JP2020007343A - 抗体組成物及びそのための緩衝系 - Google Patents
抗体組成物及びそのための緩衝系 Download PDFInfo
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- JP2020007343A JP2020007343A JP2019163553A JP2019163553A JP2020007343A JP 2020007343 A JP2020007343 A JP 2020007343A JP 2019163553 A JP2019163553 A JP 2019163553A JP 2019163553 A JP2019163553 A JP 2019163553A JP 2020007343 A JP2020007343 A JP 2020007343A
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- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
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- 239000001257 hydrogen Substances 0.000 description 1
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- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
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- AKMJJGSUTRBWGW-UHFFFAOYSA-N pyridine-2-carboxylic acid Chemical compound OC(=O)C1=CC=CC=N1.OC(=O)C1=CC=CC=N1 AKMJJGSUTRBWGW-UHFFFAOYSA-N 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
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Abstract
Description
実施例1.ラテラルフローアッセイ
ラテラルフロー試験ストリップ(4mm幅)を、ヤギ抗−ウサギIgG(試験ライン)及びビオチン−BSA(対照ライン)でスジ状としたニトロセルロース膜(Millipore Hi Flow plus 90)から調製した。ストリップをプラスチックカートリッジに収容し、金コンジュゲート試料(80ul)を、試料パッドに添加した。カートリッジを30分間静置し、次にESE−Quant GOLDリーダー−QIAGENを使用して読み取った。各試料に、対照ラインに結合する一定量のストレプトアビジン−金(Innova biosciences−製品コード:250−0200)を添加(spiked)して、各試験ストリップの動作が正確であること、及び試験ラインデータが有効であることをチェックした。
黒色96ウェルプレート(Greiner Bio−One(コード655077))を、ウサギIgG50ul(1ug/ウェル)と共に4℃において一晩インキュベートした。プレートをTBS/0.1%BSAで室温で1時間ブロッキングし、TBS(洗浄バッファー)で5回洗浄した。蛍光性抗体コンジュゲートを、TBS/0.1%BSAで連続希釈し、一定分量50ulをウェルに三連で添加した。室温で1時間インキュベートした後、プレートを5回洗浄し、蛍光発光を、Tecan Infinite M200プレートリーダーとi−Control 1.4ソフトウェアを使用して、適切な励起/発光設定(フルオレセイン、490nm/535nm;フィコエリトリン(phycoeythrin)、535nm/575nm)で読み取った。蛍光データを、コンジュゲート希釈関数としてプロットした。異なる実験で試験した添加剤に関する結果を比較するために、データを対照値(すなわち任意の試験添加剤が存在しない状態で調製したコンジュゲートのシグナル)の百分率に変換した。
ヤギ抗−ウサギIgG(GAR)10μgを、10.0ODにおいてカルボキシル官能基を有するコーティングされた40nmのナノ粒子(InnovaCoat GOLD、Innova Biosciences)1ml当たり、最終濃度50mMの添加剤が存在する状態で使用した。典型的に、以下の添加剤を用いて反応を引き起こした。40nmのInnovaCoat(カルボキシル化)金ナノ粒子(nanopartcles)40μlを、0.1mg/mlのGAR4μl、水20μl、及び100mMの添加剤、pH5.0を含有する100mMのMESバッファー80μlと共に5分間インキュベートした。次に、1mMのEDC16μlを添加し、22℃で10分間インキュベーションした後、20μlの10×TBS/1%Tween20及び水20μlを添加して、2ODのコンジュゲートを得た。
反応は、添加剤がヤギ−抗ラビットIgGのコンジュゲーション反応では最大50mMの濃度で含まれていたことを除いて、製造者の標準プロトコルに従って、フルオレセインLightning−Linkキット(製品コード707−0010、Innova Biosciences、UK)を使用して実施した。コンジュゲートを、実施例2に記載のプロトコルに従って試験した。グリシンベタインが存在する状態で調製したコンジュゲートの用量応答曲線を図1に示す。以下の化合物が存在する状態で調製したコンジュゲート(1/100希釈)の対照値に対する%(括弧内の値)は、添加剤を含まない対照(100%)と比較して、プロリン(53%)、グリシンベタイン(98%)、N,N−ジメチルグリシン(96%)、2ピコリン酸(83%)であった。
この方法は、ヤギ抗−ウサギIgGを、Lightning−Link PEコンジュゲーションキット(703−0005)を使用して蛍光性タンパク質フィコエリトリン(PE)にコンジュゲートしたことを除いて、実施例4に記載した通りであった。トリシンが存在する状態で調製したフィコエリトリンコンジュゲートの用量応答曲線を、図2に示す。トリシンは、試験濃度範囲では、Lightning−Link PEコンジュゲーション反応に対してほとんど効果がない。この場合、最高濃度のトリシン(50mM)で調製したコンジュゲートにより、添加剤が存在しない状態の対照値の91%というシグナルが得られた。
