JP2006523308A - 疎水性相互作用クロマトグラフィー用リガンドの製造法 - Google Patents
疎水性相互作用クロマトグラフィー用リガンドの製造法 Download PDFInfo
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- JP2006523308A JP2006523308A JP2006507945A JP2006507945A JP2006523308A JP 2006523308 A JP2006523308 A JP 2006523308A JP 2006507945 A JP2006507945 A JP 2006507945A JP 2006507945 A JP2006507945 A JP 2006507945A JP 2006523308 A JP2006523308 A JP 2006523308A
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- 150000003440 styrenes Chemical class 0.000 description 1
- 125000000446 sulfanediyl group Chemical group *S* 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 150000003568 thioethers Chemical class 0.000 description 1
- 125000005490 tosylate group Chemical group 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229920001567 vinyl ester resin Polymers 0.000 description 1
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
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- B01D15/08—Selective adsorption, e.g. chromatography
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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Abstract
Description
(a)一般式(I)で定義される1以上の骨格を準備する段階と、
(b)骨格の窒素を、残基Rに結合した反応性基Zを含む試薬で誘導体化して第一の相互作用を導入する段階と、
(c)得られた誘導体をアミノ分解することによってカルボニルとチオールの間で環構造を開環し、カルボニルに隣接して第二の相互作用を導入する段階とを含み、
第一の相互作用及び第二の相互作用の少なくとも一方が疎水性基を含み、前記相互作用がいずれもイオン交換リガンドを含まない方法である。
「分離媒体」という用語は、本明細書では、例えばクロマトグラフィーカラムの充填材として有用な材料、具体的には、ベースマトリックスに1以上のリガンドが結合したものからなる材料に用いる。ベースマトリックスは担体として作用し、リガンドはクロマトグラフィーで目標物質と相互作用する官能基を与える。
式中、Zは骨格の窒素と反応し得る基である。またRは第一の相互作用を提供する基であり、好ましくは炭素原子数約1〜20、例えば1〜10の線状、枝分れ、環状の飽和、不飽和又は芳香族炭化水素基である。
特定の実施形態では、本方法の段階(a)及び(b)は早くから行われ、既誘導体化骨格(ready−derivatised scaffold)が得られている。したがって、本発明は、骨格の誘導体化が早くから、即ち各々段階(c)及び段階(d)のアミノ分解及びベースマトリックスへの固定化とは別に、行われている方法も包含する。
段階1:
DL−ホモシステインチオラクトンIaとdi−イソプロピルアミン(DIPEA)のジクロロメタン(DCM)溶液Aを0℃に冷却した。塩化アシル又はスルホニルクロリド又は酸無水物又は活性酸をDCM中に含有する溶液Bを0℃に冷却し、これを0〜5℃に保持した溶液Aに滴下した。この混合物を室温で一晩かき混ぜた。減圧下で溶媒を除去した。必要に応じて、得られた生成物を酢酸エチルに溶解し、クエン酸溶液(10重量%の水溶液)及び炭酸カリウム溶液(10重量%の水溶液)で洗浄してもよい。有機相を水洗した後硫酸ナトリウムで乾燥し、次いで溶媒を蒸発させた。
得られた生成物をテトラヒドロフラン(THF)に溶解し、10分間窒素を吹き込んで脱気した。この溶液にTHFに溶かしたアミンを室温で加えた。反応混合物をさらに17時間かき混ぜた。減圧下で溶媒を蒸発させた後、酢酸エチル及びクエン酸溶液(10重量%の水溶液)を混合した。有機相を水洗した後硫酸ナトリウムで乾燥し、次いで溶媒を蒸発させた。
周知の手段を用いて得られた臭素化Sepharose(登録商標)6 Fast Flowを、段階2で得られた生成物のアルカリ性溶液と混合し、一晩50℃に温めた。反応後、ゲル(1容量)をろ過し、水(15容量×2回)、エタノール(15容量×2回)、0.2M酢酸(15容量×2回)及び水(15容量×2回)で洗浄した。ゲル上のリガンド濃度は、硫黄の元素分析によって測定した。
異なる基R1、R2及びR3を用いた本発明による分離媒体の生成
以下の実施例は、骨格としてD,L−ホモシステインチオラクトンIa及び既に説明した化学反応(上記のスキーム1参照)を用いている。ホモシステインチオラクトンIaを、塩化アシル、スルホニルクロリド、酸無水物又は活性酸と反応させることによってアミド又はスルホンアミドを形成した後、アミンを用いてチオラクトン環の開環を行う。得られた化合物を、さらに活性化したSepharose(登録商標)6 Fast Flow又は活性化したSepharose(登録商標)High Performanceに結合させる。
