JP5739324B2 - 骨形成タンパク質の産生増進法 - Google Patents
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Description
[0002]本発明は組換えタンパク質産生の分野に関する。特に、本発明は、骨形成タンパク質(BMP)を含むペプチド増殖因子を産生する方法に関する。
[0026]本発明の方法で使用するのに適した培地は、典型的には、培養細胞の増殖を補助するのに必要な栄養素を含有し、これには、ビタミン、ミネラル、脂肪酸、アミノ酸、炭素供給源(例えばデキストロース)、および場合によって、例えばインスリンおよびトランスフェリンを含む増殖培地、または抗生物質が含まれる。いくつかの態様において、培地は、基礎培地、例えばダルベッコの修飾イーグル培地(DMEM)、HamのF−12、Roswell Park Memorial Institute(RPMI)培地、またはその組み合わせを含む。
[0039]本発明によって提供する方法を用いて、多様なタンパク質が産生可能である。特定の態様において、タンパク質は増殖因子である。特定の態様において、増殖因子は、TGF−βスーパーファミリーのメンバーである。より詳細な態様において、TGF−βファミリーメンバーは、骨形成タンパク質(BMP)である。
[0043]当該技術分野に知られるように、BMP−2などの骨形成タンパク質は、タンパク質に基づく療法として有用である。したがって、本発明の方法は、培養から回収されるタンパク質の精製または単離の工程をさらに含んでもよい。BMP−2は、当該技術分野に知られる多様な手段によって精製可能である。特定の態様において、精製は、1以上のカラムクロマトグラフィー精製、例えばブチルセファロース樹脂精製を含む。より詳細な態様において、ブチルセファロースカラムは、CNBr活性化セファロース、例えばセファロース4Bにカップリングしたブチルアミンの樹脂を含む。いくつかの態様において、精製はヘパリン様樹脂、例えばCELLUFINETMスルフェート樹脂上のカラム精製工程をさらに含んでもよい。例えば、BMP−2を含有する培養細胞からの馴化培地をヘパリン様樹脂含有カラムに適用してもよく、BMP−2を含有する溶出物をカラムから得て、そして次いで、ブチルセファロースカラムなどの第二のカラムに適用する。例えば、本明細書に援用される国際公報第WO 99/31120号、特にその3〜7ページに記載されるように、さらなる精製が可能である。
[0045]非常に多様な細胞を用いて、本発明によって提供する方法によって組換えタンパク質を産生してもよい。関心対象のタンパク質、例えばBMPを発現する組換えDNAで形質転換可能である任意の細胞を、本発明の方法で使用可能である。細胞は、線形動物、蠕虫(worm)、昆虫、両生類、または哺乳類を含む多様な種、例えば、ヒト、霊長類、ヒツジ、ウシ、ブタ、ウマ、ネコ、イヌ、またはげっ歯類供給源由来であってもよい。特定の態様において、細胞はヒトまたはげっ歯類由来である。より詳細な態様において、細胞はハムスター由来である。
[0047]本発明の方法で使用するための細胞を、例えば、WarnockおよびAl−Rubei、Biotech. Appl. Biochem. 45:1−12(2006)で論じられるように、当該技術分野に知られる任意の手段によって培養してもよい。典型的には、細胞をバイオリアクター中で増殖させる。バイオリアクターは任意のサイズであってもよい。いくつかの態様において、バイオリアクターは、少なくとも約1L; 3L; 20L; 40L; 80L; 100L; 160L; 1,900L; 2,500L; 12,000L; 20,000L; 40,000L、またはそれより大きい。バイオリアクターは、懸濁物(すなわち係留独立増殖)中の、または支持体上に係留された細胞の増殖を補助しうる。係留依存性増殖には、例えば大きい表面積対体積比を提供する、マイクロキャリアの使用が含まれてもよい。
[0052]BMP−2産生プロセスの開発中、1,900リットルのバイオリアクターにおいて、rhBMP−2およびジヒドロ葉酸レダクターゼ(DHFR)を同時発現するCHO細胞(本明細書において、EMC−G5細胞として知られる)の高細胞密度培養で、スケールアップの問題が観察された。高細胞密度培養は、3日間バッチには0.60x106細胞/mLで、または4日間バッチには0.30x106細胞/mLで接種された。バッチ−リフィード・プロセスの最初の継代中、最終細胞密度は通常、3.0x106細胞/mLに到達するが、以下の継代の最終細胞密度は次第に低下した。例えば、3回の継代は、それぞれ、3.0x106、1.6x106、および1.0x106細胞/mLの採取細胞密度を生じるであろう。こうした増殖速度低下は、培地中、より低量のBMP−2タンパク質を生じ、そして全体の生産性は望ましいよりも低くなった。増殖速度低下は、3リットル規模では再現不能であった。この問題を研究するためのモデルとして、160リットルのバイオリアクターを用いた。採取細胞密度低下は、図1に示すように、160Lバイオリアクター中で見られた。
