Preventing disulfide bond reduction during recombinant production of polypeptides

Abstract

The present invention relates to prevention of disulfide bond reduction during recombinant production of polypeptides. The present invention concerns methods and means for preventing disulfide bond reduction 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.

Description

[0001] This application is a re-divisional application of a divisional application with an application date of July 8, 2008, Chinese application number 202011172162.5, and invention name “Preventing disulfide bond reduction during recombinant production of polypeptides” (the original application number is 200880105932.5). Technical Field

[0002] The present invention concerns methods and means for preventing disulfide bond reduction during recombinant production of disulfide-containing polypeptides. In particular, the present invention concerns preventing disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures. Background Art

[0003] In the biotechnology industry, pharmaceutical applications require the production of a variety of proteins using recombinant DNA technology. In general, recombinant proteins are produced by cell culture, using eukaryotic cells (such as mammalian cells) or prokaryotic cells (such as bacterial cells) that have been engineered to produce the protein of interest by inserting a recombinant plasmid containing a nucleic acid encoding the desired protein. In order for a protein to retain biological activity, the conformation of the protein (including its tertiary structure) must be maintained during its purification and isolation, and the protein's multiple functional groups must be protected from degradation.

[0004] Mammalian cells have become the predominant system for producing mammalian proteins for clinical applications, primarily due to their ability to generate correctly folded and assembled heterologous proteins and their ability to be post-translationally modified. Chinese hamster ovary (CHO) cells and PER from various other mammalian sources such as, for example, mouse myeloma (NS0), baby hamster kidney (BHK), human embryonic kidney (HEK-293), and human kidney cells, such as those isolated from human kidney cells, provide human glycosylation characteristics, and are capable of naturally producing antibodies that match human physiology. Cell lines obtained from R&D institutions are approved by regulatory agencies for the production of biopharmaceutical products.

[0005] Typically, to initiate the production cycle, a small number of transformed recombinant host cells are allowed to grow in culture for a few days (see e.g. Fig.23 ). Once the cells have gone through several rounds of replication, they are transferred to larger vessels where they are prepared for fermentation. The medium used to culture the cells and the levels of oxygen, nitrogen, and carbon dioxide present during the production cycle can have a significant impact on the production process. Culture parameters are determined specifically for each cell line and are frequently measured to ensure optimal growth and production conditions.

[0006] When the cells grow to a sufficient number, they are transferred to a large-scale production tank and cultured for a longer period of time. At this point in the process, the recombinant protein can be harvested. Typically, the cells are transformed into secreted polypeptides into the cell culture fluid so that the first step of the purification process is to separate the cells from the culture fluid. Typically, harvesting includes centrifugation and filtration to generate the harvested cell culture fluid (Harvested Cell Culture Fluid, HCCF). The culture fluid is then subjected to several additional purification steps to remove any cell debris, unwanted proteins, salts, minerals or other unwanted components. At the end of the purification process, the recombinant protein is highly pure and is suitable for human therapeutic use.

[0007] Although much research and improvement has been made to this process over the past few decades, the production of recombinant proteins remains fraught with difficulties. Thus, for example, during the recombinant production of polypeptides containing disulfide bonds (especially multi-chain polypeptides containing interchain disulfide bonds, such as antibodies), it is important to protect and maintain the disulfide bonds throughout the production, recovery and purification process to generate correctly folded polypeptides with the necessary biological activity. Summary of the invention

[0008] The present invention generally relates to a method for preventing the reduction of disulfide bonds in a polypeptide expressed in a recombinant host cell, comprising supplementing a pre-harvest culture fluid or a harvested culture fluid of the recombinant host cell with a thioredoxin inhibitor or a thioredoxin-like protein.

[0009] In one embodiment, the thioredoxin inhibitor is added to the culture broth prior to harvest.

[0010] In another embodiment, the thioredoxin inhibitor is added to the harvested culture fluid.

[0011] In yet another embodiment, the thioredoxin inhibitor is a direct inhibitor of thioredoxin.

[0012] In all embodiments, the thioredoxin inhibitor can be, for example, an alkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative.

[0013] In yet another embodiment, the thioredoxin inhibitor is a specific inhibitor of thioredoxin reductase.

[0014] In yet another embodiment, the thioredoxin inhibitor is a gold complex, wherein the gold complex can be, for example, aurothioglucose (ATG) or aurothiomalate (ATM). Although the effective inhibitory concentration can vary, it is generally between about 0.1 mM and 1 mM. Similarly, the minimum effective inhibitory concentration varies with the properties of the polypeptide and the overall environment, and is generally reached when the ATG or ATM concentration is at least about four times the thioredoxin concentration in the culture fluid before harvest or after harvest.

[0015] In another embodiment of this aspect of the invention, the thioredoxin inhibitor is a metal ion, wherein the metal ion can be selected from the group consisting of (but not limited to): Hg 2 +、Cu 2 +、Zn 2 +、Co 2 +, and Mn 2 +. When the metal ion is added in the form of copper sulfate, the effective inhibitory concentration is generally between about 5 μM and about 100 μM, or between about 10 μM and about 80 μM, or between about 15 μM and about 50 μM. The minimum inhibitory concentration of copper sulfate also varies, but is generally achieved when copper sulfate is added at a concentration of at least about twice the concentration of thioredoxin in the culture fluid before or after harvest.

[0016] In various embodiments, the thioredoxin inhibitor is an oxidant, e.g., an inhibitor of G6PD, such as, e.g., pyridoxal-5'-phosphate, 1-fluoro-2,4-dinitrobenzene, dehydroepiandrosterone (DHEA) or epiandrosterone (EA); cystine or cysteine. Typical effective inhibitory concentrations of DHEA are between about 0.05 mM and about 5 mM, or between about 0.1 mM and about 2.5 mM.

[0017] In yet another embodiment, the thioredoxin inhibitor is an inhibitor of hexokinase activity, including but not limited to chelators of metal ions, such as, for example, ethylenediaminetetraacetic acid (EDTA). EDTA is typically added and effective at a concentration between about 5 mM and about 60 mM, or between about 10 mM and about 50 mM, or between about 20 mM and about 40 mM.

[0018] In other preferred embodiments, the inhibitor of hexokinase activity is selected from the group consisting of sorbose-1-phosphate, polyphosphate, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, and lyxose.

[0019] Other inhibitors include cystine, cysteine, and oxidized glutathione, which are typically added at a concentration of at least about 40 times the concentration of the polypeptide in question in the pre-harvest or harvested culture medium.

[0020] In yet another embodiment, the thioredoxin inhibitor is an antibody, antisense nucleotide, or siRNA that specifically binds to thioredoxin reductase.

[0021] In another embodiment, the thioredoxin inhibitor is a measure that indirectly results in inhibition of thioredoxin activity. This embodiment includes, for example, sparging the harvested culture fluid of the recombinant host cells with air and / or lowering the pH of the harvested culture fluid of the recombinant host cells.

[0022] In various embodiments, indirect means for inhibiting thioredoxin activity, such as sparging with air and / or lowering the pH, can be combined with the use of direct thioredoxin inhibitors, such as those listed above.

[0023] In all embodiments, the polypeptide can be, for example, an antibody or a biologically functional antibody fragment. Representative antibody fragments include Fab, Fab', F(ab') 2 , scFv, (scFv) 2 , dAbs, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, miniantibodies, diabodies, and multispecific antibodies formed from antibody fragments.

[0024] Therapeutic antibodies include, but are not limited to, 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 against breast epithelial cells; antibodies that bind to colon cancer 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 antibodies; R24 against GD3 ganglioside; anti-human squamous cell carcinoma antibodies; and anti-human leukocyte antigen (HLA) antibodies, and anti-HLA DR antibodies.

[0025] In other embodiments, the therapeutic antibody is an antibody that binds to a HER receptor, VEGF, IgE, CD20, CD11a, CD40, or DR5.

[0026] In yet another embodiment, the HER receptor is HER1 and / or HER2, preferably HER2. The HER2 antibody may, for example, comprise a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, and 19.

[0027] In another embodiment, the therapeutic antibody is an antibody that binds to CD20. The anti-CD20 antibody may, for example, comprise a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 1-15.

[0028] In yet another embodiment, the therapeutic antibody is an antibody that binds to VEGF. The anti-VEGF may, for example, comprise a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 20-25.

[0029] In another embodiment, the therapeutic antibody is an antibody that binds to CD11a. The anti-CD11a antibody may, for example, comprise a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 26-29.

[0030] In yet another embodiment, the therapeutic antibody binds to the DR5 receptor. The anti-DR5 antibody can be, for example, selected from the group consisting of Apomab 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, and preferably Apomab 8.3 or Apomab 7.3, and most preferably Apomab 7.3.

[0031] In other embodiments of the methods of the present invention, the polypeptide expressed in the recombinant host cell is a therapeutic polypeptide. For example, the therapeutic polypeptide can be selected from the group consisting of growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and -β; enkephalinase; RANTES (regulated upon activation, normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Muellerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, such as beta-lactamase; DNA enzymes; IgE; cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibins; activins; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophins-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors, such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors, such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGF), such as TGF-α and TGF-β, 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 protein; CD proteins, such as CD3, CD 4. CD8, CD19, CD20, CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-α, -β, and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins;Integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens, such as HER2, HER3 or HER4 receptors; and fragments of the above polypeptides. ;

[0032] In all embodiments, the recombinant host cell can be a eukaryotic cell, such as a mammalian cell, including, for example, a Chinese Hamster Ovary (CHO) cell.

[0033] In all embodiments, the recombinant host cell can also be a prokaryotic cell, such as a bacterial cell, including but not limited to an E. coli cell.

[0034] The present application relates to the following embodiments.

[0035] 1. A method for preventing the reduction of disulfide bonds in a polypeptide expressed in a recombinant host cell, comprising supplementing a thioredoxin inhibitor to the culture fluid of the host cell before or after harvest.

[0036] 2. The method of embodiment 1, wherein the thioredoxin inhibitor is added to the culture fluid before harvesting.

[0037] 3. The method according to embodiment 1, wherein the thioredoxin inhibitor is added to the harvested culture fluid.

[0038] 4. The method of embodiment 1, wherein the thioredoxin inhibitor is a direct inhibitor of thioredoxin.

[0039] 5. The method of embodiment 4, wherein the thioredoxin inhibitor is a alkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative.

[0040] 6. The method of embodiment 1, wherein the thioredoxin inhibitor is a specific inhibitor of thioredoxin reductase.

[0041] 7. The method of embodiment 6, wherein the thioredoxin inhibitor is a gold complex.

[0042] 8. The method of embodiment 7, wherein the gold complex is aurothioglucose (ATG) or aurothiomalate (ATM).

[0043] 9. The method of embodiment 8, wherein the ATG or the ATM is added at a concentration between about 0.1 mM and about 1 mM.

[0044] 10. The method of embodiment 8, wherein the ATG or the ATM is added at a concentration that is at least about four times the concentration of thioredoxin reductase in the pre-harvest or harvested culture broth.

[0045] 11. The method of embodiment 1, wherein the thioredoxin inhibitor is a metal ion.

[0046] 12. The method of embodiment 11, wherein the metal ion is selected from the group consisting of: Hg 2 +、Cu 2 +、Zn 2 +、Co 2 + and Mn 2 +.

[0047] 13. The method of embodiment 12, wherein the metal ion is in the form of copper sulfate. 2 +.

[0048] 14. The method of embodiment 13, wherein the copper sulfate is in the form of a pentahydrate or anhydrous form.

[0049] 15. The method of embodiment 13, wherein the copper sulfate is added at a concentration between about 5 μM and about 100 μM.

[0050] 16. The method of embodiment 13, wherein the copper sulfate is added at a concentration between about 10 μM and about 80 μM.

[0051] 17. The method of embodiment 13, wherein the copper sulfate is added at a concentration between about 15 μM and about 50 μM.

[0052] 18. The method of embodiment 13, wherein the copper sulfate is added at a concentration that is at least about twice the concentration of thioredoxin in the pre-harvest or harvested culture broth.

[0053] 19. The method of embodiment 1, wherein the thioredoxin inhibitor is an inhibitor of G6PD.

[0054] 20. The method of embodiment 19, wherein the thioredoxin inhibitor is selected from the group consisting of pyridoxal-5'-phosphate, 1-fluoro-2,4-dinitrobenzene, dehydroepiandrosterone (DHEA) and epiandrosterone (EA).

[0055] 21. The method of embodiment 20, wherein the DHEA is added at a concentration between about 0.05 mM and about 5 mM.

[0056] 22. The method of embodiment 20, wherein the DHEA is added at a concentration between about 0.1 mM and about 2.5 mM.

[0057] 23. The method of embodiment 1, wherein the thioredoxin inhibitor is an inhibitor of hexokinase activity.

[0058] 24. The method of embodiment 23, wherein the thioredoxin inhibitor is a chelator of metal ions.

[0059] 25. The method of embodiment 24, wherein the metal ion chelator is ethylenediaminetetraacetic acid (EDTA).

[0060] 26. The method of embodiment 25, wherein the EDTA is added at a concentration between about 5 mM and about 60 mM.

[0061] 27. The method of embodiment 25, wherein the EDTA is added at a concentration between about 10 mM and about 50 mM.

[0062] 28. The method of embodiment 25, wherein the EDTA is added at a concentration between about 20 mM and about 40 mM.

[0063] 29. The method of embodiment 23, wherein the thioredoxin inhibitor is selected from the group consisting of sorbose-1-phosphate, polyphosphoric acid, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methyl glucose, xylose, and lyxose.

[0064] 30. The method of embodiment 1, wherein the thioredoxin inhibitor is cystine, cysteine,

[0065] or oxidized glutathione.

[0066] 31. The method of embodiment 30, wherein the cystine, cysteine ​​or oxidized glutathione is

[0067] The polypeptide is added to the culture medium at a concentration that is at least about 40 times the concentration of the polypeptide in the culture medium before or after harvest.

[0068] 32. The method of embodiment 1, wherein the thioredoxin inhibitor is an antibody, antisense nucleotide, or siRNA that specifically binds to thioredoxin reductase.

[0069] 33. The method of embodiment 1, wherein the thioredoxin inhibitor is a measure that indirectly results in inhibition of thioredoxin activity.

[0070] 34. The method of embodiment 33, wherein the step is bubbling air into the harvested culture fluid of the recombinant host cells.

[0071] 35. The method of embodiment 33, wherein the measure is to lower the pH of the harvested culture fluid of the recombinant host cell.

[0072] 36. The method according to any one of embodiments 1 to 33, further comprising the step of bubbling air into the harvested culture fluid of the recombinant host cells.

[0073] 37. The method of any one of embodiments 1-33, further comprising the step of lowering the pH of the harvested culture fluid of the recombinant host cells.

[0074] 38. The method of embodiment 1, wherein the polypeptide is an antibody or a biologically functional antibody fragment.

[0075] 39. The method of embodiment 38, wherein the antibody fragment is selected from the group consisting of Fab, Fab′, F(ab′) 2 , scFv, (scFv) 2 , dAbs, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, miniantibodies, diabodies and multispecific antibodies formed from antibody fragments.

[0076] 40. The method of embodiment 38, wherein the antibody or antibody fragment is a therapeutic antibody or a biologically functional fragment thereof.

[0077] 41. The method of embodiment 40, wherein the therapeutic antibody is selected from the group consisting of: anti-HER2 antibody; anti-CD20 antibody; anti-IL-8 antibody; anti-VEGF antibody; anti-CD40 antibody, anti-CD11a antibody; anti-CD18 antibody; anti-IgE antibody; anti-Apo-2 receptor antibody; anti-tissue factor (TF) antibody; anti-human α4β7 integrin antibody; anti-EGFR antibody; anti-CD3 antibody; anti-CD25 antibody; anti-CD4 antibody; anti-CD52 antibody; anti-Fc receptor antibody; anti-carcinoembryonic antigen (CEA) antibody; antibody against breast epithelial cells; antibody that binds to colon cancer cells; anti-CD38 antibody; anti-CD33 antibody; anti-CD22 antibody; anti-EpCAM antibody; anti-GpIIb / IIIa antibody; anti-RSV antibody; anti-CMV antibody; anti-HIV antibody; anti-hepatitis antibody; anti-CA 125 antibody; anti-αvβ3 antibody; anti-human renal cell carcinoma antibody; anti-human 17-1A antibody; anti-human colorectal tumor antibody; anti-human melanoma antibody; R24 against GD3 ganglioside; anti-human squamous cell carcinoma antibody; and anti-human leukocyte antigen (HLA) antibody and anti-HLA DR antibody.

[0078] 42. The method of embodiment 40, wherein the therapeutic antibody is an antibody that binds to a HER receptor, VEGF, IgE, CD20, CD11a, CD40 or DR5.

[0079] 43. The method of embodiment 42, wherein the HER receptor is HER1 and / or HER2.

[0080] 44. The method of embodiment 43, wherein the HER receptor is HER2.

[0081] 45. The method of embodiment 44, wherein the therapeutic antibody comprises a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NO: 16, 17, 18 and 19.

[0082] 46. ​​The method of embodiment 42, wherein the therapeutic antibody is an antibody that binds to CD20.

[0083] 47. The method of embodiment 46, wherein the therapeutic antibody comprises a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 1-15.

[0084] 48. The method of embodiment 42, wherein the therapeutic antibody is an antibody that binds to VEGF.

[0085] 49. The method of embodiment 48, wherein the therapeutic antibody comprises a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 20-25.

[0086] 50. The method of embodiment 42, wherein the therapeutic antibody is an antibody that binds to CD11a.

[0087] 51. The method of embodiment 50, wherein the therapeutic antibody comprises a heavy chain and / or light chain variable domain sequence selected from the group consisting of SEQ ID NOs: 26-29.

[0088] 52. The method of embodiment 42, wherein the therapeutic antibody binds to the DR5 receptor.

[0089] 53. The method of embodiment 52, wherein the therapeutic antibody is selected from the group consisting of Apomab 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,

[0091] 6.3, 7.3, 8.3, 9.3 and 25.3.

[0092] 54. The method of embodiment 52, wherein the therapeutic antibody is Apomab 8.3 or Apomab 7.3.

[0093] 55. The method of embodiment 54, wherein the therapeutic antibody is Apomab 7.3.

[0094] 56. The method of embodiment 1, wherein the polypeptide is a therapeutic polypeptide.

[0095] 57. The method of embodiment 56, wherein the therapeutic polypeptide is selected from the group consisting of growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulation factors, such as protein C;

[0096] atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and -β; enkephalinase; RANTES (regulated upon activation, normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-α); serum albumin,

[0097] Such as human serum albumin; Muellerian inhibitory substance; relaxin A chain; relaxin B chain;

[0098] prorelaxin; mouse gonadotropin-related peptide; microbial proteins, such as β-lactamase; DNase; IgE; cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; inhibin;

[0099] activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptors; protein A or D; rheumatoid factor; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGFs), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IG F-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD34 and CD40; erythropoietin; osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-α, -β and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins;

[0100] Integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; tumor-associated antigens, such as HER2, HER3 or HER4 receptors; and fragments of the above polypeptides.

[0101] 58. The method of embodiment 1, wherein the recombinant host cell is a eukaryotic cell.

