Prevention of disulfide bond reduction during recombinant production of polypeptides

Abstract

The invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.

1 . A method for the prevention of the reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell, comprising supplementing, following fermentation, the pre-harvest or harvested culture fluid of said recombinant host cell with a thioredoxin inhibitor. 
     
     
         2 . The method of  claim 1  wherein said thioredoxin inhibitor is added to the pre-harvest culture fluid. 
     
     
         3 . The method of  claim 1  wherein said thioredoxin inhibitor is added to the harvested culture fluid. 
     
     
         4 . The method of  claim 1  wherein said thioredoxin inhibitor is a direct inhibitor of thioredoxin. 
     
     
         5 . The method of  claim 4  wherein said thioredoxin inhibitor is an alkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative. 
     
     
         6 . The method of  claim 1  wherein said thioredoxin inhibitor is a specific inhibitor of thioredoxin reductase. 
     
     
         7 . The method of  claim 6  wherein said thioredoxin inhibitor is a gold complex. 
     
     
         8 . The method of  claim 7  wherein said gold complex is aurothioglucose (ATG) or aurothiomalate (ATM). 
     
     
         9 . The method of  claim 8  wherein said ATG or said ATM is added in a concentration between about 0.1 mM and about 1 mM. 
     
     
         10 . The method of  claim 8  wherein said ATG or said ATM is added at a concentration at least about four-times of thioredoxin reductase concentration in said pre-harvest or harvested culture fluid. 
     
     
         11 . The method of  claim 1  wherein said thioredoxin inhibitor is a metal ion. 
     
     
         12 . The method of  claim 11  wherein said metal ion is selected from the group consisting of Hg 2+ , Cu 2+ , Zn 2+ , Co 2+ , and Mn 2+ . 
     
     
         13 . The method of  claim 12  wherein said metal ion is Cu 2+  present in the form of cupric sulfate. 
     
     
         14 . The method of  claim 13  wherein said cupric sulfate is in the form of pentahydrate or in anhydrous form. 
     
     
         15 . The method of  claim 13  wherein said cupric sulfate is added in a concentration between about 5 μM and about 100 μM. 
     
     
         16 . The method of  claim 13  wherein said cupric sulfate is added in a concentration between about 10 μM to about 80 μM. 
     
     
         17 . The method of  claim 13  wherein said cupric sulfate is added in a concentration between about 15 μM and about 50 μM. 
     
     
         18 . The method of  claim 13  wherein said cupric sulfate is added at a concentration at least about two-times of thioredoxin concentration in said pre-harvest or harvested culture fluid. 
     
     
         19 . The method of  claim 1  wherein said thioredoxin inhibitor is as inhibitor of G6PD. 
     
     
         20 . The method of  claim 19  wherein said thioredoxin inhibitor is selected from the group consisting of pyridoxal 5′-phosphate, 1 fluoro-2,4 dinitrobenzene, dehydroepiandrosterone (DHEA) and epiandrosterone (EA). 
     
     
         21 . The method of  claim 20  wherein said DHEA is added in a concentration between about 0.05 mM and about 5 mM. 
     
     
         22 . The method of  claim 20  wherein said DHEA is added in a concentration between about 0.1 mM and about 2.5 mM. 
     
     
         23 . The method of  claim 1  wherein said thioredoxin inhibitor is an inhibitor of hexokinase activity. 
     
     
         24 . The method of  claim 23  wherein said thioredoxin inhibitor is a chelator of metal ions. 
     
     
         25 . The method of  claim 24  wherein said chelator of metal ions is ethylenediamine tetraacetic acid (EDTA). 
     
     
         26 . The method of  claim 25  wherein said EDTA is added in a concentration between about 5 mM and about 60 mM. 
     
     
         27 . The method of  claim 25  wherein said EDTA is added in a concentration between about 10 mM and about 50 mM. 
     
     
         28 . The method of  claim 25  wherein said EDTA is added in a concentration between about 20 mM and about 40 mM. 
     
     
         29 . The method of  claim 23  wherein said thioredoxin inhibitor is selected from the group consisting of sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, and lyxose. 
     
     
         30 . The method of  claim 1  wherein said thioredoxin inhibitor is cystine, cysteine, or oxidized glutathione. 
     
     
         31 . The method of  claim 30  wherein said cystine, cysteine or oxidized glutathione is added at a concentration at least about 40-times of the concentration of said polypeptide in said pre-harvest or harvested culture fluid. 
     
