JP3822137B2 - Additive for animal cell culture medium and method for producing protein using the same - Google Patents
Additive for animal cell culture medium and method for producing protein using the same Download PDFInfo
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- JP3822137B2 JP3822137B2 JP2002144444A JP2002144444A JP3822137B2 JP 3822137 B2 JP3822137 B2 JP 3822137B2 JP 2002144444 A JP2002144444 A JP 2002144444A JP 2002144444 A JP2002144444 A JP 2002144444A JP 3822137 B2 JP3822137 B2 JP 3822137B2
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- Prior art keywords
- meat
- mixture
- fish
- viscera
- additive
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- 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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- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- NTHWMYGWWRZVTN-UHFFFAOYSA-N sodium silicate Chemical compound [Na+].[Na+].[O-][Si]([O-])=O NTHWMYGWWRZVTN-UHFFFAOYSA-N 0.000 description 1
- 229910052911 sodium silicate Inorganic materials 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
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- 235000021335 sword fish Nutrition 0.000 description 1
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- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【0001】
【発明の属する技術分野】
本発明は、動物細胞培養用培地の添加剤、該添加剤の製造方法、該添加剤を含む動物細胞培養用培地およびそれを用いたタンパク質製造方法に関する。詳しくは、本発明は、魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を含むことを特徴とする動物細胞培養用培地への添加剤、該添加剤を含む動物細胞培養用培地およびそれを用いたタンパク質製造方法に関する。
【0002】
【従来の技術】
動物細胞を培養して該動物細胞の産生する天然型タンパク質を得ようとする場合、あるいは所望のタンパク質をコードする遺伝子を導入した動物細胞を培養して所望のタンパク質等を製造する場合には、塩類、糖類、アミノ酸類、およびビタミン類などの基礎栄養物のほかに、該動物細胞の増殖のために、通常哺乳動物由来の抽出物、具体的には牛胎児血清などの血清が5〜20%の範囲で培地に添加されている。しかしながら、かかる哺乳動物由来の血清は、培地のコストの75〜95%を占めること、品質にロット間差があるために安定した増殖が得られないという欠点がある。また、哺乳動物由来の血清はオートクレーブ等で滅菌できないので、ウイルス又はマイコプラズマ汚染される可能性があり、その多くは無害であるものの、安定生産という点からは付加的な未知の要因となりうる。更に血清には500種以上のタンパク質が含まれており、このため培養培地からの細胞産物である所望のタンパク質の単離、精製を複雑化する。このような安定生産上の問題を解決する為に,血清の代わりとして、フェツイン、インスリン、トランスフェリンなどの血清由来の純化されたタンパク質を使用する方法が行われている。また、製造コストの観点から、哺乳動物より抽出された培地成分を使用する方法も試みられている。
【0003】
しかし、近年、哺乳動物由来の成分については、狂牛病(mad cow disease)、ウシ海綿状脳症(Bovine Spongiform Encephalopathy:BSE)、感染性海綿状脳症(Transmissible Spongiform Encephalopathy:TSE)、更にはクロイツフェルト・ヤコブ病(Creutzfeld-Jakob Disease:CJD)などとの相関が懸念され、安全性の点からこれらの哺乳動物由来成分を含有しない動物細胞培養用培地の出現が望まれていた。
【0004】
しかしながら、動物細胞を培養する際、使用する培地中に上記のような哺乳動物由来の成分を添加しないと、培養の早い時期に細胞の生存率の著しい低下が生じ、培養液中の生細胞数が減少するため長期培養あるいは大量培養を行うことができないという問題があった。
【0005】
国際公開公報WO99/63058では、哺乳動物由来成分のかわりに、魚の正肉や内臓などを含む魚肉を添加することにより、動物細胞の長期培養や所望のタンパク質の大量培養が可能であることを見出している。しかしながら、WO99/63058では魚肉中の内臓と正肉の比率を変化させた場合に、タンパク質の産生量にどのような影響があるかについては言及していない。
【0006】
【発明が解決すべき課題】
本発明は魚肉抽出物又は魚肉酵素分解物を含む動物細胞培養用培地の中でも、特に効果の高い培地を提供することを目的とする。
【0007】
【課題を解決するための手段】
本発明者らは、上記のような課題を達成すべく鋭意工夫を重ねた結果、魚肉の内臓のみ又は正肉のみを添加した動物細胞培養用培地に比べて、魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を添加した動物細胞培養用培地は特に効果が高いことを見出し、本発明を完成するに至った。又、本発明者らは内臓と正肉の重量比が75:25〜40:60、好ましくは65:35〜55:45、さらに好ましくは60:40の場合に、細胞生存率及びタンパク質の生産量が向上することを見出した。
【0008】
すなわち、本発明は、魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を含むことを特徴とする動物細胞培養用培地への添加剤を提供する。