[0102] 59. The method of embodiment 58, wherein the eukaryotic cell is a mammalian cell.

[0103] 60. The method of embodiment 59, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

[0104] 61. The method of embodiment 1, wherein the recombinant host cell is a prokaryotic cell.

[0105] 62. The method of embodiment 61, wherein the prokaryotic cell is a bacterial cell.

[0106] 63. The method of embodiment 62, wherein the bacterial cell is an Escherichia coli cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 Dialysis Experiments: Digital gel-like images obtained from Bioanalyzer analysis (each lane represents a time point) demonstrate that ocrelizumab (rhuMAb 2H7-variant A) remained intact within the dialysis bag during the incubation period.

[0108] Figure 2 . Dialysis experiment: Digital gel-like images obtained from Bioanalyzer analysis (each band represents a time point) show that ocrelizumab outside the dialysis bag was reduced during the incubation. This is evidenced by the loss of intact antibody (approximately 150 kDa) and the formation of antibody fragments as depicted in the figure. At the 48 hour time point (lane 7), the reduced antibody appeared to be re-oxidized, presumably as a result of the loss of reducing activity in the harvested cell culture fluid (HCCF). The bands appearing just above the 28 kDa marker are derived from the light chains of the antibody. Significant amounts of free light chains were present in the HCCF before the start of incubation. The presence of excess free light chains and light chain dimers in the HCCF is typical for the cell lines producing ocrelizumab.

[0109] Figure 3 Free thiol levels from dialysis experiments: purified ocrelizumab in phosphate-buffered saline (PBS) was inside the dialysis bag, while HCCF containing ocrelizumab was outside the bag. Free thiols inside (squares) and outside (diamonds) the dialysis bag reached comparable levels within a few hours, indicating good exchange of small molecule components in HCCF between the inside and outside of the dialysis bag.

[0110] Figure 4 . Thioredoxin system and other reactions involving antibody reduction: The thioredoxin system (comprising thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH) functions as a hydrogen donor system for the reduction of disulfide bonds in proteins. Trx is a small monomeric protein with a CXXC active site motif that catalyzes many redox reactions via thiol-disulfide exchange. Oxidized Trx can be reduced by NADPH via TrxR. Reduced Trx can then catalyze the reduction of disulfides in proteins. The NADPH required for the thioredoxin system is provided via reactions in the pentose phosphate pathway and glycolysis.

[0111] Figure 5In vitro activity of the thioredoxin system: Digital gel-like images from Bioanalyzer analysis (each lane represents a time point), demonstrating that incubation of intact ocrelizumab (1 mg / mL) with 0.1 mM TrxR (rat liver), 5 mM Trx (human), and 1 mM NADPH in PBS resulted in complete reduction of ocrelizumab; ocrelizumab was completely reduced in less than 21 hours.

[0112] Figure 6 The in vitro activity of the thioredoxin system was inhibited by aurothioglucose: at a concentration of 1 mM Figure 5 Addition of aurothioglucose (ATG) to the same reaction mixture as described in the instructions effectively inhibited ocrelizumab reduction. This was seen in the digital gel-like images (each band represents a time point) from the Bioanalyzer analysis.

[0113] Figure 7 The in vitro activity of the thioredoxin system was inhibited by aurothiomalate: at a concentration of 1 mM Figure 5 Addition of aurothiomalate (ATM) to the same reaction mixture as described in the instructions effectively inhibited ocrelizumab reduction. This was seen in the digital gel-like images (each band represents a time point) from the Bioanalyzer analysis.

[0114] Figure 8 In vitro activity of the thioredoxin system: Digital gel-like images from Bioanalyzer analysis (each lane represents a time point) showing that incubation of intact ocrelizumab (1 mg / mL) with 0.1 mM TrxR (rat liver), 5 mM Trx (human), and 1 mM NADPH in 10 mM histidine sulfate buffer resulted in the reduction of ocrelizumab in less than 1 hour.

[0115] Fig. 9 The in vitro activity of the thioredoxin system was affected by CuSO 4 Inhibition: At a concentration of 50 μM Figure 8 The same reaction mixture as described in the instructions was added with CuSO 4 The reduction of ocrelizumab was effectively inhibited, as shown in the digital gel-like images (each band represents a time point) from the Bioanalyzer analysis.

[0116] Fig.10Ocrelizumab reduction: Digital gel-like images from Bioanalyzer analysis (each lane represents a time point) showing that ocrelizumab was reduced in incubation experiments performed with HCCF from homogenized CCF generated from a 3-L fermentor.

[0117] Fig.11 Reduction of ocrelizumab in HCCF is inhibited by aurothioglucose: Digital gel-like image from Bioanalyzer analysis (each lane represents a time point) showing the Fig.10 The same HCCF used in the incubation experiments described in the instructions supplemented with 1 mM aurothioglucose effectively inhibited ocrelizumab reduction.

[0118] Fig.12 . Reduction of ocrelizumab in HCCF is inhibited by aurothiomalate: Digital gel-like image from Bioanalyzer analysis (each lane represents a time point), indicating the Fig.10 The same HCCF used for the incubation experiments described in the instructions supplemented with 1 mM aurothiomalate effectively inhibited ocrelizumab reduction.

[0119] Fig.13 Loss of Reduction Activity in HCCF: HCCF from one of the large-scale manufacturing runs of ocrelizumab ("beta" run) (12kL Run 8) that had been subjected to several cycles of freeze-thaw demonstrated no ocrelizumab reduction when used in the incubation experiments. This was shown by Bioanalyzer analysis (each band represents a time point) and can be contrasted with the antibody reduction previously seen in freshly thawed HCCF from the same fermentation batch.

[0120] Fig.14 The reduced activity lost in HCCF was restored by adding NADPH: NADPH was added to HCCF (where the reduced activity was described above) at a concentration of 5 mM. Fig.13 After addition of NADPH (which had been eliminated under the conditions described in ), ocrelizumab reduction was again observed in the Bioanalyzer analysis (each band represents a time point).

[0121] Fig.15 The reduced activity lost in HCCF was restored by adding glucose-6-phosphate: 10 mM of glucose-6-phosphate was added to HCCF (where the reduced activity was Fig.13 After addition of G6P to the 24-well plate (where the treatment described in the previous section had been eliminated), ocrelizumab reduction was again observed in the Bioanalyzer analysis (each band represents a time point).

[0122] Fig.16 Ocrelizumab reduction: Digital gel-like image from Bioanalyzer analysis showing that ocrelizumab was reduced in an incubation experiment performed using HCCF from a large-scale manufacturing run ("α" run) (12 kL Run 9).

[0123] Fig.17 EDTA inhibits ocrelizumab reduction: Digital gel-like image from Bioanalyzer analysis (each lane represents a time point) showing that ocrelizumab reduction was inhibited in incubation experiments with HCCF from the alpha run, where HCCF (whose reducing activity is shown in Fig.16 EDTA was added.

[0124] Fig.18 The reducing activity lost in the "β" run HCCF was restored by the addition of glucose-6-phosphate, but EDTA did not inhibit reduction: when 5 mM was added to HCCF (whose reducing activity had been lost, see Fig.13 ) after addition of G6P and 20 mM EDTA, ocrelizumab reduction was observed in the Bioanalyzer analysis (each band represents a time point). Fig.17 In contrast to the results shown in , the presence of EDTA did not block ocreliumab reduction.

[0125] Fig.19 By (i) adding EDTA, (ii) adding CuSO 4 , or (iii) adjusting the pH to 5.5 to inhibit ocrelizumab reduction: All three different methods were used independently, namely (1) addition of EDTA, (2) addition of CuSO 4 , and (3) adjusting the pH to 5.5, all effectively inhibited ocrelizumab reduction. This was demonstrated by the depicted quantitative Bioanalyzer results, which showed that almost 100% of the intact (150 kDa) antibody was retained in the protein A elution pool. In contrast, ocrelizumab was completely reduced in the control HCCF after 20 hours of HCCF retention time.

[0126] Fig. 20Bubbling with air inhibits ocrelizumab reduction: Bubbling HCCF with air effectively inhibited the reduction of disulfide bonds of ocrelizumab. This was demonstrated by quantitative Bioanalyzer results showing that almost 100% intact (150 kDa) was retained in the Protein A elution pool. In contrast, ocrelizumab was almost completely reduced in the control HCCF after bubbling with nitrogen for 5 hours.

[0127] Fig.21 The V of anti-Her2 antibody (Trastuzumab) is shown L (SEQ ID NO.24) amino acid sequence.

[0128] Fig. 22 The V of anti-Her2 antibody (Trastuzumab) is shown H (SEQ ID No.25) amino acid sequence.

[0129] Fig.23 is a schematic diagram showing some of the steps of a typical large-scale manufacturing process.

[0130] Fig.24 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate.

[0131] Fig.25 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 1 mM histidine sulfate + 1 mM ATG.

[0132] Fig.26 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 0.6 μM ATG (6:1 ATG:TrxR).

[0133] Fig. 27 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 0.4 μM ATG (4:1 ATG:TrxR).

[0134] Fig.28is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 0.2 μM ATG (2:1 ATG:TrxR).

[0135] Fig.29 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 0.1 mM aurothiomalate (ATM).

[0136] Fig.30 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 0.01 mM aurothiomalate (ATM).

[0137] Fig.31 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant), in 10 mM histidine sulfate + 20 μM CuSO 4 Medium (4:1Cu 2 +:Trx).

[0138] Fig.32 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant), in 10 mM histidine sulfate + 10 μM CuSO 4 Medium (2:1Cu 2 +:Trx).

[0139] Fig.33 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant), in 10 mM histidine sulfate + 5 μM CuSO 4 Medium (1:1Cu 2 +:Trx).

[0140] Fig.34is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 532 μM cystamine (20:1 cystamine:2H7 disulfide).

[0141] Fig.35 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 266 μM cystamine (10:1 cystamine:2H7 disulfide).

[0142] Fig.36 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 133 μM cystamine (5:1 cystamine:2H7 disulfide).

[0143] Fig.37 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 26.6 μM cystamine (1:1 cystamine:2H7 disulfide).

[0144] Fig.38 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate (pH=7.6) + 2.6 mM cystine.

[0145] Fig.39 is a digital gel-like image from Bioanalyzer analysis: 2H7 (variant A) + 1 mM NADPH + 5 μM thioredoxin + 0.1 μM thioredoxin reductase (recombinant) in 10 mM histidine sulfate + 2.6 mM GSSG (oxidized glutathione).

[0146] Fig.40 . Reconstituted enzymatic reduction system. 1 mg / ml 2H7 (variant A) + 10 μg / mL hexokinase, 50 μg / mL glucose-6-phosphate dehydrogenase, 5 μM thioredoxin, 0.1 μM thioredoxin reductase, 2 mM glucose, 0.6 mM ATP, 2 mM Mg 2+, and 2 mM NADP in 50 mM histidine sulfate buffer, pH = 7.38.

[0147] Fig.41 The thioredoxin system requires NADPH. 1 mg / ml 2H7 (variant A) + 5 μM thioredoxin, 0.1 μM thioredoxin reductase, and 2 mM NADP in 50 mM histidine sulfate buffer at pH = 7.38. DETAILED DESCRIPTION

[0148] I. Definitions

[0149] In the present invention, in the context of proteins (including antibodies), in general or with respect to any particular protein or antibody, the term "reduction" is used to refer to the reduction of one or more disulfide bonds of a protein or antibody. Thus, for example, the term "ocrelizumab reduction" is used interchangeably with the term "ocrelizumab disulfide bond reduction", and the term "antibody (Ab) reduction" is used interchangeably with the term "antibody (Ab) disulfide bond reduction".

[0150] The terms "reduction" or "disulfide bond reduction" are used in the broadest sense and include complete and partial reduction as well as reduction of some or all (interchain or intrachain) disulfide bonds present in a protein (such as an antibody).

[0151] "Protein" means an amino acid sequence whose chain length is sufficient to generate a higher level of tertiary and / or quaternary structure. This is distinguished from "peptides" or other small molecular weight drugs that do not have such structure. Typically, the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassed within the definition herein include all mammalian proteins, particularly therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and proteins containing one or more disulfide bonds in general, including multichain polypeptides containing one or more interchain and / or intrachain disulfide bonds.

[0152] The term "therapeutic protein" or "therapeutic polypeptide" refers to a protein used in the treatment of a disease, regardless of its indication or mechanism of action. In order for a therapeutic protein to be useful in the clinic, it must be manufactured in large quantities. The "manufacturing scale" production of a therapeutic protein or other protein utilizes a cell culture ranging from about 400L to about 80,000L, depending on the protein produced and the demand. Typically, such manufacturing scale production utilizes a cell culture of about 400L to about 25,000L. Within this range, a specific cell culture amount is utilized, such as 4,000L, about 6,000L, about 8,000, about 10,000, about 12,000L, about 14,000L, or about 16,000L.

[0153] The term "therapeutic antibody" refers to an antibody used in the treatment of a disease. Therapeutic antibodies can have various mechanisms of action. Therapeutic antibodies can bind to and neutralize the normal function of a target associated with an antigen. For example, a monoclonal antibody that blocks the activity of a protein required for cancer cell survival causes cell death. Another therapeutic monoclonal antibody can bind to and activate the normal function of a target associated with an antigen. For example, a monoclonal antibody can bind to a protein on a cell and trigger an apoptotic signal. Yet another monoclonal antibody can bind to a target antigen that is only expressed on diseased tissue; coupling a toxic payload (effective agent) (such as a chemotherapeutic agent or a radiopharmaceutical) to a monoclonal antibody can create an agent that specifically delivers a toxic payload to diseased tissue, reducing damage to healthy tissue. A "biologically functional fragment" of a therapeutic antibody will exhibit at least one (if not some or all) biological functions attributed to a complete antibody, which at least includes specific binding to a target antigen.

[0154] The term "diagnostic protein" refers to a protein used in the diagnosis of a disease.

[0155] The term "diagnostic antibody" refers to an antibody used as a disease diagnostic reagent. Diagnostic antibodies can bind to a target antigen that is specifically associated with a particular disease or shows elevated expression in a particular disease. Diagnostic antibodies can be used, for example, to detect targets in biological samples from patients, or in diagnostic imaging of disease sites (such as tumors) in patients. A "biologically functional fragment" of a diagnostic antibody will exhibit at least one (if not some or all) biological functions attributed to the intact antibody, which function at least includes specific binding to the target antigen.

[0156] "Purified" means that the molecule is present in a sample at a concentration of at least 80-90% by weight of the sample in which it is contained.

[0157] Purified proteins (including antibodies) are preferably substantially pure, and desirably are substantially homogeneous (ie, free from contaminating proteins, etc.).

[0158] "Substantially pure" protein means that the protein composition comprises at least about 90% (by weight), preferably at least about 95% (by weight), protein based on the total weight of the composition.

[0159] "Substantially homogeneous" protein means that the protein composition comprises at least about 99% protein by weight, based on the total weight of the composition.

[0160] As mentioned above, in certain embodiments, the protein is an antibody. "Antibodies" (Ab) and "immunoglobulins" (Ig) are glycoproteins with the same structural features. While antibodies exhibit binding specificity to specific antigens, immunoglobulins include both antibodies and other antibody-like molecules that generally lack antigen specificity. The latter class of polypeptides is produced at low levels, for example, by the lymphatic system and at elevated levels by myelomas.

[0161] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), polyclonal antibodies, antibody compositions with multiple epitope specificities, bispecific antibodies, diabodies, and single-chain molecules such as scFv molecules, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv).

[0162] The term "monoclonal antibody" when used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical, except for possible mutations that may be present in very small amounts, such as naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a feature of a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies comprising a polypeptide sequence that binds to a target, wherein the target binding polypeptide sequence is obtained by a process including selecting a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a collection of numerous clones such as hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target binding sequence may be further changed, for example, to increase affinity for the target, humanize the target binding sequence, increase its yield in cell culture, reduce its immunogenicity in vivo, create multi-specific antibodies, etc., and antibodies comprising the altered target binding sequence are also monoclonal antibodies of the present invention. Unlike typical polyclonal antibody preparations comprising different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. In addition to their specificity, an advantage of monoclonal antibody preparations is that they are generally free of contamination by other immunoglobulins.

[0163] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al., Nature 256:495 (1975); Harlow et al., Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,2nd ed. 1988; Hammerling et al., at: Monoclonal Antibodies and T-Cell Hybridomas, 563-681, Elsevier, NY, 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34):12467-12472 (2004); Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals having part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; WO 91 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368:856-859(1994); Morrison, Nature368:812-813(1994); Fishwild et al., Nature Biotechnol.14:845-851(1996); Neuberger, Nature Biotechnol.14:826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0164] Monoclonal antibodies herein specifically include "chimeric" antibodies, as well as fragments of such antibodies, so long as they exhibit the desired biological activity, in which a portion of the heavy and / or light chain of the antibody is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, (U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0165] The "humanized" form of non-human (e.g., mouse) antibodies refers to chimeric antibodies that contain sequences derived from non-human immunoglobulins at a minimum. In one embodiment, humanized antibodies refer to immunoglobulins in which the hypervariable region residues in human immunoglobulins (recipient antibodies) are replaced with hypervariable region residues of non-human species (donor antibodies) (such as mice, rats, rabbits, or non-human primates) with desired specificity, affinity, and / or ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues. In addition, humanized antibodies may be included in receptor antibodies or in residues not found in donor antibodies. These modifications may be made to further improve the performance of antibodies. In general, humanized antibodies will include at least one, usually two, substantially entire variable domains as follows, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FR regions of human immunoglobulin sequences. Humanized antibodies will optionally also include at least a portion of immunoglobulin constant regions (Fc), typically the constant regions of human immunoglobulins. For more details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following reviews and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). Humanized antibodies include Primatized TM (Primatized) antibodies in which the antigen-binding region of the antibody is derived from antibodies generated by immunizing macaques with the antigen of interest.

[0166] A "human antibody" is an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and / or has been produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0167] An "affinity matured" antibody is one that has one or more changes in one or more CDR / HVRs of the antibody that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody without these changes. Preferred affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR / HVR and / or framework residues is described in the following literature: Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0168] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of an antibody heavy or light chain. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are generally the most variable parts of an antibody and contain the antigen binding site.

[0169] The term "variable" refers to the fact that certain parts of the variable domain vary widely between antibody sequences and are used for the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in the light and heavy chain variable domains. The relatively highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FRs, most of which adopt a β-pleated sheet conformation, connected by three CDRs that form a loop connection and in some cases form part of the β-pleated sheet structure. The CDRs in each chain are held together very closely by the FRs and contribute to the formation of the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, National Institute of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0170] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0171] Antibodies (immunoglobulins) can be classified into different classes according to the amino acid sequence of their heavy chain constant domain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, for example, Abbas et al., Cellular and Mol. Immunology, 4th ed. (2000). Antibodies can be part of a larger fusion molecule formed by covalent or non-covalent attachment of antibodies to one or more other proteins or peptides.

[0172] The terms "full length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody in substantially intact form rather than an antibody fragment as defined below. The term specifically refers to an antibody whose heavy chain comprises an Fc region.

