JP2017510283A - 配合培地粉末製剤および細胞培養用液体培地の調製方法 - Google Patents
配合培地粉末製剤および細胞培養用液体培地の調製方法 Download PDFInfo
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- JP2017510283A JP2017510283A JP2016561351A JP2016561351A JP2017510283A JP 2017510283 A JP2017510283 A JP 2017510283A JP 2016561351 A JP2016561351 A JP 2016561351A JP 2016561351 A JP2016561351 A JP 2016561351A JP 2017510283 A JP2017510283 A JP 2017510283A
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- 125000000466 oxiranyl group Chemical group 0.000 description 1
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- 230000002062 proliferating effect Effects 0.000 description 1
- 235000013930 proline Nutrition 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 108010025139 recombinant factor VIII SQ Proteins 0.000 description 1
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- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
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- 125000002827 triflate group Chemical group FC(S(=O)(=O)O*)(F)F 0.000 description 1
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- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
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- 230000002792 vascular Effects 0.000 description 1
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Abstract
Description
本明細書と同時に提出され、2014年4月10日に作成された「sequence_listing_ST25.txt」と名付けられた1つの(66,989バイトのASCII(Text))ファイルとして特定されるコンピューター読み取り可能な配列表は、参照によりその全体が本明細書中に組み込まれる。
本発明は、細胞生物学および細胞培養の分野に関する。
i) 0.0003〜0.003g/L ビオチン;
ii) 0.035〜0.33g/L 塩化カルシウム;
iii) 0.003〜0.03g/L 塩化コリン;
iv) 0.0002〜0.002g/L シアノコバラミン(B12);
v) 1〜10g/L D+マンノース;
vi) 0.001〜0.01g/L D−パントテン酸カルシウム;
vii) 0.3〜3.0g/L ブドウ糖(無水);
viii) 0.00003〜0.0003g/L DL−アルファ−リポ酸;
ix) 0.00002〜0.00015g/L 硝酸第二鉄9H2O;
x) 0.0001〜0.0015g/L 硫酸第一鉄7H2O;
xi) 0.001〜0.01g/L 葉酸;
xii) 0.007〜0.20g/L グリシン;
xiii) 0.001〜0.01g/L ヒポキサンチン2Na;
xiv) 0.005〜0.05g/L I−イノシトール;
xv) 0.003〜0.03g/L L−アラニン;
xvi) 0.08〜1.4g/L L−アルギニン;
xvii) 0.006〜0.16g/L L−アスパラギン;
xviii) 0.005〜0.10g/L L−アスパラギン酸;
xix) 0.005〜0.05g/L L−システインHCl H2O;
xx) 0.02〜0.2g/L L−シスチン2HCl;
xxi) 0.005〜0.15g/L L−グルタミン酸(無水);
xxii) 0.02〜1.5g/L L−グルタミン;
xxiii) 0.0003〜0.003g/L L−グルタチオン;
xxiv) 0.02〜0.2g/L L−ヒスチジンHCl;
xxv) 0.03〜0.9g/L L−イソロイシン;
xxvi) 0.03〜0.9g/L L−ロイシン;
xxvii) 0.05〜1.5g/L L−リジン;
xxviii) 0.01〜0.3g/L L−メチオニン;
xxix) 0.02〜0.6g/L L−フェニルアリン(phenylaline);
xxx) 0.008〜0.25g/L L−プロリン;
xxxi) 0.009〜0.25g/L L−セリン;
xxxii) 0.03〜0.9g/L L−スレオニン;
xxxiii) 0.006〜0.16g/L L−トリプトファン;
xxxiv) 0.03〜0.9g/L L−チロシン2Na 2H2O;
xxxv) 0.03〜0.9g/L L−バリン;
xxxvi) 0.01〜0.18g/L 塩化マグネシウム;
xxxvii) 0.02〜0.12g/L 無水硫酸マグネシウム;
xxxviii) 0.001〜0.01g/L ナイアシンアミド;
