JP2014525254A - 混合物から目的のオリゴヌクレオチドを分離するためのプロセス - Google Patents
混合物から目的のオリゴヌクレオチドを分離するためのプロセス Download PDFInfo
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Abstract
Description
第一の液体移動相と、目的のオリゴヌクレオチドに除去可能に結合する少なくとも1種類の交換体物質を含有する液体固定相とを含んでなる、二相系移動相−固定相液−液クロマトグラフィーシステムを準備すること;
液−液クロマトグラフィー装置のカラム内の液体固定相に対する、その液体固定相と接触した流れにおいて、第一の液体移動相に目的のオリゴヌクレオチドを運ばせ、目的のオリゴヌクレオチドを液体固定相中の交換体物質に結合させること;そして、
目的のオリゴヌクレオチドを前記液体固定相から、カラム内を前記固定相に対して、前記固定相と接触して流れる第二の液体移動相へと、目的のオリゴヌクレオチドを前記液体固定相から前記第二の移動相へ移すことができる置換体物質の手段により移すこと
を含んでなる。
(1)溶液中に目的のオリゴヌクレオチドと1以上の不純物を含有する第一の液体相、および目的のオリゴヌクレオチドに除去可能に結合する交換体物質を含有する第二の液体相を準備し、ここで、第一の液体相と第二の液体相は、互いに接触した際に2つの明瞭な相を形成すること;
(2)前記第二の液体相を遠心分配クロマトグラフィー装置にその中の固定液体相として導入すること;
(3)溶液中に目的のオリゴヌクレオチドと1以上の不純物を含有する前記第一の液体相を前記遠心分配クロマトグラフィー装置に第一の移動相として導入し、この第一の液体相を前記遠心分配クロマトグラフィー装置に、前記第二の液体相と接触させて流し、目的のオリゴヌクレオチドを前記第二の液体相中の交換体物質に除去可能に結合させること;
(4)前記第二の液体相と接触した際に明瞭な相を形成し、かつ、溶液中に目的のオリゴヌクレオチドを前記第二の液体相から遠心分配クロマトグラフィー装置へ移すことができる少なくとも1種類の置換体物質を含有する液体相を第二の移動相として導入し、この第二の移動相を前記遠心分配クロマトグラフィー装置に、固定液体相と接触させて流し、目的のオリゴヌクレオチドを固定相から移し、前記第二の移動相の溶液に入れること;
(5)第二の移動相から移された目的のオリゴヌクレオチドを単離すること
を含んでなる。
R1R2R3R4N+X−
式中、前記配列の第二、第三および第四、基R1、R2、R3およびR4のそれぞれ2つ、3つまたは4つは独立にC1−20アルキルもしくは置換アルキル(例えば、フルオロまたはトリフルオロメチル置換アルキル)、またはベンジルであり、残りは水素であり、X−はハリドアニオン、好適には塩素である。好適な交換体物質は、塩化トリ−(n−オクチル)メチルアンモニウムおよび塩化トリ−(n−デシル)メチルアンモニウムの混合物であり、この場合、n−オクチル化合物が優位であるのが好適である。このような混合物はAliquat 336(商標)の名称で市販されている。好適であると考えられるその他の交換体物質としては、臭化セチルトリメチルアンモニウム、塩化メチルトリオクチルアンモニウム、塩化ベンザルコニウム(塩化アルキルジメチルベンジルアンモニウムおよびADBACとしても知られる、様々なアルキル鎖長の塩化アルキルベンジルジメチルアンモニウムの混合物)、塩化ベンジルトリメチルアンモニウム、塩化テトラブチルアンモニウム、およびプロトン化型のアンバーライトLA2(遊離塩基型の液体として供給される高分子量の油溶性第二級アミン)が含まれる。
液体相を以下の手順に従って調製した。酢酸エチル、1−ブタノールおよび水を3:2:5の容量比で混合した。この二相系を、2つの明瞭な相が得られるまで静置した後、これらの相を分離した。
溶媒系はぞれぞれ容量比(3:1:4)のMe−THF/n−BuOH/水から構成された。これらの3つの液体を混合し、静置して二相とし、これらを分離した。