[0173] An "antibody fragment" comprises only a portion of an intact antibody, wherein the portion retains at least one, up to most or all of the functions normally associated with the portion when present in an intact antibody. In one embodiment, an antibody fragment comprises the antigen binding site of an intact antibody, thus retaining the ability to bind antigen. In another embodiment, an antibody fragment, such as an antibody fragment comprising an Fc region, retains at least one biological function normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, an antibody fragment is a monovalent antibody having an in vivo half-life substantially similar to that of an intact antibody. For example, such an antibody fragment may comprise an antigen binding arm and is linked to an Fc sequence that can confer in vivo stability to the fragment.

[0174] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces F(ab') 2 A fragment that has two antigen-binding sites and is still able to cross-link antigen.

[0175] The Fab fragment contains the heavy chain variable domain and the light chain variable domain, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear free thiol groups. F(ab') 2 Antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them.Other chemical couplings of antibody fragments are also known.

[0176] "Fv" is the smallest antibody fragment that contains a complete antigen binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and heavy chain are combined in a "dimer" structure similar to the two-chain Fv species. It is in this structure that the three CDRs of each variable domain interact with each other and bind to each other in V H -V L The dimer defines an antigen binding site on its surface. The six CDRs together give the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, but the affinity is lower than that of the complete binding site.

[0177] "Single-chain Fv" or "scFv" antibody fragments comprise the V H and V L domains, wherein these domains are present on one polypeptide chain. H With V L The domains further include polypeptide linkers that enable the scFv to form the desired structure for binding to the antigen. For a review of scFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315, 1994.

[0178] The term "diabodies" refers to small antibody fragments with two antigen-binding sites on the same polypeptide chain (V H -V L ) contains linked heavy chain variable domains (V H ) and the light chain variable domain (V L ). By using a linker that is too short to allow the two domains on the same chain to pair, these domains are forced to pair with the complementary domains of another chain, thereby generating two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP404,097; WO93 / 1161; Hudson et al. (2003) Nat. Med. 9:129-134; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134.

[0179] The antibodies may bind to any protein, including but not limited to members of the HER receptor family, such as HER1 (EGFR), HER2, HER3 and HER4; CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD21, CD22, and CD34; cell adhesion molecules, such as LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM and αvβ3 integrin, including its α or β subunit (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors, such as vascular endothelial growth factor (VEGF); IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; and protein C.Other exemplary proteins include growth hormone (GH), including human growth hormone (hGH) and bovine growth hormone (bGH); growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or tissue-type plasminogen activator (t-PA); bombesin; thrombin; tumor necrosis factor-α and -β; enkephalinase; RANTES (regulated upon activation, normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-α); serum albumin, such as human serum albumin (HSA); Muellerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; DNA enzyme; inhibin; activin; hormone or growth factor receptor; integrin; protein A or D; rheumatoid factor; 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 nerve growth factor, such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor, such as aFGF and bFGF F; epidermal growth factor (EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, 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 protein (IGFBP); erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; bone Morphogenetic proteins (BMPs); interferons, such as interferon-α, -β, and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factor (DAF); viral antigens, such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; immunoadhesins; antibodies; and biologically active fragments or variants of any of the above listed polypeptides. Many other antibodies and / or other proteins can be used in accordance with the present invention, and the above list is not intended to limit the present invention.

[0180] The "biologically functional fragment" of an antibody comprises only a portion of an intact antibody, wherein the portion retains at least one, up to most or all functions that are normally associated with the portion when present in the intact antibody. In one embodiment, the biologically functional fragment of an antibody comprises the antigen binding site of the intact antibody, thus retaining the ability to bind antigen. In another embodiment, the biologically functional fragment of an antibody (e.g., comprising an Fc region) retains at least one biological function that is normally associated with the Fc region when present in the intact antibody, such as FcRn binding, antibody half-life regulation, ADCC function, and complement binding. In one embodiment, the biologically functional fragment of an antibody is a monovalent antibody having an in vivo half-life that is substantially similar to that of an intact antibody. For example, such a biologically functional fragment of an antibody may comprise an antigen binding arm and is connected to an Fc sequence that can confer in vivo stability to the fragment.

[0181] The terms "thioredoxin inhibitor" and "Trx inhibitor" are used interchangeably and include all agents and measures that effectively inhibit thioredoxin activity. Thus, thioredoxin (Trx) inhibitors include all agents and measures that block any component of the Trx, G6PD and / or hexokinase system. In this context, "inhibition" includes complete elimination (blocking) and reduction of thioredoxin activity, and thus complete or partial elimination of disulfide bond reduction in proteins (such as antibodies).

[0182] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that would interfere with the research, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight of the antibody as determined, for example, by the Lowry method, and in some embodiments, greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using, for example, a spinning cup sequencer, or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions and using, for example, Coomassie blue or silver stain. Since at least one component of the antibody's natural environment will not be present, an isolated antibody includes the antibody in situ within a recombinant cell. However, an isolated antibody will typically be prepared by at least one purification step.

[0183] The terms "Protein A" and "ProA" are used interchangeably herein to encompass Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and Protein A that retains the protein A having C H 2 / C HProtein A is a variant of the ability of proteins to bind to a 3 region (such as an Fc region). Protein A can be purchased from Repligen, GE Healthcare and Fermatech. Protein A is generally immobilized on a solid support material. The term "Protein A" also refers to an affinity chromatography resin or column containing a chromatographic solid support matrix to which Protein A is covalently attached.

[0184] The term "chromatography" refers to the process of separating solutes of interest from other solutes in a mixture based on differences in the rates at which the various solutes in the mixture migrate under the influence of a mobile phase or through a stationary medium in a bind and elute process.

[0185] The terms "affinity chromatography" and "protein chromatography" are used interchangeably herein to refer to a protein separation technique in which a protein of interest or an antibody of interest is reversibly and specifically bound to a biospecific ligand. Preferably, the biospecific ligand is covalently attached to a chromatographic solid phase material and is accessible to the protein of interest in solution when the solution contacts the chromatographic solid phase material. During the chromatography step, the protein of interest (e.g., an antibody, enzyme, or receptor protein) retains its specific binding affinity for the biospecific ligand (e.g., an antigen, substrate, cofactor, or hormone), while other solutes and / or proteins in the mixture do not appreciably or specifically bind to the ligand. The binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to flow through the chromatographic medium while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then eliminated from the immobilized ligand in an active form using low pH, high pH, ​​high salt, competitive ligands, etc., and flows through the chromatography column with the elution buffer, free of the contaminating proteins or protein impurities that were previously allowed to flow through the column. Any component can be used as a ligand for purification of its corresponding specific binding protein (eg, antibody).

[0186] The terms "non-affinity chromatography" and "non-affinity purification" refer to a purification process in which affinity chromatography is not utilized. Non-affinity chromatography includes chromatographic techniques that rely on non-specific interactions between a molecule of interest (such as a protein, e.g., an antibody) and a solid matrix.

[0187] "Cation exchange resin" refers to a solid phase that is negatively charged and therefore has free cations for exchange with cations in an aqueous solution flowing through the solid phase. The negatively charged ligand attached to the solid phase to form the cation exchange resin can be, for example, a carboxylate or sulfonate. Commercial cation exchange resins include carboxymethylcellulose, sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW from GE Healthcare), and sulfonates. TM Or SP-SEPHAROSE HIGHPERFORMANCE TM) and sulfonyl groups immobilized on agarose (e.g. S-SEPHAROSE FASTFLOW from GE Healthcare TM ). "Mixed-mode ion exchange resin" refers to a solid phase covalently modified with cationic, anionic, and hydrophobic modules. Commercial mixed-mode ion exchange resins are BAKERBOND ABX TM (JT Baker, Phillipsburg, NJ), which contains weak cation exchange groups, low concentrations of anion exchange groups, and hydrophobic ligands attached to a silica solid support medium.

[0188] The term "anion exchange resin" is used herein to refer to a positively charged solid phase, for example having one or more positively charged ligands attached thereto, such as quaternary amino groups. Commercially available anion exchange resins include DEAE cellulose, QAESEPHADEX TM and FAST Q SEPHAROSE TM (GE Healthcare).

[0189] "Buffer" refers to a solution that resists pH changes through the action of its acid-base paired components. Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975) describes a variety of buffers that can be used, depending on, for example, the desired buffer pH. In one embodiment, the buffer has a pH in the range of about 2 to about 9, or about 3 to about 8, or about 4 to about 7, or about 5 to about 7. Exemplary examples of buffers that will control the pH within this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, and combinations of these buffers.

[0190] "Loading buffer" refers to a buffer for loading a composition comprising a polypeptide molecule of interest and one or more impurities onto an ion exchange resin. The loading buffer has a conductivity and / or pH such that the polypeptide molecule of interest (and generally one or more impurities) binds to the ion exchange resin or allows the protein of interest to flow through the column while the impurities bind to the resin.

[0191] "Intermediate buffer" refers to a buffer used to elute one or more impurities from an ion exchange resin prior to eluting a polypeptide molecule of interest. The conductivity and / or pH of the intermediate buffer allows one or more impurities to elute from the ion exchange resin without eluting a significant amount of the polypeptide of interest.

[0192] The term "wash buffer" as used herein refers to a buffer used to wash or re-equilibrate an ion exchange resin prior to eluting a polypeptide molecule of interest. Conveniently, the wash buffer and the loading buffer may be the same, but this is not required.

[0193] The "elution buffer" is used to elute the polypeptide of interest from the solid phase. The conductivity and / or pH of the elution buffer is such that the polypeptide of interest is eluted from the ion exchange resin.

[0194] A "regeneration buffer" can be used to regenerate an ion exchange resin so that it can be reused. The regeneration buffer has the conductivity and / or pH required to remove substantially all impurities and the polypeptide of interest from the ion exchange resin.

[0195] The phrase "substantially similar" or "substantially identical" when used herein means a sufficiently high degree of similarity between two numerical values ​​(e.g., one involving an antibody of the invention and the other involving a reference / comparison antibody) so that a person skilled in the art will consider that the difference between the two numerical values ​​has little or no biological and / or statistical significance within the context of the biological property measured by the numerical value (e.g., Kd value). As a function of the reference / comparison value, the difference between the two numerical values ​​is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and / or less than about 10%.

[0196] The phrase "substantially reduced" or "substantially different" as used herein (in relation to an amount or value, rather than referring to a chemical process of reduction) means a sufficiently high degree of difference between two values ​​(usually one associated with a molecule and the other associated with a reference / comparator molecule) that a person skilled in the art would consider the difference between the two values ​​to be statistically significant within the context of the biological property measured by the value (e.g., Kd value). The difference between the two values ​​is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value of the reference / comparator molecule.

[0197] The term "vector" when used herein means a nucleic acid molecule capable of transporting other nucleic acids connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which other DNA segments can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating along with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used forms of vectors.

[0198] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after comparing the sequences and introducing gaps, if necessary, to obtain the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparisons for the purpose of determining percentage amino acid sequence identity can be performed in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for comparing sequences, including any algorithm required to obtain maximum comparison over the entire length of the compared sequences. However, for the purposes of the present invention, % amino acid sequence identity values ​​are obtained using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted to the U.S. Copyright Office (US Copyright Office, Washington DC, 20559) together with user documentation, and is registered with U.S. Copyright Registration No. TXU510087. The public can obtain the ALIGN-2 program from Genentech (South San Francisco, California), or can compile from source code. The ALIGN2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged.

[0199] In the case of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0200] Multiply the fraction X / Y by 100

[0201] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0202] Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0203] "Percentage (%) nucleic acid sequence identity" is defined as the percentage of nucleotides identical with the nucleotides in the reference factor D coding sequence in the candidate sequence after comparing the sequences and introducing a gap when necessary to obtain the maximum percentage sequence identity. Those skilled in the art can determine the appropriate parameters for measuring the contrast, including any algorithm required for obtaining the maximum contrast for the full length of the compared sequences. The sequence identity is then calculated relative to the longer sequence, that is, even if a shorter sequence shows 100% sequence identity with a longer sequence part, the overall sequence identity will also be less than 100%.

[0204] "Treatment" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include those already suffering from the disorder as well as those in whom the disorder is to be prevented. "Treatment" or "treatment" herein encompasses both alleviation of the disease and alleviation of the signs and symptoms of a particular disease.

[0205] "Disorder" refers to any condition that would benefit from protein treatment. This includes chronic and acute conditions, or diseases that predispose the mammal to the pathological condition in question. Non-limiting examples of conditions to be treated herein include cancer and allergies.

[0206] For therapeutic purposes, "mammal" refers to any animal classified as mammals, including humans, non-human higher primates, other vertebrates, domestic and livestock animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0207] "Interfering RNA" or "small interfering RNA (siRNA)" refers to a double-stranded RNA molecule that is less than about 30 nucleotides in length and reduces the expression of a target gene. Interfering RNAs can be identified and synthesized using known methods (Shi Y., Trends in Genetics 19(1):9-12 (2003); WO 2003 / 056012; WO 2003 / 064621), and siRNA libraries can be obtained commercially, for example, from Dharmacon, Lafayette, Colorado. Typically, siRNAs can be successfully designed to target the 5' end of a gene.

[0208] II. Compositions and Methods of the Invention

[0209] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of molecular biology, etc., which are within the technical scope of the art. Such techniques are fully explained in the literature. See, e.g. Molecular Cloning: ALaboratoryManual , (J.Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989); Current Protocols in Molecular Biology (F. Ausubel et al., eds., 1987updated); EssentialMolecularBiology (T.Brown ed., IRL Press 1991); GeneExpressionTechnology (Goeddel.,Academic Press 1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwell et al., eds., Bartlett Publ. 1990); Gene Transfer and Expression (M.Kriegler, Stockton Press1990); Recombinant DNA Methodology II (R.Wu et al., eds., Academic Press 1995); PCR: A Practical Approach (M.McPherson et al., IRL Press at Oxford University Press1991); Oligonucleotide Synthesis (M. Gaited., 1984); Cell Culture for Biochemists (R.Adams ed., Elsevier Science Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology(M.Butler ed.,1991); Animal Cell Culture (J.Pollard et al.,eds.,Humana Press1990); Culture of Animal Cells ,2 nd Ed.(R.Freshney et al.,eds.,Alan R.Liss 1987); Flow Cytometry and Sorting (M.Melamed et al.,eds.,Wiley-Liss 1990);the series Methods in Enzymology (Academic Press,Inc.);Wirth M.and Hauser H.(1993); Immunochemistry in Practice ,3rd edition,A.Johnstone&R.Thorpe,BlackwellScience,Cambridge,MA,1996; Techniques in Immunocytochemistry ,(G.Bullock&P.Petrusz eds.,Academic Press 1982,1983,1985,1989);Handbook of ExperimentalImmunology,(D.Weir&C.Blackwell,eds.); Current Protocols in Immunology (J.Coliganet al.,eds.1991); Immunoassay (E.P.Diamandis&T.K.Christopoulos,eds.,AcademicPress,Inc.,1996);Goding(1986) Monoclonal Antibodies:PrinciplesandPractice (2ded)Academic Press,New York;Ed Harlow and David Lane, AntibodiesA laboratoryManual ,Cold Spring Harbor Laboratory,Cold Spring Harbor,New York,1988; AntibodyEngineering ,2 nd edition(C.Borrebaeck,ed.,Oxford University Press,1995);and the series Annual Review of Immunology ;the series Advances inImmunology 。

[0210] 1. Prevent disulfide bond reduction

[0211] The present invention concerns methods for preventing the reduction of disulfide bonds of proteins during recombinant production. Specifically, the present invention concerns methods for preventing the reduction of disulfide bonds of recombinant proteins during post-fermentation processing. The methods of the present invention are particularly valuable for large-scale production (such as manufacturing scale) of proteins containing disulfide bonds. In one embodiment, the present invention is useful for large-scale protein production with a scale exceeding 5,000 L.

[0212] It has been found experimentally that disulfide bond reduction occurs during processing of harvested cell culture fluid (HCCF) generated during the manufacture of recombinant proteins containing disulfide bonds. Typically, this reduction is observed after cell lysis, especially mechanical cell lysis during the harvesting operation, when it reaches a certain threshold, such as, for example, about 30% to about 70%, or about 40% to about 60%, or about 50% to about 60% of total cell lysis. This threshold will vary, depending on the nature of the protein (e.g., antibody) produced, the recombinant host, the production system, the production parameters used, etc., and can be easily determined experimentally.

[0213] In theory, such reduction can result from a variety of factors and conditions during the manufacturing process, and from a variety of reducing agents. The present invention is based, at least in part, on the recognition that the root cause of this reduction is the active thioredoxin (Trx) or thioredoxin-like system in HCCF.

[0214] The Trx enzyme system (composed of Trx, thioredoxin reductase (TrxR) and NADPH) is a hydrogen donor system for the reduction of disulfide bonds in proteins. Trx is a small monomeric protein with a CXXC active site motif that catalyzes many redox reactions via thiol-disulfide exchange. Oxidized Trx can be reduced by NADPH via TrxR. Reduced Trx can then catalyze the reduction of disulfides in proteins. The NADPH required for the thioredoxin system is provided via reactions in the pentose phosphate pathway and glycolysis. The results presented herein demonstrate that NADPH, which is required for the activity of the Trx system, is provided by the activity of glucose-6-phosphate dehydrogenase (G6PD), which generates NADPH from glucose and ATP via hexokinase (see Figure 4 ). Upon cell lysis, these cellular enzymes (Trx system, G6PD, and hexokinase) are released into CCF along with their substrates, allowing reduction to occur. Thus, disulfide reduction can be prevented by inhibitors of the Trx enzyme system or upstream enzyme systems that provide components for the active Trx system (such as G6PD and hexokinase activity).

