JPH01281095A - Mass production of cytokin - Google Patents
Mass production of cytokinInfo
- Publication number
- JPH01281095A JPH01281095A JP1050209A JP5020989A JPH01281095A JP H01281095 A JPH01281095 A JP H01281095A JP 1050209 A JP1050209 A JP 1050209A JP 5020989 A JP5020989 A JP 5020989A JP H01281095 A JPH01281095 A JP H01281095A
- Authority
- JP
- Japan
- Prior art keywords
- cytokin
- induction
- cells
- interleukin
- 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.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 9
- 102000004127 Cytokines Human genes 0.000 claims abstract description 7
- 108090000695 Cytokines Proteins 0.000 claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 7
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 10
- 230000000977 initiatory effect Effects 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 14
- 210000000265 leukocyte Anatomy 0.000 abstract description 9
- 235000015097 nutrients Nutrition 0.000 abstract description 9
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 abstract description 6
- 108010002350 Interleukin-2 Proteins 0.000 abstract description 5
- 102000000588 Interleukin-2 Human genes 0.000 abstract description 5
- 108010062580 Concanavalin A Proteins 0.000 abstract description 3
- 108010063738 Interleukins Proteins 0.000 abstract description 3
- 102000015696 Interleukins Human genes 0.000 abstract description 3
- 235000019270 ammonium chloride Nutrition 0.000 abstract description 3
- 210000004369 blood Anatomy 0.000 abstract description 3
- 239000008280 blood Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 210000003743 erythrocyte Anatomy 0.000 abstract description 3
- 210000002966 serum Anatomy 0.000 abstract description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 abstract description 2
- 206010018910 Haemolysis Diseases 0.000 abstract description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 2
- 230000004071 biological effect Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 239000008103 glucose Substances 0.000 abstract description 2
- 230000008588 hemolysis Effects 0.000 abstract description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 abstract description 2
- 239000011707 mineral Substances 0.000 abstract description 2
- 150000003839 salts Chemical class 0.000 abstract description 2
- 239000000725 suspension Substances 0.000 abstract description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 abstract 2
- 102000008070 Interferon-gamma Human genes 0.000 abstract 1
- 108010074328 Interferon-gamma Proteins 0.000 abstract 1
- 239000002253 acid Substances 0.000 abstract 1
- 229940104302 cytosine Drugs 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 abstract 1
- 229960003130 interferon gamma Drugs 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- 102000014150 Interferons Human genes 0.000 description 5
- 108010050904 Interferons Proteins 0.000 description 5
- 239000000411 inducer Substances 0.000 description 5
- 229940079322 interferon Drugs 0.000 description 5
- 102000000412 Annexin Human genes 0.000 description 3
- 108050008874 Annexin Proteins 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 241000711408 Murine respirovirus Species 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229940113118 carrageenan Drugs 0.000 description 2
- 235000010418 carrageenan Nutrition 0.000 description 2
- 229920001525 carrageenan Polymers 0.000 description 2
- 239000000679 carrageenan Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 239000002555 ionophore Substances 0.000 description 2
- 230000000236 ionophoric effect Effects 0.000 description 2
- 238000010979 pH adjustment Methods 0.000 description 2
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 206010015719 Exsanguination Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- -1 dexamethacin Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 150000004633 phorbol derivatives Chemical class 0.000 description 1
- 239000002644 phorbol ester Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 102000003390 tumor necrosis factor Human genes 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、誘導・生産を、異なる温度およびｐＨ値で行
なうようにして、シトキンを大量に製造する方法に関す
る。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing cytokin in large quantities, in which the induction and production are carried out at different temperatures and pH values.
（従来の技術）
シトキンは、細胞機能の調整と生体の組織との間の相互
関係において重要な役割を演じている。BACKGROUND OF THE INVENTION Cytokines play an important role in the regulation of cellular functions and the interaction between living tissues.
「シトキン」なる用語は、生細胞によって産生され、か
つそれらの周囲に分泌される生物学的活性を有するすべ
ての分子または分子グループを意味する。その代表的な
ものは、インターロイキン、インターフェロン（ＩＦＮ
−３）、リポコルチン、ｊ１瘍壊死因子などである。The term "cytokin" refers to any molecule or group of molecules with biological activity that is produced by living cells and secreted into their surroundings. The representative ones are interleukin, interferon (IFN)
-3), lipocortin, J1 tumor necrosis factor, etc.
