JP4157644B2 - Method for producing high-purity soluble thrombomodulin - Google Patents

Description

【００１３】
本発明でトロンボモジュリン（以下、トロンボモジュリンをＴＭと略することがある）とは、トロンビンによるプロテインＣ活性化を促進する作用を有する物質として定義される。ヒトＴＭについては、Ｓ．Ｙａｍａｍｏｔｏら［国際公開番号ＷＯ８８／０５０５３］によりそのｃＤＮＡが取得され、そのｃＤＮＡによりコードされるシグナル配列を有する全長のアミノ酸配列としては、５７５アミノ酸であり、このうちシグナル配列は通常１８アミノ酸と考えられており、したがって、主たるマチュアーなペプチドとしては、５５７アミノ酸が開示されている。また、このシグナル配列としては、ヒトｔＰＡ（組織プラスミノーゲンアクチベーター）のシグナルを始めとする多くの、ヒト由来、酵母由来、大腸菌由来等の公知のシグナル配列を用いることもできる。
【００１４】
本発明に記載された可溶性ＴＭとしては、界面活性剤の非存在下で水に可溶な可溶性ＴＭが例として挙げられる。可溶性の程度としては、界面活性剤の非存在下の通常の蒸留水（中性）に簡単に溶解するものであればよく、例えば、１ｍｇ／ｍｌ以上、好ましくは３ｍｇ／ｍｌ以上、特に好ましくは１０ｍｇ／ｍｌ以上の可溶性ＴＭが例示される。本発明に記載された可溶性ＴＭとしては、ヒトＴＭのアミノ酸配列を有する、またはその部分配列を有する可溶性ペプチドが好ましいが、ヒトＴＭとホモロジーの高い構造を有する可溶性ペプチドも好ましい例として挙げられる。例えばヒトＴＭとアミノ酸配列において６０％以上のホモロジーを有するペプチドが好ましい例として挙げられ、さらに好ましくは７０％以上または８０％以上、特に好ましくは９０％以上のペプチドが例示される。本発明で記載された可溶性であるＴＭとしては、例えば上記全長アミノ酸配列から、膜通過ドメイン及び細胞内ドメインを除去したＴＭが考えられるが、好ましい配列として、４９８アミノ酸の構造も本出願には記載されている。更にまた、ＴＭの活性を完全に表すことができ、かつ可溶性である好ましい配列として、１１４アミノ酸（即ち、配列番号１の１７−１３０位のアミノ酸配列）の構造も指摘されており［Ｍ．Ｚｕｓｈｉら；Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６６、１９８８６−１９８８９（１９９１）］、ＴＭの元々の活性を保持している分子種としての１１４アミノ酸の高ホモロジー変異物、またはこれら活性中心を含みかつ可溶性であるペプチドもまた好ましい。しかしながら、上記の１１４アミノ酸を完全に包含することなく若干のアミノ酸の欠失、置換若しくは付加があってもトロンボモジュリン本来の活性を程度の差はあれ保持することも大いに期待される。
【００１５】
またヒトＴＭの遺伝子には、多型性変異が存在することが知られており、開始コドンから４７３番目のアミノ酸残基に関してバリン（具体的には、配列番号１の１７−１３０位のアミノ酸配列を包含するペプチド）［山本ら；特開昭６４−６２１９号公報］とアラニン（具体的には、配列番号２の１７−１３０位のアミノ酸配列を包含するペプチド）［Ｄ．Ｗｅｎら；Ｂｉｏｃｈｅｍｉｓｔｒｙ ２６、４３５０−４３５７（１９８７）］の変異が存在するが、これらの変異はＴＭ活性には無関係であり、また血栓症等の疾患とも無関係な変異であることが明らかになっている［Ｖ．Ｄ．Ｖａｒｄｅｎら；Ｔｈｒｏｍｂｏｓｉｓ ａｎｄＨａｅｍｏｓｔａｓｉｓ、６５、５１１−５１３（１９９１）］ので、本発明においては同等に扱うことができる。
【００１６】
したがって、例えば、配列番号１の１７−１３０位のアミノ酸配列、配列番号２の１７−１３０位のアミノ酸配列、及びそれらアミノ酸配列の１又は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる群より選ばれたいずれかのアミノ酸配列を含有するアミノ酸配列、およびそれをコードする遺伝子が例示される。また、配列番号１の１−１３０位のアミノ酸配列、配列番号２の１−１３０位のアミノ酸配列、配列番号３の１−５１６位のアミノ酸配列、配列番号４の１−５１６位のアミノ酸配列、及びそれらアミノ酸配列の１又は数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる群より選ばれたいずれかのアミノ酸配列、およびそれをコードする遺伝子が好ましい例として挙げられる。これらの種々のペプチドの製法の詳細は、国際公開番号ＷＯ９８／０１１５３号に説明されている。
【００１７】
勿論本発明のＴＭは、上記の性質を有すれば特に限定されず、例えば、糖鎖を含んでいても含まなくてもよい。
本発明に用いる可溶性トロンボモジュリンおよび血清成分を含有する未精製上清としては、可溶性トロンボモジュリンを生産し得る動物細胞を、血清成分を含有する培地を用いて培養して調製された培養上清を用いることが好ましい。
【００１８】
本発明に用いる可溶性ＴＭを生産し得る細胞としては、可溶性ＴＭを生産し得る細胞であれば特に限定されず、通常、動物細胞を意味し、ヒト由来の種々の細胞やその他の動物由来の種々の細胞を用いることができる。上記のヒト由来の細胞としては、例えばヒト血管内皮細胞、胎盤組織由来の合胞体細胞、ヒト肺ガン細胞株であるＡ５４９細胞、巨核球系の細胞株であるＭｅｇ−Ｏ１細胞等が例示される。また、その他の動物由来の細胞としては、公知の細胞が知られており、後記する各種の細胞が利用できる。
【００１９】
このような可溶性ＴＭを生産し得る細胞を本発明の製造方法に用いることのできるか否かは、例えば、ヒト由来の種々の細胞やその他の動物由来の種々の細胞のＴＭ活性やＴＭと反応する抗体との反応性を調べることで、簡単に識別することが可能である。具体的には、スクリーニングしようとする細胞の培養上清や細胞自体を、実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法に準じてスクリーニングし、ＴＭ活性の検出できた細胞を本発明のＴＭを生産し得る細胞として選択する方法が例示される。また例えば細胞の培養上清や細胞の抽出物を実施例１−（４）−ｃに記載のＥＬＩＳＡに準じて、ＴＭを検出し、求める細胞を選択することや、また、細胞自体を免疫染色することにより検出する事も可能である。
【００２０】
また、本発明の未精製上清としては、細胞の膜上に発現された全長ＴＭを抽出し、例えばエラスターゼのようなプロテアーゼ処理により容易に可溶性ＴＭとなった培養液、抽出液を利用することができる。
本発明で、可溶性ＴＭを生産し得る細胞として特に好ましい細胞は、可溶性ＴＭをコードする遺伝子（ＤＮＡ配列）を導入して調製された形質転換体である。本発明で可溶性ＴＭをコードする遺伝子としては、上記で説明した可溶性ＴＭをコードすることができる遺伝子であれば特に限定されず、種々のＤＮＡ配列が含まれるが、これらは公知の方法に準じて調製することができる。例えば、このような可溶性ＴＭをコードする遺伝子、およびその調製法は、国際公開番号ＷＯ８８／０５０５３号等の公知文献に詳しく記載されている。最も簡便には、国際公開番号ＷＯ８８／０５０５３号の実施例１−（１）に記載されたｐＳＶ２ＴＭＪ２〔ＡＴＣＣ寄託番号第６７２８３号、またＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＤＨ５／ｐＳＶ２ＴＭＪ２として再度寄託（ＦＥＲＭ ＢＰ−５５７０）を利用することにより、簡単に調製することができ、遺伝子操作の常法にしたがって、種々の欠落、変異、付加、置換等の変異物の可溶性ＴＭをコードする遺伝子が調製しえる。また、特開平５−２１３９９８号公報の実施例１記載の方法を参考に調製することもできる。これらの可溶性ＴＭをコードするＤＮＡ配列は、少なくとも実質的に可溶性ＴＭをコードするＤＮＡ配列を用いればよいが、通常は、シグナル配列をコードする塩基配列を含めて用いることが好ましい。シグナル配列としては、配列番号３の１−１８または１−１６のアミノ酸配列をコードする塩基配列が例示される。即ち、実質的にＴＭをコードするＤＮＡ配列として、具体的には、例えば、配列番号１ないし４のＤＮＡ配列を導入することが好ましい例として挙げられる。
【００２１】
可溶性ＴＭをコードするＤＮＡ配列を宿主細胞へ導入する場合には、好ましくは前記可溶性ＴＭをコードするＤＮＡ配列を、ベクター、特に好ましくは、動物細胞において発現可能な発現ベクターに組み込んで導入する方法が挙げられる。発現ベクターとは、プロモーター配列、ｍＲＮＡにリボソーム結合部位を付与する配列、発現したい蛋白をコードするＤＮＡ配列、スプライシングシグナル、転写終結のターミネーター配列、複製起源配列などで構成されるＤＮＡ分子であり、好ましい動物細胞発現ベクターの例としては、Ｒ．Ｃ．Ｍｕｌｌｉｇａｎら［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．７８，２０７２（１９８１）］が報告しているｐＳＶ２−Ｘや、Ｐ．Ｍ．Ｈｏｗｌｅｙら［Ｍｅｔｈｏｄｉｎ Ｅｍｚｙｍｏｌｏｇｙ，１０１，３８７，Ａｃａｄｅｍｉｃ ｐｒｅｓｓ（１９８３）］が報告しているｐＢＰ６９Ｔ（６９−６）などが挙げられる。
【００２２】
また、本発明の製造方法に使用できる、可溶性ＴＭを生産し得る細胞を、より高生産な細胞とするための処理を行なうことも可能であって、例えば、メトトレキセイト（ＭＴＸ）耐性を与えるジヒドロ葉酸還元酵素（ＤＨＦＲ）をコードするＤＮＡ配列を細胞に導入してＭＴＸ耐性株を取得する方法や、又はＤＨＦＲをコードするＤＮＡ配列とＴＭをコードするＤＮＡ配列とを共に宿主細胞に導入した形質転換体より、ＭＴＸ耐性株を取得する方法が例示される。ＤＨＦＲとは、２経路あるＤＮＡ合成系のうち、プリン、チミジル酸、グリシンのｄｅ ｎｏｖｏ合成に必要なテトラヒドロ葉酸を生成する酵素である。ＣＨＯ細胞ＤＨＦＲ欠損株ではＤＮＡ合成はｄｅ ｎｏｖｏ経路に欠陥があるため、残存するサルベージ経路を通してのみ成される。従って、その生育にプリン、チミジル酸、グリシンを必要とする。プリン等が含まれない培地を用いれば、ＤＨＦＲ欠損株は死滅し、ＤＨＦＲを保有する細胞のみが選択され選択マーカーとなりうる。また、ＭＴＸはＤＨＦＲの阻害剤であり、細胞ＤＮＡ合成を阻害し細胞を死滅させる。細胞のＭＴＸ耐性獲得の機構としては、Ｇ．Ａ．Ｆｉｓｃｈｅｒら［Ｂｉｏｃｈｅｍ．Ｐｈａｒｍｃｏｌ．１１，１２３３−１２３７（１９６２）］の報告しているＭＴＸの細胞内移送の低下、Ｗ．Ｆ．Ｆｌｉｎｔｏｆｆら［Ｃｅｌｌ，２，２４５−２６２，（１９７６）］の報告しているＭＴＸに対する親和性の低下したＤＨＦＲ変異体、Ｒ．Ｔ．Ｓｃｈｉｍｋｅら［Ｓｃｉｅｎｃｅ，２０２、１０５１（１９７８）］の報告しているＤＨＦＲ遺伝子の増幅によるものであるが、本発明において好ましいのは、遺伝子増幅を起こすＤＨＦＲ遺伝子であり、そのようなＤＨＦＲ遺伝子であれば、ヒト、サル、ウシ、ウサギ、ハムスター、ラット、マウス等いずれの種由来のＤＨＦＲでも良いが、好ましくは、Ａ．Ｃ．Ｙ．Ｃｈａｎｇら［Ｎａｔｕｒｅ，２７５、６１７−６２４（１９７８）］やＪ．Ｈ．Ｎｕｎｂｅｒｇら［Ｃｅｌｌ，１９，３５５−３６４（１９８０）］によりその配列が明らかにされたマウスのＤＨＦＲ遺伝子である。ＤＨＦＲをコードするＤＮＡ配列を用いる場合には、実質的にＤＨＦＲをコードするＤＮＡ配列のみを含むＤＮＡを用いてもよいが、好ましくは、動物細胞において発現可能な発現ベクターに組み込んで用いる方法が挙げられ、例えば、ｐＳＶ２ＤＨＦＲ（ＡＴＣＣ３７１４６）等の動物細胞においてマウスＤＨＦＲを発現する発現ベクターが挙げられる。
【００２３】
可溶性ＴＭをコードするＤＮＡ配列を宿主細胞へ導入するに際して、可溶性ＴＭをコードする発現ベクターと、ＤＨＦＲをコードする発現ベクターとにより共形質転換した動物細胞を得る。形質転換とは、ＤＮＡを細胞の貪食能や強制的方法により細胞に取りこませ、プラスミド状態あるいは、染色体に組み込まれた状態でＤＮＡの形質を発現させることである。リン酸カルシウム法、ＤＥＡＥ−デキストラン法、プリッキング法、プロトプラスト融合法、電気窄孔法などのいずれの方法でも導入することできが、特に好ましくは、Ｍ．Ｗｉｇｌｅｒら［Ｃｅｌｌ，１４，７２５（１９７８）］によるリン酸カルシウムとＤＮＡの複合体を細胞に導入する方法である。この際のＤＮＡの形状は前述の通り、環状ＤＮＡであってもよいし、線状ＤＮＡであってもよい。
【００２４】
宿主として用いられる動物細胞としては、ＶＥＲＯ細胞、Ｈｅｌａ細胞、チャイニーズハムスター（ＣＨＯ）細胞、Ｗ１３８、ＢＨＫ、ハムスターＡＶ−１２−６６４細胞などや、ヒト２９３細胞などが挙げられる。好ましくは、ＣＨＯ細胞、更に好ましくは、Ｌ．Ｈ．Ｇｒａｆら［Ｍｏｌｅｃ．Ｃｅｌｌ Ｂｉｏｌ．２，９３ー９６（１９８２）］の報告しているＣＨＯ−ＤＸＢ１１株等のＤＨＦＲ欠損株が例示される。
【００２５】
このようにして得られた共形質転換された細胞は、ＭＴＸ濃度の漸増する濃度条件下で選択される。ＭＴＸの漸増を行うに際しては、連続的にまたは、段階的に行なえばよいが、通常は、段階的が好ましい。ＭＴＸの濃度の範囲は、通常２０ｎＭ〜１００ｍＭ、好ましくは、２０ｎＭ〜１０ｍＭである。ＭＴＸによる選択に先立ち、ＭＴＸ非添加培地による予備選択と、後記の比生産速度を指標にしてＴＭ生産性の確認を行ってもよいし、最初からＭＴＸ添加培地により選択した後に、ＴＭ生産性の確認を行ってもよい。高生産細胞を得るためには、ＭＴＸの濃度は、初期濃度の３〜５倍づつ増加させ、段階はできるだけ多く行なうのが好ましいが、少なくとも、５段階以上行うことが好ましい。さらに、各段階でＴＭの比生産速度を調べながら行なうのが好ましい。
【００２６】
かくして、本発明の製造方法に使用できる、可溶性ＴＭを生産し得る細胞を取得することができる。
本発明の可溶性ＴＭ製造用培地において、血清とは、特に限定されないが、通常、動物の血清、好ましくは牛の胎児、新生児、仔牛、成牛の血清が挙げられる。また血清に代わる増殖因子としては、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のいずれか、もしくはそれらの混合物が挙げられる。血清と増殖因子のいずれを用いるかについては、用いるＴＭを生産し得る細胞の性質に応じて適宜選択することができる。すなわち、血清非存在下でＴＭ生産細胞が増殖性を示す場合は、血清のみ、増殖因子のみ、または、血清と増殖因子の両者を用いる各方法を選択することができる。また、血清非存在下でＴＭ生産細胞が増殖性を示さない場合は、血清のみ、または、血清と増殖因子の両者を用いる各方法を選択することができる。血清か増殖因子のいずれか一方か、または混合物を用いるかにより、これらの添加量は適宜決定できるが、通常は、血清の場合には、２〜１０％（ｖ／ｖ）が例示され、増殖因子の場合には、例えば、アルブミンは約３ｍｇ、インシュリンは約５ｍｇ、トランスフェリンは約５ｍｇ、エタノールアミンは約２μＭ、亜セレン酸ナトリウムは約２０ｎＭが例示される。
【００２７】
本発明の可溶性ＴＭ製造用培地は、上記成分表１に記載の各成分から実質的になればよいが、成分表１に記載されたもののほか、通常、タイロシン、ゲンタマイシン、カナマイシン等の抗生物質を追加することも特に妨げられない。
本発明の可溶性ＴＭ製造用培地のｐＨは、細胞の培養を妨げない程度であれば特に限定されないが、通常はｐＨ６〜８、好ましくはｐＨ７付近が例示される。
【００２８】
本発明の可溶性ＴＭ製造用培地である前述の成分表１に記載された成分から実質的になる培地は、該培地の成分の組成比率が下記の成分表２に記載の組成比率であることがさらに好ましい例として挙げられる。
【００２９】
【化５】

