DE17196350T1 - Verhindern der disulfid-bindungsreduktion während der rekombinanten herstellung von polypeptiden - Google Patents
Verhindern der disulfid-bindungsreduktion während der rekombinanten herstellung von polypeptiden Download PDFInfo
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Abstract
Verfahren zur Prävention der Reduktion einer Disulfidbindung in einem in einer rekombinanten Wirtszelle exprimierten Polypeptid, umfassend das Ergänzen der Zellkulturflüssigkeit der rekombinanten Wirtszelle vor oder nach dem Gewinnen derselben mit einem Thioredoxin-Inhibitor.
Claims (25)
- Verfahren zur Prävention der Reduktion einer Disulfidbindung in einem in einer rekombinanten Wirtszelle exprimierten Polypeptid, umfassend das Ergänzen der Zellkulturflüssigkeit der rekombinanten Wirtszelle vor oder nach dem Gewinnen derselben mit einem Thioredoxin-Inhibitor.
- Verfahren nach
Anspruch 1 , wobei der Thioredoxin-Inhibitor Folgendes ist: (i) ein direkter Inhibitor von Thioredoxin; (ii) ein spezifischer Inhibitor von Thioredoxinreduktase, (iii) ein Metallion; (iv) ein Inhibitor von G6PD; (v) ein Inhibitor von Hexokinaseaktivität; (vi) Cystin, Cystein oder oxidiertes Glutathion; (vii) ein Antikörper, der spezifisch an eine Thioredoxinreduktase bindet; oder (viii) eine Maßnahme, die indirekt zur Hemmung von Thioredoxinaktivität führt. - Verfahren nach Anspruch 2(i), wobei der Thioredoxin-Inhibitor ein Alkyl-2-imidazolyldisulfid oder ein Naphthochinonspiroketal-Derivat ist.
- Verfahren nach
Anspruch 2 (ii), wobei der Thioredoxin-Inhibitor ein Goldkomplex ist. - Verfahren nach
Anspruch 4 , wobei der Goldkomplex Aurothioglucose (ATG) oder Aurothiomalat (ATM) ist und das ATG oder das ATM gegebenenfalls in einer Konzentration (a) zwischen etwa 0,1 mM und etwa 1 mM oder (b) von zumindest dem 4-Fachen der Thioredoxinreduktase-Konzentration in der Kulturflüssigkeit vor oder nach dem Gewinnen derselben zugesetzt wird. - Verfahren nach
Anspruch 2 (iii), wobei das Metallion aus der aus Hg2+, Cu2+, Zn2+, Co2+ und Mn2+ bestehenden Gruppe ausgewählt ist. - Verfahren nach
Anspruch 6 , wobei das Metallion Cu2+ ist, das in Form von Kupfer(II)-sulfat vorliegt. - Verfahren nach
Anspruch 7 , wobei das Kupfer(II)-sulfat (a) als Pentahydrat oder in wasserfreier Form vorliegt; (b) in einer Konzentration zwischen etwa 5 µM und etwa 100 µM zugesetzt wird; (c) in einer Konzentration zwischen etwa 10 µM und etwa 80 µM zugesetzt wird; (d) in einer Konzentration zwischen etwa 15 µM und etwa 50 µM zugesetzt wird; oder (e) in einer Konzentration von zumindest dem 2-Fachen der Thioredoxin-Konzentration in der Kulturflüssigkeit vor oder nach dem Gewinnen derselben zugesetzt wird - Verfahren nach
Anspruch 2 (iv), wobei der Thioredoxin-Inhibitor aus der aus Pyridoxal-5'-phosphat, 1-Fluor-2,4-dinitrobenzol, Dehydroepiandrosteron (DHEA) und Epiandrosteron (EA) bestehenden Gruppe ausgewählt ist. - Verfahren nach
Anspruch 9 , wobei das DHEA in einer Konzentration (a) zwischen etwa 0,05 mM und etwa 5 mM oder (b) zwischen etwa 0,1 mM und etwa 2,5 mM zugesetzt wird. - Verfahren nach Anspruch 2(v), wobei der Thioredoxin-Inhibitor (a) ein Metallionen-Chelatbildner ist; (b) aus der aus Sorbose-1-phosphat, Polyphosphaten, 6-Desoxy-6-fluor-glucose, 2-C-Hydroxymethylglucose, Xylose oder Lyxose bestehenden Gruppe ausgewählt ist.
