JP2009125071A - 膜イオン交換クロマトグラフィーのための媒体 - Google Patents
膜イオン交換クロマトグラフィーのための媒体 Download PDFInfo
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Abstract
【解決手段】本発明は、クロマトグラフィーに使用する媒体であって、媒体はポリエチレンイミンなどのポリマーにより表面がコートされた膜である。固定化されたポリマーコーティングを電荷修飾剤により修飾し、媒体に第4級アミンが導入する。媒体は、ウイルスのクロマトグラフィー精製に適している。
【選択図】図1
Description
1)コーティングポリマー及び架橋剤を製造し、そして、ポリマーが容易に架橋剤と反応するようにpHを調整する;
2)1)の溶液に多孔性構造を浸す;
3)溶液から多孔性構造を取り出し、そして、過剰な溶液を除去する;
4)多孔性構造を乾燥させ、架橋させる;
5)電荷修飾化合物を含んだ溶液に多孔性構造を、特定の時間、浸す;
6)修飾化合物の溶液から多孔性構造を取り出し、水で洗浄し、乾燥させる。
以下の実施例は例示目的であり、本発明はこれらに限定される訳ではない。
0.65μmの孔サイズの親水化したポリエチレン膜の6×6”(インチ)のシートを、7重量%のポリエチレンイミン(シグマアルドリッチ、Sigma-Aldrich)、0.35%のポリエチレングリコールジグリシジルエーテル(シグマアルドリッチ、Sigma-Aldrich)及び0.03Mの水酸化ナトリウムを含んだ水性溶液によりコートした。過剰な溶液を除去し、膜を一晩乾燥させた。続いて、水で洗浄し、0.1M水酸化ナトリウム及び3−ブロモプロピルトリメチル臭化アンモニウム(BPTMAB)の50%溶液の100ml中に浸した。膜を溶液中に48時間放置し、濃縮NaOHを定期的に添加し、pH13を維持した。その後、膜を溶液から取り出し、水で洗浄し、乾燥させた。
実施例1で製造した膜をアデノウイルスの精製に使用した。凍結及び解凍の複数のサイクルによって感染した細胞から最初にアデノウイルスを抽出した。上澄み液に残っている可視可能なウイルス粒子など細胞残屑を遠心分離により除去した。上澄み液をBenzonase(登録商標)によって処理した。さらに、上澄みを0.2μmの微小孔性膜フィルタに通して、精製した。溶液は、平衡化緩衝液pH8、NaCl濃度100mMにより希釈した。同様の緩衝液を膜の精製条件化に使用した。ウイルス溶液を、ウイルス粒子が吸着する膜にゆっくり流し、細胞残屑の多くはフィルタを通過させた。その後、膜をpH8、NaCl濃度200〜250mMの緩衝液で洗浄し、弱い結合で結合していた残屑を除去した。最終的に、pH8、NaCl濃度1000mMの溶出緩衝液で、ウイルスを膜から溶出させた。
実施例1に従って反応混合物中のBPTMABの濃度を様々に変化させ、修飾の割合が異なる膜を製造した。図5は、BPTMABのPEI修飾の割合が溶出するウイルスの割合に直接的に影響があることを示唆している。
Claims (14)
- 多孔を有する第一外面及び多孔を有する第二外面並びに両面の間に多孔層を有する基材を備えた多孔性収着媒体であって、
前記基材が、親水性であり、基材の固体マトリックス並びに前記第一及び第二外表面を実質的に覆う収着物質を有し、
前記収着物質が、無極性リンカーを介して結合した第4級アンモニウム官能基を有する架橋ポリマーを含む、多孔性収着媒体。 - 前記基材が微多孔膜を有する請求項1に記載の多孔性収着媒体。
- 前記膜がポリオレフィンである請求項2に記載の多孔性収着媒体。
- 前記ポリオレフィンがポリエチレンである請求項3に記載の多孔性収着媒体。
- 前記基材が超高分子量ポリエチレン膜である請求項1に記載の多孔性収着媒体。
- 前記架橋ポリマーがポリエチレンイミンを含む請求項1に記載の多孔性収着媒体。
- 前記架橋ポリマーが、前記無極性リンカーを介して前記架橋ポリマーと反応可能な部分に結合した第4級アンモニウム官能基を有する有機化合物を含む電荷修飾剤により修飾される、請求項1に記載の多孔性収着媒体。
- 前記有機化合物が式Y−Z−N(CH3)3 +X-であり、
Yは反応性脱離基であり、
Zは無極性の脂肪族又は芳香族リンカーであり、
そして、
Xは水溶性の酸の負電荷のイオンである、請求項7に記載の多孔性収着媒体。 - Yは、Br-、Cl-、I-、TsO-及びCF3SO3 -からなる群から選択され、
Zは、(CH2)nであり、nは2〜10である、請求項8に記載の多孔性収着媒体。 - 第4級アンモニウム官能基が3−ブロモプロピルトリメチル臭化アンモニウムによりポリエチレンイミンに与えられる、請求項6に記載の多孔性収着媒体。
- ウイルスを含んだ溶液を膜に透過させて、ウイルスを膜に吸着させ、
膜を緩衝液で洗浄し、膜からウイルスを溶出させる、ウイルスを精製する方法であって、
前記膜は、多孔を有する第一外面及び多孔を有する第二外面並びに両面の間に多孔層を有する基材を含み、
前記基材が、親水性であり、基材の固体マトリックス並びに前記第一及び第二外表面を実質的に覆う収着物質を有し、
前記収着物質が、無極性リンカーを介して結合した第4級アンモニウム官能基を有する架橋ポリマーを含む方法。 - 前記架橋ポリマーが、前記無極性リンカーを介して前記架橋ポリマーと反応可能な部分に結合した第4級アンモニウム官能基を有する有機化合物を含む電荷修飾剤により修飾される、請求項11に記載の方法。
- 前記有機化合物が式Y−Z−N(CH3)3 +X-であり、
Yは反応性脱離基であり、
Zは無極性の脂肪族又は芳香族リンカーであり、
そして、
Xは水溶性の酸の負電荷のイオンである、請求項12に記載の方法。 - 多孔を有する第一外面及び多孔を有する第二外面並びに両面の間に多孔層を有する基材を備えた多孔性収着媒体を含む陰イオン交換体であって、
前記基材が、親水性であり、基材の固体マトリックス並びに前記第一及び第二外表面を実質的に覆う収着物質を有し、
前記収着物質が、無極性リンカーを介して結合した第4級アンモニウム官能基を有する架橋ポリマーを含む、陰イオン交換体。
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Also Published As
Publication number | Publication date |
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ES2384479T3 (es) | 2012-07-05 |
JP2013051970A (ja) | 2013-03-21 |
US20100323430A1 (en) | 2010-12-23 |
US20090130738A1 (en) | 2009-05-21 |
CN101474552B (zh) | 2012-10-31 |
SG152982A1 (en) | 2009-06-29 |
ATE552043T1 (de) | 2012-04-15 |
CN101474552A (zh) | 2009-07-08 |
EP2060316B1 (en) | 2012-04-04 |
EP2060316A1 (en) | 2009-05-20 |
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