JP6223338B2 - 活性型TNFR−Fc融合タンパク質を製造する方法 - Google Patents
活性型TNFR−Fc融合タンパク質を製造する方法 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
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- C—CHEMISTRY; METALLURGY
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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
- 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
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- C—CHEMISTRY; METALLURGY
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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
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- C07—ORGANIC CHEMISTRY
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- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Description
組換えタンパク質発現ベクターシステムで生産された組換えタンパク質の発現レベルを確認するために、目的タンパク質としてTNFRII−Fc融合タンパク質を用いた。
一般的な組換えタンパク質発現システムであるDHFRシステムを得るために、ハムスターのDHFR(dihydrofolate reductase)遺伝子をクローニングした。
GS DNAを得るためにハムスター細胞株であるCHO DG44(Invitrogen, 12609-012)を培養し、その後TRIZOL試薬(Invitrogen)を用いて全RNAを得た。得られた全RNAを用いたRT−PCRを行ってcDNAを得て、前記得られたcDNAを鋳型とし、下記GS遺伝子を得るためのプライマー対(GS SalI-F primer, GS XbaI-R primer)を用いてPCRを行い(94℃、5分間の変性を行い、94℃、30秒間の変性、50℃、30秒間のアニーリング、72℃、90秒間の伸長を25回繰り返し、72℃、7分間の伸長を行う)PCR産物を得た。
GS SalI−F:5’−gtcgacatggccacctcagcaagttccc−3’(配列番号3)
GS XbaI−R:5’−tctagattagtttttgtattggaaaggg−3’(配列番号4)
リポフェクタミン(lipofectamine 2000; Invitrogen)を用いて、TNFRII−Fcを生産する遺伝子を含むpcDNA3.1−Kozak−TNFRII−Fc−IRES−GSベクターをチャイニーズハムスター(Chinese hamster ovary、CHO)細胞株に導入した。前期導入CHO細胞株を10%ウシ胎児血清(fetal bovine serum; FBS)を含むDMEM/F12(Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12)培地を用いて、前記遺伝子導入CHO細胞株をフラスコ又はバイオリアクターで培養した。培養液をデプスフィルタ(depth filter)で濾過して細胞を除去し、再度精密濾過法(microfiltration)を行って細胞断片を除去した。前記培養上清を回収し、プロテインAクロマトグラフィーで部分精製を行った。精製分画のpHは100mMクエン酸又はリン酸(pH2.1) を用いて3.6〜3.8の範囲内で調整した。
実験例1−1:クエン酸ナトリウムを用いた疎水性相互作用クロマトグラフィー
ガラスカラムにButyl Sepharose 4 Fast Flow(GE Healthcare)を8cm以上の高さに充填した。0.5Mクエン酸ナトリウム及び50mMリン酸ナトリウム緩衝液(pH6.8)(緩衝液Iと命名する)、及び 50mMリン酸ナトリウム緩衝液(pH6.8)(緩衝液Jと命名する)を製造した。
カラムをButyl Sepharose 4 Fast Flowで10cm以上の高さに充填した。0.72M硫酸ナトリウム及び50mMリン酸ナトリウム緩衝液(pH6.8)(以下、「緩衝液S」)及び50mMリン酸ナトリウム緩衝液(以下、「緩衝液J」)を製造した。
カラムをButyl Sepharose 4 Fast Flowで10cm以上の高さに充填した。0.75M塩化ナトリウム及び50mM トリス−HCl 緩衝液(pH7.2)(以下、「緩衝液X」)、1.0M塩化ナトリウム及び50mM トリストリス−HCl緩衝液(pH7.2)(以下、「緩衝液Y」)、1.5M塩化ナトリウム及び50mM トリス−HCl緩衝液(pH7.2)(以下、「緩衝液Z」)、及び50mM トリス−HCl緩衝液(pH7.2)(以下、「緩衝液T」)を製造した。
