JP2008503754A - 分離マトリックス及び精製法 - Google Patents
分離マトリックス及び精製法 Download PDFInfo
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- JP2008503754A JP2008503754A JP2007518010A JP2007518010A JP2008503754A JP 2008503754 A JP2008503754 A JP 2008503754A JP 2007518010 A JP2007518010 A JP 2007518010A JP 2007518010 A JP2007518010 A JP 2007518010A JP 2008503754 A JP2008503754 A JP 2008503754A
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Abstract
【解決手段】 リガンドが適宜スペーサアームを介して固定化された多孔質担体からなる分離マトリックスであって、上記リガンドが1以上の芳香族スルホンアミドを含んでいてプロトン付加性基を実質的に含まない、分離マトリックス。スルホンアミドの窒素は第二又は第三アミンである。本発明は、上記分離マトリックスを収容したクロマトグラフィーカラム、並びに分離マトリックスへの免疫グロブリン様化合物の吸着による単離法に関する。
【選択図】 なし
Description
本発明のその他の態様及び利点は以下の詳細な説明から明らかとなろう。
「抗体」及び「免疫グロブリン」という用語は、本明細書では同義に用いられる。
「芳香族」基という用語は、ヒュッケル則(4n+2)によってπ電子の数を計算できる基をいう(ただし、nは0又は正の整数である。)。
第一の態様では、本発明は、リガンドが適宜スペーサアームを介して固定化された多孔質担体からなる分離マトリックスであって、上記リガンドが1以上のスルホンアミドを含み、スルホニルのR基が1以上の芳香族基を含む分離マトリックスに関する。
(a)1種以上の抗体を含む液体を用意する段階、
(b)1種以上の抗体をマトリックスに吸着させるため、上記液体を1以上のスルホンアミド基を含む分離マトリックスと接触させる段階、及び任意段階として、
(c)1種以上の抗体を遊離させるため、溶出液を上記マトリックスに通過させる段階、及び
(d)1種以上の抗体を溶出液の画分から回収する段階
を含んでなる方法である。
図1は、本発明に係る6種類の異なる芳香族スルホンアミドリガンドの具体的リガンド構造を示す。
以下、スルホニル部分のR基が芳香族基を含む本発明に係る様々な分離マトリックスの製造について例示する。
マトリックスの体積は、沈降ベッド体積をいう。
Sepharose(商標)をアリルグリシジルエーテルで以下の通り改質した。
アミン基の窒素原子を介してアミンをマトリックスに直接導入した。マトリックスへのカップリングは、アリル基の臭素化及び塩基性条件下での求核置換反応で行った。
上述の通りアリル化したSepharose(商標)6FF(0.042又は0.124mmolアリル基/ml排水ゲル)100ml、AcONa4g及び蒸留水100mlからなる攪拌懸濁液に、黄色が持続するまで臭素を加えた。次いで、懸濁液の色が完全に消えるまでギ酸ナトリウムを加えた。反応混合物を濾過し、ゲルを蒸留水500mlで洗浄した。活性化ゲルを直ちに反応容器に移して、適当なリガンドと反応させた。
少量の6M HClを加えてpH12に調整しておいたジエチレントリアミン(24ml)と水(10ml)の溶液が入った反応バイアルに、30gの臭素活性化ゲル(0.124mmolアリル基/ml排水ゲル)を移した。反応混合物は、攪拌しながら50℃で20時間放置した。反応混合物を濾過した後、ゲルを蒸留水3×30ml、0.5 HCl水溶液3×30ml、最後に蒸留水3×30mlで順次洗浄した。置換度0.158mmolアミン/ゲルmlのジエチレントリアミンSepharose(商標)ゲルが得られた。
1)50%NaOH水溶液を2〜3滴加えてpH12.3に調整しておいたアジ化ナトリウム(2g)と水(10ml)の溶液が入った反応バイアルに、33gの臭素活性化ゲル(0.124mmolアリル基/ml排水ゲル)を移した。反応混合物は、攪拌しながら50℃で20時間放置した。反応混合物を濾過した後、ゲルを蒸留水3×60ml、DMF3×30mlで順次洗浄した。次いで、排水ゲルを、DMF(22ml)中のDTE(4.5g)とDBU(3.75ml)の溶液で処理し、混合物を室温で18時間攪拌した。反応混合物を濾過した後、ゲルをDMF3×30ml、エタノール3×30ml、最後に蒸留水3×30mlで順次洗浄した。
