JP2016521968A - ボツリヌス毒素の製造方法 - Google Patents
ボツリヌス毒素の製造方法 Download PDFInfo
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- JP2016521968A JP2016521968A JP2016506267A JP2016506267A JP2016521968A JP 2016521968 A JP2016521968 A JP 2016521968A JP 2016506267 A JP2016506267 A JP 2016506267A JP 2016506267 A JP2016506267 A JP 2016506267A JP 2016521968 A JP2016521968 A JP 2016521968A
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- Prior art keywords
- botulinum toxin
- producing
- acid
- botulinum
- anion exchange
- Prior art date
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Abstract
Description
1−1:培養に用いた培地の組成
ボツリヌス毒素の生産のためのクロストリジウムボツリヌス菌株の種培養および本培養は、2%カゼイン加水分解物(Casein hydrolysate)、1%酵母抽出物(Yeast extract)、1%グルコース(Glucose)および0.5%チオグリコール酸塩培地(Thioglycollate medium)の組成を有する培地を用いた。
実施例1−1の培地組成を有する10mlの滅菌された培地が入っているculture tubeに20ulのクロストリジウムボツリヌス(疾病管理本部管理番号:4−029−CBB−IS−001)を接種して35℃で嫌気的条件で(22〜30時間)一次種培養(静置培養)した。一次種培養で菌株の成長が確認されると同じ培地組成を有する800mlの滅菌された培地が入っている1リットルculture bottleに8mlの一次種培養液を接種して35℃で嫌気的条件で(8〜15時間)二次種培養(静置培養)した。
クロストリジウムボツリヌス菌株を培養してボツリヌス毒素を生産するために本培養を実施して、実施例1−1の組成で培地9.3リットルを調製して、10リットル培養器に入れた後、培地滅菌を実施した。嫌気的条件は、窒素を供給して作り、温度は35℃、撹はん速度は50rpmに設定して成長環境を維持させた。
2−1:硫酸沈殿工程
硫酸沈殿工程は、ボツリヌス菌を培養した後、残っている菌を全部死滅させて、多くの種類のタンパク質が含まれた培養液に硫酸を添加することによってpHを低下させてタンパク質が等電点に達することになって、沈殿させるタンパク質を分離する方法で、実施例1−3の方法で本培養を実施して、培養が完了すると10リットル培養器のpHセンサーを利用してpH3.4〜3.6になるように5N硫酸を自動ポンプ(pump)で添加した後、培養器の収穫ライン(harvest line)を介して20リットル容器(AS ONE,Cat.No AS5.372.06)も培養液を移動させて硫酸沈殿液が入っている20リットル容器を直ちに生物安全作業対(BSC)に移してBSC内で硫酸沈殿を実施した。
前記硫酸沈殿物から上澄み液を除去してpH5.3の1Mリン酸ナトリウム(sodium phosphate)バッファー700mlを添加した。その次、5N水酸化ナトリウム(NaOH)を利用してpHを5.0〜6.0に調節して、沈殿物に残っているDNAおよびRNAを除去するために、0.4Mベンザミジン塩酸(benzamidine HCl)60ml、DNase 1g、RNase 1gを添加してボツリヌス毒素を抽出するために約5時間反応させた。
前記毒素抽出培養液を4℃、12000xgで15分間遠心分離してペレット(pellet)と上澄み液を分離して、分離した上澄み液を新しい10リットルビン(AS ONE,Cat.No AS5.372.04)に移した後、1N塩酸(HCl)を添加してpH3.4〜3.6になるように4℃低温冷蔵庫で塩酸沈殿過程を行った。塩酸沈殿液を4℃、12000xgで15分間遠心分離して上澄み液を除去した後、毒素沈殿物(pellet)にpH6.5の50mMリン酸ナトリウム(sodium phosphate)バッファー50mlを添加して毒素を溶解させた。
沈殿過程完了後、ボツリヌス毒素A型を除いた多くの主な不純物を除去するためにイオン交換樹脂を利用してクロマトグラフィーを行った。陰イオン交換樹脂(Toyopearl SuperQ−650M,Tosoh Bioscience,P/N 17228)をカラムに充填した後、前記2−3工程で沈殿させた試料を注入して、50mMリン酸ナトリウム湧出緩衝溶液で毒素を湧出した。一次精製工程でボツリヌス毒素A型タンパク質は、陰イオン交換樹脂に吸着せず多くの主な不純物は吸着されて除去されて、この時、高純度ボツリヌス毒素の精製のためにpHは4.5〜7.0、伝導度(conductivity)は5〜20mS/cmが維持されるようにした。
陰イオン交換クロマトグラフィーを利用して精製されたボツリヌス毒素A型タンパク質が含まれている分画を集めて20〜40%(w/v)で硫酸アンモニウム(ammonium sulfate)を添加してボツリヌス毒素A型タンパク質を再び沈殿させて、沈殿したボツリヌス毒素A型タンパク質は、pH6.5の50mMリン酸ナトリウム(sodium phosphate)バッファーに再溶解した。