生細胞は、適当な誘導物質、例えばウィルス、レクチン
、マイトジェン、デキサメタシン、ホルボールエステル
、イオノフオア、合成リボ核酸などの作用により、イン
ビトロにおいても、シトキンを産生ずることができる。Living cells can also produce cytokin in vitro by the action of suitable inducers such as viruses, lectins, mitogens, dexamethacin, phorbol esters, ionophores, synthetic ribonucleic acids, and the like.
従って、適当な方法を用いることにより、生物学的活性
物質を、工業的規模で生産することができる。Therefore, by using appropriate methods, biologically active substances can be produced on an industrial scale.
シトキンはまた、いわゆる自然的方法、即ち遺伝子操作
を使ってもつくられる。この遺伝子操作においては、誘
導物質をヒトまたは動物の細胞に加えると、細胞が、誘
導物質の作用を受け、栄養溶液中にシトキンを産生ずる
。このシトキンを、媒体から取り出す。Cytokines can also be produced using so-called natural methods, ie genetic manipulation. In this genetic manipulation, when an inducer is added to human or animal cells, the cells are affected by the inducer and produce cytokin in a nutrient solution. This cytokin is removed from the medium.
この方法に関しては、次のような帽告がある。Regarding this method, there are the following warnings.
例えば、グルココルチコイド使用によるリポコルチン〔
ビー・ロスフート（Ｂ、 Ｒｏｔｈｈｕｔ）ら：フェブ
ス・レターズ（ＦＥＢＳ Ｌｅｔｔ、） ２１９．１６
９ページ（１９８７年）〕、ＰＭＡ使用によるＢ細胞刺
激因子（ＩＬ−４）〔ジェイ・ヒユー・リー（Ｊ、 ｆ
ｌｕ−ＬＬ） ら：ジャーナル・オブ・エクスペリメン
タル・メディシン（Ｊ。For example, lipocortin due to the use of glucocorticoids [
B. Rothhut et al.: FEBS Lett, 219.16
9 pages (1987)], B cell stimulating factor (IL-4) using PMA [J.
lu-LL) et al.: Journal of Experimental Medicine (J.
Ｅｘｐ、 Ｍｅｄ、Ｎ６５．１５７ページ（１９８７年
）〕、コンカナバリンＡ使用によるインターロイキン−
２〔ケイ・カド−（に、 Ｋａｔｏ）ら：バイオケミカ
ル・バイオフィジカル・リサーチ・コミュニケイション
ズ（Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙｓ、 Ｒｅｓ
、 Ｃｏｍｍｕｎ、）１２ヱ、 １８２ベージ（１９
８５年）〕、センダイウィルス使用によるα−インター
フェロン〔ケイ・キャンチル（Ｋ、 Ｃａｎｔｅｌｌ）
ら：メソッズ・イン・エンザイモロジ−（Ｍｅｔｈｏｄ
ｓＥｎｚｙｍｏｌ、）７２．２９ページ（１９８１年）
〕などである。Exp, Med, N65, page 157 (1987)], Interleukin by use of concanavalin A
2 [Kato et al.: Biochemical/Biophysical Research Communications (Biochem, Biophys, Res
, Commun,) 12ヱ, 182 pages (19
1985)], α-interferon using Sendai virus [K, Cantell]
et al.: Methods in Enzymology
sEnzymol, ) 72.29 pages (1981)
] etc.
上記各方法に共通する特徴は、シトキンの誘導及び生産
が、同一の条件で行なわれていることである。A common feature of each of the above methods is that cytokin induction and production are performed under the same conditions.
（発明が解決しようとする課題）
本発明の目的は、シトキンの合成を阻害する因子の影響
を抑制して、シトキンの収量を増加させることである。(Problems to be Solved by the Invention) An object of the present invention is to increase the yield of cytokin by suppressing the influence of factors that inhibit the synthesis of cytokin.
（課題を解決するための手段）
本発明は、誘導に最適な条件の下で、生成物阻害作用が
早期に進むため、これを周囲条件の適切な制限によって
、回避させることができるという認識に基づいている。(Means for Solving the Problems) The present invention is based on the recognition that under optimal conditions for induction, the product inhibitory effect proceeds early, and that this can be avoided by appropriate restriction of the ambient conditions. Based on.
本発明による方法を用いることにより、インビトロでの
さまざまな細胞からのシトキンの収率を相当に上げるこ
とができる。By using the method according to the invention, the yield of cytokin from various cells in vitro can be increased considerably.
シトキンは、通常、対応する細胞を１種類以上の誘導物
質をもって処理し、次に、それを一定時間培養すること
によって調製される。その後、栄養溶液に分泌されたシ
トキンを、通常の方法で取り出す。Cytokines are usually prepared by treating the corresponding cells with one or more inducers and then culturing them for a certain period of time. Cytkin secreted into the nutrient solution is then removed in the usual manner.
