JP2006503879A - 蛋白質非含有培地中での細胞株の取得方法およびこうして取得された細胞株 - Google Patents
蛋白質非含有培地中での細胞株の取得方法およびこうして取得された細胞株 Download PDFInfo
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Abstract
Description
蛋白質非含有培地への細胞株の2段階の適応
本方法は血清を補助添加されたもしくは血清非含有培地から蛋白質非含有培地への適応の直接的方法を実施することができない哺乳動物の細胞株を含んで成る。
Vadapt.=Δ蛋白質濃度/ΔTadapt.
として計算される、この段階における総適応時間および適応速度(Vadapt.)を決定するであろう。しかし、この段階の組み合わせは臨界濃度を含む各蛋白質濃度に対する適応に要する時間に対しては影響をもたないであろう。非臨界段階の最後および臨界蛋白質濃度を決定するためには、毎回蛋白質濃度を2倍に減少することにより、所望の蛋白質非含有培地中の細胞培養物の連続的希釈による、段階的適応を実施することが必要である(表1)。この減少は血清濃度の減少もしくは異なるレベルの何か蛋白質濃度の高い血清代用物を基礎培地に補助添加することにより実施することができる。
xi.段階(x)からの細胞を臨界前蛋白質濃度の75%の蛋白質濃度をもつ培地中に2〜6×105細胞/mLの範囲内の密度で少なくとも3ウェル中に播種する。48時間後に上澄み液の25%を新鮮な、蛋白質非含有培地により置き換え、これによりそれを前段階の濃度の75%の最終蛋白質濃度にさせる。
xiv.(xi)〜(xiii)の段階を繰り返し、各サイクル中、蛋白質濃度を前のサイクルの濃度の75%まで減少させ、次にこの方法を、細胞生存率のあらゆる喪失および集団倍加時間の減少を引き起こさない蛋白質濃度に到達するまで繰り返す。細胞をより低い蛋白質濃度をもつ培地に移し、それらは細胞生存率のあらゆる喪失および第1継代培養前の集団培養時間の減少を伴わずに増殖することができる時に、細胞は再度、非臨界段階に到達したと考えられ、蛋白質非含有培地(0mg/mLの蛋白質濃度)中にそれを直接播種する。
ヒト化抗−EGFヒト受容体hR3モノクロナール抗体の軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫NSO細胞株のトランスフェクションにより、組換え細胞株hR3を得た。この細胞株の蛋白質非含有培地への適応は培地の蛋白質含量の2段階の減少により前記の方法に従って実施した。
組換え細胞株T1hTをエピトープT抑制法によりヒト化された抗−ヒトCD6モノクロナール抗体の軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫NSO細胞株のトランスフェクションにより得た。
組換え細胞株T3Qを、ヒトリンパ球上のCD3受容体を認識するヒト化モノクロナール抗体T3Qの軽鎖および重鎖を発現するためのベクトル構造をもつ骨髄腫NSO細胞株のトランスフェクションにより得た。
Claims (20)
- 2段階:
I.細胞株生存率が80〜100%の間であり、細胞生存率が0%に低下する臨界蛋白質濃度まで連続的蛋白質濃度減少を伴う培地中で細胞が増殖される第1段階、
II.一旦臨界濃度が前以て決定された後、次に細胞増殖が可能で、かつそれが細胞培養物まで蛋白質濃度を緩徐に減少させる開始地点であるような蛋白質濃度が最初の細胞生存率および集団倍加時間に到達するように、臨界前濃度が固定される第2段階、
を含んで成る、血清および蛋白質非含有培地中での増殖に適応された哺乳動物の細胞株の取得方法。 - 第1段階が以下の段階:
i.標準細胞培地(最初の蛋白質濃度をもつ)を使用する組換え細胞株で6ウェルの培養プレート中の3ウェルに播種する。細胞密度は1〜5×105細胞/mLの範囲内になければならない。48時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより出発状態の50%の最終蛋白質濃度を与える。
ii.48時間毎に、上澄み液を出発状態の50%の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
iii.細胞をこの蛋白質濃度下で集密状態まで増殖させる。
iv.段階iii.からの細胞を出発状態の50%の蛋白質濃度をもつ培地中1〜5×105細胞/mLの範囲内の密度で少なくとも3ウェルに播種する。48時間後に、上澄み液の半分を新鮮な、蛋白質非含有培地により置き換え、それにより前の状態の50%の最終蛋白質濃度を与える。