実験1〜31の詳細を以下の表形式に示す。実験1は交換体または保持物質を用いない対照であった。実験12では、二成分系2−ブタノール−水は、乳化傾向のほとんどない2つの明瞭な相を容易に形成するような密度の違いを有する二相を形成しなかったので、固定相が保持されていなかったと考えられる。
Claims (32)
- 目的のオリゴヌクレオチドと1以上の不純物との混合物から目的のオリゴヌクレオチドを分離する方法であって、
第一の液体移動相と、目的のオリゴヌクレオチドに除去可能に結合する少なくとも1種類の交換体物質を含有する液体固定相とを含んでなる、二相系移動相−固定相液−液クロマトグラフィーシステムを準備すること;
液−液クロマトグラフィー装置のカラム内の液体固定相に対する、その液体固定相と接触した流れにおいて、第一の液体移動相に目的のオリゴヌクレオチドを運ばせ、目的のオリゴヌクレオチドを液体固定相中の交換体物質に結合させること、そして、
目的のオリゴヌクレオチドを前記液体固定相から、カラム内を前記固定相に対して、前記固定相と接触して流れる第二の液体移動相へと、目的のオリゴヌクレオチドを前記液体固定相から前記第二の移動相へ移すことができる置換体物質の手段により移すこと
を含んでなる、方法。 - 工程:
(1)溶液中に目的のオリゴヌクレオチドと1以上の不純物とを含有する第一の液体相、および目的のオリゴヌクレオチドに除去可能に結合する交換体物質を含有する第二の液体相を準備し、ここで、第一の液体相と第二の液体相は、互いに接触した際に2つの明瞭な相を形成すること;
(2)前記第二の液体相を遠心分配クロマトグラフィー装置にその中の固定液体相として導入すること;
(3)溶液中に目的のオリゴヌクレオチドと1以上の不純物とを含有する前記第一の液体相を前記遠心分配クロマトグラフィー装置に第一の移動相として導入し、この第一の液体相を前記遠心分配クロマトグラフィー装置に、前記第二の液体固定相と接触させて流し、目的のオリゴヌクレオチドを前記第二の液体固定相中の交換体物質に除去可能に結合させること;
(4)前記第二の液体相と接触した際に明瞭な相を形成し、かつ、溶液中に目的のオリゴヌクレオチドを前記第二の液体相から遠心分配クロマトグラフィー装置へ移すことができる少なくとも1種類の置換体物質を含有する第三の液体相を第二の移動相として導入し、この第二の移動相を前記遠心分配クロマトグラフィー装置に、第二の液体相と接触させて流し、目的のオリゴヌクレオチドを固定相から移し、前記第二の移動相の溶液に入れること;
(5)第二の移動相から移された目的のオリゴヌクレオチドを単離すること
を含んでなる、請求項1に記載の方法。 - 目的のオリゴヌクレオチドが10〜30塩基を含んでなる、請求項1または2に記載の方法。
- 目的のオリゴヌクレオチドが、配列5’−UCAAGGAAGAUGGCAUUUCA−3’を有する20塩基のオリゴヌクレオチドである、請求項3に記載の方法。
- 目的のオリゴヌクレオチドが、それもまたオリゴヌクレオチドである1以上の不純物から分離される、請求項1〜4のいずれか一項に記載の方法。
- 前記固定相が、水と実質的に不混和性の1以上の有機液と水と混和性の1以上の有機液との混合物を含んでなる、先行する請求項のいずれか一項に記載の方法。
- 前記第一および第二の移動相が、水と混和性の1以上の有機液と水との混合物を含んでなる、請求項6に記載の方法。
- 前記固定相および移動相が、C1−6アルキルC1−6アルカン酸エステル、C1−8アルカノールおよび水を含有する液体系の平衡相をそれぞれ2つ含んでなる、請求項6または7に記載の方法。
- 前記C1−6アルキルC1−6アルカン酸エステルが酢酸エチルであり、前記C1−8アルカノールが1−ブタノールである、請求項8に記載の方法。
- 前記固定相および移動相が、C1−8アルカノールおよび水を含有する液体系の平衡相をそれぞれ2つ含んでなる、請求項6または7に記載の方法。
- 前記C1−8アルカノールが1−ペンタノールまたは1−ブタノールである、請求項10に記載の方法。
