JPH0793880B2 - Monoclonal antibody production enhancement method - Google Patents
Monoclonal antibody production enhancement methodInfo
- Publication number
- JPH0793880B2 JPH0793880B2 JP63046897A JP4689788A JPH0793880B2 JP H0793880 B2 JPH0793880 B2 JP H0793880B2 JP 63046897 A JP63046897 A JP 63046897A JP 4689788 A JP4689788 A JP 4689788A JP H0793880 B2 JPH0793880 B2 JP H0793880B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- phosphate
- monoclonal antibody
- antibody
- antibody production
- 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.)
- Expired - Fee Related
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
【発明の詳細な説明】 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の生
産を増強する方法に関するものである。TECHNICAL FIELD The present invention relates to a method for enhancing the production of monoclonal antibodies by antibody-producing cells.
従来の技術及び発明が解決しようとする課題 従来抗体生産細胞をペトリ皿、又は培養槽などを用いて
培養し抗体を生産する場合、生産効率が悪く大量に入手
す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、その利用に限界があった。2. Description of the Related Art When producing antibody by culturing antibody-producing cells in a conventional Petri dish or a culture tank, production efficiency is poor and it is difficult to obtain a large amount. In particular, when a human-human hybridoma is used to produce a human-type monoclonal antibody, the production amount is extremely small, and its utilization is limited.
この発明は、このような問題点を解決しようとして行わ
れたものである。The present invention has been made in order to solve such a problem.
課題を解決するための手段 本発明者らは、モノクローナル抗体の生産を上昇させる
培養方法の検討を行った結果、リン酸カリウム、リン酸
ナトリウム又はアデノシン５′−リン酸がこの目的に適
合することを見出し、これに基づいて本発明を完成する
に至った。Means for Solving the Problems As a result of investigating a culture method for increasing the production of a monoclonal antibody, the present inventors have found that potassium phosphate, sodium phosphate or adenosine 5′-phosphate is suitable for this purpose. The present invention has been completed based on these findings.
以下に本発明について詳細に説明する。The present invention will be described in detail below.
（１） 抗体生産細胞 ヒト−ヒトハイブリドーマ、マウス−マウスハイブリド
ーマ、EBウィルスで形質転換した細胞等が利用できる。
特に、ヒト−ヒトハイブリドーマに有効である。(1) Antibody-producing cells Human-human hybridomas, mouse-mouse hybridomas, cells transformed with EB virus, and the like can be used.
In particular, it is effective for human-human hybridomas.
（２） リン酸カリウム、リン酸ナトリウム又はアデノ
シン５′−リン酸を添加する培地 通常用いられる無血清培地又は血清培地が利用できる。
血清培地としては10％牛胎児血清を添加した培地が例示
できるが、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が望ましい。無血清培地としては、
基本合成培地に血清アルブミン、インシュリン、トラン
スフェリン、エタノールアミン、セレニウム等の因子を
添加した培地が用いられる。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、RDF培地（RP
MI 1640、DME及びハム F−12培地を2:1:1で混合した培
地）等が用いられるが、好ましくはRDF培地が用いられ
る。(2) Medium containing potassium phosphate, sodium phosphate or adenosine 5'-phosphate A commonly used serum-free medium or serum medium can be used.
As the serum medium, a medium supplemented with 10% fetal bovine serum can be exemplified, but as the medium for producing the monoclonal antibody, a serum-free medium is preferable because of easy purification of the antibody, inexpensive medium and the like. . As a serum-free medium,
A medium in which factors such as serum albumin, insulin, transferrin, ethanolamine, and selenium are added to the basic synthetic medium is used. Dulbecco's MEM medium (DME), Ham's F-12 medium, RDF medium (RP
MI 1640, DME, and Ham's F-12 medium mixed at 2: 1: 1) and the like are used, but RDF medium is preferably used.
（３） 培養法 ペトリ皿、回転撹拌式培養槽又はホローファイバー型の
培養装置が利用できる。(3) Culture method Petri dishes, rotary stirring culture tanks or hollow fiber type culture devices can be used.
（４） リン酸カリウム、リン酸ナトリウム又はアデノ
シン５′−リン酸の添加 （２）に示した培地に、pHを7.5に調整したリン酸カリ
ウム緩衝液をリン酸イオンの終濃度で９−30mMになるよ
うに添加する。好ましくは15mMになるように培地に添加
する。(4) Addition of potassium phosphate, sodium phosphate or adenosine 5'-phosphate To the medium shown in (2), add potassium phosphate buffer adjusted to pH of 7.5 to a final concentration of phosphate ion of 9-30 mM. To be added. It is preferably added to the medium at 15 mM.