xxxix) 0.0005〜0.005g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 0.1〜1.0g/L 塩化カリウム;
xli) 0.00002〜0.0002g/L プトレッシン2HCl;
xlii) 0.001〜0.01g/L ピリドキサールHCl;
xliii) 0.00001〜0.0001g/L ピリドキシンHCl、
xliv) 0.0001〜0.001g/L リボフラビン、
xlv) 2.0〜15g/L 塩化ナトリウム、
xlvi) 0.02〜0.2g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 0.02〜0.2g/L 無水二塩基性リン酸ナトリウム、
xlviii) 0.015〜0.15g/L ピルビン酸ナトリウム、
xlix) 0.001〜0.01g/L チアミンHCl、
l) 0.0001〜0.001g/L チミジン、
li) 0.00015〜0.0015g/L 硫酸亜鉛7H2O、
lii) 0.0000006〜0.000006g/L 硫酸銅5H2O、
liii) 0.0000005〜0.000008g/L 二酸化セレン、
liv) 0.00001〜0.0001g/L リノール酸、
lv) 0.0001〜0.001g/L ベータ−メルカプトエタノール;および
lvi) 0.0003〜0.005g/L エタノールアミンFB。
i) 約0.001g/L ビオチン;
ii) 約0.11665g/L 塩化カルシウム;
iii) 約0.00998g/L 塩化コリン;
iv) 約0.00068g/L シアノコバラミン(B12);
v) 約3g/L D+マンノース;
vi) 約0.00312g/L D−パントテン酸カルシウム;
vii) 約1g/L ブドウ糖(無水);
viii) 約0.000103g/L DL−アルファ−リポ酸;
ix) 約0.00005g/L 硝酸第二鉄9H2O;
x) 約0.000417g/L 硫酸第一鉄7H2O;
xi) 約0.00366g/L 葉酸;
xii) 約0.02626g/L グリシン;
xiii) 約0.0027g/L ヒポキサンチン2Na;
xiv) 約0.01451g/L I−イノシトール;
xv) 約0.01336g/L L−アラニン;
xvi) 約0.27435g/L L−アルギニン;
xvii) 約0.0225g/L L−アスパラギン;
xviii) 約0.01995g/L L−アスパラギン酸;
xix) 約0.01756g/L L−システインHCl H2O;
xx) 約0.06256g/L L−シスチン2HCl;
xxi) 約0.02206g/L L−グルタミン酸(無水);
xxii) 約0.73g/L L−グルタミン;
xxiii) 約0.001g/L L−グルタチオン;
xxiv) 約0.07348g/L L−ヒスチジンHCl;
xxv) 約0.1057g/L L−イソロイシン;
xxvi) 約0.11096g/L L−ロイシン;
xxvii) 約0.16385g/L L−リジン;
xxviii) 約0.03224g/L L−メチオニン;
xxix) 約0.06748g/L L−フェニルアリン(phenylaline);
xxx) 約0.02875g/L L−プロリン;
xxxi) 約0.03676g/L L−セリン;
xxxii) 約0.10156g/L L−スレオニン;
xxxiii) 約0.01902g/L L−トリプトファン;
xxxiv) 約0.10771g/L L−チロシン2Na 2H2O;
xxxv) 約0.09866g/L L−バリン;
xxxvi) 約0.028g/L 塩化マグネシウム;
xxxvii) 約0.04884g/L 無水硫酸マグネシウム;
xxxviii) 約0.00302g/L ナイアシンアミド;
xxxix) 約0.0014g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 約0.31182g/L 塩化カリウム;
xli) 約0.000081g/L プトレッシン2HCl;
xlii) 約0.003g/L ピリドキサールHCl;
xliii) 約0.000031g/L ピリドキシンHCl、
xliv) 約0.000319g/L リボフラビン、
xlv) 約6.1234g/L 塩化ナトリウム、
xlvi) 約0.0625g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 約0.07099g/L 無水二塩基性リン酸ナトリウム、
xlviii) 約0.055 ピルビン酸ナトリウム、
xlix) 約0.00317g/L チアミンHCl、
l) 約0.000364g/L チミジン、
li) 約0.000432g/L 硫酸亜鉛7H2O、
lii) 約0.00000125g/L 硫酸銅5H2O、
liii) 約0.00000222g/L 二酸化セレン、
liv) 約0.000042g/L リノール酸、
lv) 約0.00039065g/L ベータ−メルカプトエタノール;および
lvi) 約0.0012g/L エタノールアミンFB。
i) 0.0005〜0.01mg/Lのモリブデン酸アンモニウム4H2O;
ii) 0.0001〜0.01mg/Lの硫酸カリウムクロム12H2O;
iii) 0.001〜0.125mg/Lの硫酸銅5H2O;
iv) 0.001〜0.1mg/Lの塩化リチウム(無水);
v) 0.00004〜0.004mg/Lの硫酸マンガンH2O;および
vi) 0.04〜4.2mg/Lのメタけい酸ナトリウム9H2O。
i) 約0.0037mg/Lのモリブデン酸アンモニウム4H2O;
ii) 約0.001mg/Lの硫酸カリウムクロム12H2O;
iii) 約0.0125mg/Lの硫酸銅5H2O;
iv) 約0.01mg/Lの塩化リチウム(無水);
v) 約0.000452mg/Lの硫酸マンガンH2O;および
vi) 約0.4263mg/Lのメタけい酸ナトリウム9H2O。
i) 0.007〜0.07g/L アスパラギンH2O;
ii) 0.25〜2.5g/L グルタミン;