段階1:11.5g(75.0ミリモル)のDL−ホモシステインチオラクトン塩酸塩を、120mlのDCM及び16.1ml(150.0ミリモル)のDIPEAに溶解した。この溶液に、DCM30mlに溶かした8.7ml(75.0ミリモル)の塩化ベンゾイルを、段階1に従って室温でゆっくり加えた。減圧下で溶媒を除去し、酢酸エチル(300ml)を加えた。有機相を、クエン酸溶液(10重量%の水溶液)及び炭酸カリウム溶液(10重量%の水溶液)で洗浄した(100ml×2回)。有機相を硫酸ナトリウムで乾燥した後溶媒を蒸発させ、13.8gの白色粉末を回収した。収率:83%。
(リガンドの計算濃度:29.1μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:53.7μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:24.8μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:41.2μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:57.0μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:18.3μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:61.6μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:46.1μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:21.9μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:55.3μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:17.1μモル/ml)。
段階1:実施例1−段階1に記載のものと同じ手順。
(リガンドの計算濃度:42.8μモル/ml)。
段階1:10.0g(65.0ミリモル)のDL−ホモシステインチオラクトン塩酸塩を、120mlのDCM及び23.9ml(136ミリモル)のDIPEAに溶解した。この溶液に、DCM30mlに溶かした7.7ml(65.0ミリモル)のバレリルクロリドを、段階1に従って室温でゆっくり加えた。減圧下で溶媒を除去し、酢酸エチル(400ml)を加えた。有機相を、クエン酸溶液(10重量%の水溶液)及び炭酸カリウム溶液(10重量%の水溶液)で洗浄した(100ml×2回)。有機相を硫酸ナトリウムで乾燥した後溶媒を蒸発させ、8.86gの白色粉末を回収した。収率:68%。
クロマトグラフィー系
すべての実験は、ソフトウェアUnicorn 3.1を備えたAKTA(登録商標)Explorer 100クロマトグラフィー系(Amersham Biosciences AB)を使用して室温で行った。
注入体積:10μl(5mMデオキシコルチコステロンのメタノール溶液)
流速:1.0ml/分
溶出液A:10mMリン酸アンモニウムのMQ Milli−Q水溶液、pH7
溶出液B:アセトニトリル
溶出:リニアグラジエント(0〜80%B)
グラジエント条件:カラム平衡=10カラム体積(CV)、グラジエント体積=20CV及び80%Bを用いてカラムを洗浄(グラジエント後)=2CV
検出波長:205、236,及び280nm
試験2:ペプチドの分離
4つの異なるpH値、pH2、3、7及び12で4種のアンギオテンシン誘導体の混合物を分離することによってクロマトグラフィー評価を行った。
アンギオテンシン−I Asp−Arg−Val−Tyr−Ile−His−Pro−Phe−His−Leu (Sigma A9650)
アンギオテンシン−III Arg−Val−Tyr−Ile−His−Pro−Phe(ICN 191237)
Ile7−アンギオテンシン−III Arg−Val−Tyr−Ile−His−Pro−Ile(Sigma A0911)
Val4アンギオテンシン−III Arg−Val−Tyr−Val−His−Pro−Phe(Sigma A6277)
注入体積:10μl(Milli−Q水で製造した試料混合物中、各ペプチド0.125mg/ml)
流速:1.0ml/分
溶出液A:0.1%TFAのMQ Milli−Q水溶液(pH2)、10mMリン酸カリウム(pH3及び7)
溶出液B:アセトニトリル
溶出:リニアグラジエント(3〜100%B)
グラジエント条件:カラム平衡=5カラム体積(CV)、グラジエント体積=10CV及び100%Bを用いてカラムを洗浄(グラジエント後)=2CV
検出波長:215nm
この場合のカラム体積は2.49mlである(この方法は鋼製カラムST4.5/150用に開発された)。
Amersham Biosciences AB製の5/5HRカラムに充填したゲル1〜2mlを1ml/分で運転する。本方法では、2M(NH4)2SO4+0.1Mリン酸Kの緩衝液A(pH7)及び0.1Mリン酸Kの別の緩衝液B(pH7)を使用する。ミオグロビン0.5mg/ml、リボヌクレアーゼA(2mg/ml)、α−キモトリプシノゲンA(0.8mg/ml)及びα−ラクトアルブミン(0.5mg/ml)の4種のタンパク質を緩衝液Aに混ぜてカラムに通液する。次いでカラムを2mlの緩衝液Aで運転し、次いでA100%からB100%へ変化する20mlのグラジエント溶出を適用した。
1:ミオグロビン
2:リボヌクレアーゼA
3:α−ラクトアルブミン
4:α−キモトリプシン
Claims (17)
- 疎水性相互作用クロマトグラフィー(HIC)用の1以上のマルチモードリガンドの製造方法であって、
(a)一般式(I)で定義される1以上の骨格を準備する段階と、
(b)骨格の窒素を、残基Rに結合した反応性基Zを含む試薬で誘導体化して第一の相互作用を導入する段階と、