【００３０】
成分表２の組成比率として記載された数値の誤差範囲として、通常１０％、好ましくは５％程度が挙げられる。なお、水分は適宜調整してもよい。
本発明の可溶性ＴＭ製造用培地として、上記の成分表２に記載の組成比率の以外の該培地の成分の組成比率も選択することができ、その場合には、下記のスクリーニング方法により、他の適宜の組成比率を検討することができ、可溶性ＴＭを生産し得る細胞や培養条件等によって、好ましい培地組成比率を選択することができる。
【００３１】
〔培地成分の組成比率に関するスクリーニング方法〕
様々に成分比率を変えたスクリーニングすべき培地へ、遠心分離した、可溶性ＴＭの製造を予定している細胞を必要数懸濁し、細胞培養用ディッシュへ分注し、炭酸ガスインキュベーターで１日〜３日間培養をする。比生産速度を測定する場合には、前述の測定法によるＴＭ活性の測定と、生細胞密度の測定を、培養１、２、３日目の培地上清に関して行い、比生産速度（細胞当たり時間当たりの生産速度）を算出する。ＴＭの最終濃度を指標とする場合には、３日間培養後のＴＭ濃度と、生細胞密度を測定する。
【００３２】
本発明において、上述の可溶性ＴＭ製造用培地を用いて、ＴＭを生産し得る細胞の製造のための培養を行なうに際しては、公知の方法にて行なえばよく、選択したＴＭを生産し得る細胞の性質に応じて適宜の条件を選択できる。例えば、細胞の接着依存の性質、即ち、付着細胞または浮遊細胞によって、付着細胞であれば、付着担体としてセファロース、セルロース、ガラス、ゼラチン、ポリスチロールそしてポリエチレンなどからできたマイクロキャリアーや、マルチトレイ、ローラーボトル、中空糸などを用いることができる。培養環境条件は細胞の性質に応じて選択でき、例えば、温度３０℃〜４０℃、好ましくは３７℃付近、撹拌が必要な場合では２０〜２００ｒｐｍ、好ましくは８０ｒｐｍ付近、溶存酸素濃度（ＤＯ）は水の空気飽和に対し約３％〜９０％、好ましくは５０％付近、ｐＨは６〜８、好ましくは７付近に調節される例が挙げられる。また培養環境条件を一定に保つため必要な培養液の混合方法として、自然対流の静置型、撹拌羽根による撹拌型、通気によるエアリフト型または循環ポンプや撹拌機による流動床型などが挙げられる。
【００３３】
本発明においては、可溶性ＴＭを製造するための培養において、本発明の可溶性ＴＭ製造用培地を用いるが、細胞を増殖させる工程においては、他の培地を使用してもよいが、本発明の可溶性ＴＭ製造用培地を細胞の増殖からＴＭの製造のための培養に一貫して使用することも好ましい。細胞の増殖のために他の培地を用いる場合には、他の培地としては公知の各種培地が使用でき、例えば、血清そしてまたは増殖因子を添加したＤＭＥＭなどが例示される。増殖せしめる細胞は、凍結保存されていることも多く、このような細胞においては、３７℃の恒温槽で素早く融解し、適宜の培地に懸濁し遠心分離し、遠心後、上清は吸引除去し新鮮培地で懸濁し、スピンナーフラスコや細胞培養用ディッシュなどの培養容器に移し培養を行なうとよい。
【００３４】
細胞の増殖中の培地交換頻度は、１日〜６日たびに１培養量相当の上清を連続的もしくは間欠的に除き、除去した上清と等量の新鮮培地を連続的もしくは間欠的に添加し培地交換を行なうことができる。
そして可溶性ＴＭを製造するための培養に必要な細胞密度に達した後、好ましくは飽和密度に達した後、１日〜３日たびに好ましくは毎日１培養量相当の上清を連続的もしくは間欠的に除き、上清除去と等量の新鮮培地を連続的もしくは間欠的に添加し培地交換を行なうことができる。
【００３５】
また培地交換を実施せずに可溶性ＴＭを製造するための培養においては、適当な期間培養し、好ましくは最大の累積生産性になるまで培養し、その培養液全てを目的産物を含む培養物として収穫することができる。
培養上清の回収方法としてはバッチ式、繰り返しバッチ式または連続式などが挙げられ、細胞と培養上清の分離として、重力沈降、遠心分離、膜分離が挙げられる。可溶性ＴＭを含有する培養上清を以後の精製工程に用いる。これらの培養上清を保存する場合には、例えば、１０℃から−８０℃にて保存することが例示される。
【００３６】
次いで上記により取得された培養上清からの可溶性ＴＭの精製方法は、通常用いられる手法〔堀尾武一編集；蛋白質・酵素の基礎実験法〕に準じて行なうことができる。例えば、可溶性ＴＭと逆の電荷を持つ官能基を固定化したクロマトグラフィー担体と、可溶性ＴＭの間の相互作用を利用したイオン交換クロマトグラフィーの使用も好ましい。また、可溶性ＴＭの分子量サイズを利用した、ゲル濾過クロマトグラフィーや限外濾過が挙げられる。そしてまた、疎水性基を固定化したクロマトグラフィー担体と、可溶性ＴＭのもつ疎水性部位との間の疎水結合を利用した疎水性クロマトグラフィーが挙げられる。これらの手法は適宜組み合わせることができる。また更に、主精製工程としては、可溶性ＴＭとの特異的親和性を利用したアフィニティークロマトグラフィーが好ましい例として挙げられる。吸着体の好ましい例として、ＴＭのリガンドであるトロンビンや可溶性ＴＭの抗体を利用する例が挙げられる。これらの抗体としては、適宜の性質、或いは適宜のエピトープを認識する可溶性ＴＭの抗体を利用することができ、例えば、特公平５−４２９２０号公報、特開昭６４−４５３９８号公報、特開平６−２０５６９２号公報などに記載された例が挙げられる。精製の程度は、使用目的等により選択できるが、例えば電気泳動、好ましくはＳＤＳ−ＰＡＧＥの結果が単一バンドとして得られるか、もしくは単離精製品のゲル濾過ＨＰＬＣまたは逆相ＨＰＬＣの結果が単一のピークになるまで純粋化することが望ましい。
【００３７】
しかしながら可溶性ＴＭを医薬用途として用いるためには、上記の通常用いられる精製手法を単に組み合わせただけの精製工程では、安全性の面で問題を有する可能性のある夾雑蛋白質を実質的に含有しないレベルまで除去することは困難である。こうした問題を有する混入蛋白質としては、細胞培養による増殖、可溶性ＴＭ産生のために用いられる培地成分中に含まれる例えばウシの血清成分、生産基材である細胞として例えばＣＨＯ細胞を用いた場合にはチャイニーズハムスター由来の成分、更には主精製に抗体アフィニティーカラムを用いた場合には、例えばマウス抗ＴＭ抗体成分といった異種蛋白があげられる。これらの異種蛋白は基本的にヒトに対して抗原性を有し、その混入は、医薬として投与した際に、アナフィラキシー等の予期せぬ事態を引き起こしかねない。また最近では特にウシ血清由来物中に含まれる、プリオンに由来するクロイツフェルト・ヤコブ病の危険性も指摘されている。これら混入異種蛋白は元々多成分の混合物であることから、工業的な可溶性ＴＭ製造プロセスの中で、簡便な手法によりそれぞれを実質的に含有しないレベルまで除去することは非常な困難を招いている。
【００３８】
精製法を具体的に例示すれば、ＴＭ活性、例えばトロンビンによるプロテインＣ活性化の促進活性を指標に精製する方法が挙げられ、例えばイオン交換カラムのＱ−セファロースＦＦで培養上清を粗精製しＴＭ活性を有する画分を回収し、ついでアフィニティーカラムのマウス抗ＴＭモノクローナル抗体で精製し、ＴＭ活性が強い画分を回収し、回収画分を濃縮し、ゲルろ過にかけＴＭ活性画分を取得する精製方法［五味ら；Ｂｌｏｏｄ、７５、１３９６−１３９９、１９９０］が挙げられる。この際抗ＴＭ抗体カラムによる精製の後、本発明による陽イオン交換体、好ましくは強陽イオン交換体である、スルホプロピル基を有する陽イオン交換体、例えばＳＰ−セファロースＦＦ（ファルマシア社）を最適な条件下で用いることにより、培地由来のウシ血清由来物及びマウス抗体由来物を１工程で同時に除去し、簡便に高純度なＴＭを取得することが可能である。
【００３９】
好ましい精製法を例示すると以下の通りである。
可溶性ＴＭと良好な吸着条件を有する適当なイオン交換樹脂を選定し、イオン交換クロマト精製を行なう。特に好ましい例としては、０．１８Ｍ ＮａＣｌを含む０．０２Ｍトリス塩緩衝液（ｐＨ７．４）で平衡化したＱ−セファロースＦＦを用いる方法である。適宜洗浄後、例えば０．３Ｍ ＮａＣｌを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．４）で溶出し粗精製品の可溶性ＴＭを得ることができる。
【００４０】
次に、例えば可溶性ＴＭと特異的親和性を持つ物質を樹脂に固定化しアフィニティークロマト精製を行なうことができる。好ましい例としてＤＩＰ−トロンビン−アガロースカラムの例と、抗ＴＭモノクローナル抗体カラムの例が挙げられる。ＤＩＰ−トロンビン−アガロースカラムは、予め、例えば、１００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で平衡化せしめ、上記の粗精製品をチャージして、適宜の洗浄を行い、例えば、１．０Ｍ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で溶出し精製品のＴＭを取得することができる。また抗ＴＭモノクローナル抗体カラムにおいては、予めＣＮＢｒにより活性化したセファロース４Ｂ（ファルマシア社）に、抗ＴＭモノクローナル抗体を溶解した０．５ＭＮａＣｌ含有０．１Ｍ ＮａＨＣＯ3 緩衝液（ｐＨ８．３）に接触させ、セファロース４Ｂに抗ＴＭモノクローナル抗体をカップリングさせた樹脂を充填したカラムを、予め例えば１．０Ｍ ＮａＣｌ含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化し、適宜の洗浄の後、例えば、０．３Ｍ ＮａＣｌ含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．０）にて溶出せしめる方法が例示される。
【００４１】
次に得られた上記精製可溶性トロンボモジュリン溶液を、塩濃度、ｐＨの測定精度及びＴＭの分子種にもよるが、通常例えば比伝導度２５〜３４ｍｓ／ｃｍ、ｐＨ３〜４の条件下にて平衡化せしめた、陽イオン交換体、好ましくは強陽イオン交換体であるＳＰ−セファロースＦＦ（ファルマシア社）にチャージする。上記の比伝導度としては、３０±３ｍｓ／ｃｍが、より好ましく、またｐＨはｐＨ３．０〜３．７が好ましく、さらに好ましくはｐＨ３．５±０．１の条件が例示される。これらの条件は、適当な濃度の塩を添加した緩衝液を使用することが好ましく、種々の態様が考えられるが、例えば０．２５〜０．３２Ｍ、好ましくは０．３±０．０１Ｍの食塩を含む５０〜１５０ｍＭのｐＨ３〜４の緩衝液が例示される。緩衝液の種類としては特に限定されないが、例えば、グリシン−塩酸、クエン酸−リン酸２ナトリウム、クエン酸−クエン酸ナトリウム、酢酸−酢酸ナトリウムが例示される。上記の条件をさらに具体的に示せば、３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）が例示される。比伝導度は、例えば、ポータブル伝導度計（東亜電波工業社製、ＰシリーズＣＭ−１１Ｐ、換算基準温度２５度）により簡単に測定することができる。
【００４２】
上記のカラムを例えば３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５、比伝導度３１ｍｓ／ｃｍ）で洗浄を開始し、素通り画分の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの素通り画分を得、適当な緩衝液で中和し、血清由来物及び抗体由来物を実質的に含有しない程度まで精製された可溶性ＴＭを高純度精製品として取得することができる。後記する通り、このカラムを使用すると血清由来物や抗体由来物を効率および再現性よく除去できることが確認される。勿論、これらは、適宜限外濾過により濃縮することができる。
【００４３】
さらに、ゲル濾過による緩衝液交換を行なうことも好ましい。例えば、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化せしめたＳｅｐａｈｃｒｙｌ Ｓ−３００カラムもしくはＳ−２００カラムに、限外濾過により濃縮した高純度精製品をチャージし、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で展開分画し、トロンビンによるプロテインＣの活性化の促進活性の確認を行ない活性画分を回収し緩衝液交換した高純度精製品を取得することができる。
【００４４】
目標とする純度が得られ、さらに緩衝液交換された可溶性ＴＭ高純度精製品は、凍結乾燥を行い製剤化することができる。例えば、配列番号３に記載のアミノ酸配列をコードする遺伝子を導入して得られる形質転換体より取得される可溶性トロンボモジュリンの凍結乾燥方法は、特開平６−３２１８０５号公報に記載の方法に従い実施できる。製剤化した可溶性ＴＭは遮光条件、例えば暗室、箱、褐色ビンのなかで保存することができる。
【００４５】
公知の通常の培地、例えば、大量培養に好んで用いられるダルベッコ改変イーグル培地（以下ＤＭＥＭ）では、細胞の高密度化にともない、時間当たり細胞当たりのＴＭの生産速度（以下、比生産速度）が著しく低下するのに対して、本発明の可溶性ＴＭ製造用培地によれば、効率的に可溶性ＴＭの生産が行われ、極めて好ましい結果を与えた。
【００４６】
【発明の実施の態様】
以下実施例によって、本発明をより具体的に説明するが、本発明は何らこれらによって限定されるものではない。
【００４７】
実施例１
細胞の作製
（１）プラスミドｐＳＶ２ＴＭＤ１２３の構築
国際特許出願（国際公開番号ＷＯ８８／０５０５３）の実施例１−（１）に記載されたｐＳＶ２ＴＭＪ２〔ＡＴＣＣ寄託番号第６７２８３号、またＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＤＨ５／ｐＳＶ２ＴＭＪ２として再度寄託している（ＦＥＲＭ ＢＰ−５５７０）〕をＮｃｏＩで完全消化した後、切断末端をＥ．ｃｏｌｉＤＮＡポリメラーゼを用いて平滑末端にした。次いでＨｉｎｄIII で完全消化して約１９００ｂｐのＤＮＡ断片を得た。得られたＤＮＡ断片をＴＭＪ３と称した。一方、ファージＭ１３ｍｐ１９（宝酒造製、日本、カタログ番号３１１９）をＨｉｎｄIII 及びＨｉｎｃIIで消化してベクターを調製した。このベクターにＤＮＡ断片ＴＭＤ３を挿入して組換え体プラスミドＭ１３ｍｐ１９ＴＭＪ３を得た。
【００４８】
また別途、下記に示す削除用ＤＮＡプローブ〔以下“ディリーター（ｄｅｌｅｔｅｒ）”と称する〕を有機合成した：５’−ＧＧＡＧＧＣＣＧＣＴＣＡＧＣＣＣＧＡＡＴＧＣＡＣＧ−３’（２５ｍｅｒ、（配列表５）。この合成ディリーターをＴＭＤと称した。このようにして作成したディリーターＴＭＤを用い、メソッド イン エンザイモロジー（Ｍｅｔｈｏｄ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ）、第１００巻、４６８頁、（１９８３年）、アカデミックプレス（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ）に記載の方法に従って部位特異的変異の手法で前記の如く得られた組換え体プラスミドＭ１３ｍｐ１９ＴＭＪ１３の１７７塩基からなる部分の削除を行った。即ち、２５ｐｍｏｌのディリーターＴＭＤおよび１０ｐｍｏｌのＭ１３プライマーＭ３（ユニバーサルプライマー、宝酒造製、カタログ番号３８３１）の５′末端をＴ4 キナーゼを用いてリン酸化した後、０．５ｐｍｏｌの組換え体プラスミドＭ１３ｍｐ１９ＴＭＪ３のシングルストランドＤＮＡを加え、９５℃で５分間加熱後、室温にまで冷却した。
【００４９】
次いで５単位のＥ．ｃｏｌｉ ＤＮＡポリメラーゼＩ（Ｋｌｅｎｏｗ Ｆｒａｇｍｅｎｔ）、及び１０単位のＴ4 ＤＮＡリガーゼを混合物に加えて３７℃で３０分間インキュベートして混合物中に組換え体プラスミドを生成させた。得られた混合物を大腸菌（Ｅ．ｃｏｌｉ）ＪＭ１０５（ファルマシア社製、スウェーデン、カタログ番号２７−１５５０）に加えた。それによって、この大腸菌を組換え体プラスミドでトランスフェクションした。３７℃で一夜培養して生じた寒天培地上のプラークをニトロセルロースフィルターに移しとり、８０℃で２時間加熱後、プレハイブリダイゼーションを行った。プレハイブリダイゼーションは６×ＳＥＴ〔０．９Ｍ ＮａＣｌ、１８０ｍＭトリス緩衝液（ｐＨ８．０）、６ｍＭ ＥＤＴＡ〕、５×Ｄｅｎｈａｒｔｓ’〔０．１％（ｗ／ｖ）フィコール（Ｆｉｃｏｌｌ）、０．１％（ｗ／ｖ）ポリビニルピロリドン、０．１％（ｗ／ｖ）、ウシ血清アルブミン（ＢＳＡ）〕、０．１％ＳＤＳ，１００μｇ／ｍｌ変性サケ精子ＤＮＡを含む溶液中で５５℃、２時間加温することにより実施した。
【００５０】
次いで、上記の溶液中の変性サケ精子ＤＮＡのかわりに³²ＰでラベルしたＴＭＤを加えた溶液を用いてハイブリダイゼーション反応を５５℃、２時間実施した。次いで６×ＳＳＣ（０．９Ｍ食塩、０．０９Ｍクエン酸三ナトリウムの水溶液）を用いてニトロセルロースフィルターを洗浄した。洗浄は室温で、５分間、２回洗った後、５５℃、６５℃、７５℃、と段階的に温度を上げていって、それぞれ５分間２回ずつ洗った。Ｘ線フィルムＸＡＲ−５（イーストマン コダック社製、米国）を得られたニトロセルロースフィルターに密着させて−８０℃、一夜露出させたところ、Ｘ線フイルム上に強く露光した黒いスポットが数１０個検出された。各スポットは組換え体プラスミドで感染したクローンに対応するものである。そのうち、６クローンを選択し、各クローンの組換え体プラスミドを単離して制限酵素解析、及び塩基配列の解析を行ったところ、これらのクローンの保有する組換え体プラスミドは制限部位と塩基配列がそれぞれ同一であることがわかった。得られた組換え体プラスミドをＭ１３ＴＭＤ１２３と称した。さらにこの組換え体プラスミドＭ１３ＴＭＤ１２３は、配列表５に示した開始コドン（ＡＴＧ）から始まる５１６個のアミノ酸からなるヒトトロンボモジュリンをコードする塩基配列を含む塩基配列を含有するＤＮＡ断片を有することがわかった。この組換え体プラスミドＭ１３ＴＭＤ１２３に含まれるＤＮＡ断片をＴＭＤ１２３と称した。
【００５１】
この組換え体プラスミドＭ１３ＴＭＤ１２３をＨｉｎｄＩＩＩおよびＢａｍＨＩで完全消化してＴＭＤ１２３の約１９００ｂｐのＤＮＡ断片を単離した。一方、プラスミドｐＳＶ２ＤＨＦＲ（ＡＴＣＣ ３７１４６）をＨｉｎｄＩＩＩ及びＢｇｌIIで完全消化してベクター断片を得た。このベクター断片と、先のＤＮＡ断片ＴＭＤ１２３とをＴ4 ＤＮＡリガーゼを用いて継ぎあわせ、プラスミドｐＳＶ２ＴＭＤ１２３を得た。
【００５２】
（２）プラスミドｐＳＶ２ＴＭＤ１２３とプラスミドｐＳＶ２−ＤＨＦＲによるＣＨＯ細胞の形質転換
上記の実施例１−１で得られたプラスミドｐＳＶ２ＴＭＤ１２３、約４０μｇとプラスミドｐＳＶ２ＤＨＦＲ、約３．５μｇを混合し、エタノール沈殿した。沈殿物を風乾後、４５０μｌのＴＥ（ｐＨ７．９、１ｍＭトリス塩酸緩衝液、０．１ｍＭ ＥＤＴＡ）に溶解し、５０μｌの２．５Ｍ ＣａＣｌ2 を滴下し、５００μｌの２×ＨＢＳ（５０ｍＭ ＨＥＰＥＳ、２８０ｍＭ ＮａＣｌ、１．５ｍＭ Ｎａ2 ＨＰＯ4 、ｐＨ７．１２）を加えて混合した。次いで氷上で１０分間放置した後、１ｍｌの１０％（ｖ／ｖ）ウシ胎児血清（以下”ＦＣＳ”と略する）及び１ｖ／ｖ％ペニシリン−ストレプトマイシン（米国、フローラボラトリー社製、カタログ番号１６−７００−４９）を含有するＨａｍ’ｓＦ−１２培地（米国、フローラボラトリー社製、カタログ番号１０−４２１−２０）に懸濁した。一方、１０％（ｖ／ｖ）ＦＣＳ及び１％（ｖ／ｖ）ペニシリン−ストレプトマイシンを含有するＨａｍ’ｓＦ−１２培地に直径６ｃｍの組織培養用プレートにプレート１枚当たり細胞数約５×１０⁵ 程度播種したＣＨＯ−ＤＸＢ１１株（米国コロンビア大、Ｄｒ．Ｌ．Ａ．Ｃｈａｓｉｎより分与）を１夜培養し、培地を新鮮な培地に交換し、更に４時間培養した。このＣＨＯ−ＤＸＢ１１株に前述のＣａＣｌ2 を滴下したプラスミドＤＮＡ溶液を重層し、３７℃で約８時間培養した。５ｍｌのＰＢＳ（−）（米国、フローラボラトリー社製、カタログ番号２８−１０３−０５）を用いて３回洗浄し、さらに、５ｍｌの前述の培地で洗浄後、新鮮な培地を５ｍｌ加えて３日間さらに培養した。
【００５３】
（３）プラスミドｐＳＶ２ＴＭＤ１２３とプラスミドｐＳＶ２−ＤＨＦＲにより形質転換したＣＨＯ細胞からＤＨＦＲ産生株の選択
上記実施例１−（２）で形質転換した細胞を０．２５％トリプシン、０．０２％ＥＤＴＡ溶液を用いてはがし、直径１０ｃｍの組織培養プレート４枚に広げて培養した。２４時間後、培地を選択培地に交換した。選択培地の組成は１０％（ｖ／ｖ）透析ＦＣＳ、１５０μｇ／ｍｌプロリン、１％（ｖ／ｖ）ペニシリン−ストレプトマイシンを含むＤＭＥＭ（米国、フローラボラトリー社製、カタログ番号１７−１０１−２２）である。３〜４日おきに培地交換を行いながら約２週間培養して、トランスフォームした細胞をクローニングし、５９株のＤＨＦＲ産生株を得た。
【００５４】
（４）ＤＨＦＲ産生株からのＴＭ生産株の選択
前述の実施例３で得られたＤＨＦＲ産生株の各クローンを９６穴組織培養用プレートで培養し、コンフルエントになった時点で培地を１５０μｇ／ｍｌプロリンを含むＤＭＥＭ １００μｌに交換した。その後２４時間培養し、その培養上清を回収した。このうち５０μｌを９６穴の平底ＥＬＩＳＡ用プレート（米国、ＤＹＮＡＴＥＣＨ社製）で４℃で一晩インキュベートした。この培養液中のＴＭ量は、抗ヒトトロンボモジュリンポリクローナル抗体を用いたエンザイムインムノアッセイ（以下”ＥＬＩＳＡ”と略す）により定量した。詳しくは以下に述べる。
【００５５】
（４）−ａ 免疫源の精製
抗ヒトトロンボモジュリンポリクローナル抗体の免疫源は、以下のようにしてヒト肺より抽出して得た。公立病院より提供されたヒト肺標本約８００ｇを挟みで約１ｃｍ四方程度の大きさに細切りした後、得られた組織片に１ｍＭのＤＦＰ（Ｄｉｉｓｏｐｒｏｐｙｌ ｆｌｕｏｒｏｐｈｏｓｐｈａｔｅ）を含む４℃に冷却した５００ｍｌの生理食塩水を加え、ワーリングブレンダーとしてＡｃｅ Ｈｏｍｏｇｅｎｉｚｅｒ ＡＭ−１型（日本精器会社製、日本）を用いて４℃で５分間、ホモジナイズした。ホモジナイズ後、混合物を氷中で５分間冷却した。次に混合物を更に４℃で５分間、ホモジナイズし氷中で５分間冷却した。上記のホモジナイズ及び冷却操作を更に３回くり返した。得られたホモジェネートを１２０００ｇで４℃において３０分間遠心分離にかけて上澄液とペレットに分け、ペレットを集める。これに０．５％（ｖ／ｖ）トリトンＸ−１００、０．２５Ｍ庶糖、１ｍＭベンズアミジン塩酸、０．５ｍＭ ＣａＣｌ2 を含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）１００ｍｌに懸濁し、ワーリングブレンダーを用いて４℃で５分間、５回ホモジナイズして細胞抽出物を得た。
【００５６】
得られた抽出物を３５，０００ｇ、１０℃で６０分間遠心分離にかけて上澄液を集めた。Ｎ．Ｌ．Ｅｓｍｏｎら〔Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２５７巻、８５９頁（１９８２年）〕の方法に従って作成したＤＩＰ−トロンビン〔ジイソプロピルホスフォロトロンビン（ｄｉｉｓｏｐｒｏｐｙｌｐｈｏｓｐｈｏｒｏ−ｔｈｒｏｍｂｉｎ）を、Ｐ．Ｃｕａｔｒｅｃａｓａｓの方法〔Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２４５巻、３０５９頁（１９７０年）〕に従ってブロムシアン化したアガロースに結合させて、ＤＩＰ−トロンビン−アガロースを作成した。
【００５７】
次にＤＩＰ−トロンビン−アガロースを２．５ｃｍφ×１０ｃｍの大きさのカラムに充填してＤＩＰ−トロンビン−アガロースカラムを作成し、室温で０．１Ｍ ＮａＣｌ、０．５ｍＭ ＣａＣｌ2 、１ｍＭベンズアミジン塩酸、０．５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸ（半井科学薬品製、日本）を含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）でカラムを平衡化した。次いで、上記の抽出上澄液をカラムに供した。カラムを０．３Ｍ ＮａＣｌ、０．５ｍＭ ＣａＣｌ2 、１ｍＭベンズアミジン塩酸、０．５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で洗浄した後、１Ｍ ＮａＣｌ、０．１ｍＭ ＥＤＴＡ、１ｍＭベンズアミジン塩酸０．５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で溶出して２．０ｍｌずつフラクシヨンを集めた。溶出によって得られる各フラクションについてトロンビンによるプロテインＣ活性化を促進する活性を測定した。即ち、それぞれのフラクションを定量用希釈液（２０ｍＭトリス塩酸、１００ｍＭ ＮａＣｌ、０．１％ ルブロールＰＸ、０．１％アジ化ナトリウム、０．００１％ＢＳＡ、ｐＨ７．５）で希釈した。この液を５μｌ、トロンビン（米国、シグマ社、カタログ番号Ｔ−６７５９、２０ｎｇ／μｌ）３μｌ、１０×アッセイ緩衝液（１Ｍ ＮａＣｌ、３０ｍＭ ＣａＣｌ2 、１％牛血清アルブミン含有、０．５Ｍ トリス塩酸緩衝液、ｐＨ８．５）５μｌ、及び蒸留水２９．５μｌを混合し、３７℃５分間放置後、プロテインＣ（牛由来、０．２μｇ／μｌ）７．５μｌを加え、３７℃で３０分間反応させた。
【００５８】
反応は、ストップ液（１００ｍＭ ＮａＣｌ、０．３Ａ２８０アンチトロンビンＩＩＩ、１００μｇ／ｍｌヘパリン含有２０ｍＭトリス塩酸緩衝液、ｐＨ７．５）を６．２５μｌ加えることにより止めた。活性化プロテインＣの活性は、Ｂｏｃ−Ｌｅｕ−Ｓｅｒ−Ｔｈｒ−Ａｒｇ−ＭＣＡ（日本、ペプチド研究所、５ｍｇ／ｍｌ）１０μｌ、５Ｍ ＣｓＣｌ ５μｌ、基質反応緩衝液（１００ｍＭ ＮａＣｌ含有５０ｍＭトリス塩酸緩衝液、ｐＨ８．５）４９５μｌを加え、３７℃、２０分間反応させ、酢酸５５μｌを加え反応を止めた後、遊離したＡＭＣ（７−アミノ−７−メチル−クマリン）濃度を励起波長３８０ｎｍ，発光波長４４０ｎｍで蛍光分光光度計（島津製作所 ＲＦ−５４０）により測定した。得られた蛍光強度を既知濃度のＡＭＣの蛍光強度と比較して、遊離したＡＭＣ量を求めた。このＡＭＣ量から精製品を含まない水溶液を加えたときのＡＭＣ量を差し引いた値がサンプルのトロンビンによるプロテインＣ活性化を促進する活性の強さを表す。１分間に反応液１ｍｌあたり１ｎＭの活性化プロテインＣを生成する活性を１ｕとした。また、同時に島津製作所（日本）製スペクトロフォトメーターＵＶ−２４０を用いて各フラクションの波長２８０ｎｍにおける吸光度（Ａ２８０）を測定した。
【００５９】
プロテインＣ活性化を促進する活性のある画分を回収し、０．１Ｍ ＮａＣｌ、０．５ｍＭ ＣａＣｌ2 、０．０５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で透析した。得られた透析液を２回目のＤＩＰ−トロンビン−アガロースカラムクロマトグラフィーに供した。即ち、透析液を１．５ｃｍφ×１０ｃｍの大きさのＤＩＰ−トロンビン−アガロースカラムに供し、０．４Ｍ ＮａＣｌ、０．５ｍＭ ＣａＣｌ2 、０．１％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で洗浄後、さらに０．４Ｍ ＮａＣｌ、０．１ｍＭ ＥＤＴＡ、０．１％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で洗浄し、次いで１Ｍ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ、０．１％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で溶出した。
【００６０】
プロテインＣ活性化を促進する活性のある画分を回収し、さらに０．１Ｍ ＮａＣｌ、０．０５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で透析した。得られた透析液を３回目のＤＩＰ−トロンビン−アガロースカラムクロマトグラフィーに供した。カラムの大きさ、洗浄条件および溶出条件は２回目のＤＩＰ−トロンビン−アガロースカラムクロマトグラフィーの条件と全く同じ条件で行なった。なお、溶出して得られるフラクションは２ｍｌずつ集めた。プロテインＣ活性化を促進する活性のある画分を回収し、０．１Ｍ ＮａＣｌ、０．０５％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で透析した後、０．９ｃｍφ×８ｃｍの大きさの４回目のＤＩＰ−トロンビン−アガロースカラムクロマトグラフィーに供した。０．３５Ｍ ＮａＣｌ、０．５ｍＭ ＣａＣｌ2 、０．１％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で洗浄後、１Ｍ ＮａＣｌ、０．５ｍＭ ＥＤＴＡ、０．１％（ｖ／ｖ）Ｌｕｂｒｏｌ ＰＸを含む０．０２Ｍトリス塩酸緩衝液（ｐＨ７．５）で溶出した。溶出して得られたフラクションは１．９ｍｌずつ集めた。この第４回目のＤＩＰ−トロンビン−アガロースカラムクロマトグラフィーのプロテインＣ活性化を促進する活性のある画分を回収し、免疫源とした。
【００６１】
このようにして精製されたフラクションの吸光度から、得られた精製品の分子吸光係数を一般的な蛋白質の分子吸光係数にならない１０．０（Ｅ１％１ｃｍ・２８０ｎｍ＝１０．０）と規定してそれに基づき本精製品の量を計算したところ約５００μｇであった。なお、得られた精製画分をポリアクリルアミドゲル濃度５〜１０％のグラジェントを用いるＳＤＳ−ポリアクリルアミドゲル電気泳動を５０Ｖの電圧で２時間行ない、銀染色によってバンドを観察したところ単一バンドのみ確認された。さらにこの精製画分には、トロンビンによるプロテインＣ活性化を促進する効果が認められた。
【００６２】
（４）−ｂ 抗ヒトトロンボモジュリンポリクローナル抗体の作製
抗ヒトトロンボモジュリンポリクローナル抗体（ウサギ抗体）は、前述の免疫源を用いて、成書〔Ｌ．ｈｕｄｓｏｎｅｔ、Ｐｒａｃｔｉｃａｌ Ｉｍｍｕｎｏｌｏｇｙ、９頁（１９７６年）、Ｂｌａｃｋｗｅｌｌ ＳｃｉｅｎｔｉｆｉｃＰｕｂｌｉｃａｔｉｏｎｓ〕に従って作製した。
【００６３】
この抗体が、上記免疫源と反応することを以下の様にして確認した。すなわち、実施例１−（４）−ａで得た免疫源（精製ヒトトロンボモジュリン蛋白）の約１０ｎｇをニトロセルロースのフィルターにスポットする。よく風乾した後、この抗体を一次抗体としてニトロセルロースフィルター上のタンパクと反応させ、次いでヤギで調製したビオチン化抗ウサギＩｇＧ（ザイメットラボラトリー社製、米国、カタログ番号６２−１８４０）を二次抗体として反応させた後、アビジン・ビオチン化した西洋ワサビ由来パーオキシダーゼ（アマシャムジャパン社製、日本、カタログ番号ＲＰＮ．１０５１）を作用させる方法で発色させると黒褐色のスポットを与えた。
【００６４】
（４）−ｃ ＥＬＩＳＡ
培養液の入った９６穴の平底ＥＬＩＳＡ用プレートの穴を２００μｌのＰＢＳ（日本、日水製）で５回洗浄する。その後、０．５％ＢＳＡ含有ＰＢＳを１００μｌ／穴となるように加え、室温で２時間静置する。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、上記実施例１−４−（ｂ）で作製した抗ヒトＴＭポリクローナル抗体を１％ＢＳＡ含有ＰＢＳで１０００倍希釈し、１００μｌ／穴で各穴に加え、一次抗体として反応させる。その後、プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、酵素標識された抗ラビットＩｇＧ抗体（カッペル社、カタログ番号５０−７４１５−１６）を１％ＢＳＡ含有ＰＢＳで１０００倍希釈し、１００μｌ／穴で各穴に加え、室温で１時間反応させた。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、発色液（３０ｍｇオルトフェニレンジアミンを２０ｍｌクエン酸燐酸緩衝液に３０％過酸化水素液を７０μｌ添加した液）１００μｌ添加し、適度な発色が得られた時点で５０μｌの４．１Ｍ硫酸液を添加し反応を止め、４９２ｎｍの測定波長で測定し、ヒトトロンボモジュリン生産株を選択した。
【００６５】
（５）プラスミドｐＳＶ２ＴＭＤ１２３及びプラスミドｐＳＶ２ＤＨＦＲにより形質転換されたＣＨＯ細胞株のＭＴＸによるＴＭ生産性の増幅
上述の実施例１−（４）の結果より得られたＴＭの高生産株を選別し、６穴の組織培養用プレートでコンフルエントになるまで培養し、７／１０、３／１０の割合で直径１０ｃｍの組織培養用プレートに継代した。７／１０で継代したプレートは、１５０μｇ／ｍｌプロリンと１０％透析ＦＣＳを含むＤＭＥＭでコンフルエントになるまで培養した後、生産性測定培地（１５０μｇ／ｍｌプロリン含有ＤＭＥＭ）１０ｍｌに交換し、２４時間後に培養液を回収し、ＭＴＸ無添加細胞生産液とした。
【００６６】
また、３／１０で継代したプレートは、翌日２０ｎＭのＭＴＸ含有選択培地に交換し培養を続けた。コンフルエントになった後、生産性測定培地１０ｍｌに交換し、２４時間後に培養液を回収し、２０ｎＭ ＭＴＸ選択細胞生産液とした。この回収された生産液は、以下に述べるサンドイッチ法ＥＬＩＳＡで生産液中のＴＭの定量を行い、細胞１×１０⁶ 個が２４時間当たりに生産するＴＭの生産量を評価した。
【００６７】
抗ヒトトロンボモジュリンモノクローナル抗体の作製は、Ｍａｒｕｙａｍａらの方法［Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６０，１５４３２（１９８５）］に従った。即ち、実施例１−（４）−ａで精製したヒト肺由来トロンボモジュリンを抗原とし、Ｂａｌｂ／Ｃマウスに数回免疫後、マウスの脾臓細胞液を調製し、適当なラインからのマウス骨髄細胞種とポリエチレングリコール等の融合促進剤の使用により、細胞融合させる。
【００６８】
細胞融合に用いるマウス骨髄細胞は、例えば、Ｐ３−Ｘ６３−Ａｇ８−Ｕ１細胞（Ｐ３−Ｕ１）［Ｙｅｌｔｏｎら、Ｃｕｒｒｅｎｔ．Ｔｏｐｉｃｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｏｇｙ ａｎｄ Ｉｍｍｕｎｏｌｏｇｙ，８１，１（１９７８）］等が用いられる。融合した細胞を適当な選択培地、例えば、ＨＡＴ（ヒポキサンチン・アミノプテリン・チミジン）培地を用いて選択する。このようにしてハイブリドーマ細胞が検出された後、その培養上清を採取し、トロンボモジュリンに対する抗体についてトロンボモジュリンを固相抗原としたＥＬＩＳＡ（酵素免疫定量法）によりスクリーニングする。トロンボモジュリンに対する抗体を産生するハイブリドーマ細胞を適当な方法、例えば限外希釈法によりクローン化する。その結果、２種の抗ヒトトロンボモジュリンモノクローナル抗体が得られ、それぞれ抗ＴＭモノクローナル抗体Ａ及び、Ｂと命名した。
【００６９】
上述の抗ＴＭモノクローナル抗体Ａの精製品を２００μｇ／ｍｌとなるように０．１Ｍ重炭酸ナトリウム緩衝液（ｐＨ９．２）で希釈し、５０μｌ／穴となるように９６穴の平底ＥＬＩＳＡ用プレートに分注する。室温で２時間静置後、プレートの穴を２００μｌのＰＢＳ（日本、日水製）で５回洗浄する。その後、０．５％ＢＳＡ含有ＰＢＳを１００μｌ／穴となるように加え、室温で２時間静置する。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、回収した培養液を段階的に希釈して５０μｌ／穴となるように添加し、室温で２時間静置する。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、ビオチン標識した抗ＴＭモノクローナル抗体Ｂを２０μｇ／ｍｌとなるように、１％ＢＳＡ含有ＰＢＳで１０００倍希釈し、１００μｌ／穴で加え、室温で１時間反応させた。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、アビジンＤ（米国、Ｖｅｃｔｏｒ社製，カタログ番号Ａ−２００４）を１％ＢＳＡ含有ＰＢＳで５０００倍希釈し、１００μｌ／穴で各穴に加え、室温で３０分反応させた。プレートの各穴を０．０５％Ｔｗｅｅｎ８０含有ＰＢＳで５回洗浄後、発色液（３０ｍｇオルトフェニレンジアミンを２０ｍｌクエン酸燐酸緩衝液に３０％過酸化水素液を７０μｌ添加した液）１００μｌ添加し、適度な発色が得られた時点で５０μｌの４．１Ｍ硫酸液を添加し反応を止め、４９２ｎｍの測定波長で測定した。なお、標準品には、上記実施例１−（４）−ａで精製したヒト肺トロンボモジュリンを用いた。
【００７０】
ＭＴＸ無添加細胞生産液と２０ｎＭ ＭＴＸ選択細胞生産液のサンドイッチ法ＥＬＩＳＡの結果を比較して、２０ｎＭ ＭＴＸ選択細胞生産液の方が高生産であった株を選択した。これらの株は、さらに、１×１０⁴ 、５×１０⁴ 細胞／プレートの細胞密度で直径１０ｃｍの組織培養用プレートに継代し、翌日２００ｎＭ ＭＴＸ含有選択培地に交換し培養を続けた。出現してきた２００ｎＭ ＭＴＸ耐性株をクローニングし、９６穴の組織培養用プレートに継代しコンフルエントになるまで培養した後、１５０μｇ／ｍｌプロリン含有ＤＭＥＭ１０ｍｌに交換し、２４時間後に培養液を回収した。回収した培養液は、上記のサンドイッチＥＬＩＳＡでＴＭの生産性を評価した。高生産株は上記で述べたように６穴の組織培養用プレートに継代しさらに直径１０ｃｍの組織培養用プレートに継代し、２４時間後培養上清を回収し、ヒトトロンボモジュリン生産性ををサンドイッチ法ＥＬＩＳＡで定量した。
【００７１】
高生産株を選択し、上記と同様の操作を繰り返して、ＭＴＸの濃度を、２００ｎＭから、２μＭ、２０μＭ、４０μＭ、１００μＭ、５００μＭと上げていった。こうしてＭＴＸによる遺伝子増幅によりＴＭの生産性が上昇した株を取得した。ＭＴＸ濃度の上昇により効率的にヒトトロンボモジュリンの生産性が増幅され、４０μＭ ＭＴＸ濃度で、約１７ｐｇ／細胞・日、１００μＭ ＭＴＸ濃度で、約２０ｐｇ／細胞・日、５００μＭで４７ｐｇ／細胞・日という高生産の細胞株が樹立された。
【００７２】
（６）ＴＭ生産安定性の検討
実施例１−（５）で得られた１００μＭ ＭＴＸ耐性株について、各耐性濃度のＭＴＸ含有非選択培地とＭＴＸを含有しない非選択培地（１０％（ｖ／ｖ）ＦＣＳ、１５０μｇ／ｍｌプロリン含有ＤＭＥＭ）で長期間継代培養を行い、サンドイッチ法ＥＬＩＳＡで培養終了後細胞当りのＴＭ生産量を評価した。２０μＭＭＴＸ耐性株については、１カ月間の継代培養と２カ月間の継代培養を行った。１００μＭ ＭＴＸ耐性株は、ＭＴＸ無しの培養を２カ月行っても、生産性は維持され、安定なＴＭ生産株が得られた。
【００７３】
（７）浮遊培養馴化株の取得
実施例１−（６）で得られた１００μＭ ＭＴＸ耐性株について、１０％（ｖ／ｖ）ＦＣＳ、１５０μｇ／ｍｌプロリン含有ＤＭＥＭを用い５０ｍｌスケールの浮遊攪拌培養を行なった。１×１０⁵ ｃｅｌｌｓ／ｍｌで培養を開始し、１×１０⁶ ｃｅｌｌｓ／ｍｌの細胞密度に達するまで培養した。比生産速度の維持を確認しつつ同様の培養を５回繰り返した結果、浮遊培養に馴化したＴＭ生産株が得られた。
【００７４】
実施例２
浮遊培養繰り返しバッチ培養でのＤＭＥＭと本発明のＴＭ製造用培地の比較
本発明のＴＭ製造用培地の調製方法は、前述の成分表２の各成分（但し、水、血清そして増殖因子は除く）を混合し、所定量の脱イオン限外濾過水へ加え、更に硫酸カナマイシン（明治製菓製）、タイロシン（三樂製）を加え溶解後、さらに牛血清（ハイクロン）を４％（ｖ／ｖ）添加し、濃塩酸（和光純薬）でｐＨを７．１に調製した後、０．２２μｍのフィルター（ミリポア製）で濾過し、４℃保存した（以下、本発明のＴＭ製造用培地の名称をＨＭ−０４培地と略することがある）。また血清を添加したＤＭＥＭの調製方法は、粉末培地のＤＭＥＭ（ライフテック社）を所定量の脱イオン限外濾過水へ加え、更に重曹（和光純薬）、硫酸カナマイシン（明治製菓製）、タイロシン（三樂製）を加え溶解後、牛血清（ハイクロン）を４％（ｖ／ｖ）添加し濃塩酸（和光純薬）でｐＨを７．１に調製した後、０．２２μｍのフィルター（ミリポア製）で濾過し、４℃保存した。何れの培地も、使用直前に３７℃水浴恒温槽で３７℃に加温し使用した。
【００７５】
培養の開始では２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地５０ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。
【００７６】
培養条件は、３７℃恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）、溶存酸素濃度（ＤＯ）５０％制御（ＤＯ電極（エイブル社）、ＤＯコントローラー（東亜化学））、通気流量：空気１００ｍｌ／分、二酸化炭素ガス１０ｍｌ／分、ＤＯ制御用酸素ガス３００ｍｌ／分 で培養を実施した。培養は浮遊生細胞密度が平衡に達するまで行なった。
【００７７】
培養中の培地交換は５日目、９日目に行なった。また、９日目以降は毎日行なった。培地交換は、培養液全量を５０ｍｌ遠心チューブ（ファルコン社）に入れ遠心分離（２０００ｒｐｍ、２分）し、上清除去後、予め３７℃に加温したそれぞれの５０ｍｌ新鮮培地で懸濁し培養容器に移し培養を再開した。遠心除去した培養上清は−２０℃保存した。この培養上清から一定量をサンプリングした。また別に６日目〜８日目の培養液より、毎日一定量をサンプリングし、遠心除去して得た培養上清からも一定量をサンプリングした。これらのサンプリングした培養上清に含まれるＴＭ濃度は、実施例１−（４）−ａ記載の方法（トロンビンによるプロテインＣ活性化を促進する活性の測定方法）により測定した。なお、ＴＭの比活性を８万Ｕ／ｍｇとして換算した。
【００７８】
浮遊生細胞密度の測定は、サンプリングした培養液と等量のトリパンブルー染色液（フロ−）を混合し、血球計算盤を用い顕微鏡で非染色細胞を生細胞として測定し、浮遊生細胞密度を測定した。