iii) 0.5〜5.0g/L ヒスチジン、遊離塩基;および
iv) 0.01〜0.1g/L セリン。
i) 約 0.0225g/L アスパラギンH2O;
ii) 約0.73g/L グルタミン;
iii) 約1.552g/L ヒスチジン;および
iv) 約0.03676g/L セリン。
i) 0.3〜3g/L MOPS遊離酸;および
ii) 1.0〜10g/L MOPS Na。
i) 0.0003〜0.003g/L ビオチン;
ii) 0.035〜0.33g/L 塩化カルシウム;
iii) 0.003〜0.03g/L 塩化コリン;
iv) 0.0002〜0.002g/L シアノコバラミン(B12);
v) 1〜10g/L D+マンノース;
vi) 0.001〜0.01g/L D−パントテン酸カルシウム;
vii) 0.3〜3.0g/L ブドウ糖(無水);
viii) 0.00003〜0.0003g/L DL−アルファ−リポ酸;
ix) 0.00002〜0.00015g/L 硝酸第二鉄9H2O;
x) 0.0001〜0.0015g/L 硫酸第一鉄7H2O;
xi) 0.001〜0.01g/L 葉酸;
xii) 0.007〜0.20g/L グリシン;
xiii) 0.001〜0.01g/L ヒポキサンチン2Na;
xiv) 0.005〜0.05g/L I−イノシトール;
xv) 0.003〜0.03g/L L−アラニン;
xvi) 0.08〜1.4g/L L−アルギニン;
xvii) 0.006〜0.16g/L L−アスパラギン;
xviii) 0.005〜0.10g/L L−アスパラギン酸;
xix) 0.005〜0.05g/L L−システインHCl H2O;
xx) 0.02〜0.2g/L L−シスチン2HCl;
xxi) 0.005〜0.15g/L L−グルタミン酸(無水);
xxii) 0.02〜1.5g/L L−グルタミン;
xxiii) 0.0003〜0.003g/L L−グルタチオン;
xxiv) 0.02〜0.2g/L L−ヒスチジンHCl;
xxv) 0.03〜0.9g/L L−イソロイシン;
xxvi) 0.03〜0.9g/L L−ロイシン;
xxvii) 0.05〜1.5g/L L−リジン;
xxviii) 0.01〜0.3g/L L−メチオニン;
xxix) 0.02〜0.6g/L L−フェニルアリン(phenylaline);
xxx) 0.008〜0.25g/L L−プロリン;
xxxi) 0.009〜0.25g/L L−セリン;
xxxii) 0.03〜0.9g/L L−スレオニン;
xxxiii) 0.006〜0.16g/L L−トリプトファン;
xxxiv) 0.03〜0.9g/L L−チロシン2Na 2H2O;
xxxv) 0.03〜0.9g/L L−バリン;
xxxvi) 0.01〜0.18g/L 塩化マグネシウム;
xxxvii) 0.02〜0.12g/L 無水硫酸マグネシウム;
xxxviii) 0.001〜0.01g/L ナイアシンアミド;
xxxix) 0.0005〜0.005g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 0.1〜1.0g/L 塩化カリウム;
xli) 0.00002〜0.0002g/L プトレッシン2HCl;
xlii) 0.001〜0.01g/L ピリドキサールHCl;
xliii) 0.00001〜0.0001g/L ピリドキシンHCl、
xliv) 0.0001〜0.001g/L リボフラビン、
xlv) 2.0〜15g/L 塩化ナトリウム、
xlvi) 0.02〜0.2g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 0.02〜0.2g/L 無水二塩基性リン酸ナトリウム、
xlviii) 0.015〜0.15g/L ピルビン酸ナトリウム、
xlix) 0.001〜0.01g/L チアミンHCl、
l) 0.0001〜0.001g/L チミジン、
li) 0.00015〜0.0015g/L 硫酸亜鉛7H2O、
lii) 0.0000006〜0.000006g/L 硫酸銅5H2O、
liii) 0.0000005〜0.000008g/L 二酸化セレン、
liv) 0.00001〜0.0001g/L リノール酸、
lv) 0.0001〜0.001g/L ベータ−メルカプトエタノール;および
lvi) 0.0003〜0.005g/L エタノールアミンFB。
i) 約0.001g/L ビオチン;
ii) 約0.11665g/L 塩化カルシウム;
iii) 約0.00998g/L 塩化コリン;
iv) 約0.00068g/L シアノコバラミン(B12);
v) 約3g/L D+マンノース;
vi) 約0.00312g/L D−パントテン酸カルシウム;
vii) 約1g/L ブドウ糖(無水);
viii) 約0.000103g/L DL−アルファ−リポ酸;
ix) 約0.00005g/L 硝酸第二鉄9H2O;
x) 約0.000417g/L 硫酸第一鉄7H2O;
xi) 約0.00366g/L 葉酸;
xii) 約0.02626g/L グリシン;
xiii) 約0.0027g/L ヒポキサンチン2Na;
xiv) 約0.01451g/L I−イノシトール;
xv) 約0.01336g/L L−アラニン;
xvi) 約0.27435g/L L−アルギニン;
xvii) 約0.0225g/L L−アスパラギン;
xviii) 約0.01995g/L L−アスパラギン酸;
xix) 約0.01756g/L L−システインHCl H2O;
xx) 約0.06256g/L L−シスチン2HCl;
xxi) 約0.02206g/L L−グルタミン酸(無水);
xxii) 約0.73g/L L−グルタミン;
xxiii) 約0.001g/L L−グルタチオン;
xxiv) 約0.07348g/L L−ヒスチジンHCl;
xxv) 約0.1057g/L L−イソロイシン;
xxvi) 約0.11096g/L L−ロイシン;
xxvii) 約0.16385g/L L−リジン;