【0009】
本発明はさらに、以下の工程を含む動物細胞培養用培地への添加剤の製造方法を提供する:
(a)魚肉の内臓と正肉を混合する工程、及び
(b)上記混合物を酵素分解して酵素分解物を得るか、又は前記混合物から抽出物を得る工程。
【0010】
本発明はさらに、以下の工程を含む動物細胞培養用培地への添加剤の製造方法を提供する:
a)魚肉の内臓を酵素分解して酵素分解物を得るか、又は内臓から抽出物を得る工程、
(b)魚肉の正肉を酵素分解して酵素分解物を得るか、又は正肉から抽出物を得る工程、及び
(c)上記(a)工程で作製した物質と、上記(b)工程で作製した物質を混合する工程。
【0011】
本発明はさらに、前記添加物を含む動物細胞培養用培地を提供する。
本発明はさらに、動物細胞を培養して所望のタンパク質を製造する方法において、該培養を魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を含むことを特徴とする動物細胞培養用培地を使用して行うタンパク質の製造方法を提供する。
【0012】
本発明はさらに、上記方法で製造されたタンパク質を提供する。
【0013】
【発明の実施の形態】
本発明の動物細胞培養用培地への添加剤では、内臓と正肉との重量比が75:25〜40:60、好ましくは65:35〜55:45、さらに好ましくは60:40である。
【0014】
本発明の添加剤を含む培地を用いると、哺乳動物由来の成分を添加しなくとも特に良好に動物細胞を培養することが可能である。また、良好なタンパク質の産生量が得られる。
【0015】
本発明で使用する魚肉の内臓及び正肉を採取する魚としては、鰹、ソウダガツオ、鮪、鯖、秋刀魚、鰯、鯵、鮭などの赤身魚や、鯛、鱈、平目、鰈、鱸などの白身魚が挙げられ、好ましくは鰹、ソウダガツオ、鯖、鱈、鮭、鰯であり、特に好ましくは鰹、鯖である。
【0016】
混合する正肉と内臓は同じ魚種のものを用いてもよいが、正肉と内臓で異なる魚種のものを用いてもよい。
本発明で使用する添加剤には、魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を用いることができる。前記混合物の酵素分解物は、前記混合物又は混合物抽出物をタンパク質分解酵素で処理して得ることができる。
【0017】
あるいは、魚肉の内臓を酵素分解して酵素分解物を得るか、又は内臓から抽出物を得て、別途魚肉の正肉を酵素分解して酵素分解物を得るか、又は正肉から抽出物を得た後、これらを混合することによって添加剤を製造してもよい。
【0018】
本発明で使用する魚肉混合物の抽出物は、例えば、上記魚肉の内臓と正肉を一緒にあるいは別々に適当な断片に切断したり、あるいはミンチにしてペースト状にして、その可溶性成分を熱水、例えば90-95℃の熱水で数十分から数十時間抽出することによって得ることができる。具体的には、鰹節の製造時の鰹煮汁や缶詰製造時のクックドレンなどが挙げられる。
【0019】
また、魚肉混合物の酵素分解物は、例えば、魚肉の内臓と正肉を一緒にあるいは別々に煮たものをそのまま、あるいは、ミンチにしてペースト状にしたもの、あるいは上記のようにして得られた抽出物に適量の水を加え、必要に応じて加熱してタンパク変性させた後、タンパク質分解酵素で処理し、適宜遠心分離や濾過等により油分や不溶化物を除去することによって得ることができる。かかる魚肉混合物の抽出物あるいは酵素分解物はpHを7-7.4程度に調製して使用するのが好ましい。
【0020】
本発明で使用するタンパク質分解酵素としては、プロテイナーゼ及び/又はペプチダーゼが挙げられる。本発明では、プロテイナーゼの語は、タンパク質を基質としてタンパク質を加水分解する酵素をいい、ペプチダーゼの語は、ペプチドを基質とするペプチド結合加水分解酵素をいう。すなわち、プロテアーゼのタンパク質基質に対する活性をプロテイナーゼ活性、ペプチド基質に対する活性をペプチダーゼ活性として区別することができる。タンパク質基質に対するプロテアーゼの活性によって、ペプチド結合鎖の中程からの切断を触媒するときにはプロテイナーゼという用語を用い、従ってエンドペプチダーゼは本明細書ではプロテイナーゼの1種として使用している。本発明で使用するプロテイナーゼ又はペプチダーゼは哺乳動物由来でないことが好ましい。
【0021】
具体的には、パパイン、キモパパイン、プロメライン、フィシンなどの植物起源の酵素およびカビ、細菌、酵母などの微生物起源の酵素で、エンドペプチダーゼ、エキソペプチダーゼ、アミノペプチダーゼ、カルボキシペプチダーゼ、ジペプチダーゼなどの酵素等が挙げられる。これらの酵素は単独あるいは併用して使用することができる。併用する場合は、それらを同時に添加してもよいし、段階的に添加してもよい。
【0022】
本発明における魚肉混合物の酵素分解物としては、上記のプロテイナーゼで処理し、次いでペプチダーゼで処理して得られる魚肉混合物の酵素分解物が好ましい。
【0023】
酵素処理は、使用する酵素の種類によって異なるが、通常、pH2−12、好ましくはpH4−8で、30−90℃、好ましくは40−65℃の温度で、30分−72時間、好ましくは3−24時間行う。その際、酵素は基質としてのタンパク質の0.01−10%、好ましくは0.5−5%、さらに好ましくは1−3%程度使用する。
【0024】
このようにして得られた魚肉混合物の酵素分解物中の酵素を加温などによって失活させた後、遠心分離や濾過等を適宜行って油分や不溶化物を除去することによって酵素分解物を調製することができる。
【0025】
得られた酵素分解物は必要に応じて濃縮したり、スプレードライやフリーズドライなどの乾燥、粉化技術を擁して粉末にし、動物培養培養用培地への添加剤にすることも可能である。
【0026】
本発明における魚肉の内臓としては、胃、幽門垂、腸、肝臓、胆のう、精巣、卵巣、心臓などを挙げることができる。本発明における魚肉の正肉とは、魚から上記の内臓及び骨、頭などを除いた正肉部分をいう。
【0027】
本発明の動物細胞培養用培地への添加物は魚肉の内臓と正肉が含まれていればよく、その比率は特に限定されないが、細胞の生存率及びタンパク質の産生量の点からは、好ましくは内臓と正肉との重量比が75:25〜40:60、好ましくは65:35〜55:45、さらに好ましくは60:40である。
【0028】
なお、本発明では、魚肉の内臓と正肉からなる混合物を製造するときに、魚の骨、頭などの部分が混入してもよい。
本発明の動物細胞培養用培地には、本発明の魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を含むことを特徴とする動物細胞培養用培地への添加物を含み、さらに他の成分として通常動物細胞培養培地で使用されている各成分を適宜使用できる。これらにはアミノ酸、ビタミン類、脂質因子、エネルギー源、浸透圧調節剤、鉄源、pH緩衝剤を含む。上記成分のほか、例えば、微量金属元素、界面活性剤、増殖補助因子、ヌクレオシドなどを添加しても良い。
【0029】
