JP4630280B2 - イムノグロブリン様(Ig−様)ドメインを含む非イムノグロブリンタンパク質の精製方法 - Google Patents
イムノグロブリン様(Ig−様)ドメインを含む非イムノグロブリンタンパク質の精製方法 Download PDFInfo
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Description
a)目的のタンパク質を含有する生物学的液体に疎水性電荷クロマトグラフィー(HCIC)樹脂を接触させる工程、
b)アンバウンド(unbound)のコンタミナントを除去するために樹脂を洗う工程、
c)酸性pHを有する溶液または有機溶媒を含む溶液により樹脂を処理することにより目的のタンパク質を溶出させる工程
を含む、生物学的液体から1と10の間のイムノグロブリン様(Ig−様)ドメインを有する目的の非イムノグロブリンタンパク質の精製または捕獲方法に関する。
a)目的のタンパク質を含有する生物学的液体に疎水性電荷クロマトグラフィー(HCIC)樹脂を接触させる工程、
b)アンバウンドのコンタミナントを除去するために樹脂を洗う工程、
c)酸性pHを有する溶液または有機溶媒を含む溶液により樹脂を処理することにより目的のタンパク質を溶出させる工程
を含む生物学的液体から1と10の間のイムノグロブリン様(Ig−様)ドメインを有する目的の非イムノグロブリンタンパク質を捕獲するためのHCICの使用を提供する。
a)目的のタンパク質を含有する生物学的液体に疎水性電荷クロマトグラフィー(HCIC)樹脂を接触させる工程、
b)アンバウンドのコンタミナントを除去するために樹脂を洗う工程、
c)酸性pHを有する溶液または有機溶媒を含む溶液により樹脂を処理することにより目的のタンパク質を溶出させる工程
を含む、生物学的液体から1と10の間のイムノグロブリン様(Ig−様)ドメインを有する目的の非イムノグロブリンタンパク質の精製および/または捕獲に関する。
r−hIL−18BPの捕獲工程。
r−hIL−18BPは、2%血清含有培養培地において組換えCHOクローンにより産生された。r−hIL−18BPは高度にグリコシル化されており、そして強酸性タンパク質である(等電点は約3)。
r−hIL−18BP捕獲における種々のMEP HyperCel樹脂バッチの試験。
MEP HyperCel樹脂の性能のバッチ間(batch-to-batch)の一貫性を調べるために、3つの異なるMEP HyperCel樹脂バッチをr−hIL−18BP捕獲において試験した(バッチは#A112、#A113、#A130)。評価されたパラメーターは、a)樹脂キャパシティー、b)純度、およびc)精製因子である。カラム(5ml HR10カラム、ベッド高さは7cm)に1ml樹脂あたり2.5〜3.5mgのr−hIL−18BPであるIL−18BP濃度でCMMをロードした(実施例1のランニング条件を使用)。
IL−18BPの捕獲におけるMEP HyperCelカラムへの粗精製物のローディングに最適なpHの評価。
先に得られた実験上のキャパシティー(実施例2)は、樹脂1mlあたりr−hIL−18BPは2.1〜2.4mgの範囲であった(表4)。MEP HyperCelカラムを用いるとイムノグロブリンのフラクションはpH6.1で溶出しはじめ、pH4でカラムから完全に溶質される(文献1および2)のに対して、我々の予備実験によると、IL−18BPはそのようなpH範囲においてはカラムに結合したままである。我々は、この特徴はIL−18BPに対するカラムのキャパシティーを増加させるためにMEP−HyperCelカラムにCMMをロードする間に利用できる、すなわちイムノグロブリンの樹脂に対する吸着をより少なくし、結果として樹脂に対するr−hIL−18BPのより多くの吸着を可能とする、と推論した。したがって、pH7.2のかわりにpH6.1でCMMをロードすることの効果を調査した。r−hIL−18BPを含有するCMMのpHをMEP HyperCelカラムへの適用の前にpH6.1に調整した。また、ローディングの前に、カラムをpH6.1の緩衝液PBSで平衡化し、ローディングの後に同じ緩衝液で最初の洗浄を行った。
