JP7414874B2 - カプリル酸を用いてタンパク質を精製する方法 - Google Patents
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Classifications
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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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/12—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the preparation of the feed
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1892—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns the sorbent material moving as a whole, e.g. continuous annular chromatography, true moving beds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3847—Multimodal interactions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
Landscapes
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
i. 固定相にタンパク質含有サンプルをロードし、前記固定相に標的タンパク質が結合されるようにする工程;
ii. 結合された標的タンパク質を有する固相をカプリル酸溶液に曝露する工程であって、前記カプリル酸溶液が遊離カプリル酸を生成するようなpHであり、前記カプリル酸溶液が、遊離カプリル酸に関して測定される濃度が1~50 mMであるカプリル酸緩衝液である、工程;
iii. 前記標的タンパク質を溶出する工程
を含む、サンプルから標的タンパク質を精製するための方法を提供する。
i. 固定相にタンパク質含有サンプルをロードし、前記固定相に標的タンパク質が結合されるようにする工程;
ii. 固相をカプリル酸溶液に曝露する工程;及び
iii. 前記標的タンパク質を溶出する工程
を含む、サンプルから標的タンパク質を精製するための方法を対象とする。
ii. 結合された標的タンパク質を有する固相をカプリル酸溶液に曝露する工程であって、前記カプリル酸溶液が、遊離カプリル酸を生成するpHであり、遊離カプリル酸に関して測定される濃度が1~50 mMであるカプリル酸緩衝液である、工程;及び
iii. 前記標的タンパク質を溶出する工程
を含む、サンプルから標的タンパク質を精製するための方法。
i. アフィニティークロマトグラフィー用の固定相に1つ以上の前記タンパク質を含むサンプルをロードする工程;
ii. 遊離カプリル酸に関して測定される濃度が2~10 mM、例えば2~8 mM、例えば2~6 mMで、4.9<pH<6.2のpHのカプリル酸溶液に、固相を曝露し、ウイルスを不活性化し洗い流す工程;
iii. 標的タンパク質を溶出する工程
を含む、方法。
組換えヒトFab断片を含む細胞株培地を、0.22 μmで濾過し、その後eMuLVウイルスを混入させた。Capto L媒体(GE-Healthcare)を充填したカラムに混入培地をアプライした。ロード後、カラムをまず平衡緩衝液(20 mM ホスフェート、150 mM NaCl、pH 7.2)を用いて洗浄し、その後pH 5.5の35 mM カプリル酸緩衝液(5.8 mM 遊離カプリル酸)を用いて洗浄した。カプリル酸緩衝液を用いた30分間の洗浄の後、pH 3.5の20 mM ギ酸を用いてFab断片をカラムから溶出させた。プロセス温度は15~25℃であった。Fab断片の収率は97%であり、Fab断片を含む溶出画分中にeMuLVは検出されなかった(表1)。
組換えヒト抗体(IgG1)を含む細胞株培地を、0.22 μmフィルターで濾過し、eMuLVウイルスを混入させた。プロテインA(Mab Select SuRe、Ge Healthcare)を充填したカラムに混入培地をロードした。ロード後、カラムをまず平衡緩衝液(50 mmol/kg ホスフェート、300 mmol/kg NaCl、pH 7.0)を用いて洗浄し、その後pH 5.8の32 mmol/kg カプリレート緩衝液(2.9 mM 遊離カプリル酸)を用いて洗浄した。最悪条件(低カプリル酸濃度)におけるこの工程の有効性を示すために、低濃度のカプリレート及び高pHを用いた。pHが安定化された後、洗浄を30分間続けた。さらなる3回の洗浄(平衡緩衝液、50 mmol/kg ホスフェート、1 mol/kg NaCl、そしてその後平衡緩衝液)後、ギ酸緩衝液、pH 3.5を用いてカラムからmAbを溶出させた。mAbの収率は100%であり、mAbを含む溶出画分中にeMuLVは検出されなかった(表2)。プロセス温度は15~25℃であった。
組換えヒト抗体(IgG4)を含む細胞株培地を、0.22 μmフィルターを通して濾過し、eMuLVウイルスを混入させた。プロテインA(Mab Select SuRe、Ge Healthcare)を充填したカラムに混入培地をロードした。ロード後、カラムをまず平衡緩衝液(50 mmol/kg ホスフェート、300 mmol/kg NaCl、pH 7.0)を用いて洗浄し、その後pH 5.8の32 mmol/kg カプリレート緩衝液(2.9 mM 遊離カプリル酸)を用いて洗浄した。pHが安定化された後、洗浄を30分間続けた。さらなる3回の洗浄(平衡緩衝液、50 mmol/kg ホスフェート、1 mol/kg NaCl)、そしてその後平衡緩衝液)後、ギ酸緩衝液、pH 3.5を用いてカラムからmAbを溶出させた。mAbの収率は90%であり、mAbを含む溶出画分中にeMuLVは検出されなかった(表3)。プロセス温度は15~25℃であった。
組換えヒト抗体(IgG1)を含む細胞株培地を、0.22 μmフィルターを通して濾過し、eMuLVウイルスを混入させた。プロテインA(Mab Select SuRe、Ge Healthcare)を充填したカラムに混入培地をロードした。ロード後、カラムをまず平衡緩衝液(50 mmol/kg ホスフェート、300 mmol/kg NaCl、pH 7.0)を用いて洗浄し、その後pH 5.7の35 mmol/kg カプリレート緩衝液(3.9 mM 遊離カプリル酸)を用いて洗浄した。pHが安定化された後、洗浄を30分間続けた。さらなる3回の洗浄(平衡緩衝液、50 mmol/kg ホスフェート、1 mol/kg NaCl、そしてその後平衡緩衝液)後、ギ酸緩衝液、pH 3.5を用いてカラムからmAbを溶出させた。