具体的には、例えば、L-アラニン、L-アルギニン、L-アスパラギン、L-アスパラギン酸、L-システイン、L-シスチン、L-グルタミン、L-グルタミン酸、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-オルニチン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-チロシン、L-バリン等、好ましくはL-アラニン、L-アルギニン、L-アスパラギン、L-アスパラギン酸、L-シスチン、L-グルタミン、L-グルタミン酸、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-チロシン、L-バリン等のアミノ酸類;i−イノシトール、ビオチン、葉酸、リポ酸、ニコチンアミド、ニコチン酸、p-アミノ安息香酸、パントテン酸カルシウム、塩酸ピリドキサール、塩酸ピリドキシン、リボフラビン、塩酸チアミン、ビタミンB12、アスコルビン酸等、好ましくはビオチン、葉酸、リポ酸、ニコチン酸アミド、パントテン酸カルシウム、塩酸ピリドキサール、リボフラビン、塩酸チアミン、ビタミンB12、アスコルビン酸等のビタミン類;塩化コリン、酒石酸コリン、リノール酸、オレイン酸、コレステロール等、好ましくは塩化コリン等の脂質因子;グルコース、ガラクトース、マンノース、フルクトース等、好ましくはグルコース等のエネルギー源;塩化ナトリウム、塩化カリウム、硝酸カリウム等、好ましくは塩化ナトリウム等の浸透圧調節剤;EDTA鉄、クエン酸鉄、塩化第一鉄、塩化第二鉄、硫酸第一鉄、硫酸第二鉄、硝酸第二鉄等、好ましくは塩化第二鉄、EDTA鉄、クエン酸鉄等の鉄源類;炭酸水素ナトリウム、塩化カルシウム、リン酸二水素ナトリウム、HEPES、MOPS等、好ましくは炭酸水素ナトリウム等のpH緩衝剤を含む培地を例示できる。
【0030】
上記成分のほか、例えば、硫酸銅、硫酸マンガン、硫酸亜鉛、硫酸マグネシウム、塩化ニッケル、塩化スズ、塩化マグネシウム、亜ケイ酸ナトリウム等、好ましくは硫酸銅、硫酸亜鉛、硫酸マグネシウム等の微量金属元素;Tween80、プルロニックF68等の界面活性剤;および組換え型インシュリン、組換え型IGF、組換え型EGF、組換え型FGF、組換え型PDGF、組換え型TGF-α、塩酸エタノールアミン、亜セレン酸ナトリウム、レチノイン酸、塩酸プトレッシン等、好ましくは亜セレン酸ナトリウム、塩酸エタノールアミン、組換え型IGF、塩酸プトレッシン等の増殖補助因子;デオキシアデノシン、デオキシシチジン、デオキシグアノシン、アデノシン、シチジン、グアノシン、ウリジン等のヌクレオシドなどを添加してもよい。なお上記本発明の好適例においては、ストレプトマイシン、ペニシリンGカリウム及びゲンタマイシン等の抗生物質や、フェノールレッド等のpH指示薬を含んでいても良い。
【0031】
本発明の動物細胞培養用培地を具体的に調製するには、市販の動物細胞培養用培地、例えば、BME培地、MEM培地、DMEM培地、F10培地、F12培地などの培地に、哺乳動物由来の成分に代えて、本発明の添加物を添加して調製してもよい。
【0032】
本発明においては、培地中Brix 5%以下、好ましくは、Brix 0.5-3%、特に好ましくはBrix 1-2%程度の濃度となるように培地に魚肉混合物抽出物又は魚肉混合物の酵素分解物を添加する。なお、上記濃度は屈折糖度計により測定した可溶性固形分を指標にした濃度である。
【0033】
また、培地中のその他の成分の含量は、アミノ酸は0.05−1500mg/L、ビタミン類は0.001−10mg/L、脂質因子は0−200mg/L、エネルギー源は1−20g/L、浸透圧調節剤は0.1−10000mg/L、鉄源は0.1−500mg/L、pH緩衝剤は1−10000mg/L、微量金属元素は0.00001−200mg/L、界面活性剤は0−5000mg/L、増殖補助因子は0.05−10000μg/Lおよびヌクレオシドは0.001−50mg/Lの範囲であり、培養する動物細胞の種類、所望のタンパク質の種類などにより適宜決定できる。
【0034】
培地のpHは培養する細胞により異なるが、一般的にはpH6.8〜7.6、多くの場合pH7.2〜7.4である。
本発明の培地は特に限定されることなく種々の動物細胞を好適に培養するのに使用できる。例えば、遺伝子工学的操作によって所望の抗体あるいは生理活性物質の遺伝子を組み込んだCOS細胞やCHO細胞、あるいは、抗体を産生するマウス−ヒト、マウス−マウス、マウス−ラット等のハイブリドーマに代表される融合細胞を培養することが可能である。特に、CHO細胞の培養に好適である。勿論、本発明の動物細胞培養用培地は、動物細胞を培養して該動物細胞の産生する天然型タンパク質を得ようとする場合にも使用でき、上述した細胞の他に、BHK細胞、HeLa細胞などの培養にも使用できる。
【0035】
培養条件は使用する細胞の種類によって異なるので、適宜好適な条件を決定すればよい。例えばCHO細胞であれば通常、気相のCO2濃度が0−40%、好ましくは、2−10%の雰囲気下、30−39℃、好ましくは、37℃程度で、1−14日間培養すればよい。
【0036】
また、動物細胞培養用の各種の培養装置としては、例えば発酵槽型タンク培養装置、エアーリフト型培養装置、カルチャーフラスコ型培養装置、スピンナーフラスコ型培養装置、マイクロキャリアー型培養装置、流動層型培養装置、ホロファイバー型培養装置、ローラーボトル型培養装置、充填槽型培養装置等を用いて培養することができる。
【0037】
本発明の動物細胞培養用培地中で培養を行うことにより、動物細胞により生産されたタンパク質を培地中に得ることができる。動物細胞のタンパク質の生産は、単にそれを培養するのみで良いものや、特殊な操作を必要とするものも存在するがそれらの操作又は条件等は培養する動物細胞により適宜決定すれば良い。例えば遺伝子工学的操作によりマウス−ヒトキメラ抗体をコードする遺伝子を含むベクターでトランスフォームされたCHO細胞では、前記のような条件下で培養を実施することにより、1−14日間、好ましくは7−10日間程度で所望のタンパク質を培地中に得ることができる。これを常法(例えば、抗体工学入門、地人書館、p.102-104;Affinity Chromatography Principles & Methods、アマシャム ファルマシア バイテク(株)、p.56-60など参照)に従い単離、精製することによって、所望のタンパク質を得ることができる。
【0038】
上記の製造方法によって、抗ヒトIL-6レセプター抗体などの組換え抗体(キメラ抗体、ヒト化抗体、ヒト型化抗体を含む)、顆粒球コロニー刺激因子(G-CSF)、顆粒球マクロファージコロニー刺激因子(GM-CSF)、エリスロポエチン、インターフェロン、IL-1やIL-6等のインターロイキン、t-PA、ウロキナーゼ、血清アルブミン、血液凝固第VIII因子等の遺伝子組換えタンパク質を製造することができる。
【0039】
【実施例】
以下に本発明を更に詳細に説明するための実施例を示すが、本発明はこれら実施例に限定されるものではない。当業者にとっては種々の変更、改変が可能であり、これらも本発明の範囲に含まれる。