リン酸ナトリウムについては、13-30mMの濃度になるよ
うに培地に添加する。好ましくは15mMの濃度を用いる。Sodium phosphate is added to the medium at a concentration of 13-30 mM. Preferably a concentration of 15 mM is used.
アデノシン５′−リン酸については、0.5-25mg/mlの濃
度になるように培地に添加する。好ましくは5mg/mlの濃
度を用いる。Adenosine 5'-phosphate is added to the medium at a concentration of 0.5-25 mg / ml. Preferably a concentration of 5 mg / ml is used.
発明の効果 抗体生産性ハイブリドーマをリン酸カリウム添加培地で
培養することにより、ハイブリドーマのモノクローナル
抗体生産量は2.7倍に上昇した。また、リン酸ナトリウ
ム添加培地を用いた場合には2.1倍に、アデノシン５′
−リン酸添加培地を用いた場合には1.5倍に、それぞれ
抗体生産量が上昇した。EFFECTS OF THE INVENTION By culturing antibody-producing hybridomas in a medium containing potassium phosphate, the amount of monoclonal antibody produced by the hybridomas increased 2.7-fold. In addition, when a sodium phosphate-containing medium was used, the adenosine 5 ′ was increased 2.1 times.
-When the phosphate-supplemented medium was used, the antibody production amount increased 1.5 times.
従って、抗体生産細胞を用いて抗体生産を行う場合に
は、リン酸カリウム、リン酸ナトリウム及びアデノシン
５′−リン酸を単独又はそれらを組み合わせることによ
り、抗体を効率よく生産できるようになる。Therefore, when antibody production is carried out using antibody-producing cells, potassium phosphate, sodium phosphate and adenosine 5'-phosphate can be used alone or in combination to efficiently produce antibodies.
以下本発明の実施例を示す。Examples of the present invention will be shown below.
実施例１ IgM型抗体生産性ヒト−ヒトハイブリドーマHB4C5〔イン
・ビトロ・セルラー・アンド・デイベロップメンタル・
バイオロジー（In Vitoro Cell.Develop.Biol.）、21、
593（1985）〕を、35mmプラスチックペトリ皿に５×105
細胞/mlになるように培地中に植え込んだ。培地として
は、リン酸カリウム非添加培地及び添加培地を用いた。
非添加培地はRDF培地に下記のものを添加する 血清アルブミン （ヒト） 2mg/ml インシュリン （ウシ） 5μg/ml トランスフェリン （ヒト） 35μg/ml エタノールアミン 20μＭ セレニウム 2.5nM リン酸カリウム添加培地は非添加培地にpH7.5に調整し
た。1Mリン酸カリウム緩衝液を用いて、リン酸イオン濃
度が15mMになるように添加する。Example 1 IgM type antibody-producing human-human hybridoma HB4C5 [in vitro cellular and developmental
Biology (In Vitoro Cell.Develop.Biol.), 21 ,
593 (1985)] in a 35 mm plastic Petri dish at 5 × 10 5
Cells were seeded in medium to give cells / ml. As the medium, a potassium phosphate-free medium and a supplemented medium were used.
For non-supplemented medium, add the following to RDF medium Serum albumin (human) 2 mg / ml Insulin (bovine) 5 μg / ml Transferrin (human) 35 μg / ml Ethanolamine 20 μM Selenium 2.5 nM Potassium phosphate-supplemented medium is non-supplemented medium The pH was adjusted to 7.5. Using a 1M potassium phosphate buffer, add phosphate to a concentration of 15 mM.
37℃の５％炭酸ガスインキュベーター中で４日間培養し
て、培地中の抗体濃度を測定したところ、非添加培地は
0.71μg/mlであり、添加培地は1.9μg/mlであった。す
なわち非添加培地に比べてリン酸カリウム添加培地は2.
7倍の抗体生産の増強が認められた。この増強度は、再
現性よく得られる。After culturing for 4 days in a 5% carbon dioxide incubator at 37 ℃ and measuring the antibody concentration in the medium,
It was 0.71 μg / ml and the addition medium was 1.9 μg / ml. That is, compared to the non-supplemented medium, the potassium phosphate-supplemented medium is 2.
A 7-fold increase in antibody production was observed. This enhancement is obtained with good reproducibility.
実施例２ 実施例１と同様の方法で実験を行った。実施例１の非添
加培地にpH7.4に調整した1Mリン酸ナトリウム緩衝液を
リン酸イオン濃度が15mMになるように添加し、リン酸ナ
トリウム添加培地を作製した。Example 2 An experiment was performed in the same manner as in Example 1. A 1 M sodium phosphate buffer adjusted to pH 7.4 was added to the non-addition medium of Example 1 so that the phosphate ion concentration was 15 mM to prepare a sodium phosphate-added medium.