浮遊生細胞密度（×１０⁵ ｃｅｌｌｓ／ｍｌ）を縦軸に、培養日数（日）を横軸にプロットしたものが、図１である。この結果によれば、浮遊生細胞密度の推移は、培養初期の低密度での増殖速度はＤＭＥＭ、ＨＭ−０４培地ともに同程度であったが、約７×１０⁵ ｃｅｌｌｓ／ｍｌから増殖速度に差が現れ、ＤＭＥＭは最終到達密度が約４５×１０⁵ ｃｅｌｌｓ／ｍｌであったが、ＨＭ−０４培地は約１００×１０⁵ ｃｅｌｌｓ／ｍｌまで増殖した。
【００７９】
交換した培地の容量とＴＭ濃度を考慮して、各培養日数におけるＴＭの累積生産量（ｍｇ）を算出し、縦軸に、各培養日数（日）を横軸にプロットし、図２を得た。
図２によれば、培養開始後６日目から２６日目までの２０日間の累積生産性は、ＨＭ−０４培地を用いた培養法がＤＭＥＭを用いた培養法に比べ約３倍高い値を得た。したがって、本発明の培地によれば、ＴＭの大量生産が容易であり、好ましいものであることが確認された。
【００８０】
各培養日数におけるＴＭの１日当りの生産量を、浮遊生細胞密度で割って算出された比生産速度（ｐｇ／ｃｅｌｌ／ｄａｙ）を、縦軸に、各培養日数（日）を横軸にプロットし、図３を得た。
図３によれば、比生産速度の経時変化は、ＨＭ−０４培地を用いた培養方法では比較的安定していたが、ＤＭＥＭを用いた培養方法では経時的に低下した。したがって、本発明の培地によれば、長期生産が可能であり、好ましいものであることが確認された。
【００８１】
各培養日数における比生産速度（ｐｇ／ｃｅｌｌ／ｄａｙ）を、縦軸に、各培養日数（日）における浮遊生細胞密度（×１０⁵ ｃｅｌｌｓ／ｍｌ）を横軸にプロットし、図４を得た。
図４によれば、浮遊生細胞密度に対する比生産速度の変化は、ＨＭ−０４培地を用いた培養ではなかったが、ＤＭＥＭを用いた培養では浮遊生細胞密度の増加により低下した。したがって、本発明の培地によれば高密度培養においても生産性が変化しない好ましいものであることが確認された。
【００８２】
実施例３
浮遊培養での繰り返しバッチ培養
培地作製は、実施例２と同様に行なった。但し血清添加濃度は、ＴＭ生産細胞を増殖せしめる培養（以下、増殖培養）に用いる培地では１０％（ｖ／ｖ）添加、ＴＭを製造するための培養（以下、生産培養とも略す）に用いる培地では２％（ｖ／ｖ）添加し、作製した。
【００８３】
増殖培養の開始では、２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地１００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。増殖培養は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。増殖培養は浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで継代した。継代の培養スケールは１００ｍｌ、５００ｍｌ、２Ｌ、８Ｌと順次拡大した。継代では継代後の培養スケールから継代前の培養スケールを差し引いた値と同じ量の新鮮培地を加え拡大した。
【００８４】
培養スケール８Ｌでの増殖培養で浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところでＤＯ制御を開始した。制御条件はＤＯ５０％制御（ＤＯ電極（エイブル社）、ＤＯコントローラー（東亜化学））、通気流量：空気３００ｍｌ／分、二酸化炭素ガス３０ｍｌ／分、ＤＯ制御用酸素ガス９００ｍｌ／分で行った。
【００８５】
培養スケール８Ｌでの増殖培養での培地交換は、５、９、１１日目に、連続遠心分離機（アルファ−ラバル社、製品名ＣＣ−１００）を用い培養液の遠心分離を行ない予め３７℃に加温した新鮮培地へ交換した。
培養スケール８Ｌでの増殖培養の１２日目から生産培養を開始した。培地交換は毎日、連続遠心分離機（アルファ−ラバル社、製品名ＣＣ−１００）を用い培養液の遠心分離を行ない上清を取得し、細胞は予め３７℃に加温した新鮮培地と懸濁し生産培養を開始した。遠心分離した上清のうち８００ｍｌをサンプリングし−２０℃で凍結保存した。生産培養の条件は、３７℃恒温室内、撹拌１００ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）、溶存酸素濃度（ＤＯ）５０％制御（ＤＯ電極（エイブル社）、ＤＯコントローラー（東亜化学））、通気流量：空気１００ｍｌ／分、二酸化炭素ガス１０ｍｌ／分、ＤＯ制御用酸素ガス１０００ｍｌ／分で培養を２０日間行なった。浮遊生細胞密度は実施例２と同様に測定した。また、生産培養の上清中のＴＭ濃度は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
【００８６】
生産培養結果は、平均生細胞密度３．１×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は２７ｍｇ／Ｌ／ｄａｙであった。
【００８７】
実施例４
浮遊培養でのバッチ培養
培地作製は実施例２と同様に行なった。但し血清添加濃度は１０％（ｖ／ｖ）で作製した。
細胞は実施例３の８Ｌスケールの増殖培養５日目から培養液２０ｍｌをサンプリングし、新鮮培地８０ｍｌに懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。培養は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、培養液全量を遠心分離（２０００ｒｐｍ、２分、国産化学）し、５００ｍｌの新鮮培地に懸濁し、スピナーフラスコ（カルスター５００、柴田ハリオ社製）に移し、３７℃１０％炭酸ガス恒温室内、撹拌１２０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）でバッチ培養を開始し生産培養とした。浮遊生細胞密度は実施例２と同様に測定した。また、生産培養の上清中のＴＭ濃度は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
【００８８】
生産培養は８日間培養し、到達生細胞密度１．８×１０⁶ ｃｅｌｌｓ／ｍｌで、累積生産性は３５ｍｇ／Ｌであった。
【００８９】
実施例５
浮遊培養での連続培養
培地作製は、実施例２と同様に行なった。但し血清添加濃度は増殖培養用培地では８％（ｖ／ｖ）、生産培養用培地では４％（ｖ／ｖ）で調製した。
増殖培養の開始では、２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地１００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。増殖培養は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。増殖培養は浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで継代した。継代の培養スケールは１００ｍｌ、５００ｍｌ、１．５Ｌと順次拡大した。継代では継代後の培養スケールから継代前の培養スケールを差し引いた値と同じ量の新鮮培地を加え拡大した。
【００９０】
培養スケール１．５Ｌでの増殖培養で浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところでＤＯ制御を開始した。制御条件はＤＯ５０％制御（ＤＯ電極（エイブル社）、ＤＯコントローラー（東亜化学））、通気流量：空気３００ｍｌ／分、二酸化炭素ガス３０ｍｌ／分、ＤＯ制御用酸素ガス９００ｍｌ／分で行なった。
【００９１】
培養スケール１．５Ｌでの増殖培養での連続培地交換は、分離モジュールを用いた連続濾過で実施した。
培養槽外に設置した循環ポンプ（ハワード社）で培養液を循環させ、その循環ライン中に培養上清分離モジュール（旭化成工業）を設置し、ペリスターポンプ（ギルソン社）でモジュール２次側を陰圧にし定量濾過（濾過速度は１．５Ｌ／日に設定）を行ない、細胞を分離した培養上清を抜きだした。抜きだした培養上清は４℃に冷却保存し、その中から毎日５０ｍｌずつサンプリングし−２０℃凍結保存した。新鮮培地の添加は抜きだした培養上清と等量を培養槽内へペリスターポンプにより定量移送した。
【００９２】
２０×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、増殖培地を生産培地に切り換え、培養スケール１．５Ｌでの増殖培養での連続培地交換と同様な方法で生産培養を開始し、２０日間生産培養を行なった。但し、通気流量：空気１００ｍｌ／分、二酸化炭素ガス１０ｍｌ／分、ＤＯ制御用酸素ガス１０００ｍｌ／分、撹拌１２０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）。浮遊生細胞密度は実施例２と同様に測定した。また、生産培養の上清中のＴＭ濃度はトロンビンによる実施例１−（４）−ａ記載のプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
【００９３】
生産培養結果は、平均生細胞密度１２．５×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は７６ｍｇ／Ｌ／ｄａｙであった。
【００９４】
実施例６
浮遊培養での連続培養
培地作製は、実施例２と同様に行なった。但し血清添加濃度は増殖培養用培地では８％（ｖ／ｖ）、生産培養用培地では４％（ｖ／ｖ）で作製した。
増殖培養の開始では、２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地１００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。増殖培養は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。増殖培養は浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで継代した。継代の培養スケールは１００ｍｌ、５００ｍｌ、１．５Ｌと順次拡大した。継代では継代後の培養スケールから継代前の培養スケールを差し引いた値と同じ量の新鮮培地を加え拡大した。
【００９５】
培養スケール１．５Ｌでの増殖培養で浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、培養液全量をＢＩＯＳＴＡＴ−Ｍ（ビーブラウン社製培養装置）に移送した。
培養スケール１．５Ｌでの増殖培養での連続培地交換は、槽内に設置された分離チューブ（親水性多孔質テフロン、ビーブラウン社）を用いた連続濾過で実施した。槽内分離チューブをペリスターポンプ（ギルソン社）で陰圧にし定量濾過（濾過速度は１．５Ｌ／日に設定）を行ない、分離チューブにより培養上清を抜きだした。培養上清は４℃に冷却保存し、その中から毎日５０ｍｌずつサンプリングし−２０℃凍結保存した。新鮮培地の添加は抜きだした培養上清と等量を培養槽内へペリスターポンプにより定量移送した。
【００９６】
増殖培養条件は、槽内温度３７℃、ＤＯ＝５０％、ｐＨ＝７．１に制御した。
２０×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、増殖培地を生産培地に切り換え、培養スケール１．５Ｌでの増殖培養での連続培地交換と同様な方法で生産培養を開始し、２０日間培養を行なった。
浮遊生細胞密度は実施例２と同様に測定した。
【００９７】
また、生産培養の上清中のＴＭ濃度は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
生産培養結果は、平均生細胞密度１２．０×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は７８ｍｇ／Ｌ／ｄａｙであった。
【００９８】
実施例７
浮遊培養での連続培養培養
（１）血清培地
培地作製は、実施例２と同様に行なった。但し血清添加濃度は増殖培養用培地では８％（ｖ／ｖ）、生産培養用培地では４％（ｖ／ｖ）で作製した。
【００９９】
増殖培養の開始では、２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地１００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。
【０１００】
増殖培養条件は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。増殖培養は浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで継代した。継代の培養スケールは１００ｍｌ、５００ｍｌ、１．５Ｌと順次拡大した。継代では継代後の培養スケールから継代前の培養スケールを差し引いた値と同じ量の新鮮培地を加え拡大した。
【０１０１】
培養スケール１．５Ｌでの増殖培養で浮遊生細胞密度が５×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、培養液全量をＢＩＯＳＴＡＴ−Ｍ（ビーブラウン社製培養装置）に移送した。
培養スケール１．５Ｌでの増殖培養での連続培地交換は、槽内に設置された分離ステンレスメッシュフィルター（孔径２０μｍ、ビーブラウン社）を用いた連続濾過で実施した。
【０１０２】
槽内分離ステンレスメッシュフィルター内に差し込んだ抜きだしパイプをペリスターポンプ（ギルソン社）で陰圧にし定量濾過（濾過速度は１．５Ｌ／日に設定）を行ない、ステンレスメッシュフィルターにより分離した培養上清を抜きだした。培養上清は４℃に冷却保存し、その中から毎日５０ｍｌずつサンプリングし−２０℃凍結保存した。新鮮培地の添加は抜きだした培養上清と等量を培養槽内へペリスターポンプにより定量移送した。
【０１０３】
増殖培養条件は、槽内温度３７℃、ＤＯ＝５０％、ｐＨ＝７．１に制御した。２０×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、増殖培地を生産培地に切り換え、培養スケール１．５Ｌでの増殖培養での連続培地交換と同様な方法で生産培養を開始し、２０日間培養した。
浮遊生細胞密度は実施例２と同様に測定した。
【０１０４】
また、生産培養の上清中のＴＭ濃度は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
生産培養結果は、平均生細胞密度１１．２×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は７６ｍｇ／Ｌ／ｄａｙであった。
【０１０５】
（２）増殖因子培地
実施例７−（１）の方法に従い培養を行った。但し生産培養用培地には血清を添加せず、増殖因子としてアルブミン３ｍｇ、インシュリン５ｍｇ、トランスフェリン５ｍｇ、エタノールアミン２μＭ、亜セレン酸ナトリウム２０ｎＭを添加し生産培養用培地を調製した。
【０１０６】
本細胞は完全に血清非依存性で無いためか、飽和細胞密度は実施例７−（１）の約半分の５×１０⁶ ｃｅｌｌｓ／ｍｌ程度であった。
生産培養結果は、平均生細胞密度４．８×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は４２ｍｇ／Ｌ／ｄａｙであった。
【０１０７】
実施例８
付着培養での繰り返しバッチ培養
培地作製は、実施例２と同様に行った。但し血清添加濃度は増殖培養用培地では１０％（ｖ／ｖ）、生産培養用培地では２％（ｖ／ｖ）で作製した。
増殖培養の開始では、２×１０⁷ ｃｅｌｌｓ／アンプルで凍結保存した細胞を、３７℃恒温槽で素早く融解し、それぞれの培地に懸濁し遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地１００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）に入れ培養を開始した。
【０１０８】
増殖培養条件は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。浮遊生細胞密度が２×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、培養液を遠心分離（２０００ｒｐｍ、２分、国産化学）した。遠心後、上清は吸引除去しそれぞれの新鮮培地２００ｍｌで懸濁し、培養容器（カルスター１００、柴田ハリオ社製）２基にそれぞれ１０ｍｌづつ入れた。そして直ちにそれぞれの培養器に９０ｍｌの新鮮培地に懸濁した３ｇ／Ｌの表面付着型のサイトデクス１（ファルマシア製）と多孔質性の内部付着型の旭化成マイクロキャリア（旭化成工業製）を添加した（サイトデクス１はファルマシアバイオテクの添付マニュアル、旭化成マイクロキャリアは旭化成工業の添付マニュアルに従い準備した）。
【０１０９】
付着担体を用いた１００ｍｌ撹拌増殖培養は、３７℃１０％炭酸ガス恒温室内、撹拌８０ｒｐｍ（マグネティックスターラー、柴田ハリオ社製）で実施した。
培地交換は、培養の撹拌を停止し付着担体が沈降後、上清をピペットで吸引除去し等量の新鮮培地を加え培養を再開した。培地交換は、５、８、１０日目実施した。
【０１１０】
２０×１０⁵ ｃｅｌｌｓ／ｍｌに達したところで、増殖培地を生産培地に切り換え、培地交換は同様な方法で毎日行ない、生産培養は２５日間行なった。培地交換で得た培養上清１００ｍｌは−２０℃で冷凍保存した。
浮遊生細胞密度は実施例２と同様に測定した。
担体付着細胞密度は、サンプリングした培養液の担体沈澱上清を除去し、除去した上清と等量の脱核染色液（クリスタルバイオレット、ＴＷＥＥＮ８０、クエン酸）を加え、脱核後、血球計算盤で核数を測定し、核密度を付着細胞密度とした。
【０１１１】
また、生産培養の上清中のＴＭ濃度は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により比活性を８万Ｕ／ｍｇとして算出した。
生産培養結果は、平均細胞密度はサイトデクス１使用の培養で２．５×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は２６ｍｇ／Ｌ／ｄａｙであり、また平均細胞密度は旭化成マイクロキャリア使用の培養で５．５×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は４６ｍｇ／Ｌ／ｄａｙであった。
【０１１２】
実施例９
強陰イオン交換樹脂カラムによる粗精製
実施例３で取得した−２０℃凍結培養上清１１Ｌを溶解し、０．２μｍのメンブレンフィルター（ミリポア社、ミリパック２０）で濾過した。
濾過した培養上清については、１５０ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で平衡化したＱ−Ｓｅｐｈａｒｏｓｅ（ファルマシア社）カラム（直径９０ｍｍ、高さ６．５ｃｍ）に供した。次に１８０ｍＭ ＮａＣｌを含む２０ｍＭ酢酸緩衝液で洗浄し、更に１８０ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で洗浄を行ない、３００ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりからの０．５カラムボリューム容量の溶出液を粗精製品として取得した。
【０１１３】
実施例１０
アフィニティーカラム（抗体Ｂ）による主精製
アフィニティーカラムは以下のように作製した。即ち、上記実施例１−（５）で得られた抗ＴＭモノクローナル抗体Ｂは、その抗体を産生するハイブリドーマを培養することにより得た培養上清、あるいは、ハイブリドーマを組織適合性動物、ヌードマウス等の腹腔内にて増殖させて得た腹水より、塩析、イオン交換クロマトグラフィー、プロテインＡカラム等の分離精製操作により精製した。次に、ファルマシア社のマニュアル（Ａｆｆｉｎｉｔｙ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ ｐｒｉｎｃｉｐｌｅｓ ＆ ｍｅｔｈｏｄｓ）に従い、精製抗ＴＭモノクローナル抗体Ｂを０．５Ｍ ＮａＣｌ含有０．１Ｍ ＮａＨＣＯ3 緩衝液（ｐＨ８．３）に溶解し、ＣＮＢｒ−ａｃｔｉｖａｔｅｄ Ｓｅｐｈａｒｏｓｅ ４Ｂ（ファルマシア社、５２−１１５３−００−ＡＩ）と接触反応させ、セファロース４Ｂに抗ＴＭモノクローナル（抗体Ｂ）をカップリングし、抗ＴＭモノクローナル（抗体Ｂ）結合Ｓｅｐｈａｒｏｓｅ ４Ｂを作製した。次いでこの抗ＴＭモノクローナル（抗体Ｂ）結合Ｓｅｐｈａｒｏｓｅ ４Ｂをカラムに充填しモノクローナル（抗体Ｂ）カラムを作製した。
【０１１４】
実施例９で得られた溶出画分４００ｍｌを、１．０Ｍ ＮａＣｌ含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化したモノクローナル抗体（抗体Ｂ）カラム（直径５０ｍｍ、高さ６ｃｍ）に供した。１．０Ｍ ＮａＣｌ含む２０ｍＭりん酸緩衝液（ｐＨ７．３）を流し、更に１００ｍＭ酢酸緩衝液（ｐＨ５．０）を流し洗浄し、０．３Ｍ ＮａＣｌ含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．０）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの溶出液を主精製品として取得した。
【０１１５】
実施例１１
強陽イオン交換樹脂カラムによる高純度精製
実施例１０で得られた溶出液１８０ｍｌを１．０Ｍグリシン塩酸緩衝液（ｐＨ２．０）にてｐＨ３．５に調製した液を、３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で平衡化したＳＰ−Ｓｅｐｈａｒｏｓｅ（ファルマシア社）カラム（直径２６ｍｍ、高さ３ｃｍ）に供した。３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で洗浄を開始し、吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの素通り画分を得、直ちに５００ｍＭりん酸緩衝液（ｐＨ７．３）でｐＨ７に中和し、高純度精製品として取得した。
【０１１６】
実施例１２
ポリスルフォン中空糸による高純度精製品の濃縮
実施例１１で得られた高純度精製品をそれぞれ、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で湿潤化した１ｍのポリスルフォン中空糸（旭化成工業製）を用い濃縮し、それぞれ５ｍｌの濃縮液を取得した。
【０１１７】
実施例１３
ゲル濾過カラムによる高純度精製品の緩衝液交換
実施例１２で得られたそれぞれの濃縮液５ｍｌを、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化したそれぞれのＳｅｐａｈｃｒｙｌ Ｓ−３００カラム（ファルマシア社、直径１６ｍｍ、高さ９０ｃｍ）に供した。５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で展開し分画した。各画分は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定法方により活性の確認を行ない活性画分を回収し緩衝液交換した高純度精製品を取得した。
【０１１８】
実施例１４
アフィニティーカラム（トロンビンカラム）を用いた精製
実施例９で得られた溶出画分４００ｍｌを１００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）に対して透析した。透析後、１００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で平衡化したＤＩＰ−トロンビン−アガロース（ＰＡＥＳＥ ＬＯＲＥＩ社、０６−１４８−１０３５、直径５０ｍｍ、高さ６ｃｍ）に供した。２００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で洗浄後、１．０Ｍ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの溶出液を主精製品として取得した。
【０１１９】
得られた溶出液２００ｍｌに１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）を加え希釈した液を、次に１．０Ｍグリシン塩酸緩衝液（ｐＨ２．０）でｐＨ３．５に調製した。この希釈ｐＨ調製した溶出液を３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で平衡化したＳＰ−Ｓｅｐｈａｒｏｓｅ（ファルマシア社）カラム（直径２６ｍｍ、高さ３ｃｍ）に供した。３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で洗浄を開始し、吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの素通り画分を得、直ちに５００ｍＭりん酸緩衝液（ｐＨ７．３）でｐＨ７に中和し、高純度精製品として取得した。
【０１２０】
この高純度精製品を、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で湿潤化した１ｍのポリスルフォン中空糸（旭化成工業製）を用い濃縮し、５ｍｌの濃縮液を取得した。
次にゲル濾過カラムによる高純度精製品の緩衝液交換を行った。即ち、得られた濃縮液５ｍｌを、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化したそれぞれのＳｅｐａｈｃｒｙｌ Ｓ−３００カラム（ファルマシア社、直径１６ｍｍ、高さ９０ｃｍ）に供した。５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で展開し分画した。各画分は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定方法により活性の確認を行ない、活性画分を回収し緩衝液交換した高純度精製品を取得した。
【０１２１】
実施例１５
凍結した生産培養上清、すなわち実施例４の培養上清、実施例５の培養上清、実施例６の培養上清、実施例７−（１）の培養上清、実施例８の培養付着担体としてサイトデクスを用いた培養の培養上清、そして実施例８の培養付着担体として旭化成マイクロキャリアを用いた培養の培養上清それぞれを、精製の出発材料にした。
【０１２２】
精製は、実施例９のイオン交換クロマトによる粗精製を行ない、次に粗精製品を実施例１０のアフィニティークロマトで精製した。そしてこの精製品を実施例１１のイオン交換クロマトで高純度精製し、実施例１２の濃縮方法で高純度精製品を濃縮し、実施例１４のゲル濾過を行ない緩衝液置換された高純度精製品を得た。
【０１２３】
実施例１６
高純度精製品の物性
実施例１３、１４、１５で得られた緩衝液置換された高純度精製品について、以下の検討をした。
高純度精製品は、界面活性剤の非存在下でｐＨ中性の注射用蒸留水に少なくとも１０ｍｇ／ｍｌは溶解せしめることができた。
【０１２４】
また、高純度精製品を、還元、非還元の両条件でＳＤＳ−ＰＡＧＥ電気泳動を行ない、クマシー染色で検出した結果、還元で約９４ｋＤａ、非還元で約７３ｋＤａの１本のバンドが検出された。
また、パーキンエルマー社製のプロテインシーケンサーＰｒｏｃｉｓｅ４９２型を用いて、パルスリキッド法で、高純度精製品のＮ末端アミノ酸解析を行なった。その結果、Ｎ末端のアミノ酸はアラニンから始まるものとフェニルアラニンから始まる２種類の蛋白からなり、これはそれぞれ配列番号３の１９−５１６及び配列番号３の１７−５１６に相当するアミノ酸配列と考えられた。そしてアラニンから始まるもの含量比率は、それぞれの実施例の生産培養上清由来について、実施例３：６２％、実施例４：６８％、実施例５：６９％，実施例６：５９％、実施例７：６３％、実施例８の培養付着担体としてサイトデクス１（ファルマシアバイオテク社製）を用いた培養：６５％、そして実施例８の培養付着担体として旭化成マイクロキャリア（旭化成工業製）を用いた培養：５９％であった。
【０１２５】
高純度精製品の翻訳後修飾解析
以下の実施例において表記されているアミノ酸の配列位置は、配列番号３及び配列番号４に記載されたアミノ酸配列の配列位置に相当する。
（１）Ｎ結合型糖鎖結合位置の解析
実施例１３、１４、１５で得られた緩衝液置換された高純度精製品について、Ｎ結合型糖鎖の結合位置を解析した。
高純度精製品０．５ｍｇを遠心濃縮機を用いて乾固させ、トリス・尿素緩衝液・（８Ｍ尿素を含む２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）０．５ｍＬに溶解した。この溶液に２−メルカプトエタノール溶液（２−メルカプトエタノール／トリス・尿素緩衝液Ｉ＝５０μＬ／４５０μＬ）３２μＬを添加し、３７℃で１時間インキュベートした。ヨード酢酸溶液（ヨード酢酸０．３ｇを１ｍＬの２Ｍ ＮａＯＨに溶かしたもの）２７μＬを添加し、遮光して、室温で１５分間静置した。この溶液を１ｍＭ塩酸に透析した後、遠心濃縮機で乾固させたものをＲＣＭ−ＴＭとした。ＲＣＭ−ＴＭを、トリス・尿素緩衝液II（２Ｍ尿素を含む２００ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０）１８０μＬに溶解し、０．１ｍｇ／ｍＬトリプシン溶液（溶媒はトリス・尿素緩衝液II）４５μＬおよび０．１M 塩化カルシウム溶液２５μＬを添加し、３７℃で１６時間酵素消化した。
【０１２６】
この酵素消化液を０．１％ＴＦＡ／５％アセトニトリルで平衡化したＣｏｓｍｏｓｉｌ５Ｃ１８−ＡＲカラム (４．６Ｉ．Ｄ．×２５０ｍｍ、日本国、ナカライテスク社製）に添加後、０．１％ＴＦＡ／５０％アセトニトリルへの直線濃度勾配により、ペプチド断片を分画した。なお、流速は０．７ｍＬ／分、カラム温度は３５℃、検出は２１０ｎｍの吸収で行った。Ｎ結合型糖鎖の付加が予想されるコンセンサスシーケンス（Ａｓｎ−Ｘ−Ｓｅｒ／Ｔｈｒ）は、Ａｓｎ４７、Ａｓｎ１１５、Ａｓｎ１１６、Ａｓｎ３８２、Ａｓｎ４０９の５ケ所あるので、これらのアスパラギン残基を含むペプチド断片をＰＳＱ−２３、ＰＳＱ−１ (日本国、島津製作所製）または、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ Ｐｒｏｃｉｓｅ４９２（米国、パーキンエルマー社製）を用いて、メーカーの推奨する方法に従ってアミノ酸配列分析した。
【０１２７】
Ａｓｎ４７を含むペプチド断片（Ａｌａ１９−Ａｒｇ５６）のアミノ酸配列分析で、Ａｓｎ４７の位置はブランク（ＰＴＨ−Ａｓｎのピークが検出されない）であったことから、Ａｓｎ４７にはＮ結合型糖鎖が結合していることが確認された。同様に、Ａｓｎ３８２を含むペプチド断片（Ａｌａ３７２−Ａｒｇ４０３）、Ａｓｎ４０９を含むペプチド断片（Ｃｙｓ４０４−Ａｒｇ４７４）をアミノ酸配列解析した結果、Ａｓｎ３８２およびＡｓｎ４０９の位置はともにブランクであったことから、Ａｓｎ３８２およびＡｓｎ４０９にもＮ結合型糖鎖が結合していることが確認された。また、Ａｓｎ１１５およびＡｓｎ１１６を含むペプチド断片（Ｇｌｙ１０７−Ａｒｇ１２１）のアミノ酸配列を解析すると、Ａｓｎ１１５、Ａｓｎ１１６の位置はブランクではないが、検出されるＰＴＨ−Ａｓｎ量が予想される量よりそれぞれ低いことから、Ａｓｎ１１５に糖鎖が結合したもの（Ａｓｎ１１６には糖鎖が結合していない）とＡｓｎ１１６に糖鎖が結合したもの（Ａｓｎ１１５には糖鎖が結合していない）の混合物と考えられた。このことは、この断片の質量分析（ＭＡＬＤＩ−ＴＯＦＭＳ）の結果で、このペプチド断片には、Ｎ結合型糖鎖が１本結合した分子量しか認められないことからも支持された。
【０１２８】
（２）Ｎ結合糖鎖構造の解析
実施例１３、１４、１５で得られた緩衝液置換された高純度精製品について、Ｎ結合型糖鎖構造を解析した。高純度精製品０．５ｍｇを脱塩後、遠心濃縮機を用いて乾固させた。これをＨｙｄｒａｃｌｕｂＳ−２０４（日本国、ホーネン社製）を用いて、１１０℃で１時間無水ヒドラジンで処理し、糖鎖を切りだした。これを、２５０μＬの０．１Ｍ炭酸水素ナトリウムに溶解し、２５μＬの無水酢酸を加え、室温で３０分間静置した。この操作を２回繰り返し、再アセチル化した。この糖鎖を宝酒造社製のＰＡＬＳＴＡＴＩＯＮ Ｍｏｄｅｌ １０００（日本国、宝酒造社製）および糖鎖分析用試薬キットを用い、そのプロトコールに従ってピリジルアミノ（ＰＡ）化した。脱塩して遠心濃縮機で乾固させた後、２０μＬの０．２Ｍ酢酸ナトリウム緩衝液、ｐＨ５．０に溶解した。この溶液にシアリダーゼ０．０５Ｕを添加し、３７℃で終夜インキュベートして、アシアロＰＡ化糖鎖を調製した。このアシアロＰＡ化糖鎖をＴｏｍｉｙａらの方法（Ｔｏｍｉｙａ、Ｎ．，ｅｔ．ａｌ，１９８８，Ａｎａｌ．Ｂｉｏｃｈｅｍ．，１７１，７３−９０）に従って２次元マッピングし、構造を推定した。
【０１２９】
その結果、主成分はフコシルバイアンテナ型であることが推定された（８５〜９０％）。その次に多い成分はフコシルトリアンテナ型であり（１０〜１５％）、この２成分が大部分を占めていた。さらに、この主要２成分の構造は、質量分析（ＭＡＬＤＩ−ＴＯＦＭＳ、ＦＡＢ−ＭＳ）、エキソグリコシダーゼ処理（ガラクトシダーゼ、ＧｌｃＮＡｃ−ａｓｅ、フコシダーゼ）による解析においても正しいことが確認できた。
また、前述の（１）で調製した各Ｎ型糖鎖結合部位を含む糖ペプチド断片についてもそれぞれ同様に解析したが、すべての結合位置の糖鎖主成分の構造はフコシルバイアンテナ型であり、結合部位による糖鎖構造の顕著な違いは認められなかった。
【０１３０】
（３）Ｏ結合型糖鎖結合位置の解析
実施例１３、１４、１５で得られた緩衝液置換された高純度精製品について、Ｏ結合型糖鎖の結合位置を解析した。高純度精製品１ｍｇを脱塩した後、遠心濃縮機を用いて乾固させ、水３０μＬに溶解した。この溶液に１０μＬの０．２５Ｍリン酸ナトリウム緩衝液（ｐＨ８．０）とＡｓｐ−Ｎ（日本国、和光純薬工業製）１μｇを添加し、室温で３日間酵素消化した。この酵素消化液を０．１％ＴＦＡ／水で平衡化したＣｏｓｍｏｓｉｌ５Ｃ１８−３００カラム (４．６Ｉ．Ｄ．×１５０ｍｍ、日本国、ナカライテスク社製）に添加後、０．１％ＴＦＡ／９０％アセトニトリルへの直線濃度勾配により、ペプチド断片を分画した。なお、流速は０．８ｍＬ／分、検出は２２０ｎｍの吸収で行った。Ｏ結合型糖鎖は、プロリンリッチな配列付近に結合している場合が多いことから、可溶化ＴＭのＣ末端近傍に結合していることが予想された。そこでＣ末端を含むペプチド断片（Ａｓｐ４８９−Ｇｌｙ５１６）をシアリダーゼ及びガラクトシダーゼ処理した後、ＰＳＱ−２３、ＰＳＱ−１ (日本国、島津製作所製）または、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ Ｐｒｏｃｉｓｅ４９２（米国、パーキンエルマー社製）を用いて、メーカーの推奨する方法に従ってアミノ酸配列分析した。なおＳｅｒ／Ｔｈｒの分析サイクルで、ＰＴＨ−Ｓｅｒ／ＰＴＨ−Ｔｈｒの収量が低く、かつＰＴＨ−Ｓｅｒ（ＧａｌＮＡｃ）／ＰＴＨ−Ｔｈｒ（ＧａｌＮＡｃ）のピークが認められる位置を糖鎖結合位置とした。
【０１３１】
その結果、Ｏ糖鎖結合位置としてＳｅｒ４９８、Ｔｈｒ５００、Ｓｅｒ５０３、Ｔｈｒ５０４、Ｔｈｒ５０６の５カ所が確認された。だだし、この中でＳｅｒ５０３だけは部分修飾であった。Ｓｅｒ４９２についてもＰＴＨ−Ｓｅｒが検出されないことから糖鎖の修飾が予想されたが、ＰＴＨ−Ｓｅｒ（ＧａｌＮＡｃ）のピークが認められず、糖鎖構造に違いがあることが推測された。
【０１３２】
（４）Ｏ結合型糖鎖構造の解析
実施例１３、１４、１５で得られた緩衝液置換された高純度精製品について、Ｏ結合型糖鎖構造を解析した。高純度精製品１．０ｍｇを脱塩後、遠心濃縮機を用いて乾固させた。これをＨｙｄｒａｃｌｕｂＳ−２０４（日本国、ホーネン社製）を用いて、６５℃で６時間無水ヒドラジンで処理し、糖鎖を切りだした。これを、２５０μＬの０．１Ｍ炭酸水素ナトリウムに溶解し、２５μＬの無水酢酸を加え、室温で３０分間静置した。この操作を２回繰り返し、再アセチル化した。この糖鎖を宝酒造社製のＰＡＬＳＴＡＴＩＯＮ Ｍｏｄｅｌ １０００（日本国、宝酒造社製）および単糖分析用試薬キットを用い、そのプロトコールに従ってピリジルアミノ（ＰＡ）化した。遠心濃縮機で乾固させた後、０．０２５Ｍ塩酸中８０℃で１時間加熱して、アシアロＰＡ化糖鎖を調製した。このアシアロＰＡ化糖鎖をＩｗａｓｅらの方法（Ｉｗａｓｅ，ｅｔ．ａｌ，Ｊ．Ｂｉｏｃｈｅｍ．，１９９６，１２０，９２−９７）に従ってＨＰＬＣ分析を行った結果、以下に示す４種類の糖鎖構造が存在することが推測された。
１）Ｇａｌβ１，３ＧａｌＮＡｃ
２）ＮｅｕＡｃα２，３Ｇａｌβ１，３ＧａｌＮＡｃ
３）Ｇａｌβ１，３（ＮｕｅＡｃα２，６）ＧａｌＮＡｃ
４）ＮｅｕＡｃα２，３Ｇａｌβ１，３（ＮｕｅＡｃα２，６）ＧａｌＮＡｃ
【０１３３】
（５）グリコサミノグリカン鎖結合位置および構造の解析
Ｏ結合型糖鎖結合位置の解析結果から、Ｓｅｒ４９２にはＧａｌβ１，３ＧａｌＮＡｃ以外の構造の糖鎖が結合していることが推測されたので、それを含むアシアロ、アガラクト−ペプチド断片について糖組成分析を行った。すなわちＡｓｐ−Ｎ消化ペプチド断片（Ａｓｐ４８９−Ｇｌｙ５１６）を蒸留水１００μＬで溶解し、加水分解試験管に入れ１００μＬの８Ｍ ＴＦＡと混合し１００℃、３時間加水分解を行った。これを遠心濃縮乾固した後にＮ−アセチル化を行い、ピリジルアミノ化装置（パルスステーションＭｏｄｅｌ ４０００、日本国宝酒造社製）及びＰＡＬＳＴＡＴＩＯＮ Ｐｙｒｉｄｙｌａｍｉｎａｔｉｏｎ Ｋｉｔ（宝酒造社製）を用いてピリジルアミノ化を行った。これをＰＡＬＰＡＫ ＴｙｐｅＡカラム（宝酒造社製）に供し、ＰＡ−単糖を定量した。なお、流速は０．３ｍｌ／分で、カラム温度は６５℃、検出は蛍光Ｅｘ：３１０ｎｍ、Ｅｍ：３８０ｎｍで行った。
【０１３４】
その結果、Ｓｅｒ４９８、Ｔｈｒ５００、Ｓｅｒ５０３、Ｔｈｒ５０４、Ｔｈｒ５０６、Ｓｅｒ４９２に結合するＧａｌＮＡｃ以外に、キシロースとガラクトースが約１：２の割合で検出された。したがって、Ｓｅｒ４９２には、グリコサミノグリカン鎖の橋渡し４糖の構造（Ｘｙｌ−Ｇａｌ−Ｇａｌ−ＧｌｃＡ）を含むような糖鎖が結合していることが示唆された。
【０１３５】
（６）Ａｓｎ３４２のβ−ヒドロキシ化
ＳｔｅｎｆｌｏらはＥＧＦ様構造の−Ｃｙｓ−Ｘ−Ａｓｐ／Ａｓｎ−Ｘ−Ｘ−Ｘ−Ｘ−Ｔｙｒ／Ｐｈｅ−Ｘ−Ｃｙｓ−Ｘ−Ｃｙｓ−配列中のＡｓｐ／Ａｓｎがβ−ヒドロキシ化を受けることを報告している（Ｓｔｅｎｆｌｏ，Ｊ．Ｏｈｌｉｎ，ｅｔ．ａｌ，１９８７，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８４，３６８−３７２、Ｓｔｅｎｆｌｏ，Ｊ．Ｏｈｌｉｎ，ｅｔ．ａｌ，１９８８，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６３，２１−２４）Ｓｔｅｎｆｌｏらのコンセンサスシーケンスに従えば、可溶化ＴＭには２ケ所修飾が予想される位置がある（Ａｓｎ３４２、Ａｓｎ４５７）。そこで、Ａｓｎ３４２とＡｓｎ４５７を含む各ペプチド断辺のアミノ酸配列解析と質量分析を行ったところ、Ａｓｎ３４２を含むペプチド断片は、修飾されない場合の質量より１６質量数大きく、Ａｓｎがヒドロキシ化された場合の質量数とほぼ一致した（Ｍ＋Ｈ：Ａｖ．ｍａｓｓ＝３９１７．２、ヒドロキシされていない場合は３９０１．２）。この結果から、Ａｓｎ３４２はβ−ヒドロキシ化を受けていると考えられた。またその修飾率はＭＳのイオン強度比から９０％以上と予想された。
【０１３６】
（７）Ｓｅｒ３０５のグルコシル化
ＲＣＭ−ＴＭのトリプシン消化液中から、Ｓｅｒ３０５を含むペプチド断片（Ｓｅｒ２７９−Ａｒｇ３１５）を分離し、アミノ酸配列を解析した結果、Ｓｅｒ３０５の位置にはＰＴＨ−Ｓｅｒのピークは認められず、ＰＴＨ−Ａｓｐの溶出位置の近くにピークが移動していることがわかった。また、このペプチド断片を質量分析（ＭＡＬＤＩ−ＴＯＦＭＳ）した結果ｍ／ｚ４５１６にピークが認められ、ペプチド断片の予想質量より約１６２質量数だけ大きかった。この結果から、Ｓｅｒ３０５は修飾を受けており、ヘキソース（質量数＝１６２）の付加が予想された。そこで、このペプチド断片の糖組成分析を行ったところ、グルコースが検出された。
【０１３７】
ＥＧＦ様構造中のＳｅｒ残基のグルコシル化については、いくつかの血清タンパク質について報告されている（ｆａｃｔｏｒＶＩＩ、ｆａｃｔｏｒＩＸ、ｐｒｏｔｅｉｎＺ、ｔｈｒｏｍｂｏｓｐｏｎｄｉｎ）（Ｎｉｓｈｉｍｕｒａ，Ｈ．，ｅｔ．ａｌ，１９８９，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２６４，２０３２０−２０３２５）。しかし、これまでに報告されている糖鎖構造は、２糖（Ｘｙｌ−Ｇｌｃ）もしくは３糖（Ｘｙｌ−Ｘｙｌ−Ｇｌｃ）であったが、可溶化ＴＭのＳｅｒ３０５に結合する糖鎖はグルコース１糖でキシロースの付加は認められず、これまでに報告されている糖鎖構造とは異なることがわかった。また、グルコース付加のコンセンサスシーケンスはＣｙｓ−Ｘ−Ｓｅｒ−Ｘ−Ｐｒｏ−Ｃｙｓであるが、Ｓｅｒ３０５周辺のアミノ酸配列はＰｒｏ−Ｇｌｙ−Ｓｅｒ３０５−Ｔｙｒ−Ｓｅｒ−Ｃｙｓ−であり、一致しない。したがって、Ｓｅｒ３０５のグルコシル化は新しいタイプの修飾と考えられた。
【０１３８】
実施例１７
トロンボモジュリン精製工程のスケールアップとマスバランス
実施例７−（１）の培養上清１２Ｌを用い、カラムスケールを容量に合わせて増大させた他は、実質的に実施例９〜１３に記載したのと同様の方法で、高純度ＴＭを得た。このときの精製マスバランスを下記表１に示した。最終的に高純度ＴＭとして約２８５ｍｇが得られ、活性回収率は３８％であった。
【０１３９】
【表１】