xxviii) 約0.03224g/L L−メチオニン;
xxix) 約0.06748g/L L−フェニルアリン(phenylaline);
xxx) 約0.02875g/L L−プロリン;
xxxi) 約0.03676g/L L−セリン;
xxxii) 約0.10156g/L L−スレオニン;
xxxiii) 約0.01902g/L L−トリプトファン;
xxxiv) 約0.10771g/L L−チロシン2Na 2H2O;
xxxv) 約0.09866g/L L−バリン;
xxxvi) 約0.028g/L 塩化マグネシウム;
xxxvii) 約0.04884g/L 無水硫酸マグネシウム;
xxxviii) 約0.00302g/L ナイアシンアミド;
xxxix) 約0.0014g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 約0.31182g/L 塩化カリウム;
xli) 約0.000081g/L プトレッシン2HCl;
xlii) 約0.003g/L ピリドキサールHCl;
xliii) 約0.000031g/L ピリドキシンHCl、
xliv) 約0.000319g/L リボフラビン、
xlv) 約6.1234g/L 塩化ナトリウム、
xlvi) 約0.0625g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 約0.07099g/L 無水二塩基性リン酸ナトリウム、
xlviii) 約0.055 ピルビン酸ナトリウム、
xlix) 約0.00317g/L チアミンHCl、
l) 約0.000364g/L チミジン、
li) 約0.000432g/L 硫酸亜鉛7H2O、
lii) 約0.00000125g/L 硫酸銅5H2O、
liii) 約0.00000222g/L 二酸化セレン、
liv) 約0.000042g/L リノール酸、
lv) 約0.00039065g/L ベータ−メルカプトエタノール;および
lvi) 約0.0012g/L エタノールアミンFB。
i) 0.0005〜0.01mg/Lのモリブデン酸アンモニウム4H2O;
ii) 0.0001〜0.01mg/Lの硫酸カリウムクロム12H2O;
iii) 0.001〜0.125mg/Lの硫酸銅5H2O;
iv) 0.001〜0.1mg/Lの塩化リチウム(無水);
v) 0.00004〜0.004mg/Lの硫酸マンガンH2O;および
vi) 0.04〜4.2mg/Lのメタけい酸ナトリウム9H2O。
i) 約0.0037mg/Lのモリブデン酸アンモニウム4H2O;
ii) 約0.001mg/Lの硫酸カリウムクロム12H2O;
iii) 約0.0125mg/Lの硫酸銅5H2O;
iv) 約0.01mg/Lの塩化リチウム(無水);
v) 約0.000452mg/Lの硫酸マンガンH2O;および
vi) 約0.4263mg/Lのメタけい酸ナトリウム9H2O。
i) 0.007〜0.07g/L アスパラギンH2O;
ii) 0.25〜2.5g/L グルタミン;
iii) 0.5〜5.0g/L ヒスチジン、遊離塩基;および
iv) 0.01〜0.1g/L セリン。
i) 約 0.0225g/L アスパラギンH2O;
ii) 約0.73g/L グルタミン;
iii) 約1.552g/L ヒスチジン;および
iv) 約0.03676g/L セリン。
i) 0.3〜3g/L MOPS遊離酸;および
ii) 1.0〜10g/L MOPS Na。
i) 0.0003〜0.003g/L ビオチン;
ii) 0.035〜0.33g/L 塩化カルシウム;
iii) 0.003〜0.03g/L 塩化コリン;
iv) 0.0002〜0.002g/L シアノコバラミン(B12);
v) 1〜10g/L D+マンノース;
vi) 0.001〜0.01g/L D−パントテン酸カルシウム;
vii) 0.3〜3.0g/L ブドウ糖(無水);
viii) 0.00003〜0.0003g/L DL−アルファ−リポ酸;
ix) 0.00002〜0.00015g/L 硝酸第二鉄9H2O;
x) 0.0001〜0.0015g/L 硫酸第一鉄7H2O;
xi) 0.001〜0.01g/L 葉酸;
xii) 0.007〜0.20g/L グリシン;
xiii) 0.001〜0.01g/L ヒポキサンチン2Na;
xiv) 0.005〜0.05g/L I−イノシトール;
xv) 0.003〜0.03g/L L−アラニン;
xvi) 0.08〜1.4g/L L−アルギニン;
xvii) 0.006〜0.16g/L L−アスパラギン;
xviii) 0.005〜0.10g/L L−アスパラギン酸;
xix) 0.005〜0.05g/L L−システインHCl H2O;
xx) 0.02〜0.2g/L L−シスチン2HCl;
xxi) 0.005〜0.15g/L L−グルタミン酸(無水);
xxii) 0.02〜0.6g/L L−グルタミン;
xxiii) 0.0003〜0.003g/L L−グルタチオン;
xxiv) 0.02〜0.2g/L L−ヒスチジンHCl;
xxv) 0.03〜0.9g/L L−イソロイシン;
xxvi) 0.03〜0.9g/L L−ロイシン;
xxvii) 0.05〜1.5g/L L−リジン;
xxviii) 0.01〜0.3g/L L−メチオニン;
xxix) 0.02〜0.6g/L L−フェニルアリン(phenylaline);
xxx) 0.008〜0.25g/L L−プロリン;
xxxi) 0.009〜0.25g/L L−セリン;
xxxii) 0.03〜0.9g/L L−スレオニン;
xxxiii) 0.006〜0.16g/L L−トリプトファン;
xxxiv) 0.03〜0.9g/L L−チロシン2Na 2H2O;
xxxv) 0.03〜0.9g/L L−バリン;
xxxvi) 0.01〜0.18g/L 塩化マグネシウム
xxxvii) 0.02〜0.12g/L 無水硫酸マグネシウム;
xxxviii) 0.001〜0.01g/L ナイアシンアミド;