     
         32 . The method of  claim 1  wherein said thiodedoxin inhibitor is a siRNA, antisense nucleotide, or antibody specifically binding to a thioredoxin reductase. 
     
     
         33 . The method of  claim 1  wherein said thioredoxin inhibitor is a measure indirectly resulting in the inhibition of thioredoxin activity. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 33  wherein said measure is lowering the pH of the harvested culture fluid of said recombinant host cells. 
     
     
         36 . The method of  claim 1  further comprising the step of air sparging the harvested culture fluid of said recombinant host cell. 
     
     
         37 . The method of  claim 1  further comprising the step of lowering the pH of the harvested culture fluid of said recombinant host cells. 
     
     
         38 . The method of  claim 1  wherein said polypeptide is an antibody, or a biologically functional fragment of an antibody. 
     
     
         39 . The method of  claim 38  wherein said antibody fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2 , scFv, (scFv) 2 , dAb, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, diabodies, and multispecific antibodies formed from antibody fragments. 
     
     
         40 . The method of  claim 38  wherein said antibody or antibody fragment is a therapeutic antibody or a biologically functional fragment thereof. 
     
     
         41 . The method of  claim 40  wherein said therapeutic antibody is selected from the group consisting of anti-HER2 antibodies anti-CD20 antibodies; anti-IL-8 antibodies; anti-VEGF antibodies; anti-CD40 antibodies, anti-CD11a antibodies; anti-CD18 antibodies; anti-IgE antibodies; anti-Apo-2 receptor antibodies; anti-Tissue Factor (TF) antibodies; anti-human α 4 β 7  integrin antibodies; anti-EGFR antibodies; anti-CD3 antibodies; anti-CD25 antibodies; anti-CD4 antibodies; anti-CD52 antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-CD38 antibodies; anti-CD33 antibodies; anti-CD22 antibodies; anti-EpCAM antibodies; anti-GpIIb/IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies; anti-αvβ3 antibodies; anti-human renal cell carcinoma antibodies; anti-human 17-1A antibodies; anti-human colorectal tumor antibodies; anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma; and anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies. 
     
     
         42 . The method of  claim 40  wherein said therapeutic antibody is an antibody binding to a HER receptor, VEGF, IgE, CD20, CD11a, CD40, or DR5. 
     
     
         43 . The method of  claim 42  wherein said HER receptor is HER1 and/or HER2. 
     
     
         44 . The method of  claim 43  wherein the HER receptor is HER2. 
     
     
         45 . The method of  claim 44  wherein said therapeutic antibody comprises a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 16, 17, 18, and 19. 
     
     
         46 . The method of  claim 42  wherein said therapeutic antibody is an antibody that binds to CD20. 
     
     
         47 . The method of  claim 46  wherein said therapeutic antibody comprises a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 1 through 15. 
     
     
         48 . The method of  claim 42  wherein said therapeutic antibody is an antibody that binds to VEGF. 
     
     
         49 . The method of  claim 48  wherein said therapeutic antibody comprises a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 20 through 25. 
     
     
         50 . The method of  claim 42  wherein said therapeutic antibody is an antibody that binds CD11a. 
     
     
         51 . The method of  claim 50  wherein said therapeutic antibody comprises a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 26 through 29. 
     
     
         52 . The method of  claim 42  wherein said therapeutic antibody binds to a DR5 receptor. 
     
     
         53 . The method of  claim 52  wherein said therapeutic antibody is selected from the group consisting of 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, and 25.3. 
     
     
         54 . The method of  claim 52  wherein said therapeutic antibody is Apomab 8.3 or Apomab 7.3. 
     
     
         55 . The method of  claim 54  wherein said therapeutic antibody is Apomab 7.3. 
     
     
         56 . The method of  claim 1  wherein said polypeptide is a therapeutic polypeptide. 
     
     
         57 . The method of  claim 56  wherein said therapeutic polypeptide is selected from the group consisting of a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibit; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; Protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 or HER4 receptor; and fragments of said polypeptides. 
     
     
         58 . The method of  claim 1  wherein said recombinant host cell is an eukaryotic host cell. 
     
     
         59 . The method of  claim 58  wherein said eukaryotic host cell is a mammalian host cell. 
     
     
         60 . The method of  claim 59  wherein said mammalian host cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         61 . The method of  claim 1  wherein the recombinant host cell is a prokaryotic host cell. 
     
     
         62 . The method of  claim 61  wherein the prokaryotic host cell is a bacterial cell. 
     
     
         63 . The method of  claim 62  wherein the bacterial cell is an  E. coli  cell.