【０１４０】
更に実施例７−（１）の培養上清１２Ｌを用い、カラムスケールを容量に合わせて増大させた他は、実質的に実施例９〜１３に記載したのと同様の方法で、但し実施例１１に記載したＳＰイオン交換工程を実施せず精製ＴＭを得た。このときの精製マスバランスを下記表２に示した。最終的に精製ＴＭとして約２８０ｍｇが得られ、活性回収率は４０％とＳＰ−セファロースを導入した場合とほぼ同様の回収率であった。
【０１４１】
【表２】

【０１４２】
実施例１８
混入異種蛋白質量の評価
ＴＭウシ血清由来物、マウス抗体由来物及びＣＨＯ細胞由来物それぞれについて、それぞれＥＬＩＳＡによる測定系を確立した。
（１）血清由来物の評価方法
ウシ血清とフロイント完全アジュバントとの等量乳濁液１ｍｌをウサギの背皮下感作した。２週間後及び３週間後にそれぞれ背皮下に同乳濁液１ｍｌずつを投与し、最終感作１週間後に採血した。血液を約４℃で１晩静置した後、血餅を除き遠心分離した上清をとり、得られたウサギ抗ウシ血清成分抗血清より、硫安塩析法及びウシ血清成分をリガンドとしたカラムクロマトグラフ法によって、ウサギ抗ウシ血清成分抗体を得た。
【０１４３】
ウサギ抗ウシ血清成分抗体をペプシンで消化し、２Ｆ（ａｂ’）２断片を得た。２−メルカプトエチルアミン塩酸塩で還元後、Ｎ−（４−カルボキシシクロヘキシルメチル）マレイミドのＮ−ヒドロキシスクシンイミドエステルでマレイミド基を導入した西洋ワサビペルオキシダーゼと反応させ、酵素標識抗ウシ血清成分抗体を調製した。
予想されるウシ血清成分含量が０〜２５ｎｇ／ｍｌとなるように、各工程の溶出液をポリソルベート・リン酸塩緩衝食塩液で薄め、試料溶液とした。別にウシ血清成分標準品にポリソルベート・リン酸塩緩衝食塩液を加えて、その１ｍｌ中に２５〜１．２５及び０ｎｇを含む液を調製し、標準溶液とした。
【０１４４】
ポリスチレン製９６穴プレートに、１穴当たりウサギ抗ウシ血清成分抗体溶液を５０μｌずつ加えた後，２５℃で約２時間静置した。次いでリン酸塩緩衝食塩液で各穴を洗浄し，ブロッキング溶液を加え，２５℃で約１時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗った後、試料溶液及び標準溶液を１００μｌずつそれぞれの穴に加え、２５℃で約１６時間静置した。次いでポリソルベート・リン酸塩緩衝食塩液で洗浄後，酵素標識抗ウシ血清成分抗体溶液を５０μｌずつ加え、２５℃で約２時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗浄した後，酵素基質溶液を１００μｌずつ加え、２５℃で約４０分反応させた。薄めた硫酸（１→４）５０μｌずつ加えて反応を停止させた後、９６穴プレート用吸光度計で４９０ｎｍの吸光度を測定した。標準溶液による検量線から、本品１ｍｌ中のウシ血清成分の含量を求めた。標準溶液の最低濃度の１．２５ｎｇ／ｍＬを検出限界とした。
【０１４５】
（２）マウス抗体由来物の評価方法
マウス抗体とフロイント完全アジュバントとの等量乳濁液1 mlをウサギの背皮下感作した。2 週間後及び3 週間後にそれぞれ背皮下に同乳濁液1 mlずつを投与し，最終感作1 週間後に採血した。血液を約４℃で１晩静置した後，血餅を除き遠心分離した上清をとり、得られたウサギ抗マウス抗体成分抗血清より，硫安塩析法及びマウス抗体成分をリガンドとしたカラムクロマトグラフ法によって、ウサギ抗マウス抗体成分抗体を得た。
【０１４６】
ウサギ抗マウス抗体成分抗体をペプシンで消化し、Ｆ（ａｂ’）２断片を得た。２−メルカプトエチルアミン塩酸塩で還元後、Ｎ−（４−カルボキシシクロヘキシルメチル）マレイミドのＮ−ヒドロキシスクシンイミドエステルでマレイミド基を導入した西洋ワサビペルオキシダーゼと反応させ、酵素標識抗マウス抗体成分抗体を調製した。
【０１４７】
予想されるマウス抗体成分含量が０〜２５ｎｇ／ｍｌとなるように、各工程の溶出液をポリソルベート・リン酸塩緩衝食塩液で薄め、試料溶液とした。別にマウス抗体成分標準品にポリソルベート・リン酸塩緩衝食塩液を加えて、その1 ｍｌ中に２５〜１．２５及び０ｎｇを含む液を調製し、標準溶液とした。
ポリスチレン製９６穴プレートに、１穴当たりウサギ抗マウス抗体成分抗体溶液を５０μｌずつ加えた後、２５℃で約２時間静置した。次いでリン酸塩緩衝食塩液で各穴を洗浄し、ブロッキング溶液を加え、２５℃で約１時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗った後、試料溶液及び標準溶液を１００μｌずつそれぞれの穴に加え、２５℃で約１６時間静置した。次いでポリソルベート・リン酸塩緩衝食塩液で洗浄後、酵素標識抗マウス抗体成分抗体溶液を５０μｌずつ加え、２５℃で約２時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗浄した後、酵素基質溶液を１００μｌずつ加え、２５℃で約４０分反応させた。薄めた硫酸（１→４）５０μｌずつ加えて反応を停止させた後、９６穴プレート用吸光度計で４９０ｎｍの吸光度を測定した。標準溶液による検量線から、本品１ｍｌ中のマウス抗体成分の含量を求めた。標準溶液の最低濃度の１．２５ｎｇ／ｍＬを検出限界とした。
【０１４８】
（３）ＣＨＯ細胞由来物の評価方法
ＣＨＯ細胞を無血清培地で培養した上清を濃縮し、ＣＨＯ細胞由来物を得た。ＣＨＯ細胞由来物とフロイント完全アジュバントとの等量乳濁液１ｍｌをウサギの背皮下感作した。２週間後及び３週間後にそれぞれ背皮下に同乳濁液１ｍｌずつを投与し、最終感作１週間後に採血した。血液を約４℃で１晩静置した後、血餅を除き遠心分離した上清をとり、得られたウサギ抗ＣＨＯ細胞由来物成分抗血清より、硫安塩析法及びカラムクロマトグラフ法によって、ウサギ抗ＣＨＯ細胞由来物成分抗体を得た。
【０１４９】
ウサギ抗ＣＨＯ細胞由来物成分抗体をペプシンで消化し、Ｆ（ａｂ’）２断片を得た。２−メルカプトエチルアミン塩酸塩で還元後、Ｎ− (４−カルボキシシクロヘキシルメチル）マレイミドのＮ−ヒドロキシスクシンイミドエステルでマレイミド基を導入した西洋ワサビペルオキシダーゼと反応させ、酵素標識抗ＣＨＯ細胞由来物成分抗体を調製した。
【０１５０】
予想されるＣＨＯ細胞由来物成分含量が０〜５００ｎｇ／ｍｌとなるように、各工程の溶出液をポリソルベート・リン酸塩緩衝食塩液で薄め、試料溶液とした。別にＣＨＯ細胞由来物成分標準品にポリソルベート・リン酸塩緩衝食塩液を加えて、その１ｍｌ中に５００〜２５及び０ｎｇを含む液を調製し、標準溶液とした。
【０１５１】
ポリスチレン製９６穴プレートに、１穴当たりウサギ抗ＣＨＯ細胞由来物成分抗体溶液を１００μｌずつ加えた後、２５℃で約２時間静置した。次いでリン酸塩緩衝食塩液で各穴を洗浄し、ブロッキング溶液を加え、２５℃で約１時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗った後、試料溶液及び標準溶液を１００μｌずつそれぞれの穴に加え、２５℃で約１６時間静置した。次いでポリソルベート・リン酸塩緩衝食塩液で洗浄後、酵素標識抗ＣＨＯ細胞由来物成分抗体溶液を５０μｌずつ加え、２５℃で約２時間静置した。ポリソルベート・リン酸塩緩衝食塩液で洗浄した後、酵素基質溶液を１００μｌずつ加え、２５℃で約４０分反応させた。薄めた硫酸（１→４）５０μｌずつ加えて反応を停止させた後、９６穴プレート用吸光度計で４９０ｎｍの吸光度を測定した。標準溶液による検量線から、本品１ｍｌ中のＣＨＯ細胞由来物成分の含量を求めた。標準溶液の最低濃度の２５ｎｇ／ｍＬを検出限界とした。
【０１５２】
（４）各混入成分量の評価
これらの評価方法を用いて、実施例１７の表１、表２に示した高純度及び精製可溶性ＴＭの精製各段における混入量をそれぞれ評価した。結果を表３、表４に示した。ＳＰイオン交換工程の導入によって、各成分の混入量を同時に劇的に減じることができた。同様の実験を計５回実施したが、ウシ血清由来物とマウス抗体由来物に関してはいずれも検出されなかった。これに対してＳＰを用いない場合は、それぞれの含量は一定せず、５回の実験で血清由来物が１５〜１３６μｇ、血清由来物が８〜３６μｇであった。
【０１５３】
【表３】

【０１５４】
【表４】

【０１５５】
実施例１９
実施例１７でＳＰセファロース工程を用いずに精製された、精製ＴＭ溶液を用い、同イオン交換工程の溶出挙動に影響を与える因子として、食塩濃度及びｐＨを変化させ、混入異種蛋白除去効率に与える影響を調べた。用いた溶媒条件は下記の通りであった。
１、食塩濃度０．１Ｍ、ｐＨ２．５、比伝導度１６ｍｓ／ｃｍ
２、食塩濃度０．１Ｍ、ｐＨ３．０、比伝導度１３ｍｓ／ｃｍ
３、食塩濃度０．１Ｍ、ｐＨ３．５、比伝導度１２ｍｓ／ｃｍ
４、食塩濃度０．１Ｍ、ｐＨ４．０、比伝導度１１ｍｓ／ｃｍ
５、食塩濃度０．１Ｍ、ｐＨ４．８、比伝導度１１ｍｓ／ｃｍ
６、食塩濃度０．２５Ｍ、ｐＨ２．５、比伝導度３０ｍｓ／ｃｍ
７、食塩濃度０．２５Ｍ、ｐＨ３．０、比伝導度２７ｍｓ／ｃｍ
８、食塩濃度０．２５Ｍ、ｐＨ３．５、比伝導度２６ｍｓ／ｃｍ
９、食塩濃度０．２５Ｍ、ｐＨ４．０、比伝導度２６ｍｓ／ｃｍ
１０、食塩濃度０．２５Ｍ、ｐＨ４．８、比伝導度２５ｍｓ／ｃｍ
１１、食塩濃度０．３Ｍ、ｐＨ２．５、比伝導度３４ｍｓ／ｃｍ
１２、食塩濃度０．３Ｍ、ｐＨ３．０、比伝導度３２ｍｓ／ｃｍ
１３、食塩濃度０．３Ｍ、ｐＨ３．５、比伝導度３１ｍｓ／ｃｍ
１４、食塩濃度０．３Ｍ、ｐＨ４．０、比伝導度３０ｍｓ／ｃｍ
１５、食塩濃度０．３Ｍ、ｐＨ４．８、比伝導度３０ｍｓ／ｃｍ
１６、食塩濃度０．３２Ｍ、ｐＨ２．５、比伝導度３６ｍｓ／ｃｍ
１７、食塩濃度０．３２Ｍ、ｐＨ３．０、比伝導度３４ｍｓ／ｃｍ
１８、食塩濃度０．３２Ｍ、ｐＨ３．５、比伝導度３２ｍｓ／ｃｍ
１９、食塩濃度０．３２Ｍ、ｐＨ４．０、比伝導度３１ｍｓ／ｃｍ
２０、食塩濃度０．３２Ｍ、ｐＨ４．８、比伝導度３２ｍｓ／ｃｍ
【０１５６】
精製ＴＭ溶液各３２ｍｌを上記各条件に調整した１００ｍＭグリシン塩酸緩衝液に透析し、溶媒を交換した。また同じ組成の各緩衝液で平衡化したＳＰ−ＳｅｐｈａｒｏｓｅＦＦカラム（直径１６ｍｍ、高さ５ｍｍ）に供した。更に同緩衝液で洗浄を開始し、吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの素通り画分を得、直ちに５００ｍＭりん酸緩衝液（ｐＨ７．３）でｐＨ７に中和し、中和した洗浄液を精製品として取得した。
【０１５７】
得られた精製品に関して実施例１８と同様にウシ血清成分及びマウス抗体成分の混入量を評価すると共に、ＴＭの活性回収率を評価し、実質的に精製操作が可能な条件範囲を決定した。その結果を表５に示した。
【０１５８】
【表５】

【０１５９】
ウシ血清由来物およびマウス抗体由来物を同時に除去でき、かつ良好なＴＭ活性回収率を達成できる条件は、０．２５〜０．３２Ｍ、好ましくは約０．３Ｍ食塩濃度、即ち、２５〜３４ｍｓ／ｃｍ、好ましくは約３１ｍｓ／ｃｍ前後の比伝導度の条件であり、ｐＨはｐＨ３．０〜４．０、好ましくはｐＨ約３．５の条件であった。
【０１６０】
実施例２０
Ｅ４５６生産用細胞の作製
（１）プラスミドｐＳＶ２ＴＭＥ４５６の構築
実施例１記載の方法に従い、配列番号６に示すディリーターＴＭＤ２：５’−ＧＡＡＧＣＡＣＧＧＧＴＣＣＣＣＣＡＧＧＣＣＧＧＣＣ−３’（２５ｍｅｒ）を用い、実施例１−（１）で作製した組換え体プラスミドＭ１３ＴＭＤ１２３の１０５０塩基からなる部分の削除を行った。得られた組換え体プラスミドをＭ１３ＴＭＥ４５６Ｄ３と称した。更にこの組換え体プラスミドＭ１３ＴＭＥ４５６Ｄ３は、配列番号３のアミノ酸残基番号１の開始コドン（ＡＴＧ）から１６のＧｌｙ及び、３６７から５１６のアミノ酸からなるヒトトロンボモジュリンの部分配列をコードする塩基配列を含有するＤＮＡ断片を有することがわかった。さらに、配列番号７に示すディリーターＴＭＤ２：５’−ＧＧＡＧＧＣＣＧＣＴＣＡＡＣＡＧＴＣＧＧＴＧＣＣＡ−３’（２５ｍｅｒ）を用い、Ｍ１３ＴＭＥ４５６Ｄ３の１０８塩基からなる部分の削除を行った。得られた組換え体プラスミドをＭ１３ＴＭＥ４５６と称した。更にこの組換え体プラスミドＭ１３ＴＭＥ４５６は、配列番号１に示す開始コドン（ＡＴＧ）とその下流に１３０個のアミノ酸からなるヒトトロンボモジュリンの部分配列をコードする塩基配列を含有するＤＮＡ断片を有することがわかった。この組換え体プラスミドをＭ１３ＴＭＥ４５６に含まれるＤＮＡ断片をＴＭＥ４５６と称した。
【０１６１】
この組換え体プラスミドＭ１３ＴＭＥ４５６をＨｉｎｄＩＩＩおよびＢａｍＨＩで完全消化してＴＭＤ１２３の約６００ｂｐのＤＮＡ断片を単離した。一方、プラスミドｐＳＶ２ＤＨＦＲ（ＡＴＣＣ ３７１４６）をＨｉｎｄＩＩＩ及びＢｇｌIIで完全消化してベクター断片を得た。このベクター断片と、先のＤＮＡ断片ＴＭＥ４５６とをＴ4 ＤＮＡリガーゼを用いて継ぎあわせ、プラスミドｐＳＶ２ＴＭＥ４５６を得た。
【０１６２】
（２）ＴＭＥ４５６製造用高生産細胞の樹立
実施例１−（２）、（３）、（４）、（５）の方法に従い、プラスミドｐＳＶ２ＴＭＥ４５６とプラスミドｐＳＶ２−ＤＨＦＲによるＣＨＯ細胞の形質転換、形質転換したＣＨＯ細胞からＤＨＦＲ産生株の選択、Ｅ４５６生産株の選択、ＭＴＸによるＥ４５６生産性の増幅を行い、ＭＴＸ濃度の上昇により効率的にＥ４５６の生産性が増幅され、２μＭＭＴＸ濃度で、約８ｐｇ／細胞・日、２０μＭＭＴＸ濃度で、約１４ｐｇ／細胞・日、１００μＭで４０ｐｇ／細胞・日という高生産の細胞株が樹立された。また、実施例１−（６）の方法に従い、Ｅ４５６生産が安定であることを確認し、更に実施例１−（７）記載の方法に従い、浮遊培養馴化株を取得した。
【０１６３】
実施例２１
ＴＭＥ４５６高生産細胞によるＴＭを製造する培養
実施例２に記載の方法に従い、ＴＭ製造用培地を調製した。また、ＴＭ生産細胞を増殖させる培養は、実施例３に記載の方法に従い、後述するＴＭを製造する培養に必要なスケール、細胞数を満たすような継代を行った。
【０１６４】
（１）浮遊培養での繰り返しバッチ培養
実施例３記載の方法に従いＴＭを製造する培養を行った。
生産培養結果は、平均生細胞密度２．９×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は５８ｍｇ／Ｌ／ｄａｙであった。
【０１６５】
（２）浮遊培養でのバッチ培養
実施例４記載の方法に従いＴＭを製造する培養を行った。
生産培養は１０日間培養し、到達生細胞密度２．０×１０⁶ ｃｅｌｌｓ／ｍｌで、累積生産性は３８ｍｇ／Ｌであった。
【０１６６】
（３）浮遊培養での連続培養
実施例５記載の方法に従いＴＭを製造する培養を行った。
生産培養結果は、平均生細胞密度１１．４×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は１３７ｍｇ／Ｌ／ｄａｙであった。
【０１６７】
（４）浮遊培養での連続培養
実施例６記載の方法に従いＴＭを製造する培養を行った。
生産培養結果は、平均生細胞密度１１．９×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は１２８ｍｇ／Ｌ／ｄａｙであった。
【０１６８】
（５）浮遊培養での連続培養
実施例７−（１）記載の方法に従いＴＭを製造する培養を行った。
生産培養結果は、平均生細胞密度１２．３×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は１３５ｍｇ／Ｌ／ｄａｙであった。
【０１６９】
（６）付着培養での繰り返しバッチ培養
実施例８記載の方法に従いＴＭを製造する培養を行った。
生産培養結果は、平均細胞密度はサイトデクス１使用の培養で２．８×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は４８ｍｇ／Ｌ／ｄａｙであり、また平均細胞密度は旭化成マイクロキャリア使用の培養で６．２×１０⁶ ｃｅｌｌｓ／ｍｌで、平均生産性は８９ｍｇ／Ｌ／ｄａｙであった。
【０１７０】
実施例２２
ＴＭＥ４５６高生産細胞による培養上清の精製
（１）強陰イオン交換樹脂カラムによる粗精製
実施例２１−（１）で取得した−２０℃凍結培養上清５Ｌを溶解し、０．２μｍのメンブレンフィルター（ミリポア社、ミリパック２０）で濾過した。濾過した培養上清については、１５０ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で平衡化したＱ−Ｓｅｐｈａｒｏｓｅ（ファルマシア社）カラム（直径９０ｍｍ、高さ１１ｃｍ）に供した。次に１５０ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で洗浄を行ない、３００ｍＭ ＮａＣｌを含む２０ｍＭトリス塩緩衝液（ｐＨ７．４）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりからの１．０カラムボリューム容量の溶出液を粗精製品として取得した。
【０１７１】
（２）アフィニティーカラム（トロンビンカラム）による主精製
実施例２２−（１）で得られた溶出画分４００ｍｌを１００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）に対して透析した。透析後、１００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で平衡化したＤＩＰ−トロンビン−アガロース（ＰＡＥＳＥ ＬＯＲＥＩ社、０６−１４８−１０３５、直径５０ｍｍ、高さ６ｃｍ）に供した。３００ｍＭ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で洗浄後、１．０Ｍ ＮａＣｌ及び０．５ｍＭ塩化カルシウムを含む２０ｍＭトリス緩衝液（ｐＨ７．４）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの溶出液を主精製品として取得した。
【０１７２】
（３）アフィニティーカラム（抗体Ａ）による主精製
実施例１−（５）で作製した抗ＴＭモノクローナル（抗体Ａ）を実施例１１に従いカラム化し、抗ＴＭモノクローナル（抗体Ａ）カラムを作製した。次いで、実施例２２−（１）で得られた溶出画分４００ｍｌを、１．０Ｍ ＮａＣｌ含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化したモノクローナル抗体（抗体Ａ）カラム（直径５０ｍｍ、高さ６ｃｍ）に供した。１．０Ｍ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）を流し、更に１００ｍＭ酢酸緩衝液を流し洗浄し、０．３Ｍ ＮａＣｌ含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．０）で溶出を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの溶出液を主精製品として取得した。
【０１７３】
（４）強陽イオン交換樹脂カラムによる高純度精製
（４）−１ トロンビンカラム溶出液の精製
実施例２２−（２）で得られた溶出液２００ｍｌに１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）を加え希釈した液を、次に１．０Ｍグリシン塩酸緩衝液（ｐＨ２．０）でｐＨ３．５に調製した。この希釈ｐＨ調製した溶出液を３００ｍＭＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で平衡化したＳＰ−ＳｅｐｈａｒｏｓｅＦＦ（ファルマシア社）カラム（直径２６ｍｍ、高さ３ｃｍ）に供した。３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（比伝導度３１ｍｓ／ｃｍ、ｐＨ３．５）で洗浄を開始し、吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの素通り画分を得、直ちに５００ｍＭりん酸緩衝液（ｐＨ７．３）でｐＨ７に中和し、高純度精製品として取得した。
【０１７４】
（４）−２ 抗体カラム溶出液の精製
実施例２２−（３）で得られた溶出液１８０ｍｌに１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）を加え希釈した液を、次に１．０Ｍグリシン塩酸緩衝液（ｐＨ２．０）でｐＨ３．５に調製した。この希釈、ｐＨ調製した液を、３００ｍＭＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）で平衡化したＳＰ−ＳｅｐｈａｒｏｓｅＦＦ（ファルマシア社）カラム（直径２６ｍｍ、高さ３ｃｍ）に供した。３００ｍＭ ＮａＣｌを含む１００ｍＭグリシン塩酸緩衝液（ｐＨ３．５）で洗浄を開始し、溶出液の吸光度２８０ｎｍのピーク立ち上がりから立ち下がりまでの溶出液を得、直ちに５００ｍＭりん酸緩衝液（ｐＨ７．３）でｐＨ７に中和し、中和した溶出液を高純度精製品として取得した。
【０１７５】
（５）ポリスルフォン中空糸による高純度精製品の濃縮
実施例２２−（４）で得られた高純度精製品をそれぞれ、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で湿潤化した１ｍのポリスルフォン中空糸（旭化成工業製）を用い濃縮し、それぞれ５ｍｌの濃縮液を取得した。
【０１７６】
（６）ゲル濾過カラムによる高純度精製品の緩衝液交換
実施例２２−（５）で得られたそれぞれの濃縮液５ｍｌを、５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で平衡化したそれぞれのＳｅｐａｈｃｒｙｌ Ｓ−２００カラム（ファルマシア社、直径１６ｍｍ、高さ９０ｃｍ）に供した。５０ｍＭ ＮａＣｌを含む２０ｍＭりん酸緩衝液（ｐＨ７．３）で展開し分画した。各画分は実施例１−（４）−ａ記載のトロンビンによるプロテインＣ活性化を促進する活性の測定法方により活性の確認を行ない活性画分を回収し緩衝液交換した高純度精製品を取得した。
【０１７７】
実施例２３
凍結した生産培養上清、すなわち実施例２１−（２）の培養上清、実施例２２−（３）の培養上清，実施例２２−（４）の培養上清、実施例２２−（５）の培養上清、実施例２２−（６）の培養付着担体としてサイトデクスを用いた培養の培養上清、そして実施例８の培養付着担体として旭化成マイクロキャリアを用いた培養の培養上清それぞれを、精製の出発材料にした。
精製は、実施例２２−（１）のイオン交換クロマトによる粗精製を行ない、次に粗精製品を実施例２２−（３）のアフィニティークロマトで主精製した。そして主精製品を実施例２２−（４）−２のイオン交換クロマトで高純度精製し、実施例２２−（５）の濃縮方法で高純度精製品を濃縮し、実施例２２−（６）のゲル濾過を行ない緩衝液置換された高純度精製品を得た。
【０１７８】
実施例２４
高純度精製品の物性
実施例２３で得られた緩衝液置換された高純度精製品について、以下の検討をした。
高純度精製品は、界面活性剤の非存在下でｐＨ中性の注射用蒸留水に少なくとも１０ｍｇ／ｍｌは溶解せしめることができた。
【０１７９】
また、高純度精製品を、還元、非還元の両条件でＳＤＳ−ＰＡＧＥ電気泳動を行ない、クマシー染色でにより検出した結果、還元で約２６ｋＤａ、非還元で約２１−２４ｋＤａの１本のバンドが検出された。
また、パーキンエルマー社製のプロテインシーケンサーＰｒｏｃｉｓｅ４９２型を用いて、パルスリキッド法で、高純度精製品のＮ末端アミノ酸解析を行なった。その結果、Ｎ末端のアミノ酸は全ての高純度精製品サンプルでアスパラギンから始まる１種類の蛋白からなり、これは配列番号１の１７−１３０に相当するアミノ酸配列と考えられた。またいずれの高純度精製品についても、ウシ血清由来物、マウス抗体由来物の混入量は検出限界以下であった。
【０１８０】
実施例２５
高純度精製品の凍結乾燥
注射用蒸留水２ｍｌあたり１ｍｇの実施例１７で得られた高純度ＴＭを含む溶液を調製した。このＴＭ溶液にマンニトール１０ｍｇを添加した。また別にこのＴＭ溶液にアルギニン−塩酸塩０．１ｍｍｏｌを添加した。これらの溶液を２ｍｌずつガラスバイアル瓶に分注し、凍結乾燥を行った。凍結乾燥は−４０℃で１８時間予備凍結し、−４０〜＋２０℃、真空度０．０５〜０．０１ｍｍＨｇで４０時間一次乾燥し、ついで２０℃、真空度０．０５〜０．０１ｍｍＨｇで６時間二次乾燥した。得られた凍結乾燥品は、紙箱内で遮光保存した。場合によっては、褐色ガラスバイアルを用いる。
【０１８１】
【発明の効果】
本発明は、抗血液凝固剤あるいは血栓溶解剤等として有効な医薬品となりうる可溶性トロンボモジュリンの製造において、血清由来物および抗体由来を実質的に含まない、高純度トロンボモジュリンの製造方法を提供できる。そして、その培地を用いるトロンボモジュリンの製造方法を提供できる。
【０１８２】
【配列表】