xxxix) 0.0005〜0.005g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 0.1〜1.0g/L 塩化カリウム;
xli) 0.00002〜0.0002g/L プトレッシン2HCl;
xlii) 0.001〜0.01g/L ピリドキサールHCl;
xliii) 0.00001〜0.0001g/L ピリドキシンHCl、
xliv) 0.0001〜0.001g/L リボフラビン、
xlv) 2.0〜15g/L 塩化ナトリウム、
xlvi) 0.02〜0.2g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 0.02〜0.2g/L 無水二塩基性リン酸ナトリウム、
xlviii) 0.015〜0.15g/L ピルビン酸ナトリウム、
xlix) 0.001〜0.01g/L チアミンHCl、
l) 0.0001〜0.001g/L チミジン、
li) 0.00015〜0.0015g/L 硫酸亜鉛7H2O、
lii) 0.0000006〜0.000006g/L 硫酸銅5H2O、
liii) 0.0000005〜0.000008g/L 二酸化セレン、
liv) 0.00001〜0.0001g/L リノール酸、
lv) 0.0001〜0.001g/L ベータ−メルカプトエタノール;
lvi) 0.0003〜0.005g/L エタノールアミンFB;
lvii) 0.5〜5g/L MgCl2;
lviii) 0.5〜15 mg/L インスリン;
lix) 0.0005〜0.01mg/Lのモリブデン酸アンモニウム4H2O;
lx) 0.0001〜0.01mg/Lの硫酸カリウムクロム12H2O;
lxi) 0.001〜0.125mg/Lの硫酸銅5H2O;
lxii) 0.001〜0.1mg/Lの塩化リチウム(無水);
lxiii) 0.00004〜0.004mg/Lの硫酸マンガンH2O;
lxiv) 0.04〜4.2mg/Lのメタけい酸ナトリウム9H2O;
lxv) 0.007〜0.07g/L アスパラギンH2O;
lxvi) 0.25〜2.5g/L グルタミン;
lxvii) 0.5〜5.0g/L ヒスチジン、遊離塩基;
lxviii) 0.01〜0.1g/L セリン;
lxix) 0.3〜3g/L MOPS遊離酸;
lxx) 1.0〜10g/L MOPS Na;および
lxxi) 0.1〜10g/L Pluronic F68。
第VIII因子を生産するための現行の培地調製方法は、それらの細胞培養培地を構築するために数種のアドバック(add−back)溶液および乾燥粉末成分/混合物の添加を伴う。増殖および性能特性が許容されるより複雑な(すなわちアドバックのより少ない)粉末製剤を特定するため、アドバック(DPMへと粉砕されたもの)のうちのいくつかまたは全てを包含する数種の関係する粉末バリエーションを評価した。ゴールは、培地製剤プロセスを合理化し、(表1中に見られるような)サプライチェーンの複雑性を減らすことである。
材料
天秤はMSA124Sであり;浸透圧計はAdvanced Instruments、Model 3300であり;pHの示度はThermoOrion Model 720Aで取得し;濁度の示度はHach 2100N Turbidimeter上で測定し;インスリンのUPLC分析はAcquity H−Class、Acquity BEH300 C4カラムによって行い;インスリンのELISA定量はMillipore ELISAキットを用いて行い;アミノ酸の定量はWaters HPLC、Zorbax Eclipse AAAカラムによって行い;ICP分析はAgilent 720−ES ICP−OES上で行った。
DPMのバージョンならびに全ての関連したアドバック溶液および粉末はSAFC’s Immediate Advantage部門により製造された。アドバック粉末および溶液は表1中にまとめられている。バージョン、粉末の明細、およびSAFC製品番号は表2中にまとめられている。
以下の特性についてこれらのバージョンを試験した:
1)水和された粉末のpHおよび浸透圧
2)アミノ酸濃度の分析
3)Fe濃度の分析
4)インスリン濃度の分析
表3Aおよび表3Bは、試験された非PEG化FVIIIバージョンについてのデータをまとめている。
非PEG化FVIIIの最終バージョンは、Version 3.3であった。これは、製剤中の大部分(97.1%)の鉄および全EDTAを抜いた完全培地である。この鉄/EDTAを含まない乾燥粉末製剤(鉄/EDTAの液体アドバックを利用する)は、Version 2.1(DPMへと粉砕された鉄およびEDTAを含有する)は水和すると鉄を適切にキレート(すなわち可溶化)しなかったという発見に起因して製剤として選択されたものであり、したがって鉄はろ過により溶液から取り除かれていた(表4)。
この例は、本発明の配合培地製剤にさらに配合することにより強化することができる例示的な基礎培地を示す。この例において、指示薬、例えばフェノールレッドなどが包含されているが、指示薬は省くことができる。
Claims (44)
- 基礎培地粉末および細胞培養培地補充物を含む配合細胞培養培地粉末製剤であって、細胞培養培地補充物が
i) 1または複数の塩;
ii) 1または複数の増殖因子;
iii) 1または複数の無機イオン;
iv) アスパラギン、グルタミン、ヒスチジンおよびセリンのうちの1または複数を含むアミノ酸補充物;
v) 1または複数のバッファー;ならびに
vi) 1または複数の消泡剤
を含む、前記配合細胞培養培地粉末製剤。 - 基礎培地粉末が、ダルベッコ改変イーグル培地(Dulbecco’s Modified Eagle’s Medium)、ハム培地F12(Ham’s Medium F12)、イーグル最小必須培地(Eagle’s Minimal Essential Medium)、RPMI 1640 培地(RPMI 1640 Medium)およびダルベッコ改変イーグル培地(Dulbecco’s Modified Eagle’s Medium)/ハムF12培地(Ham’s F12 Medium)(DMEM/F−12;1:1の比)を含む、請求項1の配合細胞培養培地粉末製剤。