MEP HyperCelカラムを用いたr−hIL−18BP捕獲におけるローディング速度の影響。
カラムへの粗精製物のローディング速度は、カラムの捕獲性能に影響し得るのであり、たとえば、高速でのローディングは操作においてはより便利であり、一方、低速でのローディングは収率を上昇させ得る。したがって、MEP HyperCelカラムでのr−hIL−18BP捕獲におけるローディング流速の影響を評価した。樹脂1mlあたり6mgのr−hIL−18BPを得るためにCMMをカラムにロードした(先の実施例を参照)。CMMは1つ目の実験においては流速0.5ml/min、2つ目の実験では3ml/minでロードされ、標準的な流速である1ml/minと比較された(表7)。流速3ml/minにおいては、流速1ml/minで回収したときと比較して、溶出フラクション中に回収したr−hIL−18BPは相当減少していた。流速0.5ml/minでのローディングは、流速1ml/minでのローディングと比較して、溶出フラクション中のr−hIL−18BPの量を顕著な増加はみられなかった。
SDS−PAGE分析
精製した5μgのr−hIL−18BPまたは20μlのクロマトグラフィーのフラクションをサンプル緩衝液4:1に希釈した。サンプルを95℃で5分間インキュベートし、還元条件下または非還元条件下、12%トリス−グリシン SDS−PAGE調製済みゲルで分離した。ゲルは20mAの定電流で〜1.5時間流され、クマシーブルーまたはゲルコード(GELCODE)染色された。
r−hIL−18BPを検出するためのELISA
4℃で一晩、コーティング緩衝液中IL−18BPに対し作製した5μg/mlのモノクローナル抗体(モノクローナルは国際公開第02/092008号パンフレットで記述したとおりに作製され、プロテインGカラムでアフィニティー精製された)でマイクロプレートをコートした。プレートを洗浄緩衝液で3回洗浄し、ブロッキング緩衝液でブロックした(下記を参照)。洗浄後、100μlのr−hIL−18BPサンプルの等分(aliquots)をウエルに加え、振とうしながら37℃で1時間インキュベートした。プレートを再度洗浄し、そしてアッセイ緩衝液で1:5000に希釈されたウサギ抗血清100μlを各ウエルに加えた。振とうしながら37℃で1時間インキュベートした後、洗浄工程を繰り返し、そして結合した抗体をアッセイ緩衝液で1:10000に希釈されたヤギ抗ウサギに結合された(cojugated)HRPで検出した。100μ/ウェルのHRP−コンジュゲート(HRP-conjugate)をプレートに加え、振とうしながら37℃で1時間インキュベートした。ついでプレートを洗浄し、発色反応(colour reaction)を125μl/ウェルのOPD基質溶液を加えることで展開した。125μl/ウェルの4N HClを加えることで反応を停止させた。吸光度はELISAリーダー中、492nmで測定された。免疫精製された(immunopurified)r−hIL−18BP−Hisのバッチ由来のサンプルを、このアッセイの展開中、リファレンスサンプルとして使用した。免疫精製したリファレンススタンダードのタンパク質含有量を吸収係数(extinction coefficient)1.26OD/mgを用いて分光法によりA280の波長で測定した。検量線は標準r−hIL−18BPをそれぞれのアッセイのためのアッセイ緩衝液において20ng/mlから0.3ng/mlまでの範囲でアッセイ緩衝液を用いて系列希釈することで作製した。
逆相HPLC(RP−HPLC)分析
r−hIL−18BPフラクションの同定および精製は、RP−HPLC(RP−HPLCカラム スーペルコシル(Supelcosil)LC308、カタログ番号5810、4.6mm、IDX5cm、ロット#C175、スーペルコ(Supelco)社製、米国)カラムを用いて次のように行った:20μgのr−hIL−18BPをカラムにインジェクトし、0.1%のTFA(トリフルオロ酢酸、ベーカー(Baker)社製、カタログ番号9470−01)中、10〜100%プロパノールのグラディエントを用い分離した。