【図面の簡単な説明】
【図１】本発明の実施例２における浮遊生細胞密度（×１０⁵ ｃｅｌｌｓ／ｍｌ）の経時変化を示すグラフである。
【図２】本発明の実施例２におけるＴＭの累積生産量（ｍｇ）の経時変化を示すグラフである。
【図３】本発明の実施例２におけるＴＭの比生産速度（ｐｇ／ｃｅｌｌ／ｄａｙ）の経時変化を示すグラフである。
【図４】本発明の実施例２におけるＴＭの浮遊生細胞密度（×１０⁵ ｃｅｌｌｓ／ｍｌ）に対する比生産速度（ｐｇ／ｃｅｌｌ／ｄａｙ）の変化を示すグラフである。[0001]
[Industrial application fields]
The present invention relates to a method for producing highly pure soluble thrombomodulin substantially free of serum-derived and antibody-derived products.
[0002]
[Prior art]
Thrombomodulin is conventionally known as a substance having an action of promoting protein C activation by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation / fibrinolysis system, and is activated by the action of thrombin to become active protein C. Active protein C is involved in the production of plasminogen activator which inactivates active factor V and active factor VIII of blood coagulation factor in vivo and has thrombolytic activity [Koji Suzuki, Ayumi Medicine, Vol. 125, 901 (1983)]. Thrombomodulin promotes the activation of protein C by thrombin to produce a large amount of active protein C exhibiting anticoagulant action and thrombolytic action. Therefore, thrombomodulin greatly contributes to anticoagulation and thrombolysis in the living body.
[0003]
Conventional uses of thrombomodulin include, for example, myocardial infarction, thrombosis (eg, acute or chronic cerebral thrombosis, arterial or venous acute or chronic peripheral thrombosis, etc.), embolism (eg, acute or chronic phase) Cerebral embolism, arterial or venous acute or chronic peripheral embolism, etc.), peripheral vascular occlusion (eg, Buerger's disease, Raynaud's disease, etc.), obstructive arteriosclerosis, functional disorder secondary to cardiac surgery, transplanted organ Complications, intravascular blood coagulation syndrome (DIC), angina pectoris, transient cerebral ischemic attack, pregnancy toxemia, diabetes, liver VOD (Liver Veno-occlusive disease); hepatic vein after fulminant hepatitis or bone marrow transplantation It is expected to be used for the treatment and prevention of diseases such as obstruction) and deep vein thrombosis (DVT).
[0004]
Human-derived thrombomodulin has been found as a membrane protein present on the cell surface of vascular endothelial cells [W. G. Owen et al. Biol. Chem. 256, 5532-5535 (1981)], Yamamoto et al. Succeeded in cloning the human thrombomodulin gene (Japanese Patent Laid-Open No. 64-6219), and it is now possible to obtain various recombinant thrombomodulins by genetic manipulation techniques. It was. In particular, thrombomodulin prepared by removing the amino acid sequence at the membrane binding site is soluble in water even in the absence of a surfactant, and is considered to be extremely suitable for use in pharmaceuticals.
[0005]
[Problems to be solved by the invention]
Needless to say, it is necessary to obtain thrombomodulin in large quantities and at a lower cost as a premise for use in pharmaceuticals, but in recent years, especially from the production of heterogeneous proteins, especially from cows, etc. It has been pointed out that it has an effect on the safety of antibodies and antibodies derived from mice. (ICH Q6B February 6, 1998 p7 and p25)
Therefore, establishment of a highly safe method for producing thrombomodulin that is sufficiently efficient as an industrial level has been desired. In order to increase the production of thrombomodulin, a method of culturing cells at a high density is generally employed. However, if culture is performed using a normal medium, the density of the cells may have a negative effect. There has been a problem that the production rate per hour per cell (hereinafter referred to as the specific production rate) is lowered, and the expected improvement in productivity due to the increase in density cannot be obtained.
[0006]
When thrombomodulin is produced efficiently by culturing and growing using animal cells that can produce soluble thrombomodulin, especially animal cells using transformants obtained by genetic recombination, generally bovine serum components, etc. However, the final purified product usually contains a trace amount of serum-derived substances. In addition, affinity chromatography using an antibody against thrombomodulin as a ligand can be used as a means to efficiently remove impurities such as serum-derived substances and greatly improve the purity. It was necessary to check the mixing of ingredients sufficiently.
[0007]
These heterologous proteins are basically antigenic to humans, and it has been pointed out that their contamination may cause an unexpected situation such as anaphylaxis when administered as a medicine. Recently, the risk of prion-derived Creutzfeldt-Jakob disease, etc., particularly contained in bovine serum components has also been pointed out ("Revised Preferential Measurements to Reduced the Risk of Produced Bistoberfed-D-Bando-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bedo-Bredo-Bredo-Bredo-Bredo-Bedo-Bredo-Bedo-Bd" “CBER Memorandum 12/11/96, Guidelines for Minimizing the Risk of Transmitting Agents Causing Sponsorge environmental medicine product CP No MP19”. Since these contaminating heterogeneous proteins are originally a multi-component mixture, it is very difficult to remove them to a level that does not substantially contain them by a simple method in an industrial thrombomodulin production process. Yes.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have intensively studied various conditions in order to achieve efficient production of soluble thrombomodulin, and as a result, a production medium capable of efficient and stable production. Succeeded in finding out. However, this efficient production condition preferably requires the addition of serum components. For example, even when affinity chromatography carrying an antibody against thrombomodulin is used as the main purification step, the final thrombomodulin purified product can be obtained. It was not always easy to remove a trace amount of serum-derived substances mixed in the sample with good reproducibility. Conversely, when antibody affinity chromatography is used, it was not in a situation where a trace amount of antibody-derived material released from the carrier could be easily avoided.
[0009]
In order to solve these problems, the present inventors first considered a solution by a simple combination of ordinary purification methods. However, a very multi-step purification process was required, and the recovery rate of thrombomodulin activity was drastically reduced. I was faced with a conflicting issue.
As a result of intensive studies to solve these problems, the present inventors carried out efficient and stable production of thrombomodulin using the production medium found by the present inventors, and then in the purification step of the soluble thrombomodulin. Inventing a method for removing the serum-derived material and antibody-derived material finally mixed efficiently and reproducibly and very easily, confirming that high-purity soluble thrombomodulin is obtained, and completing the present invention It came to.
[0010]
That is, the present invention provides a method for producing a highly pure soluble thrombomodulin,
(1) obtaining an unpurified supernatant containing soluble thrombomodulin and serum components;
(2) The obtained supernatant is contacted with an antibody against the soluble thrombomodulin and then eluted to obtain a purified soluble thrombomodulin solution,
(3) In the step of contacting the obtained soluble thrombomodulin with a cation exchanger under the conditions of specific conductivity of 25 to 34 ms / cm and pH of 3 to 4, the soluble thrombomodulin is obtained as a flow-through fraction.
The present invention provides a method for producing high-purity soluble thrombomodulin substantially free of serum-derived and antibody-derived products.
[0011]
Furthermore, this invention relates to the culture medium which can manufacture efficiently the thrombomodulin which consists essentially of the component described in the following Table 1 of components.
[0012]
[Formula 4]

[0013]
In the present invention, thrombomodulin (hereinafter, thrombomodulin may be abbreviated as TM) is defined as a substance having an action of promoting protein C activation by thrombin. For human TM, see S.I. Yamamoto et al. [International Publication No. WO88 / 05053] obtained the cDNA, and the full-length amino acid sequence having a signal sequence encoded by the cDNA is 575 amino acids, of which the signal sequence is usually considered to be 18 amino acids. Therefore, 557 amino acids have been disclosed as the main mature peptide. Moreover, as this signal sequence, many known signal sequences such as those derived from human, yeast, and Escherichia coli, including human tPA (tissue plasminogen activator) signal, can also be used.
[0014]
Examples of the soluble TM described in the present invention include a soluble TM that is soluble in water in the absence of a surfactant. The degree of solubility is not particularly limited as long as it can be easily dissolved in normal distilled water (neutral) in the absence of a surfactant, for example, 1 mg / ml or more, preferably 3 mg / ml or more, particularly preferably A soluble TM of 10 mg / ml or more is exemplified. The soluble TM described in the present invention is preferably a soluble peptide having the amino acid sequence of human TM or a partial sequence thereof, but a soluble peptide having a structure having high homology with human TM is also a preferred example. For example, a peptide having 60% or more homology in the amino acid sequence with human TM can be mentioned as a preferred example, more preferably 70% or more or 80% or more, particularly preferably 90% or more. As the soluble TM described in the present invention, for example, a TM obtained by removing the transmembrane domain and the intracellular domain from the above-mentioned full-length amino acid sequence is conceivable. As a preferred sequence, the structure of 498 amino acids is also described in the present application. Has been. Furthermore, the structure of 114 amino acids (that is, the amino acid sequence at positions 17 to 130 of SEQ ID NO: 1) is also pointed out as a preferable sequence that can completely represent the activity of TM and is soluble [M. Zushi et al. Biol. Chem. 266, 1988-19889 (1991)], 114 amino acid high homology variants as the molecular species that retain the original activity of TM, or peptides that contain these active centers and are soluble. However, it is highly expected that the original activity of thrombomodulin will be retained to some extent even if there are some amino acid deletions, substitutions or additions without completely including the above 114 amino acids.
[0015]
In addition, it is known that the human TM gene has a polymorphic mutation, and valine (specifically, the amino acid sequence at positions 17 to 130 of SEQ ID NO: 1 is related to the 473rd amino acid residue from the start codon. (Yamamoto et al .; Japanese Patent Laid-Open No. 64-6219) and alanine (specifically, a peptide including the amino acid sequence at positions 17 to 130 of SEQ ID NO: 2) [D. Wen et al .; Biochemistry 26, 4350-4357 (1987)], but these mutations are unrelated to TM activity and are also unrelated to diseases such as thrombosis. [V. D. Varden et al .; Thrombosis and Haemostasis, 65, 511-513 (1991)], and can be treated equally in the present invention.
[0016]
Thus, for example, the amino acid sequence at positions 17-130 of SEQ ID NO: 1, the amino acid sequence at positions 17-130 of SEQ ID NO: 2, and the amino acid sequence in which one or several amino acids of these amino acid sequences are deleted, substituted or added An amino acid sequence containing any amino acid sequence selected from the group consisting of and a gene encoding it. Further, the amino acid sequence at position 1-130 of SEQ ID NO: 1, the amino acid sequence at position 1-130 of SEQ ID NO: 2, the amino acid sequence at position 1-516 of SEQ ID NO: 3, the amino acid sequence at position 1-516 of SEQ ID NO: 4, Preferred examples include any amino acid sequence selected from the group consisting of amino acid sequences in which one or several amino acids in the amino acid sequence are deleted, substituted or added, and a gene encoding the amino acid sequence. Details of the preparation of these various peptides are described in International Publication No. WO 98/01153.
[0017]
Of course, the TM of the present invention is not particularly limited as long as it has the above-mentioned properties. For example, the TM may or may not contain a sugar chain.
As an unpurified supernatant containing soluble thrombomodulin and serum component used in the present invention, a culture supernatant prepared by culturing animal cells capable of producing soluble thrombomodulin using a medium containing serum component is used. Is preferred.
[0018]
The cells capable of producing the soluble TM used in the present invention are not particularly limited as long as they are cells capable of producing the soluble TM, and usually mean animal cells. Various cells derived from humans and other animals derived from other animals are used. Cells can be used. Examples of the human-derived cells include human vascular endothelial cells, placental tissue-derived syncytial cells, human lung cancer cell lines A549 cells, megakaryocyte cell lines Meg-O1 cells, and the like. . As other animal-derived cells, known cells are known, and various cells described later can be used.
[0019]
Whether cells capable of producing such a soluble TM can be used in the production method of the present invention depends on, for example, the TM activity of various human-derived cells and various cells derived from other animals and the reaction with TM. Can be easily identified by examining the reactivity with the antibody. Specifically, the culture supernatant of the cells to be screened and the cells themselves are screened according to the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a. A method of selecting a cell whose activity has been detected as a cell capable of producing the TM of the present invention is exemplified. Further, for example, TM is detected from the cell culture supernatant or cell extract according to the ELISA described in Example 1- (4) -c, and the desired cell is selected, or the cell itself is immunostained. It is also possible to detect by doing.
[0020]
In addition, as the unpurified supernatant of the present invention, a full-length TM expressed on a cell membrane is extracted, and a culture solution or an extract that is easily converted to a soluble TM by a protease treatment such as elastase is used. Can do.
In the present invention, a cell particularly preferable as a cell capable of producing soluble TM is a transformant prepared by introducing a gene (DNA sequence) encoding soluble TM. The gene encoding the soluble TM in the present invention is not particularly limited as long as it is a gene capable of encoding the soluble TM described above, and includes various DNA sequences, which are according to known methods. Can be prepared. For example, a gene encoding such a soluble TM and a preparation method thereof are described in detail in known documents such as International Publication No. WO88 / 05053. Most conveniently, pSV2TMJ2 described in Example 1- (1) of International Publication No. WO88 / 05053 [ATCC Deposit No. 67283, or Escherichia coli DH5 / pSV2TMJ2 is used again (FERM BP-5570) Thus, a gene encoding a soluble TM of mutants such as various deletions, mutations, additions and substitutions can be prepared according to a conventional method of gene manipulation. Moreover, it can also prepare with reference to the method of Example 1 of Unexamined-Japanese-Patent No. 5-213998. As the DNA sequence encoding these soluble TMs, at least a DNA sequence encoding substantially soluble TM may be used, but it is usually preferable to include a nucleotide sequence encoding a signal sequence. Examples of the signal sequence include a base sequence encoding the amino acid sequence of 1-18 or 1-16 of SEQ ID NO: 3. That is, as a DNA sequence that substantially encodes TM, specifically, for example, a DNA sequence of SEQ ID NOs: 1 to 4 is preferably introduced as a preferred example.
[0021]
In the case of introducing a DNA sequence encoding a soluble TM into a host cell, a method for introducing the DNA sequence encoding the soluble TM into a vector, particularly preferably an expression vector that can be expressed in an animal cell, is preferably introduced. Can be mentioned. An expression vector is a DNA molecule comprising a promoter sequence, a sequence that gives a ribosome binding site to mRNA, a DNA sequence that encodes a protein to be expressed, a splicing signal, a termination terminator sequence for transcription termination, a replication origin sequence, etc. Examples of animal cell expression vectors include R.I. C. Mulligan et al. [Proc. Natl. Acad. Sci. U. S. A. 78, 2072 (1981)], pSV2-X and P.P. M.M. And pBP69T (69-6) reported by Howley et al. [Methodology Emzymology, 101, 387, Academic Press (1983)].
[0022]
In addition, it is possible to carry out a treatment for making a cell capable of producing soluble TM that can be used in the production method of the present invention into a higher-producing cell. For example, dihydrofolic acid that provides resistance to methotrexate (MTX) A method for obtaining a MTX-resistant strain by introducing a DNA sequence encoding reductase (DHFR) into a cell, or a transformant in which a DNA sequence encoding DHFR and a DNA sequence encoding TM are both introduced into a host cell Thus, a method for obtaining an MTX resistant strain is exemplified. DHFR is an enzyme that produces tetrahydrofolic acid necessary for de novo synthesis of purine, thymidylic acid, and glycine among two pathways of DNA synthesis. In CHO cell DHFR-deficient strains, DNA synthesis is defective only in the remaining salvage pathway because of a defect in the de novo pathway. Therefore, purine, thymidylic acid and glycine are required for its growth. If a medium containing no purine or the like is used, the DHFR-deficient strain is killed, and only cells carrying DHFR can be selected and used as a selection marker. MTX is an inhibitor of DHFR, which inhibits cellular DNA synthesis and kills cells. As a mechanism for acquiring MTX resistance of cells, G.I. A. Fischer et al. [Biochem. Pharmcol. 11, 1233-1237 (1962)], reported decreased intracellular transport of MTX, F. Flintoff et al. [Cell, 2, 245-262, (1976)] reported DHFR mutants with reduced affinity for MTX, R.M. T. T. This is based on the amplification of the DHFR gene reported by Schimke et al. [Science, 202, 1051 (1978)]. In the present invention, a DHFR gene that causes gene amplification is preferable. For example, DHFR derived from any species such as human, monkey, cow, rabbit, hamster, rat, mouse and the like may be used. C. Y. Chang et al. [Nature, 275, 617-624 (1978)] and J. Am. H. It is a mouse DHFR gene whose sequence was elucidated by Nunberg et al. [Cell, 19, 355-364 (1980)]. In the case of using a DNA sequence encoding DHFR, DNA containing substantially only a DNA sequence encoding DHFR may be used, but a method of incorporating it into an expression vector that can be expressed in animal cells is preferable. For example, an expression vector that expresses mouse DHFR in animal cells such as pSV2DHFR (ATCC37146) can be mentioned.
[0023]
When a DNA sequence encoding a soluble TM is introduced into a host cell, animal cells cotransformed with an expression vector encoding the soluble TM and an expression vector encoding DHFR are obtained. Transformation means that DNA is taken into a cell by phagocytic ability or forced method of the cell, and a DNA trait is expressed in a plasmid state or a state integrated into a chromosome. It can be introduced by any method such as calcium phosphate method, DEAE-dextran method, pricking method, protoplast fusion method, and electroporation method. This is a method of introducing a complex of calcium phosphate and DNA into cells according to Wigler et al. [Cell, 14, 725 (1978)]. As described above, the shape of the DNA at this time may be circular DNA or linear DNA.
[0024]
Examples of animal cells used as a host include VERO cells, Hela cells, Chinese hamster (CHO) cells, W138, BHK, hamster AV-12-664 cells, and human 293 cells. Preferably, CHO cells, more preferably L. H. Graf et al. [Molec. Cell Biol. 2, 93-96 (1982)], and DHFR-deficient strains such as CHO-DXB11 strain are exemplified.
[0025]
The cotransformed cells thus obtained are selected under increasing concentration conditions of MTX concentration. When MTX is gradually increased, it may be continuously or stepwise, but stepwise is usually preferable. The range of the concentration of MTX is usually 20 nM to 100 mM, preferably 20 nM to 10 mM. Prior to selection with MTX, preliminary selection with a medium not added with MTX and confirmation of TM productivity using the specific production rate described below as an index may be performed. Confirmation may be performed. In order to obtain high-producing cells, it is preferable to increase the concentration of MTX by 3 to 5 times the initial concentration and perform as many steps as possible, but it is preferable to perform at least 5 steps. Furthermore, it is preferable to carry out the process while examining the specific production rate of TM at each stage.
[0026]
Thus, cells capable of producing soluble TM that can be used in the production method of the present invention can be obtained.
In the medium for producing a soluble TM of the present invention, serum is not particularly limited, but usually includes animal serum, preferably bovine fetal, newborn, calf, and adult bovine serum. Examples of growth factors that can replace serum include albumin, insulin, transferrin, ethanolamine, sodium selenite, or a mixture thereof. Whether to use serum or growth factor can be appropriately selected according to the properties of the cells capable of producing the TM to be used. That is, when TM-producing cells are proliferative in the absence of serum, each method using only serum, only growth factor, or both serum and growth factor can be selected. Moreover, when TM production cells do not show growth in the absence of serum, each method using only serum or both serum and growth factor can be selected. Depending on whether serum or growth factor is used or a mixture is used, the amount of addition can be determined as appropriate. In the case of serum, however, 2 to 10% (v / v) is typically exemplified. In the case of factors, for example, albumin is about 3 mg, insulin is about 5 mg, transferrin is about 5 mg, ethanolamine is about 2 μM, and sodium selenite is about 20 nM.
[0027]
The soluble TM production medium of the present invention may be substantially composed of each component described in the above-mentioned component table 1. In addition to those described in component table 1, usually, antibiotics such as tylosin, gentamicin, and kanamycin are used. Addition is not particularly disturbed.
The pH of the medium for producing a soluble TM of the present invention is not particularly limited as long as it does not interfere with cell culture, but is usually pH 6 to 8, preferably about pH 7.
[0028]
The medium substantially consisting of the components described in the above-mentioned component table 1, which is the soluble TM-producing medium of the present invention, has a composition ratio of the components of the medium being the composition ratio described in the following component table 2. A further preferred example is given.
[0029]
[Chemical formula 5]