一般法
5gのアミン結合ゲルを、0.2M NaOH水溶液10ml、エタノール3×10ml、次いでDCM(ジクロロメタン)3×10mlで洗浄した。ゲルをバイアルに移し、DCM(2ml)及びDIPEA3.3当量も加え、混合物を5分間攪拌した。DCM(3ml)に溶解したアリールスルホニルクロライド3当量を滴下した後、反応混合物を室温で18時間攪拌した。
上記一般法に従ってジエチレントリアミンSepharose(商標)ゲル(0.158mmolアミン/ゲルml)をペンタフルオロベンゼンスルホニルクロライド(355μl)で処理して標記のプロトタイプを得たが、これは図1ではL1Aと表示する。
上記一般法に従ってジエチレントリアミンSepharose(商標)ゲル(0.158mmolアミン/ゲルml)を4−ニトロベンゼンスルホニルクロライド(540mg)で処理して標記のプロトタイプを得たが、これは図1ではL1Bと表示する。
上記一般法に従ってジエチレントリアミンSepharose(商標)ゲル(0.158mmolアミン/ゲルml)をp−トルエンスルホニルクロライド(460mg)で処理して標記のプロトタイプを得たが、これは図1ではL1Cと表示する。
上記一般法に従ってアンモニアSepharose(商標)ゲル(0.083mmolアミン/ゲルml)をペンタフルオロベンゼンスルホニルクロライド(200μl)で処理して標記のプロトタイプを得たが、これは図1ではL2Aと表示する。
上記一般法に従ってアンモニアSepharose(商標)ゲル(0.083mmolアミン/ゲルml)を4−ニトロベンゼンスルホニルクロライド(290mg)で処理して標記のプロトタイプを得たが、これは図1ではL2Bと表示する。
1)上記一般法に従ってアンモニアSepharose(商標)ゲル(0.026mmolアミン/ゲルml)をp−トルエンスルホニルクロライド(207mg)で処理して標記のプロトタイプ(低置換型)を得たが、これは実施例ではL2Caと表示する。
材料及び方法(一般)
本発明に係る芳香族スルホンアミドリガンドがヒト免疫グロブリン(IgG)を吸着するかどうかを試験するため、IgG及び3種類の異なるタンパク質の吸着性を様々な条件下で試験した。また、1種類のモノクロナール抗体も試験した。試験法の原理は、塩及び緩衝成分を含むA緩衝液で平衡化しておいた、スルホンアミドリガンドの固定化されたSepharose(商標)6 Fast Flowを詰めたHR5/5カラムにタンパク質(15μl)を注入し、次いでA緩衝液15mlをカラムに流す。次いで、塩を含まずに緩衝液成分を含むB緩衝液を用いて、A緩衝液からB緩衝液の直線濃度勾配5mlを適用する(以下のUNICORN法参照)。次いで、クロマトグラフィーのプロファイルを280、254及び215nmでモニターする。
3種類の吸着用緩衝液(緩衝液A#)及び2種類の脱着用緩衝液(緩衝液B#)を使用した。
緩衝液A2:0.25M Na2SO4を含むリン酸緩衝液20mM(pH7.4)
緩衝液A3:0.50M Na2SO4を含むリン酸緩衝液20mM(pH7.4)
緩衝液B1:酢酸緩衝液100mM(pH4.0)
緩衝液B2:酢酸緩衝液100mM(pH4.0)+20%(v/v)イソプロパノール
試料
使用した試料は、ウシ血清アルブミン(BSA)、リボヌクレアーゼA(RIB A)、トランスフェリン(TRANSF)及びヒト免疫グロブリン(IgG、Gammanorm Pfizer)であった。タンパク質はA緩衝液に濃度15mg/mlで溶解し、1回にカラムにかけるタンパク質は1種類だけにした。
装置(Amersham Biosciences(スウェーデン国ウプサラ))
LC系: AKTA(商標)Explorer 10 XT
ソフトウェア:UNICORN(商標)
注入ループ: Superloop 15μl
カラム: HR 5/5
機器パラメータ
流速: 0.5ml/min
検出セル: 10mm
波長: 280、254及び215nm
芳香族スルホンアミドが免疫グロブリンを吸着するかどうかを実証するために、ヒトIgGを、本発明に係る新規分離マトリックスを充填した1mlカラム(HR5/5)にかけた。本実施例では、本明細書でL2Caと呼ぶ低リガンド密度型プロトタイプL2CへのIgGの吸着及び脱着を上記の一般法に従って試験した。L2Caは、実施例1に記載の通り製造し、そのリガンド密度は26μmol/mLであった。
以下の実施例では、ウシ血清アルブミン(BSA)、リボヌクレアーゼA(RIB A)及びトランスフェリン(TRANSF)と芳香族スルホンアミドリガンドL2Caの相互作用について調べた。