- 基礎培地粉末が、以下の成分のうちの1もしくは複数またはそれらの組み合わせを含む、請求項1の配合細胞培養培地粉末製剤:
ビオチン、塩化カルシウム、塩化コリン、シアノコバラミン(B12)、D+マンノース、D−パントテン酸カルシウム、ブドウ糖(無水)、DL−アルファ−リポ酸、硝酸第二鉄9H2O、硫酸第一鉄7H2O、葉酸、グリシン、ヒポキサンチン、I−イノシトール、L−アラニン、L−アルギニン、L−アスパラギン、L−アスパラギン酸、L−システインHCl H2O、L−シスチン2HCl、L−グルタミン酸(無水)、L−グルタミン、L−グルタチオン、L−ヒスチジンFB、L−ヒスチジンHCl、L−イソロイシン、L−ロイシン、L−リジン、L−メチオニン、L−フェニルアリン(phenylaline)、L−プロリン、L−セリン、L−スレオニン、L−トリプトファン、L−チロシン、L−バリン、塩化マグネシウム、無水硫酸マグネシウム、ナイアシンアミド、O−ホスホリル−エタノールアミン、塩化カリウム、プトレッシン2HCl、ピリドキサールHCl、ピリドキシンHCl、リボフラビン、塩化ナトリウム、一塩基性リン酸ナトリウムH2O、無水二塩基性リン酸ナトリウム、ピルビン酸ナトリウム、チアミンHCl、チミジン、硫酸亜鉛7H2O、硫酸銅、二酸化セレン、リノール酸、ベータ−メルカプトエタノールおよびエタノールアミン遊離塩基FB。 - 水和させて細胞培養培地を形成させた際の基礎培地粉末の濃度が8〜30g/Lである、請求項3の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際の基礎培地粉末の濃度が約13g/Lである、請求項4の配合細胞培養培地粉末製剤。
- pH指示薬をさらに含む、請求項1〜5の配合細胞培養培地粉末製剤。
- pH指示薬がフェノールレッドNa(Phenol Red Na)であり、水和させて細胞培養培地を形成させた際にフェノールレッドNaが約0.001から約0.02g/Lの濃度で存在する、請求項6の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際にフェノールレッドNaが約0.0069g/Lの濃度で存在する、請求項7の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際の基礎培地粉末成分の終濃度が以下である、請求項3の配合細胞培養培地粉末製剤:
i) 0.0003〜0.003g/L ビオチン;
ii) 0.035〜0.33g/L 塩化カルシウム;
iii) 0.003〜0.03g/L 塩化コリン;
iv) 0.0002〜0.002g/L シアノコバラミン(B12);
v) 1〜10g/L D+マンノース;
vi) 0.001〜0.01g/L D−パントテン酸カルシウム;
vii) 0.3〜3.0g/L ブドウ糖(無水);
viii) 0.00003〜0.0003g/L DL−アルファ−リポ酸;
ix) 0.00001〜0.00015g/L 硝酸第二鉄;
x) 0.00005〜0.0015g/L 硫酸第一鉄;
xi) 0.001〜0.01g/L 葉酸;
xii) 0.007〜0.20g/L グリシン;
xiii) 0.001〜0.01g/L ヒポキサンチン2Na;
xiv) 0.005〜0.05g/L I−イノシトール;
xv) 0.003〜0.03g/L L−アラニン;
xvi) 0.08〜1.4g/L L−アルギニン;
xvii) 0.006〜0.16g/L L−アスパラギン;
xviii) 0.005〜0.10g/L L−アスパラギン酸;
xix) 0.005〜0.05g/L L−システインHCl;
xx) 0.02〜0.2g/L L−シスチン2HCl;
xxi) 0.005〜0.15g/L L−グルタミン酸(無水);
xxii) 0.02〜1.5g/L L−グルタミン;
xxiii) 0.0003〜0.003g/L L−グルタチオン;
xxiv) 0.02〜0.2g/L L−ヒスチジンHCl;
xxv) 0.03〜0.9g/L L−イソロイシン;
xxvi) 0.03〜0.9g/L L−ロイシン;
xxvii) 0.05〜1.5g/L L−リジン;
xxviii) 0.01〜0.3g/L L−メチオニン;
xxix) 0.02〜0.6g/L L−フェニルアリン(phenylaline);
xxx) 0.008〜0.25g/L L−プロリン;
xxxi) 0.009〜0.25g/L L−セリン;
xxxii) 0.03〜0.9g/L L−スレオニン;
xxxiii) 0.006〜0.16g/L L−トリプトファン;
xxxiv) 0.02〜0.9g/L L−チロシン2Na;
xxxv) 0.03〜0.9g/L L−バリン;
xxxvi) 0.01〜0.18g/L 塩化マグネシウム;
xxxvii) 0.02〜0.12g/L 無水硫酸マグネシウム;
xxxviii) 0.001〜0.01g/L ナイアシンアミド;
xxxix) 0.0005〜0.005g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 0.1〜1.0g/L 塩化カリウム;
xli) 0.00002〜0.0002g/L プトレッシン2HCl;
xlii) 0.001〜0.01g/L ピリドキサールHCl;
xliii) 0.00001〜0.0001g/L ピリドキシンHCl、
xliv) 0.0001〜0.001g/L リボフラビン、
xlv) 2.0〜15g/L 塩化ナトリウム、
xlvi) 0.01〜0.2g/L 一塩基性リン酸ナトリウム、
xlvii) 0.02〜0.2g/L 無水二塩基性リン酸ナトリウム、
xlviii) 0.015〜0.15g/L ピルビン酸ナトリウム、
xlix) 0.001〜0.01g/L チアミンHCl、
l) 0.0001〜0.001g/L チミジン、
li) 0.00006〜0.0015g/L 硫酸亜鉛、
lii) 0.0000003〜0.000006g/L 硫酸銅、