[0030]
The error range of the numerical values described as the composition ratios in the component table 2 is usually 10%, preferably about 5%. The moisture may be adjusted as appropriate.
As the medium for producing the soluble TM of the present invention, the composition ratios of the components of the medium other than the composition ratios described in the above-mentioned component table 2 can also be selected. An appropriate composition ratio can be examined, and a preferable medium composition ratio can be selected depending on cells capable of producing soluble TM, culture conditions, and the like.
[0031]
[Screening method for composition ratio of medium components]
Suspend the required number of cells for which soluble TM is scheduled to be produced in a culture medium to be screened with various component ratios, dispense into a cell culture dish, and in a carbon dioxide incubator for 1 to 3 days. Incubate for days. When measuring the specific production rate, the measurement of TM activity and the measurement of the viable cell density by the above-mentioned measurement method are performed on the culture supernatant of the cultures 1, 2, and 3, and the specific production rate (time per cell) Per production rate). When the final concentration of TM is used as an index, the TM concentration after 3 days of culture and the viable cell density are measured.
[0032]
In the present invention, the culture for producing cells capable of producing TM using the above-mentioned soluble TM production medium may be carried out by a known method, and the cells capable of producing the selected TM can be obtained. Appropriate conditions can be selected according to the properties. For example, the adhesion-dependent nature of cells, i.e., adherent cells or suspension cells, and if adherent cells, microcarriers made of Sepharose, cellulose, glass, gelatin, polystyrene, polyethylene, etc. as adherent carriers, multi-tray, A roller bottle, a hollow fiber, etc. can be used. The culture environment conditions can be selected according to the nature of the cell. For example, the temperature is 30 ° C. to 40 ° C., preferably around 37 ° C., 20 to 200 rpm when stirring is required, preferably around 80 rpm, and the dissolved oxygen concentration (DO) is Examples include adjustment to about 3% to 90%, preferably about 50%, and pH of 6 to 8, preferably about 7, with respect to water air saturation. In addition, as a method for mixing the culture solution necessary for keeping the culture environment condition constant, there are a static convection type, a stirring type using a stirring blade, an air lift type using aeration, or a fluidized bed type using a circulation pump or a stirrer.
[0033]
In the present invention, the medium for producing soluble TM of the present invention is used in the culture for producing soluble TM. In the step of growing cells, other medium may be used, but the soluble medium of the present invention is used. It is also preferred that the TM production medium be used consistently from cell growth to culture for TM production. When other media are used for cell growth, various known media can be used as the other media, and examples thereof include DMEM supplemented with serum and / or growth factors. The cells to be proliferated are often stored frozen, and in such cells, they are quickly thawed in a 37 ° C. constant temperature bath, suspended in an appropriate medium, centrifuged, and after centrifugation, the supernatant is removed by suction. It is better to suspend in a fresh medium and transfer to a culture container such as a spinner flask or a dish for cell culture to perform culture.
[0034]
The frequency of medium exchange during cell growth is such that the supernatant corresponding to one culture amount is continuously or intermittently removed every 1 to 6 days, and the same amount of fresh medium as the removed supernatant is continuously or intermittently removed. The medium can be changed by adding.
And after reaching the cell density necessary for the culture for producing soluble TM, preferably after reaching the saturation density, the supernatant corresponding to one culture amount is preferably continuously or intermittently every 1 to 3 days. The medium can be replaced by adding a fresh medium in an amount equal to the removal of the supernatant continuously or intermittently.
[0035]
In addition, in the culture for producing soluble TM without exchanging the medium, the culture is performed for an appropriate period, preferably until the maximum cumulative productivity is obtained, and the whole culture solution is used as a culture containing the target product. Can be harvested.
Examples of the method for collecting the culture supernatant include a batch method, a repetitive batch method, and a continuous method. Examples of the separation of cells and culture supernatant include gravity sedimentation, centrifugation, and membrane separation. Culture supernatant containing soluble TM is used for subsequent purification steps. When these culture supernatants are stored, for example, storage at 10 ° C. to −80 ° C. is exemplified.
[0036]
Next, the method for purifying soluble TM from the culture supernatant obtained as described above can be performed according to a commonly used technique [edited by Takeichi Horio; basic experiment method for proteins and enzymes]. For example, it is also preferable to use a chromatographic support on which a functional group having a charge opposite to that of the soluble TM is immobilized and ion exchange chromatography utilizing an interaction between the soluble TM. Moreover, the gel filtration chromatography and ultrafiltration using the molecular weight size of soluble TM are mentioned. In addition, hydrophobic chromatography using a hydrophobic bond between a chromatographic carrier on which a hydrophobic group is immobilized and a hydrophobic site of the soluble TM is mentioned. These methods can be appropriately combined. Furthermore, a preferred example of the main purification step is affinity chromatography utilizing specific affinity with soluble TM. Preferred examples of the adsorbent include thrombin, which is a TM ligand, and a soluble TM antibody. As these antibodies, antibodies having appropriate properties or soluble TM that recognize appropriate epitopes can be used. For example, JP-B-5-42920, JP-A-64-45398, and JP-A-6 -205692 gazette etc. are mentioned. The degree of purification can be selected depending on the purpose of use. For example, electrophoresis, preferably SDS-PAGE results can be obtained as a single band, or isolated purified products can be obtained by gel filtration HPLC or reverse phase HPLC. It is desirable to purify until one peak is reached.
[0037]
However, in order to use soluble TM for pharmaceutical purposes, the purification process that is simply a combination of the above-described commonly used purification methods does not substantially contain contaminating proteins that may have problems in terms of safety. Is difficult to remove. Contaminating proteins having such problems include, for example, bovine serum components contained in medium components used for cell culture growth and soluble TM production, and when CHO cells are used as production base cells, for example. In the case of using an antibody affinity column for Chinese hamster-derived components and main purification, for example, heterologous proteins such as mouse anti-TM antibody components can be mentioned. These heterologous proteins are basically antigenic to humans, and their contamination can cause unexpected situations such as anaphylaxis when administered as a medicine. Recently, the risk of Creutzfeldt-Jakob disease derived from prions, particularly in bovine serum-derived products, has also been pointed out. Since these contaminating heterogeneous proteins are originally a multi-component mixture, it is very difficult to remove them to a level that does not substantially contain them by a simple method in an industrial soluble TM production process. .
[0038]
A specific example of the purification method is a method of purification using TM activity, for example, the activity of promoting protein C activation by thrombin as an index. For example, the culture supernatant is roughly purified with Q-Sepharose FF of an ion exchange column. Fractions with TM activity are collected, then purified with an affinity column mouse anti-TM monoclonal antibody, fractions with strong TM activity are collected, the collected fractions are concentrated, and subjected to gel filtration to obtain TM active fractions A purification method [Gomi et al; Blood, 75, 1396-1399, 1990]. In this case, after purification with an anti-TM antibody column, a cation exchanger according to the present invention, preferably a cation exchanger having a sulfopropyl group, such as SP-Sepharose FF (Pharmacia), which is a strong cation exchanger, is optimal. When used under mild conditions, the bovine serum-derived material and mouse antibody-derived material derived from the medium can be simultaneously removed in one step, and a high-purity TM can be easily obtained.
[0039]
Examples of preferred purification methods are as follows.
Select an appropriate ion exchange resin having soluble TM and good adsorption conditions, and perform ion exchange chromatography purification. A particularly preferred example is a method using Q-Sepharose FF equilibrated with 0.02 M Tris salt buffer (pH 7.4) containing 0.18 M NaCl. After appropriate washing, elution is carried out with, for example, 0.02M Tris-HCl buffer (pH 7.4) containing 0.3M NaCl to obtain a soluble TM of the crude product.
[0040]
Next, for example, a substance having specific affinity for soluble TM can be immobilized on a resin and affinity chromatography purification can be performed. Preferred examples include a DIP-thrombin-agarose column and an anti-TM monoclonal antibody column. The DIP-thrombin-agarose column is pre-equilibrated with, for example, 20 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 0.5 mM calcium chloride, charged with the above crude product, and washed appropriately. For example, the purified TM can be obtained by elution with 20 mM Tris buffer (pH 7.4) containing 1.0 M NaCl and 0.5 mM calcium chloride. In the anti-TM monoclonal antibody column, Sepharose 4B (Pharmacia) previously activated with CNBr is contacted with 0.1 M NaHCO3 buffer solution (pH 8.3) containing 0.5 M NaCl and dissolved in Sepharose. A column packed with a resin in which anti-TM monoclonal antibody is coupled to 4B is previously equilibrated with, for example, 20 mM phosphate buffer (pH 7.3) containing 1.0 M NaCl, and after appropriate washing, for example, 0.3 M A method of elution with 100 mM glycine hydrochloride buffer (pH 3.0) containing NaCl is exemplified.
[0041]
Next, the purified soluble thrombomodulin solution obtained above is equilibrated, for example, under conditions of specific conductivity of 25 to 34 ms / cm and pH of 3 to 4, depending on the salt concentration, pH measurement accuracy and TM molecular species. SP-Sepharose FF (Pharmacia), which is a cation exchanger, preferably a strong cation exchanger, is charged. The specific conductivity is more preferably 30 ± 3 ms / cm, and the pH is preferably pH 3.0 to 3.7, more preferably pH 3.5 ± 0.1. For these conditions, it is preferable to use a buffer solution to which a salt of an appropriate concentration is added, and various modes are conceivable. For example, 0.25 to 0.32 M, preferably 0.3 ± 0.01 M A buffer solution having a pH of 3 to 4 and containing 50 to 150 mM is exemplified. Although it does not specifically limit as a kind of buffer solution, For example, a glycine-hydrochloric acid, a citric acid-disodium phosphate, a citric acid-sodium citrate, an acetic acid-sodium acetate is illustrated. If the above conditions are shown more specifically, a 100 mM glycine hydrochloride buffer (pH 3.5) containing 300 mM NaCl is exemplified. The specific conductivity can be easily measured by, for example, a portable conductivity meter (manufactured by Toa Denpa Kogyo Co., Ltd., P series CM-11P, conversion reference temperature 25 degrees).
[0042]
The above column is washed with, for example, 100 mM glycine hydrochloric acid buffer solution (pH 3.5, specific conductivity 31 ms / cm) containing 300 mM NaCl, and the passing fraction from the rising edge to the falling edge of the absorbance at 280 nm is measured. The soluble TM that has been obtained and neutralized with an appropriate buffer and purified to such an extent that it does not substantially contain serum-derived products and antibody-derived products can be obtained as a highly purified product. As will be described later, it is confirmed that the use of this column can remove serum-derived and antibody-derived products with high efficiency and reproducibility. Of course, these can be appropriately concentrated by ultrafiltration.
[0043]
Furthermore, it is also preferable to perform buffer exchange by gel filtration. For example, a Sephcryl S-300 column or S-200 column equilibrated with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl is charged with a highly purified product concentrated by ultrafiltration, and 50 mM NaCl is charged. Development with a 20 mM phosphate buffer solution (pH 7.3) containing, confirming the activity of promoting the activation of protein C by thrombin, collecting the active fraction, and obtaining a high-purity purified product with buffer exchange it can.
[0044]
Soluble TM high-purity purified products that have the target purity and that have been buffer exchanged can be formulated by lyophilization. For example, a method for lyophilizing soluble thrombomodulin obtained from a transformant obtained by introducing a gene encoding the amino acid sequence shown in SEQ ID NO: 3 can be performed according to the method described in JP-A-6-321805. The formulated soluble TM can be stored in light-shielding conditions, such as in a dark room, box, or brown bottle.
[0045]
In a known normal medium, for example, Dulbecco's modified Eagle medium (hereinafter referred to as DMEM), which is preferably used for mass culture, the TM production rate per hour (hereinafter referred to as specific production rate) increases as the cell density increases. In contrast to the marked decrease, the soluble TM production medium of the present invention produced soluble TM efficiently and gave very favorable results.
[0046]
BEST MODE FOR CARRYING OUT THE INVENTION
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
[0047]
Example 1
Cell production
(1) Construction of plasmid pSV2TMD123
PSV2TMJ2 described in Example 1- (1) of the international patent application (International Publication No. WO88 / 05053) [ATCC Deposit No. 67283, and again deposited as Escherichia coli DH5 / pSV2TMJ2 (FERM BP-5570) ] After complete digestion with NcoI, the digested ends were E. coli. Blunt ends were obtained using E. coli DNA polymerase. Then, it was completely digested with HindIII to obtain a DNA fragment of about 1900 bp. The obtained DNA fragment was designated TMJ3. On the other hand, phage M13mp19 (Takara Shuzo, Japan, catalog number 3119) was digested with HindIII and HincII to prepare a vector. The DNA fragment TMD3 was inserted into this vector to obtain a recombinant plasmid M13mp19TMJ3.
[0048]
Separately, a DNA probe for deletion shown below (hereinafter referred to as “deleter”) was organically synthesized: 5′-GGAGGCCCTCCAGCCCGAATGCAGG-3 ′ (25mer, (Sequence Table 5). This synthetic deleter was referred to as TMD. Site-specific mutagenesis using the thus-prepared Deleter TMD according to the method described in Method in Enzymology, Volume 100, 468, (1983), Academic Press The portion consisting of 177 bases of the recombinant plasmid M13mp19TMJ13 obtained as described above was deleted by the method described above: 25 pmol of the deleter TMD and 10 pmol of the M13 primer M3 (Univa -After phosphorylating the 5 'end of Monkey Primer (Takara Shuzo, Catalog No. 3831) using T4 kinase, 0.5 pmol of single-stranded DNA of recombinant plasmid M13mp19TMJ3 was added, heated at 95 ° C for 5 minutes, and then brought to room temperature. Until cooled.
[0049]
Then 5 units of E. coli. E. coli DNA polymerase I (Klenow Fragment) and 10 units of T4 DNA ligase were added to the mixture and incubated at 37 ° C. for 30 minutes to produce a recombinant plasmid in the mixture. The resulting mixture was added to E. coli JM105 (Pharmacia, Sweden, catalog number 27-1550). This E. coli was thereby transfected with a recombinant plasmid. Plaques on an agar medium produced by overnight culture at 37 ° C. were transferred to a nitrocellulose filter, heated at 80 ° C. for 2 hours, and then prehybridized. Prehybridization was performed using 6 × SET [0.9 M NaCl, 180 mM Tris buffer (pH 8.0), 6 mM EDTA], 5 × Denharts ′ [0.1% (w / v) Ficoll, 0.1% (W / v) polyvinylpyrrolidone, 0.1% (w / v), bovine serum albumin (BSA)], 0.1% SDS, 100 μg / ml denatured salmon sperm DNA, 55 ° C., 2 hours Performed by warming.
[0050]
Then, instead of denatured salmon sperm DNA in the above solution ³² A hybridization reaction was performed at 55 ° C. for 2 hours using a solution to which TMD labeled with P was added. The nitrocellulose filter was then washed with 6 × SSC (0.9 M sodium chloride, 0.09 M trisodium citrate aqueous solution). Washing was carried out twice at room temperature for 5 minutes, and then the temperature was raised stepwise to 55 ° C., 65 ° C., and 75 ° C., and each was washed twice for 5 minutes. When X-ray film XAR-5 (Eastman Kodak, USA) was adhered to the obtained nitrocellulose filter and exposed at −80 ° C. overnight, several tens of black spots were strongly exposed on the X-ray film. was detected. Each spot corresponds to a clone infected with the recombinant plasmid. Of these, 6 clones were selected, and the recombinant plasmids of each clone were isolated and subjected to restriction enzyme analysis and base sequence analysis. The recombinant plasmids possessed by these clones had restriction sites and base sequences. Each was found to be identical. The resulting recombinant plasmid was designated M13TMD123. Furthermore, this recombinant plasmid M13TMD123 was found to have a DNA fragment containing a base sequence containing a base sequence encoding human thrombomodulin consisting of 516 amino acids starting from the start codon (ATG) shown in Sequence Listing 5. . The DNA fragment contained in this recombinant plasmid M13TMD123 was called TMD123.
[0051]
This recombinant plasmid M13TMD123 was completely digested with HindIII and BamHI to isolate a DNA fragment of about 1900 bp of TMD123. On the other hand, plasmid pSV2DHFR (ATCC 37146) was completely digested with HindIII and BglII to obtain a vector fragment. This vector fragment and the previous DNA fragment TMD123 were spliced using T4 DNA ligase to obtain plasmid pSV2TMD123.
[0052]
(2) Transformation of CHO cells with plasmid pSV2TMD123 and plasmid pSV2-DHFR
About 40 μg of the plasmid pSV2TMD123 obtained in Example 1-1 above and about 3.5 μg of the plasmid pSV2DHFR were mixed and ethanol precipitated. The precipitate was air-dried, dissolved in 450 μl of TE (pH 7.9, 1 mM Tris-HCl buffer, 0.1 mM EDTA), 50 μl of 2.5 M CaCl 2 was added dropwise, and 500 μl of 2 × HBS (50 mM HEPES, 280 mM NaCl). 1.5 mM Na2 HPO4, pH 7.12) was added and mixed. Then, after standing on ice for 10 minutes, 1 ml of 10% (v / v) fetal calf serum (hereinafter abbreviated as “FCS”) and 1 v / v% penicillin-streptomycin (manufactured by Flow Laboratory, USA, catalog number 16- 700-49) and suspended in Ham's F-12 medium (manufactured by Flow Laboratory, USA, catalog number 10-421-20). On the other hand, a Ham's F-12 medium containing 10% (v / v) FCS and 1% (v / v) penicillin-streptomycin was added to a tissue culture plate having a diameter of 6 cm, and the number of cells per plate was about 5 × 10. ^Five The CHO-DXB11 strain (Columbia University, USA, dispensed from Dr. LA Chasin) was cultivated overnight, the medium was replaced with a fresh medium, and further cultured for 4 hours. This CHO-DXB11 strain was overlaid with the above-mentioned plasmid DNA solution in which CaCl2 was dropped, and cultured at 37 ° C. for about 8 hours. Wash 3 times with 5 ml PBS (-) (Flora Laboratories, USA, catalog number 28-103-05), and after washing with 5 ml of the above medium, add 5 ml of fresh medium for 3 days. Further culture was performed.
[0053]
(3) Selection of DHFR producing strain from CHO cells transformed with plasmid pSV2TMD123 and plasmid pSV2-DHFR
The cells transformed in Example 1- (2) were peeled off using a 0.25% trypsin and 0.02% EDTA solution, spread on four tissue culture plates having a diameter of 10 cm, and cultured. After 24 hours, the medium was replaced with selective medium. The composition of the selective medium is 10% (v / v) dialyzed FCS, 150 μg / ml proline, 1% (v / v) penicillin-streptomycin DMEM (US, Flow Laboratories, catalog number 17-101-22). is there. The cells were cultured for about 2 weeks while changing the medium every 3 to 4 days, and the transformed cells were cloned to obtain 59 DHFR-producing strains.
[0054]
(4) Selection of TM production strain from DHFR production strain
Each clone of the DHFR producing strain obtained in Example 3 was cultured on a 96-well tissue culture plate, and when it became confluent, the medium was replaced with 100 μl of DMEM containing 150 μg / ml proline. Thereafter, the cells were cultured for 24 hours, and the culture supernatant was collected. Of these, 50 μl was incubated overnight at 4 ° C. in a 96-well flat-bottom ELISA plate (manufactured by DYNATECH, USA). The amount of TM in this culture broth was quantified by enzyme immunoassay (hereinafter abbreviated as “ELISA”) using an anti-human thrombomodulin polyclonal antibody. Details are described below.
[0055]
(4) -a Purification of immunogen
The immunogen of anti-human thrombomodulin polyclonal antibody was obtained by extracting from human lung as follows. About 800 cm of human lung specimen provided by a public hospital is cut into a size of about 1 cm square, and 500 ml of physiological saline cooled to 4 ° C. containing 1 mM DFP (Diisopropyl Fluorophosphate) in the obtained tissue piece. Water was added and homogenized at 4 ° C. for 5 minutes using Ace Homogenizer AM-1 type (manufactured by Nippon Seiki Co., Ltd., Japan) as a Waring blender. After homogenization, the mixture was cooled in ice for 5 minutes. The mixture was then further homogenized at 4 ° C. for 5 minutes and cooled in ice for 5 minutes. The above homogenization and cooling operation was repeated three more times. The resulting homogenate is centrifuged at 12000 g at 4 ° C. for 30 minutes to separate the supernatant and pellets, and the pellets are collected. Suspended in 100 ml of 0.02 M Tris-HCl buffer (pH 7.5) containing 0.5% (v / v) Triton X-100, 0.25 M sucrose, 1 mM benzamidine hydrochloride, 0.5 mM CaCl 2, Waring blender Was used for 5 minutes at 4 ° C. to obtain a cell extract.
[0056]
The resulting extract was centrifuged at 35,000 g, 10 ° C. for 60 minutes, and the supernatant was collected. N. L. Esmon et al. [J. Biol. Chem. 257, 859 (1982)], DIP-thrombin (diisopropyl phospho-thrombin) was prepared according to the method described in P.C. Cuatrecasas's method [J. Biol. Chem. 245, 3059 (1970)] to form DIP-thrombin-agarose by binding to bromocyanated agarose.
[0057]
Next, DIP-thrombin-agarose was packed in a 2.5 cmφ × 10 cm column to prepare a DIP-thrombin-agarose column, and 0.1 M NaCl, 0.5 mM CaCl 2, 1 mM benzamidine hydrochloride, 0. The column was equilibrated with 0.02 M Tris-HCl buffer (pH 7.5) containing 5% (v / v) Lubrol PX (manufactured by Hanai Kagaku, Japan). Next, the extraction supernatant was applied to a column. The column was washed with 0.02 M Tris-HCl buffer (pH 7.5) containing 0.3 M NaCl, 0.5 mM CaCl 2, 1 mM benzamidine hydrochloride, 0.5% (v / v) Lubrol PX, and then 1 M NaCl, 0 Elution was performed with 0.02 M Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 1 mM benzamidine hydrochloride 0.5% (v / v) Lubrol PX, and 2.0 ml fractions were collected. Each fraction obtained by elution was measured for its activity to promote protein C activation by thrombin. That is, each fraction was diluted with a diluent for determination (20 mM Tris-HCl, 100 mM NaCl, 0.1% Lubrol PX, 0.1% sodium azide, 0.001% BSA, pH 7.5). 5 μl of this solution, 3 μl of thrombin (Sigma, USA, catalog number T-6759, 20 ng / μl), 10 × assay buffer (containing 1 M NaCl, 30 mM CaCl 2, 1% bovine serum albumin, 0.5 M Tris-HCl buffer , PH 8.5) and 5 μl of distilled water and 29.5 μl of distilled water were mixed and allowed to stand at 37 ° C. for 5 minutes. Then, 7.5 μl of protein C (derived from cattle, 0.2 μg / μl) was added and reacted at 37 ° C. for 30 minutes. .
[0058]
The reaction was stopped by adding 6.25 μl of stop solution (100 mM NaCl, 0.3A280 Antithrombin III, 20 μm Tris-HCl buffer containing 100 μg / ml heparin, pH 7.5). The activity of activated protein C is as follows: Boc-Leu-Ser-Thr-Arg-MCA (Japan, Peptide Institute, 5 mg / ml) 10 μl, 5 M CsCl 5 μl, substrate reaction buffer (100 mM NaCl-containing 50 mM Tris-HCl buffer, pH 8.5) 495 μl was added and reacted at 37 ° C. for 20 minutes. After 55 μl of acetic acid was added to stop the reaction, the free AMC (7-amino-7-methyl-coumarin) concentration was determined at an excitation wavelength of 380 nm and an emission wavelength of 440 nm. It measured with the fluorescence spectrophotometer (Shimadzu RF-540). The obtained fluorescence intensity was compared with the fluorescence intensity of AMC at a known concentration to determine the amount of released AMC. The value obtained by subtracting the amount of AMC when an aqueous solution containing no purified product is added from the amount of AMC represents the strength of the activity that promotes protein C activation by thrombin in the sample. The activity for producing 1 nM of activated protein C per ml of the reaction solution per minute was defined as 1 u. Simultaneously, the absorbance (A280) at a wavelength of 280 nm of each fraction was measured using a spectrophotometer UV-240 manufactured by Shimadzu Corporation (Japan).
[0059]
An active fraction that promotes protein C activation is collected and 0.02 M Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl, 0.5 mM CaCl2, 0.05% (v / v) Lubrol PX. ) And dialyzed. The obtained dialysate was subjected to a second DIP-thrombin-agarose column chromatography. Specifically, the dialysate was applied to a DIP-thrombin-agarose column having a size of 1.5 cmφ × 10 cm and 0.02 M Tris containing 0.4 M NaCl, 0.5 mM CaCl 2, 0.1% (v / v) Lubrol PX. After washing with hydrochloric acid buffer (pH 7.5), further washing with 0.02 M Tris hydrochloric acid buffer (pH 7.5) containing 0.4 M NaCl, 0.1 mM EDTA, 0.1% (v / v) Lubrol PX And then eluted with 0.02 M Tris-HCl buffer (pH 7.5) containing 1 M NaCl, 0.5 mM EDTA, 0.1% (v / v) Lubrol PX.
[0060]
The active fraction that promotes protein C activation was collected and dialyzed against 0.02 M Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl and 0.05% (v / v) Lubrol PX. . The obtained dialysate was subjected to the third DIP-thrombin-agarose column chromatography. The column size, washing conditions and elution conditions were exactly the same as those for the second DIP-thrombin-agarose column chromatography. In addition, 2 ml of fractions obtained by elution were collected. After collecting active fractions that promote protein C activation and dialyzing against 0.02 M Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl, 0.05% (v / v) Lubrol PX The sample was subjected to a fourth DIP-thrombin-agarose column chromatography having a size of 0.9 cmφ × 8 cm. After washing with 0.02 M Tris-HCl buffer (pH 7.5) containing 0.35 M NaCl, 0.5 mM CaCl2, 0.1% (v / v) Lubrol PX, 1 M NaCl, 0.5 mM EDTA, 0.1 Elution was performed with 0.02 M Tris-HCl buffer (pH 7.5) containing% (v / v) Lubrol PX. The fraction obtained by elution was collected in an amount of 1.9 ml. The fraction having activity that promotes protein C activation in the fourth DIP-thrombin-agarose column chromatography was collected and used as an immunogen.
[0061]
From the absorbance of the fraction purified in this way, the molecular extinction coefficient of the obtained purified product is defined as 10.0 (E1% 1 cm · 280 nm = 10.0) which does not become the molecular extinction coefficient of a general protein. Based on this, the amount of the purified product was calculated to be about 500 μg. The purified fraction obtained was subjected to SDS-polyacrylamide gel electrophoresis using a polyacrylamide gel concentration gradient of 5 to 10% for 2 hours at a voltage of 50 V, and the band was observed by silver staining. confirmed. Furthermore, this purified fraction was recognized to have an effect of promoting protein C activation by thrombin.
[0062]
(4) -b Preparation of anti-human thrombomodulin polyclonal antibody
An anti-human thrombomodulin polyclonal antibody (rabbit antibody) can be produced using the above-mentioned immunogen, and the book [L. hudsonet, Practical Immunology, p. 9 (1976), Blackwell Scientific Publications].
[0063]
It was confirmed as follows that this antibody reacts with the above-mentioned immunogen. That is, about 10 ng of the immunogen (purified human thrombomodulin protein) obtained in Example 1- (4) -a is spotted on a nitrocellulose filter. After air-drying, this antibody was reacted with a protein on a nitrocellulose filter as a primary antibody, and then a biotinylated anti-rabbit IgG prepared by goat (Zymet Laboratories, USA, catalog number 62-1840) was used as a secondary antibody. After the reaction, the color was developed by a method in which avidin-biotinylated horseradish peroxidase (manufactured by Amersham Japan, Japan, catalog number RPN.1051) was used to give a dark brown spot.
[0064]
(4) -c ELISA
The holes of the 96-well flat-bottom ELISA plate containing the culture solution are washed 5 times with 200 μl of PBS (Nissui, Japan). Thereafter, PBS containing 0.5% BSA is added to a concentration of 100 μl / well, and left at room temperature for 2 hours. After washing each well of the plate 5 times with PBS containing 0.05% Tween 80, the anti-human TM polyclonal antibody prepared in Example 1-4-4- (b) above was diluted 1000 times with PBS containing 1% BSA, and 100 μl / Add to each hole with holes and react as primary antibody. Thereafter, each well of the plate was washed 5 times with PBS containing 0.05% Tween 80, and then an enzyme-labeled anti-rabbit IgG antibody (Cappel, catalog number 50-7415-16) was diluted 1000 times with PBS containing 1% BSA. Then, 100 μl / well was added to each well and reacted at room temperature for 1 hour. Each hole of the plate was washed 5 times with PBS containing 0.05% Tween 80, and then 100 μl of color developing solution (30 mg orthophenylenediamine added to 20 ml citrate phosphate buffer and 70 μl 30% hydrogen peroxide solution) was added. When a good color was obtained, 50 μl of 4.1 M sulfuric acid solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 492 nm to select a human thrombomodulin producing strain.
[0065]
(5) Amplification of TM productivity by MTX of CHO cell lines transformed with plasmid pSV2TMD123 and plasmid pSV2DHFR
A high-producing strain of TM obtained from the result of Example 1- (4) above is selected, cultured until it becomes confluent on a 6-well tissue culture plate, and the diameter is 7/10, 3/10. Subcultured to 10 cm tissue culture plates. The plate passaged at 7/10 was cultured until it became confluent in DMEM containing 150 μg / ml proline and 10% dialyzed FCS, and then replaced with 10 ml of productivity measurement medium (DMEM containing 150 μg / ml proline) for 24 hours. Later, the culture solution was collected and used as a cell production solution without addition of MTX.
[0066]
Further, the plate passaged at 3/10 was replaced with a selective medium containing 20 nM MTX the next day, and the culture was continued. After becoming confluent, the medium was replaced with 10 ml of the productivity measurement medium, and after 24 hours, the culture solution was collected and used as a 20 nM MTX-selected cell production solution. The collected production solution was quantified for TM in the production solution by the sandwich method ELISA described below to obtain 1 × 10 cells. ⁶ The amount of TM produced per piece per 24 hours was evaluated.
[0067]
The production of anti-human thrombomodulin monoclonal antibodies was performed by the method of Maruyama et al. [J. Biol. Chem. 260, 15432 (1985)]. That is, the human lung-derived thrombomodulin purified in Example 1- (4) -a was used as an antigen, and after immunization several times with Balb / C mice, a mouse spleen cell solution was prepared, and mouse bone marrow cell species from an appropriate line And cell fusion using a fusion promoter such as polyethylene glycol.
[0068]
Mouse bone marrow cells used for cell fusion are, for example, P3-X63-Ag8-U1 cells (P3-U1) [Yelton et al., Current. Topics in Microbiology and Immunology, 81, 1 (1978)]. The fused cells are selected using an appropriate selective medium, for example, HAT (hypoxanthine / aminopterin / thymidine) medium. After hybridoma cells are thus detected, the culture supernatant is collected and screened for antibodies to thrombomodulin by ELISA (enzyme immunoassay) using thrombomodulin as a solid phase antigen. Hybridoma cells producing an antibody against thrombomodulin are cloned by an appropriate method, for example, the limiting dilution method. As a result, two types of anti-human thrombomodulin monoclonal antibodies were obtained and named anti-TM monoclonal antibodies A and B, respectively.
[0069]
The purified product of the above-mentioned anti-TM monoclonal antibody A is diluted with 0.1 M sodium bicarbonate buffer (pH 9.2) to 200 μg / ml, and placed on a 96-well flat bottom ELISA plate to 50 μl / well. Dispense. After standing at room temperature for 2 hours, the plate holes are washed 5 times with 200 μl of PBS (Nissui, Japan). Thereafter, PBS containing 0.5% BSA is added to a concentration of 100 μl / well, and left at room temperature for 2 hours. After washing each well of the plate 5 times with PBS containing 0.05% Tween 80, the collected culture solution is diluted stepwise and added to 50 μl / well, and left at room temperature for 2 hours. After washing each well of the plate 5 times with PBS containing 0.05% Tween 80, the biotin-labeled anti-TM monoclonal antibody B was diluted 1000-fold with PBS containing 1% BSA to a concentration of 20 μg / ml, and 100 μl / well In addition, the mixture was reacted at room temperature for 1 hour. Each well of the plate was washed 5 times with PBS containing 0.05% Tween 80, and avidin D (manufactured by Vector, catalog number A-2004) was diluted 5000 times with PBS containing 1% BSA, and each 100 μl / well In addition to the holes, the reaction was allowed to proceed for 30 minutes at room temperature. Each hole of the plate was washed 5 times with PBS containing 0.05% Tween 80, and then 100 μl of color developing solution (30 mg orthophenylenediamine added to 20 ml citrate phosphate buffer and 70 μl 30% hydrogen peroxide solution) was added. When a good color was obtained, 50 μl of 4.1 M sulfuric acid solution was added to stop the reaction, and measurement was performed at a measurement wavelength of 492 nm. As a standard product, human lung thrombomodulin purified in Example 1- (4) -a was used.
[0070]
The results of the sandwich ELISA between the MTX-free cell production solution and the 20 nM MTX-selected cell production solution were compared, and a strain in which the 20 nM MTX-selected cell production solution produced higher was selected. These strains are also 1 × 10 ^Four 5 × 10 ^Four The cells were subcultured to a tissue culture plate having a cell density of 10 cm in diameter at the cell density, and the culture was continued on the next day by changing to a selective medium containing 200 nM MTX. The 200 nM MTX resistant strain that had emerged was cloned, subcultured into a 96-well tissue culture plate and cultured until confluent, then replaced with 10 ml of 150 μg / ml proline-containing DMEM, and the culture medium was collected 24 hours later. The collected culture broth was evaluated for TM productivity by the sandwich ELISA described above. As described above, the high-producing strain is subcultured into a 6-well tissue culture plate and further subcultured into a tissue culture plate with a diameter of 10 cm. After 24 hours, the culture supernatant is recovered to increase human thrombomodulin productivity. Quantified by sandwich ELISA.
[0071]
A high production strain was selected, and the same operation as described above was repeated, and the concentration of MTX was increased from 200 nM to 2 μM, 20 μM, 40 μM, 100 μM, and 500 μM. In this way, a strain in which TM productivity was increased by gene amplification by MTX was obtained. Increasing MTX concentration efficiently amplifies human thrombomodulin productivity, about 17 pg / cell / day at 40 μM MTX concentration, about 20 pg / cell / day at 100 μM MTX concentration, 47 pg / cell / day at 500 μM A production cell line was established.
[0072]
(6) Examination of TM production stability
For the 100 μM MTX-resistant strain obtained in Example 1- (5), MTX-containing non-selective medium and MTX-free non-selective medium (10% (v / v) FCS, 150 μg / ml proline-containing DMEM) ) Was subjected to long-term subculture, and TM production per cell was evaluated after completion of the culture by sandwich ELISA. For the 20 μM MTX resistant strain, 1-month subculture and 2-month subculture were performed. The 100 μM MTX-resistant strain was maintained in productivity even after culturing for 2 months without MTX, and a stable TM-producing strain was obtained.
[0073]
(7) Acquisition of suspension culture habituation strain
The 100 μM MTX resistant strain obtained in Example 1- (6) was subjected to 50 ml scale floating stirring culture using DMEM containing 10% (v / v) FCS and 150 μg / ml proline. 1 × 10 ^Five Start culture at cells / ml, 1 × 10 ⁶ The cells were cultured until a cell density of cells / ml was reached. As a result of repeating the same culture five times while confirming the maintenance of the specific production rate, a TM production strain adapted to the suspension culture was obtained.
[0074]
Example 2
Comparison of DMEM and TM production medium of the present invention in suspension culture repeated batch culture
The method for preparing the TM production medium of the present invention comprises mixing each component of the above-mentioned component table 2 (excluding water, serum and growth factors), adding it to a predetermined amount of deionized ultrafiltered water, and further adding sulfuric acid. Kanamycin (manufactured by Meiji Seika) and tylosin (manufactured by Mitsumata) are added and dissolved, and then 4% (v / v) of bovine serum (hyclone) is added, and the pH is adjusted to 7.1 with concentrated hydrochloric acid (Wako Pure Chemical Industries). Then, it was filtered through a 0.22 μm filter (manufactured by Millipore) and stored at 4 ° C. (hereinafter, the name of the TM production medium of the present invention may be abbreviated as HM-04 medium). In addition, DMEM supplemented with serum is prepared by adding DMEM (Lifetech), a powdered medium, to a predetermined amount of deionized ultrafiltered water, and further baking soda (Wako Pure Chemical Industries), kanamycin sulfate (manufactured by Meiji Seika), tylosin. (Mitsubishi) was added and dissolved, 4% (v / v) of bovine serum (Hycron) was added, pH was adjusted to 7.1 with concentrated hydrochloric acid (Wako Pure Chemical Industries), and then a 0.22 μm filter (Millipore) And stored at 4 ° C. All the media were heated to 37 ° C. in a 37 ° C. water bath constant temperature bath immediately before use.
[0075]
2 × 10 at the start of culture ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 50 ml of each fresh medium, and placed in a culture container (Calstar 100, manufactured by Shibata Hario) to start culture.
[0076]
The culture conditions are 37 ° C. in a constant temperature room, stirring 80 rpm (magnetic stirrer, manufactured by Shibata Hario), dissolved oxygen concentration (DO) 50% control (DO electrode (Able), DO controller (Toa Chemical)), aeration flow rate: air Culturing was performed at 100 ml / min, carbon dioxide gas 10 ml / min, and DO control oxygen gas 300 ml / min. Culturing was carried out until the density of floating live cells reached equilibrium.
[0077]
Medium exchange during the culture was performed on the 5th and 9th days. Moreover, it carried out every day after the 9th day. To replace the medium, put the whole culture solution in a 50 ml centrifuge tube (Falcon), centrifuge (2000 rpm, 2 minutes), remove the supernatant, suspend in each fresh 50 ml medium previously heated to 37 ° C, and put it in the culture vessel. The culture was resumed. The culture supernatant removed by centrifugation was stored at -20 ° C. A certain amount was sampled from the culture supernatant. Separately, a certain amount was sampled daily from the culture solution on the 6th to 8th days, and a certain amount was also sampled from the culture supernatant obtained by centrifugation. The TM concentration contained in these sampled culture supernatants was measured by the method described in Example 1- (4) -a (method for measuring the activity of promoting protein C activation by thrombin). The specific activity of TM was converted to 80,000 U / mg.