本発明によれば、適当な時間間隔で培養条件を変化させ
ることにより、阻害因子の作用を遅らせ。According to the present invention, the action of the inhibitory factor is delayed by changing the culture conditions at appropriate time intervals.
収量を相当に上げることができる。Yields can be increased considerably.
観察したところ、シトキンの合成を遅延させる生化学的
作用は、シトキン合成の指数的間隔後に一層激しくなる
。指数的時間から開始すれば、シトキンの生産は、培養
条件の変化に左右されにくくなり、かつ、合成を緩やか
に遅らせる場合よりも阻害されなくなる。It has been observed that the biochemical effects that retard cytokine synthesis become more intense after the exponential interval of cytokine synthesis. By starting at an exponential time, cytokin production is less sensitive to changes in culture conditions and is less inhibited than when synthesis is slowly delayed.
生産の間隔は、適切な変化と、変化時点の選択とによっ
て延ばすことができる。The interval between production can be increased by appropriate changes and selection of change points.
ｐＨ調整が行なわれていない場合、そのｐｉ値を生理学
的値、即ち７．２〜７．４にする。If no pH adjustment is performed, bring the pi value to physiological values, ie 7.2-7.4.
（実施例）
以下、好適実施例に基づき、本発明による方法を詳細に
説明する。ただし、これらの実施例は、本発明を制約す
るものではない。(Example) Hereinafter, the method according to the present invention will be explained in detail based on preferred examples. However, these Examples do not limit the present invention.
率の向上
０．８３％塩化アンモニウム溶液による溶血を数回行な
い、不純物がまじっている赤血球から、軟膜（新鮮なヒ
ト血液から得られる白血球リッチな両分）を精製し、得
られた白血球を、鉱酸塩、グルコース、および１ｒｍｇ
／ｍＱの無ガンマグロブリンヒト血清を含有する適切な
栄養溶液中に、１×１０７細胞／ｒｍＱの密度で懸濁さ
せる。Improved rate of hemolysis with 0.83% ammonium chloride solution several times to purify buffy coat (white blood cell-rich fraction obtained from fresh human blood) from red blood cells mixed with impurities. Mineral salts, glucose, and 1 rmg
The cells are suspended at a density of 1 x 107 cells/rmQ in an appropriate nutrient solution containing gamma globulin-free human serum/mQ.
得られた溶液に対し、 ２００ＩＵ／ｍＱのヒト白血
球α−ＩＦＮを加え、この混合物を、３７℃にて２時間
培養する６次に、 １００ＨＡユニツト／ｒａＱのセ
ンダイウィルスを細胞に加え、その混合物を、３７℃に
て２時間培養する。To the resulting solution, 200 IU/mQ of human leukocyte α-IFN was added, and the mixture was incubated at 37°C for 2 hours. Next, 100 HA units/raQ of Sendai virus was added to the cells, and the mixture was incubated. , and culture at 37°C for 2 hours.
２時間に、 ＰＨ値が７になるような量で細胞懸濁液が
入っているビンに対し、トリス塩酸緩衝液を加えてから
、２０時間培養を行なう。次に、白血球を取り出すため
に、栄養溶液を遠心分離にかける。At 2 hours, Tris-HCl buffer is added to the bottle containing the cell suspension in an amount such that the pH value becomes 7, and then culture is carried out for 20 hours. The nutrient solution is then centrifuged to remove the white blood cells.
このようにして得られた生のＩＦＮの抗ウイルス力価は
、２００ 、０ＯＯＩＵ ／ ｍＱである。 この値は
、緩衝液によるＰＨ値調整を行なわずに得られるＩＦＮ
の力価よりも、２倍高いことになる。The antiviral titer of the raw IFN thus obtained is 200,0 OOIU/mQ. This value is based on the IFN value obtained without adjusting the pH value with a buffer solution.
The titer is twice as high as that of
実施例２
ガンマＩＦＮ及びインターロイキン−２を、 同じ細胞
培養で同時に調製する。Example 2 Gamma IFN and interleukin-2 are prepared simultaneously in the same cell culture.
新鮮な軟膜から得られた白血球、あるいはＩＦＮの調製
時に使用された白血球を、この生産のために用いる。白
血球懸濁液を、実施例１に記載の要領で調製する。Leukocytes obtained from fresh buffy coats or used during the preparation of IFN are used for this production. A leukocyte suspension is prepared as described in Example 1.