実施例1:
(1) 正肉の酵素分解物の調製
魚肉として市販の鰹を使用した。魚肉から内臓、骨、頭などを除去し、ミンチ状にカットしたもの1000gに水1500gを加え、植物由来のパパイン4gで、pH6.0、50℃の条件下で1時間インキュベートし、酵素分解を行った。次にカビ由来のエキソペプチダーゼ4gでさらに上記条件下で15時間酵素分解を行った後、95℃に加熱することで酵素を失活させた。その後遠心分離、濾過により不溶物、油分を除去、濃縮して、本発明の正肉(鰹)の酵素分解物500gを調製した。
(2)内臓の酵素分解物の調製
魚肉として市販の鰹を使用した。魚肉から内臓のみを取り出し、取り出した内臓を(1)の正肉の酵素分解物の調整と同様の方法で処理し、内臓(鰹)の酵素分解物500gを調製した。
(3)内臓と正肉の酵素分解物の混合物の調製
(1)および(2)と同様の方法で、内臓:正肉が7:3、6:4、5:5、4:6、3:7の割合で混合された酵素分解物を調製した。
実施例2:培地の調製
基本となる動物細胞培養用培地(基本培地)として、市販のDMEM/F12/培地(GIBCO BRL Product and Reference Guide, p357-358)からチミジン、ヒポキサチンを除去した培地に以下の成分を添加したものを用いた。
【0040】
市販のDMEM/F12培地からチミジン、ヒポキサチンを除去した培地に以下の成分を添加したもの:
アスコルビン酸30mg/L、
デオキシアデノシン(1H2O)10mg/L、
デオキシシチジン10mg/L、
デオキシグアノシン10mg/L、
アデノシン5mg/L、
シチジン5mg/L、
グアノシン5mg/L、
ウリジン5mg/L、
エタノールアミン(HCl)4mg/L、
プルロニックF-68 1000mg/L、
塩化第二鉄(6H2O)18.9mg/L
上記培地(基本培地)に、実施例1で調製した酵素分解物をそれぞれ固形分換算で10g/L又は17.5g/L添加し、濾過滅菌した。
実施例3:内臓と正肉の比率が細胞生存率に及ぼす効果
国際特許出願公開番号WO92/19759号公報の実施例10に記載されたヒトエロンゲ−ションファクタ−Iαプロモ−タ−を利用し、特開平8−99902号公報の参考例2に記載された方法に準じて作成したヒト型化PM−1抗体(抗ヒトIL−6レセプタ−抗体)を産生するCHO細胞株を用いて試験した。
【0041】
実施例2で調製した内臓:正肉の比率を変化させた培地(基本培地に実施例1(4)で調製した酵素分解物をそれぞれ17.5g/L添加した培地)に上記CHO細胞(1.5×105cell/ml)を添加し、シェーカーフラスコ型細胞培養装置を用いて37℃、5%CO2のインキュベーション条件下で12日間培養した。
【0042】
次いで、培養開始から7日後、10日後、12日後の細胞生存率を測定した。結果を図1〜3に示す。
培養7日後では内臓:正肉の比率が7:3、6:4、5:5、4:6の場合に生存率が高く、3:7では生存率が低いことが明らかとなった。
【0043】
培養10日後では内臓:正肉の比率が7:3、6:4の場合に生存率が高く、4:6、3:7では細胞は生存していないことが明らかとなった。
培養12日後では、内臓:正肉の比率が6:4の場合に細胞が生存しており、その他の比率では細胞は生存していないことが明らかとなった。
実施例4: 抗体産生量に及ぼす効果
本明細書の実施例2で調製した培地(基本培地+正肉酵素分解物4g/L+内臓酵素分解物6g/L(内臓:正肉の比率6:4)、基本培地+正肉酵素分解物10g/L、基本培地+内臓酵素分解物10g/L)に上記CHO細胞(1.5x105 cell/ml)を添加し、シェーカーフラスコ型細胞培養装置を用いて37℃、5%CO2のインキュベーター条件下で10日間培養した。
【0044】
次いで培地から得られた抗体タンパク質の産生量を測定した。産生量は逆相系高速液体クロマトグラフィーを使用して測定した。結果を図4に示す。
内臓と正肉の酵素分解物の混合物を添加した培地で培養した場合、内臓酵素分解物又は正肉酵素分解物のみを添加した培地で培養した場合よりも、抗体タンパク質の産生量において高い効果を示した。従って、正肉のみ又は内臓のみよりも内臓と正肉の混合物の方が効果が高いことが明らかとなった。
【0045】
以上の結果から、魚肉の内臓のみ又は正肉のみに比べて、魚肉と正肉の混合物の方が抗体産生量が多いことが明らかとなった。又、細胞生存率の点から、内臓と正肉の比率が7:3〜4:6の場合効果が高く、7:3〜6:4ではさらに高くあり、6:4の場合に最も高いことが判明した。
【0046】
【発明の効果】
本発明の動物細胞培養用培地は、魚肉の内臓と正肉からなる混合物の酵素分解物又は前記混合物の抽出物を含有することにより、牛胎児血清等の高価で品質のばらつきが大きいタンパク質を使用すること無く、安定的に動物細胞を培養することが可能である。また、本発明の培地は、魚肉の内臓のみ又は正肉のみを含む培地に比べて細胞生存率、タンパク質産生量のいずれにおいても優れている。更に本発明の細胞培養用培地で動物細胞を培養することにより、近年問題となっている異常プリオンあるいはウイルス等による汚染の危険性は除去され、安全なバイオ医薬品を製造し提供することができる。
【図面の簡単な説明】
【図1】魚肉の内臓酵素分解物と正肉酵素分解物を様々な比率で含む培地でCHO細胞を培養したときの7日後の細胞生存率を示すグラフである。
【図2】魚肉の内臓酵素分解物と正肉酵素分解物を様々な比率で含む培地でCHO細胞を培養したときの10日後の細胞生存率を示すグラフである。
【図3】魚肉の内臓酵素分解物と正肉酵素分解物を様々な比率で含む培地でCHO細胞を培養したときの12日後の細胞生存率を示すグラフである。
【図4】魚肉の内臓酵素分解物、正肉酵素分解物又はこれらの混合物を含む培地でCHO細胞を培養したときの抗体産生量に及ぼす効果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an additive for animal cell culture medium, a method for producing the additive, an animal cell culture medium containing the additive, and a protein production method using the same. Specifically, the present invention includes an enzyme degradation product of a mixture of fish meat viscera and true meat or an extract of the mixture, and an animal cell containing the additive. The present invention relates to a culture medium and a protein production method using the same.