liii) 0.0000005〜0.000008g/L 二酸化セレン、
liv) 0.00001〜0.0001g/L リノール酸、
lv) 0.0001〜0.001g/L ベータ−メルカプトエタノール;および
lvi) 0.0003〜0.005g/L エタノールアミン。 - 水和させて細胞培養培地を形成させた際の基礎培地粉末成分の終濃度が以下である、請求項9の配合細胞培養培地粉末製剤:
i) 約0.001g/L ビオチン;
ii) 約0.11665g/L 塩化カルシウム;
iii) 約0.00998g/L 塩化コリン;
iv) 約0.00068g/L シアノコバラミン(B12);
v) 約3g/L D+マンノース;
vi) 約0.00312g/L D−パントテン酸カルシウム;
vii) 約1g/L ブドウ糖(無水);
viii) 約0.000103g/L DL−アルファ−リポ酸;
ix) 約0.00005g/L 硝酸第二鉄9H2O;
x) 約0.000417g/L 硫酸第一鉄7H2O;
xi) 約0.00366g/L 葉酸;
xii) 約0.02626g/L グリシン;
xiii) 約0.0027g/L ヒポキサンチン2Na;
xiv) 約0.01451g/L I−イノシトール;
xv) 約0.01336g/L L−アラニン;
xvi) 約0.27435g/L L−アルギニン;
xvii) 約0.0225g/L L−アスパラギン;
xviii) 約0.01995g/L L−アスパラギン酸;
xix) 約0.01756g/L L−システインHCl H2O;
xx) 約0.06256g/L L−シスチン2HCl;
xxi) 約0.02206g/L L−グルタミン酸(無水);
xxii) 約0.73g/L L−グルタミン;
xxiii) 約0.001g/L L−グルタチオン;
xxiv) 約0.07348g/L L−ヒスチジンHCl;
xxv) 約0.1057g/L L−イソロイシン;
xxvi) 約0.11096g/L L−ロイシン;
xxvii) 約0.16385g/L L−リジン;
xxviii) 約0.03224g/L L−メチオニン;
xxix) 約0.06748g/L L−フェニルアリン(phenylaline);
xxx) 約0.02875g/L L−プロリン;
xxxi) 約0.03676g/L L−セリン;
xxxii) 約0.10156g/L L−スレオニン;
xxxiii) 約0.01902g/L L−トリプトファン;
xxxiv) 約0.10771g/L L−チロシン2Na 2H2O;
xxxv) 約0.09866g/L L−バリン;
xxxvi) 約0.028g/L 塩化マグネシウム;
xxxvii) 約0.04884g/L 無水硫酸マグネシウム;
xxxviii) 約0.00302g/L ナイアシンアミド;
xxxix) 約0.0014g/L O−ホスホリル(phoshphoryl)−エタノールアミン;
xl) 約0.31182g/L 塩化カリウム;
xli) 約0.000081g/L プトレッシン2HCl;
xlii) 約0.003g/L ピリドキサールHCl;
xliii) 約0.000031g/L ピリドキシンHCl、
xliv) 約0.000319g/L リボフラビン、
xlv) 約6.1234g/L 塩化ナトリウム、
xlvi) 約0.0625g/L 一塩基性リン酸ナトリウムH2O、
xlvii) 約0.07099g/L 無水二塩基性リン酸ナトリウム、
xlviii) 約0.055 ピルビン酸ナトリウム、
xlix) 約0.00317g/L チアミンHCl、
l) 約0.000364g/L チミジン、
li) 約0.000432g/L 硫酸亜鉛7H2O、
lii) 約0.00000125g/L 硫酸銅5H2O、
liii) 約0.00000222g/L 二酸化セレン、
liv) 約0.000042g/L リノール酸、
lv) 約0.00039065g/L ベータ−メルカプトエタノール;および
lvi) 約0.0012g/L エタノールアミンFB。 - 水和させて細胞培養培地を形成させた際に、請求項1のパートi)の1または複数の塩が塩化マグネシウムを0.5〜5g/Lの濃度で含む、請求項1〜10の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際の塩化マグネシウムの濃度が約1.428g/Lである、請求項11の配合細胞培養培地粉末製剤。
- 増殖因子が組換えインスリンを含む、請求項1〜12の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のインスリンの濃度が0.5〜15mg/Lである、請求項13の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のインスリンの濃度が約3mg/Lである、請求項14の配合細胞培養培地粉末製剤。
- 1または複数の無機イオンが、モリブデン酸アンモニウム、硫酸カリウムクロム、硫酸銅、塩化リチウム、硫酸マンガン、メタけい酸ナトリウムおよびそれらの組み合わせから選択される微量金属を含む、請求項1〜15の配合細胞培養培地粉末製剤。
- モリブデン酸アンモニウムがモリブデン酸アンモニウム4H2Oであり;硫酸カリウムクロムが硫酸カリウムクロム12H2Oであり、硫酸銅が硫酸銅5H2Oであり、塩化リチウムが塩化リチウム(無水)であり、硫酸マンガンが硫酸マンガンH2Oであり、メタけい酸ナトリウムがメタけい酸ナトリウム9H2Oである、請求項16の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際の微量金属の終濃度が以下である、請求項17の配合細胞培養培地粉末製剤:
i) 0.0005〜0.01mg/Lのモリブデン酸アンモニウム4H2O;
ii) 0.0001〜0.01mg/Lの硫酸カリウムクロム12H2O;
iii) 0.001〜0.125mg/Lの硫酸銅5H2O;
iv) 0.001〜0.1mg/Lの塩化リチウム(無水);