- Verfahren nach Anspruch 11(a), wobei der Metallionen-Chelatbildner Ethylendiamintetraessigsäure (EDTA) ist.
- Verfahren nach
Anspruch 12 , wobei die EDTA in einer Konzentration (i) zwischen etwa 5 mM und etwa 60 mM, (ii) zwischen etwa 10 mM und etwa 50 mM; oder (iii) zwischen etwa 20 mM und etwa 40 mM zugesetzt wird. - Verfahren nach
Anspruch 2 (vi), wobei das Cystin, Cystein oder oxidierte Glutathion in einer Konzentration von zumindest etwa dem 40-Fachen der Konzentration des Polypeptids in der Kulturflüssigkeit vor oder nach dem Gewinnen derselben zugesetzt wird. - Verfahren nach
Anspruch 2 (viii), wobei die Maßnahme das Belüften der gewonnenen Kulturflüssigkeit der rekombinanten Wirtszelle oder das Senken des pHs der gewonnenen Kulturflüssigkeit der rekombinanten Wirtszellen ist. - Verfahren nach einem der
Ansprüche 1 bis14 , das weiters den Schritt (i) des Belüftens der gewonnenen Kulturflüssigkeit der rekombinanten Wirtszelle oder (ii) des Senkens des pHs der gewonnenen Kulturflüssigkeit der rekombinanten Wirtszellen umfasst. - Verfahren zur Verhinderung der Reduktion einer Disulfidbindung eines Antikörpers oder eines biologisch funktionellen Fragments davon, der/das in einer rekombinanten Wirtszelle exprimiert wird, umfassend das Verringern des Expressionslevels eines Enyzms des Trx-Systems in der eukaryotischen Wirtszelle durch: (a) ein Nucleinsäuremolekül, das ein Antisense-Nucleotid umfasst, oder (b) eine interferierende RNA; und das Gewinnen des Antikörpers oder dessen biologisch funktionellen Fragments, wodurch die Reduktion der Disulfidbindung des Antikörpers oder dessen biologisch funktionellen Fragments im geernteten Zellkulturfluid (HCCF) verhindert wird.
- Verfahren nach
Anspruch 17 , wobei das Enzym Thioredoxinreduktase, G6PD oder Hexokinase ist. - Verfahren nach einem der
Ansprüche 1 bis16 , wobei das Polypeptid ein Antikörper oder ein biologisch funktionelles Fragment eines Antikörpers ist. - Verfahren nach einem der
Ansprüche 17 bis19 , wobei (i) das biologisch funktionelle Fragment des Antikörpers aus der aus Fab, Fab‘, F(ab‘)2, scFv, (scFv)2, dAb, Fragmenten komplementaritätsbestimmender Regionen (CDR), unverzweigten Antikörpern, Einzelketten-Antikörpermolekülen, Minibodys, Diabodys und multispezifischen Antikörpern, gebildet aus Antikörperfragmenten, bestehenden Gruppe ausgewählt ist oder (ii) der Antikörper oder das Antikörperfragment ein therapeutischer Antikörper oder ein biologisch funktionelles Fragment davon ist. - Verfahren nach
Anspruch 20 (ii), wobei der therapeutische Antikörper aus der aus Anti-HER2-Antikörpern; Anti-CD20-Antikörpern; Anti-IL-8-Antikörpern; Anti-VEGF-Antikörpern; Anti-CD40-Antikörpern; Anti-CD11a-Antikörpern; Anti-CD18-Antikörpern; Anti-IgE-Antikörpern; Anti-Apo-2-Rezeptor-Antikörpern; Anti-Gewebefaktor-(TF-) Antikörpern; Anti-Mensch-α4β7-Integrin-Antikörpern; Anti-EGFR-Antikörpern; Anti-CD3-Antikörpern; Anti-CD25-Antikörpern; Anti-CD4-Antikörpern; Anti-CD52-Antikörpern; Anti-Fc-Rezeptor-Antikörpern; Anti-karzinoembryonisches-Antigen- (CEA-) Antikörpern; Antikörpern, die gegen Brustepithelzellen gerichtet sind; Antikörpern, die an Dickdarmkarzinomzellen