[0078]
To measure the floating cell density, mix the sampled culture solution with an equal volume of trypan blue staining solution (floor), measure the unstained cells as living cells with a microscope using a hemocytometer, and determine the floating cell density. It was measured.
Suspended live cell density (× 10 ^Five FIG. 1 is a plot of cells / ml) on the vertical axis and the number of days of culture (days) on the horizontal axis. According to this result, the transition of the density of floating living cells was about 7 × 10, although the growth rate at low density in the initial stage of culture was similar in both DMEM and HM-04 medium. ^Five A difference in growth rate appears from cells / ml, and DMEM has a final density of about 45 × 10 ^Five cells / ml, but the HM-04 medium was about 100 × 10 ^Five Grows to cells / ml.
[0079]
Considering the volume of the exchanged medium and the TM concentration, the cumulative production amount (mg) of TM in each culture day is calculated, and each culture day (day) is plotted on the vertical axis, and FIG. 2 is obtained. It was.
According to FIG. 2, the cumulative productivity for 20 days from the 6th day to the 26th day after the start of culture is about 3 times higher in the culture method using HM-04 medium than in the culture method using DMEM. Obtained. Therefore, according to the culture medium of this invention, it was confirmed that mass production of TM is easy and preferable.
[0080]
The specific production rate (pg / cell / day) calculated by dividing the production amount of TM per day for each culture day by the floating cell density is plotted on the vertical axis, and each culture day (day) is plotted on the horizontal axis. 3 was obtained.
According to FIG. 3, the change over time in the specific production rate was relatively stable in the culture method using HM-04 medium, but decreased with time in the culture method using DMEM. Therefore, according to the culture medium of the present invention, it was confirmed that long-term production is possible and preferable.
[0081]
The specific production rate (pg / cell / day) for each culture day is plotted on the vertical axis with the density of viable cells in each culture day (day) (× 10 ^Five cells / ml) was plotted on the horizontal axis to obtain FIG.
According to FIG. 4, the change in the specific production rate relative to the floating living cell density was not cultivated using the HM-04 medium, but decreased by increasing the floating living cell density in the culture using DMEM. Therefore, according to the culture medium of the present invention, it was confirmed that the productivity does not change even in high-density culture.
[0082]
Example 3
Repeated batch culture in suspension culture
Medium preparation was performed in the same manner as in Example 2. However, the concentration of serum added is 10% (v / v) in the medium used for culture for growing TM-producing cells (hereinafter referred to as growth culture), and the medium used for the culture for manufacturing TM (hereinafter also referred to as production culture). Then, it was prepared by adding 2% (v / v).
[0083]
At the start of growth culture, 2 × 10 ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 100 ml of each fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario) to start culture. The growth culture was carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). Proliferation culture has a floating live cell density of 5 × 10 ^Five The cells were subcultured when reaching cells / ml. The subculture culture scale was expanded sequentially to 100 ml, 500 ml, 2 L, and 8 L. In the subculture, the same amount of fresh medium as the value obtained by subtracting the culture scale before the passage from the culture scale after the passage was expanded.
[0084]
Floating culture at 8L culture scale has a floating cell density of 5 × 10 ^Five DO control was started when cells / ml were reached. Control conditions were DO 50% control (DO electrode (Able), DO controller (Toa Chemical)), aeration flow rate: air 300 ml / min, carbon dioxide gas 30 ml / min, DO control oxygen gas 900 ml / min.
[0085]
The medium exchange in the growth culture at the culture scale of 8 L was carried out at 37 ° C. in advance by centrifuging the culture solution using a continuous centrifuge (Alfa-Laval, product name CC-100) on the 5th, 9th and 11th days. The culture medium was replaced with a fresh medium that had been heated.
Production culture was started from the 12th day of growth culture at a culture scale of 8 L. The medium is changed every day by centrifuging the culture solution using a continuous centrifuge (Alpha-Laval, product name CC-100) to obtain the supernatant, and the cells are suspended in a fresh medium preheated to 37 ° C. Production culture was started. 800 ml of the centrifuged supernatant was sampled and stored frozen at -20 ° C. The conditions of the production culture are as follows: 37 ° C constant temperature room, stirring 100 rpm (magnetic stirrer, manufactured by Shibata Hario), dissolved oxygen concentration (DO) 50% control (DO electrode (Able), DO controller (Toa Chemical)), aeration flow rate : Cultivation was carried out for 20 days with 100 ml / min of air, 10 ml / min of carbon dioxide gas, and 1000 ml / min of oxygen gas for DO control. The floating cell density was measured in the same manner as in Example 2. The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
[0086]
The result of production culture is an average viable cell density of 3.1 × 10 ⁶ The average productivity was 27 mg / L / day at cells / ml.
[0087]
Example 4
Batch culture in suspension culture
Medium preparation was performed in the same manner as in Example 2. However, the serum addition concentration was 10% (v / v).
From the 5th day of 8L scale growth culture in Example 3, 20 ml of the culture solution was sampled, suspended in 80 ml of fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario Co., Ltd.) to start the culture. The culture was carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). 5 × 10 ^Five When cells / ml are reached, the whole culture solution is centrifuged (2000 rpm, 2 minutes, domestic chemistry), suspended in 500 ml of fresh medium, transferred to a spinner flask (Calstar 500, manufactured by Shibata Hario Co., Ltd.), 10 ° C. Batch culture was started in a constant temperature carbon dioxide constant temperature chamber with stirring at 120 rpm (Magnetic Stirrer, manufactured by Shibata Hario Co., Ltd.) to obtain production culture. The floating cell density was measured in the same manner as in Example 2. The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
[0088]
Production culture is cultivated for 8 days, reaching a viable cell density of 1.8 × 10 ⁶ The cumulative productivity was 35 mg / L at cells / ml.
[0089]
Example 5
Continuous culture in suspension culture
Medium preparation was performed in the same manner as in Example 2. However, the serum concentration was 8% (v / v) for the growth culture medium and 4% (v / v) for the production culture medium.
At the start of growth culture, 2 × 10 ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 100 ml of each fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario) to start culture. The growth culture was carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). Proliferation culture has a floating live cell density of 5 × 10 ^Five The cells were subcultured when reaching cells / ml. The subculture culture scale was sequentially expanded to 100 ml, 500 ml, and 1.5 L. In the subculture, the same amount of fresh medium as the value obtained by subtracting the culture scale before the passage from the culture scale after the passage was expanded.
[0090]
Suspension live cell density is 5 × 10 in growth culture at 1.5L culture scale ^Five DO control was started when cells / ml were reached. The control conditions were DO 50% control (DO electrode (Able), DO controller (Toa Chemical)), aeration flow rate: air 300 ml / min, carbon dioxide gas 30 ml / min, DO control oxygen gas 900 ml / min.
[0091]
The continuous medium exchange in the growth culture at a culture scale of 1.5 L was performed by continuous filtration using a separation module.
Circulate the culture solution with a circulation pump (Howard) installed outside the culture tank, install a culture supernatant separation module (Asahi Kasei Kogyo) in the circulation line, and install the secondary side of the module with a peristaltic pump (Gilson). Negative pressure was applied and quantitative filtration (filtration rate was set at 1.5 L / day) was performed, and the culture supernatant from which the cells were separated was extracted. The extracted culture supernatant was cooled and stored at 4 ° C., and 50 ml was sampled daily and stored frozen at −20 ° C. For the addition of fresh medium, the same amount as the extracted culture supernatant was quantitatively transferred into the culture tank by a peristaltic pump.
[0092]
20x10 ^Five When cells / ml were reached, the growth medium was switched to the production medium, production culture was started in the same manner as the continuous medium exchange in the growth culture at a culture scale of 1.5 L, and production culture was performed for 20 days. However, aeration flow rate: air 100 ml / min, carbon dioxide gas 10 ml / min, DO control oxygen gas 1000 ml / min, stirring 120 rpm (Magnetic Stirrer, manufactured by Shibata Hario). The floating cell density was measured in the same manner as in Example 2. The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
[0093]
The production culture result shows that the average viable cell density is 12.5 × 10 ⁶ The average productivity was 76 mg / L / day at cells / ml.
[0094]
Example 6
Continuous culture in suspension culture
Medium preparation was performed in the same manner as in Example 2. However, the serum concentration was 8% (v / v) for the growth culture medium and 4% (v / v) for the production culture medium.
At the start of growth culture, 2 × 10 ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 100 ml of each fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario) to start culture. The growth culture was carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). Proliferation culture has a floating live cell density of 5 × 10 ^Five The cells were subcultured when reaching cells / ml. The subculture culture scale was sequentially expanded to 100 ml, 500 ml, and 1.5 L. In the subculture, the same amount of fresh medium as the value obtained by subtracting the culture scale before the passage from the culture scale after the passage was expanded.
[0095]
Suspension live cell density is 5 × 10 in growth culture at 1.5L culture scale ^Five When the cells / ml were reached, the entire culture solution was transferred to BIOSTAT-M (Bearn Brown's culture device).
Continuous medium exchange in growth culture at a culture scale of 1.5 L was performed by continuous filtration using a separation tube (hydrophilic porous Teflon, Bee Brown) installed in the tank. The separation tube in the tank was subjected to negative filtration with a peristaltic pump (Gilson) and quantitative filtration (filtration rate was set to 1.5 L / day) was performed, and the culture supernatant was extracted from the separation tube. The culture supernatant was cooled and stored at 4 ° C., and 50 ml of each sample was sampled daily and stored frozen at −20 ° C. For the addition of fresh medium, the same amount as the extracted culture supernatant was quantitatively transferred into the culture tank by a peristaltic pump.
[0096]
The growth culture conditions were controlled at a bath temperature of 37 ° C., DO = 50%, and pH = 7.1.
20x10 ^Five When cells / ml were reached, the growth medium was switched to the production medium, production culture was started in the same manner as the continuous medium exchange in the growth culture at a culture scale of 1.5 L, and the culture was performed for 20 days.
The floating cell density was measured in the same manner as in Example 2.
[0097]
The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
The result of production culture is an average viable cell density of 12.0 × 10 ⁶ The average productivity was 78 mg / L / day at cells / ml.
[0098]
Example 7
Continuous culture in suspension culture
(1) Serum medium
Medium preparation was performed in the same manner as in Example 2. However, the serum concentration was 8% (v / v) for the growth culture medium and 4% (v / v) for the production culture medium.
[0099]
At the start of growth culture, 2 × 10 ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 100 ml of each fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario) to start culture.
[0100]
The growth culture conditions were carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas, with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). Proliferation culture has a floating live cell density of 5 × 10 ^Five The cells were subcultured when reaching cells / ml. The subculture culture scale was sequentially expanded to 100 ml, 500 ml, and 1.5 L. In the subculture, the same amount of fresh medium as the value obtained by subtracting the culture scale before the passage from the culture scale after the passage was expanded.
[0101]
Suspension live cell density is 5 × 10 in growth culture at 1.5L culture scale ^Five When the cells / ml were reached, the entire culture solution was transferred to BIOSTAT-M (Bearn Brown's culture device).
The continuous medium exchange in the growth culture at a culture scale of 1.5 L was performed by continuous filtration using a separated stainless steel mesh filter (pore size: 20 μm, Bee Brown) installed in the tank.
[0102]
On the culture separated by a stainless mesh filter, the extraction pipe inserted into the stainless steel mesh filter in the tank is subjected to negative filtration with a peristaltic pump (Gilson) and subjected to quantitative filtration (filtration rate is set to 1.5 L / day). I'm out of Qing. The culture supernatant was cooled and stored at 4 ° C., and 50 ml of each sample was sampled daily and stored frozen at −20 ° C. For the addition of fresh medium, the same amount as the extracted culture supernatant was quantitatively transferred into the culture tank by a peristaltic pump.
[0103]
The growth culture conditions were controlled at a bath temperature of 37 ° C., DO = 50%, and pH = 7.1. 20x10 ^Five When cells / ml were reached, the growth medium was switched to the production medium, and the production culture was started in the same manner as the continuous medium exchange in the growth culture at a culture scale of 1.5 L, followed by culturing for 20 days.
The floating cell density was measured in the same manner as in Example 2.
[0104]
The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
The production culture result shows that the average viable cell density is 11.2 × 10 ⁶ The average productivity was 76 mg / L / day at cells / ml.
[0105]
(2) Growth factor medium
Culture was performed according to the method of Example 7- (1). However, no serum was added to the production culture medium, and 3 mg of albumin, 5 mg of insulin, 5 mg of transferrin, 2 μM of ethanolamine and 20 nM of sodium selenite were added as growth factors to prepare a production culture medium.
[0106]
The saturated cell density is about half that of Example 7- (1), 5 × 10 5 because this cell is not completely serum-independent. ⁶ It was about cells / ml.
The result of production culture is an average live cell density of 4.8 × 10 ⁶ The average productivity was 42 mg / L / day at cells / ml.
[0107]
Example 8
Repeat batch culture in adherent culture
The medium was prepared in the same manner as in Example 2. However, the serum concentration was 10% (v / v) for the growth culture medium and 2% (v / v) for the production culture medium.
At the start of growth culture, 2 × 10 ⁷ Cells cryopreserved in cells / ampoules were quickly thawed in a 37 ° C. constant temperature bath, suspended in each medium, and centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration, suspended in 100 ml of each fresh medium, and placed in a culture vessel (Calstar 100, manufactured by Shibata Hario) to start culture.
[0108]
The growth culture conditions were carried out in a constant temperature chamber at 37 ° C. and 10% carbon dioxide gas, with stirring at 80 rpm (Magnetic Stirrer, manufactured by Shibata Hario). The floating cell density is 2 × 10 ^Five When the cells / ml were reached, the culture was centrifuged (2000 rpm, 2 minutes, domestic chemistry). After centrifugation, the supernatant was removed by aspiration and suspended in 200 ml of each fresh medium, and 10 ml each was placed in two culture vessels (Calstar 100, manufactured by Shibata Hario). Immediately after that, 3 g / L of surface-attached cytodex 1 (Pharmacia) suspended in 90 ml of fresh medium and porous internal-attached Asahi Kasei Microcarrier (Asahi Kasei Kogyo) were added to each incubator. (Sitedex 1 was prepared according to the attached manual of Pharmacia Biotech, and Asahi Kasei Microcarrier was prepared according to the attached manual of Asahi Kasei Kogyo).
[0109]
The 100 ml stirring growth culture using the adherent carrier was carried out at 37 ° C. in a 10% carbon dioxide constant temperature chamber at 80 rpm with stirring (Magnetic Stirrer, manufactured by Shibata Hario).
In the medium exchange, the agitation of the culture was stopped and the adhering carrier was settled. Then, the supernatant was removed by suction with a pipette, and an equal amount of fresh medium was added to resume the culture. Medium change was performed on the 5th, 8th and 10th days.
[0110]
20x10 ^Five When cells / ml were reached, the growth medium was switched to the production medium, the medium was changed daily by the same method, and the production culture was performed for 25 days. 100 ml of the culture supernatant obtained by medium exchange was stored frozen at -20 ° C.
The floating cell density was measured in the same manner as in Example 2.
The carrier-attached cell density is determined by removing the carrier precipitation supernatant from the sampled culture medium, adding the same amount of enucleated staining solution (Crystal Violet, TWEEN80, citric acid) as the removed supernatant, and after enucleation, The number of nuclei was measured with, and the nuclear density was defined as the density of attached cells.
[0111]
The TM concentration in the supernatant of the production culture was calculated with a specific activity of 80,000 U / mg by the method for measuring the activity of promoting protein C activation by thrombin described in Example 1- (4) -a.
Production culture results show that the average cell density is 2.5 × 10 ⁶ cells / ml, the average productivity is 26 mg / L / day, and the average cell density is 5.5 × 10 5 in culture using Asahi Kasei Microcarrier. ⁶ The average productivity was 46 mg / L / day at cells / ml.
[0112]
Example 9
Crude purification with strong anion exchange resin column
11 L of −20 ° C. frozen culture supernatant obtained in Example 3 was dissolved and filtered through a 0.2 μm membrane filter (Millipore, Millipack 20).
The filtered culture supernatant was applied to a Q-Sepharose (Pharmacia) column (diameter 90 mm, height 6.5 cm) equilibrated with 20 mM Tris salt buffer (pH 7.4) containing 150 mM NaCl. Next, it is washed with 20 mM acetate buffer containing 180 mM NaCl, further washed with 20 mM Tris salt buffer (pH 7.4) containing 180 mM NaCl, and eluted with 20 mM Tris salt buffer (pH 7.4) containing 300 mM NaCl. Then, an eluate having a volume volume of 0.5 column from the rise of the peak of absorbance at 280 nm of the eluate was obtained as a crude product.
[0113]
Example 10
Main purification by affinity column (antibody B)
The affinity column was prepared as follows. That is, the anti-TM monoclonal antibody B obtained in Example 1- (5) above is a culture supernatant obtained by culturing a hybridoma that produces the antibody, or a hybridoma obtained from a histocompatibility animal, nude mouse, or the like. From the ascites obtained by growing in the peritoneal cavity, purification was carried out by separation and purification operations such as salting out, ion exchange chromatography, protein A column and the like. Next, according to the Pharmacia manual (Affinity Chromatography principles & methods), the purified anti-TM monoclonal antibody B was dissolved in 0.1 M NaHCO3 buffer (pH 8.3) containing 0.5 M NaCl, and CNBr-activated Sepharose 4 B (Pharmacia 4B) And 52-1533-00-AI), and the anti-TM monoclonal (antibody B) was coupled to Sepharose 4B to produce anti-TM monoclonal (antibody B) -bound Sepharose 4B. Next, this anti-TM monoclonal (antibody B) -bound Sepharose 4B was packed into a column to prepare a monoclonal (antibody B) column.
[0114]
400 ml of the elution fraction obtained in Example 9 was applied to a monoclonal antibody (antibody B) column (diameter 50 mm, height 6 cm) equilibrated with 20 mM phosphate buffer (pH 7.3) containing 1.0 M NaCl. . Pour 20 mM phosphate buffer (pH 7.3) containing 1.0 M NaCl, wash with 100 mM acetate buffer (pH 5.0), and elute with 100 mM glycine hydrochloride buffer (pH 3.0) containing 0.3 M NaCl. The eluate from the rising edge to the falling edge of the 280 nm absorbance of the eluate was obtained as the main purified product.
[0115]
Example 11
High purity purification by strong cation exchange resin column
A solution prepared by adjusting 180 ml of the eluate obtained in Example 10 to pH 3.5 with 1.0 M glycine hydrochloride buffer (pH 2.0) was added to 100 mM glycine hydrochloride buffer containing 300 mM NaCl (specific conductivity 31 ms / cm). , PH 3.5) and subjected to SP-Sepharose (Pharmacia) column (diameter 26 mm, height 3 cm). Washing was started with a 100 mM glycine hydrochloride buffer (specific conductivity 31 ms / cm, pH 3.5) containing 300 mM NaCl to obtain a flow-through fraction from the rise to the fall of the peak at an absorbance of 280 nm, and immediately a 500 mM phosphate buffer ( It was neutralized to pH 7 with pH 7.3) and obtained as a highly purified product.
[0116]
Example 12
Concentration of high purity purified products with polysulfone hollow fiber
Each of the high-purity purified products obtained in Example 11 was concentrated using 1 m polysulfone hollow fiber (Asahi Kasei Kogyo Co., Ltd.) wetted with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl, and 5 ml each. The concentrate was obtained.
[0117]
Example 13
Buffer exchange of high purity products using gel filtration column
Each Sephcryl S-300 column (Pharmacia, diameter 16 mm, height 90 cm) equilibrated with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl in 5 ml of each concentrate obtained in Example 12. It was used for. Development and fractionation were carried out with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl. Each fraction was confirmed to be active by the method of measuring the activity to promote protein C activation by thrombin described in Example 1- (4) -a, and the active fraction was collected and a high-purity purified product whose buffer solution was exchanged was obtained. I got it.
[0118]
Example 14
Purification using affinity column (thrombin column)
400 ml of the elution fraction obtained in Example 9 was dialyzed against 20 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 0.5 mM calcium chloride. After dialysis, DIP-thrombin-agarose (PAESE LOREI, 06-148-1035, diameter 50 mm, height 6 cm) equilibrated with 20 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 0.5 mM calcium chloride. Provided. After washing with 20 mM Tris buffer (pH 7.4) containing 200 mM NaCl and 0.5 mM calcium chloride, elution was started with 20 mM Tris buffer (pH 7.4) containing 1.0 M NaCl and 0.5 mM calcium chloride. The eluate from the rising edge to the falling edge of the absorbance at 280 nm of the eluate was obtained as the main purified product.
[0119]
A solution obtained by diluting 200 ml of the obtained eluate with 100 mM glycine hydrochloride buffer (pH 3.5) was adjusted to pH 3.5 with 1.0 M glycine hydrochloride buffer (pH 2.0). This diluted pH adjusted eluate was applied to an SP-Sepharose (Pharmacia) column (diameter 26 mm, height 3 cm) equilibrated with 100 mM glycine hydrochloride buffer (specific conductivity 31 ms / cm, pH 3.5) containing 300 mM NaCl. Provided. Washing was started with a 100 mM glycine hydrochloride buffer (specific conductivity 31 ms / cm, pH 3.5) containing 300 mM NaCl to obtain a flow-through fraction from the rise to the fall of the peak at an absorbance of 280 nm, and immediately a 500 mM phosphate buffer ( It was neutralized to pH 7 with pH 7.3) and obtained as a highly purified product.
[0120]
This highly purified product was concentrated using 1 m polysulfone hollow fiber (Asahi Kasei Kogyo Co., Ltd.) wetted with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl to obtain 5 ml concentrated solution.
Next, the buffer solution of the high purity purified product was exchanged with a gel filtration column. That is, 5 ml of the obtained concentrated solution was applied to each Sephcryl S-300 column (Pharmacia, diameter 16 mm, height 90 cm) equilibrated with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl. Development and fractionation were carried out with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl. For each fraction, the activity was confirmed by the method for measuring the activity to promote protein C activation by thrombin described in Example 1- (4) -a, and a high-purity purified product obtained by collecting the active fraction and exchanging the buffer solution was obtained. I got it.
[0121]
Example 15
Frozen production culture supernatant, ie, culture supernatant of Example 4, culture supernatant of Example 5, culture supernatant of Example 6, culture supernatant of Example 7- (1), culture adherence of Example 8 The culture supernatant of the culture using cytodex as the carrier and the culture supernatant of the culture using Asahi Kasei microcarrier as the culture adherent carrier of Example 8 were used as starting materials for purification.
[0122]
For the purification, the crude purification by the ion exchange chromatography of Example 9 was performed, and then the crude product was purified by the affinity chromatography of Example 10. Then, this purified product is purified to a high purity by the ion exchange chromatography of Example 11, and the purified product is concentrated by the concentration method of Example 12, and the gel filtration of Example 14 is performed to replace the buffer solution. Got.
[0123]
Example 16
Physical properties of high purity products
The following examinations were conducted on the high-purity purified products obtained by replacing the buffer solutions obtained in Examples 13, 14, and 15.
The high purity purified product was able to dissolve at least 10 mg / ml in pH-neutral distilled water for injection in the absence of a surfactant.
[0124]
In addition, as a result of SDS-PAGE electrophoresis of the highly purified product under both reducing and non-reducing conditions and detection by Coomassie staining, a single band of about 94 kDa for reduction and about 73 kDa for non-reduction was detected. .
In addition, N-terminal amino acid analysis of high-purity purified products was performed by a pulse liquid method using a protein sequencer Procise 492 type manufactured by PerkinElmer. As a result, the N-terminal amino acid consisted of two types of proteins starting with alanine and phenylalanine, which were considered to be amino acid sequences corresponding to SEQ ID NO: 3 19-516 and SEQ ID NO: 3 17-516, respectively. . The content ratio starting from alanine was derived from the production culture supernatant of each Example, Example 3: 62%, Example 4: 68%, Example 5: 69%, Example 6: 59%, Example 7: 63%, culture using Cytodex 1 (manufactured by Pharmacia Biotech) as the culture adherent carrier of Example 8: 65%, and Asahi Kasei Microcarrier (Asahi Kasei Kogyo) used as the culture adherent carrier of Example 8 Culture: 59%.
[0125]
Post-translational modification analysis of high purity products
The amino acid sequence positions described in the following examples correspond to the sequence positions of the amino acid sequences described in SEQ ID NO: 3 and SEQ ID NO: 4.
(1) Analysis of N-linked sugar chain binding position
The binding position of the N-linked sugar chain was analyzed for the high-purity purified product obtained by replacing the buffer solution obtained in Examples 13, 14, and 15.
0.5 mg of the highly purified product was dried using a centrifugal concentrator and dissolved in 0.5 mL of Tris / urea buffer solution (200 mM Tris-HCl, pH 8.0 containing 8 M urea). To this solution, 32 μL of 2-mercaptoethanol solution (2-mercaptoethanol / Tris-urea buffer I = 50 μL / 450 μL) was added and incubated at 37 ° C. for 1 hour. 27 μL of iodoacetic acid solution (0.3 g of iodoacetic acid dissolved in 1 mL of 2M NaOH) was added, protected from light, and allowed to stand at room temperature for 15 minutes. The solution was dialyzed against 1 mM hydrochloric acid and then dried with a centrifugal concentrator to obtain RCM-TM. RCM-TM is dissolved in 180 μL of Tris-urea buffer II (200 mM Tris-HCl containing 2 M urea, pH 8.0), and 45 μL of 0.1 mg / mL trypsin solution (solvent is Tris-urea buffer II) and 0 . 25 μL of 1 M calcium chloride solution was added and enzymatically digested at 37 ° C. for 16 hours.
[0126]
This enzyme digested solution was added to a Cosmosil 5C18-AR column (4.6 ID × 250 mm, manufactured by Nacalai Tesque, Japan) equilibrated with 0.1% TFA / 5% acetonitrile, and then 0.1% TFA / Peptide fragments were fractionated by a linear gradient to 50% acetonitrile. The flow rate was 0.7 mL / min, the column temperature was 35 ° C., and the detection was performed by absorption at 210 nm. Since there are five consensus sequences (Asn-X-Ser / Thr) for which N-linked sugar chains are expected to be added, Asn47, Asn115, Asn116, Asn382, and Asn409, peptide fragments containing these asparagine residues are designated as PSQ. -23, PSQ-1 (manufactured by Shimadzu Corporation, Japan) or Applied Biosystems Procedure 492 (manufactured by PerkinElmer, USA) was used for amino acid sequence analysis according to the method recommended by the manufacturer.
[0127]
In the amino acid sequence analysis of the peptide fragment containing Asn47 (Ala19-Arg56), the position of Asn47 was blank (the peak of PTH-Asn was not detected), so an N-linked sugar chain was bound to Asn47. It was confirmed. Similarly, as a result of amino acid sequence analysis of a peptide fragment containing Asn382 (Ala372-Arg403) and a peptide fragment containing Asn409 (Cys404-Arg474), the positions of Asn382 and Asn409 were both blank. Asn382 and Asn409 also It was confirmed that an N-linked sugar chain was bound. Further, when the amino acid sequence of the peptide fragment containing Asn115 and Asn116 (Gly107-Arg121) is analyzed, the positions of Asn115 and Asn116 are not blank, but the detected amount of PTH-Asn is lower than expected, respectively. It was thought to be a mixture of a sugar chain bound to Asn115 (no sugar chain bound to Asn116) and a sugar chain bound to Asn116 (no sugar chain bound to Asn115). This was supported by the fact that, as a result of mass analysis (MALDI-TOFMS) of this fragment, only a molecular weight in which one N-linked sugar chain was bound was observed in this peptide fragment.
[0128]
(2) Analysis of N-linked sugar chain structure
The N-linked sugar chain structure was analyzed for the high-purity purified products with buffer replacements obtained in Examples 13, 14, and 15. After desalting 0.5 mg of highly purified product, it was dried to dryness using a centrifugal concentrator. This was treated with anhydrous hydrazine at 110 ° C. for 1 hour using Hydraclub S-204 (manufactured by Hornen, Japan) to cut out the sugar chain. This was dissolved in 250 μL of 0.1 M sodium hydrogen carbonate, 25 μL of acetic anhydride was added, and the mixture was allowed to stand at room temperature for 30 minutes. This operation was repeated twice to reacetylate. This sugar chain was converted to pyridylamino (PA) according to the protocol using PALSTATION MODEL 1000 (Takara Shuzo Co., Ltd., Japan) and a reagent kit for sugar chain analysis. After desalting and drying with a centrifugal concentrator, it was dissolved in 20 μL of 0.2 M sodium acetate buffer, pH 5.0. Sialidase 0.05U was added to this solution and incubated overnight at 37 ° C. to prepare asialo-PA sugar chain. This asialo-PA sugar chain was two-dimensionally mapped according to the method of Tomiya et al. (Tomiya, N., et. Al, 1988, Anal. Biochem., 171, 73-90) to estimate the structure.
[0129]
As a result, it was estimated that the main component was fucosyl bi-antenna type (85-90%). The next most common component was the fucosyltri antenna type (10 to 15%), and these two components occupied the majority. Furthermore, it was confirmed that the structure of the two main components was correct in the analysis by mass spectrometry (MALDI-TOFMS, FAB-MS) and exoglycosidase treatment (galactosidase, GlcNAc-ase, fucosidase).
In addition, the glycopeptide fragment containing each N-type sugar chain binding site prepared in the above (1) was also analyzed in the same manner, but the structure of the main sugar chain at all binding positions was fucosyl biantenna type, There was no significant difference in sugar chain structure depending on the binding site.
[0130]
(3) Analysis of O-linked sugar chain binding position
The binding position of the O-linked sugar chain was analyzed for the buffer-purified purified product obtained in Examples 13, 14, and 15. After desalting 1 mg of the highly purified product, it was dried using a centrifugal concentrator and dissolved in 30 μL of water. To this solution, 10 μL of 0.25 M sodium phosphate buffer (pH 8.0) and 1 μg of Asp-N (manufactured by Wako Pure Chemical Industries, Japan) were added, followed by enzyme digestion at room temperature for 3 days. This enzyme digested solution was added to a Cosmosil 5C18-300 column (4.6 ID × 150 mm, manufactured by Nacalai Tesque, Japan) equilibrated with 0.1% TFA / water, and then 0.1% TFA / 90%. Peptide fragments were fractionated by a linear gradient to acetonitrile. The flow rate was 0.8 mL / min, and the detection was performed by absorption at 220 nm. Since the O-linked sugar chain is often bound near the proline-rich sequence, it was predicted that the O-linked sugar chain was bound near the C-terminus of the solubilized TM. Therefore, after treating the peptide fragment containing C-terminal (Asp489-Gly516) with sialidase and galactosidase, PSQ-23, PSQ-1 (manufactured by Shimadzu Corporation, Japan) or Applied Biosystems Procedure 492 (manufactured by PerkinElmer, USA) was used. The amino acid sequence was analyzed according to the method recommended by the manufacturer. In the Ser / Thr analysis cycle, the position where the yield of PTH-Ser / PTH-Thr was low and the peak of PTH-Ser (GalNAc) / PTH-Thr (GalNAc) was observed was defined as the sugar chain binding position.
[0131]
As a result, five positions, Ser498, Thr500, Ser503, Thr504, and Thr506, were confirmed as O sugar chain binding positions. However, only Ser503 was a partial modification. As for Ser492, modification of the sugar chain was predicted since PTH-Ser was not detected, but no peak of PTH-Ser (GalNAc) was observed, suggesting that there was a difference in sugar chain structure.
[0132]
(4) Analysis of O-linked sugar chain structure
The O-linked sugar chain structure was analyzed for the purified high purity purified products obtained in Examples 13, 14, and 15. After desalting 1.0 mg of highly purified product, it was dried to dryness using a centrifugal concentrator. This was treated with anhydrous hydrazine at 65 ° C. for 6 hours using Hydraclub S-204 (manufactured by Honen, Japan) to cut out the sugar chain. This was dissolved in 250 μL of 0.1 M sodium hydrogen carbonate, 25 μL of acetic anhydride was added, and the mixture was allowed to stand at room temperature for 30 minutes. This operation was repeated twice to reacetylate. This sugar chain was converted to pyridylamino (PA) according to its protocol using PALSTATION MODEL 1000 (Takara Shuzo Co., Ltd., Japan) and a monosaccharide analysis reagent kit. After drying with a centrifugal concentrator, the mixture was heated in 0.025 M hydrochloric acid at 80 ° C. for 1 hour to prepare an asialo-PA sugar chain. As a result of HPLC analysis of this asialo-PA sugar chain according to the method of Iwase et al. (Iwase, et.al, J. Biochem., 1996, 120, 92-97), the following four types of sugar chain structures exist. Was speculated to be.
1) Galβ1,3GalNAc
2) NeuAcα2,3Galβ1,3GalNAc
3) Galβ1,3 (NueAcα2,6) GalNAc
4) NeuAcα2,3Galβ1,3 (NueAcα2,6) GalNAc
[0133]
(5) Analysis of glycosaminoglycan chain binding position and structure
From the analysis result of the O-linked sugar chain binding position, it was speculated that Ser492 has a sugar chain with a structure other than Galβ1, 3GalNAc, and the sugar composition analysis of asialo and agaracto-peptide fragments containing it was conducted. went. That is, an Asp-N digested peptide fragment (Asp489-Gly516) was dissolved in 100 μL of distilled water, placed in a hydrolysis test tube, mixed with 100 μL of 8M TFA, and hydrolyzed at 100 ° C. for 3 hours. After centrifugal concentration and drying, N-acetylation was performed, and pyridylamination was performed using a pyridylamination apparatus (Pulse Station Model 4000, manufactured by Takara Shuzo Co., Ltd.) and PALSTATION Pyridylation Kit (manufactured by Takara Shuzo Co., Ltd.). This was applied to a PALPAK Type A column (Takara Shuzo Co., Ltd.), and PA-monosaccharide was quantified. The flow rate was 0.3 ml / min, the column temperature was 65 ° C., and the detection was performed with fluorescence Ex: 310 nm and Em: 380 nm.
[0134]
As a result, in addition to GalNAc binding to Ser498, Thr500, Ser503, Thr504, Thr506, and Ser492, xylose and galactose were detected at a ratio of about 1: 2. Therefore, it was suggested that Ser492 is linked to a sugar chain containing a bridged tetrasaccharide structure (Xyl-Gal-Gal-GlcA) of a glycosaminoglycan chain.
[0135]
(6) β-hydroxylation of Asn342
Stenflo et al. Asp / Asn in the -Cys-X-Asp / Asn-X-X-X-Tyr / Phe-X-Cys-X-Cys-sequence of EGF-like structure undergoes β-hydroxylation. (Stenflo, J. Ohlin, et.al, 1987, Proc. Natl. Acad. Sci. USA, 84, 368-372, Stenflo, J. Ohlin, et.al, 1988, J. Biol. Chem., 263, 21-24) According to the consensus sequence of Stenflo et al., There are two positions in the solubilized TM that are expected to be modified (Asn342, Asn457). Therefore, when amino acid sequence analysis and mass analysis of each peptide stump containing Asn342 and Asn457 were performed, the peptide fragment containing Asn342 was 16 mass numbers larger than the mass without modification, and the mass when Asn was hydroxylated. It almost coincided with the number (M + H: Av. Mass = 3917.2, 3901.2 when not hydroxy). From this result, Asn342 was considered to have undergone β-hydroxylation. The modification rate was predicted to be 90% or more from the ionic strength ratio of MS.
[0136]
(7) Glucosylation of Ser305
A peptide fragment containing Ser305 (Ser279-Arg315) was isolated from the tryptic digest of RCM-TM, and the amino acid sequence was analyzed. As a result, no PTH-Ser peak was observed at the Ser305 position. It was found that the peak moved near the elution position. As a result of mass analysis (MALDI-TOFMS) of this peptide fragment, a peak was observed at m / z 4516, which was about 162 mass numbers larger than the expected mass of the peptide fragment. From this result, Ser305 was modified and hexose (mass number = 162) was expected to be added. Then, when the sugar composition analysis of this peptide fragment was performed, glucose was detected.
[0137]
Glycosylation of Ser residues in the EGF-like structure has been reported for several serum proteins (factorVII, factorIX, proteinZ, thrombospondin) (Nishimura, H., et.al, 1989, J. Biol. Chem). , 264, 20320-20325). However, the sugar chain structure reported so far has been disaccharide (Xyl-Glc) or trisaccharide (Xyl-Xyl-Glc), but the sugar chain bound to Ser305 of solubilized TM is glucose 1 sugar. In addition, no addition of xylose was observed, and it was found that it was different from the sugar chain structure reported so far. Moreover, although the consensus sequence of glucose addition is Cys-X-Ser-X-Pro-Cys, the amino acid sequence around Ser305 is Pro-Gly-Ser305-Tyr-Ser-Cys-, which does not match. Thus, glucosylation of Ser305 was considered a new type of modification.
[0138]
Example 17
Scale-up and mass balance of thrombomodulin purification process
Except that the culture supernatant 12L of Example 7- (1) was used and the column scale was increased in accordance with the volume, the high purity TM was obtained in the same manner as described in Examples 9-13. Obtained. The purified mass balance at this time is shown in Table 1 below. Finally, about 285 mg was obtained as high-purity TM, and the activity recovery rate was 38%.
[0139]
[Table 1]

