JP5883849B2 - 陰イオン交換樹脂を用いる二価カチオン結合タンパク質の精製方法 - Google Patents
陰イオン交換樹脂を用いる二価カチオン結合タンパク質の精製方法 Download PDFInfo
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- JP5883849B2 JP5883849B2 JP2013506675A JP2013506675A JP5883849B2 JP 5883849 B2 JP5883849 B2 JP 5883849B2 JP 2013506675 A JP2013506675 A JP 2013506675A JP 2013506675 A JP2013506675 A JP 2013506675A JP 5883849 B2 JP5883849 B2 JP 5883849B2
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- divalent cation
- binding protein
- buffer
- anion exchange
- exchange resin
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Images
Classifications
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- C07K1/18—Ion-exchange chromatography
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
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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
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
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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
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- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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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
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- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6437—Coagulation factor VIIa (3.4.21.21)
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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
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- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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Description
本発明は、以下の工程を含む二価カチオン結合タンパク質の精製方法に関する:
(a)陰イオン交換樹脂物質に二価カチオン非存在下でローディング緩衝液中の二価カチオン結合タンパク質をロードし、所望により、二価カチオン非存在下でロードした陰イオン交換樹脂物質を洗浄緩衝液で洗浄し、
(b)該二価カチオン結合タンパク質を少なくとも1の二価カチオンを含む溶離剤で溶出し、該二価カチオン結合タンパク質を含む溶出液を形成する、
ここで、工程(b)の溶離剤は、工程(a)の洗浄緩衝液、または洗浄工程を行わない場合は工程(a)のローディング緩衝液のpHより高いpHを有する。
ある局面において、本発明は、以下の工程を含む二価カチオン結合タンパク質の精製方法に関する:
(a)陰イオン交換樹脂物質に二価カチオン非存在下でローディング緩衝液中の二価カチオン結合タンパク質をロードし、所望により、二価カチオン非存在下でロードした陰イオン交換樹脂物質を洗浄緩衝液で洗浄し、
(b)該二価カチオン結合タンパク質を少なくとも1の二価カチオンを含む溶離剤で溶出し、該二価カチオン結合タンパク質を含む溶出液を形成する;
ここで、工程(b)の溶離剤は、工程(a)の洗浄緩衝液、または洗浄工程を行わない場合は工程(a)のローディング緩衝液のpHより高いpHを有する。
実施例1
rFIXのQ-Sepharose FFによる精製
表1:rFIXのQ-Sepharose FFによる精製
表2:rFIIXのQ-Sepharose FFによる精製
実施例4
ラン1:ロード 中性pH、洗浄 低pH6.5、溶出 高pH7.9、伝導度 洗浄および溶出緩衝液と同じ。
ラン2:ロード 中性pH、洗浄 低pH6.0、溶出 高pH8.0、洗浄緩衝液の伝導度 溶出緩衝液より低い。
ラン2.1:ラン2と同様に洗浄の塩濃度を増加させた。
表3
表4:2回のクロマトグラフィーrFIX精製ランの緩衝液条件
表5:2回のクロマトグラフィーrFIX精製ランの結果
Claims (9)
- 以下の工程を含む二価カチオン結合タンパク質の精製方法:
(a)二価カチオン非存在下でローディング緩衝液中の二価カチオン結合タンパク質を陰イオン交換樹脂物質にロードし、該ロードした陰イオン交換樹脂物質を二価カチオン非存在下で洗浄緩衝液で洗浄し、
(b)該二価カチオン結合タンパク質を少なくとも1の二価カチオンを含む溶離剤で溶出し、該二価カチオン結合タンパク質を含む溶出液を形成する;
ここで、工程(b)の溶離剤のpHは、工程(a)の洗浄緩衝液のpHより少なくとも0.5pH単位高いpHを有し、該洗浄緩衝液中の塩の濃度は≦200mMであり、二価カチオン非存在下で少なくとも1のさらなる洗浄工程を行う。 - 工程(a)の洗浄緩衝液が、工程(b)の溶離剤の伝導度と同じかまたはそれより低い伝導度を有する、請求項1記載の方法。
- 工程(b)中の少なくとも1の二価カチオンがCa2+、Be2+、Ba2+、Mg2+、Mn2+、Sr2+、Zn2+、Co2+、Ni2+、およびCu2+、またはその組み合わせからなる群から選ばれる請求項1または2に記載の方法。
- 該陰イオン交換樹脂物質が、ジエチルアミノエタン(DEAE)、ジメチルアミノエタン(DMAE)、トリメチルアミノエチル(TMAE)、ポリエチレンイミン(PEI)、第4アミノアルキル、第4アミノエタン(QAE)、および第4アンモニウム(Q)からなる群から選ばれる正荷電基を有する、請求項1〜3のいずれかに記載の方法。
- 該陰イオン交換樹脂物質が、アミノヘキシル、ベンズアミジン、リジン、およびアルギニンからなる群から選ばれる第一アミンをリガンドとして保持する請求項1〜4のいずれかに記載の方法。
- 該二価カチオン結合タンパク質がカルシウム結合タンパク質である請求項1〜5のいずれかに記載の方法。
- 該二価カチオン結合タンパク質がビタミンK依存性タンパク質である請求項1〜6のいずれかに記載の方法。
- 該二価カチオン結合タンパク質が、第IX因子、第VII因子、およびアネキシンVからなる群から選ばれる、請求項1〜7のいずれかに記載の方法。
- 請求項1〜8のいずれかに記載の方法を実施するための手段を含むキットであって、該キットは、二価カチオン結合タンパク質を緩衝液中の陰イオン交換樹脂物質から溶出するのに適した少なくとも1の二価カチオンを含む溶離剤を含み、該緩衝液が洗浄緩衝液であり、該溶離剤が該洗浄緩衝液のpHより少なくとも0.5単位高いpHを有するものであり、該洗浄緩衝液中の塩の濃度が≦200mMである、キット。
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WO2013053888A1 (en) * | 2011-10-14 | 2013-04-18 | Baxter International Inc. | Protein purification by anion exchange chromatography |
CN103509082B (zh) * | 2012-06-15 | 2016-03-02 | 郭怀祖 | 一种糖基化蛋白和非糖基化蛋白的分离方法 |
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GB2552853B (en) * | 2015-09-17 | 2018-11-21 | Annexin Pharmaceuticals Ab | Purification of Annexin A5 from a composition that includes a calcium ion chelator |
GB2552724B (en) * | 2015-09-17 | 2020-05-06 | Annexin Pharmaceuticals Ab | Purification of Annexin A5 by anion exchange and affinity chromatography |
CN114040813A (zh) * | 2019-07-02 | 2022-02-11 | 日本医事物理股份有限公司 | 含226Ra溶液的纯化方法、226Ra靶的制造方法及225Ac的制造方法 |
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