[0215] For further details on these enzyme systems or other details on protein production see, for example: Babson, AL and Babson, SR (1973) Kinetic Colorimetric Measurement of Serum Lactate Dehydrogenase Activity. Clin.Chem. 19:766-769; Michael W. Laird et al., “Optimization of BLyS Production and Purification from Eschericia coli,” Protein Expression and Purification 39:237-246(2005); John C.Joly et al., "Overexpression of Eschericia coli Oxidoreductases Increases RecombinantInsulin-like Growth Factor-I Accumulation," Proc.Natl.Acad.Sci.USA 95:2773-2777(March 1998); Dana C.Andersen et al., "Production Technologies forMonoclonal Antibodies and Their Fragments,” Current Opinion inBiotechnology 15:456-462(2004); Yariv Mazor et al., "Isolation ofEngineered,Full-lengthAntibodies from Libraries Expressed in Escherichia coli," Nature Biotech. 25,563-565(01Jun 2007); Laura C.Simmons et al., "Expression of Full-length Immunoglobulins in Escherichia coli: Rapid and Efficient Production ofAglycosylated Antibodies," Journal of Immunological Methods263:133-147(2002);Paul H.Bessette et al.,“Efficient Folding of Proteins with MultipleDisulfideBonds in the Escherichia coli cytoplasm,”Proc.Natl.Acad.Sci.96(24):13703-08(1999);Chaderjian,W.B.,Chin,E.T.,Harris,R.J.,and Etcheverry,T.M.,(2005)“Effect of copper sulfate on performance of a serum-free CHO cellcultureprocess and the level of free thiol in the recombinant antibodyexpressed,” Biotechnol.Prog.21:550-553;Gordon G.,Mackow M.C.,and Levy H.R.,(1995)“On the mechanism of interaction of steroids with human glucose 6-phosphatedehydrogenase,”Arch.Biochem.Biophys.318:25-29;Gromer S.,Urig S.,andBecker K.,(2004)“The Trx System-From Science to Clinic,”MedicinalResearchReviews,24:40-89;Hammes G.G.and Kochavi D.,(1962a)“Studies ofthe EnzymeHexokinase.I.Steady State Kinetics at pH 8,”J.Am.Chem.Soc.84:2069-2073;HammesG.G.and Kochavi D.,(1962b)“Studies of the EnzymeHexokinase.III.The Role ofthe Metal Ion,”J.Am.Chem.Soc.84:2076-2079;Johansson C.,Lillig C.H.,andHolmgren A.,(2004)“Human MitochondrialGlutaredoxin Reduces S-GlutathionylatedProteins with High Affinity AcceptingElectrons from Either Glutathione orThioredoxin Reductase,”J.Biol.Chem.279:7537-7543;Legrand,C.,Bour,J.M.,Jacob,C.,Capiaumont J.,Martial,A.,Marc,A.,Wudtke,M.,Kretzmer,G.,Demangel,C.,Duval,D.,and Hache J.,(1992)“Lactate Dehydrogenase(LDH)Activity of the NumberofDead Cells in theMedium of Cultured Eukaryotic Cells as Marker,”J.Biotechnol.,25:231-243;McDonald,M.R.,(1955)“Yeast Hexokinase:ATP+Hexose-->Hexose-6-phosphate+ADP,”Methods in Enzymology,1:269-276,AcademicPress,NY;Sols,A.,DelaFuente,G.,Villar-Palasi,C.,and Asensio,C.,(1958)“SubstrateSpecificity and Some Other Properties of Bakers'Yeast Hexokinase,”BiochimBiophys Acta 30:92-101;Kirkpatrick D.L.,Kuperus M.,DowdeswellM.,Potier N.,Donald L.J.,Kunkel M.,Berggren M.,Angulo M.,and Powis G.,(1998)“Mechanisms ofinhibition of the Trx growth factor system by antitumor2-imidazolyldisulfides,”Biochem.Pharmacol.55:987-994;Kirkpatrick D.L.,Watson S.,KunkelM.,Fletcher S.,Ulhaq S.,and Powis G.,(1999)“Parallel syntheses of disulfideinhibitors of the Trx redox system as potential antitumor agents,”AnticancerDrug Des.14:421-432;Milhausen,M.,and Levy,H.R.,(1975)“Evidence for anEssential Lysine in G6PD from Leuconostoc mesenteroides,”Eur.J.Biochem.50:453-461; Pleasants, JC, Guo, W., and Rabenstein, DL, (1989) "Acomparative study of the kinetics of selenol / diselenide and thiol / disulfide exchangereactions," J.Am.Chem.Soc.111:6553-6558; Whitesides, GM, Lilburn, JE, and Szajewski, RP, (1977) "Rates of thioldisulfide interchange reactions between mono-and dithiols and Ellman's reagent," J.Org.Chem.42:332-338; and Wipf P., Hopkins TD, Jung JK, Rodriguez S., Birmingham A., Southwick EC, Lazo JS, and Powis G, (2001) “New inhibitors of the Trx–TrxR system based on anaphthoquinone spiroketal natural product lead,". Bioorg.Med.Chem.Lett .11:2637-2641.

[0216] According to one aspect of the invention, disulfide bond reduction can be prevented by blocking any component of the Trx, G6PD and hexokinase enzyme systems. Inhibitors of these enzyme systems are collectively referred to herein as "thioredoxin inhibitors" or "Trx inhibitors". Typically, Trx inhibitors are added to cell culture fluid (CCF) containing recombinant host cells and culture fluid and / or harvested cell culture fluid (HCCF) obtained after harvesting by centrifugation, filtration, or similar separation methods. HCCF lacks intact cells, but typically contains host cell proteins and other contaminants, including DNA, which are removed in subsequent purification steps. Thus, Trx inhibitors can be added before and / or during harvest, preferably before harvest.

[0217] Alternatively or additionally, other non-specific methods may be used to prevent disulfide bond reduction after fermentation during recombinant production of recombinant proteins, such as air sparging or pH adjustment. Certain reduction inhibition methods contemplated herein are listed in Table 1 below.

[0218] Table 1: Reduction Inhibition Methods

[0219]

[0220]

[0221] 1 Apply to CCF before harvest or to HCCF immediately after harvest.

[0222] 2 Not yet commercialized.

[0223] "Trx inhibitors" for use in the methods of the present invention include, but are not limited to (1) direct inhibitors of Trx, such as alkyl-2-imidazolyl disulfides and related compounds (e.g., 1-methylpropyl-2-imidazolyl disulfide) (Kirkpatrick et al., 1998 and 1999, supra) and naphthoquinone spiroketal derivatives (e.g., palmarumycin CP 1 )(Wipf et al., 2001, supra); (2) specific inhibitors of TrxR, including gold complexes such as aurothioglucose (ATG) and aurothiomalate (ATM) (see, e.g., review by Gromer et al., 2004), which are examples of irreversible inhibitors of TrxR; (3) metal ions such as Hg 2 +、Cu 2 +、Zn 2 +、Co 2 +, and Mn 2 +, which can readily form complexes with thiols and selenols and thus can be used as inhibitors of TrxR or Trx in embodiments of the present invention; (4) inhibitors of G6PD, such as, for example, pyridoxal-5'-phosphate and 1-fluoro-2,4-dinitrobenzene (Milhausen and Levy 1975, supra), certain steroids, such as dehydroepiandrosterone (DHEA) and epiandrosterone (EA) (Gordon et al., 1995, supra); and (4) inhibitors of hexokinase activity (thereby generating G6P for G6PD), including metal ions (e.g., Mg 2+) chelating agents (such as EDTA) and compounds that react with SH groups, sorbose-1-phosphate, polyphosphoric acid, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methyl glucose, xylose and lyxose (Sols et al., 1958, supra; McDonald, 1955, supra); other hexokinase inhibitors are disclosed in U.S. Pat. No. 5,854,067, entitled "Hexokinase Inhibitors". It should be understood that these inhibitors are listed by way of example only. Other Trx inhibitors exist and can be used alone or in various combinations in the methods of the present invention.

[0224] "Trx inhibitors" for use in the methods of the present invention also include agents that reduce or prevent the reduction of recombinantly produced antibodies or proteins by reducing the levels of enzymes of the Trx system, pentose phosphate pathway, or hexokinase at various points during the production activity. In some embodiments, the reduction in enzyme levels can be achieved by using targeted siRNA, antisense nucleotides, or antibodies. In order to design targeted siRNA or antisense nucleotides for genes found in CHO cells, these gene sequences can be obtained from public databases to select sequences for targeting enzymes in different organisms. For examples of genes for E. coli and mouse Trx systems, see Example 9 below.

[0225] In addition to the use of the inhibitors described above, in certain embodiments of the present invention, it is also possible to prevent the reduction of the recombinant protein to be purified by bubbling air into the HCCF to maintain the oxidative redox potential in the HCCF. This is an indirect measure that can deplete glucose, G6P, and NADPH by continuously oxidizing and reducing Trx and TrxR. Bubbling air into the HCCF tank can be implemented, for example, with an air flow of about 100 liters per minute to about 200 liters per minute, such as, for example, 150 liters per minute. Bubbling air can be implemented to reach an end point saturation percentage; for example, air bubbling can be continued until the HCCF is about 100% saturated with air, or air bubbling can be continued until the HCCR is about 30% saturated with air, or until between about 100% saturated with air and about 30% saturated with air. The minimum dissolved oxygen (dO) required for the desired inhibitory effect is 2 ) also depends on the antibody or other recombinant protein produced. Thus, for example, about 10% dO 2 (or a continuous flow of about 10 sccm) would have the desired effect during the production of antibody 2H7 (variant A), while Apomab may require a higher (about 30%) dO 2 .

[0226] In other embodiments of the invention, another indirect method useful for blocking the reduction of recombinant proteins is to lower the pH of HCCF. This embodiment takes advantage of the particularly slow thiol-disulfide exchange at lower pH values ​​(Whitesides et al., 1977, supra; Pleasants et al., 1989, supra). Therefore, the activity of the Trx system at pH values ​​below 6 is significantly lower, thus inhibiting the reduction of recombinant proteins (such as ocrelizumab).

[0227] Indirect approaches can also be combined with each other and / or with the use of one or more Trx inhibitors.

[0228] After the cell culture process is completed, preferably before harvesting for CCF or immediately after harvesting for HCCF, disulfide bond reduction can be inhibited (i.e., partially or completely blocked) by using one or more Trx inhibitors and / or applying indirect methods. The optimal application time and mode and effective amount depend on the nature of the protein to be purified, the recombinant host cell, and the specific production method used. The determination of the optimal parameters is well within the technical scope of ordinary technicians in this field.

[0229] For example, in mammalian cell culture processes, such as the CHO antibody production process described in the Examples herein, if copper sulfate (CuSO 4 , pentahydrate or anhydrous form) as a Trx inhibitor, then add it to supplement CCF or HCCF in a concentration range of about 5 μM to about 100 μM, such as about 10 μM to about 80 μM, preferably about 15 μM to about 50 μM. Since some cell cultures already contain copper (e.g., about 0.04 μM CuSO in the CHO cell culture used in the Examples herein), the amount of copper added to the CCF or HCCF is not limited to the amount of copper added to the CCF or HCCF. 4 ), so this amount is in addition to the copper already present in the cell culture (if any). Any copper(II) salt can be used instead of CuSO 4 , as long as solubility is not an issue. For example, copper acetate and copper chloride (both of which are soluble in water) can be used in place of CuSO 4 The minimum effective concentration may also depend on the antibody generated and the stage at which the inhibitor is used. Thus, for example, when copper sulfate was added before lysis, the minimum effective concentration for antibody 2H7 (variant A) was about 30 μM, for Apomab was about 75 μM, and for antibody variant C (see Table 2) was about 50 μM. When copper sulfate was added in CC medium, the minimum effective concentration for antibody 2H7 (variant A) was about 15 μM, for Apomab was about 25 μM, and for antibody variant C was about 20 μM. A typical minimum CuSO 4 The inhibitor concentration was twice the Trx concentration (or Trx equivalent).

[0230] EDTA can be used in a wide range of concentrations, depending on the degree of cell lysis, the recombinant host cells used, and other parameters of the production process. For example, when using CHO or other mammalian cells, typically, EDTA can be added at a concentration between about 5mM to about 60mM, such as about 10mM to about 50mM or about 20mM to about 40mM, depending on the degree of cell lysis. For lower degrees of cell lysis, lower EDTA concentrations will be sufficient, while for about 75%-100% cell lysis, the required EDTA concentration is higher, such as, for example, about 20mM to about 40mM. The minimum effective concentration may also depend on the antibody generated. Thus, for example, for antibody 2H7 (variant A), the minimum effective EDTA concentration is about 10mM.

[0231] Typically, DHEA is effective as a Trx inhibitor at lower concentrations, such as, for example, in a concentration range of about 0.05 mM to about 5 mM, preferably about 0.1 mM to about 2.5 mM.

[0232] Other Trx inhibitors, such as aurothioglucose (ATG) and aurothiomalate (ATM), inhibit disulfide bond reduction in the μM concentration range. Thus, for example, ATG or ATM can be added at a concentration between about 0.1 mM and about 1 mM. Although the minimum inhibitory concentration varies with actual conditions, for ATG and ATM, it is typically around 4 times the TrxR concentration.

[0233] Note that all inhibitors can be used in excess, therefore, it is not always necessary to know the amount of Trx or TrxR in the system.

[0234] In a preferred embodiment, the mammalian cells used in the manufacturing process are Chinese hamster ovary (CHO) cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)). Other mammalian cells include, but are not limited to, monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; human hepatoma line (Hep G2); and myeloma or lymphoma cells (e.g., Y0, J558L, P3 and NS0 cells) (see U.S. Pat. No. 5,807,715).

[0235] A preferred cell for producing the polypeptides herein is the CHO cell line DP12 (CHO K1dhfr - This is one of the best known CHO cell lines and is widely used in laboratory practice (see, for example, EP 0,307,247 published on March 15, 1989). In addition, other CHO-K1 (dhfr - ) cell lines and can be used in the methods of the present invention.

[0236] Mammalian cells for producing peptides, polypeptides and proteins can be cultured in a variety of culture media. Commercial culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM, Sigma) are suitable for culturing cells. In addition, any culture medium described in the following literature can be used as a culture medium for cells: Ham and Wallace (1979), Meth.in Enz.58:44; Barnes and Sato (1980), Anal.Biochem.102:255; U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90 / 03430; WO 87 / 00195; U.S. Patent No. Re. 30,985; or U.S. Patent No. 5,122,469, all of the disclosures of which are incorporated herein by reference. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin TM Drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that will be known to those skilled in the art. The culture conditions (such as temperature, pH, etc.) are those previously selected for expression in the cell, as will be apparent to the ordinarily skilled artisan.

[0237] The scheme for generating, recovering and purifying recombinant antibodies in mammalian (such as CHO) cells may include the following steps: cells can be cultivated in a stirred tank bioreactor system, and a fed-batch culture protocol is adopted. In a preferred fed-batch culture, mammalian cells and culture medium are first provided to the culture vessel, and other culture nutrients are supplemented to the culture continuously or in discrete increments during the incubation period, with or without regular cell and / or product harvests before the culture is terminated. Fed-batch culture may include, for example, semi-continuous fed-batch culture, in which all cultures (including cells and culture medium) are regularly removed and replaced with fresh culture medium. The difference between fed-batch culture and single batch culture is that all components (including cells and all culture nutrients) of cell culture are provided to the culture vessel at the beginning of the incubation process. The difference between fed-batch culture and perfusion culture is that there is no supernatant removed from the culture vessel during the process (in perfusion culture, cells are restricted in the culture by, for example, filtering, encapsulating, anchoring to microcarriers, etc., and culture medium is introduced into the culture vessel continuously or intermittently and the culture vessel is removed from the culture vessel).

[0238] In addition, the cells of the culture can be propagated according to any scheme or custom suitable for the specific cells and specific production plans covered. Therefore, a single step or multi-step culture protocol can be adopted. In a single step culture, cells are inoculated into a culture environment, and the process is adopted in a single production stage of cell culture. Alternatively, multi-stage culture can be used. In multi-stage culture, cells can be cultured in many steps or stages. For example, cells can be cultured in the first step or growth stage, wherein cells (possibly taken from storage) are inoculated into a suitable culture medium for promoting growth and high viability. In the growth stage, cells are maintained for a suitable period of time by adding fresh culture medium to cell culture.

[0239] In certain embodiments, fed-batch or continuous cell culture conditions can be designed to enhance the growth of mammalian cells during the growth phase of cell culture. During the growth phase, the conditions and time for culturing cells maximize growth. Culture conditions (such as temperature, pH, dissolved oxygen (dO) 2 ) etc.) are those that are suitable for a particular host and will be apparent to a person of ordinary skill. In general, an acid (e.g., CO 2 ) or bases (such as Na 2 CO 3 The pH is adjusted to a level between about 6.5 and 7.5 using dHO or NaOH. A suitable temperature range for culturing mammalian cells such as CHO cells is between about 30°C and 38°C, and a suitable dHO 2 Between 5-90% air saturation.

[0240] At a particular stage, the cells may be used to inoculate a production phase or step of cell culture. Alternatively, as described above, the production phase or step may be continuous with the inoculation or growth phase or step.

[0241] The cell culture environment during the production phase of cell culture is typically controlled. Thus, if glycoproteins are produced, factors that affect the cell specific productivity of mammalian cells can be manipulated so that the desired sialic acid content is achieved in the resulting glycoprotein. In a preferred aspect, the production phase of the cell culture process is preceded by a transition phase of cell culture, which involves parameters for the production phase of cell culture. For further details on this process, see U.S. Pat. No. 5,721,121 and Chaderjian et al., Biotechnol.Prog. 21(2):550-3(2005), the entire disclosure of which is expressly incorporated herein by reference.

[0242] After fermentation, purify protein. The procedure for purifying protein from cell debris first depends on the position of protein expression. Some proteins can be directly secreted into the surrounding culture medium from cells, and other proteins are prepared intracellularly. For the latter class of proteins, the first step of the purification process involves lysing cells, which can be carried out by a variety of methods, including mechanical shearing, osmotic shock or enzyme treatment. This type of destruction releases the entire contents of the cell into the homogenate, and generates subcellular fragments that are difficult to remove due to the small size. These are generally removed by differential centrifugation or by filtration. Due to the natural death of cells and the release of intracellular cellular proteins and components in the process of protein production operation, there is the same problem in the directly secreted protein, but to a lesser extent.

[0243] Once a clear solution containing a protein of interest is obtained, a combination of different chromatography steps is usually used to attempt to separate it from other proteins produced by the cell. These techniques are based on the charge, hydrophobicity degree or size of the protein and their mixture is separated. Each of these techniques can utilize several different chromatography resins, thereby allowing for precise tailoring of purification schemes for the specific protein involved. The essence of each of these separation methods is to enable proteins to move downward along a long column at different rates, thereby achieving the physical separation that increases as they move further downward along the column, or to enable protein selective adhesion to a separation medium, and then to use different solvent differential elutions. In some cases, when impurity specificity adheres to a column and the protein of interest does not adhere to the column, the desired protein is separated from the impurity, that is, the protein of interest is present in the "effluent". In this way, the cell culture purification of recombinant proteins from mammalian cells may include one or more affinity (e.g., protein A) and / or ion exchange chromatography steps.

[0244] Ion exchange chromatography is a chromatographic technique commonly used to purify proteins. In ion exchange chromatography, the charged patches on the surface of the solute are attracted by the opposite charges attached to the chromatography matrix, provided that the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e., conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH, thereby changing the charge of the solute, is another way to achieve solute elution. Changes in conductivity or pH can be gradual (gradient elution) or stepwise (step elution). In the past, these changes were gradual; that is, pH or conductivity increased or decreased in a single direction.

[0245] For further details on industrial purification of therapeutic antibodies see, for example, Fahrner et al., Biotechnol.Genet.Eng.Rev. 18:301-27 (2001), the entire disclosure of which is expressly incorporated herein by reference.

[0246] In addition to mammalian cells, other eukaryotic organisms can be used as cells for expressing recombinant proteins. For expression in yeast cells (such as commonly used baker's yeast or Saccharomyces cerevisiae), suitable vectors include episomal replicating vectors based on 2 micron plasmids, integration vectors, and yeast artificial chromosome (YAC) vectors. Other yeasts suitable for recombinant production of heterologous proteins include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 2: 968 975 (1991)) such as, for example, Kluyveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 737 (1983)), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 12,424). 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265 278 (1988)); Candida; Trichoderma reesia (EP 402,226); 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci.USA, 76: 5259 5263 (1979)); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi, such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284 289 (1983); Tilburn et al., Gene, 26: 205 221 (1983); Yelton et al., Gene, 26: 205 221 (1983); al., Proc. Natl. Acad. Sci. USA, 81:1470 1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475 479 (1985)). Methylotrophic yeasts are suitable herein, including but not limited to yeasts capable of growing on methanol, selected from the following genera: Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of exemplary specific species of such yeasts can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). Expression systems for the listed and other yeasts are well known in the art and / or are commercially available. .