硫酸アンモニウム沈殿過程完了後、ボツリヌス毒素A型を除いた少数の不純物を除去するために、イオン交換樹脂を利用したクロマトグラフィーを再度行った。陰イオン交換樹脂(Toyopearl SuperQ−650M,Tosoh Bioscience,P/N 17228)をカラムに充填した後、前記2−5工程で再溶解した硫酸アンモニウム沈殿物を注入して、50mMリン酸ナトリウム湧出緩衝溶液で毒素を湧出した。この時、ボツリヌス毒素A型タンパク質は吸着せず少数の不純物は吸着されて除去される。
前記2−6工程の陰イオン交換クロマトグラフィーを利用して精製されたボツリヌス毒素A型タンパク質が含まれている分画を集めて、0.2um除菌ろ過して原液を製造して、製造された原液は−70℃以下に保管した。製造されたボツリヌス毒素A型タンパク質はDWP450と命名した。
HPLC(Waters社e2695)は、SEC(size exclusion chromatography)法で行った。移動相はpH6.5の100mMリン酸ナトリウム溶液を用いて、P/N 08542に対するガードカラム(Tosoh Bioscience社、P/N 08543)にTSKgel G4000SWXL(Tosoh Bioscience社、P/N 08542)カラムを連結してボツリヌスA型毒素タンパク質20μgをローディング(loading)して1mL/minで30分間流した。その結果、一次陰イオン交換クロマトグラフィーを行った後のボツリヌス毒素の純度は98.38%で(図2)、二次陰イオン交換クロマトグラフィーを行った後のボツリヌス毒素の純度は98.99%であることを確認した(図3)。
実施例2で精製されたボツリヌス毒素A型タンパク質(DWP450)の安全性を確認するために、アラガン(Allergan)社のBOTOX(登録商標)と安全性を比較する実験を行った。
実施例2で精製されたボツリヌス毒素A型タンパク質(DWP450)の効能を確認するために、複合筋活動電位(compound muscle action potential:CMAP)振幅および伝導速度(conduction velocities;tC)を測定した。
Claims (14)
- 下記の工程を含むボツリヌス毒素の製造方法:
(a)ボツリヌス毒素生産菌株培養液を酸で処理して、ボツリヌス毒素を酸沈殿させる工程;
(b)前記沈殿したボツリヌス毒素に緩衝溶液を添加した後、プロテアーゼ抑制剤(protease inhibitor)および核酸分解酵素を処理してボツリヌス毒素を抽出する工程;
(c)前記抽出されたボツリヌス毒素を酸で処理してボツリヌス毒素を酸沈殿させた後、沈殿物を緩衝溶液に溶解させる工程;および
(d)陰イオン交換クロマトグラフィーを利用してボツリヌス毒素を精製する工程。 - ボツリヌス毒素生産菌株は、クロストリジウムボツリヌス(Clostridium botulinum)であることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 精製されたボツリヌス毒素は、純度98%以上のボツリヌス毒素A型タンパク質であることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(a)工程での酸沈殿は、pHが3.0〜4.5になるように硫酸または塩酸を添加することによって行うことを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(b)工程でのプロテアーゼ抑制剤(protease inhibitor)は、ベンザミジン塩酸(benzamidine HCl)であることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(b)工程での核酸分解酵素は、DNaseおよびRNaseであることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(b)工程でのボツリヌス毒素抽出は、pH4.5〜6.5で行うことを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(c)工程での酸沈殿は、pHが2.5〜4.5になるように硫酸または塩酸を添加することによって行うことを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(c)工程での緩衝溶液は、リン酸ナトリウムであることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(d)工程の陰イオン交換クロマトグラフィーは、pH3.5〜7.5および伝導度(conductivity)3〜30mS/cmの条件で行われることを特徴とする請求項1に記載のボツリヌス毒素の製造方法。
- 前記(d)工程以後に、
(e)ボツリヌス毒素が含まれている陰イオン交換クロマトグラフィー分画を硫酸アンモニウム((NH4)2SO4)で処理して沈殿させた後、沈殿物を緩衝溶液に溶解させる工程;および
(f)陰イオン交換クロマトグラフィーを利用してボツリヌス毒素を精製する工程をさらに含むことを特徴とする請求項1〜10のいずれかに記載のボツリヌス毒素の製造方法。 - 前記(e)工程での硫酸アンモニウムは、 濃度が10〜50%(w/v)になるように添加されることを特徴とする請求項11に記載のボツリヌス毒素の製造方法。
- 前記(e)工程での緩衝溶液は、リン酸ナトリウムであることを特徴とする請求項11に記載のボツリヌス毒素の製造方法。
- 前記(e)工程の陰イオン交換クロマトグラフィーは、pH3.5〜7.5および伝導度(conductivity)3〜30mS/cmの条件で行われることを特徴とする請求項11に記載のボツリヌス毒素の製造方法。
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