , and glutamine are combined.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06927191A JP3375140B2 (en) | 1991-03-08 | 1991-03-08 | Method for producing medium for producing monoclonal antibody and kit therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP06927191A JP3375140B2 (en) | 1991-03-08 | 1991-03-08 | Method for producing medium for producing monoclonal antibody and kit therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04281797A JPH04281797A (en) | 1992-10-07 |
| JP3375140B2 true JP3375140B2 (en) | 2003-02-10 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP06927191A Expired - Fee Related JP3375140B2 (en) | 1991-03-08 | 1991-03-08 | Method for producing medium for producing monoclonal antibody and kit therefor |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999028455A1 (en) | 1997-12-03 | 1999-06-10 | Roche Diagnostics Gmbh | Method for preparing polypeptides with appropriate glycosilation |
| DK1543106T3 (en) | 2002-07-15 | 2007-08-13 | Immunex Corp | Methods and media for controlling sialylation of proteins produced by mammalian cells |
-
1991
- 1991-03-08 JP JP06927191A patent/JP3375140B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 「新生化学実験講座１８ 細胞培養技術」日本生化学会編 １９９０年１０月２０日 第１版発行 第２５〜３ |
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| Publication number | Publication date |
|---|---|
| JPH04281797A (en) | 1992-10-07 |
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