１ｍｇ／ｎ＋ｕの無ガンマグロブリンヒト血清を含む通
常の栄養溶液中に、１×ｌＯ７〜２Ｘ１０’細胞／ｍＱ
の濃度範囲細胞点球を懸濁させる。1 x lO7 to 2 x 10' cells/mQ in normal nutrient solution containing 1 mg/n+u gamma globulin-free human serum.
Suspend the cell dot spheres at a concentration range of .
１０〜２０μｇ／ｍＱのコンカナバリンＡか、 １〜１
０μｇ／ｍＱの植物凝集素か、２０〜ｌＯｎｇ／ｍｎの
メセレイン（ｍｅｓｅｒｅｉｎｅ）か、Ｏ−５〜２ μ
ｇ／ｍＱのＡ２３１８フイオノフオアか、または５〜３
０ｎｇ／ｍｕのホルボールミリステートアセテートを加
え、３７℃で誘導を行なう。Concanavalin A at 10-20μg/mQ or 1-1
0μg/mQ of phytoagglutinin, 20~lOng/mn of mesereine, O-5~2μ
g/mQ of A2318 ionophore or 5-3
Add 0 ng/mu of phorbol myristate acetate and perform induction at 37°C.
誘導物質を加えてから３時間後、白血球の人っているビ
ンを、３０℃に保たれている恒温槽に移し、ｐｌｉ値を
７に調整してから、混合物を培養する。Three hours after adding the inducer, the bottle containing the leukocytes is transferred to a constant temperature bath maintained at 30°C, the pli value is adjusted to 7, and the mixture is incubated.
培養後、ガンマ−ＩＦＮおよびインターロイキン−２（
几−２）を含む栄養溶液から、遠心分離によって細胞を
取り除く。After culturing, gamma-IFN and interleukin-2 (
Cells are removed from the nutrient solution containing 几-2) by centrifugation.
ガンマ−ＩＦＮの抗ウイルス力価は１５，０ＯＯＩＵ／
ｍＱ、ＩＬ−２の抗ウイルス力価は１３００１０／＋
ａＲである。これらの値は、緩衝液によるｐＨ調整をせ
ずに３７℃の一定温度で得られる生成物の力価よりも、
２〜２．５倍高くなっている。The antiviral titer of gamma-IFN is 15,0 OOIU/
The antiviral titer of mQ and IL-2 is 130010/+
It is aR. These values are higher than the product titer obtained at a constant temperature of 37°C without pH adjustment with buffers.
It is 2 to 2.5 times higher.
ガンマ−ＩＦＮとＩＬ−２とは、別の精製操作で分離さ
れる。Gamma-IFN and IL-2 are separated in separate purification operations.
新鮮なヒト血液から得られた軟膜を、０．８３％塩化ア
ンモニウム溶液による数回にわたる瀉血によす、不純物
がまじっている赤血球から精製する。Buffy coat obtained from fresh human blood is purified from contaminated red blood cells by several rounds of exsanguination with 0.83% ammonium chloride solution.
次に、得られた純粋な白血球画分を血清の含まれていな
い栄養溶液中に懸濁させ、１＋ｍＭ当りの細胞数をｌＸ
ｌ０’個とし、更に、１μＮのデキサメタシンによる処
理を行なってから、３７℃で２時間培養する。それから
、培養温度を３０〜３２℃の範囲に調節し、その温度で
更に１５〜２０時間培養を続ける。The resulting pure leukocyte fraction was then suspended in a serum-free nutrient solution and the number of cells per 1+mM
After treatment with 1 μN of dexamethacin, the cells were cultured at 37° C. for 2 hours. Then, the culture temperature is adjusted to a range of 30-32°C, and culture is continued at that temperature for an additional 15-20 hours.
その後、栄養溶液から白血球を取り出すために。Then to remove the white blood cells from the nutrient solution.
遠心分離にかける。Centrifuge.
このようにして得られた上澄み液は、粗調製のリボコル
チンであるが、カラゲニン誘発炎症に対して強力な抑制
作用を発揮する。The supernatant thus obtained is crudely prepared ribocortin, but it exhibits a strong inhibitory effect on carrageenan-induced inflammation.
温度を一定に保つことなく生成された粗リボコルチンの
炎症抑制作用は、一定温度で生成されるリポコルチンよ
りも、２〜２．５倍強力である。The anti-inflammatory effect of crude ribocortin produced without constant temperature is 2-2.5 times more potent than that of lipocortin produced at constant temperature.