binden; Anti-CD38-Antikörpern; Anti-CD33-Antikörpern; Anti-CD22-Antikörpern; Anti-EpCAM-Antikörpern; Anti-GpIIb/IIIa-Antikörpern; Anti-RSV-Antikörpern; Anti-CMV-Antikörpern; Anti-HIV-Antikörpern; Anti-Hepatitis-Antikörpern; Anti-CA125-Antikörpern; Anti-αvβ3-Antikörpern; Anti-Human-Nierenzellenkarzinom-Antikörpern; Anti-Humen-17-1A-Antikörpern; Anti-Humen-Kolorektaltumor-Antikörpern; Anti-Human-Melanom-Antikörper R24, der gegen GD3-Gangliosid gerichtet ist; Anti-Human-Plattenepithelzellkarzinom-; und Anti-Human-Leukozytenantigen- (HLA-) Antikörpern und Anti-HLA-DR-Antikörpern bestehenden Gruppe ausgewählt ist. - Verfahren nach
Anspruch 20 (ii), wobei der therapeutische Antikörper ein Antikörper ist, der an Folgendes bindet: (a) einen HER-Rezeptor, wobei der HER-Rezeptor gegebenenfalls HER2 ist und der therapeutische Antikörper eine Sequenz einer variablen Schwer- und/oder Leichtkettendomäne umfasst, die aus der aus Seq.-ID Nr. 16, 17, 18 und 19 bestehenden Gruppe ausgewählt ist; (b) VEGF, wobei der therapeutische Antikörper gegebenenfalls eine Sequenz einer variablen Schwer- und/oder Leichtkettendomäne umfasst, die aus der aus Seq.-ID Nr. 20 bis 55 bestehenden Gruppe ausgewählt ist; (c) IgE; (d) CD20, wobei der therapeutische Antikörper gegebenenfalls eine Sequenz einer variablen Schwer- und/oder Leichtkettendomäne umfasst, die aus der aus Seq.-ID Nr. 1 bis 15 bestehenden Gruppe ausgewählt ist; (e) CD11a, wobei der therapeutische Antikörper gegebenenfalls eine Sequenz einer variablen Schwer- und/oder Leichtkettendomäne umfasst, die aus der aus Seq.-ID Nr. 26 bis 29 bestehenden Gruppe ausgewählt ist; (f) CD40; oder (g) DR5, wobei der therapeutische Antikörper gegebenenfalls aus der aus den Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3 und 25.3 bestehenden Gruppe ausgewählt ist. - Verfahren nach einem der
Ansprüche 1 bis16 , wobei das Polypeptid ein therapeutisches Polypeptid ist und wobei das therapeutische Polypeptid gegebenenfalls aus der aus einem Wachstumshormon, einschließlich des menschlichen Wachstumshormons und des Rinderwachstumshormons; dem Wachstumshormon-freisetzenden Faktor; Parathormon; Schilddrüsenstimulationshormon; Lipoproteinen; α-1-Antitrypsin; Insulin-A-Kette; Insulin-B-Kette; Proinsulin; follikelstimulierendem Hormon; Calcitonin; luteinisierendem Hormon; Glucagon; Gerinnungsfaktoren wie Faktor VIIIC, Faktor IX, Gewebefaktor und von-Willebrand-Faktor; Anti-Gerinnungsfaktoren wie Protein C; atrialem natriuretischem Faktor; Lungentensid; einem Plasminogenaktivator wie Urokinase oder menschlichem Urin- oder Gewebetyp-Plasminogen-Aktivator (t-PA); Bombesin; Thrombin; hämatopoietischem Wachstumsfaktor; Tumornekrosefaktor-α und -β; Enkephalinase; RANTES (reguliert bei Aktivierung, normalerweise T-Zellen-exprimiert und -sekretiert); menschlichem Makrophagenentzündungsprotein (MIP-1-α); einem Serumalbumin wie menschlichem Serumalbumin; Müllerscher Inhibitionssubstanz; Relaxin-A-Kette; Relaxin-B-Kette; Prorelaxin; Mäuse-Gonadotropin-assoziiertem Peptid; einem mikrobiellen Protein wie β-Lactamase; DNase; IgE; einem zytotoxischen T-Lymphozyten-assoziierten Antigen (CTLA) wie CTLA-4; Inhibin; Activin; Gefäßendothelwachstumsfaktor (VEGF); Rezeptoren für Hormone oder Wachstumsfaktoren; Protein A oder D; rheumatoiden Faktoren; einem neurotrophen Faktor wie knochenabgeleiteter