JP6892563B1 - 帯電物質の精製方法 - Google Patents
帯電物質の精製方法 Download PDFInfo
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- JP6892563B1 JP6892563B1 JP2020554568A JP2020554568A JP6892563B1 JP 6892563 B1 JP6892563 B1 JP 6892563B1 JP 2020554568 A JP2020554568 A JP 2020554568A JP 2020554568 A JP2020554568 A JP 2020554568A JP 6892563 B1 JP6892563 B1 JP 6892563B1
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- adsorbent
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- buffer solution
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Images
Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
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- B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
- B01J20/048—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium containing phosphorus, e.g. phosphates, apatites, hydroxyapatites
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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Abstract
Description
帯電した部分を有する化合物を精製するための精製方法であって、前記精製方法は、
帯電した部分を有する化合物を含む組成物を準備する工程と、
表面がハイドロキシアパタイト又はハイドロキシアパタイトが有する水酸基の少なくとも一部がフッ素原子で置換されたフッ素アパタイトで構成された吸着剤を準備する工程と、
HEPESとアルコールとを含む保存用緩衝剤溶液を準備する工程と、
前記保存用緩衝剤溶液中に前記吸着剤を保存する保存工程と、
前記吸着剤に前記組成物を接触させることにより、前記吸着剤に前記化合物を吸着させる吸着工程と、
勾配溶出により前記吸着剤から前記化合物を分取する分取工程と、
を含む、精製方法。
[A2]
前記帯電物質が、負に帯電した部分を有する、[A1]に記載の精製方法。
[A3]
前記帯電物質が、正に帯電した部分を有する、[A1]に記載の精製方法。
[A4]
[A1]から[A3]のいずれかに記載の精製方法により、帯電した部分を有する化合物を精製する精製工程を含む、帯電物質の生産方法。
[A5]
帯電した部分を有する化合物を精製するために用いられ、かつ、表面がハイドロキシアパタイト又はハイドロキシアパタイトが有する水酸基の少なくとも一部がフッ素原子で置換されたフッ素アパタイトで構成された吸着剤を保存するために用いられる保存用緩衝剤溶液であって、
前記保存用緩衝剤溶液は、HEPESとアルコールとを含む、
保存用緩衝剤溶液。
図1は、本発明の精製方法を適用し得る吸着剤を備える吸着装置の一例を示す縦断面図である。なお、以下の説明では、図1中の上側を「流入側」、下側を「流出側」と言う。
次に、このような吸着装置1を用いたタンパク質の精製方法について説明する。
まず、分離(精製)すべきタンパク質と、分離すべきタンパク質以外の物質(混合物)とを含有する試料液(組成物)を準備する。
次に、得られた試料液を、流入管24およびフィルタ部材4を介して吸着剤3に供給して、カラム2(吸着装置1)内を通過させて、吸着剤3に接触させる。
次に、流入管24からカラム2内に、タンパク質を溶出させるための溶出液として例えばリン酸系緩衝液を供給する。そして、カラム2内から流出管25を介して流出する流出液を、所定量ずつ分画(採取)する。これにより、吸着剤3に吸着しているタンパク質および混入物は、それぞれが有する吸着剤3に対する吸着力の差に応じて異なるタイミングでそれぞれ流出し、それぞれが、各分画内に溶解した状態で回収(分離・精製)される。
[4A] 保存工程
