JP2016532457A - 代謝が最適化された細胞培養 - Google Patents
代謝が最適化された細胞培養 Download PDFInfo
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Abstract
Description
本願は、2013年10月11日に出願された米国仮特許出願第61/889,815号への米国特許法第119条第(e)項の下での利益を主張する。米国仮特許出願第61/889,815号は、本明細書で、その全体が参考として具体的に援用される。
本発明は、細胞培養においてラクテート消費へと代謝がシフトする細胞に関する。シードトレイン培養(seed train culture)におけるラクテート消費代謝プロフィールへの切り替わりは、生産培養(production culture)に対して有益な効果を有する。生産用リアクターの接種の際に、細胞は、哺乳動物細胞流加培養において、低いラクテート生産速度、低いピークラクテートレベル、ラクテート消費への早期の切り替わり、およびその後の増大した生産性を有するより効率的なラクテート代謝を示す。従って、細胞培養におけるタンパク質および/もしくはポリペプチドの大規模生産のための改善された方法が提供される。
生物学的製剤、特にタンパク質およびポリペプチドは、新規な薬学的生成物としてより頻繁に開発されている。非常に高レベルの目的の特定のタンパク質を生産する操作された細胞は、これら薬学的介入物の成功裡の商業的生産のために極めて重要になっている。細胞培養条件の制御および最適化は、培養において生産される治療用タンパク質のレベルおよび品質を変動させ、これに対して大きな影響を有する。
細胞のフィードの制御は、より効率的な代謝表現型に到達しようとする努力において利用されている(Europa, A. F., et al., 2000, Biotechnol. Bioeng. 67:25-34; Cruz et al., 1999, Biotechnol Bioeng, 66(2):104−113; Zhou et al., 1997, Cytotechnology 24, 99-108; Xie and Wang, 1994, Biotechnol Bioeng, 43:1174−89)。しかし、これは、高細胞密度流加培養において認められる、栄養素欠乏、ならびに急速な変化(例えば、アンモニウム濃度における変化)がアポトーシス(「プログラムされた細胞死」)を誘発し得るという事実によって複雑である(Newland et al., 1994, Biotechnol. Bioeng. 43(5):434−8)。よって、一般的な最適化アプローチは、流加(fed−batch)において中程度に高密度まで細胞を増殖させ、その後、例えば、温度もしくはpH変化によって長期間の生産的な静止期を意図的に誘発することである(Quek et al., 2010, Metab Eng 12(2):161−71. doi: 10.1016/j.ymben.2009.09.002. Epub 2009 Oct 13).
最適化技術(例えば、前出で考察されるもの)は、流加細胞培養に焦点を当てており、この栄養素依存プロセスは、目的のポリペプチドの生産のために操作された各宿主細胞に適合されなければならない。培養において細胞をラクテート消費者へと適合させるための方法は、生物学的治療剤を製造するプロセスにおいて非常に望まれている。ある代謝表現型を有する細胞株をラクテート消費に最適化することは、ポリペプチドの商業的生産のために有益であることが判明する。
本発明は、ラクテート消費へと代謝がシフトした細胞およびこの細胞を培養する方法を提供する。代謝が適合した細胞は、大規模タンパク質生産に理想的である。
本発明が記載される特定の方法および実験条件に限定されないことは理解されるべきである。なぜならこのような方法および条件は変動し得るからである。同様に、本明細書で使用される用語法は、特定の実施形態を記載する目的に過ぎず、限定することを意図していないこともまた理解されるべきである。なぜなら本発明の範囲は、特許請求の範囲によって定義されるからである。
「バッチ培養(バッチ培養物)」(batch culture)もしくは「バッチモード」は、本明細書で使用される場合、細胞および最初の完全作業体積の培地(これは一度も交換されない)で満たされたユニット(例えば、培養容器)をいう語句である。このようなバッチ培養において、細胞培養のための全成分は、培養プロセスの開始時に培養容器へと供給される。培養は通常、その栄養素が使い果たされるまで、もしくは老廃物が毒性レベルに達して細胞が増殖するのを止めるまで、行われる(run)。
語句「代謝シフト」とは、本明細書で使用される場合、細胞代謝、もしくは炭素栄養源の使用の、ラクテート生産から正味のラクテート消費への変化をいう。いずれか1つの理論には拘束されないが、無血清培地において最も一般的な炭素栄養源は、グルコースおよびグルタミンであり、これらは、迅速な細胞増殖を支える。グルコースは、CO2およびH2Oへと完全に酸化され得るか、または酸素の利用能に基づいて、ラクテートへと(例えば、好気性解糖系では)変換され得る。速く増殖する細胞は、グルコースおよびグルタミンを迅速に消費し、不完全な酸化的代謝、従って、過剰なラクテート生産をもたらす。炭水化物代謝は、ラクテート消費へと切り替わり得、従って、ラクテートの蓄積を低下させ得る。