[0247] For expression in insect cells (such as Sf9 cells), suitable vectors include baculovirus vectors. For expression in plant host cells (particularly dicotyledonous hosts, such as tobacco), suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.

[0248] The method of the present invention also extends to the cultivation of prokaryotic cells. Prokaryotic cells suitable for expressing antibodies and other proteins to be protected by the means of the present invention include archaea and true bacteria, such as gram-negative or gram-positive organisms. Examples of useful bacteria include Escherichia (Escherichia) (e.g., Escherichia coli / Escherichia coli (E. coli)), Bacilli (Bacilli) (e.g., Bacillus subtilis (B. subtilis)), Enterobacteria (Enterobacteria), Pseudomonas (Pseudomonas) species (e.g., Pseudomonas aeruginosa (P. aeruginosa)), Salmonella typhimurium (Salmonella typhimurium), Serratia marcescans (Serratia marcescans), Klebsiella (Klebsiella), Proteus (Proteus), Shigella (Shigella), Rhizobia (Rhizobia), Vitreoscilla (Vitreoscilla) or Paracoccus (Paracoccus). In one embodiment, gram-negative cells are used. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC deposit No. 27,325) and derivatives thereof, including strain 33D3 (U.S. Pat. No. 5,639,635) having the genotype W3110ΔfhuA (ΔtonA) ptr3 lac Iq lacL8ΔompTΔ (nmpc-fepE) degP41 kanR. Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and non-limiting. Methods for constructing derivatives of any of the above-mentioned bacteria having a specified genotype are known in the art, see, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select suitable bacteria taking into account the replicability of the replicon in the bacterial cell. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to provide the replicon, Escherichia coli, Serratia, or Salmonella species may be suitable for use as hosts. Generally, the host cell should secrete minimal amounts of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors in the cell culture.

[0249] Methods for producing, recovering and purifying recombinant proteins from non-mammalian cells are also well known in the art. If the polypeptide is produced in a non-mammalian cell, e.g., a microorganism such as a fungus or E. coli, the polypeptide is recovered either inside the cell or in the periplasmic space (Kipriyanov and Little, 1996). Molecular Biotechnology ,12:173 201(1999);Skerra and Pluckthun, Science , 240:1038 1040 (1988)). Therefore, proteins must be released from cells into the extracellular medium by extraction, such as cell lysis. Such disruption releases the entire contents of the cells into the homogenate and additionally generates subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration.

[0250] Cell lysis is usually achieved using mechanical disruption techniques, such as homogenization or head milling. Although generally effective in liberating the protein of interest, such techniques have several disadvantages (Engler, Protein Purifica tionProcessEngineering , Harrison eds., 37 55 (1994)). The temperature increase that often occurs during processing can lead to protein inactivation. In addition, the resulting suspension contains a wide spectrum of contaminating proteins, nucleic acids, and polysaccharides. Nucleic acids and polypeptides increase solution viscosity, which may be accompanied by subsequent processing by centrifugation, cross-flow filtration, or chromatography. The complex association of these contaminants with the protein of interest can complicate the purification process and result in unacceptably low yields. Improved methods for purifying heterologous proteins from microbial fermentation broths or homogenates are described, for example, in U.S. Pat. No. 7,169,908, the entire disclosure of which is expressly incorporated herein by reference.

[0251] It is emphasized that the fermentation, recovery and purification methods described herein are for illustrative purposes only. The methods of the present invention can be combined with any manufacturing process developed for the production, recovery and purification of recombinant proteins.

[0252] 2. Antibodies

[0253] In a preferred embodiment, the method of the present invention is used to prevent the reduction of interchain and / or intrachain disulfide bonds of antibodies (including therapeutic and diagnostic antibodies). Antibodies within the scope of the present invention include, but are not limited to: anti-HER2 antibodies, including Trastuzumab (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat. No. 5,725,856); anti-CD20 antibodies, such as chimeric anti-CD20 "C2B8", such as U.S. Pat. No. 5,736,137 Chimeric or humanized variants of 2H7 antibodies, such as those in U.S. Pat. No. 5,721,108B1 or Tositumomab Anti-IL-8 (St John et al., Chest, 103:932 (1993) and International Publication No. WO 95 / 23865); anti-VEGF antibodies, including humanized and / or affinity matured anti-VEGF antibodies, such as humanized anti-VEGF antibody huA4.6.1 (Kim et al., Growth Factors, 7:53-64 (1992); International Publication No. WO 96 / 30046; and WO 98 / 45331 published on October 15, 1998); anti-PSCA antibodies (WO01 / 40309); anti-CD40 antibodies, including S2C6 and humanized variants thereof (WO00 / 75348); anti-CD11a (U.S. Pat. No. 5,622,700; WO 98 / 23761; Steppe et al., Transplant Intl. 4:3-7 (1991); and Hourmant et al., Transplantation 58:377-380 (1994)); anti-IgE (Presta et al., Transplant Intl. 4:3-7 (1991); and Hourmant et al., Transplantation 58:377-380 (1994)); al., J. Immunol. 151:2623-2632 (1993); and International Publication No. WO95 / 19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued April 22, 1997; or WO 97 / 26912, published July 31, 1997); anti-IgE, including E25, E26 and E27 (U.S. Pat. No. 5,714,338, issued February 3, 1998; U.S. Pat. No. 5,091,313, issued February 25, 1992; WO 97 / 26912, published March 4, 1993); 93 / 04173; or International Application No. PCT / US98 / 13410 filed June 30, 1998; U.S. Patent No. 5,714,338); anti-Apo-2 receptor antibodies (WO 98 / 51793 published November 19, 1998); anti-TNF-α antibodies, including cA2 CDP571 and MAK-195 (see U.S. Patent No. 5,672,347 issued on September 30, 1997; Lorenz et al., J. Immunol. 156(4):1646-1653 (1996); and Dhainaut et al., Crit. Care Med. 23(9):1461-1469 (1995)); anti-tissue factor (TF) (European Patent No. 0 420 937 B1 issued on November 9, 1994); anti-human α 4 β 7 Integrin (WO 98 / 06248 published February 19, 1998); anti-EGFR (chimeric or humanized 225 antibody, such as in WO 96 / 40210 published December 19, 1996); anti-CD3 antibodies, such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies, such as CHI-621 and (See U.S. Pat. No. 5,693,762, issued Dec. 2, 1997); anti-CD4 antibodies, such as cM-7412 antibody (Choy et al., Arthritis Rheum 39(1):52-56 (1996)); anti-CD52 antibodies, such as CAMPATH-1H (Riechmann et al., Nature 332:323-337 (1988)); anti-Fc receptor antibodies, such as M22 antibody directed against FcγRI, as in Graziano et al., J. Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies, such as hMN-14 (Sharkey et al., Cancer Res.55(23Suppl):5935s-5945s(1995)); antibodies against breast epithelial cells, including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al., Cancer Res.55(23):5852s-5856s(1995); and Richman et al., Cancer Res.55(23Supp):5916s-5920s(1995)); antibodies that bind to colon cancer cells, such as C242 (Litton et al., Eur J.Immunol.26(1):1-9(1996)); anti-CD38 antibodies, such as AT 13 / 5 (Ellis et al., J.Immunol.155(2):925-937(1995)); anti-CD33 antibodies, such as HuM195 (Jurcic et al., al., Cancer Res 55 (23 Suppl): 5908s-5910s (1995)) and CMA-676 or CDP771; anti-CD22 antibodies, such as LL2 or LymphoCide (Juweid et al., Cancer Res 55 (23 Suppl): 5899s-5907s (1995)); anti-EpCAM antibodies, such as 17-1A Anti-GpIIb / IIIa antibodies, such as abciximab or c7E3 Fab Anti-RSV antibodies, such as MEDI-493 Anti-CMV antibodies, such as Anti-HIV antibodies, such as PRO542; Anti-hepatitis antibodies, such as anti-Hep B antibodies Anti-CA125 antibody OvaRex; Anti-idiotypic GD3 epitope antibody BEC2; Anti-αvβ3 antibody Anti-human renal cell carcinoma antibodies, such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human melanoma antibody, R24 against GD3 ganglioside; anti-human squamous cell carcinoma antibody (SF-25 antibody); and anti-human leukocyte antigen (HLA) antibodies, such as Smart ID10 and anti-HLADR antibody Oncolym (Lym-1). Preferred target antigens of the antibodies herein are: HER2 receptor, VEGF, IgE, CD20, CD11a, and CD40.

[0254] Many of these antibodies are widely used in clinical practice to treat various diseases, including cancer.

[0255] In certain specific embodiments, the methods of the present invention are used to produce the following antibodies and recombinant proteins.

[0256] Anti-CD20 antibody

[0257] Rituximab It is a genetically engineered chimeric mouse / human monoclonal antibody against the CD20 antigen. Rituximab is the antibody referred to as "C2B8" in U.S. Patent No. 5,736,137 (Anderson et al.), issued on April 7, 1998. Rituximab is indicated for the treatment of patients with relapsed or refractory low-grade or follicular CD20-positive B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have demonstrated that rituximab binds to human complement and lyses lymphoid B cell lines via complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83(2):435-445 (1994)). In addition, it has significant activity in an antibody-dependent cellular cytotoxicity (ADCC) assay. Recently, rituximab was shown to have antiproliferative effects (in a tritiated thymidine incorporation assay) and to directly induce apoptosis, whereas other anti-CD19 and CD20 antibodies do not (Maloney et al., Blood 88(10):637a(1996)). Synergistic effects between rituximab and chemotherapy and toxins have also been observed experimentally. Specifically, rituximab sensitized drug-resistant human B-cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin, and ricin (Demidem et al., Cancer Chemotherapy&Radiopharmaceuticals 12(3):177-186(1997)). In vivo preclinical studies have shown that rituximab depletes B cells from the peripheral blood, lymph nodes, and bone marrow of rhesus monkeys, presumably through complement- and cell-mediated processes (Reff et al., Blood83(2):435-445(1994))。

[0258] Patents and patent publications of interest for CD20 antibodies include U.S. Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and 5,843,439, and U.S. Patent Application Nos. US 2002 / 0197255A1, US2003 / 0021781A1, US2003 / 0082172A1, US2003 / 0095963A1, US2003 / 0147885A1 (Anderson et al.); U.S. Pat. No. 6,455,043B1 and WO00 / 09160 (Grillo-Lopez, A.); WO00 / 27428 (Grillo-Lopez and White); WO00 / 27433 (Grillo-Lopez and Leonard); WO00 / 44788 (Braslawsky et al.); et al.); WO01 / 10462 (Rastetter, W.); WO01 / 10461 (Rastetter and White); WO01 / 10460 (White and Grillo-Lopez); U.S. Application Nos. US2002 / 0006404 and WO02 / 04021 (Hanna and Hariharan); U.S. Application Nos. US2002 / 0012665A1 and WO01 / 74388 (Hanna, N.); U.S. Application No. US2002 / 0058029 A1 (Hanna, N.); U.S. Application No. US2003 / 0103971 A1 (Hariharan and Hanna); U.S. Application No. US2002 / 0009444A1, and WO01 / 80884 (Grillo-Lopez, A.); WO01 / 97858 (White, C.); U.S. Application No. US2002 / 0128488A1 and WO02 / 34790 (Reff, M.); WO02 / 060955 (Braslawsky et al.); WO2 / 096948 (Braslawsky et al.); WO02 / 079255 (Reff and Davies); U.S. Patent No. 6,171,586B1, and WO98 / 56418 (Lam et al.) al.); WO98 / 58964 (Raju, S.); WO99 / 22764 (Raju, S.); WO99 / 51642, U.S. Patent No. 6,194,551B1, U.S. Patent No. 6,242,195B1, U.S. Patent No. 6,528,624B1 and U.S. Patent No. 6,538,124 (Idusogie et al.); WO00 / 42072 (Presta, L.); WO00 / 67796 (Curd et al.); WO01 / 03734 (Grillo-Lopez et al.); U.S. Application Nos. US 2002 / 0004587A1 and WO01 / 77342 (Miller and Presta); U.S. Application No. US2002 / 0197256 (Grewal, I.); U.S. Application No. US2003 / 0157108 A1 (Presta, L.); U.S. Patent Nos. 6,090,365B1, 6,287,537B1, 6,015,542, 5,843,398, and 5,595,721, (Kaminski et al. al.); U.S. Patent Nos. 5,500,362, 5,677,180, 5,721,108, and 6,120,767 (Robinson et al.); U.S. Patent No. 6,410,391 B1 (Raubitschek et al.); U.S. Patent No. 6,224,866 B1 and WO00 / 20864 (Barbera-Guillem, E.); WO01 / 13945 (Barbera-Guillem, E.); WO00 / 67795 (Goldenberg); U.S. Application Nos. US 2003 / 01339301 A1 and WO00 / 74718 (Goldenberg and Hansen); WO00 / 76542 (Golay et al.); al.); WO01 / 72333 (Wolin and Rosenblatt); U.S. Patent No. 6,368,596 B1 (Ghetie et al.); U.S. Application No. US2002 / 0041847 A1, (Goldenberg, D.); U.S. Application No. US2003 / 0026801 A1 (Weiner and Hartmann); WO02 / 102312 (Engleman, E.); U.S. Patent Application No. 2003 / 0068664 (Albitar et al.); WO03 / 002607 (Leung, S.); WO 03 / 049694 and US2003 / 0185796 A1 (Wolin et al.); WO03 / 061694 (Sing and Siegall); US2003 / 0219818 A1 (Bohen et al.); al.); US2003 / 0219433A1 and WO 03 / 068821 (Hansen et al.), each of which is expressly incorporated herein by reference. See also U.S. Patent No. 5,849,898 and European Patent Application No. 330,191 (Seed et al.); U.S. Patent No.4,861,579 and EP332,865A2 (Meyer and Weiss); U.S. Patent No. 4,861,579 (Meyer et al.) and WO95 / 03770 (Bhat et al.).

[0259] Publications focusing on therapy with rituximab include: Perotta and Abuel "Response of chronic relapsing ITP of 10years duration to Rituximab" Abstract #3360 Blood 10(1)(part 1-2):p.88B(1998); Stashi et al., "Rituximab chimeric anti-CD20monoclonal antibody treatment for adults with chronic idopathic thrombocytopenic purpura" Blood 98(4):952-957(2001); Matthews, R. "MedicalHeretics" New Scientist (7Apr., 2001); Leandro et al., "Clinical outcome in 22patients with rheumatoid arthritis treated with B lymphocyte depletion" Ann Rheum Dis 61:833-888(2002); Leandro et al., "Lymphocyte depletion in rheumatoidarthritis: early evidence for safety, efficacy and dose response. Arthritis & Rheumatism 44(9):S370(2001); Leandro et al., "An open study of Blymphocytedepletion in systemic lupus erythematosus", Arthritis & Rheumatism46(1):2673-2677(2002);Edwards and Cambridge"Sustained improvement in rheumatoidarthritis following a protocol designed to deplete B lymphocytes" Rheumatology 40:205-211(2001);Edwards et al.,"B-lymphocyte depletion therapy in rheumatoidarthritis and other autoimmune disorders" Biochem.Soc.Trans. 30(4):824-828(2002);Edwards et al.,"Efficacy and safety of Rituximab,a B-cell targetedchimeric monoclonal antibody:Arandomized,placebo controlled trial in patientswith rheumatoid arthritis. Arthritis & Rheumatism 46(9):S197(2002);Levine andPestronk"IgM antibody-related polyneuropathies:B-cell depletion chemotherapyusing Rituximab" Neurology 52:1701-1704(1999);DeVita et al.,"Efficacy ofselective B cell blockade in the treatment of rheumatoid arthritis" Arthritis & Rheumatism46:2029-2033(2002); Hidashida etal., "Treatment of DMARD-Refractoryrheumatoid arthritis with rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology; October 24-29; New Orleans, La. 2002; Tuscano, J. "Successful treatment of Infliximab-refractory rheumatoidarthritis with rituximab" Presented at the Annual Scientific Meeting of the American College of Rheumatology; October 24-29; New Orleans, La. 2002. Sarwal et al., N.Eng.J.Med. 349(2):125-138 (July 10, 2003) reported molecular heterogeneity in acute renal allograft rejection identified by DNA microarray profiling.

[0260] In various embodiments, the present invention provides a pharmaceutical composition comprising a humanized 2H7 anti-CD20 antibody. In a specific embodiment, the humanized 2H7 antibody is an antibody listed in Table 2.

[0261] Table 2: Humanized anti-CD20 antibodies and their variants

[0262]

[0263] Each of the antibody variants A, B and I in Table 2 comprises a light chain variable sequence (V L ):

[0264]

[0265] and

[0266] Heavy chain variable sequence (V H ):

[0267]

[0268] Each of the antibody variants C, D, F and G in Table 2 comprises a light chain variable sequence (V L ):

[0269]

[0270] and

[0271] Heavy chain variable sequence (V H ):

[0272]

[0273] Antibody variant 2 of Table 2 comprises a light chain variable sequence (V L ) and heavy chain variable sequence (V H ):

[0274]

[0275] Antibody variants A, B and I of Table 2 each comprise a full-length light chain sequence:

[0276]

[0277] Variant A of Table 2 comprises the full-length heavy chain sequence:

[0278]

[0279] Variant B of Table 2 comprises the full-length heavy chain sequence:

[0280]

[0281]

[0282] Variant 1 of Table 2 comprises the full-length heavy chain sequence:

[0283]

[0284] Antibody variants C, D, F, G and H of Table 2 each comprise a full-length light chain sequence:

[0285]

[0286] Variant C of Table 2 comprises the full-length heavy chain sequence:

[0287]

[0288] Variant D of Table 2 comprises the full-length heavy chain sequence:

[0289]

[0290] Variant F of Table 2 comprises the full-length heavy chain sequence:

[0291]

[0292] Variant G of Table 2 comprises the full-length heavy chain sequence:

[0293]

[0294]

[0295] Variant H of Table 2 comprises the full-length heavy chain sequence:

[0296]

[0297] In certain embodiments, the humanized 2H7 antibodies of the present invention further comprise amino acid changes in IgG Fc and exhibit a binding affinity to human FcRn that is increased by at least 60-fold, at least 70-fold, at least 80-fold, more preferably at least 100-fold, preferably at least 125-fold, even more preferably at least 150-fold to about 170-fold compared to an antibody with a wild-type IgG Fc.