MRP HyperCelカラムによるr−IL6−IL6Rキメラの捕獲
IL6−IL6Rキメラ(または「IL6R/IL6」もしくは「IL−6キメラ」とも呼ばれる)はキメラ分子であって、インターロイキン6に融合されたIg−様ドメインを有するインターロイキン6受容体の可溶性部位からなる(チェバスら(1997年)およびコレットら(1999年))。MEP HyperCelカラムへのr−hIL6−IL6Rキメラの結合を調査した。ローディングに用いた原料は、浄化および10kDaの膜による20倍濃縮後に得られた2%のFBS中CHOプロジューサー細胞の粗精製収穫物である(CCM)。
MEP HyperCelカラムによるr−β−ガラクトシダーゼの捕獲。
もう1つの実験において、Ig−様ドメインを有するバクテリアのタンパク質、β−ガラクトシダーゼのMEP HyperCelカラムへの結合を調べた。大腸菌β−ガラクトシダーゼ(ロッシュ・ダイアグノスティックス社製、カタログ番号567779)を、無血清培地(SFM)ProCHO−5に0.625mcg/mlで加えた。
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Claims (32)
- a)目的のタンパク質を含有する生物学的液体に疎水性電荷誘導クロマトグラフィー(HCIC)樹脂を接触させる工程であって、該樹脂がメルカプト−エチル−ピリジンリガンドを含む工程、
b)アンバウンドのコンタミナントを除去するために緩衝液で樹脂を洗う工程、および
c)有機溶媒を含む緩衝液により樹脂を処理することにより目的のタンパク質を溶出させる工程であって、該有機溶媒がプロピレングリコールである工程
を含む、生物学的液体から1と10の間のイムノグロブリン様(Ig−様)ドメインを有する目的の非イムノグロブリンタンパク質の精製または捕獲方法。 - 溶液中のプロピレングリコール濃度が、25%と50%の間にある請求項1記載の方法。
- 工程a)が、3と6.8の間のpHで実施される請求項1または2記載の方法。
- 洗浄工程b)が、3と6.8の間のpHを有する溶液で実施される請求項1、2または3記載の方法。
- 生物学的液体が、細胞調製培養培地、細胞溶解物、細胞抽出物、組織抽出物、血漿、血清、乳汁、尿、腹水、脳脊髄液、野菜ジュース、植物抽出物または初期クロマトグラフィー分離工程由来のフラクションから選択される請求項1、2、3または4記載の方法。
- 目的のタンパク質が、1〜7のIg−様ドメインを有する請求項1、2、3、4または5記載の方法。
- 目的のタンパク質が、IL−18結合タンパク質(IL−18BP)、NCAM、フィブロネクチンタイプIII、ICAM−1、madCAM−1、PE CAM−1、VCAM−1、タイチン、カドヘリン、ニューロカン、LIFR、CNTFR、IL−1R、IL−3R、IL5R、IL−6R、IL−12R、GM−CSFR、オンコスタチンM受容体(OSMR)、VEGF受容体、FGF受容体、hPDGF受容体、T細胞受容体、MHCタンパク質、ミクログロブリン−β、CTLA4、B7活性化因子、ニューレグリン、凝集因子XIII、NF−kB、IL6−IL6R、β−ガラクトシダーゼおよびスーパーオキシドディスムターゼ、または少なくとも1つのIg−様ドメインを含むそれらのアイソフォーム、ムテイン、融合タンパク質またはフラグメントから選択される請求項1、2、3、4、5または6記載の方法。
- タンパク質が、IL−18結合タンパク質(IL−18BP)である請求項7記載の方法。
- タンパク質が、IL−6−IL6Rキメラである請求項7記載の方法。
- タンパク質が、β−ガラクトシダーゼである請求項7記載の方法。