[0002]
[Prior art]
When culturing animal cells to obtain a natural protein produced by the animal cells, or when culturing animal cells introduced with a gene encoding a desired protein to produce a desired protein, In addition to basic nutrients such as salts, saccharides, amino acids, and vitamins, extracts from mammals, specifically serum such as fetal calf serum, are usually used for the growth of the animal cells. % Is added to the medium. However, such mammal-derived serum has the disadvantage that it accounts for 75 to 95% of the cost of the culture medium and that stable growth cannot be obtained due to the difference in lot quality. In addition, since mammal-derived serum cannot be sterilized by autoclaving or the like, it may be contaminated with virus or mycoplasma, many of which are harmless, but may be an additional unknown factor in terms of stable production. Furthermore, serum contains more than 500 kinds of proteins, which complicates the isolation and purification of desired proteins, which are cell products from the culture medium. In order to solve such a problem in stable production, a method of using a purified protein derived from serum such as fetuin, insulin, transferrin or the like instead of serum has been performed. In addition, from the viewpoint of production cost, an attempt has been made to use a medium component extracted from a mammal.
[0003]
In recent years, however, the components derived from mammals are mad cow disease, bovine spongiform encephalopathy (BSE), infectious spongiform encephalopathy (Transmissible Spongiform Encephalopathy: TSE), and even Creutzfeldt. -Correlation with Creutzfeld-Jakob Disease (CJD) and the like is concerned, and from the viewpoint of safety, the emergence of animal cell culture media that do not contain these mammal-derived components has been desired.
[0004]
However, when cultivating animal cells, if the above-mentioned components derived from mammals are not added to the medium to be used, the cell viability is significantly reduced at an early stage of culture, and the number of viable cells in the culture solution Therefore, there is a problem that long-term culture or mass culture cannot be performed.
[0005]
In International Publication No. WO99 / 63058, it is found that long-term culture of animal cells and large-scale culture of desired proteins are possible by adding fish meat including fish meat and viscera instead of mammal-derived components. ing. However, WO99 / 63058 does not mention what kind of influence is exerted on the amount of protein produced when the ratio of viscera and true meat in fish meat is changed.
[0006]
[Problems to be Solved by the Invention]
An object of the present invention is to provide a medium that is particularly effective among animal cell culture media containing fish extract or fish enzyme degradation product.
[0007]
[Means for Solving the Problems]
As a result of intensive efforts to achieve the above-mentioned problems, the inventors of the present invention are composed of fish internal organs and meat compared to animal cell culture media containing only fish internal organs or only meat. It has been found that an animal cell culture medium to which an enzyme degradation product of the mixture or an extract of the mixture has been added is particularly effective, and the present invention has been completed. In addition, the inventors of the present invention have a cell viability and protein production when the weight ratio of viscera to meat is 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40. The amount was found to improve.
[0008]
That is, the present invention provides an additive to an animal cell culture medium, comprising an enzymatic degradation product of a mixture of fish meat viscera and meat, or an extract of the mixture.
[0009]
The present invention further provides a method for producing an additive in an animal cell culture medium comprising the following steps:
(A) a step of mixing the internal organs and the true meat of fish meat, and (b) a step of enzymatically decomposing the mixture to obtain an enzymatic degradation product, or obtaining an extract from the mixture.
[0010]
The present invention further provides a method for producing an additive in an animal cell culture medium comprising the following steps:
a) Enzymatic degradation of fish internal organs to obtain an enzymatic degradation product, or obtaining an extract from the internal organs,
(B) Enzymatic degradation of fish meat to obtain an enzymatic degradation product, or obtaining an extract from the true meat, and (c) the substance produced in the step (a) and the step (b) The process of mixing the produced substance.
[0011]
The present invention further provides an animal cell culture medium containing the additive.
The present invention further includes a method for producing a desired protein by culturing animal cells, wherein the culture includes an enzymatic degradation product of a mixture of fish internal organs and meat or an extract of the mixture. Provided is a method for producing a protein using a cell culture medium.
[0012]
The present invention further provides a protein produced by the above method.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
In the additive for animal cell culture medium of the present invention, the weight ratio of viscera to meat is 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40.
[0014]
When a medium containing the additive of the present invention is used, it is possible to cultivate animal cells particularly well without adding a mammal-derived component. In addition, a good protein production amount can be obtained.
[0015]
Fish for collecting the internal organs and true meat of the meat used in the present invention include red fish such as salmon, soda bonito, salmon, salmon, sword fish, salmon, salmon, salmon, and white meat such as salmon, salmon, flat eyes, salmon, salmon Examples include fish, preferably salmon, soda bonito, salmon, salmon, salmon, salmon, and particularly preferably salmon and salmon.