[0298] The N-glycosylation site in IgG is located at C H 2 domain. The humanized 2H7 antibody compositions of the present invention include any of the aforementioned compositions of humanized 2H7 antibodies with Fc regions, wherein about 80-100% (preferably about 90-99%) of the antibodies in the composition comprise a mature core carbohydrate structure lacking fucose attached to the Fc region of the glycoprotein. Such compositions are demonstrated herein to exhibit unexpected improvements in binding to FcγRIIIA(F158), which is less effective than FcγRIIIA(V158) in interacting with human IgG. FcγRIIIA(F158) is more common than FcγRIIIA(V158) in normal, healthy African Americans and Caucasians. See Lehrnbecher et al., Blood 94:4220(1999). Historically, antibodies produced in Chinese hamster ovary cells (CHO), one of the most commonly used industrial hosts, have been found to be approximately 2-6% non-fucosylated. However, YB2 / 0 and Lec13 can produce antibodies that are 78-98% non-fucosylated. Shinkawa et al., JBio.Chem.278(5),3466-347(2003) reported that antibodies produced in YB2 / 0 and Lec13 cells with low FUT8 activity showed significantly increased ADCC activity in vitro. The production of antibodies with reduced fucose content is also described in, for example, Li et al., (GlycoFi) "Optimization of humanized IgGsin glycoengineered Pichia pastoris" in Nature Biology online publication 22Jan.2006; Niwa R.et al., Cancer Res. 64(6):2127-2133(2004); US2003 / 0157108 (Presta); US 6,602,684 and US2003 / 0175884 (Glycart Biotechnology); US 2004 / 0093621, US2004 / 0110704, US2004 / 0132140 (all of Kyowa Hakko Kogyo).

[0299] Bispecific humanized 2H7 antibodies encompass antibodies wherein one arm of the antibody has at least an antigen binding region of the H and / or L chain of the humanized 2H7 antibody of the present invention, and the other arm has a V region binding specificity for a second antigen. In a specific embodiment, the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.

[0300] Anti-HER2 antibodies

[0301] A recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or U.S. Pat. No. 5,821,337) clinical history in patients with metastatic breast cancer that overexpress HER2 who have received extensive prior anticancer therapy (Baselga et al., J. Clin. Oncol. 14: 737-744 (1996)). Trastuzumab received marketing approval from the Food and Drug Administration (FDA) on September 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress HER2 protein. In November 2006, the FDA approved Herceptin as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel as an adjuvant treatment for patients with HER2-positive, node-positive breast cancer.

[0302] In one embodiment, the anti-HER2 antibody comprises the following V L and V H Domain sequence:

[0303] Humanized 2C4 version 574 antibody V L (SEQ ID NO:16)

[0304]

[0305] and humanized 2C4 version 574 antibody V H (SEQ ID NO:17)

[0306]

[0307] In another embodiment, the anti-HER2 antibody comprises Fig.21 and Fig. 22 The V of trastuzumab is shown respectively L (SEQ ID NO: 18) and V H (SEQ ID NO: 19) domain sequence.

[0308] Other HER2 antibodies with various properties are described in Tagliabue et al., Int.J.Cancer 47:933-937(1991); McKenzie et al., Oncogene 4:543-548(1989); Maier et al., Cancer Res .51:5361-5369(1991); Bacus et al., Molecular Carcinogenesis 3:350-362(1990); Stancovski et al., PNAS(USA) 88:8691-8695(1991); Bacus et al., Cancer Research 52:2580-2589(1992); Xu et al., Int. J. Cancer 53:401-408(1993); WO94 / 00136; Kasprzyk et al., Cancer Research 52:2771-2776(1992); Hancock et al., Cancer Res. 51:4575-4580(1991); Shawver et al., Cancer Res.54:1367-1373(1994);Arteaga et al.,Cancer Res.54:3758-3765(1994);Harwerth et al., J.Biol.Chem. 267:15160-15167 (1992); U.S. Patent No. 5,783,186; and Klapper et al., Oncogene 14:2099-2109(1997).

[0309] Anti-VEGF Antibodies

[0310] The anti-VEGF antibody may comprise, for example, the following sequence:

[0311] In one embodiment, the anti-VEGF antibody comprises the following V L Sequence (SEQ ID NO:20):

[0312] and

[0313] The following V H Sequence (SEQ ID NO:21):

[0314]

[0315] In another embodiment, the anti-VEGF antibody comprises the following V L Sequence (SEQ ID NO:22):

[0316]

[0317] and

[0318] The following V H Sequence (SEQ ID NO:23):

[0319]

[0320] In a third embodiment, the anti-VEGF antibody comprises the following V L Sequence (SEQ ID NO:24):

[0321]

[0322] and

[0323] The following V H Sequence (SEQ ID NO:25):

[0324]

[0325] Anti-CD11a antibody

[0326] Humanized anti-CD11a antibody efalizumab or (U.S. Patent No. 6,037,454) received marketing approval from the Food and Drug Administration on October 27, 2003 for the treatment of psoriasis. One embodiment provides an anti-human CD11a antibody comprising V of HuMHM24 below: L and V H sequence:

[0327] V L (SEQ ID NO:26):

[0328]

[0329] and

[0330] V H (SEQ ID NO:27):

[0331]

[0332] The anti-human CD11a antibody may comprise V of SEQ ID NO: 27 H and the full-length L chain of HuMHM24, the latter having the sequence:

[0333]

[0334] or

[0335] The above L chain and the H chain having the following sequence:

[0336]

[0337]

[0338] Antibodies against the DR5 receptor (anti-DR5 antibodies) can also be generated according to the present invention. Such anti-DR5 antibodies specifically include all antibody variants disclosed in PCT Publication No. WO 2006 / 083971, such as anti-DR5 antibodies called Apomab 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, especially Apomab 8.3 and Apomab 7.3, preferably Apomab 7.3. The entire content of WO 2006 / 083971 is expressly incorporated by reference.

[0339] 3. Other disulfide-containing proteins

[0340] In addition to antibodies, the methods of the present invention can be used to make other polypeptides that include disulfide bonds. Representative examples of such polypeptides include, but are not limited to, the following therapeutic proteins: tissue plasminogen activator (t-PA), such as human tissue plasminogen activator (htPA, alteplase, ), a thrombolytic drug used to treat myocardial infarction; TNKase TM , a ht-PA variant with extended half-life and fibrin specificity for single bolus administration; recombinant human growth hormone (rhGH, somatropin, ); and recombinant human deoxyribonuclease I (DNase I) for the treatment of cystic fibrosis (CF).

[0341] Examples of biologically important proteins containing disulfides include growth hormones, 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; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated upon activation, normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; Muellerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin adenosine-related peptide; a microbial protein, such as β-lactamase; a DNA enzyme; an IgE; a cytotoxic T-lymphocyte-associated antigen (CTLA), such as CTLA-4; an inhibin; an activin; a vascular endothelial growth factor (VEGF); a hormone or growth factor receptor; a protein A or D; a rheumatoid factor; 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; a platelet-derived growth factor (PDGF); a synergist Fibroblast growth factors, such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGF), such as TGF-α and TGF-β, 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 protein; CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; Osteoinductive factors; immunotoxins; bone morphogenetic proteins (BMPs); interferons, such as interferon-α, -β, and -γ; colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factors; viral antigens, such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM;Tumor-associated antigens, such as HER2, HER3 or HER4 receptors; and fragments of any of the above-listed polypeptides. ;

[0342] 4. General methods for recombinant production of antibodies

[0343] The antibodies and other recombinant proteins herein can be produced by well-known techniques of recombinant DNA technology. Thus, in addition to the antibodies specifically identified above, a skilled practitioner can generate antibodies against an antigen of interest, for example using the techniques described below.

[0344] Antigen selection and preparation

[0345] The antibodies herein are directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or condition can produce a therapeutic benefit in the mammal. However, antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also contemplated. If the antigen is a polypeptide, it can be a transmembrane molecule (e.g., a receptor) or a ligand (such as a growth factor). Exemplary antigens include those proteins described in section (3) below. Exemplary molecular targets of antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD19, CD20, CD22, CD34, CD40; members of the ErbB receptor family such as EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM and αv / β3 integrin, including its α or β subunit (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, or any other antigen described herein. The antigens to which the antibodies listed above are directed are explicitly included within the scope of the present invention.

[0346] Soluble antigens or fragments thereof (optionally coupled to other molecules) can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments thereof (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines), or can be cells transformed by recombinant technology to express transmembrane molecules.

[0347] Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art.

[0348] Polyclonal antibodies

[0349] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 or R 1 N=C=NR (where R and R 1 It may be useful to couple the antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0350] Animals are immunized against antigens, immunogenic conjugates or derivatives by mixing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5-1 / 10 of the initial amount of antigen or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, the blood of the animals is collected and the antibody titer of the serum is determined. The animals are boosted until the titer reaches a plateau. Preferably, the animals are boosted with the same antigen but with different proteins and / or conjugates obtained by coupling with different cross-linking agents. Conjugates can also be prepared as protein fusions in recombinant cell culture. Similarly, coagulants such as alum are appropriately used to enhance the immune response.

[0351] Monoclonal antibodies

[0352] Monoclonal antibodies may be produced using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be produced by recombinant DNA methods (US Pat. No. 4,816,567).

[0353] In the hybridoma method, mice or other suitable host animals, such as hamsters or macaques, are immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986).

[0354] The hybridoma cells thus prepared are seeded and cultured in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0355] Preferred myeloma cells are those that fuse efficiently, support stable and high-level production of antibodies by the selected antibody-producing cells, and are sensitive to culture media such as HAT culture media. Among them, preferred myeloma cell lines are mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (Salk Institute Cell Distribution Center, San Diego, California USA) and SP-2 or X63-Ag8-653 available from the American Type Culture Collection (American Type Culture Collection, Rockville, Maryland USA). Human myeloma and mouse-human heteromyeloma cell lines are also documented for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

[0356] The culture medium in which the hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0357] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1986). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo in animals as ascites tumors.

[0358] The monoclonal antibodies secreted by the subclones can be suitably separated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Preferably, the protein A chromatography procedure described herein is used.

[0359] The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the monoclonal antibodies). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin proteins, such as Escherichia coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the monoclonal antibody in the recombinant host cell.

[0360] The DNA also may be modified, for example, by replacing the homologous murine sequences with the coding sequences for human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0361] Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site with specificity for one antigen and another antigen-combining site with specificity for a different antigen.

[0362] In another embodiment, monoclonal antibodies can be isolated from antibody phage libraries constructed using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) respectively describe the use of phage libraries to separate mouse and human antibodies. Subsequent publications describe chain shuffling (Marks et al., Bio / Technology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993)), generating high affinity (nM range) human antibodies. Thus, these techniques are viable alternatives to traditional hybridoma techniques for isolation of monoclonal antibodies.

[0363] Humanization and human antibodies

[0364] Humanized antibodies have one or more amino acid residues from non-human sources introduced therein. These non-human amino acid residues are often referred to as "import" residues, which are usually taken from the "import" variable domain. Humanization can be carried out essentially following the method of Winter and his colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), i.e., rodent CDR or CDR sequence is substituted for the corresponding sequence of human antibodies. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which essentially less than the entire human variable domain is substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted with residues from similar sites in rodent antibodies.

[0365] The selection of human variable domains for preparing humanized antibodies, including light chains and heavy chains, is very important for reducing antigenicity. According to the so-called "best-fit" method, the entire library of known human variable domain sequences is screened with the variable domain sequence of rodent antibodies. The human sequence closest to the rodent sequence is then selected as the human FR of the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).

[0366] More importantly, the antibody maintains high affinity to the antigen and other favorable biological properties after humanization. To achieve this goal, according to a preferred method, humanized antibodies are prepared by using a three-dimensional model of the parent and humanized sequence to analyze the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are usually available and are familiar to those skilled in the art. A computer program that illustrates and displays the possible three-dimensional conformational structure of the selected candidate immunoglobulin sequence can also be obtained. Checking these display images can analyze the possible role of residues in the functioning of the candidate immunoglobulin sequence, i.e., analyzing the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the receptor and the input sequence and combined to obtain the desired antibody characteristics, such as the affinity to the target antigen is improved. Generally, CDR residues are directly and most substantially involved in the impact on antigen binding.

[0367] Alternatively, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transferring a large number of human germline immunoglobulin genes in such germline mutant mice will result in the generation of human antibodies upon antigen attack. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993); and Duchosal et al., Nature, 355: 258 (1992). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)).

[0368] Antibody fragments

[0369] A variety of techniques have been developed for generating antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be generated directly by recombinant host cells. For example, antibody fragments can be isolated from the phage antibody libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 Fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab') can be isolated directly from recombinant host cell culture. 2 Fragments. Other techniques for generating antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO 93 / 16185).

[0370] Multispecific Antibodies

[0371] Multispecific antibodies have binding specificities for at least two different antigens. Although such molecules typically bind only two antigens (ie, bispecific antibodies, BsAbs), this expression as used herein encompasses antibodies with additional specificities, such as trispecific antibodies.

[0372] Methods for constructing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy chains-light chains, in which the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random distribution of immunoglobulin heavy chains and light chains, these hybridomas (quadromas) generate a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecules usually carried out by affinity chromatography steps is quite cumbersome and the product yield is low. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0373] According to another method of recording in WO96 / 27011, the interface between a pair of antibody molecules can be transformed to maximize the percentage of heterodimers to be recovered from recombinant cell culture. The preferred interface comprises at least a portion of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is produced on the interface of the second antibody molecule. This provides a mechanism for improving heterodimer output compared to other undesirable end products such as homodimers.

[0374] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other antibody can be coupled to biotin. Such antibodies have been suggested for use, for example, in targeting immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for treating HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art along with many cross-linking techniques and are disclosed in U.S. Patent No. 4,676,980.

[0375] The literature also describes techniques for generating bispecific antibodies from antibody fragments. For example, chemical linkage can be used to prepare bispecific antibodies. Brennan et al., Science, 229:81 (1985) described the generation of F(ab') by proteolytic cleavage of intact antibodies. 2 The fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then restored to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a selective immobilization reagent for enzymes.

[0376] Recent progress has led to the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) described a fully humanized bispecific antibody F(ab') 2 Generation of molecules. Each Fab' fragment was secreted separately by E. coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibodies thus formed were able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0377] A variety of techniques for directly generating and isolating bispecific antibody fragments from recombinant cell culture have also been described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provides an alternative mechanism for constructing bispecific antibody fragments. The fragment comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a linker, and the linker is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains on one fragment are forced to pair with the complementary VL and VH domains on the other fragment, thereby forming two antigen binding sites. Another strategy for constructing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994). Alternatively, the antibody may be a "linear antibody" as described in Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995). In brief, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0378] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol., 147:60 (1991).

[0379] Immunoadhesin

[0380] The simplest and most direct immunoadhesin design combines the binding domain of the adhesin (e.g., the extracellular domain (ECD) of a receptor) with the hinge and Fc region of an immunoglobulin heavy chain. Typically, when preparing the immunoadhesins of the invention, the nucleic acid encoding the adhesin binding domain is fused to the C-terminus of the nucleic acid encoding the N-terminus of the immunoglobulin constant domain sequence, however, N-terminal melting is also possible.

[0381] Typically, in such fusions, the encoded chimeric polypeptide will retain at least a functionally active immunoglobulin heavy chain constant region hinge, C H2 and C H The 3 domains can also be fused to the C-terminus of the Fc portion of the constant domain, or immediately adjacent to the heavy chain C H The precise site at which the fusion is made is not critical; specific sites are well known and can be selected to optimize the biological activity, secretion or binding characteristics of the immunoadhesin.

[0382] In a preferred embodiment, the adhesin sequence is fused to the immunoglobulin G 1 (IgG 1 ) N-terminal of the Fc domain. It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, it is more preferred to use a sequence that begins in the hinge region immediately upstream of the papain cleavage site that chemically defines the IgG Fc (i.e., residue 216, with the first residue of the heavy chain constant region being 114) or a similar site of other immunoglobulins in the fusion. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to the (a) hinge region, C H 2 and C H 3 or (b) C H 1. Hinge, C H 2 and C H 3 domain fusion.

[0383] For bispecific immunoadhesins, the immunoadhesins are assembled into polymers, particularly heterodimers or heterotetramers. In general, these assembled immunoglobulins will have a known unit structure. The basic four-chain structural unit is the form in which IgG, IgD and IgE exist. The four-chain unit is repeated in higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four-chain basic units held together by disulfide bonds. IgA globulins and occasional IgG globulins can also be present in serum in the form of polymers. In the case of polymers, each four-chain unit can be the same or different.

[0384] The following schematically lists various exemplary assembled immunoadhesins within the scope of this invention:

[0385] AC L -AC L ;

[0386] AC H -(AC H , A.C. L -AC H , A.C. L -V H C H , or V L C L -AC H );

[0387] AC L -AC H -(AC L -AC H , A.C. L -V H C H , V L C L -AC H , or V L C L -V H C H );

[0388] AC L -V H C H -(AC H , A.C. L -V H C H , or V L C L -AC H );

[0389] V L C L -AC H -(AC L -V H C H , or V L C L -AC H );and

[0390] (AY)n-(V L C L -V H C H ) 2 ,

[0391] wherein each A represents the same or different adhesin amino acid sequence;

[0392] V L is the immunoglobulin light chain variable domain;

[0393] V H is the immunoglobulin heavy chain variable domain;

[0394] C L is the immunoglobulin light chain constant domain;

[0395] C H is the immunoglobulin heavy chain constant domain;

[0396] n is an integer greater than 1;

[0397] Y represents the residue of a covalent cross-linker.

[0398] For brevity, the above structures show only the key features; they do not indicate the joining (J) or other domains of the immunoglobulin, nor do they show disulfide bonds. However, if such domains are required for binding activity, they should be constructed to be present in the usual positions they occupy in the immunoglobulin molecule.

[0399] Alternatively, the adhesin sequence can be inserted between the immunoglobulin heavy chain and light chain sequences to obtain an immunoglobulin comprising a chimeric heavy chain. In this embodiment, the adhesin sequence is fused to the 3' end of the immunoglobulin heavy chain in each arm of the immunoglobulin, or between the hinge and C H 2 domains, or in C H 2 and C H A similar construct has been reported by Hoogenboom et al., Mol. Immunol. 28: 1027-1037 (1991).

[0400] Although immunoglobulin light chains are not required to be present in the immunoadhesins of the present invention, immunoglobulin light chains may be present, either covalently bound to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to an adhesin. In the former case, DNA encoding immunoglobulin light chains is usually coexpressed with DNA encoding an adhesin-immunoglobulin heavy chain fusion protein. After secretion, the hybrid heavy chain and light chain will covalently bind to provide an immunoglobulin-like structure comprising two pairs of disulfide-linked immunoglobulin heavy chains-light chains. Methods suitable for preparing such structures are disclosed in, for example, U.S. Patent No. 4,816,567, published on March 28, 1989.

[0401] The most convenient is to construct immunoadhesin by fusing the cDNA sequence encoding the adhesin part with the immunoglobulin cDNA sequence in a form that conforms to the reading frame. However, fusion with genomic immunoglobulin fragments can also be used (see, for example, Aruffo et al., Cell 61:1303-1313 (1990); Stamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. The cDNA encoding the constant region of the IgG heavy chain can be separated from a cDNA library derived from spleen or peripheral blood lymphocytes according to the published sequence, by hybridization or by polymerase chain reaction (PCR) technology. The cDNA encoding the "adhesin" of the immunoadhesin and the immunoglobulin part are inserted in series into a plasmid vector that guides efficient expression in a selected host cell.

