JP2011507551A - 細胞培養プロセス - Google Patents
細胞培養プロセス Download PDFInfo
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Abstract
Description
本発明は、哺乳動物細胞、具体的には、異種タンパク質および/または組み換え体タンパク質を分泌する哺乳動物細胞、そしてさらに具体的には、血液凝固第VIII因子(本明細書中以後、「第VIII因子」または単に「FVIII」)、ADAMTS−13、フューリン、あるいは凝固第VII因子(本明細書中以後、「第VII因子」または単に「FVII」のような血中タンパク質を分泌する哺乳動物細胞を培養するためのプロセスに関する。
比=(A405nm第VIIa因子の変異体)/(A405nm第VIIa因子野生型)。
基本的な細胞培養
FVIIIの生産
典型的な培養物は、Kaufmanら(1989)Mol.Cell.Biol.9:1233−1242および米国特許第5,250,421号に記載されている、第VIII因子とフォン・ヴィレブランド因子とを同時に発現するように形質転換された10A1C6 CHO細胞株のサブクローンを使用して、バイオリアクターの中で確立される。特定のサブクローンを、動物に由来する生成物を含まない標準的な培地への適応と、マイクロプレートの中でのサブクローニングによって得た。
DMEM/Ham’s F12 50/50 11.76g/kg
これには以下を添加した:
L−グルタミン 0.6g/kg
エタノールアミン 1.53mg/kg
Synperonic F68 0.25g/kg
NaHCO3 2g/kg
大豆ペプトン 4g/kg
CuSO4.5H2O 17.02mg/kg 。
CHO DUKX−B11細胞を、ADAMTS−13遺伝子を導入するためにリン酸カルシウム共沈法を使用してトランスフェクトした。細胞をネオマイシン選択条件下で培養し、メトトレキセートとG418での処理の後で選択した。無血清への適応後、細胞をサブクローニングし、サブクローン640−2を生産用クローン(production clone)として選択した。
CHO DUKX−B11細胞を、フューリン遺伝子を導入するために、リン酸カルシウム共沈法を使用してトランスフェクトした。細胞を、ヒポキサンチン、チミジン、およびグリシンを含まないDHFR培地で選択した。生産用クローン488−3は、サブクローニングと100nMのメトトレキセートを含む培地中での選択によって同定し、その後、無血清に適応させた。
CHO DUKX−B11細胞を、FVIIとVKORC(ビタミンKエポキシドレダクターゼ複合体)を同時発現することができる2シストロン性のベクターでトランスフェクトした。遺伝子発現はCMVプロモーターによって駆動され、内部リボゾーム侵入部位(IRES)はFVII遺伝子とVKORC遺伝子との間に配置されている。細胞を、リン酸カルシウム共沈法を使用してトランスフェクトした。選択培地には、200μg/mlのハイグロマイシンBを含めた。細胞を無血清条件下でサブクローニングし、高発現サブクローン1E9を生産用クローンとして選択した。1E9を、連続培養条件下での増殖、生産性、および安定性に関して有利な特性を有しているとして選択した。安定性は、ケモスタット様式で2ヶ月間評価した。
5Lの連続培養
培地を、37℃でCO2インキュベーター(5%〜15%のCO2)の中で数時間、予め馴化させた。培養物を確立させるために、少なくとも1つの1mlのバイアル(107個のCHO細胞/ml)を解凍し、細胞を、Rouxフラスコ(200ml)中の60mlの予め馴化させた培地の中に希釈し、そして37℃でCO2インキュベーター中で培養した。約3日後、60mlの培養物を、1つのローラーボトル(1.8L)中の140mlの新鮮な培地に添加した。このローラーボトルに15%のCO2をスパージングし、回転させながら37℃で培養した。2日後、細胞を1/3に分け、2つのローラーボトルの中で新鮮な培地の中で培養した(200mlの培地+100mlの培養物=1つのローラーボトルあたり300ml)。さらに2日または3日後、細胞を再び1/3に分け、全部で6個のローラーボトルの中で上記のように新鮮な培地の中で培養した。およそ1×106細胞/mlの必要な細胞密度に達したら、1800mlの接種材料を、上記のように予め馴化させた3.2Lの培地に接種し、5Lのバイオリアクターの中で培養した。標準的な条件下で、5Lのバイオリアクターを、pH7.2、1.4×106細胞/mlの細胞密度、および37℃の温度で操作した。標準的な条件下で、培養物に、10μmの気泡の大きさを有しているO2を、0.25VVHの速度(1時間あたりの培養物の容量あたりのO2の容量)でスパージングした。