[0140]
Furthermore, except that the culture supernatant 12L of Example 7- (1) was used and the column scale was increased in accordance with the volume, the method was substantially the same as that described in Examples 9 to 13, except that Example The purified TM was obtained without carrying out the SP ion exchange step described in No. 11. The purified mass balance at this time is shown in Table 2 below. Finally, about 280 mg of purified TM was obtained, and the activity recovery rate was 40%, almost the same recovery rate as when SP-Sepharose was introduced.
[0141]
[Table 2]

[0142]
Example 18
Assessment of contaminating foreign protein mass
A measurement system by ELISA was established for each of TM bovine serum-derived material, mouse antibody-derived material and CHO cell-derived material.
(1) Evaluation method of serum-derived substances
Rabbits were subcutaneously sensitized with 1 ml of an equal volume of bovine serum and Freund's complete adjuvant. Two weeks and three weeks later, 1 ml of the same emulsion was administered subcutaneously in the back of the skin, and blood was collected one week after the final sensitization. The blood was allowed to stand at about 4 ° C. overnight, the clot was removed, and the centrifuged supernatant was collected. From the obtained rabbit anti-bovine serum component antiserum, an ammonium sulfate salting-out method and a column using bovine serum components as ligands Rabbit anti-bovine serum component antibodies were obtained by chromatographic methods.
[0143]
The rabbit anti-bovine serum component antibody was digested with pepsin to obtain 2F (ab ′) 2 fragment. After reduction with 2-mercaptoethylamine hydrochloride, an enzyme-labeled anti-bovine serum component antibody was prepared by reacting with horseradish peroxidase into which a maleimide group was introduced with N-hydroxysuccinimide ester of N- (4-carboxycyclohexylmethyl) maleimide.
The eluate of each step was diluted with polysorbate / phosphate buffered saline so that the expected bovine serum component content would be 0 to 25 ng / ml to obtain a sample solution. Separately, polysorbate / phosphate buffered saline was added to a bovine serum component standard, and a solution containing 25 to 1.25 and 0 ng in 1 ml thereof was prepared as a standard solution.
[0144]
After adding 50 μl of a rabbit anti-bovine serum component antibody solution per well to a polystyrene 96-well plate, the plate was allowed to stand at 25 ° C. for about 2 hours. Next, each hole was washed with a phosphate buffered saline, a blocking solution was added, and the mixture was allowed to stand at 25 ° C. for about 1 hour. After washing with polysorbate / phosphate buffered saline, 100 μl of sample solution and standard solution were added to each well and allowed to stand at 25 ° C. for about 16 hours. Next, after washing with polysorbate / phosphate buffered saline, 50 μl of enzyme-labeled anti-bovine serum component antibody solution was added and allowed to stand at 25 ° C. for about 2 hours. After washing with polysorbate / phosphate buffered saline, 100 μl of enzyme substrate solution was added and reacted at 25 ° C. for about 40 minutes. After adding 50 μl of diluted sulfuric acid (1 → 4) to stop the reaction, the absorbance at 490 nm was measured with a 96-well plate absorptiometer. The content of bovine serum components in 1 ml of this product was determined from a calibration curve using a standard solution. The lowest concentration of the standard solution, 1.25 ng / mL, was taken as the detection limit.
[0145]
(2) Evaluation method of mouse antibody-derived material
Rabbits were subcutaneously sensitized with 1 ml of an equal volume of mouse antibody and Freund's complete adjuvant. Two weeks later and 3 weeks later, 1 ml of the same emulsion was administered subcutaneously in the back of the skin, and blood was collected one week after the final sensitization. The blood is allowed to stand at about 4 ° C. overnight, the clot is removed, and the centrifuged supernatant is collected. From the obtained rabbit anti-mouse antibody component antiserum, ammonium sulfate salting-out and a column using the mouse antibody component as a ligand Rabbit anti-mouse antibody component antibodies were obtained by chromatographic methods.
[0146]
Rabbit anti-mouse antibody component antibody was digested with pepsin to obtain F (ab ′) 2 fragment. After reduction with 2-mercaptoethylamine hydrochloride, an enzyme-labeled anti-mouse antibody component antibody was prepared by reacting with horseradish peroxidase into which a maleimide group was introduced with N-hydroxysuccinimide ester of N- (4-carboxycyclohexylmethyl) maleimide.
[0147]
The eluate of each step was diluted with polysorbate / phosphate buffered saline so that the expected mouse antibody component content was 0 to 25 ng / ml, and used as a sample solution. Separately, polysorbate / phosphate buffered saline was added to a mouse antibody component standard, and a solution containing 25 to 1.25 and 0 ng in 1 ml thereof was prepared as a standard solution.
After adding 50 μl of a rabbit anti-mouse antibody component antibody solution per well to a polystyrene 96-well plate, the plate was allowed to stand at 25 ° C. for about 2 hours. Next, each hole was washed with a phosphate buffered saline, a blocking solution was added, and the mixture was allowed to stand at 25 ° C. for about 1 hour. After washing with polysorbate / phosphate buffered saline, 100 μl of sample solution and standard solution were added to each well and allowed to stand at 25 ° C. for about 16 hours. Next, after washing with polysorbate / phosphate buffered saline, 50 μl of enzyme-labeled anti-mouse antibody component antibody solution was added and allowed to stand at 25 ° C. for about 2 hours. After washing with polysorbate / phosphate buffered saline, 100 μl of enzyme substrate solution was added and reacted at 25 ° C. for about 40 minutes. After adding 50 μl of diluted sulfuric acid (1 → 4) to stop the reaction, the absorbance at 490 nm was measured with a 96-well plate absorptiometer. The content of the mouse antibody component in 1 ml of this product was determined from a calibration curve using a standard solution. The lowest concentration of the standard solution, 1.25 ng / mL, was taken as the detection limit.
[0148]
(3) Method for evaluating CHO cell-derived material
The supernatant obtained by culturing CHO cells in a serum-free medium was concentrated to obtain a CHO cell-derived product. Rabbits were subcutaneously sensitized with 1 ml of an equal volume of CHO cell-derived material and Freund's complete adjuvant. Two weeks and three weeks later, 1 ml of the same emulsion was administered subcutaneously in the back of the skin, and blood was collected one week after the final sensitization. The blood was allowed to stand at about 4 ° C. overnight, the clot was removed, and the centrifuged supernatant was collected. From the obtained rabbit anti-CHO cell-derived component antiserum, ammonium sulfate salting-out and column chromatography were used. Rabbit anti-CHO cell-derived product component antibodies were obtained.
[0149]
Rabbit anti-CHO cell-derived product component antibody was digested with pepsin to obtain F (ab ′) 2 fragment. After reduction with 2-mercaptoethylamine hydrochloride, reaction with horseradish peroxidase into which a maleimide group has been introduced with N-hydroxysuccinimide ester of N- (4-carboxycyclohexylmethyl) maleimide to prepare an enzyme-labeled anti-CHO cell derived component antibody did.
[0150]
The eluate of each step was diluted with polysorbate / phosphate buffered saline so that the expected content of CHO cell-derived components was 0 to 500 ng / ml, and used as a sample solution. Separately, polysorbate / phosphate buffered saline was added to the CHO cell-derived component standard, and a solution containing 500 to 25 and 0 ng in 1 ml thereof was prepared as a standard solution.
[0151]
After adding 100 μl of rabbit anti-CHO cell-derived component antibody solution per well to a polystyrene 96-well plate, the plate was allowed to stand at 25 ° C. for about 2 hours. Next, each hole was washed with a phosphate buffered saline, a blocking solution was added, and the mixture was allowed to stand at 25 ° C. for about 1 hour. After washing with polysorbate / phosphate buffered saline, 100 μl of sample solution and standard solution were added to each well and allowed to stand at 25 ° C. for about 16 hours. Subsequently, after washing with polysorbate / phosphate buffered saline, 50 μl of enzyme-labeled anti-CHO cell-derived component antibody solution was added and allowed to stand at 25 ° C. for about 2 hours. After washing with polysorbate / phosphate buffered saline, 100 μl of enzyme substrate solution was added and reacted at 25 ° C. for about 40 minutes. After adding 50 μl of diluted sulfuric acid (1 → 4) to stop the reaction, the absorbance at 490 nm was measured with a 96-well plate absorptiometer. From the calibration curve with the standard solution, the content of the CHO cell-derived component in 1 ml of this product was determined. The lowest concentration of the standard solution, 25 ng / mL, was taken as the detection limit.
[0152]
(4) Evaluation of each mixed component amount
Using these evaluation methods, the amounts of high purity and purified soluble TM mixed in each stage of Example 17 shown in Tables 1 and 2 were evaluated. The results are shown in Tables 3 and 4. By introducing the SP ion exchange process, the amount of each component mixed can be drastically reduced at the same time. The same experiment was carried out 5 times in total. However, none of the bovine serum-derived material and mouse antibody-derived material was detected. On the other hand, when SP was not used, the respective contents were not constant, and the serum-derived material was 15 to 136 μg and the serum-derived material was 8-36 μg in five experiments.
[0153]
[Table 3]

[0154]
[Table 4]

[0155]
Example 19
Using the purified TM solution purified without using the SP Sepharose process in Example 17, the salt concentration and pH are changed as factors affecting the elution behavior of the ion exchange process, and the mixed foreign protein removal efficiency is given. The effect was investigated. The solvent conditions used were as follows:
1. Salt concentration 0.1M, pH 2.5, specific conductivity 16ms / cm
2, salt concentration 0.1M, pH 3.0, specific conductivity 13ms / cm
3. Salt concentration 0.1M, pH 3.5, specific conductivity 12ms / cm
4. Salt concentration 0.1M, pH 4.0, specific conductivity 11ms / cm
5. Salt concentration 0.1M, pH 4.8, specific conductivity 11ms / cm
6. Salt concentration 0.25M, pH 2.5, specific conductivity 30ms / cm
7. Salt concentration 0.25M, pH 3.0, specific conductivity 27ms / cm
8. Salt concentration 0.25M, pH 3.5, specific conductivity 26ms / cm
9. Salt concentration 0.25M, pH 4.0, specific conductivity 26ms / cm
10, salt concentration 0.25M, pH 4.8, specific conductivity 25ms / cm
11. Salt concentration 0.3M, pH 2.5, specific conductivity 34ms / cm
12, salt concentration 0.3M, pH 3.0, specific conductivity 32ms / cm
13. Salt concentration 0.3M, pH 3.5, specific conductivity 31ms / cm
14. Salt concentration 0.3M, pH 4.0, specific conductivity 30ms / cm
15, salt concentration 0.3M, pH 4.8, specific conductivity 30ms / cm
16, salt concentration 0.32M, pH 2.5, specific conductivity 36ms / cm
17. Salt concentration 0.32M, pH 3.0, specific conductivity 34ms / cm
18, salt concentration 0.32M, pH 3.5, specific conductivity 32ms / cm
19, salt concentration 0.32M, pH 4.0, specific conductivity 31ms / cm
20, salt concentration 0.32M, pH 4.8, specific conductivity 32ms / cm
[0156]
Each 32 ml of the purified TM solution was dialyzed against a 100 mM glycine hydrochloride buffer adjusted to the above conditions, and the solvent was exchanged. Moreover, it used for SP-SepharoseFF column (diameter 16mm, height 5mm) equilibrated with each buffer solution of the same composition. In addition, washing was started with the same buffer solution, and a pass-through fraction from the rise to the fall of the absorbance at 280 nm was obtained, immediately neutralized to pH 7 with 500 mM phosphate buffer (pH 7.3), and the neutralized wash solution was purified. Obtained as a product.
[0157]
In the same way as in Example 18, the amount of the bovine serum component and mouse antibody component mixed in the obtained purified product was evaluated, and the activity recovery rate of TM was evaluated to determine the range of conditions under which purification operation could be substantially performed. The results are shown in Table 5.
[0158]
[Table 5]

[0159]
Conditions under which bovine serum-derived material and mouse antibody-derived material can be simultaneously removed and a good TM activity recovery rate can be achieved are 0.25 to 0.32 M, preferably about 0.3 M salt concentration, ie 25 to 34 ms / The specific conductivity was about cm, preferably about 31 ms / cm, and the pH was about 3.0 to 4.0, preferably about 3.5.
[0160]
Example 20
Production of cells for E456 production
(1) Construction of plasmid pSV2TME456
In accordance with the method described in Example 1, a portion consisting of 1050 bases of the recombinant plasmid M13TMD123 prepared in Example 1- (1) using the diretar TMD2: 5′-GAAGCACGGGTCCCCCAGGCCGCC-3 ′ (25mer) shown in SEQ ID NO: 6 Was deleted. The resulting recombinant plasmid was designated M13TME456D3. Further, this recombinant plasmid M13TME456D3 contains a base sequence encoding a partial sequence of human thrombomodulin consisting of 16 lys from the start codon (ATG) of amino acid residue No. 1 of SEQ ID NO: 3 and 367 to 516 amino acids. It was found to have a DNA fragment. Further, using a dereitter TMD2: 5′-GGAGGCCGCTCAACAGTCCGGTCCCA-3 ′ (25mer) shown in SEQ ID NO: 7, a portion consisting of 108 bases of M13TME456D3 was deleted. The resulting recombinant plasmid was designated M13TME456. Furthermore, this recombinant plasmid M13TME456 was found to have a DNA fragment containing a base sequence encoding a partial sequence of human thrombomodulin consisting of 130 amino acids downstream of the initiation codon (ATG) shown in SEQ ID NO: 1. . A DNA fragment contained in this recombinant plasmid in M13TME456 was designated as TME456.
[0161]
This recombinant plasmid M13TME456 was completely digested with HindIII and BamHI to isolate a DNA fragment of about 600 bp of TMD123. On the other hand, plasmid pSV2DHFR (ATCC 37146) was completely digested with HindIII and BglII to obtain a vector fragment. This vector fragment and the previous DNA fragment TME456 were spliced using T4 DNA ligase to obtain plasmid pSV2TME456.
[0162]
(2) Establishment of high production cells for production of TME456
In accordance with the method of Example 1- (2), (3), (4), (5), transformation of CHO cells with plasmid pSV2TME456 and plasmid pSV2-DHFR, selection of DHFR producing strain from transformed CHO cells, E456 E456 productivity is amplified by selecting the production strain and MTX, and by increasing the MTX concentration, the productivity of E456 is efficiently amplified. At 2 μMMTX concentration, about 8 pg / cell · day, at 20 μMMTX concentration, about 14 pg / cell A high-producing cell line of 40 pg / cell / day was established at 100 μM / day. Moreover, according to the method of Example 1- (6), it confirmed that E456 production was stable, and also according to the method of Example 1- (7), the suspension culture acclimatization strain was acquired.
[0163]
Example 21
Culture to produce TM with TME456 high production cells
According to the method described in Example 2, a medium for TM production was prepared. Moreover, the culture | cultivation which expands a TM production cell performed the subculture which satisfy | fills the scale and cell number required for the culture | cultivation which manufacture TM mentioned later according to the method as described in Example 3.
[0164]
(1) Repeated batch culture in suspension culture
According to the method described in Example 3, culture for producing TM was performed.
The result of production culture is an average live cell density of 2.9 × 10 ⁶ The average productivity was 58 mg / L / day at cells / ml.
[0165]
(2) Batch culture in suspension culture
Culture for producing TM was performed according to the method described in Example 4.
The production culture is cultured for 10 days, and the reached viable cell density is 2.0 × 10 ⁶ The cumulative productivity was 38 mg / L at cells / ml.
[0166]
(3) Continuous culture in suspension culture
Culture for producing TM was performed according to the method described in Example 5.
The result of production culture is an average live cell density of 11.4 × 10 ⁶ The average productivity was 137 mg / L / day at cells / ml.
[0167]
(4) Continuous culture in suspension culture
According to the method described in Example 6, culture for producing TM was performed.
The production culture result shows that the average viable cell density is 11.9 × 10 ⁶ The average productivity was 128 mg / L / day at cells / ml.
[0168]
(5) Continuous culture in suspension culture
Culture for producing TM was performed according to the method described in Example 7- (1).
The result of production culture is an average viable cell density of 12.3 × 10 ⁶ The average productivity was 135 mg / L / day at cells / ml.
[0169]
(6) Repeated batch culture in adherent culture
Culture for producing TM was performed according to the method described in Example 8.
Production culture results show that the average cell density is 2.8 × 10 for the culture using Cytodex 1 ⁶ cells / ml, the average productivity is 48 mg / L / day, and the average cell density is 6.2 × 10 6 in culture using Asahi Kasei Microcarrier. ⁶ The average productivity was 89 mg / L / day at cells / ml.
[0170]
Example 22
Purification of culture supernatant by TME456 high-producing cells
(1) Crude purification using strong anion exchange resin column
5 L of the −20 ° C. frozen culture supernatant obtained in Example 21- (1) was dissolved and filtered through a 0.2 μm membrane filter (Millipore, Millipack 20). The filtered culture supernatant was applied to a Q-Sepharose (Pharmacia) column (diameter 90 mm, height 11 cm) equilibrated with 20 mM Tris salt buffer (pH 7.4) containing 150 mM NaCl. Next, it was washed with 20 mM Tris salt buffer (pH 7.4) containing 150 mM NaCl, and elution was started with 20 mM Tris salt buffer (pH 7.4) containing 300 mM NaCl. From the peak of absorbance of the eluate at 280 nm. 1.0 column volume volume of eluate was obtained as a crude product.
[0171]
(2) Main purification by affinity column (thrombin column)
400 ml of the elution fraction obtained in Example 22- (1) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 0.5 mM calcium chloride. After dialysis, DIP-thrombin-agarose (PAESE LOREI, 06-148-1035, diameter 50 mm, height 6 cm) equilibrated with 20 mM Tris buffer (pH 7.4) containing 100 mM NaCl and 0.5 mM calcium chloride. Provided. After washing with 20 mM Tris buffer (pH 7.4) containing 300 mM NaCl and 0.5 mM calcium chloride, elution was started with 20 mM Tris buffer (pH 7.4) containing 1.0 M NaCl and 0.5 mM calcium chloride. The eluate from the rising edge to the falling edge of the absorbance at 280 nm of the eluate was obtained as the main purified product.
[0172]
(3) Main purification by affinity column (antibody A)
The anti-TM monoclonal (antibody A) prepared in Example 1- (5) was columnized according to Example 11 to prepare an anti-TM monoclonal (antibody A) column. Subsequently, a 400 ml elution fraction obtained in Example 22- (1) was equilibrated with a 20 mM phosphate buffer solution (pH 7.3) containing 1.0 M NaCl (antibody A) column (diameter 50 mm, high 6 cm). A 20 mM phosphate buffer solution (pH 7.3) containing 1.0 M NaCl is flowed, a 100 mM acetate buffer solution is further washed and washed, and elution is started with a 100 mM glycine hydrochloride buffer solution (pH 3.0) containing 0.3 M NaCl. The eluate from the rising edge to the falling edge of the absorbance at 280 nm of the eluate was obtained as the main purified product.
[0173]
(4) High purity purification by strong cation exchange resin column
(4) -1 Purification of thrombin column eluate
The diluted solution obtained by adding 100 mM glycine hydrochloride buffer (pH 3.5) to 200 ml of the eluate obtained in Example 22- (2) was then diluted with 1.0 M glycine hydrochloride buffer (pH 2.0) to pH 3.5. Prepared. The diluted pH adjusted eluate was applied to an SP-SepharoseFF (Pharmacia) column (diameter 26 mm, height 3 cm) equilibrated with 100 mM glycine hydrochloride buffer (specific conductivity 31 ms / cm, pH 3.5) containing 300 mM NaCl. did. Washing was started with a 100 mM glycine hydrochloride buffer (specific conductivity 31 ms / cm, pH 3.5) containing 300 mM NaCl to obtain a flow-through fraction from the rise to the fall of the peak at an absorbance of 280 nm, and immediately a 500 mM phosphate buffer ( It was neutralized to pH 7 with pH 7.3) and obtained as a highly purified product.
[0174]
(4) -2 Purification of antibody column eluate
The diluted solution obtained by adding 100 mM glycine hydrochloride buffer (pH 3.5) to 180 ml of the eluate obtained in Example 22- (3) was then diluted with 1.0 M glycine hydrochloride buffer (pH 2.0) to pH 3.5. Prepared. The diluted and adjusted pH solution was applied to an SP-Sepharose FF (Pharmacia) column (diameter 26 mm, height 3 cm) equilibrated with 100 mM glycine hydrochloride buffer (pH 3.5) containing 300 mM NaCl. Washing was started with 100 mM glycine hydrochloride buffer (pH 3.5) containing 300 mM NaCl to obtain an eluate from the rising edge to the falling edge of the absorbance at 280 nm of the eluate, and immediately with 500 mM phosphate buffer (pH 7.3). Neutralized to pH 7, and the neutralized eluate was obtained as a highly purified product.
[0175]
(5) Concentration of high purity purified products using polysulfone hollow fiber
Each of the high-purity purified products obtained in Example 22- (4) was concentrated using 1-m polysulfone hollow fiber (Asahi Kasei Kogyo Co., Ltd.) wetted with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl. In each case, 5 ml of concentrated liquid was obtained.
[0176]
(6) Exchange of buffer solution of high purity purified product by gel filtration column
Each Sephcryl S-200 column (Pharmacia, diameter 16 mm, equilibrated with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl in each concentrated solution 5 ml obtained in Example 22- (5). (Height 90 cm). Development and fractionation were carried out with 20 mM phosphate buffer (pH 7.3) containing 50 mM NaCl. Each fraction was confirmed to be active by the method of measuring the activity to promote protein C activation by thrombin described in Example 1- (4) -a, and the active fraction was collected and a high-purity purified product whose buffer solution was exchanged was obtained. I got it.
[0177]
Example 23
Frozen production culture supernatant, ie, culture supernatant of Example 21- (2), culture supernatant of Example 22- (3), culture supernatant of Example 22- (4), Example 22- (5 Culture supernatant of Example 22- (6), culture supernatant using cytodex as the culture adherent carrier, and culture supernatant using Asahi Kasei microcarrier as the culture adherent carrier of Example 8. Was the starting material for purification.
For purification, crude purification by ion exchange chromatography of Example 22- (1) was performed, and then the crude product was mainly purified by affinity chromatography of Example 22- (3). The main purified product is purified to high purity by the ion exchange chromatography of Example 22- (4) -2, and the high purity purified product is concentrated by the concentration method of Example 22- (5). Example 22- (6) A high-purity purified product substituted with a buffer solution was obtained.
[0178]
Example 24
Physical properties of high purity products
The following examination was performed on the high-purity purified product substituted in the buffer obtained in Example 23.
The high purity purified product was able to dissolve at least 10 mg / ml in pH-neutral distilled water for injection in the absence of a surfactant.
[0179]
In addition, as a result of detecting high purity purified product by SDS-PAGE electrophoresis under both reducing and non-reducing conditions and by Coomassie staining, a single band of about 26 kDa for reduction and about 21-24 kDa for non-reduction was obtained. was detected.
In addition, N-terminal amino acid analysis of high-purity purified products was performed by a pulse liquid method using a protein sequencer Procise 492 type manufactured by PerkinElmer. As a result, the N-terminal amino acid consisted of one kind of protein starting with asparagine in all high purity purified products, which was considered to be an amino acid sequence corresponding to SEQ ID NO: 1 17-130. In any high-purity product, the amount of bovine serum-derived product or mouse antibody-derived product was below the detection limit.
[0180]
Example 25
Freeze drying of high purity products
A solution containing 1 mg of the high-purity TM obtained in Example 17 per 2 ml of distilled water for injection was prepared. 10 mg of mannitol was added to this TM solution. Separately, 0.1 mmol of arginine-hydrochloride was added to this TM solution. 2 ml of these solutions were dispensed into glass vials and lyophilized. Freeze-drying is pre-freezing at −40 ° C. for 18 hours, primary drying at −40 to + 20 ° C. and a vacuum degree of 0.05 to 0.01 mmHg for 40 hours, and then 6 ° C. at 20 ° C. and a vacuum degree of 0.05 to 0.01 mmHg. Secondary dried for hours. The obtained freeze-dried product was stored in a paper box in the dark. In some cases, a brown glass vial is used.
[0181]
【The invention's effect】
INDUSTRIAL APPLICABILITY The present invention can provide a method for producing high-purity thrombomodulin that is substantially free from serum-derived substances and antibodies in the production of soluble thrombomodulin that can be an effective pharmaceutical as an anticoagulant or thrombolytic agent. And the manufacturing method of thrombomodulin using the culture medium can be provided.
[0182]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the floating cell density (× 10) in Example 2 of the present invention. ^Five It is a graph which shows a time-dependent change of cells / ml).
FIG. 2 is a graph showing changes over time in the cumulative production amount (mg) of TM in Example 2 of the present invention.
FIG. 3 is a graph showing the change with time in the specific production rate (pg / cell / day) of TM in Example 2 of the present invention.
FIG. 4 shows the floating cell density of TM in Example 2 of the present invention (× 10 ^Five It is a graph which shows the change of the specific production rate (pg / cell / day) with respect to cells / ml).

Claims (9)

 In the method for producing high-purity soluble thrombomodulin, (1) an unpurified supernatant containing soluble thrombomodulin and serum components is obtained, and (2) the obtained supernatant is contacted with an antibody against the soluble thrombomodulin and then eluted. The purified soluble thrombomodulin solution is obtained by the step of, and (3) the obtained soluble thrombomodulin is mixed with the cation exchanger under the conditions of a specific conductivity of 25 to 34 ms / cm and a pH of 3 to 4. A method for producing high-purity soluble thrombomodulin substantially free of serum-derived material and antibody-derived material, wherein the soluble thrombomodulin is obtained as a flow-through fraction in the contacting step.  The production method according to claim 1, wherein the unpurified supernatant is a culture supernatant prepared by culturing animal cells capable of producing soluble thrombomodulin using a medium containing a serum component.  The production method according to claim 1 or 2, wherein the serum component is a bovine serum component.  The production method according to any one of claims 1 to 3, wherein the antibody to the soluble thrombomodulin is a mouse anti-human thrombomodulin monoclonal antibody.  The production method according to claim 1, wherein the cation exchanger is a carrier for column chromatography having a sulfopropyl group. The production method according to any one of claims 1 to 5, wherein the medium containing the serum component is a medium substantially comprising the components described in the following Component Table 1.

The production method according to any one of claims 1 to 6, wherein the composition ratio of the components of the medium containing the serum component is a medium having the composition ratio shown in the following component table 2.

 An animal cell capable of producing the soluble thrombomodulin has an amino acid sequence at positions 17-130 of SEQ ID NO: 1, an amino acid sequence at positions 17-130 of SEQ ID NO: 2, and one or several amino acids of those amino acid sequences; 8. A transformant obtained by introducing a gene encoding an amino acid sequence containing any amino acid sequence selected from the group consisting of a substituted or added amino acid sequence. Manufacturing method.  An animal cell capable of producing the soluble thrombomodulin is an amino acid sequence at positions 1-130 of SEQ ID NO: 1, an amino acid sequence at positions 1-130 of SEQ ID NO: 2, an amino acid sequence at positions 1-516 of SEQ ID NO: 3, SEQ ID NO: 4 A gene encoding any amino acid sequence selected from the group consisting of the amino acid sequence at positions 1 to 516 and amino acid sequences in which one or several amino acids of the amino acid sequence have been deleted, substituted or added; The production method according to any one of claims 1 to 8, which is a transformant obtained in the above manner.

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