３日間培養して抗体濃度を測定したところ、非添加培地
は0.94μg/mlであり、添加培地は2.0μg/mlであった。
すなわち非添加培地に比べてリン酸ナトリウム添加培地
は2.1倍の抗体生産の増強が認められた。この増強度
も、再現性よく得られる。When the antibody concentration was measured by culturing for 3 days, the non-supplemented medium was 0.94 μg / ml and the supplemented medium was 2.0 μg / ml.
That is, a 2.1-fold increase in antibody production was observed in the sodium phosphate-added medium as compared with the non-added medium. This enhancement is also obtained with good reproducibility.
実施例３ 実施例１と同様の方法で実験を行った。実施例１の非添
加培地にアデノシン５′−リン酸を5mg/mlになるように
添加し、アセノシン５′−リン酸添加培地を作製した。Example 3 An experiment was conducted in the same manner as in Example 1. Adenosine 5'-phosphate was added to the non-supplemented medium of Example 1 at 5 mg / ml to prepare an asenosin 5'-phosphate-supplemented medium.
３日間培養して抗体濃度を測定したところ、非添加培地
は0.46μg/mlであり、添加培地は0.68μg/mlであった。
すなわち非添加培地に比べてアデノシン５′−リン酸添
加培地は1.5倍の抗体生産の増強が認められた。この増
強度も、再現性よく得られる。When the antibody concentration was measured after culturing for 3 days, the non-supplemented medium was 0.46 μg / ml and the supplemented medium was 0.68 μg / ml.
That is, the adenosine 5'-phosphate-added medium was found to have a 1.5-fold increase in antibody production as compared to the non-added medium. This enhancement is also obtained with good reproducibility.
フロントページの続き (56)参考文献 特開 昭63−22183（ＪＰ，Ａ) 特開 昭58−13389（ＪＰ，Ａ) 特開 昭58−23784（ＪＰ，Ａ) 富山・安東編「単クローン抗体実験マニ ュアル」（1987−11−20）講談社Ｐ．162 −174，Ｐ．85−90Continuation of the front page (56) References JP 63-22183 (JP, A) JP 58-13389 (JP, A) JP 58-23784 (JP, A) Toyama and Ando ed. Experimental Manual "(1987-11-20) Kodansha P. 162-174, p. 85-90
Claims (3)
終濃度で９−30mMとなるように添加することを特徴とす
るモノクローナル抗体生産増強法。1. A method for enhancing monoclonal antibody production, which comprises adding potassium phosphate to a medium so that the final concentration of phosphate ions is 9-30 mM.
の終濃度で13−30mMとなるように添加することを特徴と
するモノクローナル抗体生産増強法。2. A method for enhancing monoclonal antibody production, which comprises adding sodium phosphate to a medium so that the final concentration of phosphate ions is 13-30 mM.
ることを特徴とするモノクローナル抗体生産増強法。3. A method for enhancing monoclonal antibody production, which comprises adding adenosine 5'-phosphate to the medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63046897A JPH0793880B2 (en) | 1988-02-29 | 1988-02-29 | Monoclonal antibody production enhancement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63046897A JPH0793880B2 (en) | 1988-02-29 | 1988-02-29 | Monoclonal antibody production enhancement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01222793A JPH01222793A (en) | 1989-09-06 |
| JPH0793880B2 true JPH0793880B2 (en) | 1995-10-11 |
Family
ID=12760155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63046897A Expired - Fee Related JPH0793880B2 (en) | 1988-02-29 | 1988-02-29 | Monoclonal antibody production enhancement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0793880B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062600C (en) * | 1996-09-10 | 2001-02-28 | 中国科学院长春应用化学研究所 | Preparation of selenic antibody enzyme with activity higher than that of natural enzyme |
| KR20220097421A (en) * | 2019-11-05 | 2022-07-07 | 아지노모토 가부시키가이샤 | Methods for making proteins |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5813389A (en) * | 1981-07-16 | 1983-01-25 | Kyowa Hakko Kogyo Co Ltd | Medium for cell cultivation |
| JPS5823784A (en) * | 1981-08-04 | 1983-02-12 | Ajinomoto Co Inc | Culture medium for mammalian cell |
| FR2600076A1 (en) * | 1986-06-12 | 1987-12-18 | Fond Ctre Nal Transfusion | CULTURE MEDIUM COMPRISING HUMAN ALBUMIN, PROCESS FOR PREPARING A PRODUCT INJECTABLE THEREFROM, PRODUCT OBTAINED AND USE THEREOF, COMPOSITION OBTAINED |
-
1988
- 1988-02-29 JP JP63046897A patent/JPH0793880B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 富山・安東編「単クローン抗体実験マニュアル」（１９８７−１１−２０）講談社Ｐ．１６２−１７４，Ｐ．８５−９０ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01222793A (en) | 1989-09-06 |
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