JP5058159B2 - 分離マトリックスの製造方法 - Google Patents
分離マトリックスの製造方法 Download PDFInfo
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- JP5058159B2 JP5058159B2 JP2008519222A JP2008519222A JP5058159B2 JP 5058159 B2 JP5058159 B2 JP 5058159B2 JP 2008519222 A JP2008519222 A JP 2008519222A JP 2008519222 A JP2008519222 A JP 2008519222A JP 5058159 B2 JP5058159 B2 JP 5058159B2
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- polysaccharide
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Classifications
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- B01J20/30—Processes for preparing, regenerating, or reactivating
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- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
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Description
「粒子状」分離マトリックスという用語は、本明細書では、粒子、例えば実質的に球状の粒子又はそれほど規則的な形状をもたない粒子からなる分離マトリックスを意味する。
(a)ゲル化した未変性多糖類からなる粒子の水性懸濁液を用意し、
(b)1種以上の塩を加えて上記懸濁粒子を塩処理し、
(c)塩処理した粒子を架橋する
ことを含んでなる分離マトリックスの製造方法に関する。
(i)ゲル化性未変性多糖類の水溶液を用意し、
(ii)多糖類溶液の温度を、そのゲル化温度未満の温度まで下げ、
(iii)工程(ii)で得られた多糖類に、1種以上の塩を加えて塩処理を行い、
(vi)塩処理した多糖類を架橋させる
ことを含む分離マトリックスの製造方法である。
なお、実施例7〜10は、96℃での塩処理の効果を示す参考例である。
粒子の特性
プロトタイプのKd評価に使用したデキストランは、以下の通りである。
M=196300 10mg/ml
M=12360 10mg/ml
M=66700 10mg/ml
M=43500 10mg/ml
M=4440 10mg/ml
M=1080 10mg/ml。
有効性試験は、AKTA(商標)explorer(ID 494)(GE Healthcare社(スウェーデン国ウプサラ))を用いて、1%アセトン溶液の注入によって実施した。この試験では、カラム充填の質についての値が得られる。合格とされる非対象性は0.75〜1.25、段数は>1000Nmである。
Kdの評価は、オートサンプラーを装備したAKTA(商標)explorer(ID 494)を用いて実施した。(株)島津製作所製の屈折率検出器(RID−10A)をAKTA(商標)に接続してデキストラン試料を検出した。以下の条件を用いた。
移動相:0.2M NaCl
インジェクションループ:100μl。
当技術分野で周知の方法でデキストランピークを求めた。Kd値を、有効細孔表面の指標として、以下の通り計算した。
Ve=溶出デキストランの保持体積(ml)
V0=空隙体積(未変性デキストラン保持体積)(ml)
Vc=計算カラム体積(ベッド高さ(cm)×カラム表面積(cm2))(ml)
Vt=合計液体体積(NaCl保持体積)(ml)。
塩処理: 約1.5gの非架橋Sepharose(商標)HP(Amersham Biosciences社(スウェーデン国ウプサラ))を、10ベッド体積の脱イオン水で洗浄し、ゲル粒子の75%水性懸濁液を得た。次いで、以下の各プロトタイプの塩を懸濁液に加えた。反応混合物を撹拌しながら各プロトタイプについて以下に示す温度まで30分間加熱した後、氷浴中で30℃未満に冷却した。次に、ガラス濾過器上でゲルを10ベッド体積の水で洗浄して、懸濁液中の塩を除去した。
市販のSepharoseマトリックスを出発材料として使用し、塩の種類、処理回数及び処理時間を変えて、複数のプロトタイプを上述の通り製造した。結果を以下の表に示す。
上記の実施例2〜4で出発材料として用いた2種類のSepharose(商標)マトリックスについて、塩処理工程を行わずに、2つの比較例を実施した。
実施例7: アガロース溶液中でのメンブランの含浸−メンブランA
0.3gのアガロースを、30mLの水(95℃)に3時間00分間溶解することによって、1%アガロース溶液を調製した。再生セルロースメンブラン(Sartorius社製、0.45μm、製品番号3S18406−047N)を、上記アガロース溶液(95℃)に1時間00分間浸漬した。次に、メンブランをアガロース溶液から引き上げ、メンブラン構造内でアガロースがゲル化できるように室温まで放冷した。
実施例7に記載の通り製造したハイブリッドメンブランAを、硫酸アンモニウム((NH4)2SO4)(1M溶液)(50mL)中に96℃で1時間00分間浸漬した後、溶液から引き上げて放冷した。メンブランを、水で十分に洗浄した。