組換えヒト抗体(IgG1)を含む170リットルの細胞株培地を、パイロットプラントにおいて二等分し、その後、別個に、自動クロマトグラフィーシステム(AKTA pilot、GE Healthcare)のプロテインA(Mab Select SuRe、Ge Healthcare)を含むカラムに通した。
種々のカプリル酸濃度及び種々のpHにおけるMuLVの不活性化を調査した(表6)。5.0~6.0の範囲のpHにおいて、遊離カプリル酸濃度が少なくとも約4 mMに維持されるように、pHを上昇させるとともにカプリル酸濃度を高めた場合、5分間後にMuLVの完全な不活性化が達成された。約2 mMのカプリル酸濃度では、反応時間を増加させ、これらの最も困難なウイルスについて30分間後に部分的な不活性化が得られた。1mM未満では、不活性化は観察されなかった。
Claims (9)
- i. 固定相にタンパク質含有サンプルをロードして、該固定相に標的タンパク質を結合させる工程;
ii. 結合された前記標的タンパク質をカプリル酸溶液に曝露する工程であって、前記カプリル酸溶液が、1~10 mMの遊離カプリル酸を含む、工程;
iii. 前記標的タンパク質を溶出する工程
を含む、前記標的タンパク質及びエンベロープウイルスを含むサンプルから前記標的タンパク質を精製するための方法であって、
前記標的タンパク質の精製が、前記サンプル中での、前記カプリル酸処置による、エンベロープウイルスの不活性化を含み、
前記カプリル酸溶液のpHが、4<pH≦6の範囲であり、
前記標的タンパク質が、抗体又はその断片であり、
前記固定相が、クロマトグラフィーカラムである、
方法。 - 前記サンプルが、発酵培養液、細胞培養物、細胞株培地、腹水、組織培養培地、血漿画分を含むヒト又は動物の血漿から成る群から選択される、請求項1に記載の方法。
- 前記サンプルが、イオン交換クロマトグラフィーカラム、アフィニティークロマトグラフィーカラム、ミックスモードクロマトグラフィーカラム、高速タンパク質液体クロマトグラフィーカラム、及び吸着流動床(EBA)クロマトグラフィーカラムから選択されるクロマトグラフィーカラムにロードされる、請求項1又は2に記載の方法。
- 前記カプリル酸溶液が、4.5<pH≦6、例えば4.6≦pH<6、例えば約pH 4.6、約pH 4.7、約pH 4.8、約pH 4.9、約pH 5.0、約pH 5.1、約pH 5.2、約pH 5.3、約pH 5.4、約pH 5.5、約pH 5.6、約pH 5.7、約pH 5.8、及び約pH 5.9、例えば4.9<pH<6のpHを有する、請求項1~3のいずれか一項に記載の方法。
- 前記カプリル酸溶液が、遊離カプリル酸に関して測定される濃度が2~10 mM、例えば2~8 mM、例えば2~6 mMの濃度のカプリル酸緩衝液である、請求項1~4のいずれか一項に記載の方法。
- 自動化されているか、又は自動化されたタンパク質精製プロセスの一部である、請求項1~5のいずれか一項に記載の方法。
- 前記抗体のアイソタイプが、ヒトIgG1、IgG2、IgG3又はIgG4抗体である、請求項1~6のいずれか一項に記載の方法。
- 前記抗体のアイソタイプが、ヒトIgG2、IgG3又はIgG4抗体である、請求項7に記載の方法。
- さらにマイコプラズマを不活性化する、請求項1~8のいずれか一項に記載の方法。
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WO2014186350A1 (en) * | 2013-05-15 | 2014-11-20 | Medimmune Limited | Purification of recombinantly produced polypeptides |
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US10654887B2 (en) | 2016-05-11 | 2020-05-19 | Ge Healthcare Bio-Process R&D Ab | Separation matrix |
US10889615B2 (en) | 2016-05-11 | 2021-01-12 | Cytiva Bioprocess R&D Ab | Mutated immunoglobulin-binding polypeptides |
EP3455243B1 (en) | 2016-05-11 | 2021-03-24 | Cytiva BioProcess R&D AB | Separation matrix |
US10703774B2 (en) | 2016-09-30 | 2020-07-07 | Ge Healthcare Bioprocess R&D Ab | Separation method |
US10730908B2 (en) | 2016-05-11 | 2020-08-04 | Ge Healthcare Bioprocess R&D Ab | Separation method |
JP7106187B2 (ja) | 2016-05-11 | 2022-07-26 | サイティバ・バイオプロセス・アールアンドディ・アクチボラグ | 分離マトリックスを保存する方法 |
EP3455241B1 (en) | 2016-05-11 | 2022-02-23 | Cytiva BioProcess R&D AB | Method of cleaning and/or sanitizing a separation matrix |
AU2016231646B2 (en) * | 2016-09-26 | 2021-04-08 | Instituto Grifols, S.A. | Method for the preparation of immunoglobulins |
CN109134648A (zh) * | 2018-09-14 | 2019-01-04 | 武汉伊莱瑞特生物科技股份有限公司 | 一种抗体纯化方法 |
CN112409477B (zh) * | 2019-08-21 | 2022-11-08 | 广东菲鹏生物有限公司 | IgM纯化方法 |
CN111991571B (zh) * | 2020-08-12 | 2022-04-05 | 湖州师范学院 | 一种柱上低pH病毒灭活的方法 |
CN115873810B (zh) * | 2022-12-26 | 2024-02-09 | 苏州良辰生物医药科技有限公司 | 一种鼠白血病病毒的纯化方法 |
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