[0056]これらの微量金属評価中、先の高密度培養において生じたrhBMP−2由来のサイズ排除クロマトグラム(CEX)の第二のピーク上に異常な肩が観察された。培地中で低レベルのデキストラン硫酸(200mg/Lの代わりに10mg/L)を用いると、リン酸とのインキュベーション後、より高い割合の肩ピークが生じることが見出された(それぞれ、およそ4%に対しておよそ12%)。デキストラン硫酸用量反応実験によって、細胞培地中のデキストラン硫酸レベルがより高いと、用量依存方式で、観察された肩ピーク量が減少することが示された。デキストラン硫酸は、以前、BMP−2産生中、タンパク質収量を増加させることが示されてきている。例えば、米国特許第5,318,898号および第5,516,654号を参照されたい。
[0057]鉄および銅濃度が、培地中で濃縮されている場合、実験室規模バイオリアクター中であっても、細胞増殖が安定でないことが時折観察された。図3を参照されたい。さらなる金属が、他の培地構成要素(単数または複数)と反応し、そして不安定な培養増殖を生じうると仮定された。
[0059]さらなる研究において、増殖速度欠陥を誘発する培地A1を、増殖速度欠陥を誘発しない培地A2に比較した。培地A1は、主にピリドキサールを含有する一方、培地A2はピリドキシンのみを含有することが注目された。BMP−2 EMC G5細胞を、異なる鉄濃度であり、そしてピリドキサールを含む、および含まない、培地A1および培地A2中で増殖させた。培地A1およびA2の鉄およびビタミンB6含量を表2に要約する。
[0062]培地からピリドキサールを除いたさらなる培地修飾後(培地B2を生じる)、細胞増殖およびBMP−2生産性は一貫しており、そして以前のプロセスに比較して、小規模バイオリアクター中、ならびに商業規模バイオリアクター中で、生産性に2倍の増加を示した。図6および図7を参照されたい。したがって、本出願に記載する培地に対する修飾は、商業規模への組換えタンパク質産生のスケールアップ中に生じる増殖欠陥を排除した結果、高品質組換えタンパク質の収量が実質的に増加する。
表3
Claims (45)
- BMP−2(骨形成タンパク質−2)産生法であって:
i)少なくとも2.25μMの濃度の鉄、ポリアニオン性化合物、および15mMから40mMの総アミノ酸濃度を含み、そしてピリドキサールが存在している場合、ピリドキサールが培地中のビタミンB6のモル濃度の50%未満を構成する培地中で、BMP−2をコードするDNA分子を含む適切な宿主細胞を培養し;そして
ii)関心対象のタンパク質を回収する
工程を含み、
宿主細胞を少なくとも160Lの容量を有するバイオリアクター中で培養する、前記方法。 - 鉄が少なくとも5μMの濃度である、請求項1の方法。
- 培地が少なくとも2×106細胞/mLの採取密度を補助しうる、請求項1の方法。
- 培地がさらに、ピリドキシン、ピリドキサミン、ピリドキシン5’−リン酸、ピリドキサミン5’−リン酸からなる群より選択される、少なくとも1つのビタミンB6を含む、請求項1の方法。
- 培地が少なくとも15μMの総ビタミンB6濃度を有する、請求項4の方法。
- 培地が1.2未満のピリドキサール対ピリドキシン比を有する、請求項4の方法。
- ピリドキサール対ピリドキシン比が0.4未満である、請求項6の方法。
- 培地が少なくとも10nMの濃度の銅をさらに含む、請求項1の方法。
- 培地が少なくとも0.2μMの濃度の亜鉛をさらに含む、請求項1の方法。
- ポリアニオン性化合物がデキストラン硫酸である、請求項1の方法。
- デキストラン硫酸が少なくとも10mg/Lの濃度で存在する、請求項10の方法。
- デキストラン硫酸が少なくとも100mg/Lの濃度で存在する、請求項10の方法。
- デキストラン硫酸が、5,000〜500,000g/モルの間の分子量を有する、請求項11の方法。
- デキストラン硫酸が7,000g/モルの分子量を有する、請求項13の方法。
- 培地が少なくとも0.5mMの濃度のL−システインを含む、請求項1の方法。