- 溶出タンパク質の精製因子が、11倍と94倍の範囲にある請求項1、2、3、4、5、6、7、8、9または10記載の方法。
- 溶出タンパク質の精製因子が、94倍である請求項11記載の方法。
- 溶出タンパク質の濃縮因子が、1.5倍と3.1倍の範囲にある請求項1、2、3、4、5、6、7、8、9、10、11または12記載の方法。
- 溶出タンパク質の濃縮因子が、3.1倍である請求項13記載の方法。
- 溶出タンパク質の収率が、73%と98%の範囲にある請求項1、2、3、4、5、6、7、8、9、10、11、12、13または14記載の方法。
- 溶出タンパク質の収率が、85%である請求項15記載の方法。
- a)目的のタンパク質を含有する生物学的液体に疎水性誘導電荷クロマトグラフィー(HCIC)樹脂を接触させる工程であって、該樹脂がメルカプト−エチル−ピリジンリガンドを含む工程、
b)アンバウンドのコンタミナントを除去するために緩衝液で樹脂を洗う工程、および
c)有機溶媒を含む緩衝液により樹脂を処理することにより目的のタンパク質を溶出させる工程であって、該有機溶媒がプロピレングリコールである工程、
からなる生物学的液体から1と10の間のイムノグロブリン様(Ig−様)ドメインを有する目的の非イムノグロブリンタンパク質を捕獲するためのHCICの使用。 - 溶液中のプロピレングリコール濃度が、25%と50%の間にある請求項17記載の使用。
- 工程a)が、3と6.8の間のpHで実施される請求項17または18記載の使用。
- 洗浄工程b)が、3と6.8の間のpHを有する溶液で実施される請求項17、18または19記載の使用。
- 生物学的液体が、細胞調製培養培地、細胞溶解物、細胞抽出物、組織抽出物、血漿、血清、乳汁、尿、腹水、脳脊髄液、野菜ジュース、植物抽出物または初期クロマトグラフィー分離工程由来のフラクションから選択される請求項17、18、19または20記載の使用。
- 目的のタンパク質が、1〜7のIg−様ドメインを有する請求項17、18、19、20または21記載の使用。
- 目的のタンパク質が、IL−18結合タンパク質(IL−18BP)、NCAM、フィブロネクチンタイプIII、ICAM−1、madCAM−1、PE CAM−1、VCAM−1、タイチン、カドヘリン、ニューロカン、LIFR、CNTFR、IL−1R、IL−3R、IL5R、IL−6R、IL−12R、GM−CSFR、オンコスタチンM受容体(OSMR)、VEGF受容体、FGF受容体、hPDGF受容体、T細胞受容体、MHCタンパク質、ミクログロブリン−β、CTLA4、B7活性化因子、ニューレグリン、凝集因子XIII、NF−kB、IL6−IL6R、β−ガラクトシダーゼおよびスーパーオキシドディスムターゼ、または少なくとも1つのIg−様ドメインを含むそれらのアイソフォーム、ムテイン、融合タンパク質またはフラグメントから選択される請求項17、18、19、20、21または22記載の使用。
- タンパク質が、IL−18結合タンパク質(IL−18BP)である請求項23記載の使用。
- タンパク質が、IL−6−IL6Rキメラである請求項23記載の使用。
- タンパク質が、β−ガラクトシダーゼである請求項23記載の使用。
- 溶出タンパク質の精製因子が、11倍と94倍の範囲にある請求項17、18、19、20、21、22、23、24、25または26記載の使用。
- 溶出タンパク質の精製因子が、94倍である請求項27記載の使用。
- 溶出タンパク質の濃縮因子が、1.5倍と3.1倍の範囲にある請求項17、18、19、20、21、22、23、24、25、26、27または28記載の使用。
- 溶出タンパク質の濃縮因子が、3.1倍である請求項29記載の使用。
- 溶出タンパク質の収率が、73%と98%の範囲にある請求項17、18、19、20、21、22、23、24、25、26、27、28、29または30記載の使用。
- 溶出タンパク質の収率が、85%である請求項31記載の使用。
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