[0402] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0403] All patents and literature references cited in this specification are hereby incorporated by reference in their entirety.

[0404] Example

[0405] Unless otherwise indicated, commercial reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified by ATCC numbers in the following examples and throughout the specification is the American Type Culture Collection (Manassas, Virginia).

[0406] Example 1: Description of Materials and Methods

[0407] The following materials and methods were used in Examples 2-8.

[0408] Material

[0409] The materials and equipment used in the experiments described in the experimental examples included: stainless steel vials (Micro Tanks, Flow Components, Dublin, CA; short (50 cc) and tall (55 cc)); dialysis tubing (Spectra / Por, 6-8000 MWCO, catalog #132645); 0.22 μm filters (Millipore Millipak Gamma Gold, catalog #MPGL04GH2); phosphate buffered saline (PBS, EMD, catalog #6506); ethylenediaminetetraacetic acid (EDTA, Sigma, catalog #E4884); alpha-nicotinamide adenine dinucleotide phosphate (NADPH, Calbiochem, catalog #481973); dehydroepiandrosterone (DHEA, TCI, catalog #D0044); copper sulfate (Sigma, catalog #C8027); glucose-6-phosphate (G6P, Calbiochem, catalog #346764); aurothioglucose (ATG, USP, catalog #1045508); aurothiomalate (ATM, Alfa Aesar, catalog #39740); reduced glutathione (GSH, JTBaker, catalog #M770-01); monobromobimane (mBB, Fluka, catalog #69898); histidine (JTBaker, catalog #2080-05); sodium sulfate (JTBaker, catalog #3897-05); Trx (Sigma, catalog #T8690); TrxR (Sigma, catalog #T9698). All chemicals and reagents were used as received without further purification. EDTA (250 mM, pH 7.5), CuSO 4 Stock solutions of ATG (10 mM), ATG (30 mM), ATM (30 mM), NADPH (75 mM), and G6P (300 mM) were prepared for use in the minitank time course studies.

[0410] Generation of cell culture fluid (CCF)

[0411] To generate ocrelizumab CCF for various antigen studies, a representative small-scale fermentation procedure similar to that described previously (Chaderjian et al., 2005) was used. Briefly, a 3-liter glass stirred tank equipped with a pitched blade impeller was used. Bioreactors and ocrelizumab medium components were used for inoculum-train and production cultures. The bioreactors were equipped with calibrated dissolved oxygen (DO), pH, and temperature probes. A digital control unit was used to control DO, pH, temperature, and agitation rate to the specified parameters of the ocrelizumab manufacturing process. The working volume for both inoculum-train and production cultures was 1.5 L. Daily samples were analyzed on a NOVA Bioprofile blood gas analyzer to ensure the accuracy of online values ​​for pH and dissolved oxygen and to monitor glucose, lactate, ammonium, glutamine, glutamate, and sodium concentrations in the cultures. Daily samples were also collected to monitor cell growth, viability, and titer. Cell growth was measured using ViCell both by viable cell counts and on a packed cell volume (PCV) basis. Culture viability was determined on the ViCell instrument by trypan blue exclusion. Supernatant samples were analyzed by an HPLC-based method to measure ocrelizumab titer values.

[0412] Harvested Cell Culture Fluid (HCCF) Preparation

[0413] Complete lysis of CCF was achieved by high pressure homogenization using a Microfluidics HC-8000 homogenizer. The pressure regulator of the instrument was set to 4,000-8,000 psi, and CCF was passed through the homogenizer to obtain complete cell lysis (membrane rupture) after a single pass. Once water was removed through the system, the CCF homogenate was collected. The homogenate was transferred to a centrifuge bottle and centrifuged at 4,500 rpm for 30 minutes at 20 ° C in a Sorval RC-3B rotor centrifuge. The centrifuge was poured out, then deep filtered, followed by sterile filtration using a peristaltic pump and silicon tube 0.22 μm to generate final HCCF from homogenized CCF (100% cell lysis). Alternatively, CCF from a fermenter was directly centrifuged without any homogenization, and the centrifuge was then filtered with a sterile 0.22 μm filter to generate HCCF.

[0414] Micro tank operation

[0415] A laminar flow hood was used when handling all microtanks, and all materials used in HCCF incubation experiments were either autoclaved or rinsed with 70% isopropanol to minimize bacterial contamination.

[0416] Lactate dehydrogenase assay

[0417] For the lactate dehydrogenase assay, see Babson & Babson (1973) and Legrand et al., (1992), which are incorporated herein by reference.

[0418] Dialysis experiment

[0419] Dialysis experiments were performed to determine whether the component causing ocrelizumab reduction was a small molecule or a macromolecule (i.e., an enzyme). The 3mL sample of purified and formulated ocrelizumab (30.2mg / mL) was dialyzed for 24 hours against 1L phosphate buffered saline (PBS, 10mM, pH 7.2), and PBS was replaced after 8 hours. The concentration of the ocrelizumab sample was then adjusted to 1mg / mL using 280nm absorbance. The aliquots were stored at -70°C before use. The dialysis tubing was hydrated overnight in 0.05% azide solution and rinsed with sterile water before use. The HCCF obtained by homogenization of the CCF from the 3L fermenter was melted, and a peristaltic pump was used to filter via a 0.22μm Millipak filter. 30mL HCCF was each loaded to six short miniature tanks. 500μL ocrelizumab samples in the sealed dialysis tubing were added to each miniature tank. The mini cans were sealed and loaded into a benchtop mixer (Barnstead Lab-Line MAX Q4000) operating at 35 rpm at ambient temperature. For each time point, one mini can was removed from the mixer and aliquots of HCCF (in the mini can) and ocrelizumab samples (in the dialysis bag) were collected and stored at -70°C until analysis by the free thiol assay and the Bioanalyzer assay (described below).

[0420] Testing inhibitors for reduction in small-scale in vitro systems

[0421] 27mL HCCF is filled to high micro tank. According to the experimental design, various reagents (NADPH, G6P, G6PD inhibitor or TrxR) are added to the desired concentration, and the final volume in the micro tank is made to reach 30mL with PBS (10mM, pH 7.2). The micro tank is sealed, and loaded into a tabletop mixer operated at 35rpm in ambient temperature. At each time point of sampling, the outside of the micro tank is sterilized with 70% IPA, and opened in a laminar flow hood to take out aliquots. The micro tank is then resealed and loaded into the tabletop mixer again. All aliquots are stored at -70°C until analyzed with free thiol assay and Bioanalyzer assay (described below).

[0422] In vitro Trx / Trx reductase studies

[0423] A commercial TrxR (rat liver) solution (4 μM) was diluted with water to produce a 2.86 μM solution. Lyophilized Trx (human) was reconstituted with PBS (10 mM, pH 7.2) to produce a 500 μM solution. A 20 mM NADPH solution and 10 mM ATG and ATM solutions were prepared in water.

[0424] In a black polypropylene 1.5mL microcentrifuge tube, 437μL PBS, 25μL NADPH, 16μL prepared ocrelizumab solution (30.2mg / mL) and 5μL Trx were gently mixed. The reaction was started by adding 17.5μL TrxR. The reaction was incubated at room temperature for 24 hours. A 20μL aliquot was taken out at each sampling time point and stored at -70°C until analyzed by the Bioanalyzer assay (see below). Controls were implemented to determine whether the enzyme pathway was active when the enzyme was omitted by replacing Trx and / or TrxR in the reaction mixture with an equal volume of PBS.

[0425] Inhibition of the Trx system was demonstrated using the same reaction conditions as above with the addition of 5 μL ATG or ATM. 2 + For inhibition of the Trx system, add 2.5 μL CuSO to the reaction mixture 4 (10 mM), using the same enzyme but different buffer (10 mM histidine, 10 mM Na 2 SO 4 , 137 mM NaCl, 2.5 mM KCl, pH 7.0) to prevent insoluble Cu 3 (PO 4 ) 2 form.

[0426] Free thiol determination method

[0427] A standard curve for GSH was generated in PBS (10 mM, pH 6.0 ± 0.05). From a 110 mM GSH solution, standards at concentrations of 0, 5.5, 11, 22, 44, 55, 110 and 550 μM were prepared by serial dilution. From an acetonitrile stock solution of mBB (10 mM, stored at -20°C), a 100 μM solution of mBB was prepared in PBS (10 mM, pH 10.0 ± 0.05) and stored in the dark.

[0428] In a black flat-bottom 96-well plate, 100 μL of mBB was dispensed into each well. For the standard curve, 10 μL of standard GSH solution was added to produce a working pH of 8.0 ± 0.2. For samples, 10 μL of sample was added to the well. All wells were prepared in triplicate. The plate was incubated in the dark at room temperature for 1 hour and then read using a fluorescence plate reader (Molecular Devices). Gemini XS) ​​readings were performed using an excitation wavelength of 390 nm and an emission wavelength of 490 nm. A linear standard curve was generated using the average results of three standard wells plotted against GSH concentration. The free thiol levels in the samples were calculated from the linear equation of the standard curve using the average of the three sample wells.

[0429] Bioanalyzer assay

[0430] Capillary electrophoresis measurements were obtained using an Agilent 2100 Bioanalyzer. Sample preparation was performed as described in the Agilent Protein 230 assay protocol (manual part number G2938-90052) with slight changes. Prior to preparation, HCCF samples were diluted 1:4 with water and protein A samples were diluted to 1.0 g / L. For the HCCF samples of the denaturation step, 24 μL of 50 mM iodoacetamide (IAM), 0.5% SDS solution was added in addition to the 2 μL of denaturation solution provided. For protein A samples, 0.5% SDS without IAM and 2 μL of denaturation solution were used. Digital gel-like images were generated using Agilent 2100 Expert software.

[0431] Stock solutions for HCCF retention time studies

[0432] Three different stock solutions were used in the bench-scale HCCF retention time studies: (1) 250 mM EDTA stock solution (pH 7.4) prepared using disodium EDTA dihydrate (Mallinckrodt, catalog #7727-06 or Sigma, catalog #E-5134) and tetrasodium EDTA dihydrate (Sigma, catalog #E-6511); (2) copper sulfate pentahydrate (CuSO 4 , Sigma, catalog # C-8027); and (3) 1 M acetic acid solution (Mallinckrodt, catalog # V193).

[0433] Inhibitor addition and cell culture fluid (CCF) spiking

[0434] Before homogenization, add 250 mM EDTA or 50 mM CuSO 4Stock solutions of 100 μg / mL were added to CCF to evaluate the final concentration range that prevented antibody disulfide reduction. Once the final HCCF was generated from homogenized CCF, these solutions were mixed with HCCF generated from unhomogenized CCF (also containing EDTA or CuSO 4 ) to dilute and reduce the overall level of cell lysis to below 100% maximum. Alternatively, a stock solution of 1 M acetic acid was added to the final blended HCCF solution (homogenized CCF and non-homogenized CCF) to lower the pH of the solution to prevent antibody disulfide reduction.

[0435] In a 50 mL 316L stainless steel vial, approximately 30-50 mL of each HCCF solution (containing EDTA, CuSO 4 , acetic acid, or no addition as a control). The vials were sealed with clamps and the solutions were not aerated or stirred. The vials were stored at room temperature (18-22°C). At predetermined time points, the solutions were removed and purified on a laboratory scale protein A affinity resin.

[0436] Similar results can be obtained with other oxidizing agents such as, for example, cystine and oxidized glutathione.

[0437] Blow in air

[0438] In order to evaluate the bubbling of air into the HCCF generated from the homogenized CCF to prevent the reduction of antibody disulfide, a 3L glass or 15L stainless steel container was used. Approximately 1-5L HCCF was aseptically filtered through 0.22 μm into each sterilized container. The experimental conditions were maintained at 18-22 ° C and 50 (15L fermenter) or 275rpm (3L fermenter) agitation, with or without pH control by adding carbon dioxide. Either air was bubbled into the solution to increase the dissolved oxygen level to air saturation, or nitrogen (control) was bubbled to remove any dissolved oxygen in the solution. The airflow to each container was variable, depending on whether a constant aeration rate was used or a minimum dissolved oxygen level was maintained. At a predetermined time point, 25-50mL samples were taken out from both containers and purified on a laboratory scale protein A affinity resin before analysis.

[0439] Protein A Processing

[0440] Specific affinity chromatography resins can be used to capture and purify antibodies in harvested cell culture fluid samples. Protein A resin (Millipore, Prosep-vA High Capacity) was selected as the affinity resin for antibody purification. The resin was filled into a glass column with a bed height of 14 cm and an inner diameter of 0.66 cm (final column volume of 4.8 mL). Chromatography was performed using an AKTAExplorer 100 chromatography system (GE Healthcare).

[0441] The resin is exposed to a buffer and HCCF with a linear flow rate between 350-560cm / hr. The resin is balanced with 25mM Tris, 25mM NaCl, 5mM EDTA, pH 7.1. For each purification, 5-15mg of antibody is loaded to the resin per mL of resin. The antibody concentration in HCCF is determined using an immobilized protein A HPLC column (Applied Biosystems, POROS A). After loading, the resin is cleaned with 25mM Tris, 25mM NaCl, 5mM EDTA, 0.5M TMAC, pH 7.1, and then the antibody is eluted using 0.1M acetic acid, pH 2.9. The elution is combined based on the 280nm UV absorbance measured online after the column. The elution combined solution after purification is adjusted to pH 5.0-5.5 using 1M HEPES sodium. After regenerating the resin with 0.1M phosphoric acid, subsequent purification of other HCCF solutions is performed using the same or similar filled resin.

[0442] The antibody concentration of the purified Protein A pool was measured using 280 nm UV spectrometry. The purified Protein A elution pool was analyzed by Bioanalyzer assay to quantify the percentage of intact antibody of 150 kDa molecular weight.

[0443] Example 2: Dialysis Experiment

[0444] A dialysis experiment was designed and performed to determine whether the reduction of ocrelizumab was caused by a reducing small molecule or a macromolecule (e.g., an enzyme). In this dialysis experiment, the purified intact ocrelizumab was placed in a dialysis bag with a molecular weight cutoff (MWCO) of 7000 and the dialysis bag was incubated in HCCF containing ocrelizumab in a stainless steel microtank. Figure 1 and Figure 2 As shown, the ocrelizumab in the bag was not reduced after the incubation period ( Figure 1 ), while ocrelizumab outside the HCCF pocket was significantly reduced soon after the start of incubation. This was demonstrated by the loss of intact ocrelizumab (approximately 150 kDa) and the formation of ocrelizumab fragments (various heavy and light chain combinations) ( Figure 2 ). Mass spectrometry analysis of ocrelizumab in the protein A elution pool from the reduced manufacturing run indicated that the observed fragments were formed by reduction of only the interchain disulfide bonds.

[0445] Free thiol measurements showed that no free thiol was present inside the dialysis bag at the beginning of the incubation; however, less than 5 hours after the start of the incubation, the free thiol levels inside and outside the dialysis bag became comparable, indicating that the small molecule components in HCCF were completely equilibrated inside and outside the dialysis bag ( Figure 3 ). Since reduction was only observed outside the dialysis bag with MWCO 7000Da, but not inside the dialysis bag, the molecular weight of the reducing molecule must be greater than 7000Da. Thus, the enzymatic reaction leads to the reduction of ocrelizumab.

[0446] Example 3: Reduction of Ocrelizumab (rhuMAb 2H7, variant A) by Trx / TrxR in vitro

[0447] The Trx system was tested for its ability to reduce ocrelizumab in vitro by incubating intact ocrelizumab with Trx, TrxR, and NADPH. Bioanalyzer results indicated that ocrelizumab was reduced by the Trx system in vitro ( Figure 5 The reduction rate in this in vitro system appears to be slower than that of HCCF (e.g. Figure 2 This may be due to the lower concentrations of enzymes (Trx and Trx-R) and / or the buffer system used in the in vitro reactions, since the reaction rate of the Trx system depends on the enzyme concentration and the buffer system.

[0448] Example 4: Inhibitors of the Trx system

[0449] (i) Inhibition of reduction of recombinant antibodies by copper sulfate

[0450] Copper sulfate is known to have the ability to provide redox potential and has been used to minimize free thiol levels (i.e., minimize unpaired cysteines) in recombinant antibody molecules during cell culture (Chaderjian et al., 2005, supra). Copper sulfate was tested for its efficacy in inhibiting the Trx system and subsequent ocrelizumab reduction in vitro. In this in vitro reduction experiment, the buffer system was changed from PBS to histidine sulfate to avoid insoluble Cu 3 (PO 4 ) 2 form. Figure 8 It was shown that ocrelizumab was readily reduced by the Trx system in histidine sulfate buffer (even faster than in PBS buffer). Addition of CuSO 4 Significantly inhibited ocrelizumab reduction ( Fig. 9 ).

[0451] (ii) Inhibition of recombinant antibody reduction in HCCF by ATG and ATM

[0452] Two commercially available specific inhibitors of TrxR, aurothioglucose (ATG) and aurothiomalate (ATM), were tested for their ability to inhibit the Trx system and ocrelizumab reduction in vitro. Both ATG and ATM effectively inhibited ocrelizumab reduction in the assay described above (see Figure 6 and Figure 7 ). Figure 5 This indicates that the addition of aurothioglucose or aurothiomalate to the same reaction mixture effectively inhibited the reduction of ocrelizumab, as shown in the digital gel-like images from the Bioanalyzer analysis.

[0453] If the Trx system is active in HCCF and reduces ocrelizumab, as observed in manufacturing runs or bench-scale experiments resulting in reduced antibody molecules, then both gold compounds (ATG and ATM) should be able to inhibit ocrelizumab reduction in HCCF. Fig.10 It was shown that ocrelizumab was readily reduced in HCCF from homogenized CCT (which was generated from a 3L fermentor) after a period of incubation. However, when 1 mM ATG or ATM was added to HCCF, the ocrelizumab reduction event was completely inhibited ( Fig.11 and Fig.12 ). These results demonstrate that the Trx system is active in HCCF and directly causes ocrelizumab reduction.

[0454] Example 5: Source of NADPH for Trx system activity and the role of G6P and glucose in the reduction mechanism

[0455] The reduction of disulfides by the Trx system requires reducing equivalents from NADPH ( Figure 4 ). The major cellular metabolic pathway that provides NADPH for all reductive biosynthetic reactions is the pentose phosphate pathway. In order for antibody reduction events to occur, the enzymes in this pathway must remain active in HCCF to keep the Trx system active. At a minimum, the first step in the pentose phosphate pathway (catalyzed by G6PD) must be active to reduce NADP + NADPH is converted to G6P simultaneously to 6-phosphogluconolactone. In addition, G6P is most likely generated in HCCF from glucose and adenosine-5'-triphosphate (ATP) by hexokinase activity. Figure 4 The overall mechanism of ocrelizumab reduction was summarized.

[0456] Reducing activity in HCCF appears to be transient in some cases and can be inhibited over time under certain storage conditions or after multiple cycles of freeze-thaw. HCCF completely devoid of reducing activity provides an opportunity to explore the role of NADPH and G6P in the reduction of ocrelizumab by the Trx system.