(c)得られた誘導体をアミノ分解することによってカルボニルとチオールの間で環構造を開環し、カルボニルに隣接して第二の相互作用を導入する段階とを含み、
第一の相互作用及び第二の相互作用の少なくとも一方が疎水性基を含み、前記相互作用がいずれもイオン交換リガンドを含まない方法。 - 式(I)において、A、B及びXが炭素原子であり、mが1である、請求項1記載の方法。
- 式(I)において、骨格がホモシステインチオラクトンである、請求項2記載の方法。
- 段階(b)で使用される試薬が一般式(II)で定義される、請求項1乃至請求項3のいずれか1項記載の方法。
−Z−R− (II)
式中、Zは骨格の窒素と反応し得る基であり、Rが線状、枝分れ、環状の飽和、不飽和又は芳香族炭化水素基である。 - 段階(b)がアルキル化、アシル化又はスルホニル化である、請求項1乃至請求項4のいずれか1項記載の方法。
- 段階(b)が、アシル化又はスルホニル化、アルキル化とアシル化の組合せ、或いはアルキル化とスルホニル化の組合せを含む、請求項5記載の方法。
- 段階(c)から得られる生成物のチオールを、反応性基を含むベースマトリックスと反応させる段階(d)も含む、請求項1乃至請求項6のいずれか1項記載の方法。
- 誘導体化骨格のチオール基がベースマトリックスのアリル基と結合する、請求項7記載の方法。
- ベースマトリックスがアガロースなどの多糖類から作製され、分離媒体がHIC媒体である、請求項7又は請求項8記載の方法。
- ベースマトリックスがジビニルベンゼン又はスチレンなどの合成高分子から作製され、分離媒体がRPC媒体である、請求項7又は請求項8記載の方法。
- 複数のマルチモードリガンドを含み、各リガンドが標的分子と疎水性相互作用し得る1以上の基と、同一の標的分子と相互作用し得る1以上の別の基とを含む、疎水性相互作用クロマトグラフィー用分離媒体の製造における、ホモシステインチオラクトンの使用。
- 標的分子と疎水性相互作用し得る1以上の基と、同一の標的分子と相互作用し得る1以上の別の基とを含む、疎水性相互作用クロマトグラフィー(HIC)用マルチモードリガンド。
- 請求項1乃至請求項10のいずれか1項記載の方法で製造された、請求項12記載のリガンド。
- ベースマトリックスに固定化された複数のリガンドを含み、各リガンドが標的分子と疎水性相互作用し得る1以上の基と、同一の標的分子と相互作用し得る1以上の別の基とを含む、疎水性相互作用クロマトグラフィー(HIC)用分離媒体。
- 請求項1乃至請求項10のいずれか1項記載の方法で製造された、請求項14記載の分離媒体。
- 請求項14又は請求項15において定義された分離媒体を充填した、疎水性相互作用クロマトグラフィー(HIC)用クロマトグラフィーカラム。
- 請求項14又は請求項15において定義された分離媒体を提供する段階と、前記媒体を液体と接触させて目標物質をリガンドへ吸着させる段階とを含む、液体から目標物質を分離する方法。
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JPH04503157A (ja) * | 1989-01-05 | 1992-06-11 | アクゾ・ノベル・ナムローゼ・フェンノートシャップ | 核酸の入手および検出のためのポリアクリルアミド固形支持体に対するオリゴヌクレオチドの末端の付着 |
JP2000351776A (ja) * | 1999-04-08 | 2000-12-19 | Kuraray Co Ltd | 光学活性ホモシステインチオラクトン塩の製造方法およびその中間体 |
WO2001038227A2 (en) * | 1999-11-22 | 2001-05-31 | Amersham Biosciences Ab | A method for anion-exchange adsorption and anion-exchangers |
WO2002053288A2 (en) * | 2000-12-31 | 2002-07-11 | Amersham Biosciences Ab | A method for the manufacture of compositions containing low concentrations of salts |
WO2003024588A1 (en) * | 2001-09-14 | 2003-03-27 | Amersham Biosciences Ab | Generation of ion exchanger media |
WO2004076475A1 (en) * | 2003-02-28 | 2004-09-10 | Amersham Biosciences Ab | A method of generating metal chelating affinity ligands |
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US4837348A (en) * | 1986-04-30 | 1989-06-06 | Varian Associates, Inc. | Bonded phase chromatographic supports |
US5652348A (en) * | 1994-09-23 | 1997-07-29 | Massey University | Chromatographic resins and methods for using same |
GB2384640B (en) * | 2002-01-29 | 2004-04-14 | Simpleware Ltd | Image processing method |
SE0300624D0 (sv) * | 2003-03-05 | 2003-03-05 | Amersham Biosciences Ab | A method of preparing affinity ligands |