v) 0.00004〜0.004mg/Lの硫酸マンガンH2O;および
vi) 0.04〜4.2mg/Lのメタけい酸ナトリウム9H2O。 - 配合細胞培養培地粉末製剤が、モリブデン酸アンモニウム4H2O、硫酸カリウムクロム12H2O、硫酸銅5H2O、塩化リチウム(無水)、硫酸マンガンH2Oおよびメタけい酸ナトリウム9H2Oの組み合わせを含む、請求項1〜18の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際の微量金属の終濃度が以下である、請求項19の配合細胞培養培地粉末製剤:
i) 約0.0037mg/Lのモリブデン酸アンモニウム4H2O;
ii) 約0.001mg/Lの硫酸カリウムクロム12H2O;
iii) 約0.0125mg/Lの硫酸銅5H2O;
iv) 約0.01mg/Lの塩化リチウム(無水);
v) 約0.000452mg/Lの硫酸マンガンH2O;および
vi) 約0.4263mg/Lのメタけい酸ナトリウム9H2O。 - アミノ酸補充物が、アスパラギンH2O、グルタミン、ヒスチジンおよびセリンの組み合わせを含む、請求項1〜20の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のアミノ酸補充物の終濃度が以下である、請求項1〜21の配合細胞培養培地粉末製剤:
i) 0.007〜0.07g/L アスパラギンH2O;
ii) 0.25〜2.5g/L グルタミン;
iii) 0.5〜5.0g/L ヒスチジン、遊離塩基;および
iv) 0.01〜0.1g/L セリン。 - 水和させて細胞培養培地を形成させた際のアミノ酸補充物の終濃度が以下である、請求項1〜22の配合細胞培養培地粉末製剤:
i) 約 0.0225g/L アスパラギンH2O;
ii) 約0.73g/L グルタミン;
iii) 約1.552g/L ヒスチジン;および
iv) 約0.03676g/L セリン。 - 1または複数のバッファーが、3−(N−モルフォリノ)プロパンスルホン酸(MOPS)遊離酸、3−(N−モルフォリノ)プロパンスルホン酸(MOPS)Na、ヒドロキシエチルピペラジンエタンスルホン酸(HEPES)および炭酸水素ナトリウムよりなる群から選択される、請求項1〜23の配合細胞培養培地粉末製剤。
- 製剤が、MOPS遊離酸およびMOPS Naの組み合わせを含む、請求項1〜24の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のバッファーの終濃度が以下である、請求項1〜25の配合細胞培養培地粉末製剤:
i) 0.3〜3g/L MOPS遊離酸;および
ii) 1.0〜10g/L MOPS Na。 - 消泡剤が、エチレンオキシドおよびプロピレンオキシドをベースとしたポリオールコポリマーを含む、請求項1〜26の配合細胞培養培地粉末製剤。
- 消泡剤がPluronic F68である、請求項1〜27の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のPluronic F68の濃度が0.1〜10g/Lである、請求項1〜28の配合細胞培養培地粉末製剤。
- 水和させて細胞培養培地を形成させた際のPluronic F68の濃度が約1g/Lである、請求項29の配合細胞培養培地粉末製剤。
- 基礎培地粉末;塩化マグネシウム;インスリン;1または複数の微量金属;アスパラギン、グルタミン、ヒスチジンおよびセリンのうちの1または複数を含むアミノ酸補充物;1または複数のバッファー;ならびに1または複数の消泡剤を合わせることを含む、請求項1〜30の配合細胞培養培地粉末製剤を作製する方法。
- 請求項1〜30の配合細胞培養培地粉末製剤を水と合わせ、それによって哺乳動物細胞を増殖させるための細胞培養培地を作製することを含む、哺乳動物細胞を増殖させるための細胞培養培地を作製する方法。
- FeSO4 7H2Oおよびキレート剤を含む溶液を合わせることをさらに含む、請求項32の方法。
- キレート剤がエチレンジアミン四酢酸(EDTA)である、請求項33の方法。
- 細胞培養培地中のFeSO4 7H2OおよびEDTAの終濃度が以下である、請求項34の方法:
i) 0.004〜0.04g/L FeSO4 7H2O;および
ii) 0.006〜0.06g/L EDTA。 - 細胞培養培地中のFeSO4 7H2OおよびEDTAの終濃度が以下である、請求項35の方法:
i) 約0.0138g/L FeSO4 7H2O;および
ii) 約0.018625g/L EDTA。 - 細胞培養培地を哺乳動物細胞と接触させることをさらに含む、請求項32〜36の方法。
- 哺乳動物細胞が、チャイニーズハムスター卵巣(Chinese Hamster Ovary、CHO)細胞、DP12 CHO細胞、DG44 CHO細胞、ヒト胎児腎(Human Embryonic Kidney、HEK)細胞、HEK293細胞およびベビーハムスター腎(BHK)細胞よりなる群から選択される、請求項37の方法。
- 哺乳動物細胞が目的タンパク質を組換えにより発現する、請求項38の方法。
- 目的タンパク質が、第VIII凝固因子(FVIII)、そのバリアントおよび断片よりなる群から選択される、請求項39の方法。
- FVIIIが、野生型FVIII、B−ドメインを欠損したFVIIIおよび生体適合性ポリマーとコンジュゲートしたFVIIIから選択される、請求項40の方法。
- 生体適合性ポリマーがポリエチレングリコール(PEG)である、請求項41の方法。
- PEGが、第VIII因子のアミノ酸位置81、129、377、378、468、487、491、504、556、570、711、1648、1795、1796、1803、1804、1808、1810、1864、1903、1911、2091、2118および2284のうちの1または複数においてこのポリペプチドに共有結合的に付着している、請求項42の方法。
- 哺乳動物細胞がBHK細胞である、請求項38〜43の方法。
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