[0457] HCCF from a large-scale manufacturing run ("β" run) was subjected to several cycles of freeze-thaw and used in experiments designed to measure reduction; no ocrelizumab reduction was observed ( Fig.13 ), despite its ability to induce antibody reduction as seen previously in freshly thawed HCCF from the same fermentation. Addition of NADPH at a concentration of 5 mM to this non-reducing HCCF restored the reduction event ( Fig.14 ). In addition, the Trx system is still intact and active in HCCF where reduction no longer occurs and is able to reduce proteins and / or antibodies in the presence of cofactors. In addition, reducing activity is lost over time because the NADPH source is depleted (presumably due to oxidation of NADPH by all reductive reactions competing for NADPH), rather than because the Trx system is degraded or inactivated.

[0458] This was confirmed by another experiment. 10 mM G6P was added to HCCF from the beta run that had been subjected to repeated freeze-thaw cycles. This G6P addition reactivated the Trx system, which subsequently reduced ocrelizumab in the HCCF incubation experiment ( Fig.15 ). This demonstrates that the reduction of ocrelizumab in HCCF is caused by the activity of both the Trx system and G6PD. In addition, G6PD is still active in the β-run HCCF after repeated freeze-thaw cycles; the loss of reducing activity in the β-run HCCF after repeated freeze-thaw cycles appears to be due to the depletion of G6P, thus eliminating NADP + The transition to NADPH.

[0459] In our study, we observed that EDTA effectively inhibited the reduction of ocrelizumab in HCCF incubation experiments. Fig.16 As shown in Figure 2, ocrelizumab was reduced after incubation of HCCF from a 12,000 L scale ocrelizumab manufacturing run (without repeated freeze-thaw cycles and without loss of reducing activity) at ambient temperature for more than 19 hours. However, when 20 mM EDTA was added to 12 kL HCCF and maintained in a separate stainless steel microtank, reduction was completely inhibited ( Fig.17). In the first step of glycolysis, hexokinase catalyzes the transfer of phosphate groups from Mg2+-ATP to glucose, i.e., a reaction that requires Mg2+ to complex with ATP (Hammes & Kochavi, 1962a & 1962b, supra). Since EDTA is a metal ion chelator, especially for Mg2+, it will be an effective inhibitor of hexokinase. The observation that excess EDTA can effectively block reduction indicates that hexokinase is involved in the mechanism of ocrelizumab reduction (i.e., providing G6P). Without being limited to this or any other theory, EDTA blocks ocrelizumab reduction by eliminating hexokinase activity and thereby reducing the level of G6P available to G6PD and the level of NADPH available to the subsequent Trx system.

[0460] Although EDTA effectively blocked ocrelizumab reduction in fresh HCCF every time, it was incapable of preventing ocrelizumab reduction in beta-run HCCF that had lost Trx system activity and then reactivated by the addition of G6P. For example, ocrelizumab reduction was observed in HCCF incubation experiments in which 5 mM G6P and 20 mM EDTA (final concentration) were added to beta-run HCCF that had completely lost its reducing activity ( Fig.18 ). However, no reduction was seen in control incubations without the addition of G6P and EDTA. Without being limited to this or any other theory, EDTA used in this manner may therefore not inhibit the Trx system or G6PD, and may function as an inhibitor of hexokinase that generates G6P for G6PD. Without G6P, the Trx system would not receive the necessary NADPH supply to achieve activity.

[0461] Example 6: Inhibition of DHEA on the reduction of recombinant antibodies

[0462] Dehydroepiandrosterone (DHEA) blocks G6PD activity as effectively as other similar G6PD inhibitors (Gordon et al., 1995, supra). G6PD inhibitors also prevent the reduction of antibodies (e.g., ocrelizumab) in HCCF by blocking NADPH generation. The ability of DHEA to inhibit the reduction of orcelizumab was demonstrated in HCCF incubation experiments. Adding DHEA to HCCF prevents antibody reduction.

[0463] DHEA is generally used in a concentration range of about 0.05 mM to about 5 mM. DHEA is also generally used in a concentration range of about 0.1 mM to about 2.5 mM.

[0464] Example 7: Inhibition of reduction of recombinant antibodies by adding (i) EDTA, (ii) copper sulfate, and (iii) acetic acid

[0465] Four different HCCFs were stored and maintained in stainless steel vials. The amount of cell lysis in each solution was similar and was generated by diluting HCCF from homogenized CCF with HCCF from unhomogenized CCF. For example, 150 mL of the first lysis solution was mixed with 50 mL of the second solution. The four HCCF mixtures evaluated in this study contained either: (1) 20 mM EDTA, (2) 30 μM CuSO 4 , (3) 15 mM acetic acid (pH 5.5), and (4) no chemical inhibitors were added as control solutions. Ocrelizumab antibody was purified from all four mixtures immediately (t = 0 hr) using protein A chromatography and then stored again in stainless steel vials for 20 hr and 40 hr. The purified protein A elution pool was analyzed by Bioanalyzer assay to quantify the percentage of intact antibody (150 kDa). The results showed that more than 90% of intact antibody ( Fig.19 ). However, at the 20 hr time point, no intact antibody was detected in the control mixture (without any addition), indicating reduction of antibody disulfide bonds. In the other three mixtures, more than 90% of intact antibody was still detected at both the 20 hr and 40 hr time points, demonstrating that all three inhibitors tested prevented disulfide bond reduction.

[0466] Example 8: Inhibition of Reduction of Recombinant Antibodies by Bubbling Air into HCCF

[0467] A portion of the HCCF mixture generated from the homogenized CCF was stored and maintained in two separate 10L stainless steel fermenters. Air was blown into one container, while nitrogen was blown into the other container. The ocrelizumab antibody was purified from the initial mixture using protein A chromatography immediately (t=0 hr). At the selected time point, 50 mL samples were taken from each container and the antibody was purified using protein A chromatography. The purified protein A elution pool was then analyzed by the Bioanalyzer assay to quantify the percentage of the 150 kDa complete antibody. The results showed that there was approximately 85% complete antibody ( Fig. 20 ), indicating some early reduction of antibody disulfide bonds prior to exposure to oxygen (i.e., air bubbled in the fermenter). Once the mixture was bubbled with air for 2 hours, more than 90% intact antibody was measured for the remainder of the 36-hour study. In contrast, when nitrogen was bubbled into the mixture, antibody reduction events continued to occur, as measured at 2 hours (28% 150 kDa peak) and 6 hours (5% 150 kDa peak). These results demonstrate that disulfide bond reduction in the antibody is prevented when the HCCF mixture generated from homogenized CCF is exposed to oxygen.

[0468] Example 9: Design of targeted siRNA or antisense nucleotide Trx inhibitors

[0469] The design of targeted siRNAs or antisense nucleotides for genes found in CHO cells can be performed using publicly available sequences, such as E. coli thioredoxin TrxA (SEQ ID NO: 30), E. coli thioredoxin reductase TrxB (SEQ ID NO: 31); mouse thioredoxin 1 (SEQ ID NO: 32), mouse thioredoxin 2 (SEQ ID NO: 33), mouse thioredoxin reductase 1 (SEQ ID NO: 34), and mouse thioredoxin reductase 2 (SEQ ID NO: 35). One of ordinary skill in the art can use these sequences to select sequences for designing Trx inhibitors targeting enzymes in different organisms and / or cells (such as CHO cells).

[0470] The sequence of E. coli thioredoxin TrxA is:

[0471]

[0472] The sequence of E. coli thioredoxin TrxB is:

[0473]

[0474]

[0475] The sequence of mouse thioredoxin 1 is:

[0476]

[0477] The sequence of mouse thioredoxin 2 is:

[0478]

[0479]

[0480] The sequence of mouse thioredoxin reductase 1 is:

[0481]

[0482]

[0483] The sequence of mouse thioredoxin reductase 2 is:

[0484]

[0485]

[0486] Example 10: In vitro Trx / Trx reductase studies

[0487] Measurement and methods

[0488] A commercial TrxR (rat liver) solution (4 μM) was diluted with water to produce a 2.86 μM solution. Lyophilized Trx (human) was reconstituted with PBS (10 mM, pH 7.2) to produce a 500 μM solution. A 20 mM NADPH solution and 10 mM ATG and ATM solutions were prepared in water.

[0489] In a black polypropylene 1.5 mL microcentrifuge tube, gently mix 437 μL of reaction buffer (10 mM histidine, 10 mM Na 2 SO 4 , 137mM NaCl, 2.5mM KCl, pH 7.0), 25μL NADPH, 16μL prepared ocrelizumab solution (30.2mg / mL) and 5μL Trx. The reaction was started by adding 17.5μL TrxR. The reaction was incubated at room temperature for 24 hours. A 20μL aliquot was taken at each sampling time point and stored at -70°C until analysis by Bioanalyzer assay.

[0490] Inhibition of the Trx system was demonstrated using the same reaction conditions described above with the addition of various inhibitors.

[0491] 1. In vitro activity of the thioredoxin system

[0492] Fig.24 A digital gel-like image from a Bioanalyzer analysis (each lane represents a time point) is shown showing the incubation time of intact ocrelizumab ("2H7", a humanized anti-CD20 antibody, referred to above as "variant A") (1 mg / mL) with 0.1% iodine in 10 mM histidine sulfate buffer. μ M TrxR (rat liver), 5 μ Incubation of M Trx(human) with 1 mM NADPH resulted in reduction of ocrelizumab in less than 1 hour.

[0493] 2. In vitro activity of the thioredoxin system inhibited by aurothioglucose

[0494] Aurothioglucose (ATG) was added to the ocrelizumab mixture described above at the following concentrations: 1 mM; 0.6 μ M (6:1 ATG:TrxR); 0.4 μM (4:1 ATG:TrxR); and 0.2 μM (2:1 ATG:TrxR).

[0495] like Figure 25-27 As demonstrated by the digital gel-like images from the Bioanalyzer analysis shown, aurothioglucose added at concentrations of 1 mM, 0.6 μM, and 0.4 μM effectively inhibited the reduction of ocrelizumab by the thioredoxin system. Fig.28 As shown, under these experimental conditions, aurothioglucose added at a concentration of 0.2 μM was unable to inhibit ocrelizumab reduction after 24 hours.

[0496] 3. In vitro activity of the thioredoxin system inhibited by aurothiomalate

[0497] Aurothiomalate (ATM) was added to the ocrelizumab mixture described above at concentrations of 0.1 mM and 0.01 mM. Fig.29 and 30 As demonstrated by the digital gel-like images shown from Bioanalyzer analysis, ATM effectively inhibited the reduction of ocrelizumab by the thioredoxin system at both concentrations tested.

[0498] 4. CuSO 4 Inhibition of the in vitro activity of the thioredoxin system

[0499] 20 μM (4:1Cu 2 +:Trx); 10μm(2:1Cu 2 +:Trx); and 5 μM (1:1Cu 2 CuSO was added to the ocrelizumab mixture described above at a concentration of Trx. 4 .like Figure 31-33 As shown, CuSO 4 At concentrations of 20 μM and 10 μM, ocrelizumab effectively inhibited thioredoxin-induced reduction of Fig.31 and 32 ), but 5 μM concentration was insufficient to cause complete inhibition of reduction ( Fig.33 ).

[0500] 5. In vitro activity of the thioredoxin system inhibited by cystamine

[0501] Cystamine was added to the ocrelizumab mixture described above at the following concentrations: 532 μM (20:1 cystamine:2H7 (variant A) disulfide); 266 μM (10:1 cystamine:2H7 (variant A) disulfide); 133 μM (5:1 cystamine:2H7 disulfide); and 26.6 μM (1:1 cystamine:2H7 (variant A) disulfide). Figure 34-37As shown, cystamine effectively inhibited thioredoxin-induced ocrelizumab reduction at concentrations of 532 μM (20:1 cystamine:2H7 (variant A) disulfide) and 266 μM (10:1 cystamine:2H7 (variant A)). Fig.34 and 35 ), but concentrations of 133 μM (5:1 cystamine:2H7(variant A) disulfide) and 26.6 μM (1:1 cystamine:2H7(variant A) disulfide) were insufficient to inhibit ocrelizumab reduction after 24 hours ( Fig.36 and 37 ).

[0502] 6. In vitro activity of the thioredoxin system inhibited by cystine

[0503] Cystine was added to the ocrelizumab mixture described above at a concentration of 2.6 mM. Fig.38 As shown, at this concentration, cystine effectively inhibits the reduction of ocrelizumab by the thioredoxin system. It should be noted that the minimum effective concentration of cystine (just as the effective minimum concentration of other inhibitors) depends on the actual situation and can be different for different proteins (such as antibodies) and can vary with the time of addition. Thus, for example, if cystine is added before lysis, the minimum effective concentration for antibody 2H7 (variant A) is about 1.3 mM, for Apomab is about 1 mM and for antibody variant C is about 4.5 mM. When cystine is added to the cell culture medium, the minimum effective concentration is generally higher and is about 5.2 mM for 2H7 (variant A), 6 mM for Apomab and 9 mM for antibody variant C. Typically, for cystine, cystamine and oxidized glutathione (see below), the minimum effective inhibitory concentration is about 40 times the antibody concentration (in μM).

[0504] 7. In vitro activity of the thioredoxin system inhibited by oxidized glutathione (GSSG)

[0505] GSSG was added to the ocrelizumab mixture described above at a concentration of 2.6 mM. Fig.39 As shown, at this concentration, GSSG effectively inhibits the reduction of ocrelizumab by the thioredoxin system. However, it should be noted that the minimum effective concentration of oxidized glutathione (as for other inhibitors) depends on the actual situation, such as, for example, the nature of the protein (e.g., antibody) generated and the time of addition. For example, for antibody 2H7 (variant A), the minimum effective concentration added before cleavage is about 1.3 mM.

[0506] 8. In vitro activity of the enzymatic reduction system

[0507] Fig.40Figure 2 shows a digital gel-like image (each lane represents a time point) from a Bioanalyzer analysis showing the incubation of intact ocrelizumab ("2H7", a humanized anti-CD20 antibody, variant A) (1 mg / mL) with 10 μg / mL hexokinase, 50 μg / mL glucose-6-phosphate dehydrogenase, 5 μM thioredoxin, 0.1 μM thioredoxin reductase, 2 mM glucose, 0.6 mM ATP, 2 mM Mg in 50 mM histidine sulfate buffer, pH 7.38. 2 +, incubation with 2 mM NADP resulted in reduction of ocrelizumab in approximately 1 hour. Addition of 0.1 mM HDEA, a known inhibitor of glucose-6-phosphate dehydrogenase, did not inhibit the reduction.

[0508] 9. The in vitro activity of the enzymatic reduction system requires NADPH

[0509] like Fig.41 As shown in the digital gel-like image from the Bioanalyzer analysis, incubation of intact ocrelizumab (1 mg / mL) with 5 μM thioredoxin, 0.1 μM thioredoxin reductase, and 2 mM NADP in 50 mM histidine sulfate buffer pH 7.38 did not result in reduction of the ocrelizumab antibody. Ocrelizumab reduction cannot occur in the absence of hexokinase and glucose-6-phosphate dehydrogenase and their substrates to generate NADPH.

[0510] The invention described illustratively herein can be implemented in the absence of any one or more components, one or more limitations not explicitly disclosed herein. Thus, for example, in each case herein, the terms "comprise", "include", "contain", etc. should be read as open and without limitation. In addition, the terms and expressions adopted herein are used as descriptive terms rather than restrictions, and the use of such terms and expressions is not intended to exclude any equivalent scheme or part thereof of the invention shown, but it is recognized that various changes within the scope of the claimed invention are possible. Thus, it should be understood that, although the present invention has been clearly disclosed by preferred embodiments and optional features, modifications and changes of the invention disclosed herein can be easily made by those skilled in the art, and such modifications and changes are considered to be within the scope of the invention disclosed herein. The present invention has been described broadly and generally herein. Each narrower category and subcategory grouping falling within the general disclosure also constitutes a part of these inventions. This includes a proviso or negative limitation within the general description of each invention, which allows any subject to be eliminated from the category, regardless of whether the material to be eliminated is specifically described. In addition, when describing features or aspects of the invention in terms of Markush groups, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group. In addition, when referring to a range of individual members listed in one aspect of the invention, such as, for example, "SEQ ID NO: 1 to SEQ ID NO: 100 (including both ends)", it is equivalent to listing each member of the list individually, and it should be understood that each individual member can be excluded or included individually in the claims.

[0511] The steps described and / or used in the methods herein may be implemented in a different order than that described and / or described. These steps are merely illustrative of the order in which these steps may occur. These steps may occur in any order desired so that it still performs the purpose of the claimed invention.

[0512] According to the description of the present invention herein, it is obvious that various equivalent schemes can be used to carry out the concept of the present invention without departing from its scope. In addition, although the present invention has been described with specific reference to certain embodiments, those of ordinary skill in the art will recognize that changes can be made in form and detail without departing from the spirit and scope of the present invention. The described embodiments are considered illustrative and non-restrictive in all respects. It should also be understood that the present invention is not limited to the specific embodiments described herein, but many equivalent schemes, rearrangements, improvements and replacements can be carried out without departing from the scope of the present invention. In this way, other embodiments are also within the scope of the present invention and within the appended claims.

[0513] All U.S. patents and applications; foreign patents and applications; scientific articles; books; and publications mentioned herein are incorporated herein by reference in their entirety as if each individual patent or publication were specifically and individually indicated to be incorporated by reference, including any figures, as if fully set forth.

[0514] SCORE placeholder page for IFW content

[0515] Application Number: PCT / US08 / 69395 Document Date: 07 / 08 / 2008

[0516] The presence of this form in the IFW record indicates that the following document types were received in electronic format on the date identified above. This content is stored in the SCORE database.

[0517] Design drawing

[0518] Since this is submitted electronically, there is no physical artifact folder, no artifact folder is recorded in PALM, and no paper files or physical media exist. The TIFF images in the IFW record are created from the original files stored in SCORE.

[0519] To access the files in the SCORE database, see the instructions prepared by SIRA.

[0520] At file entry (as described above):

[0521] Examiners can access SCORE content through the eDAN interface.

[0522] Other USPTO employees can bookmark the current SCORE URL

[0523] ( http: / / es / ScoreAccessWeb / ).

[0524] External users can access SCORE content via the Public and Private PAIR interface.

[0525] Table revised on: February 8, 2006.

Claims

1. A method for preventing the reduction of disulfide bonds in a polypeptide expressed in a recombinant host cell, comprising supplementing a thioredoxin inhibitor to the culture fluid of the host cell before or after harvest.

2. The method of claim 1, wherein the thioredoxin inhibitor is added to the culture broth prior to harvest.

3. The method of claim 1, wherein the thioredoxin inhibitor is added to the harvested culture fluid.

4. The method of claim 1, wherein the thioredoxin inhibitor is a direct inhibitor of thioredoxin.

5. The method of claim 4, wherein the thioredoxin inhibitor is a alkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative.

6. The method of claim 1, wherein the thioredoxin inhibitor is a specific inhibitor of thioredoxin reductase.

7. The method of claim 6, wherein the thioredoxin inhibitor is a gold complex.

8. The method of claim 7, wherein the gold complex is aurothioglucose (ATG) or aurothiomalate (ATM).

9. The method of claim 8, wherein said ATG or said ATM is added at 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 the concentration of thioredoxin reductase in said pre-harvest or harvested culture broth.

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