SE0300612D0 (sv) * | 2003-03-05 | 2003-03-05 | Amersham Biosciences Ab | A method of preparing ligands for hydrophobic interaction chromatography |
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2003
- 2003-03-05 SE SE0300612A patent/SE0300612D0/xx unknown
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2004
- 2004-03-05 WO PCT/SE2004/000316 patent/WO2004078311A1/en active Application Filing
- 2004-03-05 EP EP04717913A patent/EP1599265A1/en not_active Withdrawn
- 2004-03-05 CA CA2517757A patent/CA2517757C/en not_active Expired - Fee Related
- 2004-03-05 JP JP2006507945A patent/JP4570052B2/ja not_active Expired - Fee Related
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JPS59104377A (ja) * | 1982-11-08 | 1984-06-16 | アマーシャム インターナショナル ピーエルシー | アミノ酸チオラクトン |
JPH04503157A (ja) * | 1989-01-05 | 1992-06-11 | アクゾ・ノベル・ナムローゼ・フェンノートシャップ | 核酸の入手および検出のためのポリアクリルアミド固形支持体に対するオリゴヌクレオチドの末端の付着 |
JP2000351776A (ja) * | 1999-04-08 | 2000-12-19 | Kuraray Co Ltd | 光学活性ホモシステインチオラクトン塩の製造方法およびその中間体 |
WO2001038227A2 (en) * | 1999-11-22 | 2001-05-31 | Amersham Biosciences Ab | A method for anion-exchange adsorption and anion-exchangers |
WO2002053288A2 (en) * | 2000-12-31 | 2002-07-11 | Amersham Biosciences Ab | A method for the manufacture of compositions containing low concentrations of salts |
WO2003024588A1 (en) * | 2001-09-14 | 2003-03-27 | Amersham Biosciences Ab | Generation of ion exchanger media |
WO2004076475A1 (en) * | 2003-02-28 | 2004-09-10 | Amersham Biosciences Ab | A method of generating metal chelating affinity ligands |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20190039144A (ko) * | 2016-08-16 | 2019-04-10 | 리제너론 파아마슈티컬스, 인크. | 혼합물로부터 개별 항체들을 정량하는 방법 |
KR102369014B1 (ko) | 2016-08-16 | 2022-03-02 | 리제너론 파아마슈티컬스, 인크. | 혼합물로부터 개별 항체들을 정량하는 방법 |
KR20220025943A (ko) * | 2016-08-16 | 2022-03-03 | 리제너론 파아마슈티컬스, 인크. | 혼합물로부터 개별 항체들을 정량하는 방법 |
US11571636B2 (en) | 2016-08-16 | 2023-02-07 | Regeneron Pharmaceuticals, Inc. | Methods for quantitating individual antibodies from a mixture |
KR102511050B1 (ko) * | 2016-08-16 | 2023-03-17 | 리제너론 파아마슈티컬스, 인크. | 혼합물로부터 개별 항체들을 정량하는 방법 |
US11850535B2 (en) | 2016-08-16 | 2023-12-26 | Regeneron Pharmaceuticals, Inc. | Methods for quantitating individual antibodies from a mixture |
Also Published As
Publication number | Publication date |
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WO2004078311A1 (en) | 2004-09-16 |
CA2517757A1 (en) | 2004-09-16 |
US20060175258A1 (en) | 2006-08-10 |
SE0300612D0 (sv) | 2003-03-05 |
JP4570052B2 (ja) | 2010-10-27 |
EP1599265A1 (en) | 2005-11-30 |
CA2517757C (en) | 2011-07-19 |
US7320755B2 (en) | 2008-01-22 |
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