本例では、上述の通り製造した(26μmol/mL)L2CaでのBSAの吸着及び脱着を上記一般法に従って試験した。図3にみられる通り、280nmでのUV応答(青線)は、BSA試料は緩衝液A3を用いて吸着されずに、7.5〜9mLで溶出されることを示している。緩衝液A3=0.50M Na2SO4を含む20mMリン酸緩衝液(pH7.4)、緩衝液B1=100mM酢酸緩衝液(pH4.0)。
本例では、上述の通り製造した(26μmol/mL)L2CaでのRIBの吸着及び脱着を上記一般法に従って試験した。図4にみられる通り、280nmでのUV応答(青線)は、RIB試料は緩衝液A3を用いて吸着されずに、7.5〜9mLで溶出されることを示している。緩衝液A3=0.50M Na2SO4を含む20mMリン酸緩衝液、緩衝液B1=100mM酢酸緩衝液(pH4.0)。
本例では、上述の通り製造した(26μmol/mL)L2CaでのTRANSFの吸着及び脱着を上記一般法に従って試験した。図5にみられる通り、280nmでのUV応答(青線)は、TRANSF試料は緩衝液A3を用いて吸着されずに、7.5〜9mLで溶出されることを示している。緩衝液A3=0.50M Na2SO4を含む20mMリン酸緩衝液(pH7.4)、緩衝液B1=100mM酢酸緩衝液(pH4.0)。
Claims (22)
- リガンドが適宜スペーサアームを介して固定化された多孔質担体からなる分離マトリックスであって、上記リガンドが1以上の芳香族スルホンアミドを含んでいてプロトン付加性基を実質的に含まない、分離マトリックス。
- 前記スルホンアミドの窒素が第一又は第二アミンである、請求項1記載のマトリックス。
- 前記リガンドがモノアミンである、請求項1又は請求項2記載のマトリックス。
- 前記リガンドがポリアミンである、請求項1乃至請求項3のいずれか1項記載のマトリックス。
- 各ポリアミンが2〜6個のアミンを含む、請求項4記載のマトリックス。
- 前記リガンドが、担体に固定化されたポリマーの繰返し単位として存在する、請求項1乃至請求項5のいずれか1項記載のマトリックス。
- 前記ポリマーがポリエチレンイミンである、請求項6記載のマトリックス。
- 前記ポリマーが2種以上の異なるリガンド基を提示する、請求項6又は請求項7記載のマトリックス。
- 担体が架橋多糖類である、請求項1乃至請求項8のいずれか1項記載のマトリックス。
- 請求項1乃至請求項9のいずれか1項記載の分離マトリックスが充填されたクロマトグラフィーカラム。
- 抗体精製用マトリックスの製造方法であって、スルホンアミドをアミンのNを介して又はスルホニルのSを介して多孔質担体に固定化する第一段階を含んでなる方法。
- 液体からの抗体の単離方法であって、
(a)1種以上の抗体を含む液体を用意する段階、
(b)1種以上の抗体をマトリックスに吸着させるため、上記液体を1以上のスルホンアミド基を含む分離マトリックスと接触させる段階、及び任意段階として、
(c)1種以上の抗体を遊離させるため、溶出液を上記マトリックスに通過させる段階、及び
(d)1種以上の抗体を溶出液の画分から回収する段階
を含んでなる方法。 - 段階(a)で用意される液体が1種以上の他のタンパク質も含む、請求項12記載の方法。
- 段階(b)の分離マトリックスがクロマトグラフィーカラム内に収容されている、請求項12又は請求項13記載の方法。
- 段階(b)の分離マトリックスが請求項1乃至請求項9のいずれか1項記載のものである、請求項12乃至請求項14のいずれか1項記載の方法。
- 段階(b)が中性付近のpH、例えばpH7.2〜pH7.6、好ましくは約7.4で実施される、請求項15記載の方法。
- 段階(c)が、塩濃度の漸減する溶出液を分離マトリックスに添加することによって実施される勾配溶出である、請求項12乃至請求項16のいずれか1項記載の方法。
- 段階(b)が中性よりも高いpHで実施され、段階(c)が、pHの漸減する溶出液を添加することによって実施される勾配溶出である、請求項12乃至請求項17のいずれか1項記載の方法。
- 段階(d)で回収される抗体がヒトの又はヒト化抗体である、請求項12乃至請求項18のいずれか1項記載の方法。
- 段階(d)で回収される抗体が免疫グロブリンG(IgG)である、請求項12乃至請求項19のいずれか1項記載の方法。
- 前記抗体がモノクロナール抗体である、請求項12乃至請求項20のいずれか1項記載の方法。
- 抗体の量の測定方法であって、請求項12乃至請求項20のいずれか1項記載の方法と、さらに抗体の量を分光光度法で測定する段階(f)とを含んでなる方法。