ここで、この吸着装置1を用いた従来の分離方法では、上述したタンパク質の分離(精製)の後に、期間を空けることなく、高濃度の緩衝液(リン酸系緩衝液等)を用いて、吸着剤3に吸着する他の物質(夾雑タンパク質等)を溶出させて洗浄し、その後、再度、タンパク質の分離に吸着装置1を供することで、タンパク質の分離が繰り返して実施されていた。
1.吸着装置の製造
吸着剤(充填剤)としてハイドロキシアパタイト(BIO−RAD社製、CHT 40μm Type I)を、ステンレスカラム(内径4.0mm×長さ100mm)の充填空間にほぼ満量となるように充填することにより、吸着装置を製造した。
(実施例1)
2−1.保存液による保存前の吸着装置によるタンパク質の分離
<1A> まず、酸性タンパク質としてのBSA(牛血清アルブミン)の含有量が10mg/mL、塩基性タンパク質としてのα−キモトリプシノーゲンAの含有量が5mg/mLとなるように、BSAおよびα−キモトリプシノーゲンAを、それぞれ、1.0mMリン酸ナトリウム緩衝液(pH6.8)に溶解させて混合液を得た。その後、混合液を0.22μmのフィルタで濾過することで試料液を得た。
<4A>次いで、20%エタノールを含む10mM HEPES緩衝液(pH8.0)を、通液速度1.0mL/minで吸着装置に通液することで、吸着剤を上記HEPES緩衝液中に浸漬させた。そして、この状態で、吸着剤を30日間保存した。
<1B> 次に、吸着装置を、クロマト装置に装着した。その後、10mMリン酸ナトリウム緩衝液(pH6.5、温度25℃)を、通液速度1.0mL/minで吸着装置に通液することで、吸着装置を平衡化した。
上記工程4Aにおいて、20%エタノールを含む10mM HEPES緩衝液(pH8.0)に代えて、1M NaOHを用いて吸着剤を保存し、工程4Aの終了後かつ上記工程1Bを行う前に、0.4M NaP pH6.5で吸着装置の保存液を置換するステップを追加したこと以外は、実施例1と同様に試験を行った。なお、比較例1では、吸着剤をリン酸緩衝液に浸漬して静置するような従来技術で行われている工程は行われていない。比較例1における溶出の結果を図3に示す。保存後に酸性タンパク質の溶出位置が変化することがわかった。
上記工程4Aにおいて、20%エタノールを含む10mM HEPES緩衝液(pH8.0)による保存工程に代えて、1M NaOHを用いて吸着剤を保存し、工程4Aの終了後に、吸着装置を10mm NaP pH6.5で置換し、1時間以上の各所定時間に亘って静置するステップを追加したこと以外は、実施例1と同様にして試験を行った。比較例2における溶出の結果を図4に示す。比較例2より、静置時間を長くすると緩和効果が大きくなることがわかった。
上記工程4Aにおいて、20%エタノールを含む10mM HEPES緩衝液(pH8.0)による保存工程に代えて、20%エタノールを含む10mm NaP pH6.5を用いて吸着剤を保存したこと以外は、実施例1と同様に試験を行った。比較例3における溶出の結果を図5に示す。カラムの保存前と比較して、酸性タンパク質の溶出位置は変化しなかったが、塩基性タンパク質の溶出位置が変化してしまった。
2 カラム
3 吸着剤
4 フィルタ部材
5 フィルタ部材
20 吸着剤充填空間
21 カラム本体
22 キャップ
23 キャップ
24 流入管
25 流出管
Claims (5)
- 帯電した部分を有する化合物を精製するための精製方法であって、前記精製方法は、
帯電した部分を有する化合物を含む組成物を準備する工程と、
表面がハイドロキシアパタイト又はハイドロキシアパタイトが有する水酸基の少なくとも一部がフッ素原子で置換されたフッ素アパタイトで構成された吸着剤を準備する工程と、
HEPESとアルコールとを含む保存用緩衝剤溶液を準備する工程と、
前記保存用緩衝剤溶液中に前記吸着剤を保存する保存工程と、
前記吸着剤に前記組成物を接触させることにより、前記吸着剤に前記化合物を吸着させる吸着工程と、
勾配溶出により前記吸着剤から前記化合物を分取する分取工程と、
を含む、精製方法。 - 前記帯電物質が、負に帯電した部分を有する、請求項1に記載の精製方法。
- 前記帯電物質が、正に帯電した部分を有する、請求項1に記載の精製方法。
- 請求項1から3のいずれか1項に記載の精製方法により、帯電した部分を有する化合物を精製する精製工程を含む、帯電物質の生産方法。
- カラムと、
前記カラムに収容され、HEPESとアルコールとを含む緩衝剤溶液と、
前記緩衝剤溶液中に保存され、表面がハイドロキシアパタイト又はハイドロキシアパタイトが有する水酸基の少なくとも一部がフッ素原子で置換されたフッ素アパタイトで構成された吸着剤と、
とを備える吸着装置。
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