JPWO2017014257A1 - 酸性pH領域での抗体結合能が低下した抗体結合性タンパク質 - Google Patents
酸性pH領域での抗体結合能が低下した抗体結合性タンパク質 Download PDFInfo
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Abstract
Description
回収率(%)=(溶出した免疫グロブリンのFc領域を含むタンパク質の濃度(mg/mL)×溶出した液量(ml))÷(負荷した免疫グロブリンのFc領域を含むタンパク質の濃度(mg/mL)×負荷した液量(ml))×100
プロテインAのCドメイン体にG29A変異を導入したC−G29A.2d(配列番号6)をコードするDNAの5’末端にPstI認識サイト、3’末端にXbaI認識サイトを付与したDNA(配列番号7)に、表1に記載のアミノ酸置換変異を導入した改変型C−G29A.2dの人工合成遺伝子を、外注によって全合成した(ユーロフィンジェノミクス社製)。このサブクローニング後の発現プラスミドを、制限酵素PstIおよびXbaI(タカラバイオ社製)で消化し、取得したDNA断片を、同じ制限酵素で消化したブレビバチルス発現用ベクターpNCMO2(タカラバイオ社製)へライゲーションし、改変型C−G29A.2dのアミノ酸配列をコードするDNAがブレビバチルス発現用ベクターpNCMO2に挿入された発現プラスミドを調製した。なお、プラスミドの調製にはエシェリヒア・コリJM109株を用いた。
担体:IgG Sehparose FF(GEヘルスケア社製)
カラム:オムニフィットカラム(ディバ・インダストリーズ社製)、カラム径0.66cm、ベッド高6.4cm、カラムボリューム:2.19mL
流速:0.8mL/min、接触時間2.7min
負荷量:470μL(リガンド濃度1.3mg/mL)
平衡化buffer:50mM トリス塩酸、150mM NaCl buffer pH7.5
溶出条件:50mM クエン酸buffer pH6.0→50mM クエン酸buffer pH3.0(20CV)
表面プラズモン共鳴を利用したバイオセンサーBiacore 3000(GEヘルスケア社製)を用いて、実施例1で取得した各種タンパク質の免疫グロブリンとの親和性を解析した。本実施例では、ヒト血漿から分画したヒト免疫グロブリンG製剤(以後は、ヒトIgGと記する)を利用した。
プロテインAのBドメイン体にG29A変異を導入したB−G29A.2d(配列番号8)をコードするDNAの5’末端にPstI認識サイト、3’末端にXbaI認識サイトを付与したDNA(配列番号9)に表3に記載のアミノ酸置換変異を導入した改変型B−G29A.2dの人工合成遺伝子を、外注によって全合成した(ユーロフィンジェノミクス社製)。実施例1と同様にして、組換え発現し、得られた培養上清についてIgG固定化担体を用いて溶出試験を実施した。
実施例3で取得した各種タンパク質の免疫グロブリンとの親和性を実施例2と同様にして解析した。結果を表4に示す。改変型B−G29A.2dのヒトIgGに対する結合パラメータは、B−G29A.2d(対照)と同程度であった。いずれの改変型B−G29A.2dも中性pH領域では変異導入前のB−G29A.2dと同程度の抗体結合能を有していた。
実施例1と同様に培養して得られた培養物を遠心分離して菌体を分離し、培養上清に酢酸を添加してpHを4.5に調整後、一時間静置し、目的のタンパク質を沈殿させた。遠心分離で沈殿を回収し、緩衝液(50mM Tris−HCl、pH8.5)にて溶解した。次に、HiTrap Qカラム(GEヘルスケアバイオサイエンス株式会社製)を利用した陰イオン交換クロマトグラフィーにて目的タンパク質を精製した。具体的には、目的タンパク質溶液を、陰イオン交換用緩衝液A(50mM Tris−HCl、pH8.0)にて平衡化したHiTrap Qカラムに添加し、陰イオン交換用緩衝液Aで洗浄後、陰イオン交換緩衝液Aと陰イオン交換緩衝液B(50mM Tris−HCl、1M NaCl、pH8.0)を利用した塩濃度勾配にて、途中に溶出される目的タンパク質を分取した。分取した目的タンパク質溶液を超純水に透析し、透析後の水溶液を最終精製サンプルとした。なお、カラムを用いたクロマトグラフィーによるタンパク質精製は、全てAKTA avantシステム(GEヘルスケアバイオサイエンス株式会社製)を利用して実施した。
カラム:プレパックカラムHitrap NHS activated HP」1mL(GEヘルスケア社製)(各リガンドを固定化した担体を含むカラム)
流速:0.33mL/min、接触時間3.0min
負荷液:ガンマグロブリン「ニチヤク」(日本製薬社製)5mL(リガンド濃度1mg/mL)
平衡化buffer:ダルベッコリン酸緩衝生理食塩水(シグマ・アルドリッチ社製)
溶出条件:
溶出1:50mM クエン酸buffer(4CV)、試験A:pH4.0、試験B:pH3.75、試験C:pH3.5
溶出2:50mM クエン酸buffer pH3.0(4CV)
プロテインAのCドメイン体にG29A変異を導入したC−G29A.2d(配列番号6)をコードするDNAの5’末端にPstI認識サイト、3’末端にXbaI認識サイトを付与したDNA(配列番号7)に、表6に記載のアミノ酸置換変異を導入した改変型C−G29A.2dの人工合成遺伝子を、外注によって全合成した(ユーロフィンジェノミクス社製)。
表面プラズモン共鳴を利用したバイオセンサーBiacore 3000(GEヘルスケア社製)を用いて、実施例6で取得した各種タンパク質の、免疫グロブリンとの親和性を、実施例2と同じ条件で解析した。免疫グロブリンとして、ヒトIgGを使用した。