上述の通り製造・処理したメンブランを、直径15mmの漏斗に装着し、0.7バールの真空に引いた。水50mLを漏斗に注ぎ、ストップウォッチを始動させた。水の全量がメンブランを通過するまでの時間を記録した。測定は3回行った。
Claims (21)
- 分離マトリックスの製造方法であって、当該方法が、
(a)ゲル化した未変性多糖類からなる粒子の水性懸濁液を用意する工程と、
(b)1種以上の塩を加えて上記懸濁粒子を塩処理する工程と、
(c)塩処理した粒子を架橋する工程と、
(d)ゲル化した多糖類の水酸基にクロマトグラフィーリガンドを結合させる工程と
を含んでおり、前記多糖類がアガロースであって、懸濁液の塩濃度が0.5〜1.3Mとなるまで塩が加えられ、塩処理時の温度が94〜98℃である、方法。 - 工程(b)で加える塩の陰イオンが硫酸塩である、請求項1記載の方法。
- 塩の陽イオンがMg、Na、K、Li、Cuからなる群から選択される、請求項1又は請求項2記載の方法。
- 塩の陽イオンがMg及びNaからなる群から選択される、請求項3記載の方法。
- 塩がMgSO4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。
- 塩処理時の温度が96℃である、請求項1乃至請求項5のいずれか1項記載の方法。
- 工程(a)の多糖類粒子が有機溶媒中での乳化によって調製したものである、請求項1乃至請求項6のいずれか1項記載の方法。
- 粒子を乳化後に洗浄する、請求項7記載の方法。
- 工程(c)の架橋工程が架橋剤を添加することを含む、請求項1乃至請求項8のいずれか1項記載の方法。
- 請求項1乃至請求項9のいずれか1項記載の方法で製造した架橋多糖類分離マトリックス。
- 有効細孔容積が110kDデキストランに対する0.5以上のKav値に対応する、請求項10記載のマトリックス。
- 請求項10又は請求項11記載の分離マトリックスを含むクロマトグラフィーカラム。
- 分離マトリックスの製造方法であって、当該方法が、
(i)ゲル化性未変性多糖類の水溶液を用意する工程と、
(ii)多糖類溶液の温度を、そのゲル化温度よりも低い温度まで下げる工程と、
(iii)工程(ii)で得られた多糖類に、1種以上の塩を加えて塩処理を行う工程と、
(iv)塩処理した多糖類を架橋させる工程と、
(v)ゲル化した多糖類の水酸基にクロマトグラフィーリガンドを結合させる工程と
を含んでおり、前記多糖類がアガロースであって、溶液中の塩濃度が0.5〜1.3Mとなるまで塩が加えられ、塩処理時の温度が94〜98℃である、方法。 - 請求項13記載の方法で製造した架橋多糖類分離マトリックス。
- 当該マトリックスがメンブラン又はモノリスである、請求項13記載のマトリックス。
- 分離マトリックスの製造方法であって、当該方法が、
(i)多孔質メンブラン担体を用意して、ゲル化性未変性多糖類の水溶液と接触させる工程と、
(ii)多糖類溶液の温度をそのゲル化温度よりも低い温度まで下げる工程と、
(iii)1種以上の塩を加えて塩処理を行う工程と、
(iv)塩処理した多糖類を架橋させる工程と、
(v)ゲル化多糖類コーティングの水酸基にクロマトグラフィーリガンドを結合させる工程と
を含んでおり、前記多糖類がアガロースであって、溶液中の塩濃度が0.5〜1.3Mとなるまで塩が加えられ、塩処理時の温度が94〜98℃である、方法。 - 前記メンブラン担体が親水性表面を有する、請求項16記載の方法。
- 前記メンブラン担体がセルロースからなる、請求項16又は請求項17記載の方法。
- 得られた架橋分離マトリックスを滅菌することを含む、請求項16乃至請求項18のいずれか1項記載の方法。
- 液体から生体分子又は有機分子を精製、単離及び/又は除去するための、請求項10、請求項11、請求項14及び請求項15のいずれか1項記載の分離マトリックスの使用。
- 分離マトリックスに加えられる移動相の流速が300cm/h以上である、請求項20記載の使用。
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CN101218023B (zh) | 2012-02-08 |
US20080154031A1 (en) | 2008-06-26 |
RU2007146247A (ru) | 2009-08-20 |
JP2008544847A (ja) | 2008-12-11 |
CN101218023A (zh) | 2008-07-09 |
BRPI0612720B1 (pt) | 2016-02-10 |
KR101326438B1 (ko) | 2013-11-07 |
EP1904226B1 (en) | 2016-11-23 |
AU2006266529B2 (en) | 2010-12-16 |
US8309709B2 (en) | 2012-11-13 |
WO2007004947A1 (en) | 2007-01-11 |
EP1904226A4 (en) | 2013-08-07 |
KR20080027326A (ko) | 2008-03-26 |
RU2411081C2 (ru) | 2011-02-10 |
BRPI0612720A2 (pt) | 2011-02-15 |
CA2609093C (en) | 2014-12-30 |
CA2609093A1 (en) | 2007-01-11 |
AU2006266529A1 (en) | 2007-01-11 |
ES2608638T3 (es) | 2017-04-12 |
EP1904226A1 (en) | 2008-04-02 |
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