[0016]
The meat and viscera to be mixed may be the same fish species, but the fish and viscera may be different fish species.
As the additive used in the present invention, an enzymatic degradation product of a mixture of fish meat viscera and true meat or an extract of the mixture can be used. The enzyme degradation product of the mixture can be obtained by treating the mixture or mixture extract with a proteolytic enzyme.
[0017]
Alternatively, the internal organs of fish meat can be enzymatically decomposed to obtain an enzymatic decomposition product, or an extract can be obtained from the internal organs, and the fish meat can be enzymatically decomposed to obtain an enzymatic decomposition product, or an extract from the true meat can be obtained. After obtaining, an additive may be manufactured by mixing these.
[0018]
The extract of the fish meat mixture used in the present invention includes, for example, the above-mentioned fish meat viscera and the true meat cut together or separately into appropriate pieces, or minced into a paste form, and the soluble component is heated with hot water. For example, it can be obtained by extraction with hot water of 90 to 95 ° C. for several tens of minutes to several tens of hours. Specific examples include boiled simmered broth at the time of bonito manufacture and cook drain at the time of canned manufacture.
[0019]
In addition, the enzymatic decomposition product of the fish meat mixture was obtained by, for example, cooking the fish internal organs and the true meat together or separately, or as a paste made from mince, or obtained as described above. It can be obtained by adding an appropriate amount of water to the extract, heating it as necessary to denature the protein, treating it with a proteolytic enzyme, and removing oils and insolubilized materials by centrifugation or filtration as appropriate. It is preferable to use the extract or enzymatic degradation product of such a fish meat mixture after adjusting the pH to about 7-7.4.
[0020]
Examples of proteolytic enzymes used in the present invention include proteinases and / or peptidases. In the present invention, the term proteinase refers to an enzyme that hydrolyzes a protein using the protein as a substrate, and the term peptidase refers to a peptide-bonded hydrolase that uses the peptide as a substrate. That is, the activity of a protease on a protein substrate can be distinguished as proteinase activity, and the activity on a peptide substrate can be distinguished as peptidase activity. The term proteinase is used to catalyze the cleavage of the peptide bond chain from the middle by the activity of a protease on a protein substrate, and thus endopeptidase is used herein as a type of proteinase. The proteinase or peptidase used in the present invention is preferably not derived from a mammal.
[0021]
Specifically, enzymes of plant origin such as papain, chymopapain, promelain, ficin and enzymes of microorganism origin such as mold, bacteria, yeast, etc., enzymes such as endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, dipeptidase Etc. These enzymes can be used alone or in combination. When used in combination, they may be added simultaneously or stepwise.
[0022]
The enzymatic decomposition product of the fish meat mixture in the present invention is preferably an enzymatic decomposition product of the fish meat mixture obtained by treating with the above proteinase and then treating with the peptidase.
[0023]
The enzyme treatment varies depending on the type of enzyme used, but is usually pH 2-12, preferably pH 4-8, 30-90 ° C, preferably 40-65 ° C, 30 minutes-72 hours, preferably 3 -Perform for 24 hours. At that time, the enzyme is used in an amount of 0.01-10%, preferably 0.5-5%, more preferably 1-3% of the protein as a substrate.
[0024]
After deactivating the enzyme in the enzymatic decomposition product of the fish meat mixture obtained in this way by heating, etc., the enzymatic decomposition product is prepared by removing the oil and insolubilized material by appropriate centrifugation, filtration, etc. can do.
[0025]
The obtained enzyme degradation product can be concentrated as necessary, or can be powdered by using drying and powdering techniques such as spray drying and freeze drying, and can be used as an additive to a culture medium for animal culture.
[0026]
Examples of the internal organs of fish meat in the present invention include stomach, pylorus, intestine, liver, gallbladder, testis, ovary, heart and the like. The true meat of the fish meat in the present invention refers to a real meat portion obtained by removing the above-mentioned internal organs, bones, heads and the like from fish.
[0027]
The additive to the animal cell culture medium of the present invention is not particularly limited as long as it contains fish viscera and true meat, but is not particularly limited in terms of cell viability and protein production. Has a weight ratio of viscera to true meat of 75:25 to 40:60, preferably 65:35 to 55:45, and more preferably 60:40.
[0028]
In addition, in this invention, when manufacturing the mixture which consists of the internal organs and true meat of a fish meat, parts, such as a fish bone and a head, may mix.
The animal cell culture medium of the present invention contains an additive to the animal cell culture medium characterized by containing the enzymatic degradation product of the mixture of the fish viscera and the meat of the present invention or the extract of the mixture. In addition, other components that are usually used in animal cell culture media can be appropriately used as other components. These include amino acids, vitamins, lipid factors, energy sources, osmotic pressure regulators, iron sources, pH buffers. In addition to the above components, for example, trace metal elements, surfactants, growth cofactors, nucleosides and the like may be added.
[0029]
Specifically, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, etc., preferably L-alanine , L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L- Amino acids such as phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine; i-inositol, biotin, folic acid, lipoic acid, nicotinamide, nicotinic acid, p- Aminobenzoic acid, calcium pantothenate, pyridoxal hydrochloride, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, ascorbic acid, etc., preferably biotin, folic acid, lipoic acid, nicotinamide, calcium pantothenate, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride Vitamins such as vitamin B12 and ascorbic acid; choline chloride, choline tartrate, linoleic acid, oleic acid, cholesterol, etc., preferably lipid factors such as choline chloride; energy such as glucose, galactose, mannose, fructose, preferably glucose Source: Sodium chloride, potassium chloride, potassium nitrate, etc., preferably osmotic pressure regulator such as sodium chloride; EDTA iron, iron citrate, ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, nitric acid Ferric iron, etc. Preferably iron sources such as ferric chloride, EDTA iron, iron citrate; sodium bicarbonate, calcium chloride, sodium dihydrogen phosphate, HEPES, MOPS, etc., preferably including pH buffer such as sodium bicarbonate A culture medium can be illustrated.