neurotropher Faktor (BDNF), Neurotrophin-3, -4, -5 oder -6 (NT-3, NT-4, NT-5 oder NT-6) oder einem Nervenwachstumsfaktor wie NGF-β; plättchenabgeleitetem Wachstumsfaktor (PDGF); Fibroblastenwachstumsfaktor wie aFGF und bFGF; Epidermiswachstumsfaktor (EGF); transformierendem Wachstumsfaktor (TGF) wie TGF-α und TGF-β, einschließlich TGF-β1, TGF-β2, TGF-β3, TGF-β4 oder TGF-β5; insulinähnlichem Wachstumsfaktor-I und -II (IGF-I und IGF-II); des(1-3)-IGF-I (Hirn-IGF-I); Bindungsproteinen für insulinähnlichen Wachstumsfaktor; CD-Proteinen wie CD3, CD4, CD8, CD19, CD20, CD34 und CD40; Erythropoietin; osteoinduktiven Faktoren; Immuntoxinen; einem knochenmorphogenetischen Protein (BMP); einem Interferon wie Interferon-α, -β und -γ; koloniestimulierenden Faktoren (CSFs), z.B. M-CSF, GM-CSF und G-CSF; Interleukinen (ILs), z.B. IL-1 bis IL-10; Superoxiddismutase; T-Zellen-Rezeptoren; Oberflächenmembranproteinen; Abbaubeschleunigungsfaktor; viralen Antigenen wie z.B. eines Teils der AIDS-Hülle; Transportproteinen; Homing-Rezeptoren; Addressinen; Regulationsproteinen; Integrinen wie CD11a, CD11b, CD11c, CD18, einem ICAM, VLA-4 und VCAM; einem tumorassoziierten Antigen wie HER2-, HER3- oder HER4-Rezeptor; und Fragmenten der Polypeptide bestehenden Gruppe ausgewählt ist. - Verfahren nach einem der
Ansprüche 1 bis23 , wobei die rekombinante Wirtszelle (i) eine eukaryotische Wirtszelle oder (ii) eine prokaryotische Wirtszelle ist. - Verfahren nach
Anspruch 24 , wobei die eukaryotische Wirtszelle eine Säugetier-Wirtszelle ist und gegebenenfalls eine Chinahamster-Eierstock- (CHO-) Zelle ist oder die prokaryotische Wirtszelle eine Bakterienzelle ist und gegebenenfalls eine E.-coli-Zelle ist.
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US94867707P | 2007-07-09 | 2007-07-09 | |
US948677P | 2007-07-09 | ||
EP17196350.7A EP3327026B1 (de) | 2007-07-09 | 2008-07-08 | Verhindern der disulfid-bindungsreduktion während der rekombinanten herstellung von polypeptiden |
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DE17196350T1 true DE17196350T1 (de) | 2018-12-27 |
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US (14) | US20090053786A1 (de) |
EP (8) | EP4245766A3 (de) |
JP (5) | JP5687057B2 (de) |
KR (6) | KR101643514B1 (de) |
CN (3) | CN101932591B (de) |
AU (2) | AU2008275229B2 (de) |
BR (1) | BRPI0812603B1 (de) |
CA (2) | CA2691692C (de) |
DE (1) | DE17196350T1 (de) |
DK (2) | DK3597659T3 (de) |
ES (4) | ES2659829T3 (de) |
FI (1) | FI3597659T3 (de) |
HR (3) | HRP20230461T3 (de) |
HU (4) | HUE061746T2 (de) |
IL (6) | IL202934B (de) |
LT (2) | LT3327026T (de) |
MX (2) | MX2010000196A (de) |
PL (4) | PL3327026T3 (de) |
PT (2) | PT3327026T (de) |
SG (3) | SG10201406411XA (de) |
SI (4) | SI3327026T1 (de) |
TR (1) | TR201802088T4 (de) |
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EP2064314A4 (de) | 2006-09-13 | 2009-12-30 | Abbott Lab | Verbesserungen an zellkulturen |
US8911964B2 (en) | 2006-09-13 | 2014-12-16 | Abbvie Inc. | Fed-batch method of making human anti-TNF-alpha antibody |
CN101932591B (zh) | 2007-07-09 | 2018-02-16 | 健泰科生物技术公司 | 在多肽的重组生产期间防止二硫键还原 |
TWI472339B (zh) | 2008-01-30 | 2015-02-11 | Genentech Inc | 包含結合至her2結構域ii之抗體及其酸性變異體的組合物 |
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