v.48時間毎に、上澄み液を前の状態の50%の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
vi.細胞をこの蛋白質濃度下で集密状態まで増殖させる。
vii.(iv)〜(vi)の段階を繰り返し、各サイクル中に、前のサイクルの濃度の50%まで蛋白質濃度を減少させる。細胞死をもたらす蛋白質濃度に到達するまでこの方法を繰り返す。
により構成される請求項1記載の方法。 - 第2段階が以下の段階:
viii.2〜6×105細胞/mLの範囲内の密度で少なくとも3ウェル中に臨界前蛋白質濃度中に増殖する、80%以上の生存率を伴う細胞培養物からの細胞を播種する。細胞は臨界前蛋白質濃度中で増殖し、48時間後に上澄み液の25%を新鮮な、蛋白質非含有培地により置き換え、これによりそれを臨界前蛋白質濃度の75%の最終蛋白質濃度にさせる。
ix.48時間毎に上澄み液を臨界前蛋白質濃度の75%の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
x.細胞をこの蛋白質濃度下で集密状態まで増殖させる。
xi.段階(x)からの細胞を臨界前蛋白質濃度の75%の蛋白質濃度をもつ培地中に2〜6×105細胞/mLの範囲内の密度で少なくとも3ウェル中に播種する。48時間後に上澄み液の25%を新鮮な、蛋白質非含有培地により置き換え、これによりそれを前段階の濃度の75%の最終蛋白質濃度にさせる。
xii.48時間毎に上澄み液を段階(x)の濃度の75%の蛋白質濃度をもつ新鮮な培地により完全に置き換える。
xiii.細胞をこの蛋白質濃度下で集密状態まで増殖させる。
xiv.(xi)〜(xiii)の段階を繰り返し、各サイクル中、蛋白質濃度を前のサイクルの濃度の75%まで減少させ、次にこの方法を、細胞生存率のあらゆる喪失および集団倍加時間の減少を引き起こさない蛋白質濃度に到達するまで繰り返す。細胞をより低い蛋白質濃度をもつ培地に移し、それらは細胞生存率のあらゆる喪失および第1継代培養の前の集合培養時間の減少を伴わずに増殖することができる時に、細胞は再度、非臨界段階に到達したと考えられ、蛋白質非含有培地(0mg/mLの蛋白質濃度)中に直接播種する。
により構成される、請求項1記載の方法。 - 細胞が最初に播種される血清および蛋白質含有培地が5%〜10%の間の胎児ウシ血清を含んで成る請求項1〜3に記載の方法。
- 血清および蛋白質非含有培地中での増殖に適応された哺乳動物の細胞株が骨髄腫である請求項1〜4に記載の方法。
- 骨髄腫がNSO細胞株である請求項5記載の方法。
- 前記NSO細胞株が組換えポリペプチドもしくは組換え蛋白質をエンコードする配列を含む請求項6記載の方法。
- 組換えポリペプチドもしくは組換え蛋白質をエンコードする配列が組換え抗体もしくはその断片をコード化する請求項7記載の方法。
- 前記細胞株が血清および蛋白質非含有培地中での増殖に適応されている、請求項1〜8の方法により得られる哺乳動物の細胞株。
- 前記細胞株が骨髄腫である請求項9記載の哺乳動物の細胞株。
- 前記細胞株がNSO細胞株である請求項10記載の哺乳動物の細胞株。
- NSO細胞株が組換えポリペプチドもしくは組換え蛋白質をエンコードする配列を含む請求項11記載の哺乳動物の細胞株。
- 組換えポリペプチドもしくは組換え蛋白質をエンコードする配列が組換え抗体もしくはその断片をコード化する請求項12記載の哺乳動物細胞株。
- 配列がヒト化組換え抗体抗−EGF−R hR3もしくはその断片をコード化する請求項13記載の哺乳動物細胞株。
- 配列がヒト化組換え抗−CD6抗体T1hTもしくはその断片をコード化する請求項13記載の哺乳動物細胞株。
- 配列がキメラ組換え抗−CD3抗体T3Qもしくはその断片をコード化する請求項13記載の哺乳動物細胞株。
- 血清および蛋白質非含有培地中での増殖に適応された哺乳動物細胞株を取得するための請求項1〜8記載の方法の使用。
- 請求項1〜8の方法により得られる細胞株により分泌されるEGF−R hR3もしくはその断片に対するヒト化モノクロナール抗体。
- 請求項1〜8の方法により得られる細胞株により分泌されるCD6抗原T1hTもしくはその断片に対するヒト化モノクロナール抗体。
- 請求項1〜8の方法により得られる細胞株により分泌されるCD3抗原T3Qもしくはその断片に対するキメラモノクロナール抗体。
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