- 培地が最大で0.3mMの濃度のL−グルタミン酸を含む、請求項15の方法。
- 培地が260〜360mOsmの間の初期モル浸透圧濃度を有する、請求項1の方法。
- BMP−2が組換えヒトBMP−2(hBMP−2)である、請求項1の方法。
- 適切な宿主細胞が、COS細胞、CHO細胞、BHK細胞、Balb/c 3T3細胞、または293細胞からなる群より選択される、請求項1の方法。
- 適切な宿主細胞がCHO細胞である、請求項19の方法。
- CHO細胞がDHFR遺伝子の減少した発現を有する、請求項20の方法。
- バイオリアクターが少なくとも500Lの容量を有する、請求項21の方法。
- バイオリアクターが少なくとも2,500Lの容量を有する、請求項22の方法。
- バイオリアクターが少なくとも12,000Lの容量を有する、請求項23の方法。
- 宿主細胞をバッチ・リフィード(batch refeed)、流加(fed batch)、または灌流によって培養する、請求項24の方法。
- 宿主細胞をバッチ・リフィードによって培養する、請求項25の方法。
- 宿主細胞を一定の温度で培養する、請求項26の方法。
- 温度が36〜38℃である、請求項27の方法。
- ブチルセファロース樹脂上でBMP−2を精製する工程をさらに含む、請求項1の方法。
- 精製が、BMP−2を含有する馴化培地をヘパリン様樹脂に適用し、BMP−2を含有する溶出物を得て、そして溶出物をブチルセファロース樹脂に適用する工程をさらに含む、請求項29の方法。
- BMP−2(骨形成タンパク質−2)産生法であって:
i)少なくとも2.25μMの濃度の鉄、ポリアニオン性化合物、15mMから40mMの総アミノ酸濃度、および少なくとも15μMのビタミンB6を含み、そしてピリドキサールが存在している場合、ピリドキサールが培地中のビタミンB6のモル濃度の50%未満を構成する培地中で、BMP−2タンパク質をコードするDNA分子を含む適切な宿主細胞を培養し;そして
ii)BMP−2タンパク質を回収する
工程を含み、
宿主細胞を少なくとも160Lの容量を有するバイオリアクター中で培養する、前記方法。 - 培地が少なくとも10nMの濃度の銅をさらに含む、請求項31の方法。
- 培地が少なくとも0.2μMの濃度の亜鉛をさらに含む、請求項31の方法。
- 宿主細胞がCHO細胞である、請求項31の方法。
- BMP−2が組換えヒトBMP−2(rhBMP−2)である、請求項31の方法。
- ポリアニオン性化合物が、少なくとも10mg/Lの濃度のデキストラン硫酸である、請求項31の方法。
- 培地が少なくとも0.5mMの濃度のL−システインを含む、請求項31の方法。
- 培地が最大で0.3mMの濃度のL−グルタミン酸をさらに含む、請求項37の方法。
- 培地が260〜360mOsmの間の初期モル浸透圧濃度を有する、請求項31の方法。
- BMP−2(骨形成タンパク質−2)産生法であって:
i)少なくとも2.25μMの濃度の鉄、少なくとも10nMの濃度の銅、少なくとも20mMの総濃度のアミノ酸、少なくとも0.5mMの濃度のL−システイン、少なくとも10mg/Lの濃度のデキストラン硫酸、15mMから40mMの総アミノ酸濃度、および少なくとも15μMの濃度のビタミンB6を含み、そしてピリドキサールが存在している場合、ピリドキサールが培地中のビタミンB6のモル濃度の50%未満を構成する培地中で、BMP−2タンパク質をコードするDNA分子を含むCHO細胞を、バッチ・リフィード・プロセスで培養し;そして
ii)BMP−2タンパク質を回収する
工程を含み、
宿主細胞を少なくとも160Lの容量を有するバイオリアクター中で培養する、前記方法。 - 培地が少なくとも0.2μMの濃度の亜鉛をさらに含む、請求項40の方法。
- BMP−2(骨形成タンパク質−2)産生法であって:
i)少なくとも2.25μMの濃度の鉄、ポリアニオン化合物、および15mMから40mMの総アミノ酸濃度を含み、そしてピリドキサールが存在している場合、15μM未満の濃度で存在する培地中で、BMP−2をコードするDNA分子を含む適切な宿主細胞を培養し;そして
ii)関心対象のタンパク質を回収する
工程を含み、
宿主細胞を少なくとも160Lの容量を有するバイオリアクター中で培養する、前記方法。 - 請求項1〜42の方法のいずれかによって産生される産物。
- 請求項43の産物を含む薬学的配合物。
- 骨組織の欠陥、傷害、疾患、または障害を持つ患者を治療するための薬剤の調製における、請求項43の産物の使用。
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