10mg/mlのウサギIgG40μgを、50mMの添加物(1つはグリシンベタイン、クエン酸又はグリシンから)を含有する80mMのCHESバッファーpH9.0中、50μMのFITCと共に、暗室中22℃で一晩インキュベートした。コンジュゲートを、TBSバッファー中、PD10カラム(GE Healthcare)で脱塩し、溶出された溶液の吸光度値を、280nm及び495nmで決定した。様々な添加剤が存在する状態の抗体色素コンジュゲートに関する色素とタンパク質のモル比を、図3に示す。グリシンは、著しい干渉を示したが、グリシンベタイン及びクエン酸は、対照(すなわち添加剤なし)の結果に類似した結果を示した。
10mg/mlのウサギIgG40μgを、50mMの様々な添加物の1つ(グリシンベタイン、クエン酸又はグリシンから)を含有する80mMのリン酸ナトリウムpH8.0中、50μMのフルオレセイン−NHS色素と共に、暗室中22℃で30分間インキュベートした。コンジュゲートを、TBSバッファー中で平衡化したPD10カラムで脱塩し、溶出された溶液の吸光度値を、280nm及び495nmで決定した。様々な添加剤が存在する状態の抗体色素コンジュゲートに関する色素とタンパク質のモル比を、図4に示す。グリシンベタイン及びクエン酸は、対照(添加剤なし)の結果に類似した結果を示したが、グリシンは、実質的な干渉を示した。
80ug/mlのDTNB及び50mMの添加剤(グリシンベタイン、クエン酸又はグリシン)を含有する100mMのリン酸ナトリウムpH8.0、200μlの試料を、透明96ウェルプレート(Greiner Bio−One、コード655077)に三連で添加した。2mMの2−イミノチオラン20μlを各ウェルに添加し、速やかに混合し、吸光度値を、Tecan Infinite M200プレートリーダーとi−Control 1.4ソフトウェアを使用して、405nmで30分間かけて毎分読み取った。2−イミノチオランからのチオールの放出を、図5に示す。図は、2−イミノチオランの開環反応におけるチオールの放出を示している。対照反応では、試薬が水によって加水分解されるので、吸光度は着実に線形増加する。ベタイン又はクエン酸が存在する状態では、この比率は増大しない(対照の曲線と重ね合わせた曲線)。グリシンが存在する状態では、急速な加水分解及び2−イミノチオランの開環が生じる。グリシンベタインもクエン酸も、対照反応(添加剤なし)ほど急速にチオールを放出しなかったことが観測された。ベースラインの上昇は、アルカリpH値における加水分解に対する2−イミノチオランの公知の感度に寄与し得る。
グリシンベタイン3ml、pH2.25に、「キャッチ」バッファー(1MのHepes30ul、pH7.5)を添加した後、高pKa成分(0.5MのCHESバッファー、pH9.5)10ulの添加を反復した。pHを、各添加後に測定した。pH9.5のCHESバッファー70ul、80ul及び90ulを添加した後、混合物のpH値は、7.27、7.45、及び7.6であった。図6は、キャッチバッファーが存在する場合及び存在しない場合の完全なpHプロファイルを示している。図は、キャッチバッファー(1MのHepes30ul、pH7.5)が存在する状態(黒丸)又は存在しない状態(白丸)のいずれかで、CFEB(ベタイン、pH2.25)3mlを中和した後、0.5MのCHESバッファー10ul、pH9.5の添加を反復したことを示している。キャッチバッファーを添加すると、pHは約1pH単位上昇したので、曲線は異なる出発位置を有している。より重要なことに、キャッチバッファー(pKa7.5)は、pH7及びpH8の間のpH変化に抵抗するので、pH9.5の中和バッファーを添加しても、pHをpH7.5近くの任意の値に調節することが容易になる。キャッチバッファーが存在しない状態では、CHESバッファー10ulを1回増やすと(90ulから100ulまで)、pH4.87からpH8.19への急激な移行が見られる。
ヤギ抗−マウスセファロースビーズを、使い捨てのカラムに充填した(充填体積0.5ml)。TBS中、マウスIgGを含有する試料と共に2時間インキュベートした後、カラムを10mMのリン酸ナトリウム/150mMのNaCl、pH8.0で洗浄し、次に10mMのベタイン、pH2.3で溶出した。100ulの画分を収集し、50mMのHepes(キャッチバッファー)及び90mMのCHES(高pKa成分)を含む10×バッファーA11.1ulですぐに中和した。10×バッファーAは、適切な体積の1MのHepes、pH7.5、0.5MのCHES、pH9.5、及び水を混合することによって調製した。抗体(ベタイン中)と中和バッファーの体積比によって、最終キャッチバッファー濃度5mM、及びpH7.1が得られる。或いは、溶出された画分を、10×バッファーB(30mMのHepes及び105mMのCHESバッファー)11.1ulで中和して、最終pHが7.55の、より弱く緩衝化された抗体調製物(すなわち3mMのキャッチバッファー)を得た。
Claims (1)
- 単離された抗体を標識又は誘導体化試薬にコンジュゲートする方法であって、グリシン以外の、α位又はβ位にアミン置換基を有するモノカルボン酸バッファー化合物を含む緩衝系中で、前記抗体を、活性化標識若しくは活性化誘導体化試薬と接触させるか、又は前記抗体を、抗体コンジュゲーション試薬が存在する状態で前記標識若しくは誘導体化試薬と接触させる工程を含み、
前記モノカルボン酸バッファー化合物が、ベタイン、アラニン、β−アラニン、2−アミノ酪酸、プロリン、トリシン、N−メチルグリシン、N,N−ジメチルグリシン、及び2−ピコリン酸から選択される少なくともいずれかであることを特徴とする方法。
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WO2007068906A2 (en) * | 2005-12-12 | 2007-06-21 | Innova Biosciences Ltd | Production of conjugates |
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