接種材料のプールは、原則として5Lの連続培養に関して上記に記載したように得た。しかし、このプールはおよそ5Lであり、6個のローラーボトルからではなく18個のローラーボトルから得た。BR−40バイオリアクターを洗浄し、操作前に滅菌し、そしておよそ8Lの培地を、接種前にBR−40に移した。およそ5Lの接種物のプールを、プールタンクからバイオリアクターに、移送ラインを介して、およそ13Lの全培養容量に達するまで移した。細胞密度が≧9×105細胞/mLに達したら、培養物を培地で希釈した(1:3)。さらに1〜3日の後、細胞濃度は再び≧9×105細胞/mLに達し、320Lのバイオリアクターへの接種材料の移送を行った。
BR−320バイオリアクターを洗浄し、操作前に滅菌し、そしておよそ80Lの培地を、接種の前にBR−320に移した。およそ40Lの接種材料を、BR−40バイオリアクターからBR−320バイオリアクターに、移送ラインを介して、およそ120Lの最初の培養容量に達するまで移した。細胞密度が≧9×105細胞/mLに達したら、培養物を培地で希釈した(1:3)。(全部で)3〜6日の後、細胞濃度は再び≧9×105細胞/mLに達し、2500Lのバイオリアクターへの接種材料の移送を行った。
構築
およそ320Lの接種材料を、BR−320からすでにおよそ630Lの培地を含むBR1バイオリアクターに、移送ラインを介して、およそ950Lの最初の培養容量に達するまで移した。細胞密度が≧9×105細胞/mLに達したら、培養物を、およそ2500Lの最終容量となるように培地で希釈した(〜1:3)。(全部で)4〜7日の後、細胞濃度は再び≧9×105細胞/mLに達し、およそ1150Lの接種材料を、BR1からおよそ1350Lの培地を含むBR2バイオリアクターに移した。移送後、およそ1150Lの培地を、バイオリアクターBR1に、およそ2500Lの最終培養容量に達するまで添加した。
「ケモスタット」培養様式は、それぞれのバイオリアクター中での細胞濃度が≧1.2×106細胞/mLに達するとすぐに開始した。1日あたりおよそ1250Lの培地を、それぞれのバイオリアクターに対して連続様式で添加した。細胞濃度は、個々の2500Lのバイオリアクター中で9×105細胞/mL〜1.6×106細胞/mLの間とした。およそ1250L/日/バイオリアクターの複数の回収物を、滅菌バッグの中で2℃〜8℃で保存した。培養は、ケモスタット様式で約50日〜57日間維持した。標準的な条件下では、pHは7.2に設定し、温度は37℃に設定し、そして培養物には、10μmの気泡の大きさを有しているO2を、0.02VVHの速度(1時間あたりの培養物の容量あたりのO2の容量)をスパージングした。
FVIIIの生産性に対する様々なパラメーターの変更の影響
実施例1に記載したFVIIIとvWFとを発現するCHO細胞クローンのFVIII生産性を、様々な培養条件下で決定した。
FVIIIの生産性に対する細胞密度と銅濃度の影響
実施例1に記載したFVIIIとvWFとを発現するCHO細胞クローンのFVIIIの生産性を、様々な培養条件下で決定した。
vWFの生産性に対する細胞密度と銅濃度の影響
実施例3を繰り返したが、vWFの容量生産性を測定した。1.6×106細胞/mlおよび4ppbの銅(36℃、pH7.2)では、生産性は対照の124%であった。2.0×106細胞/mlおよび6ppbの銅では、生産性は182%であった。
細胞密度、pH、およびdCO2の影響
実施例1に記載したFVIIIとvWFとを発現するCHO細胞クローンのFVIIIの生産性を、様々な培養条件下で決定した。
フューリンの生産に対するpHと温度の影響