[0030]
In addition to the above components, for example, trace metal elements such as copper sulfate, manganese sulfate, zinc sulfate, magnesium sulfate, nickel chloride, tin chloride, magnesium chloride, sodium silicate and the like, preferably copper sulfate, zinc sulfate, magnesium sulfate and the like; Surfactants such as Tween80 and Pluronic F68; and recombinant insulin, recombinant IGF, recombinant EGF, recombinant FGF, recombinant PDGF, recombinant TGF-α, ethanolamine hydrochloride, selenite Sodium, retinoic acid, putrescine hydrochloride, etc., preferably sodium selenite, ethanolamine hydrochloride, recombinant IGF, putrescine hydrochloride and other growth cofactors; deoxyadenosine, deoxycytidine, deoxyguanosine, adenosine, cytidine, guanosine, uridine, etc. The nucleoside may be added. The preferred embodiment of the present invention may contain antibiotics such as streptomycin, penicillin G potassium and gentamicin, and a pH indicator such as phenol red.
[0031]
In order to specifically prepare the animal cell culture medium of the present invention, a commercially available animal cell culture medium such as BME medium, MEM medium, DMEM medium, F10 medium, F12 medium, etc. It may replace with a component and may add and prepare the additive of this invention.
[0032]
In the present invention,
[0033]
In addition, the content of other components in the medium is 0.05-1500 mg / L for amino acids, 0.001-10 mg / L for vitamins, 0-200 mg / L for lipid factors, 1-20 g / L for energy sources, osmotic pressure regulation 0.1-10000mg / L for iron, 0.1-500mg / L for iron source, 1-10000mg / L for pH buffer, 0.00001-200mg / L for trace metal elements, 0-5000mg / L for surfactant, growth factor Is in the range of 0.05 to 10000 μg / L and nucleoside is in the range of 0.001 to 50 mg / L, and can be appropriately determined depending on the type of animal cells to be cultured, the type of desired protein, and the like.
[0034]
The pH of the medium varies depending on the cells to be cultured, but is generally pH 6.8 to 7.6, and in many cases pH 7.2 to 7.4.
The medium of the present invention is not particularly limited and can be used for suitably culturing various animal cells. For example, fusion represented by COS cells and CHO cells into which a desired antibody or physiologically active substance gene is incorporated by genetic engineering operations, or hybridomas such as mouse-human, mouse-mouse, mouse-rat, etc. that produce antibodies Cells can be cultured. In particular, it is suitable for CHO cell culture. Of course, the animal cell culture medium of the present invention can also be used when culturing animal cells to obtain a natural protein produced by the animal cells. In addition to the cells described above, BHK cells, HeLa cells It can also be used for culturing.
[0035]
Since culture conditions differ depending on the type of cells used, suitable conditions may be determined as appropriate. For example, in the case of CHO cells, it is usually cultured for 1-14 days at 30-39 ° C., preferably about 37 ° C., in an atmosphere with a CO 2 concentration in the gas phase of 0-40%, preferably 2-10%. That's fine.
[0036]
In addition, as various culture apparatuses for animal cell culture, for example, fermenter tank culture apparatus, air lift culture apparatus, culture flask culture apparatus, spinner flask culture apparatus, microcarrier culture apparatus, fluidized bed culture Culture can be performed using an apparatus, a holofiber type culture apparatus, a roller bottle type culture apparatus, a filled tank type culture apparatus, or the like.
[0037]
By culturing in the animal cell culture medium of the present invention, the protein produced by the animal cells can be obtained in the medium. The production of protein in animal cells includes those that can be simply cultured, and those that require special operations, but these operations or conditions may be appropriately determined depending on the animal cells to be cultured. For example, in a CHO cell transformed with a vector containing a gene encoding a mouse-human chimeric antibody by genetic engineering, the culture is carried out under the conditions as described above for 1-14 days, preferably 7-10. The desired protein can be obtained in the medium in about days. By isolating and purifying this according to conventional methods (eg, Introduction to Antibody Engineering, Jinshokan, p.102-104; Affinity Chromatography Principles & Methods, Amersham Pharmacia Biotech, p.56-60, etc.) The desired protein can be obtained.
[0038]
Recombinant antibodies such as anti-human IL-6 receptor antibody (including chimeric antibodies, humanized antibodies, humanized antibodies), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimuli Genetically modified proteins such as factor (GM-CSF), erythropoietin, interferon, interleukins such as IL-1 and IL-6, t-PA, urokinase, serum albumin, blood coagulation factor VIII and the like can be produced.
[0039]
【Example】
Examples for explaining the present invention in more detail are shown below, but the present invention is not limited to these examples. Various changes and modifications can be made by those skilled in the art, and these are also included in the scope of the present invention.
Example 1:
(1) Preparation of Enzyme Decomposition Product of True Meat Commercially available salmon was used as fish meat. Remove the internal organs, bones, heads, etc. from the fish meat, add 1500 g of water to 1000 g of minced cut, and incubate with 4 g of plant-derived papain for 1 hour at pH 6.0, 50 ° C. went. Next, after further enzymatic degradation with 4 g of mold-derived exopeptidase under the above conditions for 15 hours, the enzyme was inactivated by heating to 95 ° C. Thereafter, insoluble matters and oil were removed by centrifugation and filtration, and concentrated to prepare 500 g of an enzymatic decomposition product of the true meat (rice cake) of the present invention.
(2) Preparation of Visceral Enzymatic Degradation Products Commercial salmon was used as fish meat. Only the internal organs were taken out from the fish meat, and the extracted internal organs were treated in the same manner as the preparation of the enzymatic decomposition product of the true meat of (1) to prepare 500 g of the enzymatic decomposition product of the internal organs (sardine).
(3) Preparation of a mixture of visceral and meat digests
In the same manner as in (1) and (2), an enzyme degradation product was prepared in which viscera: meat were mixed at a ratio of 7: 3, 6: 4, 5: 5, 4: 6, 3: 7.
Example 2 Preparation of Medium As a basic animal cell culture medium (basic medium), a medium obtained by removing thymidine and hypoxatin from commercially available DMEM / F12 / medium (GIBCO BRL Product and Reference Guide, p357-358) What added these components was used.