- 前記固定相および移動相が、C1−8アルカノール、ジ−(C1−8アルキル)ケトンおよび水を含有する液体系の平衡相をそれぞれ2つ含んでなる、請求項6または7に記載の方法。
- 前記C1−8アルカノールが1−ブタノールであり、前記ジ−(C1−8アルキル)ケトンがメチルイソブチルケトンである、請求項12に記載の方法。
- 前記固定相および移動相が、ジ−(C1−8アルキル)ケトンおよび水を含有する液体系の平衡相をそれぞれ2つ含んでなる、請求項6または7に記載の方法。
- 前記ジ−(C1−8アルキル)ケトンがメチルイソブチルケトンである、請求項14に記載の方法。
- 前記固定相および移動相が、C1−6アルキルC1−6アルキルエーテルまたはC4−10環状エーテル、C1−8アルカノールおよび水を含有する液体系の平衡相をそれぞれ2つ含んでなる、請求項6または7に記載の方法。
- 前記C1−6アルキルC1−6アルキルエーテルおよびC4−10環状エーテルがメチル−第三級ブチルエーテルおよび2−メチルテトラヒドロフランから選択され、前記C1−8アルカノールが1−ペンタノールおよび1−ブタノールから選択される、請求項16に記載の方法。
- 前記第一の移動相がpH7〜14を有する、先行する請求項のいずれか一項に記載の方法。
- 前記カラム中の目的のオリゴヌクレオチドの濃度が200mg〜80g/Lである、先行する請求項のいずれか一項に記載の方法。
- 前記交換体物質が有機アミンの対アニオンとの塩である、先行する請求項のいずれか一項に記載の方法。
- 前記塩が第二級、第三級または第四級アンモニウム塩である、請求項20に記載の方法。
- 前記交換体物質が、塩化トリ−(n−オクチル)メチルアンモニウムと塩化トリ−(n−デシル)メチルアンモニウムとの混合物、臭化セチルトリメチルアンモニウム、塩化メチルトリオクチルアンモニウム、塩化ベンザルコニウム(塩化アルキルジメチルベンジルアンモニウムおよびADBACとしても知られる、様々なアルキル鎖長の塩化アルキルベンジルジメチルアンモニウムの混合物)、塩化ベンジルトリメチルアンモニウム、塩化テトラブチルアンモニウム、およびプロトン化型のアンバーライトLA2(遊離塩基型の液体として供給される高分子量の油溶性第二級アミン)から選択される、請求項20または21に記載の方法。
- 前記交換体物質が、塩化トリ−(n−オクチル)メチルアンモニウムと塩化トリ−(n−デシル)メチルアンモニウムとの混合物、および塩化ベンザルコニウムから選択される、請求項22に記載の方法。
- 前記固定相中の交換体物質の濃度が5〜500mMである、先行する請求項のいずれか一項に記載の方法。
- 前記第二の液体移動相が前記第一の移動相と同じ液体組成を有する、先行する請求項のいずれか一項に記載の方法。
- 前記固定相が主としてC1−8アルカノールとC1−6アルキルC1−6アルカン酸エステルとの混合物を含んでなり、前記第二の移動相が水とC1−8アルカノールとの混合物を含んでなる、請求項25に記載の方法。
- 前記固定相が主としてC1−6環状エーテルとC1−8アルカノールとの混合物を含んでなり、前記第二の移動相が主として水とC1−8アルカノールとの混合物を含んでなる、請求項25に記載の方法。
- 前記第二の移動相のpHがpH7〜14の範囲である、先行する請求項のいずれか一項に記載の方法。
- 前記置換体物質が、一価であっても多価であってもよいアニオン部分を組み込んでいる、先行する請求項のいずれか一項に記載の方法。
- 前記置換体物質が、ヨウ化ナトリウムまたはヨウ化カリウム、サッカリンナトリウム塩、サンセットイエローおよびアマランスから選択される、請求項29に記載の方法。
- 前記第二の移動相中の置換体物質の濃度が5〜30mMである、先行する請求項のいずれか一項に記載の方法。
- 目的のオリゴヌクレオチドが次に前記第二の移動相から単離される、先行する請求項のいずれか一項に記載の方法。
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