フューリンを発現するCHO細胞を、ケモスタット様式で2.5Lのバイオリアクターの中で培養した。細胞密度は、連続する5日間の間中、個々の培養物について1.52×106細胞/ml〜1.78×106細胞/mlの間の平均で維持した。溶存酸素は、全ての実験において、20%の空気飽和度の設定値で制御した。溶存CO2濃度は、バイオリアクターの上部空間にCO2を重層することによって、5%〜6%の間で維持した。
フューリンの生産に対するdCO2の影響
2種類の醗酵操作を、2.5Lのバイオリアクターの中でケモスタット様式で平行して行った。一方は、およそ7.5%のCO2濃度で操作し、そして他方はおよそ12%のCO2濃度で操作した。CO2濃度は、上部空間を流れるCO2の割合を変化させることによって調整した。醗酵は、37℃、7.15のpHで、そして20%のpO2を用いて行った。細胞数は、高CO2培養物中では12日間にわたり、およそ1.07×106細胞/mlとし、そして低CO2培養物中では1.49×106細胞/mlとした。
FVIIの生産に対するpHと温度の影響
FVIIを発現するCHO細胞を、ケモスタット様式で2.5Lのバイオリアクターの中で培養した。ここでは、細胞密度は、連続する4週間にわたり、それぞれの培養物について約2.5×106細胞/ml(2×106細胞/mlと3×106細胞/mlとの間)の平均で維持した。溶存酸素は、全ての実験において20%の空気飽和の設定値で制御した。溶存CO2濃度は、バイオリアクターの上部空間にCO2を重層することによって、4%〜7%の間で維持した。
FVIIの生産に対するdCO2の影響
4種類の異なるCO2濃度(5.0%、6.3%、7.6%、および8.9%)のFVIIの生産性に対する効果を、小規模の連続培養において試験した。
ADAMTS−13の生産に対する温度とpHの影響
組み換え体ADAMTS−13を発現するトランスフェクトしたCHO細胞を、1.5Lのバイオリアクターの中でケモスタット培養で培養した。
Claims (51)
- 細胞培養上清中で異種タンパク質を分泌する哺乳動物細胞を培養する方法であって、ここでは、該細胞培養上清は、X±0.9℃に設定される温度で維持され、ここでは、Xは35.1から36.5までの値を有し、ただし該温度は37℃未満に設定される、方法。
- 前記温度が36±0.9℃に設定される、請求項1に記載の方法。
- 前記温度が36±0.5℃に設定される、請求項1に記載の方法。
- 前記温度が36±0.2℃に設定される、請求項1に記載の方法。
- 前記温度が36℃に設定される、請求項1に記載の方法。
- 前記温度が35.1±0.9℃に設定される、請求項1に記載の方法。
- 前記温度が35.1±0.5℃に設定される、請求項1に記載の方法。
- 前記温度が35.1±0.2℃に設定される、請求項1に記載の方法。
- 前記温度が35.1℃に設定される、請求項1に記載の方法。
- 前記温度が36.5±0.9℃に設定される、請求項1に記載の方法。
- 前記温度が36.5±0.5℃に設定される、請求項1に記載の方法。
- 前記温度が36.5±0.2℃に設定される、請求項1に記載の方法。
- 前記温度が36.5℃に設定される、請求項1に記載の方法。
- 細胞培養上清中で異種タンパク質を分泌する哺乳動物細胞を培養する方法であって、ここでは、該細胞培養上清は、X±0.05に設定されるpHで維持され、ここではXは、7.15から7.20までの値を有し、ただし該pHは7.10より大きく設定される、方法。
- 前記pHが7.20±0.05に設定される、請求項14に記載の方法。
- 前記pHが7.20±0.03に設定される、請求項14に記載の方法。
- 前記pHが7.20±0.01に設定される、請求項14に記載の方法。
- 前記pHが7.20に設定される、請求項14に記載の方法。
- 前記pHが7.15±0.05に設定される、請求項14に記載の方法。
- 前記pHが7.15±0.03に設定される、請求項14に記載の方法。
- 前記pHが7.15±0.01に設定される、請求項14に記載の方法。
- 前記pHが7.15に設定される、請求項14に記載の方法。