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SE0401665A SE0401665D0 (sv) | 2004-06-24 | 2004-06-24 | Purification of immunoglobulins |
SE0401665-5 | 2004-06-24 | ||
PCT/SE2005/001002 WO2006001771A1 (en) | 2004-06-24 | 2005-06-22 | Separation matrix and method of purification |
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JP2014507649A (ja) * | 2011-01-18 | 2014-03-27 | バクスター・インターナショナル・インコーポレイテッド | ヒト血液中の抗βアミロイド抗体の測定 |
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JP4677623B2 (ja) * | 2005-05-16 | 2011-04-27 | 独立行政法人産業技術総合研究所 | 液体クロマトグラフィー用担体、該担体を充填したクロマトグラフィー用カラム、及び該カラムを用いた有機物の分離方法 |
WO2007064281A1 (en) * | 2005-12-02 | 2007-06-07 | Ge Healthcare Bio-Sciences Ab | Hydrophobic interaction chromatography |
US8092683B2 (en) * | 2007-01-10 | 2012-01-10 | Ge Healthcare Bio-Sciences Ab | Multi-modal ion exchange chromatography resins |
US9044740B2 (en) | 2007-03-30 | 2015-06-02 | Ge Healthcare Bio-Sciences Ab | Purification of immunoglobulins |
CN104812491B (zh) | 2012-03-08 | 2018-06-19 | 生物辐射实验室股份有限公司 | 阴离子交换-疏水性混合模式 |
EP2986625B1 (en) * | 2014-03-14 | 2020-03-11 | Bio-rad Laboratories, Inc. | Mixed mode ligands |
CN106102903A (zh) * | 2014-03-14 | 2016-11-09 | 通用电气医疗集团生物工艺研发股份公司 | 用于生物颗粒纯化的分离基质 |
CN104725559B (zh) * | 2015-03-13 | 2017-06-06 | 安徽师范大学 | 嗜硫色谱材料及其制备方法和应用 |
WO2017174422A1 (en) * | 2016-04-06 | 2017-10-12 | Ge Healthcare Bioprocess R&D Ab | Chromatography matrix |
EP3442988A4 (en) * | 2016-06-15 | 2020-02-26 | Agency for Science, Technology and Research | METHOD FOR IMPROVING THE PERFORMANCE OF CHROMATOGRAPHY PROCESSES FOR THE PURIFICATION OF PROTEINS |
CN112480246A (zh) * | 2020-12-22 | 2021-03-12 | 中国科学院过程工程研究所 | 一种犬免疫球蛋白的分离纯化方法及其应用 |
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US20070196858A1 (en) | 2007-08-23 |
SE0401665D0 (sv) | 2004-06-24 |
EP1759196A1 (en) | 2007-03-07 |
JP4890453B2 (ja) | 2012-03-07 |
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CA2570624A1 (en) | 2006-01-05 |
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