[0040]
A medium obtained by removing thymidine and hypoxatin from a commercially available DMEM / F12 medium, with the following components added:
Ascorbic acid 30mg / L,
Deoxyadenosine (1H 2 O) 10 mg / L,
Deoxyguanosine 10mg / L,
Adenosine 5mg / L,
Cytidine 5mg / L,
Guanosine 5mg / L,
Uridine 5mg / L,
Ethanolamine (HCl) 4mg / L,
Pluronic F-68 1000mg / L,
Ferric chloride (6H 2 O) 18.9mg / L
10 g / L or 17.5 g / L of the enzyme degradation product prepared in Example 1 was added to the medium (basic medium) in terms of solid content, respectively, and sterilized by filtration.
Example 3: Effect of visceral to true meat ratio on cell viability Using the human elongation factor-Iα promoter described in Example 10 of International Patent Application Publication No. WO92 / 197559, The test was carried out using a CHO cell line producing humanized PM-1 antibody (anti-human IL-6 receptor antibody) prepared according to the method described in Reference Example 2 of Kaihei 8-99902.
[0041]
The above-mentioned CHO cells (1.5 ×) were prepared in a medium in which the visceral: meat ratio prepared in Example 2 was changed (a medium obtained by adding 17.5 g / L of the enzyme degradation product prepared in Example 1 (4) to the basic medium). 10 5 cell / ml) was added, and the cells were cultured for 12 days under an incubation condition of 37 ° C. and 5% CO 2 using a shaker flask type cell culture apparatus.
[0042]
Subsequently, the cell viability after 7 days, 10 days, and 12 days from the start of the culture was measured. The results are shown in FIGS.
After 7 days of culture, the viability was high when the visceral: real ratios were 7: 3, 6: 4, 5: 5, 4: 6, and the viability was low at 3: 7.
[0043]
After 10 days of culture, the viability was high when the visceral: real ratio was 7: 3, 6: 4, and it was revealed that the cells were not alive at 4: 6, 3: 7.
After 12 days of culture, it was clarified that cells survived when the visceral: meat ratio was 6: 4, and cells did not survive at other ratios.
Example 4: Effect on antibody production amount The medium prepared in Example 2 of the present specification (basic medium + meat digestion product 4 g / L + visceral enzyme degradation product 6 g / L (ratio of viscera: meat 6: 4) ), CHO cells (1.5x10 5 cell / ml) are added to basic medium + meat digestion product 10g / L, basic medium + visceral enzyme digestion product 10g / L), and shaker flask type cell culture device is used. The cells were cultured for 10 days under incubator conditions at 37 ° C. and 5
[0044]
Subsequently, the production amount of the antibody protein obtained from the medium was measured. The production amount was measured using reverse phase high performance liquid chromatography. The results are shown in FIG.
When cultured in a medium supplemented with a mixture of visceral and meat digests, it has a higher effect on the amount of antibody protein produced than when cultured in a medium supplemented with visceral or digested enzymes alone. Indicated. Therefore, it became clear that the viscera and the meat mixture were more effective than the meat alone or the viscera alone.
[0045]
From the above results, it became clear that the amount of antibody production was higher in the mixture of fish meat and true meat than in the internal organs of fish meat or just meat. In terms of cell viability, the effect is high when the ratio of viscera to meat is 7: 3 to 4: 6, higher at 7: 3 to 6: 4, and highest at 6: 4. There was found.
[0046]
【The invention's effect】
The medium for culturing animal cells of the present invention uses an enzyme degradation product of a mixture of fish internal organs and meat, or an extract of the mixture, so that expensive and highly variable protein such as fetal bovine serum is used. It is possible to cultivate animal cells stably without doing so. In addition, the medium of the present invention is superior in both cell viability and protein production as compared with a medium containing only fish internal organs or only meat. Furthermore, by culturing animal cells in the cell culture medium of the present invention, the risk of contamination with abnormal prions or viruses, which has become a problem in recent years, is eliminated, and safe biopharmaceuticals can be produced and provided.
[Brief description of the drawings]
FIG. 1 is a graph showing cell viability after 7 days when CHO cells are cultured in media containing various rates of visceral enzyme digest and fish enzyme digest in various ratios.
FIG. 2 is a graph showing cell viability after 10 days when CHO cells are cultured in a medium containing various rates of visceral enzyme digest of fish meat and authentic enzyme digest of meat.
FIG. 3 is a graph showing the cell viability after 12 days when CHO cells were cultured in media containing various rates of visceral enzyme digests and fish enzyme digests in various ratios.
FIG. 4 is a graph showing the effect on the amount of antibody production when CHO cells are cultured in a medium containing a visceral enzyme digest of fish meat, a digested meat digest or a mixture thereof.
Claims (18)
(a)魚肉の内臓と正肉を、内臓と正肉との重量比が75:25〜40:60の割合で混合する工程、及び
(b)上記混合物を酵素分解して酵素分解物を得る工程。A method for producing an additive to an animal cell culture medium comprising the following steps:
(A) a step of mixing the internal organs and the true meat of fish meat in a weight ratio of the internal organs and the authentic meat of 75:25 to 40:60 , and (b) an enzymatic decomposition of the above mixture to obtain an enzymatic decomposition product as that of Engineering.
(a)魚肉の内臓を酵素分解して酵素分解物を得る工程、
(b)魚肉の正肉を酵素分解して酵素分解物を得る工程、及び
(c)上記(a)工程で作製した物質と、上記(b)工程で作製した物質を混合する工程、
ただし、工程(a)及び工程(b)における内臓と正肉との重量比が75:25〜40:60である上記方法。A method for producing an additive to an animal cell culture medium comprising the following steps:
(A) the internal organs of the fish meat as obtained Ru engineering the by enzymatic degradation enzyme degradation product,
(B) a positive meat fish as enzymatic degradation and give Ru Industrial enzymatic degradation product, and (c) above (a) and material prepared by the process step of mixing the substance prepared in the above (b) step,
However, the said method whose weight ratio of the internal organs and the true meat in a process (a) and a process (b) is 75: 25-40: 60 .
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