- 細胞培養上清中で異種タンパク質を分泌する哺乳動物細胞を培養する方法であって、ここでは、該細胞培養上清が1%〜10%の溶存CO2濃度を有する、方法。
- 前記CO2濃度が4.0%〜9.0%である、請求項23に記載の方法。
- 前記CO2濃度が5.5%〜8.5%である、請求項23に記載の方法。
- 前記細胞培養上清が、X±0.9℃に設定される温度で維持され、ここでは、Xは35.1から36.5までの値を有し、ただし該温度は37℃未満に設定される、請求項23に記載の方法。
- 前記温度が36±0.9℃に設定される、請求項26に記載の方法。
- 前記温度が35.1±0.9℃に設定される、請求項26に記載の方法。
- 前記温度が36.5±0.9℃に設定される、請求項26に記載の方法。
- 前記細胞培養上清が、X±0.5に設定されるpHで維持され、ここでは、Xは7.15から7.20までの値を有し、ただし該pHは7.10より大きく設定される、請求項1、23、または26に記載の方法。
- 前記細胞培養上清が重炭酸塩で緩衝化される、請求項1〜30のいずれかに記載の方法。
- 前記上清に空気がスパージングされる、請求項1〜31のいずれかに記載の方法。
- 前記細胞密度が1.0×106細胞/ml〜5.0×106細胞/mlである、請求項1〜32のいずれかに記載の方法。
- 前記細胞密度が1.0×106細胞/ml〜3.5×106細胞/mlである、請求項33に記載の方法。
- 前記細胞密度が1.4×106細胞/ml〜2.8×106細胞/mlである、請求項34に記載の方法。
- 前記細胞密度が1.6×106細胞/ml〜2.6×106細胞/mlである、請求項35に記載の方法。
- 前記細胞密度が1.8×106細胞/ml〜2.4×106細胞/mlである、請求項36に記載の方法。
- 前記異種タンパク質が血中タンパク質である、請求項1〜37のいずれかに記載の方法。
- 前記血中タンパク質が第VIII因子である、請求項2、15、または23のいずれか1項に記載の方法。
- 前記第VIII因子がフォン・ヴィレブランド因子と同時に発現させられる、請求項39に記載の方法。
- 前記細胞培養上清中でのCu2+の濃度が少なくとも5ppbである、請求項1〜40のいずれかに記載の方法。
- 前記Cu2濃度が少なくとも7ppb、10ppb、15ppb、または25ppbである、請求項41に記載の方法。
- 前記血中タンパク質がADAMTS−13である、請求項2、19、または23のいずれか1項に記載の方法。
- 前記血中タンパク質がフューリンである、請求項6、19、または23のいずれか1項に記載の方法。
- 前記血中タンパク質が第VII因子である、請求項10、15、または23のいずれか1項に記載の方法。
- 前記哺乳動物細胞が、CHO細胞またはBHK細胞のような齧歯類の細胞である、請求項1〜45のいずれかに記載の方法。
- 前記培養がバッチ培養である、請求項1〜46のいずれかに記載の方法。
- 前記培養が流加培養である、請求項1〜45のいずれかに記載の方法。
- 前記培養が連続培養であり、特に、反復バッチ培養、ケモスタット培養、またはタービドスタット培養である、請求項1〜45のいずれかに記載の方法。
- 第VIII因子を分泌する哺乳動物細胞を細胞培養上清を含む容器中で培養する方法であって、ここでは、該細胞培養上清中の細胞の密度はインラインセンサーによって測定され、該容器への新鮮な培地の流入は、所望される範囲内に細胞の密度を維持するように自動的に制御される、方法。
- 前記細胞密度が少なくとも1.2×106細胞/mlである、請求項50に記載の方法。
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JP2017510283A (ja) * | 2014-04-10 | 2017-04-13 | バイエル・ヘルスケア・エルエルシーBayer HealthCare LLC | 配合培地粉末製剤および細胞培養用液体培地の調製方法 |
JP2017516484A (ja) * | 2014-06-03 | 2017-06-22 | ルピン・リミテッド | タンパク質生産のための細胞培養工程 |
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