JP2021506295A - 細胞培養試薬として両親媒性グラフトコポリマーを使用する細胞培養系を安定化する方法 - Google Patents
細胞培養試薬として両親媒性グラフトコポリマーを使用する細胞培養系を安定化する方法 Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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Abstract
Description
Murhammer, D.W.等:Sparged animal cell bioreactors: Mechanism of cell damage and Pluronic F- 68 protection. Biotechnology Progress, 1990, 6, 391〜397;
Murhammer, D.W. & Goochee, C.F.: Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors. Biotechnology Progress, 1990, 6, 142〜148;
Tharmalingam, T.等: Pluronic enhances the robustness and reduces the cell attachment of mammalian cells. Molecular Biotechnology, 2008, 39, 167〜177;
Hilton, M.D.: Small-scale liquid fermentations. In: Demain, A.L. and Davies, J.E. (編者): Manual of Industrial Microbiology and Biotechnology 第2版. American Society of Microbiology, Washington, 1999.
細胞株:
CHO-S-IgG1
接種密度:
3×105細胞/mL、細胞は、実験の設定の前に少なくとも5回継代するために、培地中にポロキサマー188を含めずに継代した。
接種後の体積:50mL
振盪フラスコ:500mL、バッフル付き、ナルゲン滅菌済み通気振盪フラスコ(Fisher カタログ番号10-531)
撹拌:225rpm
細胞培養培地:HyClone(商標)HyCell(商標)CHO培地(General Electric Health Care、USA、カタログ番号SH30933.01)-剪断保護剤を補給していない
稼働期間:4日
インキュベーター:Infors HT Multitron
剪断保護剤:
Pluronic(登録商標)F68(参照標準物)
市販のPEG-VCap-VAcグラフトコポリマー:57重量%のN-ビニルカプロラクタム、30重量%の酢酸ビニル及び13重量%のポリエチレングリコールPEG6000、90000から140000g/モルの範囲のMW、Soluplus(登録商標)Fa.BASF SE
剪断保護剤の濃度:0.25g/L
分析
生存細胞数、全細胞数及び%生存率:試料300μLをトリパンブルー排除法を使用してRoche CEDEX自動細胞計数機で処理した。
Ln(生存細胞密度 3、4日/生存細胞密度 0日)/3
様々な試料のアルデヒド含量を以下の方法に従って試験した。
試薬溶液:2,4-ジニトロフェニルヒドラジン(50%水で安定化している)約4gを1Lエルレンマイヤーフラスコに再計量した。水800mL及び濃縮塩酸200mLを添加した。この混合物を透明になるまで撹拌する。
固定相:Symmetry Shield RP18-5μm、Waters(直径2.1mm、ステンレススチール)
キャリブレーション溶液:アルデヒドジニトロフェニルヒドラゾン20mgを0.01mgまで正確に計量し、アセトニトリルに溶解した。希釈は、ヒドラジンの濃度が以下の範囲内にあるように調整した。
ホルムアルデヒド誘導体 0.0021〜0.43mg/10mL注入用液
アセトアルデヒド誘導体 0.0024〜0.47mg/10mL注入用液
プロピオンアルデヒド誘導体 約0.0024〜0.47mg/10mL注入用液
t/分 A[%] B[%]
0 60 40
25 30 70
35 30 70
36 60 40
45 60 40
注入量:5μL
温度:45℃
検出:UV/VIS、ラムダ=370nm
Claims (13)
- 細胞培養産生において細胞を安定化する方法であって、細胞培養培地でタンパク質を発現することができる細胞株を培養すること、及び前記細胞培養培地に、N-ビニルカプロラクタム及び酢酸ビニル部分をポリエチレングリコール骨格にグラフトしたグラフトポリマーを補給することを含む方法。
- グラフトポリマー中のグラフトしたN-ビニルカプロラクタム及び酢酸ビニル部分の量が、全重量の10から90重量%の範囲である、請求項1に記載の方法。
- グラフトポリマーが、i)20から80重量%のN-ビニルカプロラクタム部分、ii)10から50重量%の酢酸ビニル部分、及びiii)10から50重量%のポリエチレングリコールの生成物であり、但し、i)、ii)及びiii)の合計が100重量%に等しい、請求項1又は2に記載の方法。
- グラフトポリマーが、i)30から70重量%のN-ビニルカプロラクタム部分、ii)15から35重量%の酢酸ビニル部分、及びiii)10から30重量%のポリエチレングリコールの生成物である、請求項1から3のいずれか一項に記載の方法。
- グラフトポリマーが、i)40から60重量%のN-ビニルカプロラクタム部分、ii)15から35重量%の酢酸ビニル部分、及びiii)10から30重量%のポリエチレングリコールの生成物である、請求項1から4のいずれか一項に記載の方法。
- PEG-VCap-VAcグラフトポリマーが、57重量%のN-ビニルカプロラクタム、30重量%の酢酸ビニル及び13重量%のポリエチレングリコールPEG6000の生成物である、請求項1から5のいずれか一項に記載の方法。
- PEG-VCap-VAcグラフトポリマーが、補給した培養培地の体積に基づき、0.01から10重量%の量で使用される、請求項1から6のいずれか一項に記載の方法。
- 請求項1から7のいずれか一項に記載の方法によるタンパク質を発現することができる細胞を培養するための細胞培養培地における安定化剤としての、N-ビニルカプロラクタム及び酢酸ビニル部分をポリエチレングリコール骨格にグラフトしたグラフトポリマーの使用。
- グラフトポリマーが、細胞培養培地の全量に基づき、0.01から10重量%の量で存在している、請求項8に記載のN-ビニルカプロラクタム及び酢酸ビニル部分をポリエチレングリコール骨格にグラフトしたグラフトポリマーの使用。
- グラフトポリマーが、細胞培養培地の全量に基づき、0.05から5重量%の量で存在している、請求項8又は9に記載の使用。
- グラフトポリマーが、培地の全量に基づき、0.5から3重量%の量で存在している、請求項8から10のいずれか一項に記載の使用。
- グラフトポリマーを第2の安定化剤と組み合わせて使用する、請求項8から11のいずれか一項に記載の使用。
- 第2の安定化剤が剪断保護剤である、請求項8から12のいずれか一項に記載の使用。
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JP2016526391A (ja) * | 2013-07-11 | 2016-09-05 | メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung | 細胞培養培地 |
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