本発明の方法は、細胞培養において目的のタンパク質もしくはポリペプチドを生産する。本発明の方法においてタンパク質生産を可能にするために、細胞は、上記目的のポリペプチドもしくはタンパク質を組換え発現するように操作される。
細胞発現系の使用は、細胞培養におけるこのようなポリペプチドもしくはタンパク質の高生産に欠くことができない。
CHO細胞を、融合タンパク質を発現するDNAでトランスフェクトした。融合タンパク質を生産するCHO細胞株を、ダイズを含む独占培地中でシード容器培養においてインキュベートし、パラメーター(例えば、オンラインpH、オフラインラクテートおよび生細胞数)を測定および記録して、代謝状態を決定した(図1Aの#1、#2、もしくは#3を参照のこと)。塩基使用法もまたモニターし、この細胞株に関して1に基準化した(図1Aもまた参照のこと)。
抗体を生産するCHO細胞株シード容器を使用して、実施例1に類似であるが、化学的に規定された培地の中で、レプリケート生産用バイオリアクターに接種した。4種の異なる代謝状態を測定した(オフラインpH、ラクテートおよび生細胞数をモニターする−図2Aの#1、#2、#3、および#4を参照のこと)。VCCは、生産用バイオリアクターに接種した場合、シード容器インキュベーションを継続している間に増大し続けた。
Claims (18)
- 細胞を培養するための方法であって、
(a)細胞を、第1の細胞培養において培養する工程
(b)ラクテート消費への代謝シフトが該第1の細胞培養において起こったことを決定する工程、および
(c)該細胞におけるラクテート消費への該代謝シフトが起こった後に、該細胞を、第2の細胞培養に移す工程、
を包含し、ここで該第2の細胞培養におけるラクテート濃度は、正味のラクテート消費を示す、方法。 - 前記細胞は、第1の細胞培養において細胞を培養する前に、目的のポリペプチドをコードするDNAでトランスフェクトされ、前記方法は、前記第2の細胞培養を、該目的のポリペプチドの発現を可能にする条件下で維持する工程、および該目的のポリペプチドを該第2の細胞培養から採取する工程を包含する、請求項1に記載の方法。
- ラクテート消費への前記代謝シフトは、前記第1の細胞培養におけるpH、ラクテートもしくは塩基の測定によって検出される、請求項1または2に記載の方法。
- ラクテート消費への前記代謝シフトは、塩基の添加なしの前記第1の細胞培養培地におけるpH増大後に検出される、請求項1〜3のいずれか1項に記載の方法。
- 前記代謝シフトは、細胞が前記第1の細胞培養において対数期から抜け出したときに起こるか、または静止期に達したときに起こる、請求項1〜4のいずれか1項に記載の方法。
- 前記代謝シフトは、ラクテートレベルが前記第1の細胞培養においてプラトーに達したときに起こる、請求項1〜5のいずれか1項に記載の方法。
- 前記代謝シフトは、前記第1の細胞培養における細胞増殖3日間でまたはそれより後に、該第1の細胞培養において起こる、請求項1〜6のいずれか1項に記載の方法。
- 前記移された細胞は、前記第2の細胞培養において、約0.5×106細胞/mL〜約3.0×106細胞/mLの間の接種細胞密度を有する、請求項1〜7のいずれか1項に記載の方法。
- 前記代謝シフトを決定する工程は、
a.前記第1の細胞培養においてpHを測定する工程
b.塩基を添加して、pHを所定の下限より高く維持する工程、
c.該pHが該所定の下限より高いことを、間隔をあけて引き続いて決定する工程、および
d.該塩基の添加を中止する工程、
を包含し、それによって、ラクテート消費への該代謝シフトは、該第1の細胞培養において起こったことを決定する、請求項1〜8のいずれか1項に記載の方法。 - 前記第1の細胞培養は、シードトレイン培養である、請求項1〜9のいずれか1項に記載の方法。
- 前記第2の細胞培養は、生産培養である、請求項1〜10のいずれか1項に記載の方法。
- 細胞を第2の細胞培養に移す工程は、細胞を生産用バイオリアクターに移す工程を包含する、請求項1〜11のいずれか1項に記載の方法。
- 前記目的のタンパク質は、抗体、抗原結合タンパク質、および融合タンパク質からなる群より選択される、請求項1〜12のいずれか1項に記載の方法。
- 請求項1〜13のいずれか1項に記載の方法によって生産される、代謝がシフトした宿主細胞。
- 細胞ゲノムの中に安定して組み込まれた1種またはそれより多くの核酸配列を含み、ここで該核酸配列は目的のタンパク質をコードする、請求項1〜13のいずれか1項に記載の方法によって生産される、代謝がシフトした宿主細胞。
- 目的のタンパク質をコードする1種またはそれより多くの発現ベクターを含む、請求項1〜13のいずれか1項に記載の方法によって生産される、代謝がシフトした宿主細胞。
- 前記目的のタンパク質は、抗体、抗原結合タンパク質、および融合タンパク質からなる群より選択される、請求項15または請求項16に記載の宿主細胞。
- 前記細胞は、CHO細胞、COS細胞、網膜細胞、Vero細胞、CV1細胞、HEK293細胞、293 EBNA細胞、MSR 293細胞、MDCK細胞、HaK細胞、BHK21細胞、HeLa細胞、HepG2細胞、WI38細胞、MRC 5細胞、Colo25細胞、HB 8065細胞、HL−60細胞、Jurkat細胞、Daudi細胞、A431細胞、CV−1細胞、U937細胞、3T3細胞、L細胞、C127細胞、SP2/0細胞、NS−0細胞、MMT細胞、PER.C6細胞、ネズミリンパ系細胞、およびネズミハイブリドーマ細胞からなる群より選択される、請求項14〜17のいずれか1項に記載の宿主細胞。
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