実施例1−1の各ステップで得られた試料に対して疎水性相互作用HPLC(hydrophobic interaction high performance liquid chromatography)に適用した。疎水性相互作用HPLCでは、Butyl NPRカラム(Tosoh Bioscience)を使用し、移動相Aは1.8M硫酸アンモニウム、100mMリン酸ナトリウム溶液(pH7.0)を用い、移動相Bは100mMリン酸ナトリウム溶液 (pH7.0)を用いた。 初期の5分間を移動相Aの組成を1ml/minで100%となるように維持し、45分間で移動相Aの組成が100%から0%になるように線形勾配を与えた。前記結果を図6に示した。
実験例1−1の各ステップで得られた試料に対してSE−HPLCを行った。SE−HPLCは、タンパク質のサイズにより分離する方法であり、切断タンパク質や凝集体を保持時間(retention time)により分離することができる。TSK−GEL3000SWXLカラム(Tosoh Bioscience; 7.8 mm ID * 30 cm H)をPBSにより1ml/minの流速で平衡化し、20μgのタンパク質を注入してタンパク質を分析した。その結果を図7に示す。
実験例1−1のピーク2とピーク3の分画とTNF−αとの結合を確認した。ピーク2分画又はピーク3分画を適宜希釈し、抗IgFc抗体がコーティングされた固体表面に結合させた。その後、TNF−αで処理してピーク2及びピーク3分画がTNF アルファと結合することをHRPで発色させて確認した。その結果を図8に示した。
実験例1−1と同一の試料、同一の方法で、単位カラム体積当たりの試料の注入量のみ変更して分析した。実験例1−1においては実施例4で製造した溶液を12g/L bedでカラムに注入したのに対して、本実験例においてはそれぞれ9.5g/L bedと13g/L bedの容量で注入して分離分析した。その結果をそれぞれ図9及び図10に示す。
Claims (14)
- a)クエン酸ナトリウム及びリン酸ナトリウムを含む平衡化緩衝液で前平衡化された疎水性相互作用クロマトグラフィー(HIC)カラムに、哺乳類細胞から生産されたTNFR−Fc融合タンパク質の混合物を含む試料を、クロマトグラフィーレジン体積当たり10g/L bed〜14g/L bedの量で注入するステップと、
b)前記平衡化緩衝液の塩と同じ塩を含む洗浄緩衝液で前記カラムを洗浄して、TNFR−Fc融合タンパク質の混合物から切断型TNFR−Fc融合タンパク質を除去するステップと、
c)前記平衡化緩衝液よりクエン酸ナトリウムの塩濃度を減少させた溶出緩衝液で前記カラムから活性型TNFR−Fc融合タンパク質を溶出させるステップとを含む、
活性型TNFR−Fc融合タンパク質を製造する方法であって、
前記カラムのリガンドがブチル基である、
方法。 - 前記ステップa)の平衡化緩衝液が0.45M〜0.55Mクエン酸ナトリウム及び50mM〜100mMリン酸ナトリウムを含む緩衝液である、請求項1に記載の方法。
- 前記平衡化緩衝液がpH6.5〜7.0である、請求項1又は2に記載の方法。
- 前記平衡化緩衝液がpH6.7〜6.9であり、0.48M〜0.52Mクエン酸ナトリウム及び50mM〜70mMリン酸ナトリウムを含む、請求項2に記載の方法。
- 前記ステップa)のTNFR−Fc融合タンパク質がpcDNA3.1−Kozak−TNFRII−Fc−IRES−GSベクターの導入された哺乳類細胞から生産される、請求項1に記載の方法。
- 前記TNFR−Fc融合タンパク質が、カラムに注入する前にアフィニティークロマトグラフィー、イオン交換クロマトグラフィー及び脱塩からなる群から選択される少なくとも1つの方法で部分精製される、請求項1に記載の方法。
- 前記TNFR−Fc融合タンパク質の混合物を含む試料が、注入前にクエン酸ナトリウムを用いて50mS/cm〜55mS/cmの伝導度に調整される、請求項1に記載の方法。
- 前記ステップb)の洗浄緩衝液がステップa)の平衡化緩衝液と同じ組成である、請求項1に記載の方法。
- 前記ステップc)が40mS/cm〜47mS/cmの伝導度を有する溶出緩衝液で活性型TNFR−Fc融合タンパク質を溶出させるステップである、請求項1に記載の方法。
- 前記ステップc)の溶出緩衝液が0.35M〜0.4Mの範囲のクエン酸ナトリウムを含む、請求項1に記載の方法。
- 前記溶出緩衝液がpH6.5〜7.0である、請求項9又は10に記載の方法。
- 前記溶出緩衝液がpH6.7〜6.9であり、0.38M〜0.4Mクエン酸ナトリウム及び50mM〜70mMリン酸ナトリウムを含む、請求項10に記載の方法。
- 前記方法が、pHが6.5〜7.0であり、伝導度が4mS/cm〜6mS/cmの緩衝液を用いてカラムから不活性型TNFR−Fc融合タンパク質又はTNFR−Fc融合タンパク質凝集体を含む分画を分離するステップd)をさらに含む、請求項1に記載の方法。
- 前記ステップd)で使用する緩衝液が、pHが6.7〜6.9であり、50mM〜70mMリン酸ナトリウムを含む、請求項13に記載の方法。
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