ウィンター（Ｗｉｎｔθｒ）らが、プロシーデインゲス
・オブ・ソサイエティ・フォア・エクスベリメンタル・
バイオロジー・アンド・メディシン（Ｐｒｏｃ、 Ｓｏ
ｃ、 Ｅｘｐ、 Ｂｉｏｌ、 Ｍｅｄ、）、υ、１．６
４４ページ（１９６２年）に報告しているカラゲニン試
験法により。Wintour et al., Proceedings of the Society for Experimental
Biology and Medicine (Proc, So
c, Exp, Biol, Med, ), υ, 1.6
According to the carrageenan test method reported on page 44 (1962).
ラットに対する炎症抑制作用の測定を行なった。The anti-inflammatory effect on rats was measured.
Claims (5)
うことを特徴とするシトキンを大量に製造する方法。(1) A method for producing cytokin in large quantities, characterized in that induction and production are carried out at different temperatures or pH values.
時のものよりも低い値を使用する請求項（１）記載のシ
トキンの大量製造方法。(2) The method for mass-producing cytokin according to claim (1), wherein a lower culture temperature or culture pH value is used during induction than during production.
〜４０℃の範囲、好ましくは２０〜３５℃の範囲、最も
好ましくは２５〜３２℃の範囲に調節する請求項（１）
または（２）記載のシトキンの大量製造方法。(3) During production, the pH value of the culture is set to 7 and the temperature is set to 15.
Claim (1) wherein the temperature is adjusted to a range of -40°C, preferably a range of 20-35°C, most preferably a range of 25-32°C.
or (2) the method for producing cytokin in large quantities.
変化させる請求項（１）乃至（３）のいずれかに記載の
シトキンの大量製造方法。(4) The method for mass-producing cytokin according to any one of claims (1) to (3), wherein the pH value or temperature of the culture is changed after the initiation of cytokin synthesis.
値または温度を変化させる請求項（１）乃至（４）のい
ずれかに記載のシトキンの大量製造方法。(5) During the exponential time of cytokine synthesis, the pH
The method for mass-producing cytoquine according to any one of claims (1) to (4), wherein the value or temperature is changed.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HU881049A HU202594B (en) | 1988-03-04 | 1988-03-04 | Process for increasing yield of leucoquines |
| HU2251-1049/88 | 1988-03-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01281095A true JPH01281095A (en) | 1989-11-13 |
| JPH0724595B2 JPH0724595B2 (en) | 1995-03-22 |
Family
ID=10952631
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1050209A Expired - Lifetime JPH0724595B2 (en) | 1988-03-04 | 1989-03-03 | Method for producing cytokine |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPH0724595B2 (en) |
| CN (1) | CN1036794A (en) |
| AT (1) | AT392800B (en) |
| DE (1) | DE3907114A1 (en) |
| HU (1) | HU202594B (en) |
| IT (1) | IT1229193B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57105195A (en) * | 1980-10-24 | 1982-06-30 | Nat Res Dev | Production of interferon |
| JPS6296083A (en) * | 1985-10-21 | 1987-05-02 | Toray Ind Inc | Culture of antimal cell |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2906160A1 (en) * | 1979-02-17 | 1980-09-04 | Thomae Gmbh Dr K | Interferon prodn. - using stimulators to increase yields |
| US4357422A (en) * | 1980-08-14 | 1982-11-02 | Massachusetts Institute Of Technology | Method of enhancing interferon production |
-
1988
- 1988-03-04 HU HU881049A patent/HU202594B/en not_active IP Right Cessation
-
1989
- 1989-03-03 AT AT481/89A patent/AT392800B/en not_active IP Right Cessation
- 1989-03-03 IT IT8919632A patent/IT1229193B/en active
- 1989-03-03 JP JP1050209A patent/JPH0724595B2/en not_active Expired - Lifetime
- 1989-03-03 CN CN89102021.7A patent/CN1036794A/en active Pending
- 1989-03-06 DE DE3907114A patent/DE3907114A1/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57105195A (en) * | 1980-10-24 | 1982-06-30 | Nat Res Dev | Production of interferon |
| JPS6296083A (en) * | 1985-10-21 | 1987-05-02 | Toray Ind Inc | Culture of antimal cell |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1036794A (en) | 1989-11-01 |
| ATA48189A (en) | 1990-11-15 |
| DE3907114C2 (en) | 1991-12-19 |
| DE3907114A1 (en) | 1989-09-14 |
| AT392800B (en) | 1991-06-10 |
| JPH0724595B2 (en) | 1995-03-22 |
| IT8919632A0 (en) | 1989-03-03 |
| HUT50218A (en) | 1989-12-28 |
| IT1229193B (en) | 1991-07-25 |
| HU202594B (en) | 1991-03-28 |
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