実施例5で得られた、C−G29A/I31L.2dを固定化したアフィニティー分離マトリックスと、TNF受容体−Fc融合タンパク質(エタネルセプト)を含むCHO細胞培養上清(バイオセロス社製)を用いて、下記条件で抗体溶出試験を実施した。対照として同様に調製したC−G29A.2dアフィニティー分離マトリックスを用いた溶出試験も実施した。抗体回収率は、溶出液の吸光度を測定して算出した。宿主由来不純物(HCP)はCHO HCP ELISAキット(Cygnus社製)を用いて定量した。対照のC−G29A.2dアフィニティー分離マトリックスを用いた結果を図2に示し、改変型C−G29A.2dアフィニティー分離マトリックスを用いた結果を図3に示す。
カラム:プレパックカラムHitrap NHS activated HP」1mL(GEヘルスケア社製)(各リガンドを固定化した担体を含むカラム)
流速:1mL/min(サンプル負荷時のみ0.33mL/min、接触時間3.0min)
負荷液:TNF受容体−Fc融合タンパク質を含むCHO細胞培養上清(バイオセロス社製)16.1mL(濃度0.62mg/mL)
平衡化buffer:ダルベッコリン酸緩衝生理食塩水(シグマ・アルドリッチ社製)
溶出条件:
溶出1:50mM クエン酸buffer(pH6→pH3)のグラジエント溶出(20CV)
溶出2:50mM クエン酸buffer pH3.0(5CV)
Claims (19)
- 配列番号1〜5に記載のプロテインAのE、D、A、B、またはCドメインに由来するアミノ酸配列において、
Fc結合部位の疎水性アミノ酸残基を他の疎水性アミノ酸残基または極性無電荷アミノ酸残基に置換して得られるアミノ酸配列を含むタンパク質であって、
前記置換前と比較して酸性pH領域での抗体結合能が低下していることを特徴とするタンパク質。 - Fc結合部位の疎水性アミノ酸残基が、Cドメインの5位に対応するPhe、13位に対応するPhe、17位に対応するLeu、または31位に対応するIleである、請求項1に記載のタンパク質。
- 他の疎水性アミノ酸残基が、Gly、Ala、Val、Leu、Ile、Met、Phe、またはTrpである、請求項1または2に記載のタンパク質。
- 極性無電荷アミノ酸残基が、Ser、Thr、Gln、Asn、Tyr、またはCysである、請求項1または2に記載のタンパク質。
- 以下に示すアミノ酸残基;
Gln−9、Gln−10、Tyr−14、Pro−20、Asn−21、Leu−22、Gln−26、Arg−27、Phe−30、Leu−34、Pro−38、Ser−39、Leu−45、Leu−51、Asn−52、Gln−55、およびPro−57(残基番号はCドメインに対応する)のうち、
90%以上が保持されている、請求項1〜4のいずれか1項に記載のタンパク質。 - Cドメインの40位に対応するアミノ酸がVal以外のアミノ酸である、
請求項1〜5のいずれか1項に記載のタンパク質。 - 前記アミノ酸配列が、さらに、疎水性アミノ酸残基、酸性アミノ酸残基、または極性無電荷アミノ酸残基を塩基性アミノ酸残基に置換して得られるものである、
請求項1〜6のいずれか1項に記載のタンパク質。 - 請求項1〜7のいずれか1項に記載のタンパク質を2個以上連結して得られる複数ドメインからなるタンパク質。
- 請求項1〜8のいずれか1項に記載のタンパク質をコードするDNA。
- 請求項9に記載のDNAを含むベクター。
- 請求項10に記載のベクターで宿主細胞を形質転換して得られる形質転換体。
- 請求項9に記載のDNAを用いた無細胞タンパク質合成系、または、請求項11に記載の形質転換体を用いる、請求項1〜8のいずれか1項に記載のタンパク質の製造方法。
- 請求項1〜8のいずれか1項に記載のタンパク質をアフィニティーリガンドとして、水不溶性の基材からなる担体に固定化して得られる、アフィニティー分離マトリックス。
- 免疫グロブリンのFc領域を含むタンパク質に結合することを特徴とする、請求項13に記載のアフィニティー分離マトリックス。
- 免疫グロブリンのFc領域を含むタンパク質が免疫グロブリンG、または、免疫グロブリンG誘導体である、請求項14に記載のアフィニティー分離マトリックス。
- 請求項1〜8のいずれか1項に記載のタンパク質をアフィニティーリガンドとして水不溶性の基材からなる担体に固定することからなる、請求項13〜15のいずれか1項に記載のアフィニティー分離マトリックスの製造方法。
- 請求項13〜15のいずれか1項に記載のアフィニティー分離マトリックスに免疫グロブリンのFc領域を含むタンパク質を吸着させることを含む、免疫グロブリンのFc領域を含むタンパク質の精製方法。
- 下記(a)〜(b)の工程;
(a)免疫グロブリンのFc領域を含むタンパク質を含む液体を、請求項13〜15のいずれか1項に記載のアフィニティー分離マトリックスに吸着させる工程、
(b)pH3.5以上の溶出液をアフィニティー分離マトリックスと接触させ、前記免疫グロブリンのFc領域を含むタンパク質を溶出させる工程
を含む、請求項17に記載の精製方法。 - 溶出される免疫グロブリンのFc領域を含むタンパク質に含まれる、宿主由来タンパク質、および/または免疫グロブリンのFc領域を含むタンパク質の凝集体の含量が低減されている、請求項18に記載の精製方法。
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WO2011118699A1 (ja) * | 2010-03-24 | 2011-09-29 | 株式会社カネカ | 免疫グロブリンに特異的に結合するタンパク質および免疫グロブリン結合性アフィニティーリガンド |
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